Introduction

In 1993, viridiofungins A, B and C were isolated from a strain of Trichoderma viridae Pers. (Fungi, Hyphomycetes) and are considered a novel family of squalene synthases, which are of interest as antifungal agents as well as cholesterol-lowering therapeutics (Figure 1).1 They are categorized as alkyl citrate natural products, a family of naturally occurring squalene synthase inhibitors including zaragozic acids,2, 3 and possess a different aromatic α-amino acid unit: Tyr, Phe and Trp for viridiofungin A, B and C, respectively. Six additional derivatives, A1–4, B2 and Z2, were later disclosed as analogs that differ in the minor structure of the alkyl chain (oxidation level at C-13, or chain length) or lack the α-amino acid unit.4 The characteristic feature of viridiofungins that makes them unique in the alkyl citrate family is their inhibitory activity toward serine palmitoyl transferase, which is involved in the first step of sphingolipid biosynthesis at nanomolar concentrations.4, 5, 6 As sphingolipids are the major components of the lipid raft, where non-structural viral proteins of hepatitis C virus accumulate, the inhibitory effect of serine palmitoyl transferase offers a therapeutic option for the treatment of hepatitis C virus infection.7 Given these attractive biological activities, several synthetic routes of viridiofungins have been documented to date.6, 8 The introduction of an alkyl chain to a methylene group of the citric acid renders it chiral, significantly increasing the structural and synthetic complexity. The stereoselective construction of the densely functionalized polar citric acid unit of the molecule is perplexing, and the synthetic strategy for this part dictates the overall synthetic efficiency. In this review article, synthetic methodologies of viridiofungin derivatives are highlighted, with particular emphasis on the efficiency of the requisite stereochemical control.

Figure 1
figure 1

Structure of viridiofungin derivatives and citric acid.

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Synthesis via catalytic asymmetric epoxidation

In 1998, Hatakeyama and colleagues9 reported the first total synthesis of viridiofungin. This work was particularly noteworthy because of the manifestation of four stereoisomers of viridiofungin A trimethyl ester, allowing for determination of the absolute configuration of natural viridiofungin A isolated in 1993 (Figure 2).1 The key methodology used to introduce the requisite stereogenic center was Katsuki–Sharpless catalytic asymmetric epoxidation of trisubstituted allylic alcohol 1, delivering enantioenriched epoxide 2 in 92% yield and 88% ee.10, 11, 12 The synthesis commenced with O-protection and 1-C homologation with paraformaldehyde of 3-butyn-1-ol 3 to give 4, whose propargylic alcohol unit was reduced by Red-Al, and subsequent treatment of the intermediate with iodine produced the desired Z-configured iodoalkene.13 After protection of the primary alcohol as a tetrahydropyranyl ether, the addition of lithiated 5 to CO2, followed by treatment with methyl iodide gave methyl ester 6. The ester was reduced by diisobutylaluminum hydride to a primary alcohol that was protected as a p-methoxybenzyl (PMB) ether, and a tetrahydropyranyl-protecting group on the opposite side was removed by methanolysis using pyridinium p-toluene sulfonate for the subsequent key reaction, catalytic asymmetric epoxidation. The trisubstituted epoxide 2 was regioselectively opened by vinylmagnesium bromide 7 and CuI with participation of the neighboring-free hydroxyl group, thereby affording diol 8.14, 15 The free alcohols were masked as acetonide with 2,2-dimethoxypropane under acidic conditions, and subsequent sequential oxidation with OsO4 and NaIO4 converted the terminal olefin to an aldehyde as a reactive handle for introducing an alkyl chain. Wittig reaction of aldehyde 9 and phosphonium salt 10 mediated by n-butyllithium connected them with the Z-configured double bond, and removal of two PMB groups followed by protection with tert-butyldimethylsilyl (TBDPS) chloride provided mono-TBDPS ether 11. The free primary alcohol moiety of 11 was oxidized to a carboxylic acid by sequential oxidation reactions, and subsequent treatment with diazomethane afforded methyl ester 12. Deprotection/oxidation of TBDPS ether was achieved in the four steps, including hydrolytic cleavage of the acetonide, fluoride-mediated deprotection of the TBDPS ether, Jones oxidation of the primary alcohol and methyl ester formation with diazomethane.16 While the Jones oxidation preferentially proceeded at the less hindered primary alcohol to give 13 after lactonization, an undesired oxidation was also occurring, as evidenced by the formation of 14. Photoirradiation of 13 in the presence of PhSSPh allowed for olefin isomerization to give the E/Z=82/18 mixture, which was purified to give geometrically pure (E)-15 by preparative TLC. Chemoselective hydrolysis of the lactone moiety of 15 followed by diazomethane/Jones oxidation gave carboxylic acid 16.16 To determine the absolute configuration of viridiofungin, 16 was subjected to amidation with both H-Tyr-OMe and H-D-Tyr-OMe using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) hydrochloride,17 furnishing trimethyl esters of viridiofungin A and epi-1-viridiofungin A. This synthetic scheme is well designed and a slight modification of the reaction sequence enabled access to epi-9 from intermediate 8 via 17. Identical reaction sequences were applied to epi-9 to produce two other stereoisomeric trimethyl esters, epi-2-viridiofungin A and epi-3-viridiofungin A. Comparison of the optical rotations and 1H and 13C NMR spectra of these four stereoisomers with the trimethyl ester derived from natural viridiofungin A ([α]D25 –23.3°) indicated that viridiofungin A has a (3S,4S,2′S) configuration (synthetic: [α]D26 –19.1°).

