Enantioselective Synthesis of γ-Phenyl-γ-amino Vinyl Phosphonates and Sulfones and Their Application to the Synthesis of Novel Highly Potent Antimalarials

Abstract

Racemic and enantioselective syntheses of γ-phenyl-γ-amino vinyl phosphonates and sulfones have been achieved using Horner–Wadsworth–Emmons olefination of trityl protected α-phenyl-α-amino aldehydes with tetraethyl methylenediphosphonate and diethyl ((phenylsulfonyl)methyl)phosphonate, respectively, without any racemization. The present strategy has also been successfully applied to the synthesis of peptidyl vinyl phosphonate and peptidyl vinyl sulfone derivatives as potential cysteine protease inhibitors of Chagas disease, K11002, with 100% de. The developed synthetic protocol was further utilized to synthesize hybrid molecules consisting of artemisinin as an inhibitor of major cysteine protease falcipain-2 present in the food vacuole of the malarial parasite. The synthesized artemisinin–dipeptidyl vinyl sulfone hybrid compounds showed effective in vitro inhibition of falcipain-2 and potent parasiticidal efficacies against Plasmodium falciparum in nanomolar ranges. Overall, the developed synthetic protocol could be effectively utilized to design cysteine protease inhibitors not only as novel antimalarial compounds but also to be involved in other life-threatening diseases.

Introduction

Cysteine proteases (also known as thiol proteases) are protein-hydrolyzing enzymes present in all living organisms such as plants, mammals, protozoa, bacteria, fungi, and viruses.¹ The pivotal role of cysteine proteases can be found in mammalian cellular turnover² and apoptosis³ and also in the life cycle of many parasites.⁴ Cysteine proteases are further classified into three groups: papain-like, ICE-like, and picornain-like.⁵ The cysteine proteases are considered as ideal drug targets for many diseases like malaria, Chagas disease, leishmaniasis, AIDS, cancer, osteoporosis, arthritis, and immune-related diseases.^6,7 Among the several inhibitors of cysteine proteases reported in the literature,^5,7 peptidyl vinyl sulfones are considered to be the potent inhibitors as they contain the reactive sulfone moiety in addition to the peptide backbone.⁸ Similar to peptidyl vinyl sulfones, some of the peptidyl vinyl phosphonates were also reported as cysteine protease inhibitors.⁹ Several γ-alkyl-substituted vinyl sulfone and phosphonate-based cysteine protease inhibitors are reported in the literature (Figure 1).

Figure 1.

Representative examples of vinyl sulfone and vinyl phosphonate-based cysteine protease inhibitors.

The biological importance of peptidyl γ-alkyl vinyl sulfones and phosphonates has attracted the attention of organic chemists, and a few methods of synthesis of γ-alkyl vinyl sulfones and phosphonates (A) are reported in the literature.,^8d10 However, synthetic routes to γ-aryl vinyl sulfones and phosphonates (B) are less explored (Figure 2).^9b11

Figure 2.

Structures of γ-alkyl (A) and γ-aryl (B) vinyl phosphonate and sulfones. Structures of γ-phenyl-γ-amino vinyl phosphonate (5) and sulfones (6).

Enantioselective synthesis of γ-phenyl-γ-amino vinyl phosphonate (5) and sulfones (6) (Figure 2) employing Horner–Wadsworth–Emmons (HWE) olefination of α-phenyl-α-amino aldehydes and the corresponding phosphonates is a challenging task due to the racemization of α-phenyl-α-amino aldehydes under basic conditions. Despite the risk of racemization of α-phenyl-α-amino aldehydes during HWE olefination, we decided to use this strategy to synthesize γ-phenyl-γ-amino vinyl phosphonates and sulfones. Herein, we report racemic and enantioselective syntheses of γ-phenyl-γ-amino vinyl phosphonates and sulfones without any racemization employing HWE olefination of α-phenyl-α-amino aldehydes. The present methodology has also been successfully applied to synthesize peptidyl vinyl phosphonates and sulfones as potential cysteine protease inhibitors targeting the falcipain-2 enzyme.

Results and Discussion

Chemistry

Synthesis of γ-Phenyl-γ-amino Vinyl Phosphonate (5)

We had earlier reported the first racemic synthesis of γ-phenyl-γ-amino vinyl phosphonate 5 starting from cinnamaldehyde employing the Tsuji–Trost reaction as a key step.^9b However, the main drawbacks of this method were the low yield in the final deprotection step and the use of an expensive transition metal-based catalyst, Pd(OAc)₂. Moreover, the enantioselective synthesis of 5 using the asymmetric Tsuji–Trost reaction requires an expensive transition metal catalyst and chiral ligands. These drawbacks prompted us to design a general synthetic strategy for the synthesis of γ-phenyl-γ-amino vinyl phosphonate 5 and sulfones 6 without using any transition metals.

We presumed that the required γ-phenyl-γ-amino vinyl phosphonate 5 could be synthesized using an Overman rearrangement.^12a Since the Overman rearrangement yields allylic trichloroacetamides, we thought the deprotection of allylic trichloroacetamide 10 would give the required allylic amine 5 (γ-phenyl-γ-amino vinyl phosphonate). As shown in Scheme 1, allylic alcohol (hydroxy phosphonate) 8 was treated with trichloroacetonitrile under basic conditions at −35 °C to yield allylic trichloroacetimidate 9 in a 98% yield. Imidate 9 underwent rearrangement on refluxing with toluene to furnish allylic trichloroacetamide 10 in a 92% yield with the exclusive formation of the E double bond.^12b Several conditions to deprotect the trichloroacetamide group of 10 were unsuccessful and resulted in either decomposition of the starting material (30% NaOH, EtOH, 80 °C, 3 h) or the formation of enamide 11 with unwanted isomerization of the double bond (30% NH₃ in MeOH, rt, 16 h;^12b 3 N KOH, EtOH, rt, 24 h; AcOH, H₂O, 80 °C, 12 h). The NMR spectra of the enamide 11 were in full agreement with the reported data.^12b It is believed that the base initially deprotonates the benzylic position of 10, which further results in the isomerization of the double bond to form enamide 11.

Scheme 1. Attempted Synthesis of γ-Phenyl-γ-amino Vinyl Phosphonate (±)-5 using Overman Rearrangement.

Reagents and Conditions: (a) Diethyl Phosphite, Et₃N, DCM, 0 °C to rt, 3 h, 77%; (b) CCl₃CN, DBU, DCM, −35 °C, 30 min, 98%; (c) Toluene, Reflux, 24 h, 92%; and (d) 3 N KOH, EtOH, rt, 24 h, 100%.

In another approach, the synthesis of 5 was planned from 2-phenylglycinol 14, keeping in mind that the vinyl phosphonate moiety can be introduced via HWE olefination of an α-amino aldehyde with tetraethyl methylenediphosphonate. As shown in Scheme 2, the synthesis commenced from (±)-styrene oxide 12. Ring opening of epoxide 12 with sodium azide in H₂O at 60 °C yielded azido alcohol 13 in a 91% yield.¹³ α-Azido alcohol 13 was subjected to hydrogenation under Pd/C to furnish 2-phenylglycinol 14 in a 99% crude yield, which was used directly for the next step. Compound 14 was treated with (Boc)₂O and BzCl to furnish Boc and Bz-protected amino alcohols 15 and 16, respectively. Compounds 15 and 16 were oxidized with Dess–Martin periodinane to yield the corresponding α-amino aldehydes 17 and 18, respectively.¹⁴ Unfortunately, HWE olefination of both the aldehydes 17 and 18 with tetraethyl methylenediphosphonate and NaH in dry THF resulted in the unexpected double bond-isomerized olefin products 19 and 20 (enamides), respectively.

Scheme 2. Attempted Synthesis of γ-Phenyl-γ-amino Vinyl Phosphonate (±)-5 using HWE Reaction.

Reagents and Conditions: (a) NaN₃, H₂O, 60 °C, 3.5 h, 91%; (b) H₂, Pd/C, rt, 12 h, 99%; (c) (Boc)₂O or BzCl, 0 °C, 3 h; (d) Dess–Martin Periodinane, 3 h, rt; and (e) Tetraethyl Methylenediphosphonate, NaH, THF, 0 °C to rt, 3 h.

To our delight, when the trityl-protecting group was introduced to α-amino aldehyde and the HWE olefination reaction was carried out under identical reaction conditions, isomerization of the double bond was not observed (Scheme 3). Although several α-alkyl-α-(tritylamino)acetaldehydes and their subsequent utilization in HWE olefination/Julia–Kocienski olefination/Wittig olefination are reported in the literature,¹⁵ no α-aryl-α-(tritylamino)acetaldehydes are reported so far in the literature to carry out further C–C bond forming reactions.

