Cephalotaxus Alkaloids

Abstract

Cephalotaxus alkaloids represent a family of plant secondary metabolites known for 60 years. Significant activity against leukemia in mice was demonstrated for extracts of Cephalotaxus. Cephalotaxine (CET) (1), the major alkaloid of this series was isolated from Cephalotaxus drupacea species by Paudler in 1963. The subsequent discovery of promising antitumor activity among new Cephalotaxus derivatives reported by Chinese, Japanese, and American teams triggered extensive structure elucidation and biological studies in this family. The structural feature of this cephalotaxane family relies mainly on its tetracyclic alkaloid backbone, which comprises an azaspiranic 1-azaspiro[4.4]nonane unit (rings C and D) and a benzazepine ring system (rings A and B), which is linked by its C3 alcohol function to a chiral oxygenated side chain by a carboxylic function alpha to a tetrasubstituted carbon center. The botanical distribution of these alkaloids is limited to the Cephalotaxus genus (Cephalotaxaceae). The scope of biological activities of the Cephalotaxus alkaloids is mainly centered on the antileukemic activity of homoharringtonine (HHT) (2), which in particular demonstrated marked benefits in the treatment of orphan myeloid leukemia and was approved as soon as 2009 by European Medicine Agency and by US Food and Drug Administration in 2012. Its exact mechanism of action was partly elucidated and it was early recognized that HHT (2) inhibited protein synthesis at the level of the ribosome machinery. Interestingly, after a latency period of two decades, the topic of Cephalotaxus alkaloids reemerged as a prolific source of new natural structures. To date, more than 70 compounds have been identified and characterized. Synthetic studies also regained attention during the past two decades, and numerous methodologies were developed to access the first semisynthetic HHT (2) of high purity suitable for clinical studies, and then high grade enantiomerically pure CET (1), HHT (2), and analogs.

Abbreviations

acac

Acetylacetonate

AChE

Acetyl choline esterase

ACN 1,1′

Azobis(cyclohexanecarbonitrile)

ADP

Adenosine 5′-diphosphate

AIBN 2,2′

Azobisisobutyronitrile

AML

Acute myeloid leukemia

APL

Acute promyelocytic leukemia

Ara-C

Cytosine arabinoside

ATRA

All-trans-retinoic acid

AY

Average yield

BCR-ABL

Breakpoint cluster region and ABL (Abelson)

BID

Bis in die (twice daily)

Boc tert

Butyloxycarbonyl

BP

Blast phase

brsm

Based on recovered starting material

CBz

Carbobenzoxy

CCDC

Cambridge Crystallographic Data Center

CDI

Carbonyldiimidazole

CET

Cephalotaxine

CGAF

Cephalotaxus griffithi alkaloid fraction

CHR

Complete hematologic response

CMC

Cell membrane chromatography

CML

Chronic myeloid leukemia or chronic myelogenous leukemia

CP

Chronic phase

Cp

Cyclopentadiene

CSA

Camphorsulfonic acid

CSD

Cambridge Structural Database

CSE

Conventional solvent extraction

CTA

Cephalotaxinamide

dba

Dibenzylideneacetone

DBU

1,8-Diazabicyclo[5.4.0]undec-7-ene

DCC N,N′

Dicyclohexylcarbodiimide

de

Diastereoisomeric excess

DEPC

Diethyl pyrocarbonate

DIAD

Diisopropyl azodicarboxylate

DIBAL-H

Diisobutylaluminum hydride

DIPEA N,N

Diisopropylethylamine

DMAP 4

Dimethylaminopyridine

DMDO

Dimethyldioxirane

DMF

Dimethylformamide

DMP

Dess–Martin periodinane

DMPU 1,3

Dimethyl-1,3-propylurea (1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone)

DMSO

Dimethyl sulfoxide

DNR

Daunorubicin

DOX

Doxorubicin

dppe

1,3-Bis(diphenylphosphino)ethane

DTBMP 2,6

Di-tert-butyl-4-methylpyridine

EDC N-(3-Dimethylaminopropyl)-N′

ethylcarbodiimide hydrochloride

ee

Enantiomeric excess

EMA

European Medicines Agency

EMP

Embden–Meyerhof pathway

FDA

Food and Drug Administration

G-CSF

Granulocyte colony-stimulating factor

GHI

G-CSF HHT and Imatinib

GMP

Good manufacturing practice

HAA

HHT, aclacinomycin, and Ara-c

HAD

HHT, Ara-c and DNR

Hfc 3

(Heptafluoropropyl-hydroxymethylene)-d-camphorate

HHT

Homoharringtonine, Omacetaxine

H1R

Histamine 1 receptor

HMDS

Hexamethyldisilazane

HMPA

Hexamethylphosphoramide

HOBt

Hydroxybenzotriazole

HPLC

High-performance liquid chromatography

HSP

Heat shock protein

HT

Harringtonine

IBX 2

Iodoxybenzoic acid

IM

Imatinib

Im

Imidazole

IFN-α

Interferon alpha

INN

International nonproprietary name

IUCN

International Union for Conservation of Nature

KHMDS

Potassium hexamethyldisilazide

LDA

Lithium diisopropylamide

LiHMDS

Lithium hexamethyldisilazide

LCL

Long-circulating liposomes

LLS

Longest linear sequence

LOQ

Limit of quantitation

mCPBA

meta-Chloroperoxybenzoic acid

Mcl-1

Myeloid cell leukemia-1

MCyR

Major cytogenetic response

MERS-CoV

Middle East respiratory syndrome coronavirus

MDS

Myelodysplastic syndrome

MHR

Major hematologic response

MJ

Methyl jasmonate

MM

Multiple myeloma

MS

Mass spectrometry

Ms

Methanesulfonyl

MTX

Mitoxantrone

MWE

Microwave extraction

NBS

N-Bromosuccinimide

NCI

National Cancer Institute

NCS

N-Chlorosuccinimide

n.d.

Not determined

n.r.

Not reported

NIS

N-Iodosuccinimide

NMM

N-Methylmorpholine

NMO

N-Methylmorpholine N-oxide

NMR

Nuclear magnetic resonance

NOESY

Nuclear Overhauser effect spectroscopy

NS

Number of steps

NsCl 4

Nitrobenzenesulfonyl chloride

OM

Omacetaxine

OY

Overall yield

PARP

Poly(ADP-Ribose) polymerase

PCC

Pyridinium chlorochromate

PDC

Pyridinium dichromate

PEG

Polyethyleneglycol

Ph

Philadelphia (biology) or phenyl (chemistry)

Ph+

Philadephia-positive chromosome

Piv

Pivaloyl

PPA

Polyphosphoric acid

PPL

Porcine pancreas lipase

p-TsCl

p-Toluenesulfonic chloride

p-TsOH

p-Toluenesulfonic acid

pyr

Pyridine

QCC

Quaternary carbon center

RCM

Ring closing metathesis

re

Regioisomeric excess

Red-Al

Sodium bis(2-methoxyethoxy)aluminum hydride

RI

Resistance index

rt

Room temperature

SAR

Structure–activity relationship

SC

Subcutaneous

SNP

Single nucleotide polymorphism

ssHTT

Semisynthetic HHT

TBAF

Tetrabutylammonium fluoride

TBAI

Tetrabutylammonium iodide

TBAT

Tetrabutylammonium difluorotriphenylsilicate

TBDMSCl

tert-Butyldimethylsilyl chloride

TBDPSCl

tert-Butyldiphenylsilyl chloride

TCM

Traditional Chinese medicine

TEA

Triethylamine

TEMPO

(2,2,6,6-Tetramethylpiperidin-1-yl)oxy

Tf

Triflate

TFA

Trifluoroacetic acid

TFAA

Trifluoroacetic acid anhydride

TfOH

Triflic acid

THF

Tetrahydrofuran

TMEDA

Tetramethylethylenediamine

TMG

1,1,3,3-Tetramethylguanidine

TMS

Trimethylsilyl

Ts

p-Toluenesulfonyl

1. Introduction

This contribution is an updated and expanded version of the two preceding chapters published in this series in 1984¹ and 1998.² Other reviews focused on the structure and synthesis of these compounds,3, 4, 5, 6 and several reviews are available that cover the structure–activity relationships (SARs), biological, and clinical properties of these alkaloids.7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 The literature is reviewed from the last update in this series, which was covering the field till mid-1997, to early 2016. Because they usually exist under the form of unique enantiomer, we numbered the natural products by an arabical digit irrespective to their optical rotation sign, e.g., cephalotaxine (CET) (1), their antipode by ent-digit, e.g., ent-cephalotaxine ent-(1), and the corresponding racemic compound usually obtained by chemical synthesis rac-digit, e.g., rac-cephalotaxine rac-(1). In the asymmetric synthesis section, we have precised the sign of the optical rotation, e.g., CET (−)-(1), ent-CET (+)-(1), and rac-CET (±)-(1).

For more than 50 years, chemists have studied the extraction, structure determination, biogenesis, and synthesis of CET (1) (Fig. 2), the major alkaloid of this series, and related alkaloids, because of the reported biological activity of its natural esters 2–5, in view of their therapeutical use in the treatment of leukemia. Homoharringtonine (HHT) (2) and harringtonine (HT) (3), or a mixture of both, have been used in traditional Chinese medicine (TCM) since the 1970s to treat a variety of malignancies. HHT (2) being the easiest to purify and the most abundant in the plant was selected by the National Cancer Institute (NCI) for clinical development. HHT (2), also known as omacetaxine, a simple ester of CET (1), probably holds the record for the longest time to reach the market²⁰ after having experienced increasing interest since its first use in TCM, and efforts by pharmaceutical companies for its development as an antileukemic agent. Despite its clinical efficacy, natural HHT (2) was not recorded until now, simply because normal extract formulations have raised several unresolved problems, such as the reproduction of a constant product from defined extraction procedure, the purity profile, the main source of the raw material in China, and biodiversity conservation issues. The Cephalotaxus shrubs with extremely slow growth are mostly protected and endangered species. Moreover, CET (1) and its active esters are present in small quantities in the plant (roots, bark, heartwood, and leaves), which hardly makes possible to exploit it to obtain HHT (2) or its related esters. Semisynthetic HHT (ssHHT) (2) (Myelostat, Ceflatonin, or Omacetaxine mepesuccinate (OM)) is manufactured by a semisynthetic process developed by Robin from naturally occurring alkaloid CET (−)-(1) extracted from the dry leaves of Cephalotaxus harringtonia, and a synthetic precursor side chain.²¹ Although HHT (2) was investigated for the treatment of chronic myeloid leukemia (CML) in the 1990s with good results, the advent of Imatinib (IM) mesylate, a breakpoint cluster region and Abelson (BCR-ABL)1 tyrosine kinase inhibitor (TKI) at that time delayed the interest for HHT (2). However, de novo or acquired resistance to TKIs²² renewed the interest for this alternative first-in-class simple natural ester of CET (1). HHT (2) is currently approved in Europe and in the United States, due to its reported activity in patients with CML resistant to currently available TKIs. As a result, on September 2, 2004, orphan designation (EU/3/04/224) was granted by the European Union for OM (International nonproprietary name (INN), trade name Synribo)/HHT (2) for the treatment of CML to Stragen France SAS, France, for the treatment of CML. Exploitation rights were transferred to ChemGenex Europe SAS, France, in January 2009 and subsequently to Teva Pharma GmbH, Germany, in December 2012. Due to their different mechanisms of action, combinations with TKIs represent attractive treatment options. Recently, on October 26, 2012, HHT (2), Synribo (formerly Ceflatonin, Omapro), received an accelerated approval by the US Food and Drug Administration (FDA) for adult patients with chronic-phase (CP) or accelerated-phase (AP) CML with resistance or intolerance to two or more TKIs. The US FDA has granted full approval to HHT (2) (drug substance INN OM, Synribo) for injection, based on the final analysis of two phase II trials evaluating the efficacy and tolerability data of omacetaxine in 2014. This renewed interest for isolation, characterization, biological evaluation, and total synthesis of cephalotaxus alkaloids and analogs to suppress the dependance on natural source supply.

Figure 2.

Cephalotaxine (1) and its natural esters (2–5) early characterized in Cephalotaxus harringtonia var. drupacea.

2. Natural Occurrence, Isolation, and Structural Assignment

As early as 1954, Wall brought to light the presence of alkaloids in plants of the genus Cephalotaxus,²³ the only genus of the Cephalotaxaceae family composed of 11 species of evergreen small and only slowly growing conifers, which are mostly indigenous to China (Fig. 1 ). The first alkaloid characterized from Cephalotaxus harringtonia var. drupacea (Cephalotaxus drupacea) and Cephalotaxus fortunei was named CET by Paudler in 1963²⁴ and its original benzazepine nucleus fused to an azaspirononane unit established by Powell et al (Fig. 2 ).²⁵

Figure 1.

Scientific classification of Cephalotaxus genus and picture (from F. Dumas) of the fruits and leaves of Cephalotaxus fortunei at Parc Montsouris, Paris (France).

Although its biological activity and partial structure are known since 1959,²⁶ the elucidation of the structure of natural HT (3) (Fig. 2) isolated from C. harringtonia var. drupacea and antitumor activity against L1210 and P388 leukemia in mice (1 mg/kg) in 1969²⁵ stimulated the continuous search for active compounds and particularly for alkaloids from the Cephalotaxus genus. Apart from the CET series focused in this chapter, the other alkaloid part in Cephalotaxus species belongs to the class of homoerythrinane alkaloids, isolated as minor compounds with the exception of Cephalotaxus wilsoniana. Biogenesis of CET (1) postulated by Parry in 1977 showed a common origin for these alkaloids.1, 2, 27

Cephalotaxanes (6) is the generic name for the particular alkaloids of the Cephalotaxaceae family, which have a general formula derived from the cephalotaxane skeleton with either one or two units covalently linked forming dimers having two identical or different units (Fig. 3 ). Cephalotaxanes (6) include neutral compounds or salts having this basic skeleton in common and may contain various oxygenated substituents (aliphatic ethers or aromatic units, free or esterified alcohols, substituted or free enols, phenol, and others). Cephalotaxanes include CETs characterized by a free alcohol function on ring D (R = H), and HTs where the 3-OH group is esterified (R ≠ H). Isolation of cephalotaxanes and homoerythrinane alkaloids [skeleton (7)] from the same plant suggested that they might be formed through similar biogenetic routes.1, 2 Hainanensine (8) is a structurally unique Cephalotaxus alkaloid devoid of optical activity and lacking the spiranic tetrasubstituted carbon center. It was isolated from Cephalotaxus hainanensis and C. fortunei (Fig. 3) and displays a type of single rearranged CET skeleton.²⁸

Figure 3.

Cephalotaxanes (6), the skeleton (7) of minor homoerythrinane alkaloids, and the structurally unique Cephalotaxus alkaloid hainanensine (8).

Until 1997, 36 compounds related to the CET series (Table 1, Table 2 ), were described, of which 23 are natural esters and 1 is dimeric.

Table 1.

Cephalotaxus alkaloids that have been described from natural sources till mid-19971, 2

Cephalotaxine analogs

Cephalotaxine (1), C18H21NO4, mp 132–133°C, [α]D −183 (c 0.22, EtOH)

Hainanensine (8), C17H17NO4, mp 240–244°C, [α]D 0 (CH3OH)

Epicephalotaxine (9), C18H21NO4, mp 136–137°C, [α]D −150 (c 0.8, CHCI3)

Cephalotaxinone (10), C18H19NO4, mp 198–200°C, [α]D −146 (c 0.63, CHCI3)

Desmethylcephalotaxine (11), C17H19NO4, mp 109–111°C, [α]D −110 (c 0.28, CH3OH)

Demethylcephalotaxinone (12), C17H17NO4, mp 102–107°C, [α]D +2.3 (c 0.52, CH3OH)

Isocephalotaxinone (13), C18H19NO4, mp 130.5–132°C, ${{\left[\mathrm{\alpha }\right]}^{21}}_{\text{D}}$ −47 (c 0.5, CHCI3)

Oxyoenated cephalotaxines

11-Hydroxycephalotaxine (14), C18H21NO5, mp 235–242°C, [α]D −139 (c 0.56, CHCI3)

4-Hydroxycephalotaxine (15), C18H21NO5, m. 135–137°C, ${{\left[\mathrm{\alpha }\right]}^{27}}_{\text{D}}$ +120 (c 0.025, CH3OH)

Drupacine (16), C18H21NO5, mp 70–72°C, [α]D −137°C (c 0.79, CHCI3)

Demethylneodrupacine (17), C17H19NO5, mp 197–200°C

Cephalotaxinamide (18), C18H19NO5, mp 230°C

Table 2.

Natural Cephalotaxus alkaloid esters that have been described up to May 19971, 2

Harringtonines

Homoharringtonine (2), C29H39NO9, mp 144–146°C, [α]D −119 (c 0.96, CHCI3)

Harringtonine (3), C28H37NO9, mp 73–75°C, [α]D −104.6 (c 1.0, CHCI3)

Isoharringtonine (4), C28H37NO9, mp 69–72.5°C, [α]D −99.6 (c 1.06, CHCI3)

Deoxyharringtonine (5), C28H37NO8, amorphous, [α]D −125.4 (c 1.76, CHCI3)

Neoharringtonine (19), C30H33NO8, oil, [α]D −148 (c 0.48, MeOH)

Anhydroharringtonine (20) C28H35NO8,[α]D −144 (c 1.08, CHCI3)

Nordeoxyharringtonine (21), C27H36NO8, oil, [α]D −90 (c 0.07, MeOH)

Homodeoxyharringtonine (22), C29H39NO8, oil, [α]D −122 (c 1.00, MeOH)

Bishomodeoxyharringtonine (23), C30H41NO8, oil, [α]D −74 (c 0.27, CHCI3)

Homoneoharringtonine (24), C31H36NO8, oil, [α]D −114 (c 0.07, MeOH)

(2′R,3′S)-Hydroxyneoharringtonine (25), C30H33NO9, oil, [α]D −126 (c 0.20, MeOH)

Other esters of cephalotaxine

(+)-Acetylcephalotaxine (+)-(26), C20H23NO5, mp 140°C, [α]D +102 (CHCI3), [α]D +130 (EtOH)

(−)-Acetylcephalotaxine (−)-(26), C20H23NO5, mp 141–143°C, [α]D −186 (c 0.43, EtOH)

5′-Des-O-methylisoharringtonine (Isoharringtonic acid) (27), C27H35NO9, mp 224–228°C, [α]D −113(c 0.33, DMSO)

3′S-Hydroxy-5′-des-O-methylharringtonine (Cephalozemic C acid) (28), C27H35NO10, amorphous solid, [α]D −91 (c 1.00, DMSO)

Deoxyharringtonic acid (29), C27H35NO8, mp 219–220 °C, [α]D −91 (c 1.00, DMSO)

5′-Des-O-methylharringtonine (harringtonic acid) (30), C27H35NO9, amorphous solid,[α]D −113 (c 0.43, DMSO)

5′-Des-O-methylhomoharringtonine (homoharringtonic acid) (31), C27H37NO9, amorphous solid, [α]D −172 (c 0.50, MeOH)

Homoharringtonamide (32), C29H37NO9

Esters of oxygenated cephalotaxine analogs

Drupangtonine (33), C28H37NO9, oil, [α]D −24 (c 0.06, MeOH)

11α-Hydroxyhomodeoxyharringtonine (34), C29H39NO9, oil, ${{\left[\mathrm{\alpha }\right]}^{25}}_{\text{D}}$ −115 (c 0.07, MeOH)

11β-Hydroxyhomodeoxyharringtonine (35), C29H39NO9, oil, ${{\left[\mathrm{\alpha }\right]}^{25}}_{\text{D}}$ −153 (c 0.10, MeOH)

11-Hydroxydeoxyharringtonine (36), C28H37NO9, oil, ${{\left[\mathrm{\alpha }\right]}^{25}}_{\text{D}}$−77 (c 0.0085, MeOH)

Dimeric ester

Cephalotaxidine (37), C58H74N2O19, Amorphous solid, [α]D −172 (c 0.10, MeOH)

CET (1) and HHT (2) were also isolated conventionally from fruits, leaves, and branches of Cephalotaxus sinensis.²⁹ Among the alkaloids derived from CET (1), compounds possess different D rings such as cephalotaxinone (10), demethylcephalotaxine (11), and isocephalotaxinone (13) and ring C such as cephalotaxinamide (18). Oxygenated compounds with modifications on their B ring as 11-hydroxycephalotaxine (14) and 4-hydroxycephalotaxine (15) or compounds having an oxygen bridge between the rings B and D such as drupacine (16) were also described (Fig. 4 ).30, 31, 32 11-Hydroxycephalotaxine (14) was converted to drupacine (16) in acidic medium.³¹ Among the minor alkaloids isolated from the bark of C. hainanensis Li (in addition to 11 known alkaloids), CET analogs were identified as cephalotaxinone (10), cephalotaxinamide (18), and demethylneodrupacine (17), which possesses an ether bridge between C11 and C3, while drupracine (16) exhibits an ether bridge between C11 and C2. The structure of desmethylcephalotaxinone (12), isolated from Cephalotaxus harringtonia (Forbes) K. Koch var. harringtonia cv. fastigiata, was determined by semisynthesis from CET (1) via oxidation to cephalotaxinone (10) and cleavage of the enol ether thus confirming desmethyl CET (12) as a natural metabolite and biosynthetic intermediate.³³

Figure 4.

Oxidized cephalotaxines.

Repeated chromatography of an alkaloidal fraction of the ethanolic extract of Cephalotaxus fortunei Hook f.³⁴ afforded as minor component (+)-4-hydroxycephalotaxine (+)-(15) recently also identified in Cephalotaxus koreana Nakai.³⁵ Epicephalotaxine (9) is another minor metabolite of C. fortunei Hook f. accompanied by CET (1) (50%–54% of the total alkaloids), cephalotaxinone (10), (+)-acetylCET (+)-(26), demethylCET (11), HT (3), and HHT (2).³⁶

Molecules coupled with side chains bearing an ester function as in nordeoxyharringtonine (nordeoxyHT) (21), homodeoxyharringtonine (homodeoxyHT or deoxyHHT) (22), and bishomodeoxyharringtonine (bishomodeoxyHT) (23) are found among HTs (Fig. 5 ).³⁷

Figure 5.

Natural esters of cephalotaxine and cephalotaxinamide.

Including esters 2–5, these compounds are esters of CET (1) having its polycyclic core coupled to different side chains deriving from (R)-citramalic acid. Although occurrence of enantiomeric pairs in the same family of plants is unusual,³⁸ acetylcephalotaxine (acetylCET) (26) was reported under both enantiomeric forms. (−)-AcetylCET (−)-(26) was obtained by semisynthesis through acetylation of natural CET (−)-(1)²⁴ and was isolated from C. fortunei ³⁰ and C. wilsoniana. 39, 40

The opposite enantiomer (+)-acetylCET (+)-(26) was isolated in C. fortunei ⁴¹ and C. hainanensis. ⁴² Although an error cannot be excluded in literature data, these findings need to be confirmed since this natural product (+)-26 is the only one portraiting an opposite absolute configuration (AC) of alkaloid core.

Anhydroharringtonine (20) bearing an additional heterocycle in the side chain is the only natural ester of this type.⁴³ It induced 98% growth inhibition of P388 leukemia cell lines at 1 μg/mL, a level comparable to that of deoxyHT (5). There are also molecules coupled with side chains carrying a free acid group (Fig. 5): 5′-des-O-methylHT (30) (harringtonic acid), (2′R,3′S)-3′-hydroxy-5′-des-O-methylHT (28) (cephalozemic acid C), 5'-des-O-methylHHT (31) (homoharringtonic acid, also identified as a metabolite of HHT (2) in rats and rabbits) and 5′-des-O-methylisoHT (27) (isoharringtonic acid).1, 2 Others possess an ester function and an aromatic ring, as in homoneoHT (24) and (2′R,3′S)-hydroxyneoHT (25).⁴⁴ Some of these esters deliver biological activities comparable to those of the first four isolated esters 2–5, but they are available in much lower quantities in the plant. The first dimeric Cephalotaxus alkaloid, cephalotaxidine (37), was isolated from C. harringtonia var. drupacea (C. drupacea) in 1996.⁴⁵ It is a dimer of HHT (2) and homoharringtonamide (32) with an IC₅₀ of 1.8 μg/mL against P-388 murine leukemic cell lines. Drupangtonine (33)⁴⁶ was the most active (IC₅₀ 7 nM against P-388 murine leukemic cell lines) of the four esters of oxygenated CET analogs, 11α-hydroxy-homodeoxyHT (34), 11β-hydroxyhomodeoxyHT (35), and 11β-hydroxydeoxyHT (36) (IC₅₀ 0.38, 0.33, and 0.17 μg/mL, respectively, against P-388 murine leukemic cell lines) identified from C. harringtonia var. drupacea (Fig. 6 ).⁴⁷

Figure 6.

Natural esters of oxygenated cephalotaxines.

Since the last reviews in this series,1, 2 more than 30 Cephalotaxus alkaloids have been isolated from various Cephalotaxus sp. These new alkaloids include the following compounds.

2.1. Cephalotaxine and Related Alkaloids

N-oxides of CET, CET α N-oxide (38), CET β N-oxide (39), and 11β-hydroxycephalotaxine β N-oxide (40), together with isocephalotaxine (41) were identified and characterized in the ethyl acetate extract of C. fortunei fruits by Jossang et al. in 2001 (Fig. 7 ).⁴⁸ Although it was described as a synthetic compound earlier,⁴⁹ the relative configuration of isocephalotaxine (41) isolated during this work remains unknown due to the lack of Nuclear Overhauser effect spectroscopy (NOESY) correlations. These alkaloids exhibited low cytotoxicity against nasopharynx KB cells with IC₅₀ values of 30, 14, 31, and 15 μg/mL, respectively. ACs of cephalezomines G (42) and H (43) described by Kobayashi and Morita⁵⁰ from the leaves of Cephalotaxus harringtonia var. harringtonia were determined by circular dichroism (CD) as 2S,3R for cephalezomine G (42) and 2R,3R for cephalezomine H (43). However, the structure of cephalezomine H (43), initially assigned as a trans diol was revised to a syn-β,β-diol after total synthesis (Fig. 7).⁵¹ Some uncertainty remained, however, due to the divergent optical rotations of the synthetic and the natural compounds.

Figure 7.

New N-oxides and ring D analogs of cephalotaxine.

A new stereoisomer (44) of desmethylcephalotaxinone (12) has been isolated from the leaves and heartwood of Formosan C. wilsoniana (Fig. 8 ).⁵² Although the UV, 1H NMR, and ${{\left[\mathrm{\alpha }\right]}^{23}}_{\text{D}}$ of compound 44 were similar to those of natural desmethylcephalotaxinone (12), C1 to C7 and C10 signals in the ¹³C NMR spectrum differ from the one of the synthetic sample of 12.¹⁰⁰ Evidence that compound 44 was a stereoisomer of desmethylcephalotaxinone (12) was also based on H1α and H6α cross-peak in the NOESY correlation chart of 44. While this correlation seems doubtful, since only one stereogenic carbon center is present in structures 12 and 44, these findings imply that the second stereogenic center is the nitrogen atom that lost its fast inversion properties, as observed in the X-ray studies showing the rigid structure of CET (1).⁵³ Compound 44 showed no cytotoxic activity against the Hep G2, MCF-7, Hep 3B, and HT-29 human cancer cell lines.

Figure 8.

Desmethylcephalotaxinone stereoisomer and cephalancetines A–D.

A phytochemical investigation of the branches and leaves of Cephalotaxus lanceolata, a Cephalotaxus species native to Yunnan Province, China, resulted in the isolation of three new Cephalotaxus alkaloids, cephalancetines A (45) and B (46), and unsymmetrical dimer cephalancetine D (47) together with known cephalancetine C (48) isolated for the first time from this plant, along with nine other known alkaloids (Fig. 8).⁵⁴ The structure of cephalancetine C (48) was unambiguously confirmed by single-crystal X-ray diffraction as consisting of CET (1) linked at C8 to C7 (C26) of cephalotaxinamide (18) thus establishing its AC as (3S,4S,5R,8S,22S,23S,24R,26S). Cephalancetine D (47) is a dimer consisting of drupacine (16) linked at C8 to C7 (C26) of cephalotaxinamide (18). Cephalancetine C (48) was identical to the saponification product of bis-cephalezomines (see Fig. 12) with LiOH.⁵⁵

Figure 12.

Dimeric Cephalotaxus esters.

All these isolated alkaloids were tested for their cytotoxicities against four human tumor cell lines, A549, HCT116, SK-BR-3, and HepG2 that evidenced that HT (3) and HHT (2) can strongly inhibit the proliferation of these human tumor cell lines as well, however with limited potency against SK-BR-3 cells (Table 3 ).⁵⁴

Table 3.