Figure 2
figure 2

First total synthesis of viridiofungin A trimethyl ester and determination of its absolute configuration. DHP, 3,4-dihydro-2H-pyran; DIBAL, diisobutylaluminum hydride; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt, 1-hydroxybenzotriazole; MS4A, molecular sieves 4 Å; NMO, N-methylmorpholine N-oxide; NMM, N-methylmorpholine; PMB, p-methoxybenzyl; PPTS, pyridinium p-toluenesulfonate; TBDPS, tert-butyldiphenylsilyl.

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The labile nature of viridiofungin A under basic conditions hampered the hydrolysis of trimethyl ester to furnish viridiofungin A. This fact prompted the team led by Hatakeyama to devise a new synthetic route with various improvements (Figure 3).18 The revised synthesis began with the identical starting material, 3-butyn-1-ol 3, which gave structurally different trisubstituted allylic alcohol 18 for a Katsuki–Sharpless catalytic asymmetric epoxidation after six transformation steps.10, 11, 12 Once the enantioenriched (87% ee) epoxide 19 was obtained, a subsequent three-step sequence converted the primary alcohol unit to a tert-butyl ester-to allow for global deprotection under acidic conditions at the final stage. The tetrahydropyranyl ether was hydrolyzed to the hydroxyl group for a directed epoxide opening that proceeded similarly to the previous synthesis.14, 15 The thus-formed diol was protected as a cyclic carbonate by triphosgene, and 2,3-dichloro-5,6-dicyano-p-benzoquinone removed the PMB group to give primary alcohol 21. Jones oxidation16 followed by treatment with O-tert-butyl-N,N′-diisopropylisourea19 gave precursor 22 for olefin cross metathesis to install an alkyl chain with terminal olefin 23.20 Grubbs second-generation catalyst 2421 linked them with E/Z=88/12 in a reasonable yield (65%), and subsequent hydrolysis of the cyclic carbonate was fulfilled in a sequential manner; opening with allylic alcohol and palladium-catalysed reductive deallylation.22 The exposed primary alcohol unit of 25 was oxidized to a carboxylic acid and amide bond formation was achieved with H-Tyr-OtBu using EDCI hydrochloride,17 N-methylmorpholine, and 1-hydroxybenzotriazole. Global deprotection of the three tert-butyl esters readily proceeded with formic acid to furnish the tricarboxylic acid, which constitutes the first demonstration of the total synthesis of viridiofungin A.

