Synthesis and Biological Study of the Phomopsolide and Phomopsolidone Natural Products.

Abstract

The complete history of the syntheses and biological activities of the phomopsolide and phomopsolidone classes of natural products is reviewed. These efforts include the successful synthesis of four of the five phomopsolide natural products, two of the four phomopsolidone natural products and two analogues of phomopsolide E, including the 7-oxa and 7-aza analogues. In addition, the utility of these synthetic efforts to enable the initial structure activity relationship studies for these classes of natural products is also covered.

Graphical Abstract

A review of the discovery, synthesis and medicinal chemistry of the phomopsolide and phomopsolidone classes of regioisomeric polyketides is presented.

1. Introduction

The history of the phomopsolide and phomopsolidone natural products begins with the search for natural remedies for agricultural diseases caused by fungal infections. ^1,2,5 The phomopsolides were initially discovered by Grove¹ and Stierle² as part of an effort to find biologically active natural products from trees such as the pacific yew. In particular, Grove was looking for compounds with antiboring/antifeeding properties that would prevent the elm bark beetle from spreading Dutch elm disease, a devastating infection affecting ecosystems containing American elm trees. Despite its name, Dutch elm disease primarily affects the American elm tree. The elm disease is spread by the fungi Hylurgopinus rufipes and Ophiostoma ulmi.^3,4 The American elm tree was once the most popular in North America, particularly along tree-lined streets in many midwestern cities, due to its adaptability to a range of climates. Once infected, a well-established American elm can be killed within two weeks. An infestation is capable of decimating 90% of a city’s trees within a 10-year time frame. A decade later, a phomopsolide and the structurally related phomopsolidones were found in fungi associated with the grape disease esca. The isolation and structural determination of both natural product classes inspired over 25 years of synthetic efforts. Ultimately, these extensive efforts led medicinal chemists to investigate both classes of compounds for antibacterial and anticancer properties (vide infra).

The phomopsolides and the phomopsolidones are two classes of natural products that share a regioisomeric relationship (e.g., 2 and 8). They are made up of oxidized decanoic acid lactones that all share a tiglate ester at the C-4 or C-5 position. The phomopsolides consist of a group of five dihydropyranone containing structures with a C-4 tiglate ester in the (S)-configuration (Fig. 1).^1,2 All five phomopsolide pyranones are substituted with a C-5 pentyl or pent-1-enyl side chain with oxidation at the C-8 (ketone or (S)-alcohol) and C-9 ((S)-alcohol) positions.^1,2

Fig. 1.

The phomopsolide and phomopsolidone natural products and analogues.

In 2014, Abou-Mansour and his co-workers isolated related furanones, phomopsolidones A-D.⁵ Phomopsolidone A is a substituted furanone with a C-4 hex-2-enyl side chain with a tigloyloxy group at C-5, and two hydroxy groups at C-8 and C-9 (Fig. 1). Phomopsolidone B is a saturated version of phomopsolidone A with the butanolide double bond being reduced. In contrast, the E-Z-alkene isomeric phomopsolidones C and D are the result of a syn- and anti-elimination of tiglic acid from phomopsolidone A.

The structural connection between the two classes of natural products can be seen in the regioisomeric relationship between phomopsolidone A (8) and phomopsolide B (2), which can be viewed as tiglated products from a mixture of equilibrating hydroxy-lactones (e.g., C-5 hydroxybutenolide 12 and C-4 hydroxypentenolide 13, Scheme 1). Not-surprisingly, phomopsolidone A and phomopsolide B appear coincidently in nature.⁵

Scheme 1.

Proposed biosynthesis of phomopsolide B and phomopsolidone A.

In addition to the interesting structural and biosynthetic aspects of the phomopsolides and the phomopsolidones, these natural products also possess interesting biological activity. The five phomopsolides (A-E) were found to exhibit antimicrobial activity (e.g., S. aureus).¹ Phomopsolide B and the structurally related phomopsolidones were found in fungi associated with the grape disease esca. Phomopsolidones A and B showed weaker antibacterial activity than their co-metabolite phomopsolide B. In 2015, Gray et al., re-isolated phomopsolides A and C and found them to have anti-mycobacterial activity (Mycobacterium tuberculosis H37Ra, Table 3).⁶ Phomopsolide B was also found in three strains of the fungus (Phomopsis spp.) associated with the disease esca.³

Table 3.

Antibacterial activity against Mycobacterium tuberculosis H37Ra.

Compound	MIC (μM)	IC50 (μM)
phomopsolide A (1)	170	24.4
phomopsolide C (3)	680	34.3

2. Synthesis of phomopsolides

As a result of their unique structures and biological activity, the phomopsolides and phomopsolidones have garnered a fair amount of attention from the synthetic community. This includes four syntheses of phomopsolide B, two syntheses of phomopsolide C, one synthesis of phomopsolide D, one synthesis of phomopsolide E, and two syntheses of analogues of phomopsolide E. In addition, there has been two syntheses of phomopsolidone A and one synthesis of phomopsolidone B. These approaches and related biological studies are reviewed.