Cytotoxic activities {means ± S.D}⁵⁴

Compounds	IC50 (μg/mL)
	A549	HCT116	SK-BR-3	HepG2
Cephalancetine A (45)	>100	>100	>100	>100
Cephalancetine B (46)	>100	93.06 ± 13.15	>100	>100
Cephalancetine C (48)	>100	>100	74.15 ± 8.65	>100
Cephalancetine D (47)	>100	>100	>100	>100
CET(1)	85.67 ± 16.75	22.65 ± 13.86	>100	71.94 ± 12.30
HHT (2)	0.085 ± 0.018	0.001 ± 0.002	>100	0.087 ± 0.024
HT (3)	0.15 ± 0.07	0.054 ± 0.016	>100	0.38 ± 0.09
Cephalotaxinone (10)	>100	93.06 ± 5.02	>100	>100
4-HydroxyCET (15)	>100	>100	>100	>100
Drupacine (16)	28.29 ± 7.50	2.61 ± 0.78	>100	3.95 ± 0.85
Cephalotaxinamide (18)	>100	45.47 ± 4.57	>100	>100
Cephalotaxine α-N-oxide (38)	>100	>100	>100	>100
Cephalotaxine ß-N-oxide (39)	>100	51.41 ± 7.31	61.31 ± 15.37	>100
Adriamycin (doxorubicin)	0.079 ± 0.007	0.085 ± 0.008	0.039 ± 0.007	0.014 ± 0.004

CET, cephalotaxine; HT, harringtonine; HHT, homoharringtonine.

HT (3) showed remarkable cytotoxicity against A549, HCT116, and HepG2 cell lines with IC₅₀ values of 0.15, 0.054, and 0.38 μg/mL, respectively. HHT (2) also exhibited potent cytotoxicity against A549, HCT116, and HepG2 cell lines with IC₅₀ values of 0.085, 0.001 and 0.087 μg/mL, respectively, 85 times stronger than that of doxorubicin (DOX) against HCT116 cell line. Comparing the relationship between the structures with their cytotoxicities, the ester side chains at C3 of the CET skeleton seem to play the most prominent role in the cytotoxic activity of these alkaloids.

Four new cephalotaxus alkaloids, cephalotines A–D (49)–(52) were isolated from the leaves and twigs of C. lanceolata and Cephalotaxus fortunei var. alpina (Yunnan province, P.R. China) along with 24 known alkaloids (Fig. 9 ). None of these compounds showed significant activity against HeLa, SGC-7901 gastric cancer, and A-549 lung cancer cell lines (IC₅₀ > 20 μM).⁵⁶

Figure 9.

Cephalotines A–D.

2.2. Natural Cephalotaxus Esters

In 2000, Kobayashi et al. isolated from the leaves of Cephalotaxus harringtonia var. nana new cytotoxic alkaloids, cephalezomines A (53) and B (54), which are esters of drupacine (16), as well as cephalezomines C–F (55)–(58), which are esters of CET (1) (Fig. 10 ).⁵⁷ These new compounds were found together with seven known alkaloid esters, HT (3), isoHT (4), deoxyHT (5), homodeoxyHT (22), CET (1), and related CET alkaloids, demethylCET (11),³⁰ 11β-hydroxyCET (14), and drupacine (16). Their structure and stereochemistry were elucidated by spectroscopic data, CD, and X-ray diffraction analysis.

Figure 10.

Cephalozemines A–F and K–L and their yield from the natural sources in brackets.

Among the new Cephalotaxus alkaloids possessing a CET type backbone with various side chains at C3, cephalezomine D (56) is the first one with (2′R,3′R) AC. In this study⁵⁷ the team of Kobayashi also described the cytotoxicity of these alkaloids against L1210 and KB cell lines (Table 4 ). Two years later, Kobayashi and Morita reported the isolation of five new alkaloids, cephalezomines G, H, and J–L from the leaves of C. harringtonia var. nana (Sapporo, Japan). Among them, cephalezomines K (59) and L (60) are esters of CET (1) epimers at C4” (Fig. 10).⁵⁸

Table 4.

Cytotoxicity of cephalotaxines (CETs), harringtonines (HTs), and cephalezomines against L1210 and KB cell lines57, 58

Compounds	IC50 (μg/mL)
	L1210	KB
Cephalotaxine (1)	3	0.90
11-hydroxyCET (14)	2.4	0.75
DemethylCET (11)	3.8	0.87
Drupacine (16)	0.84	0.99
HT (3)	2	0.74
IsoHT (4)	0.14	0.22
DeoxyHT (5)	0.0082	0.0079
HomodeoxyHT (22)	0.014	0.010
Cephalezomine A (53)	0.067	0.020
Cephalezomine B (54)	0.030	0.024
Cephalezomine C (55)	0.88	0.078
Cephalezomine D (56)	7.6	0.40
Cephalezomine E (57)	0.68	0.18
Cephalezomine F (58)	0.10	0.084
Cephalezomine G (42)	8	>30
Cephalezomine H (43)	8.6	>30
Cephalezomine J (52)	12	5.6
Cephalezomine K (59)	1.2	0.036
Cephalezomine L (60)	3.6	0.044

In 2010, Morita et al. characterized from the leaves of Cephalotaxus harringtonia forma fastigiata a new alkaloid, cephastigiamide A (61),⁵⁹ ${{\left[\mathrm{\alpha }\right]}^{23}}_{\text{D}}$ −55 (c 0.1, MeOH), along with CET (1), cephalotaxinamide (18), bis-cephalezomine A (65), homodeoxyHT (22), and two HHT N-oxides, HHT α N-oxide (62) and HHT β N-oxide (63), previously prepared by Takano60, 61 for SAR studies (Fig. 11 ). HHT (2) and HT (3) showed pronounced antiplasmodial activity against Plasmodium falciparum 3D7 but to a lesser extent against Leishmania major (Table 5 ), while deoxyHT (5) and homodeoxyHT (22) were active in the two tests.

Figure 11.

Oxygenated Cephalotaxus esters and Cephalozemine J.

Table 5.

Biological evaluation of Cephalotaxus alkaloids against Plasmodium falciparum 3D7, Leishmania major, and human epidermoid carcinoma cells A549⁵⁹

Compounds	IC50 (μg/mL)
	P. falciparum 3D7	L. major	A549	SI
CET (1)	17.08	67.90	>31.75	>1.9
Cephalotaxinamide (18)	0.67	3.43	17.80	26.6
Cephalezomine B (54)	0.040	NT	1.78	44.5
Cephalezomine E (57)	0.071	NT	4.79	67.5
Cephalezomine F (58)	0.66	NT	4.41	6.7
Cephastigiamide A (61)	17.82	117.36	>18.35	>1
Cephalocyclidine A (72)	>31.55	NT	>63.00	>2
Bis-cephalezomine A (65)	1.99	NT	>18.30	>9.2
HHT (2)	0.012	1.36	0.084	7.0
HT (3)	0.0043	2.00	0.14	32.6
isoHT (4)	0.045	2.00	3.88	86.2
deoxyHT (5)	0.0014	0.039	0.021	15.0
HHT α N-oxide (62)	0.53	28.97	5.20	9.8
HHT ß N-oxide (63)	0.27	27.54	4.30	15.9
HomodeoxyHT (22)	0.0018	0.095	0.068	37.8
CD	0.011	–	–	–
AmB	–	0.14	–	–
Taxol	–	–	0.0014	–

AmB, Amphotericin B, Chloroquine, and Amb are positive control for the assays of antiplasmodial and antileishmanial activity respectively; CD, chloroquosine diphosphate; CET, cephalotaxine; HT, harringtonine; HHT, homoharringtonine; NT, not tested; Taxol, positive control for cytotoxicity assay, SI: selectivity index (a measure of the IC₅₀ values obtained against human A549 cell divided by the IC₅₀ against P. falciparum 3D7).

HHT α N-oxide (62) and HHT β N-oxide (63) reported for the first time as natural products, did not display any antileishmanial or cytotoxic activity against A549 cell line, despite of an esterified 3-OH function in their structures (Table 5). SAR directed toward antiplasmodial activity of isolated Cephalotaxus alkaloids was also discussed. Cephalezomine J (64) from the leaves of C. harringtonia var. nana (Fig. 11) is the unique CET conjugate with a sugar type side chain (d-glucose).⁵⁰ Although the data collected in this study are cytotoxicity values (Table 5), a selectivity index was calculated to rate this effect on human versus parasitic cell lines. In all the cases, the cytotoxicity was greater against parasites, rather on tumor cells (SI > 1).

In 2009, Kobayashi et al. isolated five new dimeric alkaloids from C. harringtonia var. nana, especially heterodimers, named bis-cephalezomines A–E (65)–(69) (Fig. 12 ).⁵⁵ The alkaloids that constitute the dimers (HHT (2), HT (3), deoxyHT (5), and cephalezomine A (53)) showed potent cytotoxic activity, whereas dimers were far less active against L1210 murine lymphoma cells, with IC₅₀ values in the range of 1.9–3.7 μg/mL.

2.3. Other Alkaloids

The new alkaloid cephalocyclidin A (72) with an unprecedented polycyclic rearranged skeleton derived from CET (1) and six contiguous asymmetric centers was isolated from fruits of C. harringtonia var. nana.⁶² ACs at C2 and C3 were assigned by the exciton chirality method as 2R and 3S, respectively, indicating 1R,2R,3S,4S,5R,11S configuration, compatible with the absolute stereochemistry of the hydrochloride of CET (1) deduced through the Flack parameter of 0.01(5), in the X-ray analysis. It could be derived biogenetically by formation of the C1–C11 link of 71 by an intramolecular aldol reaction of the putative intermediate 11-oxodemethylcephalotaxine (70) followed by reduction (Scheme 1 ). The in vitro cytotoxicity (IC₅₀) of cephalocyclidin A (72) against the L1210 mouse lymphoma and human epidermoid carcinoma cells KB is 0.85 and 0.80 μg/mL respectively.

Scheme 1.

Proposed biosynthetic pathway to cephalocyclidine A.

2.4. Production and Purification Studies

The proportion of the four major natural esters 2–5 with antitumor activity is between 0.7% in the needles of C. fortunei and 36% in the roots of C. harringtonia var. harringtonia of the total amount of alkaloids. This plant is the best natural source for these four compounds of pharmacological interest. At present, the plants of the genus Cephalotaxus are endangered species and face the threat of extinction. Only the renewable parts of the plant are used for the extraction of alkaloids. For example, the use of modern methods of extraction provides up to 2 kg of pure CET (1) from a ton of dry leaves, that is 0.2% of dry matter. Robin et al. studied a method of preparation of HHT (2) by semisynthesis from natural CET (1) by attachment of the side chain (see Section 3). Purification provided a product, the purity of which was 99.8% against 98.5% for NCI products and 95%–97% for products of Chinese origin (Table 7).⁶³ For example, 550 g of HHT (2) was injected into a preparative high-performance liquid chromatography (HPLC) system (diameter 45 cm, height 1 m containing 48 kg of ODS stationary phase). The elution was performed using a gradient of buffered solution of pH 3 methanol-water as mobile phase (flow rate 540 L/h, 1200 psi). The collected fractions (20–50 L) were separated by UV detection and analyzed by HPLC. After eliminating fractions with about 0.5% of the total content of HHT (2), fractions suitable for drug specification (purity > 99.8%) were combined. HPLC analysis of the organic phase after HHT recovery (basified to pH 8.5 and extracted with dichloromethane) showed a level of impurities less than 0.5%. HHT (2) (210 g) was then recrystallized by dissolution in 240 mL of methanol at 30°C and filtered at 0.25 microns for sterilization, and then deionized water (2.4 L) was added and methanol was distilled off. The aqueous solution of HHT (2) DS (quality “drug substance”) or “drug substance” quality (DS) was held in a decontaminated rotary evaporator until the appearance of white crystals of pure HHT (2). After filtration and drying under vacuum at 60°C for 2 days, 88% overall yield (OY) of HHT (2) was obtained when compared to its potential content in the semisynthetic gross HHT (2). This corresponds to a clinical batch allowing for the preparation of 40,000 therapeutic dosage units containing 5 mg containing less than 0.03% of impurities.⁶³

Table 7.

Impurities of commercial batches of homoharringtonine (HHT) (2) detected by He's high-performance liquid chromatography method⁷³

Batch number	Impurities %
	HT(3)	α-isoHHT (73)	Ethyl-HHT (74)	Total impurities	HHT (2) purity %
NCI 800528	0.1	1.3	0	1.4	98.6
NCI 971203	0	1.0	0	1.0	99.0
NCI 921115	0.3	3.0	0.8	4.1	95.9
NCI 960625	0	1.8	0.3	2.1	97.9
NCI 800722	0.1	0.9	0.2	1.2	98.8
NCI KS-22-130-2	0	1.5	0.9	2.4	97.6
NCI batch average	0.1	1.6	0.4	2.0	98.0
Oncopharm batch average	0.00	0.00	0.00	<0.05	>99.95

HT, harringtonine; NCI, National Cancer Institute.

Numerous methods were described for the extraction and purification of CET (1), its natural esters, and related analogs. Extraction of fruits, leaves, or branches of C. sinensis with 90% ethanol under refluxing conditions followed by acid base treatment and purification on alumina allowed for the isolation of CET (1) and HHT (2).⁶⁴

High speed countercurrent chromatography, a form of liquid–liquid partition chromatography in which the stationary liquid phase is retained in the apparatus without the use of a solid support, was performed with a pH gradient elution to shorten the duration of the separation and improve resolution, allowing a very efficient separation of crude alkaloid extract (800 mg) from C. fortunei to yield drupacine (16) (9.3 mg), wilsonine (15.9 mg), CET (1) (130.4 mg), epi-wilsonine (64.8 mg), fortuneine (12.8 mg), and acetylCET (26) (35.6 mg) with respective purities of 81.2%, 85.7%, 95.3%, 97.5%, 89.1%, and 96.2%.⁶⁵ The recovery of each alkaloid was greater than 90%. The extraction step plays an important role in the overall purification process of HHT (2). Traditional methods of isolation and purification of the chemical constituents of plant tissues have some disadvantages. These approaches require very long extraction times and usually a large amount of solvent with sometimes low efficiency. Furthermore, many natural products are thermally unstable and may degrade during hot extraction. To overcome these drawbacks, a new method of extracting HHT (2) from Cephalotaxus koreana using microwave extraction (MWE) was developed by Kim et al.⁶⁶ The MWE process of HHT (2) recovery (biomass/MeOH 1/6 m/v, 250 rpm) gave a performance of the first biomass extraction 25% higher than the conventional solvent extraction (CSE) method for which at least four extractions were required. It was possible to recover more than 99% of HHT (2) extracting once the biomass by MWE at 40°C for 15 min or 50°C for 5 min. Because the content of tars and waxy compounds were also increased, an adsorbent was added to the biomass (1/1.5 m/m) during the MWE extraction. The use of the MWE process for the extraction of HHT (2) when compared to a CSE one has many advantages such as shorter extraction time, reduced amount of solvent, and higher yield resulting in a less costly production system for a superior product. Ultrasonic-MWE of C. fortunei produced CET (1) in a simple, highly efficient, and low cost process, suitable for its industrial production in high purity after two recrystallizations from anhydrous ethanol.⁶⁷

Using a prepurification method with adsorption prior chromatography, HHT (2) with over 52% purity can be obtained simply from C. koreana biomass in good yield, minimizing the use of solvents, scale, and complexity of operations for the final HPLC purification of HHT (2) (Table 6 ).⁶⁸ This process allows increasing the purity of the raw HHT (2) from 0.5% to over 52% with three simple steps and inexpensive pretreatment of the MeOH extract.

Table 6.

The prepurification summary (Steps 2–4) of homoharringtonine (HHT) biomass (16 g)⁶⁴

Steps	HHT (2) (g)	Purity (%)	Yield/step (%)	Global yield (%)
Biomass	0.055	–	100	100.0
1. MeOH extraction	0.055	0.5	99	99.0
2. Liquid–liquid extraction	0.049	8.0	90	89.1
3. Adsorption	0.044	10.0	90	80.2
4. Low pressure chromatography	0.038	52.0	85	68.2

A recent study by Robin related a method for isolation and purification of CET (1) from the crude alkaloid extract from 10 kg dry leaves (not from the bark) of C. fortunei var alpina (Sichuan, China).⁶⁹ CET (1) purification was performed for the first time by reverse phase HPLC. The crude base (24.5 g) containing 71% of CET (1) was dissolved in a mobile phase consisting of ortho-phosphoric acid and deionized water containing 1.55% triethylamine (pH 3). The acid additive constrained the alkalization of the obtained aqueous phase and its extraction with dichloromethane. CET (1) (18 g) was fully recovered from the crude alkaloid extract as a white solid with an HPLC purity greater than 95%. The limited supply of promising secondary metabolites from their natural sources is a major hurdle to their pharmaceutical development; however, strategies to overcome this problem were developed. For instance, to increase the yield of natural anticancer drug alkaloids from natural sources, methods of extraction are developed. Recovering CET (1) from C. harringtonia, var. fastigiata with vegetable oil in the presence of an aqueous carbonate aqueous phase yielded 5% CET (1) with a purity of 26%.⁷⁰ The extraction performed with a mixture of scCO₂ (supercritical CO₂) with MeOH-10% Et₂NH yielded 0.0029% CET (1) of undescribed purity.⁷¹ HHT (2) was also obtained from C. koreana.⁷² He et al. have developed an HPLC analysis method for HHT (2) determination,⁷³ allowing to reveal three congeners present as impurities in the HHT (2) samples. Two impurities are new: α-isoHHT (73) is an isomer of HHT (2) in which the hydroxyl group is alpha to the isopropyl group and ethyl-HHT (74), the ethyl ester of HHT (2), is an isolation artifact of HHT (2) with ethyl acetate, the third being HT (3) (Table 7 ). α-IsoHHT (73) was found concentrated in the crystallization mother liquors of HHT (2) (10.7% against 1%–3% in crude extracts). The HPLC analysis according to He's protocol therefore allows the determination of HHT (2) in crude samples with a linear response from 4 to 12 μg and a limit of quantitation (LOQ) of 27 μg. It was used to assess the purity of several HHT (2) commercial batches.⁶³

A tandem HPLC-electron-spray mass spectrometry (MS–MS) method was developed for the analysis of alkaloids contained in the leaves of C. harringtonia. Nine alkaloids bearing an ester group were detected with a good sensitivity.⁷⁴ Content in HT (3) was higher than in HHT (2) in the needles of Cephalotaxus griffithii alkaloid fraction (CGAF) (122.14 and 16.79 mg/g of CGAF, respectively) by HPLC analysis.⁷⁵

Another major concern is the enantiomeric purity of CET (1), which has a direct effect on the diastereoisomeric purity of its natural esters. A process for purification of HHT (2) was early developed in view of the clinical studies,⁶³ although recently, a new process for the purification of CET (1) allowed the resolution of racemic or partially racemized samples through formation of various CET salts protonated at N9 with organic anions either chiral such as (2R)- and (2S)-malate, (2R,3R)- and (2S,3S)-tartrate, or nonchiral such as benzoate, hydrogenfumarate, hydrogensuccinate, hydrogenitaconate, hydrogenmaleate, hydrogenmalonate, hydrogentartrate, hydrogen meso-tartrate, hydrogenglutarate, or dihydrogencitrate.⁶⁹ This study also pointed out that the cristalline structure of HHT (2) deposited at the Cambridge Crystallographic Data Centre (CCDC) exhibits the reverse AC to the commonly accepted one. Eventually, selection of a Cephalotaxus cultivar grown in Europe allowed high recovering of CET (1) (up to 113–139 g of highly pure cristalline CET (1) from 25 kg of dry leaves).⁶⁹ Similarly, ammonium salts of HT (3) and HHT (2) were characterized and, as expected, protonation occurred at N9,⁷⁶ a finding in accordance with biological studies of N-oxides by Takano⁶¹ and mechanistic studies by Steitz (see Section 6).

Apart from the extraction of the natural source, plant cell cultures represent alternative, efficient, and sustainable chemical factories for the production of biologically important secondary metabolites, especially when the natural source become endangered. The production is limited by the low yields associated with harvest and the high costs are associated with complex chemical synthesis. Cephalotaxus evergreen trees are somewhat difficult to propagate and they are relatively rare. Some of them have been listed in the red list of IUCN (International Union for Conservation of Nature) as national plant of second-grade protection in China and are “near threatened” (Cephalotaxus latifolia), “vulnerable” (Cephalotaxus oliveri, Cephalotaxus mannii) or rare and “endangered” (C. lanceolata or Cephalotaxus fortunei Hook. var. lanceolata (K.M.Feng) Silba, C. hainanensis) species.⁷⁷ A variety of strategies are being developed to overcome the limitation of low product yields. A renewed interest in the study of Cephalotaxus crops⁷⁸ and the development of methods for analyzing the composition of different plants have been stimulated by the need for raw materials and the search for natural sources rich in CET (1). An HPLC protocol was used to determine the content of CET (1), HT (3), and HHT (2) in crop roots and calluses of C. harringtonia. As expected, CET (1) is the major alkaloid (10 mg/kg dry matter) while HT (3) and HHT (2) are more present in the roots (6.6 and 7.5 mg/kg, respectively).⁷⁹

In plant cell and tissue culture, cell growth and product formation are not necessarily correlated. Cell culture studies have sought to identify factors that tend to yield low yields and slow metabolic rates. Zhang et al. proposed the technique of periodic oscillation in temperature between 10 and 25°C every 12 h for 45 days to overcome this key problem.⁸⁰ The total production of HT (3), HHT (2), and isoHT (4) (1.22 mg/L) in solid cultures (SoC) of C. fortunei was enhanced by 1.8- and 1.3-fold compared to the controls at constant temperatures of 10 or 25°C. When subjected to such an oscillation every 24 h for 30 days, suspension cultures (SuC) gave a total alkaloid production of 0.18 mg/L, a 2.0- and 1.05-fold improvement compared to the SuC controls at 10 or 25°C, respectively. HT (3) represents the main alkaloid (72%–92% for SoC in 45 days, and 50%–67% for SuC for 30 days) while the HHT (2) content is only 4%–21% in SoC and 23%–40% in SuC, the remaining being isoHT (4).

In 2014, Li showed that the combination of 10 mg/L sodium fluoride (NaF) as glycometabolic regulator and 100 μmol/L methyl jasmonate (MJ) as the elicitor both promoted the biosynthesis of CET (1) in C. mannii suspension cells.⁸¹ NaF was more effective to increase cell membrane permeability and product secretion compared to MJ. The yield of HT (3) in the cells was 4.8 greater under NaF + MJ conditions than in the control, 1.7-fold greater under NaF and 1.6-fold under MJ conditions, respectively (Table 8 ). No HHT (2) was found in NaF + MJ–treated cells. The product release rates were 0%, 78%, 24%, and 62% in control, NaF, MJ, and NaF + MJ treatment, respectively. The combined NaF and MJ treatment on cells provided an efficient strategy for producing CET (1).

Table 8.

Harringtonines (HTs) production in Cephalotaxus mannii culture⁸¹

HT (3) content (mg/mL)					HHT (2)
Treatment	Intracellular	Extracellular	Total	Release rate % (mg/mL)
Control	1.506 ± 0.059	0	1.506 ± 0.059	0	–
NaF	0.917 ± 0.076	3.203 ± 0.197	4.12 ± 0.27	78 ± 0.47	–
MJ	3.37 ± 0.201	1.088 ± 0.074	4.458 ± 0.275	24 ± 0.17	–
NaF + MJ	2.735 ± 0.177	4.51 ± 0.215	7.245 ± 0.392	62 ± 0.42	0.491 ± 0.032

HHT, homoharringtonine.

Another study of the effects and action mechanisms of NaF on the growth and CET (1) production of C. mannii suspension cells indicated that NaF acted as an inhibitor of the Embden–Meyerhof pathway (EMP), not as an elicitor to promote CET (1) production.⁸²

3. Synthesis of the Alkaloid Core

The Cephalotaxus alkaloids have drawn the attention of many chemists due to their unique structure and potent antileukemic activities of certain ester derivatives they contain. Excellent review articles on this subject have been published in this series by Huang and Xue,¹ Miah et al.², and more recently among others, by Nay et al.⁶ Since 1997, 41 syntheses of CET (1) have been reported, 18 of which being enantioselective.

3.1. Overview

CET (1) can be described as the superposition of two structural units relevant for synthetic strategies, namely the pyrrolobenzazepine core ABC 75 and the synthetically challenging azaspiro unit CD 76 (Scheme 2 ). The three stereogenic centers (C3, C4, and C5) are contiguous on the D ring. Weinreb showed that the stereochemistry of the B/D ring fusion (C4/C5) is thermodynamically favored. In addition, carbonyl reduction of cephalotaxinone (10) delivers CET (1) with the natural configuration at C3.⁸³

Scheme 2.

Main strategies to access cephalotaxine (1).

33 (23 since 1997 are described in this review) syntheses of CET (1) concerned its racemic form (Table 9 ). The number of asymmetric syntheses has grown considerably from only 3 before 1997 to 18 until 2015, inflecting the development of asymmetric synthesis, and the efforts to provide a fully synthetic access to the drug (Table 10 ). The present section describes the recent synthetic strategies organized according to the main carbon–carbon bond disconnections of the central azepine ring B (Fig. 13 ). Asymmetric syntheses are also classified based on the type of asymmetric center control. The key steps are highlighted for each strategy. Most of these syntheses are formal; nevertheless, the OY of each synthesis up to CET (1) was evaluated. Eight racemic and five asymmetric syntheses of CET (1) are total syntheses, with only one racemic and three asymmetric routes described since mid-1997, with experimental OY and enantiomeric excess (ee). The OY and the number of steps (NS) are given from commercially available reagents for comparison purpose. In addition, for convergent syntheses, total number of steps (TNS), which represents the total efforts to be accomplished up to the target molecule, and the corresponding average yield (AY) are given. Synopses of the syntheses of CET (1) reported so far are presented in Table 9 (racemic) and Table 10 (asymmetric). Besides formal syntheses targeting Weinreb's enamine grouped together in Table 9, racemic syntheses are classified according to the type of disconnection of ring B (a–e, Fig. 13), the type of intermediate in the formal syntheses and then in a chronological order. The asymmetric syntheses are presented in chronological order in Table 10.

Table 9.

Overview of the racemic syntheses of rac-CET (±)-(1) arranged according to the main strategic disconnections of ring B. Formal syntheses targeting Dolby–Weinreb's enamine are grouped

Authora (year)	Ring B cut	NS	OY%	TNS	AY%	Starting material (ring A)	References
Weinreb (1972)	a	11	2.3	–	–	Piperonal (82)	2, 103
Dolby (1972)b	d	12	0.7	–	–	Piperonylic acid	106
Weinstein (1976)b	a	13	1.9	–	–	Benzodioxole (330)	2, 134
Snieckus (1976)b	a	14	1.6	–	–	Piperonal (82)	2
Danishefsky (1990)b	b	18	0.2	–	–	Piperonal (82)	2
Li (2005)b	a	17	0.4	–	–	Piperonal (82)	105
Semmelhack (1972)	a	12	12.7	18	2.0	Piperonal (82)	2, 83
Hanaoka (1986)	a	12	6.5	–	–	Homoveratric acid (143)	98
Hanaoka (1988)	a	15	3.0	–	–	3,4-Methylenedioxyacetophenone	96
Kuehne (1988)c	a	12	17.1	14	11.6	Piperonal (82)	94
Tietze (1997)	a	12	5.2	15	2.5	Piperonal (82)	84
Suga and Yoshida (2002)	a	14	2.2	17	1.5	Homopiperonylic acid (89)	86, 87
Nagasaka (2002)	a	20	3.1	22	1.8	Piperonal (82)	95
Stoltz (2007)	a	13	3.2	17	2.3	Homopiperonylic acid (89)	90
Li (2007)	a	13	4.0	–	–	Homoveratric acid (143)	97
Yang and Liu (2009)	a	15	7.1	–	–	Homoveratric acid (143)	99
Bubnov(2008)	a	16	3.8	–	–	Homoveratric acid (143)	104
Zhang and Liu (2013)	a	17	2.2	–	–	Homoveratryl amine (158)	102
Zhang and Liu (2013)	a	16	3.7	–	–	Homopiperonylic acid (89)	102
Huang (2013)	a	14	5.4	–	–	Homopiperonylic acid (89)	92
Huang and Wang (2015)	a	12	6.2	15	4.3	Homopiperonylic acid (89)	93
Hong (2015)	b	12	23.5	–	–	Homopiperonylic acid (89)	115
Hong (2015)	b	12	26.2	–	–	Homopiperonylic acid (89)	115
Fuchs (1988)	c	21	6.2	24	4.8	Piperonyl alcohol	2, 147
Mariano (1994)	c	18	5.3	–	–	Homopiperonylic acid (89)	113
Tu and Zhang (2009)	c	23	1.9	24	1.9	Homopiperonylic acid (89)	108
Li (2011)	c	13	0.4	–	–	Piperonal (82)	111
Jiang (2013)	c	15	4.3	–	–	Homopiperonylic acid (89)	112
Li (2003)	c	18	1.8	–	–	Piperonal (82)	100
Li (2005)	c	21	0.2	–	–	Piperonal (82)	105
Li (2005)	c	16	0.8	–	–	Piperonal (82)	107
Ishibashi and Ikeda (1990)	d	19	15.6	–	–	Piperonal (82)	138
Chandrasekhar (2016)	e	16	6.4	18	4.7	5-Benzodioxolol (254)	120

a, disconnection C4-C13; b, disconnection C4-C5; c, disconnection C5-N9; d, disconnection C11-C12; e, disconnection N9-C10.