Scheme 3. Synthesis of γ-Phenyl-γ-amino Vinyl Phosphonate (±)-5.

Reagents and conditions: (a) trityl chloride, Et₃N, 25 °C, 12 h, 63%; (b) Dess–Martin periodinane, DCM, 0 °C to 25 °C, 30 min; (c) tetraethyl methylenediphosphonate, NaH, THF, 0 °C to 25 °C, 3 h, 60% over two steps; and (d) TFA, DCM, 25 °C, 30 min, then sat. aq. NaHCO₃ solution, 95%.

As shown in Scheme 3, α-amino alcohol (±)-14 was protected with trityl chloride to afford trityl-protected amino alcohol (±)-23 in a 63% yield. Compound (±)-23 on oxidation with Dess–Martin periodinane furnished α-amino aldehyde (±)-24 followed by immediate HWE olefination with tetraethyl methylene diphosphonate yielding olefin (±)-25 at 60% over two steps without any isomerization of the newly formed double bond (confirmed by ¹H and ¹³C NMR, see the Supporting Information). The final deprotection of the trityl group of (±)-25 with TFA in DCM yielded the required amine (±)-5 in a quantitative yield after a basic workup with sat. aq. NaHCO₃ solution (Scheme 3).

It is believed that the carbonyl group present in −NHCOCl₃, −NHBoc, −NHBz played a significant role in the isomerization of the double bond during HWE olefination (Schemes 1 and 2). The presence of amide −NHCOCl₃ or −NHBoc or −NHBz triggers the attack of the base (NaH) at the benzylic position resulting in the isomerization of double bonds followed by protonation at the α position of -PO(OEt)₂. However, the absence of a carbonyl group in the bulky trityl protecting group resulted in the formation of the desired product without any isomerization of the double bond (Scheme 3).

Synthesis of γ-Phenyl-γ-amino Vinyl Sulfone (6)

We further extended the above mentioned synthetic strategy to synthesize γ-phenyl-γ-amino vinyl sulfone 6. So far, there is only one synthetic procedure reported in the literature for the synthesis of γ-phenyl-γ-amino vinyl sulfone 6 in eight steps by Picó et al. employing sharpless asymmetric epoxidation of cinnamyl alcohol.¹¹ We synthesized γ-phenyl-γ-amino vinyl sulfone 6 in four steps starting from commercially available (±)-2-phenylglycinol 14. As shown in Scheme 4, trityl-protected primary alcohol (±)-23 obtained from the trityl protection of (±)-2-phenylglycinol 14 was subjected to Dess–Martin oxidation followed by immediate HWE olefination with diethyl ((phenylsulfonyl)methyl)phosphonate¹⁶ and NaH affording trityl-protected vinyl sulfone (±)-26. Finally, trityl deprotection of (±)-26 with TFA in DCM yielded γ-phenyl-γ-amino vinyl sulfone (±)-6 in a quantitative yield after a basic workup with sat. aq. NaHCO₃ solution.

Scheme 4. Synthesis of γ-Phenyl-γ-amino Vinyl Sulfone (±)-6.

Reagents and conditions: (a) (i) Dess–Martin periodinane, DCM, 0 to 25 °C, 30 min and (ii) diethyl ((phenylsulfonyl)methyl) phosphonate, NaH, THF, 0 to 25 °C, 3 h, 74% over two steps and (b) TFA, DCM, 25 °C, 30 min, then sat. aq. NaHCO₃ solution, quant.

Enantioselective Synthesis of γ-Phenyl-γ-amino Vinyl Phosphonate (5) and γ-Phenyl-γ-amino Vinyl Sulfone (6)

After successfully synthesizing racemic γ-phenyl-γ-amino vinyl phosphonate 5 and γ-phenyl-γ-amino vinyl sulfone 6, we further targeted their enantioselective syntheses (Table 1). The enantiomers of 5 and 6 were synthesized using the racemic strategy as described in Schemes 3 and 4, respectively.

Table 1. Synthesis of Enantiomers of γ-Phenyl-γ-amino Vinyl Phosphonate 5 and Sulfone 6.

Commercially available (S)-and (R)-2-phenylglycinols (i.e., (S)-14 and (R)-14) were protected with a trityl group to afford trityl-protected amino alcohols (S)-23 and (R)-23, respectively. Dess–Martin oxidation of alcohols (S)-23 and (R)-23 furnished (S)-24 and (R)-24, respectively. The aldehydes (S)-24 and (R)-24 were subjected to immediate HWE olefination with tetraethyl methylenediphosphonate and NaH to afford (R)-25 and (S)-25, respectively. The final deprotection of (R)-25 and (S)-25 with TFA furnished free amines (R)-5 and (S)-5, respectively. Similarly, the aldehydes (S)-24 and (R)-24 were subjected to HWE olefination with diethyl ((phenylsulfonyl)methyl) phosphonate and NaH to afford (R)-26 and (S)-26, respectively. The structure of (S)-26 was further confirmed by single-crystal X-ray analysis (Figure 3).¹⁷ The final deprotection of (R)-26 and (S)-26 with TFA furnished free amines (R)-6 and (S)-6, respectively.

Figure 3.

ORTEP diagram of compound (S)-26.

It is pertinent to mention here that no racemization occurred as evidenced by chiral HPLC analysis during the oxidation and HWE olefination steps. The chiral HPLC analysis of all the olefin products (R)-25, (S)-25, (R)-26, and (S)-26 (see the Supporting Information) suggested the formation of a single enantiomer with >99% to 100% ee.

Synthesis of Peptidyl Phenyl Vinyl Phosphonate and Sulfone Derivatives of K11002

We next targeted the application of the present strategy to synthesize peptidyl phenyl vinyl phosphonate and sulfone derivatives of the cysteine protease inhibitor for Chagas disease, K11002 (2). The peptide backbone 27 was synthesized according to the reported procedure.¹⁸ As shown in Scheme 5, the mu-Phe-COOH 27 was subjected to individual coupling reactions with γ-phenyl-vinyl phosphonate (S)-5 and sulfone (S)-6 to afford the peptidyl phenyl vinyl phosphonate 28S and sulfone 29S, respectively, with 100% de (the products 28S and 29S were obtained as single diastereomers).

Scheme 5. Synthesis of Peptidyl Phenyl Vinyl Phosphonate 28S and Sulfone 29S Derivatives of K11002.

Reagents and conditions: (a) (S)-5, DCC, HOBt, THF, 0 to 25 °C, 12 h, 91% and (b) (S)-6, DCC, HOBt, THF, 0 to 25 °C, 12 h, 74%.

Synthesis of Artemisinin–Dipeptidyl Vinyl Sulfone Hybrid Molecules

Malaria is a vector transmitted parasitic disease, which remains to be a major medical problem in tropical and sub-tropical areas of the world, leading to approximately ∼500,000 to 1 million deaths globally every year.¹⁹ In the absence of an efficient vaccine and due to the rapid spread of drug-resistant parasite strains, there is an urgent need to identify new drug targets for the development of new drugs against the disease.

Synthesis of hybrid molecules has been a promising approach in the discovery of new lead molecules for various diseases as the hybrid molecules possess superior biological activities when compared to those of individual components.²⁰ Hybrid drugs with a lower risk of drug–drug adverse interactions may be less expensive compared to the multicomponent drugs. Another advantage of the hybrid molecule approach over combinatorial chemistry is that it may generate a diverse class of new chemical entities (NCEs) with high structural diversity and different biological activities. Also, the partner drug in the hybrid molecule may be protected from the spread of resistance. The hybrid molecule approach has been utilized to develop a diverse class of compounds to treat several diseases such as AIDS, tuberculosis, cancer, and diabetes and is now gaining momentum in the area of antimalarial drug discovery. Several artemisinin-containing antimalarial hybrid molecules have been synthesized in the literature by combining with different pharmacophores.²¹ The most important challenge when designing hybrid compounds is to overcome issues associated with drug resistance, pharmacokinetics, potency, solubility, metabolism, mode of administration, and toxicity. The fact is that only one synthetic aminoquinoline–trioxaquine hybrid molecule (PA1103/SAR116242) has reached clinical trials.²² However, it was later abandoned in preclinical development. Hence, the development of novel hybrid molecules with different modes of action remains to be one of few resources for the development of effective anti-malarial drugs.