The first synthesis of phomopsolide B was by Noshita in 1994.⁷ This was followed by later syntheses of phomopsolide B by Prasad, Atmakur, and Sabitha in 2012, 2014 and 2015, respectively.^8,9,10 The first synthesis of phomopsolide C was in 2002 by O’Doherty,¹¹ which was followed three years later by a synthesis by Blechert.¹² The first synthesis of phomopsolide D was achieved in 2004 by the O’Doherty group¹³ who later accomplished the first synthesis of phomopsolide E in 2019¹⁴ In addition, several syntheses of phomopsolide analogues have been accomplished. These include the synthesis of 7-oxa-phomopsolide E,¹⁵ 7-aza-phomopsolide E¹⁶ and related C-4/C-5 stereoisomers. In 2016, the first total syntheses of phomopsolidones A and B were reported by Sabitha.^17,18 Herein we review the total syntheses of the phomopsolide and phomopsolidone natural products, as well as the approaches to various analogues.

2.1. Phomopsolide A

There have been no successful syntheses reported for phomopsolide A. This can be attributed to its instability with respect to the other phomopsolides. Specifically, the C-6/7 cis-enone portion of phomopsolide A is readily isomerized to the trans-enone of phomopsolide B.

2.2. Phomopsolide B

There have been three reported syntheses of phomopsolide B and one reported synthesis of a related diastereomer. The first synthesis of a phomopsolide was of phomopsolide B by Noshita in 1994.⁷ The second synthesis was in 2012 by Prasad,⁸ which was followed two years later by Atmakur’s synthesis of epi-phomopsolide B.⁹ Finally, the fourth synthesis of phomopsolide B was by Sabitha in 2015.¹⁰

2.2.1. Noshita’s synthesis of phomopsolide B (1994)

Noshita⁷ described his synthesis starting from chiral aldehyde 14, which can be made in four steps from D-glucose (Scheme 2).¹⁹ A NaBH₄ reduction followed by bis THP-ether protection was used to convert 14 into alkene 15. Base catalyzed methanolysis of two the acetoxy groups in 15 followed by tosylation of the terminal alcohol and deprotection of the THP-groups gave triol 16. A base-promoted epoxide ring closure was followed by reprotection with two THP-groups and a LiAlH₄ reductive ring opening to give 17. A Mitsunobu invertive esterification at C-5 was used to yield 18. A combination of basic and acidic methanolysis protocols were then followed by acetonide formation and oxidation of the allylic alcohol with active MnO₂ to provide the key aldehyde fragment 19. Starting with the protected propargyl ether 20, a lithium acetylide reaction with ethyl chloroformate followed by base-catalyzed pyrrolidine conjugate addition was used to synthesize the key vinylogous enamine fragment 21.

Scheme 2.

Synthesis of the phomopsolide B fragments.

The two key fragments were brought together to assemble phomopsolide B (2) (Scheme 3). The stereochemically unselective vinylogous aldol reaction, which commenced with the deprotonation of 21 with LDA followed by addition of the resulting anion across aldehyde 19, produced pyranone 22 as a rather complex mixture of diastereomers at both the C-5 and C-6 positions of the pyranone ring and the anomeric position of the THP-group. The C-3 pyrrolidine was removed by a conjugate reduction with NaBH₃CN and subsequent elimination upon treatment with NaHCO₃ to give 23 as a similarly complex mixture of diastereomers. An acid catalyzed deprotection in methanol removed the THP-group, which was followed by re-installation of the acetonide to provide a 1.6:1 mixture of diastereomers (5R/6S)-24 and (5S/6R)-25. An invertive Mitsunobu type esterification²⁰ was used to install the tiglic acid 26 on the major diastereomer 24, which was then followed by an acid catalyzed deprotection of the acetonide to afford phomopsolide B (2) (Scheme 3). The synthesis of phomopsolide B was accomplished in a total of 25 steps with the longest linear route requiring 17 steps.

Scheme 3.

The Noshita synthesis of phomopsolide B.

2.2.2. Prasad’s synthesis of phomopsolide B (2012)

In 2012, Prasad⁸ reported a convergent synthesis of phomopsolide B from two chiral synthons 29 and 33,²¹ which were prepared from lactic acid 27 and tartaric acid 30 respectively (Scheme 4). The synthesis of the keto-phosphonate 29 was accomplished in three steps from lactic acid. This started with the conversion of lactic acid to its ethyl ester followed by TBS-protection. Finally, a condensation of the TBS-protected ester with BuLi/28 gave the keto-phosphonate 29. The synthesis of aldehyde 33 was accomplished in four steps from the ethyl ester of D-tartaric acid. This involved a benzylidene protection of the 2,3-diol and a chloroallane reduction to a 2-benzyl-protected 1,2,3,4-tetraol, which was then benzylidene protected to give primary alcohol 31. Finally, an IBX oxidation²² of the primary alcohol gave aldehyde 33.

Scheme 4.

Synthesis of the phomopsolide B fragments.

The synthesis of phomopsolide B from 29 and 33 was accomplished in a 12-step procedure (Scheme 5). The coupling commenced in the presence of Cs₂CO₃ in acetonitrile to give enone 34. A Felkin-Ahn type diastereoselective 1,2-reduction of 34 was accomplished with NaBH₄ and CeCl₃ followed by deprotection to a mono-Bn-protected tetraol. The resulting triol was per-TBS protected and then selectively deprotected to give primary alcohol 35. The pyranone was introduced by a three-step sequence of IBX oxidation of the primary alcohol, cis-selective olefination with the Still-Gennari reagent 36 and selective TBS-deprotection and lactonization to give 37. An iron trichloride de-benzylation and acetonide formation converted 37 to acetonide 38, which was converted into a tiglic ester and deprotected to give phomopsolide B (2). The Prasad convergent synthesis of phomopsolide B was accomplished in 19 total steps with 12 longest linear synthesis.