AY, average yield accounting for the TNS; NS, number of steps (longest linear sequence); OY, overall yield; TNS, total number of steps from boths precursors to CET.

^aName in bold style, total synthesis; name in plain style, formal synthesis.

^bFormal synthesis through Weinreb's enamine.

^cPreparative HPLC of intermediate required.

Table 10.

Overview of the asymmetric syntheses of (−)- and (+)-cephalotaxine

Authora(year)	B Cycle cut	NS	OY% (ee%)	TNS	AY%	SMb	Chiral source	References
Weinreb-Merk (1972)c	a	15	5.3 (98.5)	–	–	82	l-tartaric acid	2
Zhong (1994)c	a	16	(nr)	–	–	82	l-tartaric acid	2
Mori (1995)c	a	18	1,6 (86)	19	1,6	143	d-proline (R)-(140)	85
Nagasaka (1997)c,d,e	a	16	5.8 (99)	18	4.5	89	(2R,3R)-butanediol (404)	133
Ikeda (1999)c	d	19	5.1 (88)	–	–	330	d-proline (R)-(140)	137
Tietze (1999)c	a	13	3.1 (87)	16	1.7	82	Oxazaborolidine 267	121
El Bialy (2002)c	d	20	<7.5% (89)	–	–	82	l-malic acid (409)	140
El Bialy (2002)c	a	14	12.2 (89)	20	4.3	330	d-proline (R)-(140)	140
Royer (2004)c	a	16	7.5 (98)	18	5.8	89	(S)-1-Naphtylethylamine (298)	132
Dumas, d'Angelo (2005)c	a	20	5.1 (>95)	22	3.9	317	(R)-1-Phenylethylamine (309)	135
Stoltz (2007)c,d	a	14	0.2 (97.5)	19	0.03	82	(−)-Ephedrine (412)	90
Mariano 1 (2006)c	a	19	0.2 (51)	–	–	143	AchE (electric eel)	127
Mariano 2 (2006)c	a	19	0.5 (95)	23	0.3	89	AchE (electric eel)	127
Hayes 1 (2008)c	a	23	0.2 (87)	–	–	89	l-proline (S)-(140)	141
Djaballah, Gin (2008)c	a	25	1.0 (nr)	28	0.7	330	d-ribose (365)	144
Ishibashi (2008)c	a	17	1.3 (99.6)	22	0.7	82	Diethyl d-tartrate (384)	145
Hayes 2 (2008)c	a	20	0.5 (91)	22	0.45	89	l-proline (S)-(140)	142
Tu (2012)c	a	14	1.7 (99) or 3.3 (80)	18	1.3	82	Silver phosphate (R)-273	122
Renaud (2012)c	a	26	0.9 (97)	29	0.6	89	Noyori catalyst (S,S)-281	124
Renaud(2012)c	a	18	1.8 (96.5)	20	1.4	89	PPL	124
Trost (2012)c	d	19	13.9 (91)	21	8.4	82	Phosphoramidite 296	129

(143), homoveratric acid; (317), safrole; (330), benzodioxole; (82), piperonal; (89), homopiperonytic acid.

AchE, Acetyl choline esterase; nr, not reported; PPL, Porcine Pancreatic Lipase.

^aName in bold, total synthesis; name in plain style, formal synthesis.

^bFor ring A.

^cSynthesis of (−)-1.

^dSynthesis of (+)-1.

^eIncluding one step of separative HPLC.

Figure 13.

Main disconnections used for the construction of cephalotaxine ring B.

It is apparent from Table 9 that most of the syntheses of rac-CET (±)-(1) are linear (only 10 being convergent) and proceed predominantly (19 of 33) by the C4–C13 disconnection a. The shortest access to CET (1) was described in the first synthesis of CET (1) by Weinreb et al.¹⁰³ in 11 steps including the preparation of homopiperonylic acid (89) in two steps from piperonal (82).¹³⁴ The most effective total syntheses were described by Kuehne et al.⁹⁴ in only 11 steps and 11.2% OY (average 82% yield per step) or 12 steps and 17.1% OY, by Ishibashi and Ikeda et al.¹³⁸ in 19 steps and 15.6% OY (average >90% yield per step), and by Chandrasekhar et al.¹²⁰ (6.4% OY in 16 steps, 4.7% AY in 18 TNS). The recent formal synthesis by Hong et al. in 12 steps and 23.5% OY is the most efficient route.¹¹⁵ The favorite choices as ring A precursors are piperonal (82) and homopiperonylic acid (89) with C11 and the methylenedioxy group already at the right position.

Table 10 shows that proline (140) is a widely used chiral source (5 over 21 syntheses), either in its natural L form, or in its unnatural D one. Herein, 15 syntheses are convergent and 6 are linear. The most efficient total syntheses of CET (−)-(1) (OY > 5%) were reported by Weinreb et al.¹⁰³ (5.3% OY in 15 steps, 98.5% ee) and Royer et al.¹³² (7.5 OY in 16 steps, 98% ee; 5.8% AY in 18 TNS). Formal syntheses by Nagasaka et al.¹³³ (5.8% OY and 4.5% AY in 16–18 steps, 99% ee), Ikeda et al.¹³⁷ (5.1% OY and AY in 19 steps, 88% ee), Dumas and d’Angelo¹³⁵ (5.4% OY in 20 steps, >95% ee; 4.1% AY in 22 TNS), El Bialy et al.¹⁴⁰ (12.2% OY in 14 steps, 89% ee; 4.3% AY in 20 TNS) and Trost et al.¹²⁹ (13.9% OY in 19 steps, 91% ee; 8.4% AY in 21 TNS) were also efficient.

3.2. Racemic Syntheses

3.2.1. C4–C13 Ring Closure a

The ring closure C4–C13 a (Fig. 14 ) to form the azepine cycle was frequently adopted after Semmelhack's early extensive developments in the second synthesis of rac-CET (±)-(1).⁸³

Figure 14.

C4–C13 bond disconnection a for elaboration of cephalotaxine (1) B ring.

3.2.1.1. Tietze's Formal Synthesis (1997)

In 1997, Tietze et al.⁸⁴ reported an efficient synthesis of Mori's intermediate (±)-88 ⁸⁵ in which rings B and C were constructed by two consecutive intramolecular palladium-catalyzed reactions (Scheme 3 ). The precursor of the D ring and C6–C7–C8 fragment was prepared from 1,3-cyclopentanedione (77) via its enol ether 78. Addition of the Grignard reagent (±)-81 and elimination of ethanol followed by tosylation, reduction, and acetylation gave the allylic acetate (±)-81. Alkylation of the bromoamine 84 prepared in four steps from piperonal 82 with the intermediate iodide derivative generated in situ from tosylate (±)-81 provided the secondary amine (±)-85 bearing the AD ring units, which contain all carbon atoms and functionalities required for synthesis completion.

Scheme 3.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Tietze (1997).

A palladium-catalyzed Tsuji–Trost allylation furnished the spiro compound (±)-86 containing the A and CD ring units. The B ring was formed by a highly stereoselective intramolecular Heck reaction using Herrmann–Beller catalyst 87. The selective formation of Mori's tetracyclic intermediate (±)-88 can be explained by an oxidative addition of the palladium complex to the double bond syn to the nitrogen atom followed by a syn elimination of PdH. Mori's procedure⁸⁵ would allow to complete the synthesis of rac-CET (±)-(1) in 12 steps (longest linear sequence (LLS)) and 5.2% OY from 1,3-cyclopentanedione (77) (2.5% AY in 15 TNS). It should be mentioned that Mori's protocols differ for the synthesis of racemic or optically active CET (1). Indeed, the transformation of desmethyl CET (12) into cephalotaxinone (10) using 2,2-dimethoxypropane and p-TsOH led to racemization of the product (76% yield). On the contrary, when p-TsOH and methyl orthoformate were used for this transformation at room temperature (rt), limited racemization occurred (ee 88%), but the yield went down to 47%.

3.2.1.2. Suga and Yoshida's Formal Synthesis (2002, 2006)

Suga and Yoshida developed an approach to the azaspiro-compound 101 via ring-closing metathesis (RCM) of a functionalized pyrrolidine 100 generated by electrochemical oxidation of α,α-bis(trimethylsilyl)pyrrolidine 95 (Scheme 4 ).86, 87

Scheme 4.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Suga and Yoshida (2002).

First, homopiperonylic acid (89) was used to prepare the A unit 91 bearing a nosylate and an aromatic iodide suitable for ring B annulation, relying on Semmelhack's procedure.83, 88 The two silyl groups were introduced by Beak's methodology⁸⁹ (deprotonation with sec-BuLi and quenching with TMSCl) furnishing compound 94, which was protected into N-methoxycarbonyl compound 95. Its anodic oxidation at low temperature yielded N-acyliminium ion 96, reacting in situ with homoallylmagnesium bromide (97) furnishing pyrolidine 98. This latter compound was next submitted to a second anodic oxidation followed by direct trapping with the organozinc reagent 99 to provide the diolefin 100. Diene 100 was converted to azaspiro CD unit 101 via ring closing metathesis promoted by Grubbs I catalyst. After deprotection using TMSI, the spiranic amine intermediate reacted with nosylate 91 obtained from homopiperonylic acid (89) following Semmelhack's procedure.⁸⁸

The so-formed aryl iodide (±)-102 was submitted to an intramolecular Heck-type ring closing reaction according to Tietze⁸⁴ to give Mori's intermediate (±)-88. Applying Mori's procedure to complete the synthesis from intermediate (±)-88 would provide an access to rac-CET (±)-(1) as a linear sequence of 14 steps and 2.2% OY from pyrrolidine 92.

3.2.1.3. Stoltz's Formal Synthesis (2007)

Stoltz et al. proposed a concise formal route to rac-CET (±)-(1) in which the key spirocyclic amine (±)-110 was built through palladium(II)-catalyzed aerobic oxidation (Scheme 5 ).⁹⁰ This oxidative cyclization and condensation with ring A-containing aldehyde 104 by reductive amination were the basis to elaborate both enantiomers of CET (1) and (−)-drupacine (16) (see Section 3.3.4.3, Scheme 40 ).

Scheme 5.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Stoltz (2007).

Scheme 40.

Asymmetric syntheses of (−)-drupacine (16) and formal asymmetric syntheses of cephalotaxine (−)-(1) and ent-cephalotaxine (+)-(1) by Stoltz (2007).

Regioselective bromination of homopiperonylic acid (89) yielded bromocarboxylic acid 103 converted to Weinreb's amide,2, 83 and then reduced by DIBAL to furnish aldehyde 104. Different routes were proposed to prepare the ACD ring–containing aryl bromide 86. The best one starts from β-ketoester 105 transformed by reduction to allylic alcohol 106,⁹¹ which was treated with triethyl orthoacetate under Johnson orthoester–Claisen conditions to give ethyl ester 107. Afterward, ester 107 was converted in three steps into sulfonamide 108 by successive reduction by LiAlH₄, tosylation, and nucleophilic displacement by tosylamine. The oxidative palladium(II)-catalyzed heterocyclization of 108 provided efficiently the spirocyclic tosylamide 109 (scale up to 1.1 g) whose reductive cleavage of the tosyl group with LiAlH₄ furnished the desired spirocyclic amine 110. This compound and the aldehyde 104 were coupled by reductive amination. The resulting tertiary amine (±)-86 was converted to the target intermediate (±)-88 using stereoselective Heck conditions described by Tietze.⁸⁴ Including the four steps of Mori's procedure, this formal synthesis of rac-CET (±)-(1) would consist in 17 steps and 3.1% OY from 89 (AY 2.3% in 17 TNS).

3.2.1.4. Huang's Formal Synthesis (2013)

An efficient synthesis of 1-azaspiro[4.4]nonenes in four steps from γ-butyrolactams (e.g., 113 → 102, Scheme 6 ) was developed by Huang et al. in 2013⁹² and applied to the synthesis of rac-CET (1), involving Tietze's Heck ring closure⁸⁴ of aromatic iodide (±)-102 ⁸⁶ (Scheme 6).

Scheme 6.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Huang (2013).

The γ-butyrolactam 113 was prepared from 2-arylethanol 90 in 65% OY by Mitsunobu's substitution of the primary alcohol, followed by partial reduction of the crude succinimide 112 with NaBH₄ and reductive dehydroxylation of the resulting hemiaminal. Huang's methodology, namely activation of compound 113 by triflic anhydride (2,6-di-tert-butyl-4-methylpyridine (DTBMP)) was the best base for this reaction, followed by successive addition of buten-1-yl Grignard (97) and vinyl Grignard reagents provided the desired pyrrolidine 114. The hydrochloride salt of the dienyl pyrrolidine 114 was next transformed into the known spiro-pyrrolidine (±)-102 under RCM conditions (Grubbs's generation II catalyst). The formal synthesis by Huang et al. was achieved with the synthesis of Mori's intermediate (±)-88 using the palladium-catalyzed Heck-type cyclization of aryl iodide (±)-102 developed by Tietze⁸⁴ and employed by Suga and Yoshida.⁸⁶ Once again, four steps (Mori's protocol)⁸⁵ have to be performed to obtain rac-CET (±)-(1), to give a 14-steps pathway in 5.4% estimated OY from homopiperonylic acid (89).

3.2.1.5. Huang and Wang's Formal Synthesis (2015)

In 2015, Huang and Wang proposed a rapid construction of 1-azaspiro[4.4]nonenone (±)-121 via a nitroso-ene cyclization (Scheme 7 ).⁹³

Scheme 7.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Huang and Wang (2015).

Reaction of 1,2-epoxycyclopentane 116 with in situ generated dimethylsulfonium methylide gave allylic alcohol 106, which was directly used for the subsequent Johnson–Claisen rearrangement. The resulting ester 107 was subjected to amidation reaction conditions with hydroxylamine to afford hydroxamic acid 117. The nitroso-ene cyclization was accomplished on oxidation with tetrapropylammonium periodate via a highly reactive acylnitroso intermediate 118 delivering N-hydroxy-1-azaspiro[4.4]non-2-one 119, which was trapped as an acetate 120 yielding in moderate yield spiro-lactam (±)-121 ⁹⁰ on SmI₂ reduction. Alkylation of the sodium salt of (±)-121 with iodoaryl tosylate 115 gave quantitatively (±)-122. The subsequent Heck reaction promoted by Herrmann–Beller catalyst 87 proceeded smoothly to deliver the ABCD Kuehne's intermediate⁹⁴ (±)-123, and the corresponding regioisomeric product (±)-124 in 82% combined yield, based on recovered starting material (brsm) as an inseparable 54:46 mixture. rac-CET (±)-(1) could be obtained in 12 steps from cyclopentene oxide (116) and 6.2% OY provided that separation of the regioisomers is effective and four supplementary steps applied to transform the ABCD intermediate (±)-123 (TNS 15 steps, AY 2.5%, 4.3% brsm). Although Huang and Wang described a rapid access to the CD spirocyclic amide (±)-121, Heck ring closure of ring B led to a mixture of two regioisomers and decreasing dramatically the yield.

3.2.1.6. Nagasaka's Formal Synthesis (2002)

In 2002, Nagasaka et al. reported a formal synthesis of CET (1),⁹⁵ through a new access to Hanaoka's precursor⁹⁶ (±)-139 (Scheme 8 ), building successively rings C and B. Ring B was installed by ring expansion of pyrroloisoquinoline 132, which resulted from the cyclization of N-acyliminium ion intermediate 131. Alkyne 127 that contains C5, C6, C7, and C8 carbon atoms of ring C and C2, C3, and C4 carbon atoms of the CET skeleton was prepared in four steps (63% OY) from 4-pentyn-1-ol 125. Coupling of 127 with homopiperonylamine 83 (which could be obtained from piperonal 80 in two steps and 64% OY), gave amide 128 whose treatment with LiHMDS and catalytic amounts of silver triflate gave the AC enamide 129 by intramolecular N-heterocyclization. Oxidation of 129 with dimethyldioxirane delivered the unstable methoxylactam 130 immediately treated with BF₃·OEt₂ producing pyrroloisoquinoline 132 via the N-acyliminium ion 131. Ring-expansion reaction of the six-membered ring elaborated the seven-membered ring B (132 → 133). Aldehyde 134 was isolated after debenzylation of 133 followed by Dess–Martin oxidation. The missing C1 carbon atom in aldehyde 134 was introduced by aldol reaction with the lithium enolate of benzyl acetate. Subsequent oxidation of the resulting aldol gave β-keto-ester 135. Formation of ring D was accomplished via Mannich cyclization of N-acyliminium ion 136 and elimination of hydroiodic acid to produce the enone 138. The final compound of this formal synthesis, spiroketone (±)-139 reported by Hanaoka⁹⁶ was isolated after decarbobenzyloxylation followed by catalytic reduction. Application of Hanaoka's procedure (3 steps, 34% OY) to complete the synthesis from compound (±)-139 would provide an access to rac-CET (±)-(1) in 20 steps and 3.1% OY from 4-pentyn-1-ol 125.

Scheme 8.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Nagasaka (2002).

3.2.1.7. Li's Formal Synthesis (2007)

In 2007, Li developed a formal synthesis of rac-CET (±)-(1) based on a facile Friedel–Crafts cyclization of the amido-spiro-cyclopentenone precursor 148 mediated by triflic acid (Scheme 9 ).⁹⁷

Scheme 9.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Li (2007).

Reaction of acid chlorides 144 and 145 prepared from homopiperonylic acid 86 and homoveratric acid 143, with proline-derived spirocyclic amino enone (±)-142, led to the amido spirocyclopentenones (±)-146 and (±)-148, respectively. Friedel–Craft cyclization of the methylenedioxy precursor (±)-146 did not led to compound (±)-147 under various acidic conditions due to an electron-deficient aryl system. On the contrary, an efficient Friedel–Craft cyclization of the dimethoxy derivative (±)-148 was observed on treatment with triflic acid yielding Hanaoka's intermediate⁹⁸ (±)-149 isolated in 93% yield. In summary, Li described here a concise formal synthesis of rac-CET (±)-(1) in 13 steps from l-proline (S)-140 and 4% evaluated OY.

3.2.1.8. Yang and Liu's Formal Synthesis (2009)

Yang and Liu reported two strategies to access to intermediate (±)-148 involving a [2,3]-Stevens rearrangement and an acid lactonization sequence as key transformations.⁹⁹ We describe here the best route that led to Li's intermediate⁹⁷ (±)-148 via the spirolactone 154 (Scheme 10 ).

Scheme 10.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Yang and Liu (2009).

Spirolactone 154 was obtained from methyl prolinate (±)-150 by N-allylation with allyl bromide, leading to allylamine 151, which underwent a [2,3]-Stevens rearrangement of the resulting quaternary ammonium salt by reaction with benzyl bromide providing N-benzyl pyrrolidine 152. After lactonization of 152 under acidic conditions followed by debenzylation through catalytic hydrogenation, spirolactone 154 was acylated with 3,4-dimethoxyphenacetyl chloride (145) to produce the amide spirolactone 155 containing the AC ring units, which was selectively reduced with LiBH₄ to diol 156 in 63.9% OY. Swern oxidation of diol 156 furnished the amido keto-aldehyde 157, which was treated with potassium carbonate to give the amido spirocyclopentenone (±)-148 with the ACD ring units. This formal synthesis is especially suitable for the production of (±)-148 at a scale up to 10–50 g. Li⁹⁷ and Hanaoka's⁹⁸procedures (6 steps, 15% OY) would allow to complete the synthesis of rac-CET (±)-(1) in 15 steps and 7.1% OY from rac-proline (±)-(140).

3.2.1.9. Zhang and Liu's Formal Synthesis (2013)

In 2013, Zhang et al. proposed two pathways for the formal synthesis of rac-CET (±)-(1) targeting Li's enone (±)-174 ¹⁰⁰ and Hanaoka's enone (±)-180 ⁹⁶ based on conventional alkylations and aldol condensations from known benzazepines¹⁰¹ 165 and 166 obtained in four steps from aryl amine 83 (bearing a methylenedioxy substituent) or 158 (bearing two methoxy substituents), respectively (Scheme 11 ).¹⁰²

Scheme 11.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Zhang and Liu, the “dimethoxy” route (2013).

The C ring was elaborated via an N- and C-alkylating annulation by treatment of benzazepine 165 with 1,3-dibromopropane (167) and Cs₂CO₃ giving product 168 in one step. ABC dione 172 was prepared by alkylation either with allyl bromide followed by Wacker oxidation or more efficiently with 2-methoxyallyl bromide (170) followed by acidic hydrolysis (Scheme 11). Aldol condensation efficiently constructed the CET (1) core in (±)-173. The dimethoxy groups were transformed into the required methylenedioxy group by the usual protocol⁸⁵ to afford ABCD enone (±)-174 reported by Li in 2003.¹⁰⁰ Application of Li and Weinreb's¹⁰³ procedures would end up a synthesis of rac-CET (±)-(1) in 17 steps from homoveratryl amine (158) and 2.2% calculated OY.

In an alternative synthesis, these authors also studied the use of methylenedioxybenzazepine 166 as starting material (Scheme 12 ).

Scheme 12.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Zhang and Liu, the “methylenedioxy” route (2013).

Ring C was elaborated by acylation/alkylation of benzazepine 166 with 3-chloropropanoyl chloride (175) to furnish the ABC ketone 176. Treatment with sodium hydride and 2-methoxyallyl bromide (170) produced the enol ether 177 (74%) as a major O-alkylated product along with the desired C-alkylation derivative 178 (22%). Claisen rearrangement allowed conversion of 177 to 178, which was exposed to acid hydrolysis to give dione 179. Intramolecular aldol condensation provided the known amido enone (±)-180 ⁹⁶ in 95% yield. Formally, rac-CET (±)-(1) could be obtained following Hanaoka’ synthesis⁹⁶ in a total of 16 steps from homopiperonylic acid (89) and 2.5% estimated OY.

3.2.1.10. Bubnov's Formal Synthesis (2008)

Bubnov et al. developed a convenient and practical methodology for the preparation of various spiro-β-amino alcohols. This procedure was applied to a formal synthesis of rac-CET (±)-(1) involving allylboration and RCM to prepare the CD ring containing azaspirobicyclic compound 183 (Scheme 13 ).¹⁰⁴

Scheme 13.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Bubnov (2008).

2-Pyrrolidone (181) was converted to 2,2-diallylated pyrrolidine 182 by addition of triallylborane. After amine protection, RCM was accomplished with Grubbs I catalyst providing azaspirocyclic carbamate 183 in high yield (three steps, 87% OY). Treatment of 183 with N-bromosuccinimide (NBS) produced tricyclic bromoamide 184. After t-butoxide mediated dehydrobromination affording the spiro olefin 185, an allylic isomerization in the presence of MgBr₂ gave 186 as the sole isomer. Hydrolysis of 186 and acylation with acid chloride 145 were performed in a one-pot procedure leading to 187. Mori's ACD intermediate, generated by reduction of amide 187 with LiAlH₄, was allowed to cyclize into ABCD compound (±)-188 by treatment with polyphosphoric acid (PPA). Then, the synthesis of rac-CET (±)-(1) in 16 steps from 2-pyrrolidone (181) and 3.8% estimated OY could be completed using Mori's sequence⁸⁵ as the final steps.

3.2.2. N9–C5 Ring Closure c

3.2.2.1. Li's Formal Syntheses (2003–2005)

From 2003 to 2005, Li et al. reported three formal syntheses, two of them via the same intermediate 192 (Scheme 14 ).100, 105 Formation of ring B and D was achieved by transannular reductive skeletal rearrangements leading to the formation of N9–C5 bond (Fig. 15).

Figure 15.

C5–N9 bond disconnection c for construction of ring B of cephalotaxine (1).

Scheme 14.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Li (2003).

Scheme 15.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Li (2003): alternative synthesis of ketone (±)-197.

Starting from piperonyl amine 83, diester 189 was obtained in four steps via a Bishler–Napieralski cyclization followed by hydrogenation and N-alkylation with ethyl 4-bromobutyrate. A second N-alkylation with allyl bromide led to the ammonium salt intermediate 190, which was transformed via a 2,3-sigmatropic Stevens rearrangement into diester 191. Eventually, compound 193 was obtained by a Dieckmann cyclization with subsequent decarboxylation followed by Wacker oxidation. Aldol condensation of the dione 193 provided the spirocyclic AD enone 194 (Scheme 14). CET (1) B and C rings were formed by reductive rearrangement in acetic acid via the aziridinium ion 195. The obtained ABCD ketone (±)-197 (revised stereochemistry at C4 as drawn) was oxidized with phenyliodonium diacetate to give acetal (±)-198, which, after hydrolysis, was submitted to autoxidative dehydrogenation with potassium t-butoxide and oxygen to provide the desmethylcephalotaxinone (±)-12. Formally synthesis of rac-CET (±)-(1) could be achieved according to Weinreb¹⁰³ using two additional steps, leading to a formal total synthesis in 1.8% calculated OY over 18 steps from piperonal (82).

An alternative approach to ketone (±)-197 was described from allyl ketone 192. Clemmensen reductive rearrangement of this ketone first led to benzazepine derivative 199 (Scheme 15 ).¹⁰⁰

After Wacker oxidation of compound 199, application of Polonovski–Potier reaction conditions to 200 gave the enamine 201. It was previously reported that cyclization of this intermediate to (±)-174 (formally an endocyclic enamine annulation) could only be realized in low yield. Li found that an aerobic treatment of 201 with Fe²⁺ salt in acetic acid promoted the cyclization into ABCD enone (±)-174 in good yield. This cyclization can be seen as an unusual azo-Nazarov-type cyclization. A catalytic hydrogenation eventually gave ketone (±)-197. This synthesis in six steps and 14.5%–17% OY from 192 is not advantageous when compared to the three steps and 40.5% OY version presented in Scheme 14. Thus, formal synthesis of rac-CET (±)-(1) following this procedure could be theoretically achieved in 21 steps and 0.7%–0.8% OY from piperonal (82).

In 2005, Li et al. described a formal synthesis of the Dolby–Weinreb enamine103, 106 203 from dione 193, with a mild Clemmensen–Clemo–Prelog–Léonard reductive rearrangement of 193 as the key step (Scheme 16 ).¹⁰⁵

Scheme 16.

Formal synthesis of rac-Cephalotaxine (±)-(1) via Dolby–Weinreb enamine 203 by Li (2005).

Dehydrogenation of the benzazepine 202 by Hg(OAc)₂ gave enamine 203, which could be likely transformed in rac-CET (±)-(1) by Weinreb's strategy¹⁰³ in expected 0.4% OY for 17 steps from piperonal (82). Reductive rearrangement of aminodione 193 also produced aminoketone 200 in 15% yield, which could be processed into demethylcephalotaxinone (12) and then into CET (1) through a nine-stage sequence. This constitutes another formal synthesis of CET (1) with an expected OY of 0.17% in 21 steps from piperonal (82).

In 2005, Li reported a novel synthesis of the amino enone 194 involving annulation of the readily available aminodione 193 (Scheme 17 ).¹⁰⁷

Scheme 17.

Formal synthesis of rac-Cephalotaxine (±)-(1) via amino enone 194 by Li (2005).