A major class of cysteine proteases (named falcipains) of Plasmodium falciparum (P. falciparum) is involved in the degradation of host hemoglobin for the survival of the malarial parasite.²³ Since the cysteine proteases of malaria parasites named falcipain-2, -2′, and -3 play a vital role in the host hemoglobin degradation, the inhibition of the falcipain-2 enzyme can be considered as an ideal drug target for the development of new antimalarial drugs. The peptidyl vinyl sulfones are well-known reported potent inhibitors of the falcipain-2 protease enzyme of P. falciparum.^8b,21i,24 In principle, falcipain-2 inhibitors require a hydrophobic interaction in P₁ and P₂ pockets and an electrophilic center at the active site of the enzyme (Figure 4A).^6b

Figure 4.

Design of artemisinin–dipeptidyl vinyl sulfone hybrid molecules targeting the falcipain-2 enzyme.

In continuation of our new antimalarial drug discovery,²⁵ we designed and synthesized artemisinin–dipeptidyl vinyl sulfone hybrid molecules by combining dihydroartemisinin with a vinyl sulfone falcipain-2 inhibitor using suitable amino acid-based linkers (Figure 4B). We further aimed to study the effect of the falcipain-2 inhibitor on the antimalarial activities of these hybrid molecules. The hybrid molecules 33, 34, and 35 were synthesized by coupling dipeptidyl carboxylic acids 30, 31, and 32 with γ-phenyl-vinyl sulfone (±)-6, respectively (Scheme 6). The hybrid molecules 33, 34, and 35 are obtained as a mixture of diastereomers that cannot be separated by silica gel chromatography. The dipeptidyl carboxylic acid precursors 30, 31, and 32 were synthesized from dihydroartemisinin by following our reported procedure.^25a

Scheme 6. Synthesis of Artemisinin–Dipeptidyl Vinyl Sulfone Hybrid Molecules 33, 34, and 35.

Biology

In Vitro Antimalarial Activity of Artemisinin–Dipeptidyl Vinyl Sulfone Hybrid Molecules 33, 34, and 35

The hybrid molecules 33, 34, and 35 were assayed for their in vitro inhibition activity against the falcipain-2 protease enzyme as well as P. falciparum parasite growth. To test the inhibitory activities of artemisinin–dipeptidyl vinyl phosphonate hybrid molecules, we expressed and purified recombinant falcipain-2 and HDP proteins by protocols previously described.²⁶ The recombinant falcipain-2 activity was analyzed by quantitatively measuring the blue fluorescence generated as a result of the release of free AMC after the hydrolysis of Z-FR-AMC (benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin hydrochloride) by the enzyme (Figure S1; see the Supporting Information). The hybrid compounds showed potent inhibition activities with IC₅₀ in the nanomolar range against the falcipain-2 enzyme (Table 2). All three hybrid compounds were subsequently assessed for their antimalarial activities in a P. falciparum 3D7 culture. The three hybrid compounds displayed anti-parasiticidal efficacy with EC₅₀ in the nanonmolar range, being more active than artemisinin. The EC₅₀ of the three compounds was about sevenfold better than that of artemisinin (Table 2). This result strongly suggests that the anti-falcipain-2 activity exhibited by vinyl sulfone peptide pharmacophore provides synergistic efficacy to the anti-plasmodial activity of the artemisinin core.

Table 2. In Vitro Falcipain-2 Inhibition and Parasite Growth Inhibitions by Compounds 33, 34, and 35.

Compound.	IC50 (nM) on falcipain-2 activity	EC50 (nM) on parasite growth
33	136.8 ± 69	3.9 ± 0.5
34	13.5 ± 2.5	4.0 ± 0.2
35	17.1 ± 3.2	3.0 ± 0.2
artemisinin	N.A.	27 ± 0.3

Conclusions

In conclusion, we have developed an efficient method for the enantioselective synthesis of γ-phenyl-γ-amino vinyl phosphonate and sulfones using HWE olefination of the trityl-protected α-amino aldehyde with tetraethyl methylenediphosphonate and diethyl ((phenylsulfonyl)methyl) phosphonate, respectively. It is pertinent to mention here that no racemization was observed as evidenced by chiral HPLC. The developed methodology was applied to the synthesis of peptidyl phenyl vinyl phosphonate 28S and sulfone 29S derivatives of the cysteine protease inhibitor of Chagas disease, K11002, with 100% de. The methodology was also successfully utilized to synthesize artemisinin–dipeptidyl vinyl sulfone hybrid molecules 33, 34, and 35 to study their potential as antimalarials. The hybrid molecules 33, 34, and 35 showed potent in vitro falcipain-2 inhibition activity along with anti-parasiticidal activity against P. falciparum in nanomolar ranges. We presume that the present developed methodology would allow easy access to the synthesis of peptidyl phenyl vinyl phosphonates and sulfones for their further utilization in the design and synthesis of cysteine protease inhibitors to target various diseases.

Experimental Section

General Methods

All dry reactions were carried out under an argon atmosphere, and flash chromatography was performed with CombiFlash Rf 200i with UV/vis and ELSD, Isco Teledyne Inc., U.S.A., using a RediSep column (SiO₂). Commercially available reagents and solvents were used as received. Dry solvents were prepared following the standard procedures. All the melting points were recorded on a Büchi melting point apparatus in open capillaries and are uncorrected. ¹H NMR spectra were recorded on 500 or 400 or 200 MHz spectrometers, and ¹³C NMR spectra were recorded at 125 or 100 or 50 MHz. Chemical shifts were reported as δ values (ppm) relative to internal standard tetramethylsilane in CDCl₃. HRMS (ESI) were recorded on an Orbitrap (quadrupole plus ion trap) and TOF mass analyzer. Optical rotations were recorded on a JASCO P-1020 polarimeter. HPLC was performed with an Agilent HPLC system (UV detection at 215/220/254 nm, chiral column: Chiralpak-IA or IB (250 × 4.6 mm), mobile phase: EtOH in ⁿhexane or isopropyl alcohol (IPA) in ⁿhexane, flow rate: 1 mL/min).

(E)-1-(Diethoxyphosphoryl)-3-phenylallyl 2,2,2-Trichloroacetimidate (9)

To a stirred solution of hydroxyphosphonate 8 (4.0 g, 1.0 equiv) and CCl₃CN (4.5 mL, 3.0 equiv) in DCM (60 mL) was added a catalytic amount of DBU (1.1 mL, 0.5 equiv) under Ar at −35 °C. Stirring was continued at this temperature for 30 min. After completion of the reaction (TLC), solvent and volatile compounds were removed in vacuo. The residue was then immediately purified by flash chromatography on silica gel to afford pure trichloroacetimidate 9 as a pale yellow syrup (6.0 g, 98%).^12bR_f = 0.35 (EtOAc–petroleum ether, 2:3); ¹H NMR (200 MHz, CDCl₃): δ 8.59 (s, 1H), 7.49–7.21 (m, 5H), 6.86 (dd, J = 16.0, 4.0 Hz, 1H), 6.43–6.22 (m, 1H), 6.06 (dd, J = 14.0, 6.9 Hz, 1H), 4.37–4.09 (m, 4H), 1.37–1.30 (m, 6H); ¹³C NMR (50 MHz, CDCl₃): δ 161.3 (d, ³J_PC = 9.2 Hz, C=NH), 135.8 (d, ⁴J_PC = 2.2 Hz, i-C_arom), 135.0 (d, ³J_PC = 12.1 Hz, C-3), 129.0, 128.6, 128.4, 127.2, 126.9, 123.4, 119.5 (d, ²J_PC = 4.4 Hz, C-2), 114.1, 91.0 (CCl₃), 73.9 (d, ¹J_PC = 169.8 Hz, PCH), 63.7 (d, ²J_PC = 7.3 Hz, POCH₂), 63.4 (d, ²J_PC = 6.2 Hz, POCH₂), 16.5, 16.4; ESI-LCMS: m/z 437.9 (M + Na)⁺; HRMS (ESI): m/z for C₁₅H₁₉O₄NCl₃NaP (M + Na)⁺: calcd 436.0009, found 435.9997.