Scheme 5.

The Prasad synthesis of phomopsolide B.

2.2.3. Atmakur’s synthesis of bis-epi-phomopsolide B (2014)

In 2014, Atmakur⁹ reported an alternative approach to the phomopsolides with a synthesis of the C-4/5 bis-epimer of phomopsolide B (50). The synthesis was a convergent approach from fragments 42 and 45. Like the Prasad synthesis, the Atmakur synthesis prepared one of the fragments from a tartrate (Scheme 6). This time, the synthesis used dimethyl L-tartrate to establish the C-8 and C-9 stereocenters. Specifically, dimethyl L-tartrate was protected as an acetonide then reduced to a C₂-symmetric diol. The symmetry of the diol was broken by a mono-tosylation and reductive displacement with NaBH₄ to give alcohol 40.²³ A TEMPO oxidation and in situ Wittig olefination,²⁴ followed by DIBAL reduction was used to prepare allylic alcohol 41. Finally, an IBX oxidation was used to prepare enal 42. The other chiral reagent 45 was prepared by acylation of the Evans auxiliary 44 with a PMB-protected glycolic acid 43.

Scheme 6.

Synthesis of the C-4,5-bis-epi-phomopsolide B fragments.

The two fragments were brought together to give 46 by a boron enolate aldol reaction between 45 and 42. A TBS-protection of the aldol product 46 and reductive removal of the chiral auxiliary gave primary alcohol 47. A Dess-Martin oxidation²⁵ and Still-Gennari olefination²⁶ gave cis-enoate 48. A TBS-deprotection with HF•Py and a titanium tetraisopropoxide promoted lactonization gave pyranone 49. The PMB group was oxidatively removed with DDQ and a tiglic ester was installed at the C-4 position to give the acetonide protected target compound. Finally, the acetonide protecting group was removed with Amberlyst-15 in MeOH to give the C-4/5 bis-epimer of phomopsolide B (50). The synthesis was accomplished in 18 total steps with a 10 step longest linear route.

2.2.4. Sabitha’s synthesis of phomopsolide B (2015)

In 2015, Sabitha¹⁰ also reported a convergent synthesis of phomopsolide B. Retrosynthetically, the target molecule was conceived as being derived from two chiral fragments, pyranone 58 and diol 59 (Scheme 8). In the Sabitha synthesis, both fragments were prepared from L-ascorbic acid 51, which was used to control the C-4/5 and C-8/9 stereochemistry of alkenol 53 and alkene 56, respectively.

Scheme 8.

Synthesis of the phomopsolide B fragments.

The Sabitha synthesis commenced with the acetonide protection of L-ascorbic acid, which was oxidatively cleaved to a four-carbon acid with hydrogen peroxide. Methylation of the carboxylic acid gave hydroxy-ester 52. TBS-protection of the alcohol in 52 was followed by selective DIBAL reduction to an aldehyde, which was then treated with the Wittig reagent to form alkene 53. Acetonide deprotection with aqueous TFA gave a diol of which the primary alcohol was TBS-protected. A base promoted benzyl-ether protection (BnBr/NaH) gave an inseparable mixture of regioisomeric alkenes 54 and 55. Selective deprotection of the primary TBS-group with PPTS in EtOH produced a similar mixture of two primary alcohols 56 and 57 in a 7:3 ratio that were separated by silica gel column chromatography.

The major alcohol isomer 56 was oxidized with IBX in CH₃CN followed by Still-Gennari olefination to form a cis-α,β-unsaturated ester (Scheme 8). The TBS-group was removed along with concomitant lactonization with TsOH and then the benzyl ether was removed with TiCl₄ to give pyranone 58. Returning to alkene 53, the TBS and acetonide groups were deprotected with aqueous TFA. The resulting triol was selectively sulfonylated at the primary alcohol and the tosylate group was reductively removed with LiAlH₄ to give diol 59.

With the two key fragments in hand, Sabitha turned to an olefin cross-metathesis reaction to join the two fragments (Scheme 9). Thus, exposure of a mixture of pyranone 58 and diol 59 to the 2^nd generation Grubbs catalyst (60)²⁷ produced a 1:2 mixture of three products. The mixture consisted of the desired hetero-dimer product along with lesser amounts of the two homo-dimers. After acetonide protection, the major isomer 38 was isolated. Esterification of the free alcohol with tiglic acid/DCC followed by deprotection of acetonide with PTSA and MeOH gave phomopsolide B (2). The Sabitha synthesis of phomopsolide B was accomplished in 21 total steps with a 14 step longest linear route.

Scheme 9.

The Sabitha synthesis of phomopsolide B.

2.3. Phomopsolide C

There have been two syntheses of phomopsolide C (Schemes 10-14). The first synthesis of phomopsolide C was reported in 2002 by O’Doherty et al. and the second by Blechert in 2005.^11,12 The two approaches were thematically quite different. While both prepared the C-8-10 portion of the molecule from lactic acid, the two approaches used different coupling strategies and chemistry to prepare the pyranone ring system. The O’Doherty synthesis was unique from all other approaches to the phomopsolides in that it used asymmetric catalysis to stereoselectively install the pyranone.