Bischler–Napierralski condensation of amine 83 with δ-valerolactone (204) mediated by POCl₃ gave the iminium ion 205 in 74% yield. Treatment of 205 with aqueous NaOH generated a labile enamine 206, which was alkylated with bromoacetone to give the keto enamine 207 (20%) and the iminium bromide 208 (50%). Both were transformed by treatment with 70% perchloric acid into iminium perchlorate 209, which on exposure to NaOH furnished the unexpected dione 193 (25%) and annulated cis-cyclopentanone 210. Both compounds can be transformed to 194 by standard procedures (aldol condensation from 193, selenoxide elimination from 210 (15%). Formally, rac-CET (±)-(1) could be obtained according to Li's and Weinreb's protocols100, 103 with a calculated 0.8% OY for 16 steps from piperonal (82). The ABCD ring construction by rearrangement can be regarded as a biomimetic synthesis.

3.2.2.2. Tu and Zhang's Formal Synthesis (2009)

In 2009, Tu and Zhang developed a synthesis of Li's intermediate dione¹⁰⁰ 193 based on an intramolecular Schmidt reaction of symmetric azido dione 221 (Scheme 18 ).¹⁰⁸

Scheme 18.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Tu and Zhang (2009).

The synthesis started with compound 213 (ring A precursor) obtained from homopiperonylic acid (89) by reduction, bromation, and chlorination,¹⁰⁹ and iodocyclopentenone 215 (ring D precursor) prepared by iodination of cyclopentenone (214) (Scheme 18).¹¹⁰ Suzuki–Kumada coupling of an aryl boronic acid prepared from 213 with 2-iodocyclopentenone (215) afforded enone 216 in 81% yield. Alcohol 217 obtained by Grignard addition on enone 216 was transformed to the azide 220 on treatment with sodium azide in the presence of sodium iodide. Epoxidation and protection with TMSCl led to siloxy epoxy azide 219 in 63% OY from 216. Treatment of epoxide 219 with TiCl₄ afforded β-hydroxy ketone 220, which was oxidized with Dess–Martin's reagent (70% yield, two steps). The key intramolecular Schmidt reaction was applied to dione 221 giving the amide 222. The terminal double bond in 222 was converted into a methylketone by Wacker oxidation. Selective reduction of an intermediate thiolactam, prepared from amide 223 with Lawesson's reagent, using Raney Ni afforded the expected tertiary amine 193 described by Li.100, 107 An aldol cyclization gave the key enone 194 in 80% yield. rac-CET (±)-(1) could be obtained by Li's and Weinreb's procedures in 23 steps from homopiperonylic acid (89) and 1.9% evaluated OY.

3.2.2.3. Li's Formal Synthesis (2011)

The new synthetic strategy described by Li et al. in 2011 was based on a facile reductive oxy-Nazarov (RON) cyclization as a key step (Scheme 19 ).¹¹¹ Condensation of norhydrastinine (224), prepared in four steps from piperonal (22.8% OY), with iodo enolsilane 225 afforded the aldehyde 226, which was exposed to 2,2,2-trichloroethyl chloroformate (227) to give the macrocyclic enal 228. Wittig olefination with triphenylphosphoranylidene dioxolanone 229 furnished the 10-membered amide 230 with the exocyclic (E)-olefin in 68% isolated yield, along with the separable (Z)-isomer (14%). Mild hydride reduction led to hydroxy enone 232 presumably via the oxonium ion 231, which could have underwent rapid and effective 4π electrocyclization. Acetylation of the hydroxyl function of the resulting compound 232 followed by selective cleavage of the N-Troc group by zinc reduction led to the spontaneous formation of ABCD unit 233 as a single diastereomer by an intramolecular aza-Michael addition. Air-oxidation in the presence of potassium t-butoxide gave rac-demethylcephalotaxinone (±)-(12). Application of Weinreb's method¹⁰³ would allow to synthesize rac-CET (±)-(1) in 13 steps from piperonal (82) and 0.4% OY.

Scheme 19.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Li (2011).

3.2.2.4. Jiang's Formal Synthesis (2013)

In 2013, Jiang et al. described the construction of the cyclopentenone D ring of rac-CET (±)-(1) by an intermolecular Pauson–Khand reaction (Scheme 20 ).¹¹² Iodo alcohol 90 was first protected as a mesylate and submitted to mild Sonogashira conditions to afford the alkyne 235. The intermolecular Pauson–Khand reaction of alkyne 235 with ethylene could generate two regioisomeric products. Only the desired isomer 236 (58% yield) was obtained under optimized conditions using n-butyl methyl sulfide as a promoter with dimethyl sulfoxide as the oxidant. Oxidation of alcohol 236 followed by reductive amination produced the amine 238, which was advanced to the 10-membered ring compound 239 by intramolecular N-alkylation. Mariano's intermediate113, 114 240 was prepared successively by deprotection and reprotection with a t-butoxycarbonyl group.

Scheme 20.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Jiang (2013).

This strategy led quickly and efficiently to macrocyclic precursor 240 in eight steps and 20% OY. With this compound 240 in hand, a formal synthesis of rac-CET (±)-(1) was thus attained, and additional five-step sequence113, 114 would give rac-CET (±)-(1) over 15 steps from homopiperonylic acid (89) and 4.3% estimated OY.

3.2.3. Other Strategies for Ring B Formation

3.2.3.1. C4–C5 Ring Closure b: Hong's Formal Synthesis (2015)

Recently, Hong et al. reported two formal syntheses¹¹⁵ involving an unusual C4–C5 disconnection b 116, 117, 118 for ring B formation (Fig. 16 ). The key steps were first an N-acyliminium ion cyclization, which established stereogenic centers C5 and C4 and the formation of B ring. Then an RCM furnished the spiro-ring D. The two syntheses started from iodo alcohol 90 prepared from homopiperonylic acid (89) in two steps.

Figure 16.

C4–C5 bond disconnection b for elaboration of ring B of rac-cephalotaxine (±)-(1).

Sonogashira coupling of aryl iodide 90 with propargyl trimethylsilane catalyzed by palladium(II) and copper(I) iodide delivered alkyne 242 in excellent yield (97%) (Scheme 21 ). After a catalyst screening, partial hydrogenation was carried out with “P-2 Nickel” and ethylenediamine (Brown catalyst) leading to (Z)-243 in quantitative yield (dr > 15/1). Mitsunobu reaction with succinimide, followed by allylation next gave the hemiaminal 246, which, treated with TiCl₄ at low temperature in mesitylene, allowed for the generation of an N-acyliminium ion that cyclized to furnish the ABC unit 247 (cis/trans 2.5/1). Zhan's catalyst-1B¹¹⁹ was the most effective for the RCM reaction to deliver Kuehne intermediate⁹⁴ (±)-123. Formally, synthesis of rac-CET (±)-(1) could be achieved by Kuehne's protocol⁹⁴ in 12 steps from homopiperonylic acid (89) and 23.5% calculated OY.

Scheme 21.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Hong via Kuehne's intermediate (±)-123 (2015).

Since iminium ion cyclization showed a moderate diastereoselectivity and the separation of the two diastereomers by silica gel chromatography failed, this route was less attractive. To improve it, authors investigated cyclization from the E-allylsilane 249 obtained by Suzuki coupling between compound 248 and aryl iodide 90 catalyzed by palladium (Scheme 22 ). As previously seen, a Mitsunobu reaction gave 250. Subsequently, 250 was treated with a Grignard reagent and cyclized by a Hosomi–Sakurai reaction to 251.

Scheme 22.

Formal synthesis of rac-Cephalotaxine (±)-(1) by Hong via Weinreb's intermediate (±)-12 (2015).

Through a TiCl₄-promoted iminium ion cyclization, the ABC pyrrolobenzazepine 251 was obtained with a ratio of 20/1 in favor of the trans-isomer. The RCM reaction of the trans-isomer of amide 251 did not happen whatever the catalysts used. Reduction of the amide led to a less rigid tertiary amine 252, suitable for the desired cyclization. Metathesis proceeded smoothly in the presence of camphor sulfonic acid (1 equiv.) to protonate the amine and Zhan-1B's catalyst (1 mol %). The ABCD ring system 253 was obtained with inverted relative stereochemistry at C4. Dihydroxylation and Swern oxidation afforded the known demethylcephalotaxinone (±)-(12). Application of an improved Weinreb's procedure¹⁰³ would generate rac-CET (±)-(1) in 12 steps and 26.2% expected OY from homopiperonylic acid (89).

3.2.3.2. N9–C10 Ring Closure e: Chandrasekhar's Total Synthesis (2016)

In 2016, Chandrasekhar et al. developed a total synthesis of rac-CET (±)-(1) via an aryne insertion from the benzyne precursor 255 into β-ketoester 257 as a key step to build the complete set of carbon atoms and functionalities to end up the synthesis of rac-CET (±)-(1) (Scheme 23 ).¹²⁰ An unusual N9–C10 bond formation through an amidation reaction closed B ring (Fig. 17) .

Scheme 23.

Total synthesis of rac-cephalotaxine (±)-(1) by Chandrasekhar (2016).

Figure 17.

N9–C10 bond disconnection e for construction of cephalotaxine (1) B ring.

Aldehyde 256 derived in five steps from l-proline (140) was transformed into β-ketoester 257 by Roskamp conversion. Then, β-ketoester 257 was submitted to [2 + 2] cycloadditon with benzyne generated from aryne precursor 255 easily prepared in a one pot sequence involving involving O-silylation, metal–halogen exchange, O- to C-silyl migration, and entrapment of the phenoxide with triflic anhydride. The unstable benzocyclobutane 258 underwent C–C bond cleavage leading to compound 259. At this point, all carbons atoms of the CET (1) skeleton were introduced. The synthesis of ABCD enone 262 was accomplished by Wacker oxidation, aldol condensation, ester hydrolysis, Butyloxycarbonyl (Boc) deprotection and amidation. Reduction followed by oxidation gave Hanaoka's intermediate⁹⁸ (±)-147. From proline (140), rac-CET (±)-(1) was obtained through intermediate 147 applying literature conditions⁹⁸ in a total of 16 steps and 6.4% OY. Including the preparation of aromatic unit 255 from 5-benzodioxolol (254), rac-CET (±)-(1) was obtained in 18 TNS and 4.7% AY.

3.3. Syntheses of Enantioenriched Cephalotaxine

Synthesis of optically pure CET (1) remains a challenge for organic chemists. Three contiguous asymmetric centers including a tetrasubstituted one on D ring have to be controlled. It was early noticed by Weinreb¹⁰³ that racemic syntheses led to only one diastereoisomer without any required separation. This means that the configurations of the three asymmetric centers are mutually controlled. Strategies using disconnection C4–C5 for B ring formation exemplify this phenomenon. During the cyclization step, the thermodynamically more stable BD ring junction is cis, which is the relative C4–C5 configuration found in natural CET. Moreover, Weinreb also showed that NaBH₄ reduction of cephalotaxinone (10) is stereoselective (Scheme 24 ), delivering the required relative stereochemistry of the hydroxyl function at C3 of CET (1), which is assumed to be formed as a result of hydride attack from the convex face of 10.

Scheme 24.

Stereoselective reduction of cephalotaxinone (10).

It is thus possible to control successively the C4 center via the C5 tetrasubstituted center; then the C3 center via the C4 stereocenter, and consequently, control of the tetrasubstituted C5 stereocenter is the only necessary one to synthetize enantiomerically pure CET (1). Most of asymmetric syntheses are based on the control of this spiro center followed by a stereoselective cyclization affording the B ring and C4 control, a final reduction establishing the desired configuration at C3. The asymmetric syntheses of CET (1) described below are classified according to the type of stereochemical control (asymmetric catalysis, use of chiral auxiliaries, incorporation of chiral building blocks, and resolution strategies) and deconnection of ring B, related strategies then in chronological order in each section.

3.3.1. Asymmetric Catalysis

3.3.1.1. Tietze's Formal Synthesis (1999)

In 1999, Tietze et al. reported an asymmetric version of their previous synthesis⁸⁴ using Corey's oxazaborolidine 267 to reduce enantioselectively ketone 266 (Scheme 25 ).¹²¹ Ketone 266 was prepared by bromination of cyclopentane-1,3-dione (77) with potassium bromate followed by treatment with a large excess of Grignard reagent prepared from chloroalcohol 265 and tosylation (three steps, 40% OY). Chiral bromoacetate 268 corresponding to the future D ring was obtained by reduction with diborane in the presence of catalytic amounts of the chiral oxazaborolidine 267, followed by acetylation of the allylic alcohol. A 87% ee was measured by NMR using Eu(hfc)₃. Formation of the B and C rings was achieved by the strategy described for rac-CET (±)-(1), employing two successive Pd-catalyzed transformations. The optically active form of Mori's intermediate⁸⁵ (−)-88 was isolated. The synthesis of CET (−)-(1) could be likely achieved in 13-steps linear sequence, 3.1% calculated OY, and 87% expected ee from cyclopentane-1,3-dione (77).

Scheme 25.

Formal asymmetric synthesis of cephalotaxine (−)-(1) by Tietze (1999).

3.3.1.2. Tu's Formal Synthesis (2012)

In 2012, Tu et al. developed a formal synthesis of CET (−)-(1) targeting Mori's intermediate⁸⁵ (−)-88, which was built from 1-azaspirocycle (−)-109 by a tandem hydroamination/semipinacol rearrangement reaction.¹²² At the same time, chirality was introduced using a chiral silver phosphate (R)-273 catalyst (Scheme 26 ).

Scheme 26.

Formal asymmetric synthesis of cephalotaxine (−)-(1) by Tu (2012).

The anion of butyne 270 prepared from trimethylsilylacetylene (269) reacted with cyclobutanone to give 272, which was submitted to a cyclization rearrangement. The key reaction was readily performed using chiral silver phosphate 273 to give azaspirocycle 275 in very good yield and 80% ee. The ketone 275 was transformed into vinyl triflate 276, followed by reduction to lead to the key intermediate (R)-109 reported by Stoltz et al. (see Section 3.2.1.3).⁹⁰ Elimination of the tosyl group generated the amine (R)-110, which reacted with bromo aldehyde⁹⁰ 104 (see Scheme 5) under reductive amination conditions to give Tietze's intermediate¹²¹ (R)-86. Application of the Tietze's procedure led to Mori's alkene (−)-88 in 80% ee determined by chiral HPLC. Surprisingly, during its purification by column chromatography on silica gel, authors found that enantiomers could be separated leading to (−)-88 in 35.5% yield with >99% ee. This phenomenon called “self-dispropornation of enantiomers” results from different affinities of aggregates of compound 88 on silica gel.¹²³ Tu thus accomplished a formal synthesis of CET (−)-(1) in 14 steps, 3.3% calculated OY and 80% ee (18 TNS, 2.5% OY). The high purity material (estimated 99% ee) could be obtained in 1.7% calculated OY from trimethylsilylacetylene (269).

3.3.1.3. Renaud's Formal Synthesis (2012)

In 2012, Renaud et al. reported two variants for the formal synthesis of CET (−)-(1).¹²⁴ Both syntheses were based on a stereoselective carboazidation of methylene cyclopentanol derivatives. In the first approach, which will be described later (see Section 3.3.5.2, Scheme 43 ), the starting material was obtained from the racemate by resolution. The second approach was more efficient and started with (S)-cyclopentenol (284) obtained in 96% ee set up via enantioselective Noyori's hydrogenation of propargylic ketone 280 using (S,S)-1,1′-bi-2-naphthol 281 to give 282. Compound 282 was obtained in 64.5% yield from 4-pentenoic acid (277) (Scheme 27 ).¹²⁵

Scheme 43.

Formal asymmetric synthesis of cephalotaxine (−)-(1) via Nagasaka's intermediate by Renaud (2012).

Scheme 27.

Alternative formal asymmetric synthesis of cephalotaxine (−)-(1) by Renaud (2012).

Dihydroxylation of silyl enolate (S)-285 afforded the diol 286 as a single diastereoisomer, which was protected as an acetonide. Desilylation, oxidation, and Wittig methylenation afforded the bicyclic alkene 287, which was submitted to radical carboazidation. The azido ester 289 was obtained in high diastereoselectivity (dr > 97:3) resulting from exo-selective azidation of the bicyclic intermediate radical 288. The spirolactam 290 was obtained by reduction of the azide. First, the stereogenic center at C5 (CET numbering) of (S)-cyclopentenol 284 was used to control the configuration of the newly formed diol at C1–C2, and then it was destroyed on double bond formation in 287. This chiral center was eventually controlled during the carboazidation step leading to 289. This sequence corresponds to the concept of self-regeneration of stereogenic centers.¹²⁶ Reduction of the lactam 290 gave an amine, which was N-alkylated with nosylate 91 (see Scheme 4), to afford the ACD aryl iodide 291. After hydrolysis of the acetonide, the diol was converted to thionocarbonate 292, which on treatment with Corey–Hopkins’ reagent afforded the desired alkene (R)-102. It was noteworthy that no racemization took place during the synthesis of alkene (R)-102. Optically active alkene (R)-102 was already prepared by Mariano127, 128 (Scheme 42 ) and, in its racemic form, by Suga and Yoshida⁸⁶ (Scheme 4).

Scheme 42.

Formal asymmetric synthesis of cephalotaxine (−)-(1) by Mariano via Suga and Yoshida's intermediate (−)-102 (2006).

Formation of ring B via Heck intramolecular reaction according to Tietze's procedure afforded Mori's intermediate⁸⁵ (−)-88. Synthesis of CET (−)-(1) could be likely achieved in 97% ee within 26 steps from 4-pentenoic acid (277) and 0.9% expected OY.

3.3.1.4. Trost's Formal Synthesis (2012)

In 2012, Trost et al. developed an enantioselective palladium-catalyzed [3 + 2] cycloaddition of trimethylenemethane with a nitroalkene and described an application to the synthesis of the enantiopure AD unit (+)-297 (Scheme 28 ),¹²⁹ which racemic form is an intermediate in Ikeda's synthesis.¹³⁰

Scheme 28.

Formal asymmetric synthesis of ent-cephalotaxine (+)-(1) by Trost (2012).

The phosphoramidite ligand 296 provided the chirality during the [3 + 2] cycloaddition, delivering compound (+)-297 in 91% ee (chiral HPLC). Transformation of compound (±)-297 into rac-CET (1) had been previously reported by Ikeda, and formally, ent-CET (+)-(1) (enantiomer of the natural product) could be obtained in 91% expected ee and 14% calculated OY over a 19 steps in the LLS from piperonal (82).

3.3.2. Use of Chiral Auxiliaries

3.3.2.1. Royer's Total Synthesis (2004)

In 2002, Royer et al. described an asymmetric synthesis of azaspiro compound (−)-303, containing the CD ring fragment exploiting a stereoselective semipinacolic reaction as the key step.¹³¹ In 2004, these authors developed an enantioselective synthesis of (−)-CET (−)-(1) from spirolactam ketal (−)-306,¹³² relying on Kuehne⁹⁴ and Nagasaka's¹³³ procedures (Scheme 29 ). The immolative removal of the chosen chiral inducer, (S)-1-(1-naphthyl)ethylamine (298), did not allow the recovery of this chiral material.

Scheme 29.

Asymmetric synthesis of cephalotaxine (−)-(1) by Royer (2004).

The silyloxypyrrole 300 prepared from 2,5-dimethoxy-2,5-dihydrofuran (299) over two steps using (S)-1-(1-naphthyl)ethylamine (299) as the chiral source, was subjected to a vinylogous Mukaiyama aldol reaction with cyclobutanone (271) affording the α,β-unsaturated γ-lactam 301. On treatment with concentrated HCl, a semipinacolic rearrangement of 301 furnished the desired spiro compound (−)-303. The N-acyliminium ion 302 was attacked preferably anti to the naphtyl group, delivering compound (−)-303 with a good diastereoisomeric excess (de 80%). The major diastereoisomer was recrystallized and also easily recovered by chromatography, and thus azaspirononanedione (−)-303 was obtained in 70% yield and de surpassing 99%. The ketone function of (−)-303 was protected as a cyclic ketal, and then reductive cleavage of the N-benzylic bond provided spirolactam ketal (−)-304. N-alkylation of its sodium salt with the tosylate 305 of 3,4-methylenedioxypheny-2-ethanol^{133, 134} followed by ketal hydrolysis afforded the enantiopure ACD intermediate (−)-306 described by Kuehne in its racemic form.⁹⁴ Following Kuehne's procedure, compound (−)-306 was transformed into (−)-CET (1) with improved formation of enone (−)-307 using a stoichiometric amount of Pd(OAc)₂ without benzoquinone (83% yield). A further modification consisted in the use of AlH₃ ⁹⁶ at 0°C instead of LiAlH₄ in refluxing tetrahydrofuran (THF) for the reduction of (−)-308 to avoid any racemization of sensitive CET (1). CET (−)-(1) was thus obtained in 98.7% ee and 7.5% OY over a 16-steps linear sequence from 2,5-dimethoxy-2,5-dihydrofuran (299) using (S)-1-(1-naphthyl)ethylamine (298) as a chiral source.

3.3.2.2. Dumas and d’Angelo's Formal Synthesis (2005)

After having explored ABC → ABCD synthetic strategies,116, 117 in 2005, Dumas and d’Angelo et al. reported a novel enantioselective synthesis of the azaspiro unit (−)-304 containing CD rings described by Royer¹³² based on an asymmetric Michael reaction involving a chiral enamine prepared from cheap and recyclable enantiopure (R)-1-phenylethylamine 309 (Scheme 30 ).¹³⁵ As a consequence of stereogenic control of the quaternary carbon center in the resulting (R)-ketodiester 311, the two asymmetric centers C3 and C4 of CET (−)-(1) were formed with their natural ACs. Similarly, use of the antipodal (S)-1-phenylethylamine (309) could afford ent-CET (+)-(1), although it would lead to biogolically inactive esters.¹³⁶ The key step was a Curtius rearrangement of acyl azide 314, which allowed installation with perfect stereochemical fidelity and high efficiency of an α-nitrogen substituent at the tetrasubstituted center in ketodiester 311 to set the tetrasubstituted spirocyclic center.

Scheme 30.

Formal asymmetric synthesis of cephalotaxine (−)-(1) by Dumas and d’Angelo (2005).

The ketodiester (R)-311 was elaborated in 85% yield and an ee ≥ 95% through Michael addition of enamino ester (R)-310 to methyl acrylate. Sequential highly stereoselective Luche reduction of the ketone at low temperature followed by saponification and acidification provided an intermediary hydroxy diacid, which on spontaneous lactonization generated the diastereomerically pure lactone acid 313 obtained in 75% yield. The two ester functions of 311 were differentiated at this stage. The acid 313 was first converted into acyl azide 314, which was directly rearranged into the benzyl carbamate 315 by treatment with benzyl alcohol at 110°C. Saponification followed by oxidation with in situ-generated ruthenium tetroxide, and then esterification of the intermediate acid by diazomethane afforded the ketoester (R)-316. This compound was converted into ketal and then submitted to hydrogenolysis to deliver the target spiroketal amide (−)-304. By relying on Royer¹³² and Kuehne's procedures,⁹⁴ using tosylate 305 obtained in two steps and 76.5% OY from safrole (317)117, 118 via 2-arylethanol 211, the synthesis of CET (−)-(1) could be achieved in expected ee ≥ 95%, 20 steps from β-ketoester 105 and calculated 5.1% OY (Scheme 30).

3.3.3. Incorporation of Chiral Sources

3.3.3.1. Ikeda's Formal Synthesis (1999)

In 1999, Ikeda et al. developed an asymmetric version¹³⁷ of their racemic synthesis.130, 138 The intermediate ABCD ketone (+)-149 was prepared from unnatural d-proline (R)-140 used to furnish the C ring with C4 atom and to control the C5 tetrasubstituted stereogenic center set in 319 (Scheme 31 ).

Scheme 31.

Formal asymmetric synthesis of cephalotaxine (−)-(1) by Ikeda (1999).

Compound 318 obtained from d-proline (R)-140 according to Seebach¹³⁹ was stereoselectively allylated to give oxalolidinone 319, which reacted with 5-lithiobenzodioxole (320), generated from 4-bromobenzodioxole (itself prepared from benzodioxole 330) and n-BuLi at −78°C. Then the intermediate aminoketone was protected to give the AC ring unit found in 321. Wacker oxidation, followed by aldol condensation of dione 322 generated the D ring of CET (1) characteristic of the α,β-unsaturated ketone 323. Catalytic diastereoselective hydrogenation provided the cyclopentanone 324 with the desired AC at C4 and C5. Eventually, Ikeda applied his racemic strategy130, 137 to build the B ring in six steps using a Pummerer reaction for closure of B ring (327 → 328). Hanaoka's intermediate (+)-147 was obtained in two steps by treatment with K₂CO₃ and Swern oxidation. Ikeda proposed a formal synthesis of CET (−)-(1) in 88% estimated ee (HPLC), 19 steps from d-proline (R)-140 and 5.1% expected OY.

3.3.3.2. El Bialy's Formal Synthesis (2002)

In 2002, El Bialy described a formal synthesis of CET (−)-(1), where formation of the B ring involved a palladium-catalyzed cyclization leading to Hanaoka's⁹⁸ ketolactam intermediate (+)-147 (Scheme 32 ).¹⁴⁰ The optically active ACD iodoaryl enone 329 was prepared from d-proline (R)-140 by Seebach's method¹³⁹ relying on Ikeda's procedure. Using 20 mole % Pd(OAc)₂, PPh₃, Et₃N, and formic acid in THF allowed the intramolecular reductive Heck reaction to furnish the ketolactam (+)-147 in 76% yield, a noticeable improvement compared to Ikeda's method130, 137 whose catalysis implying Pd(OAc)₂ in the presence of PBu₃, 1,3-bis(diphenylphosphino)propane (dppp), Ag₂CO₃ in DMF led to (+)-147 in a modest 7% yield.

Scheme 32.

Asymmetric formal synthesis of cephalotaxine (−)-(1) by El Bialy (2002).

This formal synthesis would deliver CET (−)-(1) in expected 89.3% ee over a 14-steps linear sequence from d-proline (R)-140 and an evaluated 12.2% OY.

3.3.3.3. Hayes's Formal Syntheses (2008)

In 2008, Hayes et al. developed a formal synthesis of CET (−)-(1) based on an alkylidene carbene 1,5-CH insertion reaction as a key step to construct the 1-azaspiro[4.4]nonane CD ring system (338 → 339).¹⁴¹ In a first approach, vinyl chloride 338 was chosen as a carbene precursor prepared in seven steps and 50% OY from l-proline (S)-(140) via N-Boc-proline methyl ester 331 (Scheme 33 ).

Scheme 33.

Formal synthesis of cephalotaxine (−)-(1) by Hayes via Mori's intermediate (−)-88 (2008).

DIBAL-H reduction of 331 followed by Wittig reaction with 1-triphenylphosphoranylidene-2-propanone (332) gave an intermediate E-enone. Catalytic hydrogenation and a second Wittig reaction with chloromethylenephosphorane (334) afforded the vinyl chloride 335. After deprotection of the amine, amide formation with the carboxylic acid 89 and reduction of the resulting amide 337 led to the pivotal alkylidene carbene precursor 338. Its treatment with potassium hexamethyldisilazide caused the key alkylidene carbene 1,5-CH insertion reaction leading to the desired azaspiro[4.4]nonane derivative 339 in good yield (65%–79%). Regioselective iodination of the aromatic ring system gave the Heck-cyclization precursor 340. Its X-ray crystal structure allowed the confirmation of its absolute stereochemistry, thus demonstrating that as expected the 1,5-CH insertion reaction had proceeded with retention of configuration. The Heck cyclization under modified Fu's conditions (Pd(OAc)₂, HBF₄·PtBu₃, Cs₂CO₃) delivered the ABCD olefin 341 in 61% yield, together with enamine 342 (30%). The last stage involved removal of the extra carbon atom at C3 on ring D by decarbonylation of aldehyde 347. Toward this end, alkene 341 was epoxidized using dimethyldioxirane in the presence of BF₃·Et₂O. The resulting epoxide 343 was submitted to a regioselective Yamamoto rearrangement to lead to an allylic alcohol protected as an acetate 345, which was transposed by treatment with aqueous HCl to primary alcohol 346 and then oxidized (Swern) into aldehyde 347, which, in the presence of Wilkinson's catalyst, afforded the key Mori's intermediate (−)-88. CET (−)-(1) could be likely obtained using Mori's protocol⁸⁵ in this way in 87% expected ee, 23 steps and 0.06%–0.2% calculated OY from l-proline (S)-140.

In their second formal synthesis of CET (−)-(1), an alkylidene carbene 1,5-CH insertion reaction was also used to establish the key tetrasubstituted stereocenter as previously described (Scheme 34 ).¹⁴²

Scheme 34.

Second formal asymmetric synthesis of cephalotaxine (−)-(1) by Hayes (2008).