Diethyl (E)-(3-Phenyl-3-(2,2,2-trichloroacetamido)prop-1-en-1-yl)phosphonate (10)

A solution of trichloroacetimidate 9 (3.0 g) in toluene (80 mL) was refluxed under Ar for 24 h. After complete disappearance of the starting material (TLC), the solvent was removed in vacuo, and the residue was subjected to flash chromatography on silica gel to afford trichloroacetamide 10 as a colorless crystalline solid (2.76 g, 92%).^12bR_f = 0.35 (EtOAc-petroleum ether, 1:1); ¹H NMR (500 MHz, CDCl₃): δ 7.57–7.46 (m, 1H), 7.44–7.29 (m, 5H), 6.95 (t, J = 17.9 Hz, 1H), 5.89 (t, J = 17.7 Hz, 1H), 5.69 (brs, 1H), 4.15–3.95 (m, 4H), 1.38–1.18 (m, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 161.3 (C=O), 148.6 (d, ²J_PC = 6.7 Hz, C-2), 137.2, 129.3, 128.8, 127.3, 119.0 (d, ¹J_PC = 186.9 Hz, C-1), 92.4 (CCl₃), 62.1 (d, ²J_PC = 5.7 Hz, POCH₂), 57.1 (d, ³J_PC = 22.9 Hz, CHN), 16.4 (d, ³J_PC = 3.8 Hz, CH₃), 16.3 (d, ³J_PC = 3.8 Hz, CH₃); ESI-LCMS: m/z 437.9 (M + Na)⁺; HRMS (ESI): m/z for C₁₅H₁₉O₄NCl₃NaP (M + Na)⁺: calcd 436.0009, found 436.0002.

Diethyl (Z)-(3-Phenyl-3-(2,2,2-trichloroacetamido)allyl)phosphonate (11)

Trichloroacetamide 10 (150 mg, 0.361 mmol, 1.0 equiv) was dissolved in 3.5 mL of EtOH and was added to 3 N KOH solution (670 μL). The whole mixture was stirred for 24 h at rt. After the completion of the reaction (TLC), the reaction mixture was extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo to give the crude product, which was further purified by flash chromatography on silica gel to furnish pure amine 11 as a colorless syrup (97 mg, 100%). R_f = 0.42 (EtOAc-petroleum ether, 1:1); ¹H NMR (500 MHz, CDCl₃): δ 10.04 (brs, 1H), 7.49–7.43 (m, 2H), 7.42–7.33 (m, 3H), 5.81–5.73 (m, 1H), 4.21–4.11 (m, 4H), 2.76 (dd, ²J_PH = 22.5 Hz, J = 8.0 Hz, 2H, PCH₂), 1.35 (t, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 160.6 (d, ⁵J_PC = 2.9 Hz, C=O), 139.4 (d, ³J_PC = 13.4 Hz, C-3), 135.7 (d, ⁴J_PC = 4.7 Hz, i-C_arom), 128.8, 128.6, 125.7 (d, ⁵J_PC = 1.9 Hz, o-CH_arom), 112.4 (d, ²J_PC = 13.4 Hz, C-2), 92.8 (CCl₃), 62.8 (d, ²J_PC = 7.6 Hz, POCH₂), 25.8 (d, ¹J_PC = 139.2 Hz, PCH₂), 16.4 (d, ³J_PC = 5.7 Hz, CH₃).

tert-Butyl (Z)-(3-(Diethoxyphosphoryl)-1-phenylprop-1-en-1-yl)carbamate (19)

In a round-bottom flask, tetraethyl methylenediphosphonate (1.83 g, 7.37 mmol, 1.7 equiv) was taken up in THF (8 mL). The reaction mixture was cooled to 0 °C in an ice bath. NaH (60% dispersion in mineral oil, 260 mg, 6.50 mmol, 1.5 equiv) was added to the reaction mixture. The solution was stirred at 0 °C for 15 min. N-Boc-protected α-amino aldehyde 17 (1.02 g, 4.33 mmol, 1.0 equiv) was taken up in dry THF (7 mL) and was added to the reaction mixture. The reaction mixture was then warmed to rt and stirred for 3 h. After completion of the reaction (TLC), the reaction mixture was concentrated in vacuo. The residue was dissolved in water and extracted with DCM (3 × 80 mL). The combined organic layers were washed with water and brine, dried over anhydrous Na₂SO₄, and concentrated to give the crude product, which was purified by column chromatography on a silica gel column with EtOAc–petroleum ether as eluents to give pure olefin 19 as a pale yellow syrup (0.953 g, 59%). R_f = 0.31 (EtOAc–petroleum ether, 2:3); ¹H NMR (500 MHz, CDCl₃): δ 7.45–7.40 (m, 2H), 7.37–7.28 (m, 3H), 5.43–5.33 (m, 1H), 4.21–4.08 (m, 4H), 2.77 (dd, ²J_PH = 22.2 Hz, J = 8.0 Hz, 2H, PCH₂), 1.48–1.25 (m, 15H); ¹³C NMR (125 MHz, CDCl₃): δ 153.5, 140.1, 128.4, 128.2, 128.1, 126.1, 108.4, 80.2, 62.4 (d, ²J_PC = 7.6 Hz, POCH₂), 28.1, 27.7, 26.1 (d, ¹J_PC = 140.2 Hz, PCH₂), 16.5 (d, ³J_PC = 5.7 Hz, CH₃); ESI-LCMS: m/z 370.0 (M + H)⁺; HRMS (ESI): m/z for C₁₈H₂₈O₅NNaP (M + Na)⁺: calcd 392.1597, found 392.1586.

Diethyl (Z)-(3-benzamido-3-phenylallyl)phosphonate (20)

The same HWE olefination procedure as described for 19 was followed for the synthesis of olefin 20 using N-Bz-protected α-amino aldehyde 18 and tetraethyl methylenediphosphonate as starting materials. Colorless syrup; yield: 60%; R_f = 0.15 (EtOAc–petroleum ether, 2:3); ¹H NMR (400 MHz, CDCl₃): δ 9.96 (brs, 1H), 8.07 (d, 2H), 7.58–7.42 (m, 5H), 7.36–7.26 (m, 3H), 5.58 (q, J = 7.6 Hz, 1H), 4.19–4.05 (m, 4H), 2.77 (dd, ²J_PH = 22.1 Hz, J = 8.4 Hz, 2H, PCH₂), 1.31 (t, J = 6.9 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 165.6 (d, ⁵J_PC = 1.9 Hz, C=O), 141.5 (d, ³J_PC = 12.5 Hz, C-3), 137.3 (d, ⁴J_PC = 4.8 Hz, i-C_arom), 133.5, 132.0, 128.7, 128.6, 128.4, 128.3, 127.8, 127.5, 126.0 (d, ⁵J_PC = 1.9 Hz, o-CH_arom), 109.4 (d, ²J_PC = 13.4 Hz, C-2), 62.9 (d, ²J_PC = 7.7 Hz, POCH₂), 25.9 (d, ¹J_PC = 139.0 Hz, PCH₂), 16.5 (d, ³J_PC = 5.8 Hz, CH₃); ESI-LCMS: m/z 374.0 (M + H)⁺; HRMS (ESI): m/z for C₂₀H₂₄O₄NNaP (M + Na)⁺: calcd 396.1335, found 396.1326.

General Procedure for the Synthesis of 2-Phenyl-2-(tritylamino)ethan-1-ol ((±)-23 or (R)-23 or (S)-23)

To a mixture of 2-phenylglycinol (±)-14, (R)-14, or (S)-14 (1.0 g, 1.0 equiv) and triphenylmethyl chloride (2.03 g, 1.0 equiv) in dichloromethane (25 mL) was added triethylamine (1.01 mL, 1.0 equiv). The resulting mixture was stirred at 25 °C for 12 h. The mixture was diluted with ethyl acetate (75 mL) and washed with water and brine. The ethyl acetate fraction was dried (anhydrous Na₂SO₄), filtered, and concentrated. The solid was purified by chromatography on a silica gel column with EtOAc–petroleum ether as eluents to furnish the pure product (±)-23 or (R)-23 or (S)-23, respectively, as a colorless viscous syrup or foaming solid.

2-Phenyl-2-(tritylamino)ethan-1-ol ((±)-23)

Colorless viscous syrup (1.6 g, 58%); R_f = 0.43 (EtOAc–petroleum ether, 1:4).

(R)-2-Phenyl-2-(tritylamino)ethan-1-ol ((R)-23)

Colorless viscous syrup (1.71 g, 62%); R_f = 0.43 (EtOAc–petroleum ether, 1:4); [α]_D²⁴ −97.3 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 7.58–7.45 (m, 6H), 7.33–7.12 (m, 14H), 3.80 (t, J = 4.6 Hz, 1H), 3.21 (dd, J = 10.7, 3.8 Hz, 1H), 2.78 (dd, J = 10.7, 5.3 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 146.6, 143.6, 129.0, 128.4, 128.0, 127.9, 127.2, 126.7, 126.5, 71.9, 67.0, 58.6.