Figure 3
figure 3

Revisited synthesis of viridiofungin A by Hatakeyama et al. DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt, 1-hydroxybenzotriazole; NMM, N-methylmorpholine; PPTS, pyridinium p-toluenesulfonate.

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Synthesis via catalytic asymmetric hydrogenation

Ghosh et al. reported a different approach utilizing a Mukaiyama aldol reaction23, 24, 25 to construct the densely functionalized polar part of viridiofungin A.26 Dihydrofuran 28 was used as an optically active nucleophile prepared from γ-ketoester 26, involving Corey–Bakshi–Shibata reduction27 using oxazaborolidine catalyst 27 (Figure 4).28, 29, 30 The aldol reaction of 28 with engineered α-ketoester 29 mediated by TiCl4 presumably proceeded through oxonium cation intermediate 30 with minimum steric constraint, which was in situ reduced by Et3SiH to give 31 with a fully saturated tetrahydrofuran ring. An efficient chirality transfer from 28 was manifested, as evidenced by the high diastereomeric ratio of 31 (>20/1). A hydroboration/oxidation sequence converted the triple bond of 31 to the corresponding carboxylic acid, the ethyl ester moiety was hydrolyzed by LiOH, and treatment of the resulting dicarboxylic acid with O-tert-butyl-N,N′-diisopropylisourea19 delivered di-tert-butyl ester 32. Exposure of 32 to Cu(OTf)2 (20 mol%) in the presence of Ac2O in refluxing toluene led to tetrahydrofuran ring opening and O-acetylation, together with unexpected deprotection/cyclization of the di-tert-butyl esters to give anhydride 33. After regenerating di-tert-butyl ester 34 by sequential treatment with aqueous acetic acid and O-tert-butyl-N,N′-diisopropylisourea,19 two acetyl groups were removed by (allyl)MgBr and the resulting alcohols were protected as silyl ethers. The thus-obtained 35 was oxidized by ozone to generate the aldehyde requisite for subsequent Julia–Kocieński olefination with sulfonyltetrazole 36,31 which was originally used in Hiersemann’s synthesis in 2004 (vide infra).32 The desired coupled product 37 was obtained exclusively in the E-configuration, followed by deprotection of the silyl ethers and Jones oxidation to afford 38.16 Amide formation with H-Tyr-OtBu and deprotection of three tert-butyl esters according to Hatakeyama’s second-generation procedure18 delivered viridiofungin A.

Figure 4
figure 4

Total synthesis of viridiofungin A by Ghosh et al. EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt, 1-hydroxybenzotriazole; KHMDS, potassium hexamethyldisilazide; NMM, N-methylmorpholine; TBS, tert-butyldimethylsilyl; TES, triethylsilyl.

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Synthesis via catalytic asymmetric aldol reaction

In contrast to the two above-discussed synthetic examples, where catalytic asymmetric carbon–oxygen bond or carbon–hydrogen bond formations were exploited, the total synthesis reported by Shibasaki et al. utilizes a catalytic asymmetric carbon–carbon bond-forming reaction to enable the construction of the requisite carbon framework and introduction of the chirality in a single step (Figure 5).33 To construct the tetrasubstituted stereogenic center of viridiofungin A, α-sulfanyl lactone 40 was designed as a soft Lewis basic pronucleophile, and subjected to a direct catalytic asymmetric aldol reaction with PMB-protected glycoaldehyde 39 promoted by a soft Lewis acid (AgPF6/ligand 41)/Brønsted base (1,8-diazabicyclo[4.3.0]undec-7-ene) cooperative catalyst. The reaction efficiently proceeded with 3 mol% of catalyst loading and the desired syn-configured aldol product 42 was obtained over 20 g with high enantioselectivity (98% ee). The lactone was reduced with LiAlH4 and the resulting two primary hydroxyl groups were protected by TBDPS groups to give 43. The carbon–sulfur bond of 43 was replaced with a carbon–oxygen bond with inversion of the configuration by S-methylation with MeOTf, followed by intramolecular epoxide formation with the neighboring secondary hydroxyl group. After removing the PMB group with 2,3-dichloro-5,6-dicyano-p-benzoquinone, hydroxyl group-directed epoxide opening using vinyl cuprate derived from 45 gave a diol,14, 15 which was protected as an acetonide, and deprotection of TBDPS groups delivered bis primary alcohol 46. Sequential treatment of 46 with oxidation then tert-butyl ester formation using O-tert-butyl-N,N′-diisopropylisourea19 gave rise to the previously known intermediate 25.18 After oxidation of the primary alcohol of 25 by CrO3,16 H-Tyr-OtBu and O-1-(2-butyn)-ylated H-Tyr-OtBu 47 were coupled using (7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate as an amide-coupling reagent,17 in order to access viridiofungin A and NA808, respectively. NA808 is a druggable analog of viridiofungin A that is anticipated to block the proliferation of hepatitis C virus by inhibiting sphingolipid biosynthesis.34, 35