Scheme 10.

Asymmetric synthesis of furan alcohol 62.

Scheme 14.

The Blechert synthesis of phomopsolide C.

2.3.1. O’Doherty’s synthesis of phomopsolide C (2002)

The phomopsolide C synthesized by the O’Doherty group¹¹ was prepared in a convergent fashion from furan alcohol 62 and the stabilized Wittig reagent 69. The chiral furan alcohol 62 has served as a linchpin molecule for the synthesis of several carbohydrates,²⁸ alkaloid²⁹ and polyketide-base³⁰ products. As a result of the value of enantiomerically enriched material, the group has developed several asymmetric approaches to alcohol 62 (Scheme 10). The initial route involved the conversion of furfural 61 into a diol via a Sharpless dihydroxylation of 2-vinylfuran, which was prepared by an in situ Petersen olefination. The resulting diol can be selectively protected with a TBS-group. Alternatively, furan alcohol 62 can be prepared in either enantiomeric form by a Noyori asymmetric hydrogen transfer reaction³¹ of 67 to give 62 in excellent enantiopurity. The key achiral ketone 67 can be prepared by two routes. The first approach began with glycolic acid 63, which was converted into a pyrrolidine amide and TBS-ether. Addition of 2-lithiofuran 64 was yielded into acylfuran 67. Alternatively, commercially available 2-acetylfuran 66 can be selectively brominated and displaced with sodium acetate. The resulting acetate can be hydrolyzed and protected as a TBS-ether to give 67.

The O’Doherty second fragment 69 was prepared from the ethyl ester of lactic acid 68 (Scheme 11). The route involved a TBDPS-protection and ester hydrolysis with KOH. A DCC-coupling of the resulting acid with 2-thiopyridine gave a thioester which was reacted with the Wittig reagent to give the stabilized Wittig reagent 69.³²

Scheme 11.

Synthesis of the C-7-10 fragment of phomopsolide C.

The synthesis of the pyranone portion of the phomopsolides commenced with furan alcohol 62 (Scheme 12). Oxidation of furan alcohol 62 with aqueous NBS induced an Achmatowicz rearrangement³³ to a 6-hydroxypyran-3-one intermediate. The hydroxypyranone was further oxidized with Jones reagent to give keto lactone 70 which can be isolated by crystallization, however it is unstable to silica gel column chromatography. Instead, the crude product 70 can be selectively reduced with NaBH₄ under Luche conditions.³⁴ The resulting pyranone C-4 alcohol can be TBS-protected and the C-6 alcohol can be selectively deprotected to give 71. Dess-Martin oxidation of the primary alcohol gave an unstable aldehyde intermediate. When the crude aldehyde was exposed to the Wittig reagent 69 it was transformed into a mixture of enones trans-72 and cis-72.

Scheme 12.

The O’Doherty synthesis of phomopsolide C.

The less stable cis-enone could be readily isomerized (thiophenol/AIBN) to the more stable trans-isomer. Selective deprotection of the C-4 TBS group in trans-72 gave an alcohol, which was then tiglated. Selective deprotection of the TBDPS group gave phomopsolide C (3). Unfortunately, when cis-72 was exposed to a similar three step procedure only the trans-isomer was observed. The synthesis as described prepared phomopsolide C (3) in 18 total steps and in 14 longest linear steps.

2.3.2. Blechert’s synthesis of phomopsolide C (2005)

The Blechert group¹² also developed an approach to phomopsolide C (Schemes 13 and 14). In their convergent approach they also derived the C-4/5 stereochemistry from tartaric acid. In practice, the dimethyl ester of L-tartaric acid was protected as an acetonide and reduced with DIBAL. The resulting dialdehyde was exposed to excess Wittig reagent to form a dihydroxydiene, which when mono-protected formed trityl-ether 73. The second coupling fragment 75 was prepared from lactic acid via its ethyl ester 74. After the alcohol was TBS-protected, the ester was converted into a Weinreb amide. Exposure of the amide to vinyl-Grignard resulted in the formation of enone 75.

Scheme 13.

Synthesis of the phomopsolide C fragments.

With the two key fragments intact, the Blechert group continued their synthesis (Scheme 14) using an olefin cross metathesis reaction between 73 and 75 to form a diene product. DCC coupling with acrylic acid followed by removal of the trityl protecting group led to triene 76. Once again, they turned to the Grubbs/Hoveyda metathesis catalyst 77,³⁵ which induced a ring closing metathesis to form a pyranone ring. The pyranone was coupled with tiglic acid using DCC/DMAP and the TBS-group was removed to produce phomopsolide C (3). The synthesis as described prepared phomopsolide C (3) in 12 total steps and in 6 longest linear steps.

2.4. Phomopsolide D

The only synthesis of phomopsolide D (4) thus far was an asymmetric synthesis by the O’Doherty group (Scheme 15).¹³ In addition to being the only synthesis of phomopsolide D, this synthesis was the only phomopsolide synthesis to introduce all the chiral centers via asymmetric catalysis. Like their synthesis of phomopsolide C, their approach to phomopsolide D used a combination of Noyori asymmetric reduction and Achmatowicz chemistry to install the pyranone C-4/5 stereochemistry. However, in contrast to all the other approaches to the phomopsolides, this approach uniquely installed the C-8/9 diol stereochemistry by a Sharpless asymmetric dihydroxylation.³⁶

Scheme 15.