(S)-2-Phenyl-2-(tritylamino)ethan-1-ol ((S)-23)

Colorless viscous syrup (1.51 g, 55%); R_f = 0.43 (EtOAc–petroleum ether, 1:4); [α]_D²⁴ +96.7 (c 1.0, CHCl₃).

General Procedure for the Synthesis of Diethyl (E)-(3-Phenyl-3-(tritylamino)prop-1-en-1-yl)phosphonate ((±)-25, (R)-25, or (S)-25)

2-Phenyl-2-(tritylamino)ethan-1-ol (±)-23, (R)-23, or (S)-23 (0.5 g, 1.32 mmol, 1.0 equiv) was dissolved in DCM (10 mL) and cooled to 0 °C. To this cold solution was added Dess–Martin periodinane (0.840 g, 1.98 mmol, 1.5 equiv) portion-wise over 10 min and then stirred at 0 °C for 10 min. The reaction mixture was allowed to slowly warm to 25 °C and stirred for 30 min. After completion of the reaction (TLC), the reaction mixture was diluted with DCM. The reaction mixture was placed in an ice-water bath, and a mixture of saturated aqueous NaHCO₃ solution and saturated NaHSO₃ solution (1:1, 4 mL) was added; the cooling bath was removed, and the mixture was stirred at 25 °C until the formation of two clear layers was observed. The reaction mixture was transferred to a separatory funnel containing a saturated aqueous NaHCO₃ solution (20 mL). The aqueous layer was extracted with ethyl acetate (3 × 25 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated to give crude aldehyde (±)-24, (R)-24, or (S)-24 as a colorless foaming solid. The crude aldehyde residue was used immediately for the next step without any further purification.

Tetraethyl methylenediphosphonate (0.645 g, 2.24 mmol, 1.7 equiv) was taken up in dry THF (4 mL) and was cooled to 0 °C in an ice bath. NaH (60% dispersion in mineral oil, 80 mg, 1.98 mmol, 1.5 equiv) was added to the reaction mixture portion-wise over a period of 5–10 min. The solution was stirred at 0 °C for 15 min. Crude aldehyde (±)-24, (R)-24, or (S)-24 was taken in dry THF (4 mL) and added to the reaction mixture. The reaction mixture was then warmed to 25 °C and stirred for 3 h. After completion of the reaction (TLC), the reaction mixture was concentrated in vacuo. The residue was dissolved in water and extracted with DCM (3× 20 mL). The combined organic layers were washed with water and brine and concentrated to give the crude product, which was purified either by recrystallization from the EtOAc–petroleum ether mixture or column chromatography on a silica gel column with EtOAc–petroleum ether as an eluent to give pure olefin (±)-25, (S)-25, or (R)-25 as a colorless solid.

Diethyl (E)-(3-Phenyl-3-(tritylamino)prop-1-en-1-yl)phosphonate ((±)-25)

Colorless solid (0.404 g, 60% over two steps); mp: 187–188 °C; R_f = 0.28 (EtOAc-petroleum ether, 1:1); ³¹P NMR (162 MHz, CDCl₃): δ 19.09; ESI-LCMS: m/z 534.2 (M + Na)⁺; HPLC: Chiralpak-IA (0.46 mm ϕ × 250 mmL), 3% EtOH in hexane, flow rate 1.0 mL min^–1, UV detection at 254 nm, t_R = 9.0 min for the (R)-isomer and t_R = 10.0 min for the (S)-isomer; HRMS (ESI): m/z for C₃₂H₃₄O₃NNaP (M + Na)⁺: calcd 534.2169, found 534.2160.

Diethyl (R,E)-(3-Phenyl-3-(tritylamino)prop-1-en-1-yl)phosphonate ((R)-25)

Colorless solid (0.438 g, 65% over two steps); mp: 115–117 °C; R_f = 0.28 (EtOAc-petroleum ether, 1:1); [α]_D²³ −43.8 (c 1.0, CHCl₃); ³¹P NMR (162 MHz, CDCl₃): δ 19.09; ESI-LCMS: m/z 534.0 (M + Na)⁺; HRMS (ESI): m/z for C₃₂H₃₄O₃NNaP (M + Na)⁺: calcd 534.2169, found 534.2158; HPLC: ee 100% [Chiralpak-IA (250 × 4.6 mm), 3% EtOH in hexane, flow rate 1.0 mL min^–1, UV detection at 254 nm, t_R = 9.0 min for the (R)-isomer].

Diethyl (S,E)-(3-Phenyl-3-(tritylamino)prop-1-en-1-yl)phosphonate ((S)-25)

Colorless solid (0.430 g, 64% over two steps); mp: 115–117 °C; R_f = 0.28 (EtOAc–petroleum ether, 1:1); [α]_D²³ +41.0 (c 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.51–7.41 (m, 6H), 7.24–7.18 (m, 6H), 7.18–7.10 (m, 6H), 6.99–6.92 (m, 2H), 6.56–6.44 (m, 1H), 5.73–5.62 (m, 1H), 4.31–4.25 (m, 1H), 4.03–3.87 (m, 4H), 1.32–1.21 (m, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 155.5 (d, ²J_PC = 5.7 Hz, C-2), 146.1, 142.3, 128.9, 128.4, 127.8, 127.0, 126.8, 126.6, 114.9 (d, ¹J_PC = 186.9 Hz, C-1), 71.9, 61.7, 61.6, 61.6, 61.6, 60.4 (d, ³J_PC = 21.0 Hz, CHN), 16.4, 16.4, 16.3, 16.3; ³¹P NMR (162 MHz, CDCl₃): δ 19.09; ESI-LCMS: m/z 534.0 (M + Na)⁺; HRMS (ESI): m/z for C₃₂H₃₄O₃NNaP (M + Na)⁺: calcd 534.2169, found 534.2157; HPLC: ee 100% [Chiralpak-IA (250 × 4.6 mm), 3% EtOH in hexane, flow rate: 1.0 mL min^–1, UV detection at 254 nm, t_R = 10.0 min for the (S)-isomer].

General Procedure for the Synthesis of Diethyl (E)-(3-Amino-3-phenylprop-1-en-1-yl)phosphonate ((±)-5, (R)-5, or (S)-5)

Trityl-protected amine (±)-25, (R)-25, or (S)-25 (0.5 g, 1.0 equiv) was dissolved in DCM, and trifluoroacetic acid (150 μL, 3.0 equiv.) was added at 25 °C; the reaction mixture was stirred for 30 min. After completion of the reaction (TLC), DCM was removed under reduced pressure. Water (10 mL) was added to the residue, and the aqueous layer was washed with diethyl ether (3 × 20 mL). The remaining aqueous layer was basified with a saturated aqueous solution of NaHCO₃ until pH 8; after which, it was extracted with DCM (3 × 20 mL). The combined organic layers were dried over Na₂SO₄ and concentrated to give crude amine (±)-5, (R)-5, or (S)-5 as a pale yellow syrup in a quantitative yield, which was used for the final coupling reaction without any further purification. R_f = 0.54 (MeOH-DCM, 1:9); ¹H NMR (200 MHz, CDCl₃): δ 7.51–7.16 (m, 5H), 7.08–6.76 (m, 1H), 6.10–5.83 (m, 1H), 4.74–4.59 (m, 1H), 4.21–3.93 (m, 4H), 1.43–1.18 (m, 6H); ¹³C NMR (50 MHz, CDCl₃): δ 155.0 (d, ²J_PC = 5.1 Hz, C-2), 142.3, 128.8, 127.7, 127.5, 126.8, 125.9, 115.6 (d, ¹J_PC = 188.1 Hz, C-1), 61.8 (d, ²J_PC = 21.0 Hz, POCH₂), 58.0 (d, ³J_PC = 21.6 Hz, CHN), 16.4 (d, ³J_PC = 6.2 Hz, CH₃); ESI-LCMS: m/z 292.0 (M + Na)⁺.