However, as there is no additional carbon atom on the phosphorane 349, the previous tedious sequence to remove this very atom on ring D is no longer required (341 → (−)-88), seven steps, 6.3%–18.3% OY, Scheme 33). The carbene precursor 350 was derived from N-Boc prolinal (S)-348, obtained from l-proline (S)-140 in three steps and 94% yield.¹⁴³ The resulting spirocyclic product 352 of carbene insertion was converted by Friedel–Crafts cyclization into Mori's intermediate⁸⁵ 363 (R = Me) or (−)-88 (R = -CH₂-), which were then transformed into CET (−)-(1) following Mori's procedure (Scheme 34).

Ketone 350 was prepared by Wittig reaction of aldehyde 348 with phosporane 349. Protection of hydroxymethyl moiety with TBS was suitable for the carbene insertion, thus treatment of 350 with lithium trimethylsilyldiazomethane gave the azaspirocycle 352, which was converted to NBoc spiroamine 355 by successive deprotection, oxidation, and decarbonylation in good yield (63.2%–84.6% from 352). Because racemization occurred at the tetrasubstituted stereocenter on N-deprotection, compound 355 was first subjected to epoxidation. The epoxide 356 was not isolated and immediately treated with Lewis acid to provide 357 as the major product (2:1). Alcohol 357 was transformed into the CD amino alcohol 360 via tosylation, elimination with DBU to deliver the intermediate cyclopentene 186, and hydrolysis of the cyclic carbamate (Scheme 34). Alkylation of 360 with the known nosylates 361 and 362 provided the ACD intermediate allylic alcohols 363 and (−)-364, which were subjected to Friedel–Crafts cyclization relying on Mori's procedure⁸⁵ (R = Me, PPA, 85%) or in Kuehne's conditions⁹⁴ with tin tetrachloride to give the intermediate (−)-88 although unexpectedly in medium yield. Herein, the relative configuration of the hydroxy group of 364 is very important. Compound 187, the diastereomer of 363, with the hydroxy group of opposite configuration, did not undergo the cyclization reaction (see Scheme 13). This sequence would allow access to CET (−)-(1) in 91% ee, in 22 steps and 0.3%–0.4% OY (R = Me) or in 20 steps and 0.4%–0.5% OY (R = -CH₂-) from l-proline (S)-(140).

3.3.3.4. Djaballah and Gin's Synthesis (2008)

Three essential steps were used by Djaballah and Gin et al. for their synthesis of CET (−)-(1) (Scheme 36 ).¹⁴⁴ First, the nitrogen atom was introduced from the oxime 374 followed by Neber rearrangement. Secondly, construction of the benzazepine core relied on a strain-releasing rearrangement of N-vinyl-2-aryl aziridine 376 to 378. Eventually, assembly of the spirofused pyrrolidine core occurred via 1,3-dipolar cycloaddition of azomethine ylide derived from vinylogous amide 379. The chirality was introduced from the optically pure cyclopentenone 372 substituted on C1 and C2 (CET (1) numbering), prefiguring the D ring. Compound 372 was prepared from d-ribofuranose (365). The isopropylidene group allowed stereocontrol, so that only one diastereomer 381 was obtained.

Scheme 36.

Total asymmetric synthesis of cephalotaxine (−)-(1) by Djaballah and Gin (2008).

Synthesis began by double protection of d-ribofuranose (365), then C1 olefination followed by Parikh–Doering oxidation furnished ketone 368, which was alkylated with with vinylmagnesium bromide to provide the allylic alcohol 369 (8:1 dr) (Scheme 35 ).

Scheme 35.

Asymmetric synthesis of cephalotaxine (−)-(1) by Djaballah and Gin: formation of ring D (2008).

Cyclopentene 370 was prepared by an RCM (Grubbs II). Regioselective chloroselenylation of the alkene followed by selenide oxidation and elimination afforded the chlorocyclopentene and deprotection gave the vicinal diol 371. Eventually, the chiral β-chloroenone 372 [10 steps, 42.2% OY from d-ribofuranose (365)] was obtained by periodate-mediated oxidative cleavage of the diol 371.

After the synthesis of 372 featuring the D ring of CET (1) (Scheme 35), the aziridine 375 was prepared in a few steps (Scheme 36). Condensation of ketone 373 with hydroxylamine chlorhydrate furnished oxime 374, which was reacted with LiAlH₄ and diisopropylamine at 60°C affording the racemic aziridine 375 through a Neber-type reductive rearrangement. An addition–elimination sequence starting with the addition of aziridine 375 onto β-chloro-enone 372 furnished the N-vinyl aziridine 376 as a 1:1 diastereomeric mixture (Scheme 36). The dihydro-3-benzazepine 378 was prepared by heating the aziridine 376 with Cs₂CO₃, which generated an efficient rearrangement via opening of the aziridine. Compound 378 was N-alkylated with TMSCH₂I to give the tertiary vinylogous amide 379. O-Activation of vinylogous amide group in 379 was carried out by pivaloyl triflate (generated in situ by combination of pivaloyl chloride and AgOTf), followed by desilylation with TBAT to give azomethine ylide 380. The dipolar cycloaddition with phenyl vinyl sulfone regioselectively afforded the spirofused pyrrolidine 381. In unexpected ways, only one diastereoisomer 381 was obtained; the C1–C2 isopropylideneketal was effective as a stereodirecting element. The desired R configuration at C5 was determined by single crystal X-ray diffraction analysis. Radical desulfurization followed by hydrolysis of pivalate and reacylation with benzyl chloroformate led to 382. After diol deprotection, differentiation of the C1 and C2 oxygen substituents was achieved by using Boc₂O and Yb(OTf)₃ leading to a selective C1-O-carboxylation. The hydroxy group at C2 was oxidized to ketone using iodoxybenzoic acid (IBX). The enone 383 reacted with CrCl₂, which induced reductive deoxygenation of the O-Boc group. Subsequent benzylcarbonate hydrogenolysis provided demethylcephalotaxinone (−)-12 (Weinreb's intermediate⁸³). Sequential methyl enol ether derivatization of the C2 ketone (55%) and stereoselective reduction at C3 with NaBH₄ (95%) concluded the synthesis of (−)-CET (1) in 25 steps from d-ribofuranose (365) and 1.0% OY (ee n.r.).

3.3.3.5. Ishibashi's Total Synthesis (2008)

In 2008, Ishibashi et al. reported an asymmetric synthesis¹⁴⁵ based on a previous construction of the CET (1) skeleton using as characteristic feature an elegant radical cascade for the simultaneous construction of the B and C rings.¹⁴⁶ Diethyl D-(−)-tartrate (384) was transformed into acetonide diol 385 whose tosylation and reaction with lithium phenylacetylide gave compound 387 (Scheme 37 ). After bromination, the acetonide group was removed and the intermediate bromodiol 388 was subjected to Bu₃SnH-mediated radical cyclization to afford the cyclopentane diol 389 in 47% yield from 384 (eight steps). The two hydroxyl groups were protected and subsequent ozonolysis afforded the desired chiral cyclopentanone 390, which was condensed with amine 392 in the presence of Ti(Oi-Pr)₄ to give an intermediary imine acylated with acryloyl chloride to afford enamide 393 in 50% yield (Scheme 37).

Scheme 37.

Total asymmetric synthesis of cephalotaxine (−)-(1) by Ishibashi (2008).

Its treatment with Bu₃SnH in the presence of 1,1′-azobiscyclohexanecarbonitrile (ACN) generated a radical cascade reaction building simultaneously the two rings B and C by a 7-endo and 5-endo selective cyclization, delivering the desired ABCD compound 394 in 27% yield. Deprotection of silylated hydroxyl groups by tetrabutylammonium fluoride gave diol 395, which on treatment with trifluoroacetic acid anhydride, dimethyl sulfoxide, and Et₃N treatment afforded compound 396. Methylation of 394 and ultimate reduction using aluminum hydride96, 132 led to CET (−)-(1) in 99.6% ee, ending the total asymmetric synthesis in 1.3% OY and 17 steps from diethyl d-tartrate (−)-(384).

3.3.4. Chemical Resolutions

3.3.4.1. Nagasaka's Formal Synthesis (1997)

In 1997, Nagasaka et al. proposed the synthesis of CET (−)-(1) and ent-CET (+) (1) via resolution of chiral actetals of the spiroketone (±)-403 providing the C and D rings (Scheme 38 ).¹³³ This compound was used in the known procedure developed by Kuehne.⁹⁴ Alkylation of the Stork enamine of cyclopentanone by methyl acrylate gave the cyclopentanone ester 399. After formation of the vinyl acetate 401, a nitro group (nitrogen atom precursor) was introduced using ammonium nitrate and trifluoroacetic anhydride to yield to the nitro compound 402. Reduction of the nitro group and in situ lactamization allowed formation of the spirolactam (±)-403.

Scheme 38.

Formal asymmetric synthesis of cephalotaxine (−)-(1) by Nagasaka (1997).

Acetalization of (±)-403 with (R,R)-2,3-butanediol 404 afforded two diastereoisomers (−)-405 and (+)-406, which were easily separated by HPLC (100% combined). The optical purity was checked by chiral HPLC on compounds (−)-403 and (+)-403 obtained by hydrolysis of their respective acetals. Each enantiomer was confirmed to be enantiomerically pure. The two diastereoisomers (−)-405 and (+)-406 were alkylated with sulfonic ester 305 leading to Kuehne's intermediates (−)-306 and (+)-306. Synthesis of CET (−)-(1) and ent-CET (+) (1) in 99% estimated ee could be achieved employing Kuehne's procedure⁹⁴ in 16 steps from cyclopentanone (397) and 5.8% calculated OY for each enantiomer.

3.3.4.2. El Bialy's Formal Synthesis

In 2002, El Bialy et al.¹⁴⁰ described a resolution of ACD spiroamine (±)-408, an intermediate of Ishibashi and Ikeda's syntheses130, 137, 138 of rac-CET (±)-(1), with l-malic acid 409 (Scheme 39 ). Formal synthesis of CET (−)-(1) through Hanaoka's intermediate (+)-147 ⁹⁶ is described in Section 3.3.2.2 (Scheme 32).

Scheme 39.

Formal asymmetric synthesis of cephalotaxine (−)-(1) by El Bialy (2002).

Recrystallization of the ammonium malate of spiro compound (±)-408 gave the enantiomer (−)-408 which could be converted to unnatural ent-CET (+)-(1). The mother liquor of malate salt afforded Ikeda's intermediate (+)-408 ¹³⁰ (yield not reported), which was protected and oxidized to the known ketone (+)-324 and then transformed according to their sequence137, 138 into intermediate (+)-149 in 89.3% ee measured by HPLC. Applying Hanaoka's protocol would allow the synthesis of CET (−)-(1) in 22 steps, <7.5% OY and 89.3% estimated ee from piperonal (82).

3.3.4.3. Stoltz's Formal Syntheses (2007)

Stoltz et al. developed a formal asymmetric synthesis of the two enantiomers of CET (1) and the first total synthesis of (−)-dupracine (16) using the spirocyclic amine (±)-110 first used in their synthesis of rac-CET (±)-(1) (Scheme 5) to introduce CD rings.⁹⁰ Compound (±)-110 was coupled with the chiral nonracemic hemiacetal 414 obtained by chemical resolution (Scheme 40).⁹⁰

3,4-Methylenedioxymandelic acid (R)-(413) (97.5% ee) obtained by chemical resolution with ephedrine (412) was protected as a 1,3-dioxolan-4-one prior to bromation then reduction to deliver compound 414 as a 1/1 mixture of diastereomers. This mixture reacted with spirocyclic amine (±)-110 (see Scheme 5) under reductive amination conditions to give diastereomers 415 and 416 in a 1:1 ratio, which were smoothly separated by chromatography. Both diastereomers were submitted separately to Heck reaction conditions to produce benzazepines (+)-417 and (−)-418. The benzylic hydroxy group was removed via an ionic deoxygenation procedure to afford the two enantiomers (+)-88 and (−)-88 of Mori's intermediate,⁸⁵ respectively. Formally, both enantiomers of CET (1) could be obtained in 97.5% ee over 14 steps from β-ketoester 105, precursor of the CD unit (±)-110 (Scheme 5), in 0.2% OY for natural CET (−)-(1) and 0.7% OY for ent-CET (+)-(1). In their report, Stoltz et al. also completed the first asymmetric total synthesis of (−)-drupacine (16) in six steps from both anti and syn ABCD units (+)-417 (20.5% OY) and (−)-418 (19.3% OY) via a dynamic β-elimination/conjugate addition process of advanced intermediate a-hydroxyenones during acetalization (Scheme 5, Scheme 40). Starting from β-ketoester 105, the sequence consisted in 16 steps via spirocyclic enamine (±)-110 and delivered natural drupacine (−)-16 in a combined 1.34% OY. It is worth noting that there remains only one total synthesis of racemic drupacine (±)-(16) disclosed by Fuchs in 1990.¹⁴⁷

3.3.5. Enzymatic Resolutions

3.3.5.1. Mariano's Formal Syntheses (2006)

In 1994 and 1996 Mariano et al. reported a biomimetic synthesis of rac-CET (±)-(1) by a transannular cyclization.113, 114 In 2006, they developed two formal syntheses of CET (−)-(1) based on photochemistry of pyrrolo-fused pyridinium perchlorate 421 leading to strained aziridine (±)-422.127, 128 The first synthesis led to Mori's intermediate⁸⁵ 430 (Scheme 41 ) and the second one to Suga and Yoshida's intermediate86, 87 (−)-102 (Scheme 42).

Scheme 41.

Formal asymmetric synthesis of cephalotaxine (−)-(1) by Mariano via Mori's intermediate 430 (2006).

The pyrrolo-fused pyridinium salt 421 was irradiated to give the tricyclic aziridine 422, its regioselective ring opening with acetic acid, amine protection with Boc, then protection of alcohol as acetic ester, at last deprotection of the Boc group with TFA led to meso spirocyclic aminodiester 425 in five steps and 38% OY. Desymmetrization by enzymatic hydrolysis with electric eel acetyl cholinesterase afforded the unstable aminomonoalcohol 426 in the range of 80%–90% ee, which reacted with (3,4-dimethoxyphenyl)acetyl chloride (144) to furnish amidoalcohol 427. To remove hydroxy group at C1, compound 427 was transformed into oxalate 428, which underwent radical reduction by treatment with azobisisobutyronitrile (AIBN)/n-Bu₃SnH generating the desired product 429 in a 10% yield along with the isomerized homoallylic alcohol 431 as the major product (65%). Mori's intermediate 430 was obtained by reduction of 429 with LiAlH₄. It was also obtained through a five-step sequence (55% OY) from the rearranged homoallylic alcohol 431 involving alcohol deprotection and oxidation, α,β-unsaturated enone formation, and then ketone and amide reduction. This formal synthesis could allow the synthesis of CET (−)-(1) in 19/23 steps from 419 via ACD intermediate 430 with 0.2% expected OY (0.5% OY brsm) and 51% estimated ee. This medium ee indicates that some epimerization occurred during transformation of spirocyclic compound 426 into ACD unit 430.

The second approach by Mariano et al. described the synthesis of the ACD unit (−)-102, which is the key intermediate described by Suga and Yoshida86, 87 (Scheme 42). The azaspiroalcohol 433 was prepared earlier¹²⁸ in enantiomerically pure form by using a sequential photocyclization/aziridine ring opening/enzymatic desymmetrization sequence with >95% ee in four steps and 19% OY from pyridinium salt 421. Radical reduction with tributyltin hydride and AIBN of the mixed oxalate gave the homoallylic ester 434.

Hydrogenation of 434 with 10% Pd/C followed by hydrolysis with HCl generated the spirocyclic alkanolamine 435. Reaction with iodoarylacetyl chloride 432 furnished the spirocyclic amidoalcohol 436, which was transformed into the tosylate 437. Tosylate elimination with DBU afforded the unsaturated amide 438, which was reduced with aluminum trihydride to give the Suga and Yoshida intermediate (−)-102. This formal synthesis would give CET (−)-(1) in 95% expected ee over 19 steps in 0.5% calculated OY from alcohol 419.

3.3.5.2. Renaud's Formal Synthesis (2012)

In 2012, another formal synthesis of CET (−)-(1) was reported by Renaud.¹²⁴ Unlike the alternative approach (see Section 3.3.1.3, Scheme 27), an access to azaspiroamide 404 (Nagasaka's intermediate)¹³³ was achieved through a stereoselective carboazidation sequence as the key step (Scheme 43).

In this synthesis, the silylated derivative (S)-439 (96.5% ee) was prepared from racemic cyclopentanol¹⁴⁸ 106 via enzymatic enantioselective acetylation with porcine-pancreas lipase (PPL). Carboazidation of (S)-439 afforded azido derivative 440 in 89% yield as a 1.2:1 mixture of isomers and their separation gave the major azido ester (1R,2S)-441 in 59% yield. Then, reductive lactamization, desilylation, and oxidation gave Nagasaka intermediate (R)-403. Formally, this intermediate could be converted into CET (−)-(1) according to Kuehne⁹⁴ and Nagasaka's¹³³ protocols, in 18 steps, 1.8% calculated OY (alternatively 19 steps and 2.5% OY) and 96.5% estimated ee from methylene cyclopentanol 106 (see Scheme 38).

To conclude this part, it is interesting to compare the target intermediates in the formal syntheses of CET (1) as subtle changes in their structure may induce very different reactivities and OY of transformation into CET, either in its racemic form (±)-(1) (Fig. 18 ) or optically active ones, the natural enantiomer (−)-(1) and the distomer (+)-(1) (Fig. 19 ). In addition, this schematic view shows in some ways the interconnections between the different approaches.

Figure 18.

Target intermediates in the formal syntheses of (±)-cephalotaxine (1). Names in boxes refer to a formal synthesis.

Figure 19.

Target intermediates in the formal asymmetric syntheses of cephalotaxine (1). Names in boxes refer to a formal synthesis.

4. Synthesis and Coupling of the Side Chains of Cephalotaxus Esters

Side chains of Cephalotaxus alkaloids are very important for the biological activity of the corresponding esters since it is well known that CET itself does not exhibit interesting activity. They are characterized by a relatively simple structure with a chiral tertiary alcohol moiety. Nevertheless, coupling of the side chain to CET remains an important challenge due to the steric strain exhibited at both the alcoholic function of CET at C3 and the carboxylic function of the side chains, which precluded a classical esterification process (Scheme 44 ). Thus, in most of the syntheses of Cephalotaxus alkaloids, the esterification is done with a late, activated precursor of the side chain. It thus appeared that syntheses of the acidic side chains themselves are not useful processes to attain the Cephalotaxus esters. Nevertheless, numerous interesting syntheses of the acidic side chains have been reported. We will first report on the synthesis of the CET esters and then on the synthesis of the side chains. In each part, syntheses will be presented in chronologic order.

Scheme 44.

Coupling of the side chain onto cephalotaxine (−)-(1).

4.1. Synthesis of Cephalotaxine Esters

The previous review in this series² reported more than 15 syntheses of CET's esters. Due to the impossible coupling with the entire side chain 444, most of these syntheses dealt with the esterification of CET (1) with a precursor α-keto acid 443 as shown in Scheme 44. Formation of the α-ketoester 445 was followed by the introduction of the acetyl group through a Reformatsky reaction. Although this esterification/Reformatsky alkylation sequence is interesting, proceeded well and allowed the preparation of various HTs such as HT (3), HHT (2), deoxyHT (5), and so on, the sequence also exhibits a major drawback: the Reformatsky reaction on the prochiral ketoester part of 445 continuously gave a mixture of two epimers in a 1/1 mixture leading thus to the loss of half part of the CET. This major inconvenience was avoided in only two syntheses of HHT (2): the synthesis reported by Hudlicky¹⁴⁹ (where the presence of the second keto group allowed a chelation with the Reformatsky reagent leading to the formation of a single stereomer) and the synthesis of Cheng¹⁵⁰ (using a chiral sulfinyl ester).

Since the previous review in this series,² only four articles dealing with syntheses of CET esters have been published.

4.1.1. Robin's Synthesis of Semisynthetic Homoharringtonine (1999)

The first one by Robin²¹ in 1999 is quite interesting in opening new routes to these alkaloids. This strategy was based on the esterification of CET (1) with a cyclic precursor of the side chain, the tetrahydropyran rac-449 (Scheme 45 ), which was easily prepared in four steps from α-ketoester 446.

Scheme 45.

Hemisynthesis of semisynthetic homoharringtonine (2) by Robin (1999).

Ketoester 446 was treated with the lithium methyl acetate to give diester 447, which was fully saponified and then cyclized with formic acid to the diacid 448. The latter was selectively monoesterified with BF₃·MeOH to furnish the acid ester 449. CET (1) was coupled with rac-449 using dicyclohexylcarbodiimide or mixed anhydride method, but once again the obtained ester was a mixture of two epimers in a 6/4 ratio and had to be separated by chromatography. While the authors showed that highly enantiomerically pure HHT (2) (99.8% ee) could be obtained in a large scale process, they also developed a diastereoselective sequence. For this purpose, rac-449 was resolved through its quinine ester. Eventually, coupling of CET (−)-(1) and (−)-449 gave anhydroHHT (450), which can also be hydrolyzed to enantiopure HHT (−)-(2). This methodology using tetrahydropyran 449 was patented in 2005 and applied to synthetize various analogs.¹⁵¹

4.1.2. Marguerit's Synthesis of [¹⁴C]-Labeled Homoharringtonine (2015)

In 2015, Marguerit et al. achieved the first radiolabelled synthesis of HHT (−)-(2) (Scheme 46 ).¹⁵² This synthetic process enabled the production of Good Manufacturing Practice (GMP) compliant [¹⁴C]-HHT (−)-(2). This ¹⁴C-labeled compound was used in a human mass balance study that was a postapproval commitment to the US FDA.

Scheme 46.

Synthesis of [¹⁴C]-labeled homoharringtonine (−)-(2) by Marguerit (2015).

[¹⁴C]-Labeled HHT ([¹⁴C]-(−)-2) was obtained from [¹⁴C]-labeled CET ([¹⁴C]-(−)-(1)) according to an efficient semisynthesis described in 1999 using enantiopure Robin's acid 449.²¹ The synthetic strategy to obtain [¹⁴C]-CET (−)-(1) is based on the coupling of [¹⁴C]-labeled tosylate intermediate [¹⁴C]-305 with enantiopure spirolactam (−)-304 developed by Royer¹³² for the synthesis of CET (−)-(1). For their purpose, introduction of the [¹⁴C] label was done by reaction of benzyl chloride intermediate 451 with potassium [¹⁴C]-cyanide, a readily available source of carbon-14, yielding compound [¹⁴C]-452, followed by transformation into [¹⁴C]-tosylate 305. This convergent approach proceeds via two key unlabeled intermediates 304 and 451 synthesized in parallel. Alkylation of spirolactam (−)-304 with the tosylate [¹⁴C]-305 afforded ACD unit [¹⁴C]-(−)-306, which was transformed into radiolabelled [¹⁴C]-CET (−)-(1). Eventually, [¹⁴C]-HHT (−)-(2) was obtained using Robin's protocol by coupling [¹⁴C]-CET (−)-(1) with Robin's acid (−)-(449) and hydrolysis of the side chain of intermediate anhydroHT [¹⁴C] (−)-(450).²¹ From piperonyl alcohol obtained from piperonal (82), the 17-steps sequence provided after preparative reverse phase HPLC a 100% enantiopure product with a radiochemical purity of 98.9% and a chemical purity of 98.5% in 4.475% OY.

4.1.3. Gin's Synthesis of Deoxyharringtonine (2006)

The synthesis of deoxyHT (−)-(5) reported by Gin in 2006¹⁵³ was the most recent and also the most efficient method of synthesis of CET esters. This synthesis was based on the esterification of a cyclic acid, namely the β-lactone 456 obtained in a nonracemic form through an efficient sequence (Scheme 47 ). It started with acetal derivatization of d-malic acid D-(409) with pivalaldehyde to give dioxolanone 453. Diastereoselective alkylation of dioxolanone 453 with prenyl bromide was accomplished after deprotonation with LiHMDS to give the prenylated dioxolanone 454 in 66% yield. This sequence allowed the introduction of the chiral tertiary alcohol with complete diastereoselectivity following the studies of D. Seebach¹²⁶ and Tietze (see Scheme 51 ).¹⁵⁴ Transesterification with acetal cleavage of 454 using benzyl alcoholate provided the tertiary alcohol 455, which was cyclized to the β-lactone followed by hydrogenolysis and hydrogenation to give the acid 456. Activation of the carboxylic function with 2,4,6-trichlorobenzoyl chloride allowed the acylation of CET (−)-(1) followed by transformation into deoxyHT (−)-(5) on methanolysis.

Scheme 47.

Synthesis of deoxyharringtonine (−)-(5) by Gin (2006).

Scheme 51.

Synthesis of side chain acids of harringtonines by Tietze (2005).

4.1.4. Djaballah and Gin's Synthesis of Anhydroharringtonine, Homoharringtonine, and Homodeoxyharringtonine (2008)

Djaballah, Gin, and coworkers extended this study to the syntheses of others CET esters such as HHT (−)-(2), homodeoxyHT (−)-(22), and anhydroHT (−)-(20).¹⁴⁴ Preparation of anhydroHT (−)-(20) (Scheme 48 ) involved the intermediate (R)-455 of the previous synthesis¹⁵³ (see Scheme 47), which, after transformation into the corresponding methyl ester, was cyclized to tetrahydrofuran 457 through alkoxymercuration. Deprotection of the benzyl ester and activation as a mixed anhydride (Yamaguchi's reagent) allowed the coupling with CET (−)-(1) to give anhydroHT (−)-(20) in excellent yield.

Scheme 48.

Synthesis of anhydroHT (−)-(20) by Djaballah and Gin (2008).

Preparation of HHT (−)-(2) and homodeoxyHT (−)-(22) (Scheme 49 ) involved a synthesis based on β-lactone 458 obtained through a sequence, which mimicked the synthesis described in Scheme 47. A cross-metathesis provided the disubstituted alkene 460 in 61% yield (a dimeric bislactone was also formed). After cleavage of the benzyl ester, the activation of the carboxyl group (Yamaguchi's reagent) allowed the coupling with CET (−)-(1) to furnish 461, which was transformed into 462 on methanolysis. Preparation of each natural compound HHT (−)-(2) and homodeoxyHT (−)-(22) from this common intermediate is interesting. When allyl benzyl ether 462 was treated with H₂ (Pd/C) in MeOH followed by addition of AcOH at the last stage of the reaction, HHT (−)-(2) was isolated in 79% yield. On the other hand, 462 gave homodeoxyHT (−)-(22) in 69% yield when submitted to Pd/C catalyzed hydrogenation in glacial AcOH.

Scheme 49.

Synthesis of homoharringtonine (−)-(2) and homodeoxyharringtonine (−)-(22) by Djaballah and Gin (2008).

4.2. Synthesis of Side Chains of Cephalotaxus Esters

Though it has been clearly shown that coupling of CET (1) with entire side chains was not possible, syntheses of these side chains have been continuously prolific. All the syntheses reported since 1998 were asymmetric and are of interest due to their diversity and originality. Some of them included the preparation of already described intermediates in the synthesis of Cephalotaxus esters and then could be considered as formal syntheses of the natural products.

4.2.1. Dumas and d’Angelo's Synthesis (2001)

The first asymmetric synthesis of the side chain ester 473 of HHT (2) was reported by Dumas and d’Angelo.155, 156 In this work, the formation of the chiral tertiary alcohol center was accomplished in a highly diastereoselective manner in an early stage of the sequence. The synthesis started with the preparation of the known chiral imine 465 (Scheme 50 ), which was subjected to a Michael reaction with acetoxyacrylonitrile (466), an equivalent of the acid side chain. The adduct 467 was proved to be stereochemically homogeneous and was hydrolyzed without purification to give the more stable ketone (R,R)-468. The keto group was protected as a dioxolane and the potential aldehyde function was directly transformed into the ester 469 on treatment with MnO₂ in MeOH in the presence of NaCN. The ketal of ester 469 was cleaved using CeCl₃·7H₂O and NaI, and the so-formed keto group was converted to the silylvinyl ether 470. Ozonolysis of 470 gave the sensitive aldehyde 471. Addition of MeMgBr to the crude aldehyde and esterification of the acid furnished alcohol 472. Oxidation of the alcohol 472 to a methylketone followed by new addition of MeMgBr gave the tertiary alcohol, which eventually on hydrogenolysis led to (R)-methyl 3,7-dihydroxy-3-methylcarbonyl-7-methoxyoctanoate (473), the methyl derivative of HHT side chain.

Scheme 50.

Synthesis of homoharringtonine side chain (R)-473 by Dumas and d’Angelo (2001).