The O’Doherty synthesis of phomopsolide D.

2.4.1. O’Doherty’s synthesis of phomopsolide D (2004)

In 2004, the O’Doherty group¹³ returned to the phomopsolide structural motif with a synthesis of phomopsolide D (Scheme 15). Retrosynthetically, their asymmetric synthesis was envisioned from achiral dienone 79, which contained the C-1 to C-10 carbons of the phomopsolides. The synthesis of dienone 79 was accomplished by a two-step addition of 2-lithiofuran 64 to commercially available sorbal 78 and MnO₂ oxidation. A highly regio- and enantio-selective dihydroxylation of the most electron rich alkene in 79 lead to furan/enone 80 after acetonide protection. The Noyori reduction of the acylfuran 80 with the (S,S)-Noyori reagent 65 occurred along with the 1,4-reduction of the enone to give furan alcohol 81 as a single diastereomer. A sequential oxidation of the furan alcohol with first an Achmatowicz rearrangement (NBS/H₂O) and then a Jones oxidation was used to convert furan alcohol 81 to keto lactone 82. As is generally true for this ring system, keto lactone 82 was unstable to silica gel chromatography and, as a result, the crude material was reduced under Luche conditions (NaBH₄/CeCl₃) to give a 2.5:1 ratio of diastereomeric pyranones 83 and 84. Fortunately, both diastereomers 83 and 84 could be used for the synthesis of phomopsolide D. The major diastereomer 83 was acylated with tiglic acid/DCC in a retentive fashion to form 85. Using Mitsunobu chemistry (tiglic acid/PPh₃/DEAD), the minor diastereomer was converted into 85 via an invertive acylation reaction. Finally, an acetonide deprotection (HCl/MeOH) gave the natural product phomopsolide D (4) in excellent yield. This unique linear synthesis prepared phomopsolide D in 11 total steps and 10 longest linear steps. Thus, using the metric of total steps, one can see some of the advantages of a truly linear approach.

2.5. Phomopsolide E

There has also been only one synthesis of phomopsolide E (5), which again was an asymmetric variety by the O’Doherty group.¹⁴ The approach builds upon the approach they developed for phomopsolide D and, in fact, uses phomopsolide D as its starting material (Scheme 16).

Scheme 16.

The O’Doherty synthesis of phomopsolide E.

2.5.1. O’Doherty’s synthesis of phomopsolide E (2019)

After a long hiatus, the O’Doherty group¹⁴ returned to the phomopsolides with a report of a synthesis of phomopsolide E in 2019. The synthesis builds upon their successful synthesis of phomopsolide D in that is uses phomopsolide D as the starting material for their oxidative approach. Interestingly, their initial efforts began by exploring a selective reduction of phomopsolide C and a protected variant. Unfortunately, both materials proved to be too base sensitive.

The O’Doherty group’s approach to phomopsolide E began with the regioselective protection of synthetically derived phomopsolide D (4) with TBDPSCl. The protected compound was oxidized with Jones reagent to yield the TBDPS-protected phomopsolide D 86. Exposure of the TBDPS-ether 86 to HF•Py cleanly gave the natural product phomopsolide E (5). The synthesis was accomplished in 3 total steps from phomopsolide D (4), but also constitutes a 14 total steps/13 longest linear steps asymmetric synthesis from commercially available sorbal 78. Because the synthesis was from synthetic material with known stereochemical providence, the synthesis of phomopsolide E from D established its absolute and relative stereochemistry.

2.6. O’Doherty’s synthesis of 7-oxa-phomopsolide E (2004)

The O’Doherty group’s¹⁵ synthetic interest in the phomopsolides extends beyond total synthesis and structural determination. Their interest is also focused on the biological activity of the phomopsolides, where synthetic interest puts a high priority on synthetic efficiency. This effort began with the synthesis of a 7-oxa-analogue of phomopsolide E (6) (Scheme 17). Their convergent approach began with the synthesis of TBDPS-protected lactic acid 87, which was accomplished in two steps from ethyl lactate 68 via TBDPS-protection and ester hydrolysis. Once again, the pyranone portion of the analogue was prepared from furan alcohol 62 by means of an Achmatowicz rearrangement, Jones oxidation and subsequent reduction to give pyranone 88 with a free C-4 alcohol. A DCC promoted tiglic acid coupling of the alcohol and HF promoted TBS-deprotection gave the C-6 alcohol 90. A second DCC coupling was used to bring the two components 87 and 90 together, which after a TBDPS-deprotection gave the desired analogue 7-oxa-phomopsolide E (6). The approach to the oxa-analogue 6 was accomplished in 12 total steps and 6 longest linear steps.

Scheme 17.

The O’Doherty synthesis of 7-oxa-phomopsolide E.