General Procedure for the Synthesis of (E)-1-Phenyl-3-(phenylsulfonyl)-N-tritylprop-2-en-1-amine ((±)-26, (R)-26, or (S)-26)

2-Phenyl-2-(tritylamino)ethan-1-ol (±)-23 or (R)-23 or (S)-23 (0.5 g, 1.32 mmol, 1.0 equiv) was dissolved in DCM (10 mL) and cooled to 0 °C. To this cold solution was added Dess–Martin periodinane (0.838 g, 1.98 mmol, 1.5 equiv) portion-wise over 10 min and then stirred at 0 °C for 10 min. The reaction mixture was allowed to slowly warm to 25 °C and stirred for 30 min. After completion of the reaction (TLC), the reaction mixture was diluted with DCM. The reaction mixture was placed in an ice-water bath, and a 1:1 mixture of saturated aqueous NaHCO₃ solution and saturated NaHSO₃ solution (4 mL) was added; the cooling bath was removed, and the mixture was stirred at 25 °C until the formation of two clear layers was observed. The reaction mixture was transferred to a separatory funnel containing a saturated aqueous NaHCO₃ solution (20 mL). The aqueous layer was extracted with ethyl acetate (3× 25 mL). The combined organic layers were washed with brine (20 mL), dried over Na₂SO₄, filtered, and concentrated to give crude aldehyde (±)-24, (R)-24, or (S)-24 as a colorless foaming solid. The crude aldehyde residue was used immediately for the next step without any further purification.

Diethyl ((phenylsulfonyl)methyl)phosphonate (0.654 g, 2.24 mmol, 1.7 equiv) was taken up in dry THF (4 mL) and cooled to 0 °C in an ice bath. NaH (60% dispersion in mineral oil, 80 mg, 1.98 mmol, 1.5 equiv) was added to the reaction mixture portion-wise over a period of 5–10 min. The solution was stirred at 0 °C for 15 min. Crude aldehyde (±)-24 or (R)-24 or (S)-24 was taken in dry THF (4 mL) and added to the reaction mixture. The reaction mixture was then warmed to 25 °C and stirred for 3 h. After completion of the reaction (TLC), the reaction mixture was concentrated in vacuo. The residue was dissolved in water and extracted with DCM (3× 20 mL). The combined organic layers were washed with water and brine, dried over anhydrous Na₂SO₄, and concentrated to give the crude product, which was purified either by recrystallization from the EtOAc–petroleum ether mixture or column chromatography on a silica gel column with EtOAc–petroleum ether as eluents to furnish pure olefin (±)-26, (S)-26, or (R)-26 as a colorless solid.

(E)-1-Phenyl-3-(phenylsulfonyl)-N-tritylprop-2-en-1-amine ((±)-26)

Colorless solid (0.502 g, 74% over two steps); mp: 168–169 °C; R_f = 0.40 (EtOAc-petroleum ether, 1:4); ¹H NMR (200 MHz, CDCl₃): δ 7.74–7.44 (m, 5H), 7.43–7.30 (m, 6H), 7.24–7.07 (m, 12H), 7.05–6.92 (m, 2H), 6.67 (dd, J = 14.9, 6.2 Hz, 1H), 6.13 (d, J = 14.9 Hz, 1H), 4.26 (t, J = 5.3 Hz, 1H), 2.45 (d, J = 5.7 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 149.1, 145.8, 141.3, 140.6, 133.2, 129.2, 128.8, 127.9, 127.7, 127.4, 127.1, 126.8, 72.0, 59.0; ESI-LCMS: m/z 538.0 (M + Na)⁺; HRMS (ESI): m/z for C₃₄H₂₉O₂NNaS (M + Na)⁺: calcd 538.1811, found 538.1801; HPLC: Chiralpak-IA (250 × 4.6 mm), 5% EtOH in hexane, flow rate: 1.0 mL min^–1, UV detection at 220 nm, t_R = 11.3 min for the (R)-isomer and t_R = 12.8 min for the (S)-isomer.

(R,E)-1-Phenyl-3-(phenylsulfonyl)-N-tritylprop-2-en-1-amine ((R)-26)

Colorless solid (0.482 g, 71% over two steps); mp: 163–165 °C; R_f = 0.40 (EtOAc–petroleum ether, 1:4); [α]_D²⁵ −52.3 (c 1.0, CHCl₃); ESI-LCMS: m/z 538.0 (M + Na)⁺; HRMS (ESI): m/z for C₃₄H₂₉O₂NNaS (M + Na)⁺: calcd 538.1811, found 538.1801; HPLC: ee 100% [Chiralpak-IA (250 × 4.6 mm), 5% EtOH in hexane, flow rate: 1.0 mL min^–1, UV detection at 220 nm, t_R = 11.3 min for the (R)-isomer].

(S,E)-1-Phenyl-3-(phenylsulfonyl)-N-tritylprop-2-en-1-amine ((S)-26)

Colorless solid (0.407 g, 60% over two steps); mp: 165–166 °C; R_f = 0.40 (EtOAc-petroleum ether, 1:4); [α]_D²⁵ +59.0 (c 1.03, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.72–7.67 (m, 2H), 7.64 (t, J = 7.4 Hz, 1H), 7.56–7.50 (m, 2H), 7.44–7.38 (m, 6H), 7.26–7.12 (m, 12H), 7.06–7.00 (m, 2H), 6.71 (dd, J = 14.9, 6.5 Hz, 1H), 6.17 (dd, J = 14.9, 1.1 Hz, 1H), 4.31 (d, J = 6.1 Hz, 1H), 2.49 (brs, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 149.0, 145.8, 141.3, 140.6, 133.2, 129.2, 128.9, 128.8, 128.7, 127.9, 127.7, 127.4, 127.0, 126.7, 72.0, 59.0; ESI-LCMS: m/z 538.0 (M + Na)⁺; HRMS (ESI): m/z for C₃₄H₂₉O₂NNaS (M + Na)⁺: calcd 538.1811, found 538.1805; HPLC: ee 99.2% [Chiralpak-IA (250 × 4.6 mm), 5% EtOH in hexane, flow rate: 1.0 mL min^–1, UV detection at 220 nm, t_R = 12.6 min for the (S)-isomer].

General Procedure for the Synthesis of (E)-1-Phenyl-3-(phenylsulfonyl)prop-2-en-1-amine ((±)-6, (R)-6, or (S)-6)

Trityl-protected amine (±)-26, (R)-26, or (S)-26 (1.0 g, 1.94 mmol, 1.0 equiv) was dissolved in DCM (30 mL), and trifluoroacetic acid (445 μL, 3.0 equiv) was added at 25 °C; the reaction mixture was stirred for 30 min. After completion of the reaction (TLC), DCM was removed under reduced pressure. Water (15 mL) was added to the residue, and the aqueous layer was washed with diethyl ether (3× 20 mL). The remaining aqueous layer was basified with a saturated aqueous solution of NaHCO₃ until pH 8; after which, it was extracted with DCM (3× 20 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated to give crude amine (±)-6, (R)-6, or (S)-6 as a colorless solid in a quantitative yield, which was used for the final coupling reaction without any further purification. R_f = 0.42 (MeOH-DCM, 1:19); ¹H NMR (400 MHz, CDCl₃): δ 7.92–7.81 (m, 2H), 7.66–7.57 (m, 1H), 7.56–7.48 (m, 2H), 7.39–7.18 (m, 5H), 7.09 (dd, J = 15.3, 4.6 Hz, 1H), 6.65 (dd, J = 14.5, 1.5 Hz, 1H), 4.76–4.67 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 148.7, 141.5, 140.4, 133.5, 129.7, 129.4, 129.1, 128.2, 127.8, 126.9, 56.4; ESI-LCMS: m/z 296.0 (M + Na)⁺.

Diethyl ((S,E)-3-((S)-2-(Morpholine-4-carboxamido)-3-phenylpropanamido)-3-phenyl prop-1-en-1-yl)phosphonate (28S)