4.2.2. Tietze's Synthesis (2005)

In 2005, Tietze reported the preparation of the chiral side chain acids 480–483 of deoxyHT (5), homodeoxyHT (22), nordeoxyHT (21), and neoHT (19), respectively (Scheme 51)¹⁵⁴ based on Seebach's procedure for the alkylation of d-malic acid with self-regeneration of stereogenic centers.¹²⁶ The strategy was used later by Gin for the preparation of CET esters.¹⁵³ The dioxolanone 453 prepared from d-malic acid (D-409) was treated with two equivatents of LiHMDS followed by bromoallyl derivatives to give allylated dioxolanones 474–476 in high yield and diastereoselectivity. On hydrogenation on PtO₂, these compounds furnished dioxolanones 477–479 whose acid hydrolysis permitted the isolation of 480–482, side chains of deoxyHT (5), homodeoxyHT (22), and nordeoxyHT (21), respectively. When 453 was alkylated with benzyl bromide, the same sequence allowed the obtention of 483, the side chain of neoHT (19).

4.2.3. Russel's Synthesis (2006)

An original approach was reported by Russel et al. in 2006.¹⁵⁷ The synthesis started with chiral nonracemic aldehydes (R)-489 or (R)-491 (Scheme 52 ) whose preparation in large scale was reported by the authors from citric acid (484) and resolution of dioxolanone (±)-487.¹⁵⁸

Scheme 52.

Synthesis of harringtonines' side chains by Russel (2006).

Three compounds were described in this study: (R)-493 and (R)-496, the anhydroHT and HHT's side chains respectively, and (R)-449, the intermediate used by Robin for the synthesis of HHT (2) (see Scheme 46) thus representing a formal synthesis of HHT (2). Dioxolanone (R)-487 was obtained in three steps and resolution in 11.4% OY from citric acid (484).¹⁵⁸ The acid function of (R)-487 was selectively reduced under Rosenmund conditions to aldehyde, which reacted with a vinyl Grignard reagent (491 or 494) to give lactones 492 or 495. Lactone 492 was reduced with rearrangement to (R)-493, the side chain of anhydroHT (20) obtained in 2.3% OY (nine steps from 484), while reduction of lactone 495 gave (R)-496, the side chain of homodeoxyHT (22) in 4.7% OY over eight steps from 484. The same sequence was applied to aldehyde (R)-490 allowing preparation of Robin's intermediate (R)-449 useful for the synthesis of HTs, in 3.3% OY and 12 steps from citric acid (484).

4.2.4. Royer's Synthesis (2009)

Several Cephalotaxus alkaloid side chains were prepared by Royer through a rapid synthesis (four–five steps).¹⁵⁹ The central point of this synthesis is a chiral nonracemic epoxide 498 (Scheme 53 ). Side chains were obtained on epoxide ring opening with appropriate nucleophiles. On the other hand, it was postulated that this epoxide could be a good candidate for esterification with CET (1) while no report of such coupling was available. The diester 501, easily obtained from commercially available monomethyl itaconate (499) and methyl mandelate (500), was epoxydized with mCPBA to give a 1/1 diastereomeric mixture of 502 in good yield. Flash chromatography permitted the isolation of (+)-502 with the required (S,S) absolute configuration. Then, epoxide (+)-502 was reacted with various organocuprates to give compounds 503–507. Hydrogenolysis of 503–507 allowed the obtention of (R)-508, (R)-496, (R)-509, side chains of deoxyHT (5), homodeoxyHT (22), and neoHT (19), respectively and analog (R)-510 in 12.2%–17.8% OY. Hydroxymercuration of 507 followed by spontaneous cyclization and then hydrogenolysis of the chiral appendage delivered (R)-494, the side chain of anhydroHT (20) in 10.8% OY.

Scheme 53.

Synthesis of harringtonines' side chains by Royer (2009).

4.2.5. Yang's Synthesis (2013)

In 2013, Yang reported the [2,3]-Meisenheimer rearrangement as a general strategy for the construction of chiral tertiary alcohols.¹⁶⁰ As an illustration of this method, the synthesis of HT and HHT's side chains is shown in Scheme 54 . The synthesis began with known N-Boc ester (S)-512 of l-aspartic acid obtained through a two-step process (94%). The gem-dimethyl group was first installed by a Grignard reaction to give 513, which was transformed into the lactol 514 by IBX oxidation and concomitant cyclization. The Wittig reaction with phosphorane 515 followed by deprotection and N-bisbenzylation of the amino group furnished key allylamine (S)-516. The Meisenheimer rearrangement occurred on treatment with mCPBA on the latter to give hydroxylamine 517. This compound led to 518 on treatment with H₂ on catalytic Pd/C via double bond saturation and then N–O bond cleavage. Eventually, on oxidation of 518 the HHT's side chain acid (R)-481 was obtained whose methylation yielded the corresponding dimethyl ester (R)-473. Its optical rotation indicated a perfect stereochemical outcome of the rearrangement. A similar process allowed synthesis of a cyclic form of the HT's side chain. Starting from l-threonine (519), amino alkene 520 was prepared in seven steps. The Meisenheimer rearrangement occurred on treating 520 with mCPBA to give the sensitive hydroxylamine 521. Six steps were then needed to obtain (R)-522, a cyclic form of the HT's side chain acid.

Scheme 54.

Synthesis of harringtonines' side chains by Yang (2013).

These authors patented the preparation of HHT's side chain acid (R)-481 by this methodology.¹⁶¹ Through the same sequence, Yang and coworkers have also described the synthesis of the diacid side chain of deoxyHT (5) and homodeoxyHT (22) starting from l-valine and l-leucine respectively.¹⁶²

4.2.6. Hung's Synthesis (2014)

Hung et al.¹⁶³ recently reported the synthesis of the esters (473) and (524) of the side chains of HHT (2) and homodeoxyHT (22), respectively, by using Tietze¹⁵⁴ and Gin's methods¹⁴⁴ (Scheme 55 ). Allylation of 453 (see Scheme 51), esterification, and then cross-metathesis (see Scheme 49) with allyl alcohol 524 allowed the preparation of dioxolanone 523. The latter was subjected to different hydrogenation conditions to give after methanolysis (R)-473 or (R)-526, side chains' esters of HHT (2) and homodeoxyHT (22).

Scheme 55.

Synthesis of homoharringtonine and homodeoxyharringtonine side chains by Hung (2014).

4.2.7. Mac's Synthesis (2016)

In 2016, Tietze's method¹⁵⁴ (see Scheme 51) was also used by Mac et al. for the synthesis of a series of side chains analogs of HHT (2) (Scheme 56 ).¹⁶⁴ The dioxolanone 453 was diastereoselectively allylated to furnish compound (R,R)-523. A Mizoroki–Heck type reaction with various phenylboronic acid derivatives followed by hydrogenation and methanolysis gave arylated ester chain analogs 527a–h of HHT (2) in moderate to good yield.

Scheme 56.

Synthesis of homoharringtonine side chain's analogs by Mac (2016).

5. Synthetic Analogs and Their Biological Properties

The synthesis of analogs of bioactive natural products remains a broad area of research that can lead to very interesting new structures with improved activity. In the case of Cephalotaxus alkaloids, the exchange of the ester side chain appears to be the simplest way to access analogs and this strategy has been used and described in some patents. The structural modifications of the CET core seem more difficult but have been also scarcely addressed giving rise to interesting compounds and synthetic pathways. We will describe below the most significant reports. Works describing attempts to synthesize CET (1) or the preparation of compounds with a passing structural resemblance will not be considered in this section.

5.1. Cephalotaxine Analogs

5.1.1. Tietze's Cephalotaxine Analogs (2000, 2007)

Since 1997, several reports on the synthesis of CET's analogs have appeared. Tietze et al. in 2000¹⁶⁵ described the preparation of four analogs rac -532–535. For these compounds, variation of the size of the different rings B and C of the polycyclic core of CET (1) and ring A substitution was explored. These authors used their strategy developed for the synthesis of the natural product (Scheme 57 ).

Scheme 57.

Synthesis of cephalotaxine analogs by Tietze (2000).

Compounds 530a–h, with various chain lengths and ring sizes, were easily prepared in a few steps by alkylation of 84 and 528a–c with 81 and 529a–c using a standard S_N reaction. Compounds 530a–h were then submitted to a Pd-catalyzed Tsuji–Trost cyclization to give 531a–c,e,f,h in satisfactory yields (compounds 530d,g did not cyclize under these conditions). The final Heck cyclization required the use of Hermann–Beller's palladacycle 87 and allowed the preparation of compounds 532–535 and 88. In this study, the final compounds required further reactions to install at least an alcoholic function on ring D to allow the coupling with side chains. This drawback was eliminated in a following study published in 2007 by the same authors.¹⁶⁶ Furthermore, Cephalotaxus esters analogs were prepared by coupling the CET analogs with homodeoxyHT side chain (Scheme 58 ). The synthesis was now based on a domino reaction giving a rapid and efficient access to ACD compounds 538 from amines 536, 528a, 84 and cyclopentenone ester 537.

Scheme 58.

Synthesis of deoxyharringtonine analogs by Tietze (2007).

The Pd-catalyzed reaction of 538a–c allowed the formation of the tetracycle 539a–c. While LiAlH₄ reduction of 539a–c gave efficient access to amino alcohols 540a–c, which are analogs of CET (1), these compounds did not couple with an activated form of the side chain of homodeoxyHT (22). In contrast, it was observed that NaBH₄ reduction of 539b,c gave the amidoalcohols rac -541b,c, which allowed coupling with the imidazolide 542 of homodeoxyHT side chain furnishing after further reduction the esters 543b,c, which are analogs of homodeoxyHT(22). It should be noted that the presented synthesis is racemic and that optically pure side chains were used leading to a mixture of two diastereomers, which were separated.

5.1.2. Bubnov's Cephalotaxine Analogs (2005–2010)

In 2005, Bubnov et al.¹⁶⁷ published the synthesis of compound rac-548 exhibiting some structural analogies to CET (1) with ring B and D being bridged and not fused (Scheme 59 ). The synthetic strategy was simple and started with 2-pyrrolidone (181), which was diallylated, N N-protected, and transformed into spiro-pyrrolidine 545 through ring closure metathesis. Condensation of 545 with acyl chloride 546 gave access to 547, which was transformed to the desired product rac-548 by Heck cyclization. The same type of work was pursued by the authors in 2010¹⁶⁸ allowing the preparation of new products 549–552.

Scheme 59.

Cephalotaxine's analog synthesized by Bubnov (2005–2010).

5.1.3. Royer's Cephalotaxine Analogs (2004–2010)

Exploiting their early developed strategy, Royer et al. (see Scheme 29) prepared three C7-alkylated CET analogs from chiral nonracemic Kuehne's intermediates 123 and 553 (Scheme 60 ).¹³² Alkylation was carried out on protected lactam 554 through deprotonation using sec-BuLi and addition of MeI, BuI, or PhCH₂Br to provide diastereomeric alkylated lactams 555 and 556 in a 4/1 ratio. The new chiral center of the major isomer has the (S)-configuration. Transformation of acetonide 555 led to alkylated CETs 557–559 in a four-step sequence in 15–28% OY.

Scheme 60.

Synthesis of Cephalotaxine alkylated analogs by Royer (2004).

In 2010, these authors reported a different type of CET analogs in which the A ring was changed for a phenyl or a naphthyl group (Scheme 61 ) using the same strategy as for CET's synthesis (see Scheme 34).¹⁶⁹

Scheme 61.

Synthesis of cephalotaxine “A” analogs by Royer (2010).

N-alkylation of the spirolactam (−)-304 with the appropriate arylethyl derivative 560a,b gave 561a,b. The end of the synthesis paralleled those of the CET synthesis: functional group manipulations of 561a,b led to 562a,b. These spiro compounds 562a,b were cyclized to the ABCD compounds 563a,b, which were eventually transformed to the phenyl 564 and naphthyl 565 CET analogs.

5.1.4. Chandrasekhar's Cephalotaxine Analogs (2016)

In 2016, Chandrasekhar et al. used their aryne insertion methodology to synthetize three CET analog intermediates (Scheme 62 ).¹²⁰ In these compounds 570–572, the A ring is changed for a phenyl, dimethylphenyl, or dimethoxyphenyl ring.

Scheme 62.

Formal synthesis of cephalotaxine “A analogs” by Chandrasekhar (2016).

To conclude, it is noteworthy that if several analogs of CET (1) have been designed and synthesized, they have been coupled with side chains for only two products (in two reports), although their biological activity was not reported.

5.2. Cephalotaxus Esters With Side Chain Analogs

5.2.1. Robin's Analogs (2002–2004)

Robin et al. patented Cephalotaxus esters 575 with various side chains.170, 171 They were prepared by transesterification of HHT (2) (path B) or ring opening of intermediate 574 (Scheme 63 ) with yields between 21% and 82%. For example, HHT (2) dissolved in (E,E)-hexa-2,4-dien-1-ol was treated with sodium hydride to yield synthetic CET ester 575a in 30% yield. This compound was the most active of the tested analogs in this study with nanomolar IC₅₀ (3.5 ng/mL) while HHT (2) was less potent (IC₅₀ 14 ng/mL).

Scheme 63.

Robin's synthesis of homoharringtonine analogs at the side chain.

Analogs 576 of HT (3), 579 of drupangtonine (33), and 577 and 578 of HHT (2) were also synthetized by conventional methods (Fig. 20 ).

Figure 20.

Harringtonines analogs at the side chain (homoharringtonine (top) and drupangtonine (bottom) analogs) synthesized by Robin (2002–04).

This work delineated the ester groups that reduced the cytotoxic activity against the K562 leukemic cell line (Table 11 ). Nevertheless, a few ones gave rise to improved activity as esters 575a (n-hexadienyl), 575b (n-hexyl), 575s (n-hepthyl), 575v (4-methylbenzyl), 575w (3-methylbenzyl), 575af (2-methylthiophene), 575ag (2-methylfurane).

Table 11.

Amount and yield of Robin's harringtonines (HTs) analogs (see Scheme 63 and Fig. 18) and their cytotoxicity (K562 cell line derived from CML cells)

Compound	Quantity mg	Yield %	IC50 (ng/mL)	Compound	Quantity mg	Yield %	IC50 (ng/mL)
CET(1)	–	–	2000	575ae	62	34	8
HT (3)	–	–	30	575af	85	49	5
576	30	22	7	575ag	47	28	3
577a	11	22	13.5	575ah	100	58,6	18
578	20	21	17.5	575ai	90	54	20
HHT (2)	–	–	14	575aj	147	82	27
575a	175	30	3.5	579a	50	23,5	9
575a	158	47	5	579b	132	22	13.5
575c	171	46,5	8.5	575u	146	44	29
575d	226	54	8.5	575v	141	31	6
575e	171	46,5	10.5	575w	213	45,5	4
575f	122	30	12.5	575x	245	43,3	24
575g	107	44	13	575y	56,5	56	18
575h	289	54	14.5	5752	51,7	50	21
575i	70	31,5	20	575aa	110	60	25
575j	254	61	24	575ab	174	78	12
575k	266	48	28	575r	122	23	100
575m	118	43	50	575s	183	40	3
575n	18	76	50	575t	249	51	9
575o	120	46	80	575ac	A: 54	47	10
575p	107	44	70		B: 46	40
575I	A: 125	55	41	575q	A: 103	50	80
	B: 298	97			B: 143	39
575ad	100	55	25

CET, cephalotaxine.

5.2.2. Djaballah and Gin's Analogs (2008)

In 2008, Djaballah and Gin synthesized four CET-ester analogs, benzyldehydroHHT 580, bisnorhomodeoxyHT 581, bisnorhomodeoxyHT β-lactone 582, and deoxyHT β-lactone 583 (Fig. 21 ), using the protocol described in Section 4.1.3 (Scheme 47).¹⁴⁴ They exemplified up to 43 Cephalotaxus esters analogs, some of which being dimers of CET (1) linked by a central oxygenated chain by the C3 hydroxyl group.¹⁷²

Figure 21.

Djaballah and Gin's cephalotaxine-ester analogs (2009).

These HT structural analogs were tested against human hematopoietic and solid tumor cell lines (Table 12, Table 13 ), and compared to HHT (2), deoxyHT (5), homodeoxyHT (22), and anhydroHT (20), which are among the most active Cephalotaxus alkaloid esters.

Table 12.

Comparative antitumor effect of cephalotaxine esters against HL60 lines sensitive (HL-60) or resistant (HL-60/RV+) to vincristine

Compound	IC50 (μg/mL)		Resistance index	ClogP
	HL-60	HL-60/RV+
DeoxyHT (5)	0.02	0.22	11	1.93
HHT (2)	0.02	2.50	125	0.95
HomodeoxyHT (22)	0.03	0.10	3.3	2.33
580	0.01	0.19	19	2.80
581	0.08	1.00	12	1.21

HHT, homoharringtonine.

Table 13.

Cytotoxicity of deoxyharringtonine (5), anhydroharringtonine (20) and cephalotaxine ester analogs 580–583

IC50 (μg/mL)
Cell lines	DeoxyHT (5)	583	AnhydroHT (20)	580	582	581
HL-60	0.02	2.68	22.7	0.01	5.73	0.08
HL-60/RV+	0.22	21.8	–	0.19	40.30	0.80
JURKAT	0.04	5.71	42.99	0.03	12.01	0.19
ALL3	<0.1b	1.47	>100a	<0.01	4.24	0.16
NCEB1	0.07	8.62	>100a	0.06	39.24	0.50
JEKO	0.08	10.48	>100a	0.08	25.1	0.56
MOLT-3	0.02	2.68	26.83	0.01	6.41	0.06
SKNLP	<0.1b	6.46	5,34	<0.01	10.04	0.11
Y79	70.59	0.095	>100a	>1.00	>1.00	>100a
PC9	0.03	0.039	29.08	0.04	11.29	0.13
H1650	0.04	>1.00a	n.a.	n.a.	n.a.	n.a.
H1975	0.06	4.23	n.a.	n.a.	n.a.	n.a.
H2030	0.10	4.53	n.a.	n.a.	n.a.	n.a.
H3255	0.08	8.42	n.a.	n.a.	n.a.	n.a.
A431	0.06	n.a.	n.a.	n.a.	n.a.	n.a.
HeLa	0.04	n.a.	n.a.	n.a.	n.a.	n.a.
TC71	0.06	12	>100a	0.03	24	0.20
HTB-15	0.20	52	>100a	0.10	58	0.50
WD0082	0.10	5	>100a	0.05	11	0.20

n.a., not active.

^aHighest tested concentration.

^bLowest tested concentration giving 100% cellular death.

HHT (2) shows decreased activity by a factor of 125 (resistance index (RI) = 125), whereas esters deoxyHT 5, homodeoxyHT (22), benzyldehydroHHT (580), and bisnordeoxyHT (581) have much lower RI of 11, 3, 19, and 12 respectively, indicating that they are significantly less sensitive to multidrug resistance (MDR) and tumor cell lines can therefore be considered to be sensitive to these products. One possible explanation for the greater susceptibility of HHT (2) to MDR is its low lipophilicity due to the structure of the side chain, making it a good substrate for the efflux pumps.

Compounds having a ClogP value greater than 1.2 generally lead to a low susceptibility to MDR (i.e., IR = 19 to the esters 5, 22, 580, and 581). The exception is HHT (2), which has a relatively low value of cLogP (0.95, relatively polar) reflecting its susceptibility to MDR (i.e., IR = 125). All these compounds show a broad spectrum of cytotoxic activity, with the exception of the retinoblastoma cell line Y79 (Table 13), which can be attributed to the overexpression of the MDR gene in this line.

5.2.3. Lai's Analogs (2013)

In 2013, Lai et al. applied for two patents, one on preparation of aminated HHT derivatives 584 ¹⁷³ (Fig. 20) and the other on acylated HHT derivatives 585 and 586 (Fig. 22 ).¹⁷⁴

Figure 22.

Lai's homoharringtonine (2) aza analogs (2013).

Compounds 584a-w were prepared from HHT (2) via saponification of the methyl ester to HHT acid (31) and amidation with various amines R¹R²NH in the presence of HATU/DIPEA in 6%–44% yield (Fig. 22). Groups R¹ and R² could be H, C_1–18 alkyl, C_2–18 alkenyl, C_2–18 alkynyl, C_3–7 cycloalkyl or cycloalkenyl, aryl, heteroaryl, or saturated heterocyclic ring systems, etc. Twenty-three HHT aza analogs were tested against human hematopoietic and solid tumor cell lines (Table 14 ), and compared to HHT (2). Analogs 584c, f, n, o, and u were inactive (IC₅₀ ≥ 16 μg/mL). Compound 584b and 584i were the most active of the tested analogs in this study with nanomolar IC₅₀ (up to 42 and 57 ng/mL) while HHT (2) was more potent (IC₅₀ 6 ng/mL). The most promising analogs were tested against HeLa (cervical), CaES-17 (esophageal), CNE (nasopharyngeal), Hep2 (liver), MGC 803 (gastric), PC-3 (prostate), and SK-OV-3 (ovary) human cancer cell lines (Table 15, Table 16 ), and however, none of these compounds were more active than HHT (2).

Table 14.

Cytotoxicity (IC₅₀ and IC₉₀ in μg/mL) of Lai's homoharringtonine (HHT) aza analogs 584a-w

	K562/adr		Kasumi-1		NB4		Jurkat		H9
Compound	IC50	IC90	IC50	IC90	IC50	IC90	IC50	IC90	IC50	IC90
HHT (2)	0.035	0.98	0.005	0.024	0.006	0.012	0.007	16	0.02	0.046
584a	>16	>16	2.75	13	2.59	7.52	3.56	15.04	6.52	>16
584b	3.2	16	0.048	0.21	0.06	0.13	0.037	16	0.04	0.1
584c	>16	>16	11.06	>16	15	>16	13	>16	>16	>16
584d	0.66	4.4	0.053	0.19	0.09	0.2	0.11	16	0.17	0.43
584e	6.24	16	1.3	6.68	2.38	7.61	1.58	8	4	10.64
584f	>16	>16	5	>16	5.62	12.83	4.69	16	13.43	>16
584g	>16	>16	8.54	>16	>16	11.2	3	>16	8	>16
584h	>16	>16	8.28	>16	3.22	9.54	6.7	>16	>16	>16
584i	0.65	9.87	0.038	0.14	0.08	0.23	0.058	16	0.27	3.49
584j	>16	>16	1.33	8.61	1.29	3.7	2	>16	8.25	>16
584k	7.77	>16	0.47	2.32	0.38	1	0.88	16	2.69	>16
584I	0.72	8.99	0.041	0.18	0.08	0.17	0.12	16	0.13	0.35
584m	>16	>16	2	16	1.5	16	1.3	>16	7.26	>16
584n	>16	>16	14.58	>16	8	16	13.4	>16	>16	>16
584o	>16	>16	>16	>16	8.31	>16	>16	>16	>16	>16
584p	4.53	16	0.31	1.77	0.48	2.3	0.46	16	0.6	2.7
584q	14.1	>16	0.8	2.96	0.72	6.3	0.87	>16	5.49	>16
584r	>16	>16	1.58	16	0.7	5.08	0.41	>16	3.69	>16
584s	>16	>16	2	12.02	0.32	4.24	1.47	16	7.37	>16
584t	>16	>16	2.79	14.99	1.85	16	0.89	>16	3.2	>16
584u	>16	>16	>16	>16	7.46	15.79	15.14	>16	>16	>16
584v	5.18	16	0.38	2.25	0.25	0.64	0.48	16	1.41	16
584w	14.5	>16	0.26	2.26	0.19	0.5	0.32	15.21	0.5	7.84

Table 15.

Cytotoxicity (IC₅₀ and IC₉₀ in μg/mL) of Lai's homoharringtonine (HHT) aza analogs 584

Compound	RPMI8226		A549	PANC-1	Becap37	MG63	Huh7	RKO
	IC50	IC90	IC50	IC50	IC50	IC50	IC50	IC50	IC90
HHT (2)	0.006	0.027	0.03	0.035	0.01	0.01	0.004	0.003	0.009
584b	0.042	0.24	0.45	0.13	0.17	0.12	0.044	0.17	0.029
584d	0.092	0.24	0.72	0.15	0.14	0.12	0.12	0.14	6.97
584i	0.057	0.36	0.45	0.26	0.26	0.18	0.2	0.26	0.43
584I	0.1	0.38	0.7	0.23	0.26	0.19	0.1	0.053	0.23
584p	0.47	1.74	1.74	1.94	0.81	0.32	1.05	0.18	1.25
584v	0.48	1.76	1.91	1.91	0.71	0.8	0.94	0.16	2.37
584w	0.17	0.84	0.45	0.45	0.4	0.23	1.37	0.054	0.93

Table 16.

Cytotoxicity (IC₅₀ and IC₉₀ in μg/mL) of Lai's homoharringtonine (HHT) aza analogs 584

Compound	U87 MG		HeLa	CaES-17	CNE	Hep2	PC3	SK-OV-3	MGC 803
	IC50	IC90	IC50	IC50	IC50	IC50	IC50	IC50	IC50	IC90
HHT (2)	0.004	0.018	0.019	0.037	0.038	0.014	0.004	0.003	0.016	0.2
584b	0.12	13.03	0.45	0.29	0.13	0.97	0.31	0.21	0.096	0.25
584d	0.32	16	0.3	1.37	0.15	0.23	0.46	0.47	0.15	6
584i	0.24	1.13	0.35	0.43	0.13	0.15	0.32	0.41	0.24	6.84
584I	0.24	1.06	0.46	0.46	0.15	0.31	0.45	0.57	0.18	0.48
584p	0.89	5.74	1.97	1.91	0.49	1.29	0.91	2.81	0.81	11.81
584v	0.64	16	1.91	1.63	0.76	1.23	0.78	3.97	0.74	>16
584w	0.25	>16	1.56	0.98	0.3	0.27	0.78	1.89	0.39	8.28

Monoacyl-HHT analogs 585a–i and diacyl-HHT analogs 586a–f were prepared with activated HHT and carboxylic acids, anhydrides, or acyl chlorides via esterification in 6%–99% yield (Fig. 23 ).

Figure 23.

Lai's homoharringtonine (2) acylated analogs (2013).

The acylated analogs were compared to HHT (2) and tested against human hematopoietic (Table 17 ) and solid tumor cell lines (Table 18, Table 19 ) including K562/adr (myelogenous leukemia resistant to DOX), Kasumi-1 (acute myeloid leukemia (AML)), NB4 (acute promyelocytic leukemia (APL)), Jurkat (childhood T acute lymphoblastic leukemia (ALL)), H9 (cutaneous T-cell lymphoma), RPMI8226 (plasma cell myeloma), Huh7 (hepatoma), A549 (lung adenocarcinoma), PANC-1 (exocrine pancreas carcinoma), Becap37 (breast), MG63 (osteosarcoma), RKO (colon), U87MG (glioma), HeLa (cervical), CaES-17 (esophageal), CNE (nasopharyngeal), Hep2 (liver), PC-3 (prostate), SK-OV-3 (ovary), and MGC 803 (gastric) cell lines. Contrary to aza analogs 584 and diacylated analogs 586a–f, the monoacetate analog 585a was at least as effective as HHT (2) against all the investigated cell lines (Table 17, Table 18, Table 19). Compounds 585f and 585g were more potent than HHT (2) against 18 and 17 over the 20 cell lines tested, respectively.

Table 17.

Cytotoxicity (IC₅₀ and IC₉₀ in μg/mL) of Lai's ester analogs 585 and 586

	K562/adr		Kasumi-1		NB4		Jurkat		H9		RPMI8226
Compound	IC50	IC90	IC50	IC90	IC50	IC90	IC50	IC90	IC50	IC90	IC50	IC90
HHT (2)	0.035	0.98	0.005	0.024	0.006	0.012	0.007	16	0.02	0.046	0.006	0.027
586a	0.062	1.18	0.006	0.021	0.01	0.022	0.012	16	0.02	0.05	0.009	0.12
585a	0.015	0.31	0.002	0.007	0.002	0.006	0.003	16	0.004	0.01	0.003	0.018
586b	0.41	1.84	0.063	0.22	0.08	0.25	0.088	10.13	0.19	0.4	0.12	0.63
586d	0.32	16	0.065	0.4	0.12	0.4	0.11	7.5	0.21	0.48	0.155	1.13
585b	0.03	0.35	0.005	0.012	0.006	0.012	0.003	16	0.007	0.02	0.006	0.039
585c	0.24	12.57	0.062	0.23	0.07	0.14	0.071	16	0.14	0.25	0.062	0.25
585d	0.06	0.75	0.002	0.005	0.004	0.007	0.012	16	0.008	0.02	0.007	0.029
585e	0.03	3.18	0.0007	0.002	0.002	0.004	0.005	16	0.003	0.01	0.006	0.033
585f	0.01	0.05	0.002	0.004	0.003	0.005	0.002	16	0.003	0.009	0.002	0.014
586e	0.9	3.72	0.14	0.47	0.2	0.49	0.3	16	0.47	1.24	0.29	1.82
586f	2.5	6.5	1.35	3.56	1.19	2.47	1.4	6.4	1.3	3.9	1.15	3.84
585g	0.007	14.1	0.001	0.005	0.003	0.007	0.0038	16	0.004	0.01	0.003	0.015
585h	0.02	0.21	0.006	0.023	0.009	0.022	0.006	6.24	0.01	0.04	0.007	0.034
585i	0.01	0.05	0.004	0.012	0.009	0.022	0.001	11.37	0.01	0.03	0.004	0.024

HHT, homoharringtonine

Table 18.