2.7. O’Doherty’s synthesis of 7-aza-phomopsolide E (2018)

In addition to the synthesis of the 7-oxa-analogue (6), the O’Doherty group¹⁶ also pursued the synthesis of a related 7-aza-analogue (7) (Scheme 18). Their synthesis of 7-aza-phomopsolide E (7) was a related convergent synthesis using a protected lactic acid chloride 96 and an amino-variant of the C-6 alcohol 94, which was protected as an N-Boc amide because it is prone to rearrangement. The synthesis began with the synthesis of the key acylfuran 92, which the group developed two different approaches to. The first approach involved the α-bromination of 2-acetylfuran 65 and the base promoted displacement with t-butyl carbamate 91. The second and more direct approach involved the addition of excess of 2-lithiofuran 64 to N-Boc protected glycine 93 to give 92.

Scheme 18.

Syntheses of N-Boc-protected α-amino-acetylfuran 92.

The synthesis continued with a Noyori asymmetric reduction of acylfuran 92 (Scheme 19), which was followed by a three-step double oxidation and Luche reduction sequence to give a 3.2:1 mixture of pyranones 94 and 95. The major isomer 94 was taken forward with a DCC coupling with tiglic acid followed by a two-step deprotection of the N-Boc group and immediate coupling with acid chloride 96 to give the protected analogue 97. An HF•Py promoted deprotection of the TBDPS-group in 97 gave the desired 7-aza-phomopsolide E (7). The approach to the aza-analogue 7 was accomplished in 7 total steps and 3 longest linear steps.

Scheme 19.

The O’Doherty synthesis of 7-aza-phomopsolide E.

3. Synthesis of phomopsolidones

In addition to the phomopsolides, there have been synthetic approaches to the isomeric natural products, phomopsolidones A (8) and B (9). Both of these syntheses were accomplished by the Sabitha group (Schemes 20-22).^17,18 As with the majority of the phomopsolide syntheses, these were convergent syntheses that derived their asymmetry from the two naturally occurring chiral building blocks tartaric acid and lactic acid.

Scheme 20.

Fragment synthesis for phomopsolidone A by Sabitha.

Scheme 22.

The second Sabitha synthesis of phomopsolidone A.

3.1. Sabitha’s syntheses of phomopsolidone A (2016)

The Sabitha group¹⁷ developed two syntheses of phomopsolidone A (8). Their first synthesis of phomopsolidone A (8) was accomplish in 2016 by Sabitha et al. (Scheme 20). The convergent synthesis began with the syntheses of two chiral components, alkyne 99 and aldehyde 100. The synthesis of alkyne 99 began with the acetonide protection of diethyl tartrate 30, which was followed by reduction with LiAlH₄ and mono-TBS protection to provide primary alcohol 98. A Swern oxidation³⁷ followed by an ethyl Grignard variant of the Corey-Fuchs reaction³⁸ was used to convert 98 into the desired alkyne 99. The desired aldehyde 100 was prepared from ethyl lactate 66 by a benzyl protection and DIBAL reduction.

The Sabitha convergent synthesis continued with the addition of metalated 99 to aldehyde 100 via a chelate controlled addition to diastereoselectively give propargyl alcohol 101 (Scheme 21). A diastereoselective Red-Al reduction of the propargylic alcohol followed by a benzyl-ether protection and TBS-deprotection was used to form primary alcohol 102. An IBX oxidation to an aldehyde, Still-Gennari cis-olefination, acid catalyzed acetonide removal and lactonization led to butenolide 103. Finally, a DCC/tiglic acid esterification and bis-benzyl ether deprotection gave the natural product phomopsolidone A (8). The synthesis as described prepared phomopsolidone A (8) in 16 total steps and in 14 longest linear steps.

Scheme 21.

The first Sabitha synthesis of phomopsolidone A.

Later that same year, the Sabitha group¹⁸ reported a second-generation synthesis of phomopsolidone A (8) (Scheme 22). The synthesis also began with the acetonide protection, LiAlH₄ reduction and mono-TBS-protection of diethyl tartrate 29 to form primary alcohol 104. A Swern oxidation, Wittig olefination and TBS deprotection was used to give primary alcohol 105.³⁹ A Dess-Martin oxidation to an aldehyde followed by a Still-Gennari cis-olefination, acid catalyzed acetonide removal and lactonization led to butenolide 106. A tiglic acid/DCC coupling was used to install the C-5 tiglic ester, which was followed by a cross metathesis reaction with diol 59⁴⁰ to give phomopsolidone A (8). The second-generation synthesis was accomplished in 14 total steps and in 11 longest linear steps.

3.2. Sabitha’s synthesis of phomopsolidone B (2016)

Using a related approach to their phomopsolidone A (8) synthesis, the Sabitha group¹⁸ developed a synthesis of phomopsolidone B (9) (Scheme 23). Once again, the synthesis was convergent and derived its asymmetry for both portions from diethyl tartrate 29. As with the previous approach, the synthesis began with an acetonide protection, LiAlH₄ reduction and PMB protection to give primary alcohol 107. A Swern oxidation, HWE-olefination reagent 108 and DDQ promoted PMB-group deprotection was used to give ω-hydroxy enoate 109. An alkene hydrogenation, IBX oxidation of the primary alcohol and Wittig olefination was used to form alkene 110.⁴¹ A concomitant acid catalyzed acetonide removal and lactonization followed by DCC promoted tiglation gave γ 111. Finally, a cross metathesis reaction between 111 and 59 gave phomopsolidone B (9). This synthesis was accomplished in 21 total steps and in 12 longest linear steps.