Free acid 27 (0.1 g, 1.0 equiv), free amine (S)-5 (97 mg, 1.0 equiv), and HOBt (50 mg, 1.0 equiv) were dissolved in dry THF (4 mL), and the resulting solution was stirred in an ice-cooled water bath then DCC (90 mg, 1.2 equiv) dissolved in dry THF (3 mL) was added. Stirring was continued for 1 h at 0 °C and then an additional 12 h at 25 °C. After completion of the reaction (TLC), the solvent was removed in vacuo. Ethyl acetate (10 mL) was added to the residue, and the undissolved solid by-product was removed by filtration. The filtrate was washed with a saturated aqueous NaHCO₃ solution (3× 20 mL) and brine (1× 20 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated to give the crude peptide, which was purified by flash chromatography (MeOH-DCM mobile phase) on a silica gel column (12 g RediSep column) to furnish the pure peptide 28S (173 mg, 91%). Colorless solid; mp: 115–117 °C; R_f = 0.30 (MeOH-DCM, 1:19); ¹H NMR (400 MHz, CDCl₃): δ 7.31–7.26 (m, 3H), 7.20 (d, J = 7.6 Hz, 1H), 7.16–7.11 (m, 3H), 7.11–7.06 (m, 2H), 7.06–7.01 (m, 2H), 6.90–6.74 (m, 1H), 5.88–5.74 (m, 1H), 5.71–5.62 (m, 1H), 5.24 (d, J = 6.9 Hz, 1H), 4.58 (q, J = 7.1 Hz, 1H), 4.10–3.92 (m, 4H), 3.67–3.53 (m, 4H), 3.36–3.18 (m, 4H), 3.11–2.95 (m, 2H), 2.09–1.94 (m, 1H), 1.25 (t, 3H), 1.28 (t, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 171.2, 157.1, 150.4 (d, ²J_PC = 5.8 Hz, C-2), 138.5, 136.7, 129.4, 129.0, 128.6, 128.2, 127.5, 126.9, 117.7 (d, ¹J_PC = 186.9 Hz, C-1), 66.4, 62.0 (d, ²J_PC = 4.8 Hz, POCH₂), 55.8, 55.2 (d, ³J_PC = 23.0 Hz, CHN), 44.0, 38.6, 16.5 (d, ³J_PC = 1.9 Hz, CH₃), 16.4 (d, ³J_PC = 1.9 Hz, CH₃); ESI-LCMS: m/z 552.0 (M + Na)⁺; HPLC: de 100% [Chiralpak-IA (250 × 4.6 mm), 30% IPA in hexane, flow rate 1.0 mL min^–1, UV detection at 220 nm, t_R = 9.7 min for the 28S diastereomer]; HRMS (ESI): m/z for C₂₇H₃₆O₆N₃NaP (M + Na)⁺: calcd 552.2234, found 552.2225.

N-((S)-1-Oxo-3-phenyl-1-(((S,E)-1-phenyl-3-(phenylsulfonyl)allyl)amino)propan-2-yl) Morpholine-4-carboxamide (29S)

Free acid 27 (0.095 g, 1.0 equiv), free amine (S)-6 (94 mg, 1.0 equiv), and HOBt (46 mg, 1.0 equiv) were dissolved in dry THF (4 mL), and the resulting solution was stirred in an ice-cooled water bath then DCC (85 mg, 1.2 equiv) dissolved in dry THF (3 mL) was added dropwise. Stirring was continued for 1 h at 0 °C and then an additional 12 h at 25 °C. After completion of the reaction (TLC), the solvent was removed in vacuo. Ethyl acetate (10 mL) was added to the residue, and the undissolved solid by-product was removed by filtration. The filtrate was washed with a saturated aqueous NaHCO₃ solution (3× 20 mL) and brine solution (1× 20 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated to give the crude product, which was purified by flash chromatography (EtOAc-petroleum ether mobile phase) on a silica gel column (12 g RediSep column) to furnish the pure peptide 29S (135 mg, 74%). Colorless solid; m.p.: 183–184 °C; R_f = 0.28 (EtOAc-petroleum ether, 7:3); ¹H NMR (500 MHz, CDCl₃): δ 7.86 (d, 2H), 7.66–7.58 (m, 1H), 7.57–7.49 (m, 2H), 7.33–7.27 (m, 3H), 7.19–7.10 (m, 4H), 7.09–6.99 (m, 5H), 6.59 (d, J = 15.3 Hz, 1H), 5.76 (brs, 1H), 5.16 (d, J = 6.9 Hz, 1H), 4.55 (q, J = 7.2 Hz, 1H), 3.65–3.49 (m, 4H), 3.28–3.20 (m, 2H), 3.20–3.12 (m, 2H), 3.05–2.95 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 171.4, 157.2, 144.6, 140.2, 137.6, 136.5, 133.5, 131.2, 129.3, 129.2, 129.1, 128.6, 128.4, 127.7, 127.3, 126.9, 66.3, 56.0, 53.2, 43.8, 38.4; ESI-LCMS: m/z 556.0 (M + Na)⁺; HPLC: de 100% [Chiralpak-IA (250 × 4.6 mm), 40% IPA in hexane, flow rate: 1.0 mL min^–1, UV detection at 220 nm, t_R = 17.5 min for the 29S diastereomer]; HRMS (ESI): m/z for C₂₉H₃₁O₅N₃NaS (M + Na)⁺: calcd 556.1877, found 556.1867.

General Procedure for the Synthesis of Hybrid Molecules 33, 34, and 35

Free acid 30, 31, or 32 (1.0 equiv), free amine (±)-6 (1.0 equiv), and HOBt (1.0 equiv) were dissolved in dry THF, and the resulting solution was stirred in an ice-cooled water bath then DCC (1.2 equiv) dissolved in dry THF was added dropwise. Stirring was continued for 1 h at 0 °C and then an additional 12 h at 25 °C. After completion of the reaction (TLC), the solvent was removed in vacuo. Ethyl acetate was added to the residue and the undissolved solid by-product was removed by filtration. The filtrate was washed with a saturated aqueous NaHCO₃ solution (3×) and brine solution (1×). The organic layer was dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by flash chromatography on a silica gel column (RediSep column) to furnish the pure peptide 33, 34, or 35 as a mixture of diastereomers.

Benzyl ((2S)-1-Oxo-1-(((2S)-1-oxo-3-phenyl-1-(((E)-1-phenyl-3-(phenylsulfonyl)allyl)amino)propan-2-yl)amino)-3-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propan-2-yl)carbamate (33)

Colorless solid (yield: 75%); R_f = 0.37 (EtOAc–petroleum ether, 1:1); ¹H NMR (400 MHz, CDCl₃): δ 8.00–7.80 (m, 2H), 7.72–7.48 (m, 3H), 7.46–7.23 (m, 8H), 7.23–6.99 (m, 7H), 6.99–6.82 (m, 1H), 6.80–6.62 (m, 1H), 6.56–6.31 (m, 1H), 5.93–5.78 (m, 2H), 5.49–5.33 (m, 1H), 5.16–4.88 (m, 1H), 4.83–4.56 (m, 2H), 4.28–4.08 (m, 1H), 3.97–3.77 (m, 1H), 3.32–3.07 (m, 1H), 2.99–2.82 (m, 1H), 2.72–2.56 (m, 1H), 2.48–2.27 (m, 1H), 2.11–1.80 (m, 4H), 1.79–1.68 (m, 1H), 1.67–1.51 (m, 2H), 1.47–1.40 (m, 3 H), 1.37–1.17 (m, 5H), 1.01–0.92 (m, 3H), 0.89–0.80 (m, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 169.6, 169.5, 169.3, 156.6, 145.1, 144.5, 140.2, 137.9, 135.7, 135.6, 135.5, 133.6, 133.4, 131.3, 129.4, 129.3, 129.3, 129.1, 129.1, 129.0, 128.9, 128.6, 128.6, 128.5, 128.4, 128.1, 127.7, 127.7, 127.4, 127.4, 127.3, 127.2, 104.3, 103.4, 88.0, 80.8, 69.1, 69.0, 67.7, 67.5, 56.3, 56.0, 54.1, 53.3, 52.3, 44.0, 37.5, 36.3, 34.5, 34.1, 30.7, 29.7, 26.0, 24.7, 24.6, 23.2, 22.7, 20.3, 14.1, 12.9; ESI-LCMS: m/z 930.1 (M + Na)⁺; HRMS (ESI): m/z calcd for C₅₀H₅₇O₁₁N₃NaS [M + Na]⁺ 930.3606; found: 930.3594.