Cytotoxicity (IC₅₀ and IC₉₀ in μg/mL) of Lai's homoharringtonine (HHT) acylated analogs 585 and 586

	Huh7		A549a	PANC-1b	Becap37c	MG63		RKO
Compound	IC50	IC90	IC50	IC50	IC50	IC50	IC90	IC50	IC90
HHT (2)	0.004	0.049	0.03	0.035	0.01	0.01	1.2	0.003	0.009
586a	0.016	0.13	0.03	0.036	0.015	0.02	2.4	0.007	0.02
585a	0.0021	0.018	0.013	0.012	0.005	0.006	0.42	0.002	0.01
586b	0.13	0.99	0.23	0.69	0.23	0.24	3.25	0.044	0.35
586d	0.17	1.7	0.48	0.43	0.2	0.2	4.75	0.12	0.56
585b	0.012	0.081	0.03	0.029	0.007	0.02	3.08	0.001	0.015
585c	0.056	0.34	0.52	0.21	0.17	0.093	10.76	0.039	0.24
585d	0.004	0.049	0.04	0.044	0.014	0.012	0.77	0.005	0.025
585e	0.0016	0.023	0.03	0.026	0.02	0.0032	0.2	0.003	0.023
585f	0.0022	0.038	0.01	0.009	0.003	0.006	0.44	0.0009	0.005
586e	0.66	7.6	0.89	0.83	0.47	0.21	9.43	0.22	0.87
586f	4.8	14.5	5.4	5.8	2.9	1.34	9.88	1.4	5.24
585g	0.003	0.05	0.007	0.014	0.003	0.006	1.18	0.002	0.006
585h	0.008	0.096	0.03	0.042	0.01	0.022	1.72	0.008	0.02
585i	0.016	0.036	0.02	0.055	0.007	0.021	5.14	0.006	0.02

^aIC₉₀ > 16.

^bIC₉₀ > 12.56.

^cIC₉₀ 5.53–16.

Table 19.

Cytotoxicity (IC₅₀ and IC₉₀ in μg/mL) of Lai's homoharringtonine (HHT) acylated analogs 585 and 586

	U87 MG		HeLaa	CaES-17b	CNEc	Hep2d	PC3d	SK-OV-3d	MGC 803
Compound	IC50	IC90	IC50	IC50	IC50	IC50	IC50	IC50	IC50	IC90
HHT (2)	0.004	0.018	0.019	0.037	0.038	0.014	0.08	0.09	0.016	0.2
586a	0.018	0.11	0.026	0.062	0.062	0.026	0.029	0.12	0.03	0.66
585a	0.002	0.012	0.0077	0.02	0.024	0.0037	0.013	0.05	0.008	0.056
586b	0.16	0.59	0.33	0.61	0.21	0.22	1.37	1.33	0.16	0.76
586d	0.23	0.99	0.45	0.74	0.35	0.23	1.84	0.98	0.24	1.62
585b	0.023	0.18	0.014	0.029	0.031	0.015	0.08	0.08	0.021	0.11
585c	0.31	0.96	0.31	0.38	0.42	0.39	3.15	0.4	0.058	0.16
585d	0.02	0.1	0.026	0.058	0.028	0.015	0.11	0.12	0.015	0.07
585e	0.007	0.048	0.021	0.049	0.03	0.025	0.1	0.051	0.005	0.052
585f	0.008	0.07	0.005	0.014	0.0037	0.004	0.04	0.022	0.006	0.019
586e	0.67	3.42	0.87	1.24	0.77	0.48	0.82	1.94	0.5	12
586f	2	8.54	2.61	3.93	3.91	3.46	3.97	6.71	2.7	9.92
585g	0.012	0.08	0.0034	0.015	0.0014	0.006	0.0077	0.027	0.006	0.055
585h	0.009	0.067	0.011	0.039	0.029	0.012	0.03	0.031	0.025	0.098
585i	0.035	0.16	0.014	0.031	0.012	0.007	0.039	0.034	0.021	0.087

^aIC₉₀ 8.37–16.

^bIC₉₀ 16 or >16.

^cIC₉₀ 14.28−>16.

^dIC₉₀ > 16.

In conclusion, the study of many syntheses of CET (1) published to date can draw a number of conclusions about the key points of its structure and the way chemists have overcome the problems posed by its synthesis. The formation of the B ring often remains a crucial point in building the CET skeleton. The C4–C13 disconnection remains by far the most studied (>30 syntheses of 50). The cyclization reactions used (electrophilic aromatic substitutions, organometallic coupling, etc.) perform generally with good yields but seem susceptible to cyclization intermediates and electronic richness of the aromatic ring. This disconnection is also extremely interesting from a stereochemical point of view providing the correct relative configuration at C4 and C5, avoiding difficult separations. Other disconnections do not always give such good results and are not completely diastereoselective. The formation of the spiro CD system is also a key point in these syntheses; in fact, the control of center C5 at the start of synthesis greatly simplified stereocontrolled strategies and enabled enantioselective approach to CET (1) by asymmetric synthesis of the CD system. Subsequent stereoinduction of the C5 center on C4 center and C4 center on the C3 center facilitate the production of optically active CET (1). However, the operating conditions used after the synthesis of the spiro system must be chosen carefully because of the risk of racemization of CET (1) and certain synthetic intermediates by breaking the N9–C5 bond (See Figure 18, Figure 19, the conversion of Mori's intermediates (±)-88 and (−)-88). Last but not least, from an industrial point of view, initially the Merck Group took over the Weinreb racemic synthesis improving performance (11.7% by eliminating some purification steps). The optically active CET (1) in its natural configuration may be obtained by resolution of racemic cephalotaxine rac-CET (±)-(1) with tartaric acid, by incorporation of chiral sources, or by enantioselective synthesis. The high-purity HHT (2) used for clinical trials was obtained by extraction (NCI batches) and by semisynthesis (ssHHT) from natural CET (1) then purified by HPLC to remove impurities and minor diastereoisomers first by the French company Oncopharm SA later based in Houston (USA). A number of HHT (2) analogs were prepared; however only the monoacylated analogs 585a, 585f, and 585g showed slight increase in activity agains both hematopoietic and solid caner cell lines. The main advantage was to prepare hydrosoluble stable forms.

6. Pharmacology and Clinical Studies

6.1. Cellular Pharmacology

6.1.1. Molecular Mechanism of Action of Homoharringtonine

Molecules such as HHT (2), which works on several targets, are increasingly attractive. Protein synthesis inhibitors could affect the translation of messenger RNA (mRNA) into proteins at several different stages, such as initiation, elongation, and termination as illustrated in Fig. 24 . The main activity of HHT (2) is blocking protein synthesis during the initiation step. Mainly, it inhibits the synthesis of short-lived proteins¹⁷⁵ by binding to the A-site cleft of tRNA A-site in the peptidyl-transferase center of the ribosome. The aminoacyl-tRNAs binding site is universally conserved over prokaryotes and eukaryotes. The Nobel Prize in Chemistry 2009 was awarded jointly to Ramakrishnan, Steitz, and Yonath “for studies of the structure and function of the ribosome.” They have all generated 3D models that have shown how different antibiotics, including HHT (2),¹⁷⁶ bind to the ribosome. Also, they demonstrated by X-ray (at high resolution up to 2.9 Å) the crystals of the 80S ribosome in complexes with 12 eukaryote-specific inhibitors and four broad-spectrum inhibitors in Saccharomyces cerevisiae.¹⁷⁷

Figure 24.

Simplified diagram of protein synthesis showing the A site where homoharringtonine (HHT) binds to inhibit protein synthesis.¹⁷⁸ In the cytoplasm of the cells, ribosomes (green) bind to the mRNA containing the code translated from the switched on genes into the DNA. The 20 different amino acids carried by transfer RNA (tRNA) are recruited according to a three nuclear base code into the ribosome A site. The protein chain elongation includes the aminoacyl tRNA entry, its proofreading, the peptidyl transfer, and the ribosomal translocation along the tRNA chain. The cartoon on the right explains how HHT (2) inhibits protein synthesis through inhibition of initiation elongation step.

Both the ring system and the tail of HHT (2) participate in interactions with the ribosome that contribute to drug binding. HHT (2) interaction with the ribosome is stabilized by hydrophobic interactions and hydrogen bonds (Fig. 25 ). Its six-membered aromatic ring stacks on the base of Cytosine 2452 for Haloarcula marismortui (2487 for Escherichia coli), and it fills the A-site cleft completely, the five-membered dioxolane ring attached to it. When HHT (2) is bound to the ribosome, 73% of its surface area becomes water inaccessible. The tertiary amine of HHT (2), which is protonated at neutral pH, establishes hydrogen bond with the O2 of Cytosine 2452 H. marismortui (2487 for E.coli). The hydroxyl group at the side chain forms a hydrogen bonding to the O6 of Guanine 2058 H. marismortui (2099 for E. coli) via intermediate water molecule, and the carbonyl group at the other end of the side chain establishes hydrogen bond with the N2 of Guanine 2061 for H. marismortui (2102 for E. coli). The binding of HHT (2) to the large ribosomal subunit induces several conformational changes. The most powerful of them is roughly 50 degrees reorientation of the base of Uracil 2506 for H. marismortui (2541 for E. coli), which enables it to stack on top of the drug. In the drug complex, Uracil 2506 for H. marismortui (2541 for E. coli) forms a symmetric O4–N3/N3–O4 base pair with Uracil 2585 for H. marismortui (2620 for E. coli). In all other crystal structures of the large ribosomal subunit from H. marismortui, Uracil 2506 for H. marismortui (2541 for E. coli) forms a Guanine–Uracil base pair with G2583(2618). A number of more subtle conformational changes are seen in the A-site cleft region; C2452 of H. marismortui (2487 for E. coli) undergoes a slight rotation away from A2451 for H. marismortui (2486 for E.coli), and the base of A2453 for H. marismortui (2488 for E. coli) rotates by approximately 40 degrees about its glycosidic bond. This rotation allows A2453 for H. marismortui (2488 for E. coli) to form a base pair with U2500 H. marismortui (2535 for E. coli) and improves its stacking on C2452 for H. marismortui (2487 for E. coli).¹⁷⁶

Figure 25.

Interaction of homoharringtonine (2) with the ribosome of Haloarcula marismortui and the conformational changes associated with this binding.

One of the main actions of HHT (2) is induction of apoptosis (Fig. 25). HHT (2) has a different mechanism of action from that of tyrosine kinase inhibitors, a class of drugs used in for the treatment of CML. Accordingly its activity in CML is independent of the mutation state of BCR-ABL cells (particular CML cells that contain a fusion gene called BCR-ABL1, generated by translocation of sequences from the c-ABLgene on chromosome 9 into the BCR gene on chromosome 22).¹⁷⁹

The mechanism of apoptosis induced by HHT (2) was studied on different leukemic cell lines: U937, HL-60, HL60/MRP, HEL, THP, K562, MUTZ-1, CD34+/38^lo, CD34+, U-87MG, ΔEGFR, NB4, KU812, and KCL22 and on other diseases including systemic mastocytosis (SM) cell lines: HMC-1.2 and P815, multiple myeloma (MM) cell lines AMO-1, IM9, RPMI8226, EJM, U266, BA/F3, hematopoietic cell lines, OPM-2, and human hepatoma QGY-7703 cells.

A study was conducted on SHI-1 cell lines to prove that mitochondria are involved in the apoptosis induced by HHT (2).¹⁸⁰ Although HHT (2) induces apoptosis mediated by mitochondria (intrinsic pathway), it also induces apoptosis through death receptors (extrinsic pathway). The intrinsic pathway is induced by cleaving the proactive forms into active ones. HHT (2) activates some apoptosis mediators; it inhibits the apoptotic inhibitors and upregulates the release of other mediators through the action on genetic level. For instance apoptosis was studied in QGY-7703 cell line and 78 individual mRNAs were identified as having their transcription levels significantly changed. Those genes were mostly oncogenes, tumor suppressors, enzymes, kinases, and partially related to apoptosis, 68% being upregulated and 32% were downregulated. The following signaling apoptosis pathways were activated: TGF-β, tumor necrosis factor (TNF), FAS, p38MAPK, and p53.¹⁸¹ The head protein of intrinsic apoptosis cascade is p53. It was activated by HHT (2) in human hepatoma QGY-7703 cells¹⁸¹, but cell line with p53 mutation was more resistant to HHT (2).¹⁸² A study demonstrated rapid apoptosis of U937 and HL60 cell lines on treatment with HHT (2); this was measured by increased annexin V binding capacity, caspase-3 activation, and the cleavage of poly(adenosine 5′-diphosphate (ADP)-ribose) polymerase (PARP). Also, the expression of Bax was upregulated during cell death induced by HHT (2), while the expression of bcl-2 was only slightly decreased.¹⁸³ On treatment of human myelodysplastic syndrome (MDS) MUTZ-1 cell lines with HHT (2), Ca²⁺ translocated from endoplasmic reticulum (ER) pool to cytosol, the mitochondrial membrane potential (Dwm) decreased, and Bid protein translocated from ER to mitochondria. Ca²⁺ and Bid proteins were released from ER pool to cytosol. The activation of ER stress-associated proapoptotic factor, CHOP and ER chaperones BiP and XBP1 genes, was followed by cleavage of caspase-3 but not caspase-4.184, 185

mRNA expression levels of survivin were investigated in various hematopoietic cell lines in relation with apoptosis induced by HHT (2). It was concluded that the apoptotic effect of HHT (2) on the hematopoietic cell lines is associated with decreased level of survivin expression.¹⁸⁶ In a study of apoptosis using ssHHT (2) on HL60 and HL60/MRP (cells lacking Baxes) cell lines, ssHHT (2) was effective in a time- and dose-dependent manner, and independently of the expression of Bax. Additionally, ssHHT (2) induced apoptosis through the decrease of Dwm and the release of cytochrome c. This was explained by decreased myeloid cell leukemia-1 (Mcl-1) level and cleavage of Bcl-2, one of the proteins that governs the outer membrane of the mitochondria and has antiapoptotic activity (Fig. 26 ).¹⁸⁷ HHT (2) inhibited the telomerase content of HL-60 cells effectively and induced apoptosis.¹⁸⁸

Figure 26.

Main pathways involved in apoptosis mechanism induced by homoharringtonine (2). Yellow: apoptosis factor; Blue: apoptosis intermediator; Turquoise: dual role as intermediator and factor; Green: apoptosis inhibitor.

HHT (2) inhibits synthesis of antiapoptotic proteins of the Bcl-2 family, including Mcl-1 cell lines, leading to cell death. HHT (2) effectively induced apoptosis in primary CML stem cells (CD34+/38^lo) by downregulation of Mcl-1 protein.¹⁸⁹

The intrinsic apoptosis using transforming subfamily of growth factor-β-inducible Sp1-like proteins called transforming growth factor-beta inducible early protein gene (TIEG1) was studied. TIEG1 induced apoptosis through upregulation of Bax and Bim and downregulation of Bcl-2 and Bcl-XL, release of cytochrome c, and caspase-3 activation.¹⁹⁰ The apoptotic effect of HHT was studied in K562 cell line using gene chip technology, it was found that 17 genes were upregulated and 27 were downregulated. Most of them were found to be related to apoptosis, oncogenes, or tumor suppression. Several genes with altered gene expression were found, such as TIEG, vitamin D3 upregulated protein one gene (VDUP1), RNA binding motif protein 4 gene (RBM4), and v-myc myelocytomatosis viral oncogene homolog (C-MYC). The activated transforming growth factor-β and TNF–signaling pathways play an important role in apoptosis according to the dynamic gene expression pattern in these apoptotic cells. TIEG was significantly modified after induction of apoptosis, which is critical for apoptosis signal transmission.¹⁹¹ A Chinese team tried to identify different genes whose expression is modified during apoptosis induced by HHT (2) on leukemic HL-60 cell lines; they demonstrated that TGF-α and TNF apoptosis signaling pathways were initiated but not the Fas signaling pathway. Overexpression of TIEG and VDUP1 could induce apoptosis in some kinds of cells and they were upregulated. Other genes, such as BRD2, ACVRL1, and RBM4 were also upregulated and are probably involved in apoptosis signaling pathways.¹⁹²

The efficacy of HHT (2) against ponatinib-resistant Ph-positive cells Ba/F3 (Ba/F3 ponatinib-R) was shown by decreased protein expression levels of BCR-ABL and Crk-L. On the other hand, caspase-3 and cleaved PARP levels were significantly increased. HHT (2) treatment reduced the expression of BCR-ABL, as well as heat shock protein (HSP) 90, which stabilizes the BCR-ABL protein. It was found that HHT (2) reduced the expression level of the antiapoptotic protein Bcl-2 and of c-Myc. HHT (2) was examined on ponatinib-resistant primary Ph-positive ALL and CP CML cell lines. The synthesis of BCR-ABL, HSP90, and Bcl-2 was downregulated and was independent of caspase activity.¹⁹³ HHT (2) antagonizes specifically the p-eIF4F function, which is specifically required by cancer cell for their survival and not the eIF4F, which is required for normal cells.¹⁹⁴ HHT (2) reduces the levels of prooncogenic or prosurvival proteins (important proteins for tumorigenesis) having short half-lives, such as Mcl-1, cyclin D1, or c-Myc. This was demonstrated on a well-studied transgenic mice model Eμ-myc lymphomas harboring lesions in Pten, Tsc2, Bcl-2, or eIF4E.¹⁷⁵

Cytotoxicity of HHT (2) and cell-growth activity were investigated in three AML cell lines, HL-60, NB4, and U937, and in three CML cell lines, K562, KU812, and KCL22. AML cells showed more sensitivity than CML cells to HHT-induced cytotoxicity. Using HL-60 cells, it was revealed that HHT (2) decreased the levels of Mcl-1, X-linked inhibitor of apoptosis protein (XIAP), survivin, and B-cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins as well as the Dwm. U937, K562, KU812, and KCL22 cells that expressed B-cell lymphoma-extra-large (Bcl-xL) (antiapoptotic transmembrane protein in the mitochondria) were less responsive to HHT-induced apoptosis than HL-60 cells. It was suggested that Bcl-xL plays a more important role than Bcl-2 and Mcl-1 in protecting against HHT (2)-induced apoptosis.¹⁹⁵

In addition, it was demonstrated in leukemic cell lines K562 that HHT (2) inhibits the synthesis of vascular endothelial growth factor, an important mediator of angiogenesis in the bone marrow. This work thus demonstrated a possible antiangiogenic activity for HHT (2).¹⁹⁶

A study to identify novel biomarker that might contribute to variation in response to HHT (2) by using a panel of various human lymphoblastoid cell lines was performed. Genome-wide association analysis was applied using single nucleotide polymorphism (SNP) and mRNA expression data. The integrated analysis identified four unique SNPs that were associated with both expression and area under the curve. Functional validation using siRNA knockdown in leukemia cell lines showed that knocking down CCDC88A, CTBP2, SOCS4 genes in U937 and K562 cell lines significantly altered HHT (2) cytotoxicity.¹⁹⁷

The overexpression of EphB4 contributed to IM resistance in CML line cells. Cell lines from a patient with relapsed CML were established at the time of first diagnosis and after being relapsed. EphB4/RhoA may be a new marker for IM resistance. This is supported by the fact that HHT (2) with IM yielded more treatment advantages than IM alone by blocking EphB4/RhoA pathways.198, 199

Apoptosis varying intervals in K562 cell line treated with HHT (2) was studied. The results showed that the viability of K562 cells reduced gradually from day 1 to day 5 and ascended from day 6 to day 8 after HHT (2) treatment. At the same time, the cleaved caspase-3 expression level in K562 cell lines increased gradually from day 1 to day 7 but decreased at the day 8 (P < .05). From day 1 to day 8 after HHT (2) treatment, the BCL-2 expression level declined first and then went up (P < .05). Autophagosome was also increased at day 8 after HHT (2) treatment. It is concluded that the apoptosis level of K562 cells after being treated with HHT (2) increased at the beginning of the treatment and then it declined, which may be associated by higher autophagy level in the late stage of HHT (2) treatment.²⁰⁰ A recent study demonstrated that HHT (2) binds to the DNA with an intercalation mode.²⁰¹ Also, HHT (2) binds to the telomere G-quadruplex, which makes it a good model for drug design.²⁰²

Analysis of proteins expression profiles in K562 cell line (a CML cell line) in response to HHT (2) treatment showed that nine proteins were downregulated and four upregulated. Aside from alterations in apoptotic proteins and proteins associated with transcription and translation, changes in oxidative stress response and redox reaction–related proteins, such as HSPs, DJ-1, and thioredoxin were revealed. Specifically, these proteins were decreased after HHT (2) treatment in K562 and primary CML cell lines.²⁰³ Also, it was found that HHT (2) is an effective inhibitor of unwanted angiogenesis.204, 205

6.1.2. Potential Use for the Treatment of Other Tumors

Beside its use in the treatment of CML, HHT (2) is being investigated for other type of tumors alone and in combination with other drugs: HHT (2) significantly inhibited the proliferation of human MM cell lines and tumor cells from patients with relapsed refractory MM in a dose-dependent manner. This apoptotic process was associated with the activation of caspase-8, caspase-9, caspase-3, and PARP.²⁰⁶ The effect of HHT (2) was demonstrated on MM, on the following cell lines AMO-1, IM9, RPMI8226, EJM, U266, and OPM-2; HHT (2) significantly reduced Mcl-1, a crucial protein involved in myeloma cell survival, in all the myeloma cell lines that were examined. Also, HHT (2) reduced the levels of c-FLIPL/S, activated caspase-8, and induced active reduction of Bid. It follows that HHT-induced apoptosis appears to be mediated via both intrinsic and extrinsic apoptosis pathways. The resultant imbalance between BH3-only proteins and Mcl-1 may be pivotal for apoptosis by HHT (2). Also, HHT (2) treatment resulted in reduced levels of b-catenin and XIAP proteins, which also contribute to disease progression and resistance to chemotherapy in MM. HHT (2) enhanced the effects of melphalan, bortezomib, and ABT-737 when given in combinations with these agents. These results suggest that HHT (2) could constitute an attractive option for MM treatment through its ability to simultaneously target multiple tumor-promoting molecules.²⁰⁷ HHT (2) and Bortezomib synergistically inhibit SKM-1 MDS cell proliferation and induce apoptosis in vitro.²⁰⁸

Transmembrane receptor of the tyrosine kinase KIT oncoprotein is an important therapeutic target as part of the type III tyrosine kinases. Some mutations in this receptor (V560G, D816V, or D814Y) give it the ability of auto-dimerization and auto-phosphorylation without activation by its ligand, which can eventually lead to uncontrolled cell proliferation and a resistance to apoptosis. The gain of function associated with these mutations of the KIT receptor plays a critical role in the pathogenesis of SM and gastrointestinal stromal tumors (GISTs). The hypothesis that HHT (2), as an inhibitor of protein synthesis, decreases the protein level of KIT thereby reduces the level of phospho-KIT and repeals its constitutive signaling downstream. HHT (2) is effective both in vitro and in vivo demonstrating that it effectively inhibits tumor growth in mice and induced apoptosis of tumor cells carrying the V560G (sensitive to IM) and D816V (insensitive to IM) mutations of KIT. In addition, HHT (2) significantly prolonged the mouse survival time with aggressive SM (35 days instead of 7) though a rapid resistance to HHT (2) was observed in this case.²⁰⁹

Potential activity of HHT (2) against clear cell–type renal cell carcinoma (RCC) with mutant von Hippel–Lindau gene was revealed by high-throughput simultaneous screen and counterscreen.²¹⁰ In addition, HHT (2) has the potential for treatment of the Gefitinib-resistant non–small cell lung cancer, according to an in vivo study.²¹¹

6.1.3. Combination With Other Agents

A signal oncogene is important for some tumors for their continued activity of their malignant phenotype. The BCR-ABL fusion protein in CML is the best-studied example. HHT (2) that inhibits protein synthesis thus reduces the level of the BCR-ABL protein level. The kinase activity of BCR-ABL is directly inhibited by IM and flavopiridol (a transcription inhibitor). The median-effect method was used to evaluate combinations of flavopiridol/HHT (2) and flavopiridol/IM. The resulting analysis indicated synergistic decrease in the clonogenicity. Synergy increased when HHT (2) and IM were administered sequentially as opposed to simultaneously. Transfected IM-resistant Ba/F3 cells that expressed the E255K and T315I mutations of BCR-ABL were not cross-resistant to flavopiridol and HHT (2). These results provided a rationale for the combination of inhibitors of transcription and/or translation with specific kinase inhibitors.²¹²

The combination of HHT (2) and DOX had ratio-dependent synergistic activities, with very strong synergism observed at a molar ratio of 4/1 (DOX/HHT) within double-loaded liposomes. According to this study liposomal codelivery of DOX and HHT (2) could synergistically potentiate antitumor effects.²¹³

HL60/ADR, Kasumi-1, and primary AML cells were pretreated with Decitabine and then exposed to HAA regimen (HHT (2), aclarubicin, and cytarabine (Ara-C)). Decitabine increased cytotoxic effect of HAA regimen and enhanced apoptosis in chemoresistant AML cells.²¹⁴

A synergistic effect of Sulforaphane in human CML cell line K562 with chemotherapy drugs HHT (2) and Ara-C was found and indicated clinical potential.²¹⁵

The combination of HHT (2) and YM155 was investigated on K562 cell lines, and it was found to be synergistic. The mechanism was related to the apoptosis induced through downregulation of survivin.²¹⁶

TNF-related apoptosis-inducing ligand (TRAIL) is a proapoptotic ligand from the TNF-α family. HHT (2) represents a very efficient enhancer of TRAIL-induced apoptosis with potential application in TRAIL-based, anticancer combination therapy. The agonistic anti-TRAIL receptor antibody is a potential antitumor agent. HHT (2) was found to be a very effective (in nano-molar dose) enhancer of TRAIL-mediated apoptosis/growth suppression. Cotreatment of TRAIL-resistant RKO or HT-29 cells with HHT (2) and TRAIL led to the effective induction of apoptosis and the complete elimination of the treated cells. HHT (2) suppressed the expression of the antiapoptotic proteins Mcl-1 and cFLIP and enhanced the TRAIL-triggered activation of JNK and p38 kinases.²¹⁷ Another study suggested that HHT (2) can replace toxic substances such as cycloheximide because it activates necroptosis through the same signaling pathways (involving RIPK1/RIPK3/MLKL) to sensitize human cancer cells to TRAIL-induced necroptosis.²¹⁸

HHT (2) was combined with antiproliferatives such as alkylating agents, intercalating agents, metal coordination complexes, pyrimidine nucleosides, purine nucleosides, inhibitors of nucleic acid–associated enzymes and proteins, and agents affecting structural proteins and cytoplasmic enzymes, for the treatment of a host with a cellular proliferative disease, particularly a neoplasia.²¹⁹ When matrine combined with HHT (2) was tested on the proliferation of human AML cells HL-60, a synergistic effect on apoptosis was noticed.220, 221

6.1.4. Resistance Mechanism for Homoharringtonine

The main mechanism of HHT (2) resistance is through a pump that removes HHT (2) from tumor cells. This transporter is called drug-efflux pump P-glycoprotein, also known as MDR1. Through the action of this pump the intracellular drug levels may remain sublethal and, in the case of upregulation of MDR1, cancer cells may survive. The HHT-resistant cell line SKM-1/HHT was established and the prominent overexpression of MRD1 may be the main cause for multidrug resistance.²²² ssHHT (2) derivatives that bypass MDR1 might improve treatment efficacy.