Scheme 23.

The Sabitha synthesis of phomopsolidone B.

4. Biological activity of the phomopsolides and phomopsolidones

The initial biological evaluation of the phomopsolides and the phomopsolidones was centered upon their presumed evolved role in protecting plants (e.g., pacific yew and grapevine) as an antifeeding and/or antimicrobial agent. Of the phomopsolides (A-E), phomopsolide A had the highest antifeeding/boring activity while phomopsolide B had slightly lower activity.⁴² In a comparison of phomopsolide B with the phomopsolidones (A-D) (i.e., the grapevine extracts), phomopsolide B was the only one to induce necrosis in grapevine leaves and to inhibit callus growth on the grapevine plant.⁴² All five had moderate larvicidal activity, with phomopsolide B, phomopsolidone A and phomopsolidone B slowing larvae development times.⁴²

In addition, the phomopsolides (A-E) were evaluated for their antibacterial activity against several organisms. In a zone of inhibition study, phomopsolide B showed activity against Shigella dysenteria (50 μg, 1.2 cm).⁴³ Phomopsolides A-E were shown to have slightly better activity against Staphylococcus aureus (Table 1). In these zone of inhibition assays, phomopsolides A-C were slightly more active with antibacterial activity evident at 25 μg/mL, whereas higher concentrations were needed for phomopsolides D and E at 100 μg/mL and 50 μg/mL, respectively. Antibacterial activities against Bacillus subtilis were also found for phomopsolide B and phomopsolidones A and B.⁵ The anti-B. subtilis activity was dependent upon the stereochemistry of the pyranone ring, as the C-4/5 bis-epimer 50 had significantly reduced activity compared to phomopsoilde B, phomopsolidones A and B (Table 2). ⁹

Table 1.

Antibacterial activity against Staphylococcus Aureus.

Compound	Staphylococcus aureus
	Concentration (μg)	Zone of inhibition (mm)
phomopsolide A (1)	25	22
phomopsolide B (2)	25	20
phomopsolide C (3)	25	22
phomopsolide D (4)	100	10
phomopsolide E (5)	50	9

Table 2.

Antibacterial activity against Bacillus subtilis.

Compound	MIC
phomopsolide B (2)	0.1 nga
Epi-phomopsolide B (50)	21.1 μM
phomopsolidone A (8)	0.1 nga
phomopsolidone B (9)	0.1 nga

^aactivity measured via a bioautography method directly from the material on a thin-layer chromatography.

Although modest, phomopsolide A was found to have activity against Mycobacterium tuberculosis (Table 3)⁵ and less activity for phomopsolide C. No activity was found for phomopsolide B against Escherichia coli., Candida albicans, Saccharomyces cerevisiae, and Penicillium avellaneum (UC-4376).⁴³

The phomopsolides also showed a modicum of anticancer activity against both HeLa and UMUC3 cells with activities in the low μM range for phomopsolides A-C and E (Table 4).^44,43,9 Similar degrees of anticancer activity were found for HEK 293 cells (phomopsolide A, 6.7 μM), Y79 cells (phomopsolide C, 1.4 μM), and H3255 cells (phomopsolide C, 2.6 μM). Not surprisingly, a degree of variability in cancer cell cytotoxicity found was dependent upon the method used, with phomopsolides B and E showing no activity against HeLa or UMUC3 cells in the Alamar Blue viability assay.⁴⁴

Table 4.

Anticancer activity against HeLa and UMUC3.

Compound	IC50 (μM) against HeLa	IC50 (μM) against UMUC3
phomopsolide A (1)	6.3,a 5.0,b	6.5,a 4.8b
phomopsolide B (2)	19.2c	6.1b
phomopsolide C (3)	3.6,a 2.7b	2.2,a 1.4b
phomopsolide E (5)	7.3b	6.6b

^aAlamar Blue Viability Assay was applied

^bNuclei Count Proliferation Assay was applied

^c3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied

The 4,5-bis-epimer of phomopsolide B (50) was also screened for anticancer activity (Table 5).⁹ In contrast to the antibacterial activity, the anticancer activity was not affected by changes in pyranone stereochemistry. For example, the level of activity was retained for the 4,5-bis-epimers of phomopsolide B (50), which showed low μM cytotoxicities for four cell lines (Table 5). These cytotoxicities were on the same order as the cytotoxicity found for the other phomopsolides (Tables 4, 6-7).

Table 5.

Cancer cell cytotoxicities of C-4/5-bis-epi-phomopsolide B (50).

Cell Line	IC50 (μM)
A549 (non-small cell lung)	10.1 ± 0.22
HeLa (uterus)	6.2 ± 0.12
DU145 (prostate)	15.4 ± 0.16
HepG2 (liver)	12.1 ± 0.21

Table 6.

Cytotoxicities for phomopsolide D/E and 7-oxa- and aza-analogues.

Compound	Most sensitive cell line	IC50 (μM)	Least sensitive cell line	IC50 (μM)
phomopsolide D (4)	786–0 (renal)	1.23	SK-OV-3 (ovarian)	11.9
phomopsolide E (5)	HCT-116 (colon)	2.3	MOLT-4 (leukemia)	23.3
7-oxa-phomopsolide E (6)	786-0 (renal)	1.9	CAKl-1 (renal)	10
7-aza-phomopsolide E (7)	786-0 (renal)	11.3	DU-145 (prostate)	31.6

Table 7.