Figure 5
figure 5

Total synthesis of viridiofungin A and NA808 by Shibasaki et al. DBU, 1,8-diazabicyclo[4.3.0]undec-7-ene; DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone; DMSO, dimethylsulfoxide; HATU, (7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; PPTS, pyridinium p-toluenesulfonate; TBAF, tetrabutylammonium fluoride; TFA, trifluoroacetic acid.

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Synthesis via chiral auxiliary

The use of a chiral auxiliary for the synthesis of optically active compounds can be categorized as a classical synthetic methodology. Compared with asymmetric catalysis, the mandatory use of a stoichiometric chiral source can be a drawback, but reliable stereoselectivity as well as robustness and a broad scope of reactions make auxiliary-based approaches applicable for a myriad of practical syntheses. In particular, when inexpensive materials in the chiral pool are used as auxiliaries, at least the cost for chiral information is minimized despite being a low atom-economic process. Oxazolidinone-based chiral auxiliaries developed by Evans are representatives of such auxiliaries, readily prepared from inexpensive α-amino acids.36 The concise total synthesis of viridiofungin A–C derivatives reported by Barrett et al.37 explicitly demonstrates the usefulness of the chiral auxiliary approach in target-oriented synthesis (Figure 6). The synthesis began with a boron-mediated aldol reaction of oxazolidinone 48,19, 38 prepared from inexpensive L-phenylalanine and ethyl glyoxylate 49, affording adduct 50 with the desired vicinal stereochemistry at newly formed stereogenic centers. The oxazolidinone auxiliary and the ethyl ester were readily hydrolyzed by LiOH to give diacid 51, which was converted to acetal 52 with pivaldehyde and trimethylsilyl trifluoromethanesulfonate via a tris-trimethylsilylated intermediate.39 Introduction of the requisite acetate unit with the correct stereochemistry was achieved by following the strategy of self-reproduction of stereochemistry developed by Seebach;40 deprotonation of 52 with lithium hexamethyldisilazide and subsequent addition to tert-butyl bromoacetate delivered 53, a precursor of the cross metathesis reaction to install the long alkyl chain unit. The use of terminal alkene 23 with Grubbs second-generation catalyst 24 (Figure 3)20, 21 proved unsuccessful in the specific case of 53, and only homodimerization of 24–54 occurred. Careful experimentation identified that cross metathesis with dimer 54 using 20 mol% of Grela catalyst 55 gave the desired product 56 in moderate yield.41 With the readily cleavable five-membered acetal, viridiofungin A–C derivatives were synthesized from 56 with the corresponding amino acid tert-butyl esters using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate as an amide-coupling reagent.17

Figure 6
figure 6

Total synthesis of derivatives of viridiofungin A–C by Barrett et al. HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; KHMDS, potassium hexamethyldisilazide; TMS, trimethylsilyl.