Benzyl ((2S)-1-(((2S)-4-Methyl-1-oxo-1-(((E)-1-phenyl-3-(phenylsulfonyl)allyl)amino)pentan-2-yl)amino)-1-oxo-3-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propan-2-yl)carbamate (34)

Colorless solid (yield: 82%); R_f = 0.46 (EtOAc–petroleum ether, 1:1); ¹H NMR (400 MHz, CDCl₃): δ 8.03–7.82 (m, 2H), 7.73–7.47 (m, 3H), 7.42–7.32 (m, 6H), 7.31–7.22 (m, 3H), 7.14 (td, J = 15.1, 4.0 Hz, 1H), 6.64–6.41 (m, 2H), 5.91–5.77 (m, 1H), 5.72 (brs, 1H), 5.47–5.37 (m, 1H), 5.25–5.15 (m, 1H), 5.08–4.94 (m, 1H), 4.85–4.71 (m, 1H), 4.58–4.33 (m, 1H), 4.33–4.17 (m, 1H), 4.14–3.94 (m, 1H), 3.93–3.72 (m, 1H), 2.65 (brs, 1H), 2.47–2.30 (m, 1H), 2.11–1.96 (m, 1H), 1.96–1.85 (m, 1H), 1.84–1.67 (m, 5H), 1.66–1.47 (m, 4H), 1.45–1.39 (m, 3H), 1.37–1.19 (m, 2H), 1.09–0.80 (m, 12H); ¹³C NMR (100 MHz, CDCl₃): δ 170.6, 169.9, 156.2, 144.5, 140.1, 137.8, 135.9, 133.6, 133.5, 131.4, 129.4, 129.3, 129.2, 129.1, 128.6, 128.5, 128.4, 128.1, 127.7, 127.4, 127.3, 126.2, 104.3, 103.5, 88.0, 88.0, 80.8, 69.1, 67.7, 67.6, 55.7, 53.5, 53.3, 52.4, 52.2, 44.1, 40.1, 37.3, 36.3, 34.5, 34.4, 30.7, 26.0, 24.9, 24.6, 24.6, 22.9, 21.8, 20.3, 12.8; ESI-LCMS: m/z 896.1 (M + Na)⁺; HRMS (ESI): m/z for C₄₇H₅₉O₁₁N₃NaS (M + Na)⁺: calcd 896.3763, found 896.3752.

Benzyl (2S,4R)-2-(((2S)-4-Methyl-1-oxo-1-(((E)-1-phenyl-3-(phenylsulfonyl)allyl) amino)pentan-2-yl)carbamoyl)-4-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)pyrrolidine-1-carboxylate (35)

Colorless solid (yield: 80%); R_f = 0.47 (MeOH-DCM, 1:19); ¹³C NMR (100 MHz, CDCl₃): δ 171.6, 171.5, 171.1, 156.9, 156.6, 145.0, 144.7, 140.3, 140.2, 138.1, 138.0, 136.0, 135.7, 134.1, 133.6, 133.4, 131.4, 129.4, 129.2, 129.0, 128.7, 128.4, 128.2, 128.1, 128.0, 127.8, 127.3, 126.3, 104.4, 100.1, 91.3, 88.2, 81.0, 74.2, 74.0, 68.2, 68.0, 60.8, 60.6, 53.5, 53.4, 52.5, 52.2, 51.7, 44.2, 40.4, 39.9, 39.7, 37.6, 37.5, 36.9, 36.8, 36.4, 34.7, 34.5, 31.7, 30.5, 30.2, 29.1, 27.0, 26.2, 26.0, 25.4, 25.1, 25.1, 24.7, 24.6, 23.1, 22.7, 21.6, 21.5, 20.8, 20.4, 14.2, 12.7, 11.5; ESI-LCMS: m/z 922.1 (M + Na)⁺; HRMS (ESI): m/z for C₄₉H₆₁O₁₁N₃NaS (M + Na)⁺: calcd 922.3919, found 922.3907.

Materials and Methods

Expression and Purification of the Recombinant Falcipain-2 Enzyme

Recombinant falcipain-2 was prepared according to the method described by Shenai et al.²⁷ and Kumar et al.²⁸ with slight modifications. Briefly, Escherichia coli M15 containing pQE30-FP-2 plasmids were grown to the mid-log phase and induced with isopropyl-1-thio-β-d-galactopyranoside (IPTG, 0.5 mM) for 5 h at 37 °C. Cells were harvested, washed with ice-cold 100 mM Tris-Cl, 10 mM EDTA, pH 7.4, sonicated (12 cycles of 10 s each with cooling for 10 s between the cycles), and centrifuged at 15,000 rpm for 45 min at 4 °C. The pellet was washed twice with 2.5 M urea, 20 mM Tris-Cl, 2.5% Triton X-100, pH 8.0; centrifuged at 15,000 rpm for 45 min at 4 °C; and solubilized in 6 M guanidine HCl, 20 mM Tris-Cl, 250 mM NaCl, 20 mM imidazole, pH 8.0 (5 mL/g of inclusion body pellet) at rt for 60 min with gentle stirring. The insoluble material was separated by centrifuging at 15,000 rpm for 60 min at 4 °C. For the purification of the recombinant protein, the supernatant was incubated overnight at 4 °C with a nickel nitrilotriacetic acid (Ni-NTA) resin. The resin was loaded on a column and washed with 10 bed volumes each of 6 M guanidine HCl, 20 mM Tris-Cl, 250 mM NaCl, pH 8.0; 8 M urea, 20 mM Tris-Cl, 500 mM NaCl, pH 8.0; and 8 M urea, 20 mM Tris-Cl, 30 mM imidazole, pH 8.0. The bound protein was eluted with 8 M urea, 20 mM Tris-Cl, 1 M imidazole, pH 8.0, and quantified by the Bicinchoninic acid assay. For the refolding, the fractions containing the falcipain-2 protein were pooled in the ice-cold refolding buffer: 100 mM diluted in Tris-Cl, 1 mM EDTA, 20% glycerol, 250 mM l-arginine, 1 mM GSH, 1 mM GSSG, pH 8.0 was added in a 100-fold dilution. The mixture was incubated with moderate stirring at 4 °C for 24 h, and concentrated to 25 mL using a stirred cell with a 10 kDa cut-off membrane (Pellicon XL device, Millipore) at 4 °C. The sample was then filtered using a 0.22 mm syringe filter. The purified and concentrated protein was quantified using bicinchoninic acid assay.

Fluorometric Assay for Falcipain-2 Activity

For screening of falcipain-2 inhibitors, a 96-well plate fluorometric assay was developed following a protocol described by Kumar et al.²⁸ Briefly, the reaction was set up in a 200 mL reaction mixture containing 100 mM NaOAc, 10 mM DTT, 6 mg of the enzyme, and different concentrations of inhibitors, pH 5.5. A concentration of 10 mM of fluorogenic substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin hydrochloride (Z-FR-AMC) was added, and the release of 7-amino-4-methylcoumarin (AMC) was monitored (excitation: 355 nm; emission: 460 nm) over 30 min at rt in PerkinElmer Victor3 multilabel counter. Activities were compared as fluorescence was released over time in the assay without or with different concentrations of each compound tested. The IC₅₀ values were calculated from curve fittings by software Workout V 2.5. K_i values were derived from the Cheng–Prusoff equation relating both parameters when the substrate concentration and K_M are known:

P. falciparum Growth Inhibition Assay

The P. falciparum 3D7 strain was obtained from the Malaria Research and Reference Reagent Resource Center (MR4). The parasites were cultured in RPMI media (Invitrogen) supplemented with 0.5% albumax and 4% hematocrit using a protocol described previously.²⁹ Cultures were synchronized by repeated sorbitol treatment following a protocol described previously.³⁰ Each growth inhibition assay was performed in triplicate, and the experiment was repeated twice. Each well contained 0.5 mL of complete media [RPMI (invitrogen) with 0.5% albumax], 4% hematocrit, and the parasitemia adjusted to ∼1%; the compound was added to the parasite cultures to desired final concentrations (0–100 μM), and the same amount of solvent (DMSO) was added to the control wells. The cultures were allowed to grow further for 48 h. Parasite growth was assessed by DNA fluorescent dye-binding assay using SYBR green (Sigma) following Smilkstein et al.’s procedure.³¹ For the ring survival assay (RSA 0-3h), young ring-stage parasites were exposed to the artemisinin or selected compounds for 6 h and parasite survival is quantified 66 h later as a percentage of the control set as described earlier.

In Vitro Toxicity Assay on Mammalian Cells

The cytotoxicity assay was carried out using the mammalian cells proliferation test. The A549 human cells were seeded in triplicate at 100 μL aliquots (1 × 10⁴ cells/well) with DMEM medium in Nunclon flat-bottom 96-well plates and were allowed to grow for 24 h. Subsequently, the medium was replaced by a test medium containing each inhibitor (0–500 μM) or DMSO as a control. The CHO cells were allowed to grow for a further 24 h, and then 10 μL of the WST-1 reagent was added and plates were incubated for 30 min. The plates were read at 450 nm absorption and 630 nm reference wavelengths using a TECAN GENios Pro microplate reader. The percentage growth was calculated by comparing with the control set. The EC₅₀ values for each compound were calculated from the growth inhibition curve.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03470.

• Copies of ¹H and ¹³C NMR spectra, HRMS, HPLC chromatograms for all new target molecules and intermediates, in vitro falcipain-2 activity graphs, and crystal data of (S)-26 (PDF)

• Detailed crystallographic data of (S)-26 (CIF)

The authors declare no competing financial interest.