Short hairpin RNAs (shRNAs) were designed, constructed, and transfected into HT9 leukemia cell line, for specific silencing of the MRD1 gene. Resistance against HT (3), DOX, and curcumin was decreased alongside the decreased expression of MRD1. The study indicated that shRNA recombinant plasmids could modulate MDR in vitro.²²³

Human MDR B-cell lymphoma cell line BJAB/ADR was found to be resistant to HHT (2), daunorubicin (DNR), etoposide, and mitoxantrone (MTX).²²⁴

A study on the pharmacological effect of HHT (2) on the molecular level was conducted. CET (1) and HHT (2) were tested for resistance on the MDR cell lines. These were the 55 cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, Bethesda, Md., USA) that overexpress the conferring P-glycoprotein/MDR1 (CEM/ADR5000), which can expel HHT (2) of the cell and consequently which can attenuate its activity. In CEM/ADR5000 cells, a threefold increase to HHT (2) resistance was observed, though the other MDR cell lines did not show cross-resistance.²²⁵

6.1.5. Mechanism of Allergic Reaction

HHT (2) was screened and identified as a potential allergic factor. Histamine 1 receptor/cell membrane chromatography (H1R/CMC) model was applied for capturing membrane-retained components, which could induce H1R activation. Retention components were enriched and analyzed by H1R/CMC-HPLC/MS. HHT (2) was recognized, separated, and identified in HHT (2) medication. Ca²⁺ flux assay and p-IP3R (channels that most often mediate Ca²⁺ release from intracellular stores) expression found that HHT (2) retained by the H1R/CMC model increased phosphorylation of IP3R and promoted cytosolic free Ca²⁺ elevation in a dose-dependent manner, which further verified the activity of HHT (2) in activating H1R. This provides an indication that a patient with overexpressed H1R should be aware of possible allergic reaction when applying HHT (2) injection.²²⁶

6.1.6. Other Pharmacological Applications

From the chloroform extract of the aerial parts of the only Cephalotaxus harringtonia L. specimen grown in Egypt, five known alkaloids CET (1), HHT (2), HT (3), isoHT (4), and deoxyHT (5) were identified by HPLC–ESI–MS.²²⁷ This extract was tested in vitro for its cytotoxicity against HCT116, HepG2, and MCF-7 cell lines and resulted with IC₅₀ values of 4.77, 12.9, and 17.5 μg/mL, while DOX had IC₅₀ values of 3.74, 4.57, and 2.97 μg/mL against these cell lines, respectively.

Chikungunya virus (CHIKV) is a mosquito-transmitted virus that has reemerged as a significant public health threat in the last decade. HT (3) displayed potent inhibition of CHIKV infection (EC₅₀ 0.24 μM) with minimal cytotoxicity. HT (3) acts on the postentry stage of the CHIKV replication cycle and strongly interferes with the process of viral protein synthesis.²²⁸

Cryptosporidium parasites cause cryptosporidiosis, a diarrheal disease usually, which can result in fulminant diarrhea and death in AIDS patients and chronic infection and stunting in children. A library of drug repurposing candidates were screened against Cryptosporidium parvum among which HHT (2) showed promising in vitro activity, its IC₅₀ activity was 0.86 μM.²²⁹

HHT (2) and CET (1) were tested against the hepatitis B virus (HCV), using the bovine viral diarrhoea virus (BVDV) as surrogate for HCV. The activity relative to BVDV was determined by reduction of BVDV-RNA production and protection of infected embryonic bovine trachea (EBTr) cells. Activity against HBV was investigated by HBsAg release and HBV-DNA release from hepatoblastoma HepG2 2.2.15 cells infected with HBV. HHT (2) showed high activity against HBV (IC₅₀ was 2 μM against HBV, but it has a very low therapeutic index).²³⁰ CET (1) and HHT (2) induced toxicity in EBTr cells and had no protective effect against BVDV. In contrast, they were able to inhibit HBV production at concentrations 10- to 100-fold lower than other molecules inducing cell toxicity, which suggests that they are potentially useful in combined therapy against hepatitis B.

A library of 290 compounds was screened for antiviral activity against Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Selection of compounds for inclusion in the library was dependent on current or previous FDA approval or advanced clinical development. Some drugs that had a well-defined cellular pathway as targets were included. In total, 27 compounds with activity against both MERS-CoV and SARS-CoV were identified. The EC₅₀ activity of HHT (2) against MERS-CoV was 71.8 nM.²³¹

HT (3) was extremely effective in inhibiting human retinal glial cell proliferation. Its activity was comparable to other antiproliferative drugs such as colchicine, daunomycin, and 5-fluorouracil. HT (3) is therefore a potential drug for the treatment of proliferative vitreoretinopathy (PVR).²³²

In 2009, the inhibitory effects of HHT (2) on CML and B-ALL (B cell ALL) cell line was evaluated in vitro and showed that over 90% of leukemic stem cells were killed after treatment with HHT (2). In contrast, less than 9% or 25% of leukemic stem cells were killed after treatment with IM or dasatinib, respectively.²³³

HHT (2) has demonstrated activity against coronavirus with an IC₅₀ ranging from 11 nM to 1.2 μM.²³⁴ Also, HHT (2) when combined with the saponin digitonin showed activity against Trypanosoma brucei brucei.²³⁵

6.2. Clinical Application

HHT (2) or OM (INN) provides a treatment option with a distinct mechanism of action for patients who have not achieved an optimal outcome with TKIs, including patients with the T315I mutation.²³⁶ Recent reviews compiled clinical trials conducted first in China with mixtures of HHT (2) and HT (3), then in Western countries using purified high purity ssHHT (2) reported in 1998 by Robin (OncoPharm founder) also known as OM allowing to confirm its efficacy in CML and put the first European Medicine Agency (EMA) application in 2004 leading to its orphan drug status in 2009, and in the United States with a stable source of hydrosoluble omacetaxine reported in 2001 by Brown (ChemGenex founder).12, 237, 238, 239, 240

6.2.1. Approved Clinical Applications

HHT (2) was approved in 2009 by the EMA for t-Philadelphia chromosome positive CML in patients who have the T315I BCR-ABL kinase domain mutation and who are resistant to prior IM therapy.²⁴¹ It was approved by the FDA in October 2012 for the treatment of CML in CP or AP after failure of two or more TKIs.²⁴²

6.2.2. Different Clinical Trials for Chronic Myeloid Leukemia

To evaluate the pharmacokinetics of HHT (2), sensitive HPLC-tandem mass spectrometry (LC-MS/MS) assays for the quantification of omacetaxine and its inactive 4′-desmethyl (4′-DMHHT) and cephalotaxine metabolites in human plasma and urine were developed and validated, according to the latest FDA and EMA guidelines.²⁴³ Deuterated isotopes were used as internal standards and these assays were considered very suitable to support clinical pharmacologic studies of OM. Since omacetaxine is mainly metabolized by esterases, the plasma samples were immediately stabilized after collection with an esterase inhibitor and stored at a nominal temperature of −80°C. The validated plasma assay quantifies all analytes in the concentration range of 0.1–100 ng/mL and the urine assay in the range of 0.1–50 ng/mL. At all concentrations, the accuracies were within ±15% of the nominal concentrations and precisions were ≤15%. The developed methods have successfully been applied in a human mass balance study of omacetaxine.

Nearly 70%–90% of patients with CML in the early chronic phase (CP) who were treated with IM achieved complete cytogenetic response. With a median follow-up period of 5 years, the annual rate of resistance or progression is 3%–4%, and the mortality is 1%–2%.²⁴⁴

In 2001 a study was conducted on 37 patients ≤21 years old with recurrent refractory AML, 28 patients were evaluable for response. Four patients responded completely and one responded partially (response rate 5/28 18%); the median duration was roughly 62 days. This study showed potential use of HHT (2) for pediatrics with tolerable toxicity.²⁴⁵

Two cases of CML patients acquired the T315I mutation after IM treatment; one was treated with recombinant interferon-alpha (IFN-α), and the patient was in complete hematologic response (CHR) 10 months later, while the other patient treated with HHT (2) started at 1.25 mg/m² subcutaneously twice daily for 5 consecutive days every month and achieved a CHR after 5 months.²⁴⁶

The outcomes of 176 patients of AP CML who received treatment with IM were reviewed and compared with the outcomes of 213 historic control patients treated with other drugs including HHT (2). Treatments included INF-α (100 patients), HHT (2) (35 patients), DNR plus cytarabine (23 patients), decitabine (47 patients), and other therapies (8 patients). CHR were 82% with IM and 35% with HHT (2).²⁴⁷

In 2010, eight TKI-resistant CP CML patients unmutated and T315I-mutated BCR-ABL were enrolled in a clinical trial. They all received HHT (2) to investigate if patients will be resensitized to TKIs. Initially a rapid decline and a sustained disappearance of T315I-mutated transcripts were observed in 50% of patients. This result indicates the disappearance of the mutated clone.²⁴⁸

In 2009, a year-long study on long-term treatment with HHT (2) was carried out. A year-long study on long-term treatment with HHT (2) was carried out. One hundred and six patients with CML received 1.5 mg/m² of HHT (2) alone for 7 to 9 days every 4 weeks. 79 patients were in the control group, 31 were treated with IFN-α, and 48 with hydroxycarbamide. Seventeen patients who were treated with IFN-α failed to achieve cytogenetic response within 12 months and subsequently received HHT (2) as a salvage treatment. After 12 months of therapy, cytogenetic response rate of the HHT (2), IFN-α, and hydroxycarbamide groups were 39/106, 14/31, and 3/48, and corresponding molecular cytogenetic response rates 6/18, 3/8, and 0, respectively. Of the 17 patients who received HHT (2) as salvage treatment, 6 achieved cytogenetic response (3 major). At the 48-months follow-up, cytogenetic response was maintained in 32/39 patients treated with HHT (2). Patients who had cytogenetic response in HHT (2) group or treated with IFN-α also showed longer median chronic durations, which were 45 months (12–98 months) and 49 months (12–92 months) respectively, indicating a longer survival time.²⁴⁹

In 2015, the efficacy and safety data of long-term administration of HHT (2) were evaluated. After 24 months of follow-up, the final analysis included additional efficacy and safety analyses to assess the benefit of long-term HHT (2) administration (1.25 mg/m² twice daily for 14 days every 28 days followed by 7 days every 28 days) in CP and AP CML patients who received >3 cycles. 18% of CP-CML patients achieved a major cytogenetic response (MCyR) with a median duration of 12.5 months. Responses were maintained for 12 months in three of forteen responders, and the median overall survival (OS) was 40.3 months. Among patients with AP-CML, 14% achieved or maintained a major hematologic response (MHR) for a median of 4.7 months, MCyR was not achieved, and the median OS was 14.3 months. In patients with CP-CML and patients with AP-CML who received >3 cycles of treatment (n-50 and n-14, respectively), the median OS was 49.3 and 24.6 months respectively. Grade 3 or higher hematologic toxicities were the major side effects (79% and 73% for CP-CML and AP-CML, respectively), with discontinuation due to toxicity in 10% of CP patients and in 5% of AP patients.²⁵⁰

The efficacy and safety of HHT (2) in patients with AP-CML previously treated with tyrosine kinase inhibitors were assessed in two phase II trials. 51 patients in AP-CML and 44 in myeloid blast phase (BP-CML) received subcutaneous (SC) omacetaxine 1.25 mg/m² twice daily, days 1–14 every 28 days until hematologic response/improvement or any cytogenetic response, and then days 1–7 every 28 days until disease progression. An MHR was 37% in patients with AP-CML and 9% in patients with BP-CML (22% and 5% in those were with a history of T315I mutation).²⁵¹

In 2007, a 63-year-old male CML patient was reported harboring a T315I BCR-ABL mutation while on IM treatment that completely disappeared when he was treated with HHT (2).²⁵²

In 2013, HHT (2) produced good clinical responses with acceptable tolerance levels in patients with CML-CP who had been treated with two or more TKIs. These Data were analyzed from two phase II clinical trials of 81 patients with CML-CPs. The median age of those in the analysis was 59 years (range, 26–83 years). Eight patients completely responded. The median duration for the study was 17.7 months. Fifty-six patients (69%) achieved and/or maintained hematologic response for at least 8 weeks. The overall survival was 34 months.²⁵³

In 2015, a study was carried on 31 CML patients with complete cytogenic response to compare the immunological effect on T lymphocytes between HHT (2) (15 patients) and IM (16 patients); the study indicated that both have the same effect.²⁵⁴

6.2.3. Administration, Dosage, Metabolites, and Elimination

A recent review discussed the use and effectiveness of HHT (2) in the treatment of intractable CML, with coverage of its pharmacology, mode of action, and pharmacokinetics.²³⁷ A SC formulation demonstrated efficacy and safety in phase 1/2 trials. HHT (2) at 1.25 mg/m² SC was administered (twice daily) BID, days 1–14 every 28 days for two cycles. This mode of administration was developed because in early clinical studies short intravenous (IV) infusions (over 60–90 min) was used, but life-threatening hypotension and tachycardia were observed at dose levels above 3–4 mg/m².²⁵⁵ A stable aqueous solution for injection of HHT (2) was prepared with tartaric acid at pH around 4.²⁵⁶ It is only recently however that the site of protonation, although obvious, was unambiguously seen at N9 of CET (1) or HT (3) as established through X-ray diffraction studies of various organic salts,⁷⁶ an observation in concordance with Steitz¹⁷⁶ (see Fig. 25) and Takano's⁶⁰ results implying that N9 should be available for protonation to ensure its biological activity. Therefore, HTs seem to be active via their protonated form. The maximum tolerated dose was estimated to be 5 mg/m².²⁵⁷ It is provided in vials that contain 3.5 mg HHT (2) as a sterile, preservative-free, and white to off-white lyophilized single-use solution for injection.²⁴¹ In a recent patent HHT (2) was incorporated in phycobiliprotein nanoparticles.²⁵⁸ HHT (2) can be given as aqueous solution preferably at a pH of about 4. Also it can be provided orally.259, 260, 261, 262, 263 The protonated form of HHT (2) was patented for potential application in oral and IV dosage forms.²⁶⁴

MM remains an incurable disease in most patients. Newly developed high-proportion polyethyleneglycol (H-PEG) of long-circulating liposomes (LCL) loaded with HHT (2) (HHT-LCL-H-PEG) may be regarded as a promising nano-device to deliver anti-MM drug HHT (2) for treatment of MM patients.²⁶⁵ This HHT-LCL freeze-dried powder can significantly improve storage stability, can make HHT-LCL freeze-dried powder convenient to dilution administration, make drug in liposome-sustained release, and extend the circulation time in blood, to improve the utilization rate of medicament effective component, and reduce toxic and side effects.

The major metabolites of HHT (2) are CET (1) and 4’-desmethylHHT (31), they are detected by HPLC.²⁶⁶ In another study using ¹⁴C HHT and detection by HPLC mass spectrometry (MS) (high resolution) in combination with offline radioactivity determination, six metabolites were detected in the plasma, urine, and feces, unchanged HHT (2) was predominant.267, 268

6.2.4. Combination Therapy

Trials have been carried on combining HHT (2) with other anticancer drugs to synergize and potentiate the activities of these drugs and improve therapy outcome. In some cases, the associated drugs can be efficiently codelivered in the form of liposomes.²⁶⁹

6.2.4.1. Combination Therapy for Chronic Myeloid Leukemia

In 2002, the efficacy and safety of a combination regimen of simultaneous HHT (2) and IFN-therapy was proved with 37 patients with CP-CML who were not treated previously by either agent. The CHR rate was 85%.²⁷⁰

In 2009, a clinical trial phase II carried on 44 previously untreated patients with Philadephia-positive chromosome (Ph+) CP-CML, the treatment consisted of HHT (2) (2.5 mg/m² per day) and cytarabine (7.5 mg/m² per day). Both drugs were given together via continuous IV infusion for 7 days. 36 of 44 patients (82%) achieved a CHR. Although HHT/cytarabine was generally well tolerated, no significant amelioration was observed for patients with interferon-refractory CML and result was not nearly as high as association with IM in newly diagnosed patients.²⁷¹

In 2010 in China, 12 patients with CML in blast crisis who have failed prior single-agent therapy with IM were enrolled in clinical trial to investigate the induction therapy of granulocyte colony-stimulating factor (G-CSF) and low-dose HHT (2) as well as standard-dose IM (GHI). The outcome of this study suggested that the GHI regimen may overcome disease-poor response to conventional doses of IM followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT).²⁷²

Ninety patients with Ph+ CML in early CP received the triple regimen IFN-α, ara-C, and HHT. After a median duration of 16.5 months of therapy, 78 patients had their therapy changed to IM, 85 patients (94%) achieved CHR, and 67 patients (74%) had a cytogenetic response (Ph suppression to ≤90%).²⁷³

14 patients were treated with a regimen that combines HHT (2) and IM among which six cases failed and eight cases sub-optimally responded to IM therapy. This trial proved the efficacy and safety of combining both drugs.²⁷⁴

6.2.4.2. Combination Therapy for Acute Myeloid Leukemia

In 2009, 87 AML patients were treated with HHT (2), Ara-C, and DNR (HAD) regimen, the complete remission rate of the 87 cases was 80/87 (92%).²⁷⁵

Six hundred and twenty three newly diagnosed Chinese patients with AML were treated with induction chemotherapeutic regimens based on idarubicin, DNR, and HHT (2). Total complete remission rate was 66.5%, median survival was 18 months, and estimated survival at 3 years was 30.8%. The third year relapse rate was 55.1%.²⁷⁶

A group of 254 Chinese patients with de novo AML with glutathione S-transferase theta 1 (GSTT1) and glutathione S-transferase mu 1 (GSTM1) mutations were administered in a clinical study. As induction therapy, the patients were treated with HHT (2) 2.5 mg/m² intravenously on days 1–7, Ara-C 150 mg/m² per day continuous infusion on days 1–7, and DNR 45 mg/m² intravenously on days 1–3 (HAD regimen). The second course of HAD induction therapy was applied to patients who were not in complete remission after the initial course of induction therapy. Postremission therapy consisted of HA regimen (HHT (2) and Ara-C) (course 1 and 4), DA regimen (DNR and Ara-C) (courses 2 and 5), and MA regimen (MTX and Ara-C) or HAM (Ara-C days 1–4, combined with MTZ 8 mg/m², IV on days 5–7) (courses 3 and 6). Overall survival and disease-free survival of complete remission patients in GSTT1 and GSTM1 double present group was better than in GSTT1- and/or GSTM1-group.²⁷⁷

A regimen was investigated to explore the effect of low dose of HHT (2) and Ara-C combined with G-CSF on patients with relapsed or refractory AML: 35 out of 67 (52.2%) patients achieved complete remission and 8 out of 67 (11.9%) achieved partial remission. The overall response rate was 64.1%.²⁷⁸

In 2003 a clinical trial of combined regiments HHT (2), Ara-C, and etoposide (VP-16) was conducted on 12 pediatric patients, 9 patients achieved complete remission within 20–45 days after one course of therapy. One patient had partial remission this clinical trial gave good indication for this combination.²⁷⁹

HAA regimen, was evaluated in 46 patients with refractory and/or relapsed AML for efficacy and toxicity. 80% of patients achieved complete remission.²⁸⁰

β-Elemene emulsion had a curative effect in treatment of refractory/relapsed AML in combination with HAA regimen. This was clinically proven by comparing two groups: one using the HAA alone and one using β-elemene emulsion with HAA. The total effective rate in the β-elemene emulsion plus HAA group was 80.8%. However, the total effective rate in the HAA only group was 52.9%.²⁸¹

Sixty-seven patients with relapsed or refractory AML were enrolled in a clinical trial. They received the following treatment: HHT (2), Ara-C, and G-CSF (HAG). All the patients were treated with HAG regimen. 35 patients (52.2%) achieved complete remission and 8 (11.9%) partial remission. The overall response rate was 64.1%²⁸²

One hundred and ninety six untreated de novo AML patients were recruited in China for a clinical trial using HHT (2). Patients were treated with HHT (2) and Ara-C (HA)-based induction therapy composed of three chemotherapeutic drugs (HAD/M, D-DNR, M-MTX). The complete response rate was 153 of all cases (78.1%).²⁸³

One hundred and seventy-one pediatric patients were recruited in a trial. They received a regimen including HHT (2), cytarabine and DNR with a median age of 7.58 years. Complete response was obtained in 140 of 171 (81.9%) cases within 60 days. The 5-year event-free survival was 52.75%.²⁸⁴

Fourty-eight patients median aged 35 (14–57) years were enrolled into a clinical study to evaluate the efficacy and toxicity of HAA regimen as an induction therapy in the treatment of de novo AML. The HAA regimen was a well-tolerable and effective induction regimen for patients with de novo AML.²⁸⁵

A case was reported with primary refractory FLT3-ITD-positive AML was treated with combination of low dose HHT (2) and Sorafenib (salvage treatment); the therapy was successful and the patient was in complete remission.²⁸⁶

Ninety elderly patients with AML were chosen and divided into CAG (low-dose cytarabine and aclarubicin in combination with G-CSF) group (52 cases) and HAG (HHT (2) and Ara-C combined with G-CSF) group (38 cases). In the CAG group were 38 cases with complete remission (73.08%) and total efficiency was 84.62%; 40 cases with complete remission in HAG group (36.84%) and total efficiency was 73.68% in the HAG group. CAG regimen was more efficient than HAG in elderly patients with AML.²⁸⁷

In a recent clinical study for elderly patients with de novo AML, 56 patients were enrolled in this study. They were treated with HHT (2) and cytarabine in combination with G-CSF (HAG). Updated results showed good overall survival.²⁸⁸

A recent metaanalysis provided an overview of the effectiveness of HHT (2) combination regimens to treat AML. Twenty-one studies were analyzed including 1310 patients, 229 pediatric, and 216 elderly. HHT (2) was given in combination with cytarabine, DNR, idarubicin, aclacinomycin, MTX, or G-CSF. This retroanalysis showed the superiority of HHT (2)-containing regimens in AML treatments.

6.2.5. Other Clinical Applications

Clearly, other potential clinical applications are the other subtype of leukemia. Consequently different clinical trials with different regiments took place:

Thirty-one adult patients suffering from idiopathic thrombocytopenic purpura (ITP) were treated with low-dose combination chemotherapy consisting of cyclophosphamide and prednisone on days 1–7, combined with vincrinstine on day 1, and/or azathioprine on days 1 to 5 or 7 and/or HHT (2) on days 1, 3, 5 or on days 1–7 and/or etoposide on days 1–7.²⁸⁹ The output was as following: 13 of 31 (41.9%) patients had complete remission, 9 (29.0%) had partial remission, and 9 (29.0%) had no response.

A 45-year-old female patient was reported with de novo acute megakaryoblastic leukemia (rare type of AML) with BCR/ABL rearrangement and der(16)t(1; 16)(q21; q23) translocation but negative for t(9; 22) Ph chromosome. On HAD induction chemotherapy, the patient achieved partial hematological remission. The patient was then switched to IM plus one cycle of CAG regimen (low-dose cytarabine and aclarubicin in combination with G-CSF). She achieved complete remission, the disease recurred after 40 days and the patient eventually died of infection.²⁹⁰

Thirty-four myelodysplastic syndromes patients with MDS-RAEB and RAEB-T were recruited for a clinical trial of two regimens therapy of either HAA regimen or Idarubicin Ara-C (IA) regimen. It was suggested that the HAA regimen is more effective than IA and well tolerated.²⁹¹

Thirty-three elderly patients were enrolled in a clinical trial, 21 patients with high-risk MDS, and 12 with MDS–AML to evaluate the efficacy and toxicity of CHG (cytarabine, HHT (2), and G-CSF) induction chemotherapy (low-dose cytarabine, HHT (2) with G-CSF priming) for elderly patients with high-risk MDS or AML transformed from MDS (MDS–AML). As outcome of this study the overall response rate was 66.7% after one course of the CHG regimen with 19 patients achieved complete remission (57.6%) and 3 patients achieved partial remission (9.1%).²⁹²

In a long trial of 9 years, 53 patients were enrolled to evaluate the effect of combination therapy of HHT (2) plus all-trans-retinoic acid (ATRA)–based therapy for the treatment of APL as induction therapy followed by three courses of consolidation chemotherapy and 2 years sequential therapy. All patients achieved complete remission.²⁹³

A retrospective study on high risk 132 patients with higher risk MDS was done to compare two treatment combination therapy of low-dose CHG and Decitabine. Complete remission was not significantly different between the groups (27.1% with Decitabine vs. 30.6% with CHG).²⁹⁴

In a clinical study of total of 98 patients at the acute phase of CML with invasive pulmonary aspergillosis (IPA) were treated with Voriconazole plus a low-dose of HHT (2) and IM. This study suggested that this combination could be used as the first-line therapy for IPA patients with CML.²⁹⁵ The 49 cases in observation group were treated with the combination while the control group was treated with fluconazole, low-dose of HHT (2), and IM. The effective rates of the observation group and the control group were 95.92% and 91.84%, respectively with no significant differences. The study concluded that Voriconazole and low-dose HHT (2) and IM could be used as the first-line antifungals for the treatment of IPA patients with CML.

Thirty-three patients were enrolled in a clinical study to investigate the effectiveness of HA regimen for CML-MBC, overall improvement cytogenetic response was 60.1%, 7 patients (21.2%) had partial response. The study concluded that it is an effective treatment and that CD34⁺CD7⁺ cells may be one of the valuable clinical parameters to assess treatment effectiveness.²⁹⁶

6.2.6. Side Effects

The major side effect is myelosuppression (a condition in which bone marrow activity is decreased, resulting in fewer red blood cells, white blood cells, and platelets). Myelosuppression is a side effect of some cancer treatments,257, 271, 272, 279 with gastrointestinal manifestation including diarrhea occurring as well in some cases.²⁵² Those side effects were explained due to the effect of HHT (2) on the epithelial permeability of intestinal Caco-2 cell monolayers.²⁹⁷

6.3. Natural Extracts From Cephalotaxus sp. and Natural Chinese Medicine

The essential oil of C. griffithii inhibited proliferation and migration of human cervical cancer cells.²⁹⁸ The oil from C. griffithii needle extracted with petroleum ether was tested for its effect on proliferation of cancer cells (MTT assay on HeLa, ZR751, and HepG2 cell lines). The extract showed important phyto-components with multiple cellular targets for control of breast cancer.²⁹⁹

Patents based on Chinese traditional medicine were filed containing Cephalotaxus as an ingredient combined with different plants' extracts and are used for the following indication: treatment of phlegm stagnation type osteoarthritis,³⁰⁰ liver cancer,³⁰¹ ischemic intestinal colic,³⁰² for phthisis,³⁰³ hepatitis syndrome,³⁰⁴ for leukemia,³⁰⁵ traumatic bleeding consists,³⁰⁶ hookworm disease,³⁰⁷ ovarian cancer,308, 309 lump-resolving agent in gastric cancer,³¹⁰ treatment of gastric cancer,³¹¹ liver cancer,³¹² collateral cancer,³¹³ rectal cancer,314, 315 phlegm and fluid-retention lung-obstructing type cardiac insufficiency,³¹⁶ treating liver-depression qi-stagnation type breast cancer,³¹⁷ spleen-kidney-qi-deficiency bladder cancer,³¹⁸ and lung cancer.³¹⁹ A Cephalotaxus plant preparation inhibits the adhesion and migration of breast cancer, colorectal cancer, prostate cancer, lung cancer, gastric cancer, osteosarcoma, and other cancer cells,³²⁰ liver fats.³²¹ (Table 20 ).

Table 20.

Indications for the plant preparations incorporating Cephalotaxus extracts

Entry	Indication	Reference
1	Phlegm stagnation type osteoarthritis	300
2	Liver cancer	301
3	Ischemic intestinal colic	302
4	Phthisis	303
5	Hepatitis syndrome	304
6	Leukemia	305
7	Traumatic bleeding consists	306
8	Hookworm disease	307
9	Ovarian cancer,	308, 309
10	Lump resolving agent in gastric cancer	310
11	Gastric cancer	311
12	Liver cancer	312
13	Collateral cancer	313
14	Rectal cancer,	314, 315
15	Phlegm and fluid-retention lung-obstructing type cardiac insufficiency	316
16	Liver-depression qi-stagnation type breast cancer	317
17	Spleen-kidney-qi-deficiency bladder cancer	318
18	Lung cancer	319
19	Adhesion and migration of breast cancer, colorectal cancer, prostate cancer, lung cancer, gastric cancer, osteosarcoma and other cancer cells	320
20	Liver fats	321

A patent for dermatological application reported that HHT (2) was an active inhibitor of matrix metalloproteinase-1 that can be used for reduction and prevention of wrinkles.³²² Another preparation relates to a veterinary drug for treatment of bovine mastitis.³²³ Eventually, patents were filed using preparations that contain HHT (2) for treatment of water and purifying it from cyanobacteria.324, 325

Note Added In Proof

During the preparation of this manuscript, additional synthetic studies326, 327 and a review were published.³²⁸