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Synthesis via hplc separation of stereoisomers

Hiersemann et al.32, 42 reported a different approach to viridiofungin A triesters using [2,3]-Wittig rearrangements to construct the tertiary alcohol at the polar citric acid unit (Figure 7). Anticipating the coupling with optically pure amino acid at the last stage, the synthesis was promoted by racemic intermediates and isolation of the stereoisomers of viridiofungin A triesters relied on reversed-phase HPLC separation at the final stage. Although the overall efficiency of accessing molecules with the correct stereochemistry was diminished, a highly flexible synthetic route could be applied, offering the opportunity to provide unnatural stereoisomers for prospective biological studies. Allyl vinyl ether 58 as a precursor of the [2,3]-Wittig rearrangement was prepared from 57 in a five-step sequence.43 Enolate formation by lithium N,N-diisopropylamide-induced sigmatropic rearrangements as delineated in 59 to give tertiary alcohol (±)-60 in a racemic form, possessing the requisite functionalities for viridiofungins in the correct relative stereochemistry. The enol ether moiety was hydrolyzed to aldehyde, and subjected to oxidation/esterification using methanol, dicyclohexylcarbodiimide, and 4-(dimethylamino)pyridine to afford the methyl ester. For installation of the alkyl chain by the Julia–Kocieński reaction,31 the tertiary alcohol was protected due to the TBS ether and terminal olefin oxidizing to aldehyde by ozone. The thus-formed (±)-61 was coupled with sulfonyltetrazole 36 using potassium hexamethyldisilazide, affording (±)-(E)-62 as a single geometric isomer. Hydrogenolysis of the benzyl ether, followed by sequential oxidation reactions using tetrapropylammonium perruthenate44/N-methylmorpholine oxide and NaClO2/NaH2PO4, and subsequent removal of the TBS group furnished carboxylic acid (±)-63 for final coupling with H-Tyr-OMe. Amide formation was performed with 1H-benzotriazol-1-yloxy-tri(pylimidino)phosphonium hexafluorophosphate (PyBOP) and N-methylmorpholine to deliver a mixture of stereoisomers of viridiofungin A triester, which were isolated by reversed-phase HPLC.17 With aldehyde (±)-61, a Julia–Kocieński reaction using sulfonyltetrazoles without ketone functionality (64) or with additional 2-methylene groups (65) furnished intermediates corresponding to triesters of viridiofungin A2 and A4 that were accessed by applying similar reaction sequences.

Figure 7
figure 7

Total synthesis of derivatives of viridiofungin A triesters by Hiersemann et al. DCC, dicyclohexylcarbodiimide; DMAP, N,N-dimethylaminopyridine; HPLC, high-performance liquid chromatography; KHMDS, potassium hexamethyldisilazide; LDA, lithium N,N-diisopropylamide; NMM, N-methylmorpholine; PyBOP, 1H-benzotriazol-1-yloxy-tri(pylimidino)phosphonium hexafluorophosphate; TBS, tert-butyldimethylsilyl; TPAP, tetrapropylammonium perruthenate.

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Conclusion

Stereoselective syntheses of viridiofungins developed by five independent research groups were reviewed. As the densely functionalized polar citric acid unit is a characteristic feature of these alkyl citrate natural products, each group used a different approach to construct the carbon framework and stereogenic centers of the unit. The first three examples exploited asymmetric catalysis amenable to obtaining enantioenriched intermediates with minimal use of generally expensive and less available chiral sources. The latter two groups used a stoichiometric amount of the chiral source or HPLC separation. Although synthesis via asymmetric catalysis appears ideal in one respect, the reaction conditions pose severe limitations on the substrate scope and the operation requires strict control. The advantage of the chiral auxiliary approach is the broad scope of reaction types and substrate sets, allowing for selection of the most suitable starting material to streamline the following synthetic pathways. Indeed, the auxiliary approach presented above accomplished the synthesis in an appreciably small number of steps. These synthetic routes should be evaluated by assessing the overall efficiency and availability of reagents and catalysts, ease of the purification processes, as well as the number of steps. The attractive biological activity of vidridiofungins and their derivatives makes these alkyl citrate natural products desirable targets for the development of artful synthetic strategies, with the potential for higher efficiency and elegance.