Tissue specific average cytotoxicities for phomopsolides 4, 5, 6 and 7.

Cancer Type	4 IC50 (μM)	5 IC50 (μM)	6 IC50 (μM)	7 IC50 (μM)
melanoma	15 ± 4	24 ± 7	13 ± 3	28 ± 9
CNS	17 ± 5	26 ± 11	16 ± 5	29 ± 15
breast	11 ± 3	20 ± 6	12 ± 2	26 ± 10
prostate	18 ± 9	23 ± 15	16 ± 7	25 ± 18
renal	13 ± 4	25 ± 8	15 ± 4	30 ± 11
ovarian	18 ± 6	24 ± 10	16 ± 6	28 ± 14
leukemia	4 ± 3	16 ± 9	8 ± 5	30 ± 14
non-small cell lung	18 ± 6	26 ± 8	19 ± 5	27 ± 9

In addition to confirming the absolute and relative stereochemistry of the phomopsolides, the O’Doherty group also evaluated some of the more readily available synthetic materials as anticancer agents (Tables 6-7).¹⁶ Thus, both synthetic phomopsolides D and E, as well as the oxa- and aza-analogues of phomopsolide E, were screened for cytotoxicity against the NCI panel of 60 cancer cell lines. Depending upon the cell line, the cytotoxicity varied as much as 10-fold (Table 6). For example, phomopsolide D was most active against the renal cell line 786-0 (1.23 μM) and least active against the ovarian cancer cell line SK-OV-3 (11.9 μM); whereas, phomopsolide E was most active against the colon cell line HCT-116 (2.3 μM) and least active against the leukemia cell line MOLT-4 (23.3 μM). Interestingly, the 7-oxa- and 7-aza-phomopsolide E analogues 6 and 7 were also most active against 786-0 (1.9 and 11.3 μM, respectively).¹⁶

When the average cancer cell cytotoxicities for 4-7 are looked at by tissue types, most of the average activities range in the 10 to 30 μM range. Interestingly, the leukemia cell lines were a slight outlier to this trend, showing significantly lower average cytotoxicities for phomopsolide D (4, 4 μM) and 7-oxa-phomopsolide E (6, 8 μM).

In addition to trends for the various tissue types, trends in the anticancer activities could be seen for the different analogues in the data across the NCI-60 cell line panel. The average cytotoxicities for each compound across all the cell lines are graphically presented in Fig. 2 and displayed in Table 8. While there is clearly overlapping activity, as there was a broad range of activity depending on the cell lines tested, a general trend of phomopsolide D (4) being slightly more active than phomopsolide E (5) and 7-oxa-phomopsolide E (6) being more active than 7-aza-phomopsolide E (7) emerged. This general trend appeared to hold true when the cell lines were divided up by tissue type.

Fig. 2.

Average phomopsolide (4-7) cytotoxicity curves against the NCI cell lines.

^a Includes data from 53 of the 60 cell lines tested; ^b Includes data from 57 of the 60 cell lines tested; ^c Includes data from 58 of the 60 cell lines tested; ^d Includes data from 38 of the 60 cell lines tested

Table 8.

Average NCI 60 cell-line cytotoxicities for phomopsolides 4, 5, 6 and 7.

Compound	Average IC50 (μM)
phomopsolide D (4)a	14 ± 14
phomopsolide E (5)b	23 ± 16
7-oxa-phomopsolide E (6)c	15 ± 16
7-aza-phomopsolide E (7)d	28 ± 18

^aIncludes data from 53 of the 60 cell lines tested

^bIncludes data from 57 of the 60 cell lines tested

^cIncludes data from 58 of the 60 cell lines tested

^dIncludes data from 38 of the 60 cell lines tested

In conclusion, the isolation history of and synthetic efforts toward four of the five phomopsolide natural products (B-E) and two of the four phomopsolidone natural products (A and B) were reviewed. A novel mechanistic interconversion for the biosynthesis of these two classes of natural products was proposed (Scheme 1). In addition, the syntheses of three phomopsolide analogues, including a 7-oxa-, a 7-aza- and a diastereomeric isomer, were covered, which suggest a continuing future of scientific discovery for these novel classes of natural products. In total, the various synthetic approaches comprised thirteen unique synthetic efforts that ranged in synthetic efficiency from a maximum of 25 total steps for the synthesis of phomopsolide B by Noshita to as little as 11 total steps for the synthesis of phomopsolide D by O’Doherty. Of the thirteen syntheses, there were four routes to phomopsolide B (from 18 total steps to 25), two syntheses of phomopsolide C (from 12 total steps to 18), one synthesis of phomopsolide D (11 total steps), one synthesis of phomopsolide E (14 total steps), and two syntheses of analogues of phomopsolide E (7 and 12 steps). In addition, there have been two syntheses of phomopsolidone A (16 and 14 total steps) and one synthesis of phomopsolidone B (21 total steps). Taken in total, the syntheses of the phomopsolide, phomopsolidones and related analogues have enabled the initial structure activity relationship studies of this interesting class of polyketide natural products as both antimicrobial and anticancer agents, which one can imagine as leading to future medicinal chemistry studies.

Scheme 7.

The Atmakur synthesis of bis-epi-phomopsolide B.