Aminoacid-derived mercaptoimidazoles

Abstract

Starting from suitably protected aminoacids, mercaptoimidazoles were synthesized either from the acid or including the amine nitrogen itself. A preliminary optimisation study led to efficient conditions for the obtention of the imidazole ring. These conditions are compatible with the presence of aminoacid or dipeptide scaffolds.

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Introduction

Peptidomimetics¹ have been thoroughly studied for decades as aminoacid or peptide surrogates. Their main requirements in terms of chemical properties consist of a structural mimicry of natural peptides, though avoiding their shortcomings. Thus, the amide bond surrogate should be stable towards enzymatic hydrolysis, display a low toxicity and/or good bioavailability. Most peptidomimetics either feature a replacement of the scissile amide bond itself for instance with azapeptides² or fluoroolefins,³ or incorporate aminoacid side-chains into constrained structures such as azabicycloalkanes of various sizes.⁴ In this work, we focused our attention towards the incorporation of the N-terminus amine or C-terminus acid of aminoacids into a rigid, aromatic heterocyclic scaffold (Fig. 1). In view to explore new series of potential zinc exopeptidases inhibitors, the 4-mercaptoimidazole scaffold was chosen as a binding motif for the metal.

Fig. 1 N - or C-terminus aminoacid-derived mercaptoimidazoles.

In the past thirty years,⁵thiol inhibitors have been widely developed. The imidazole ring which is found in histidine often accounts for the chelation of the zinc cation in the binding site of the enzyme.⁶ In addition, mercaptoimidazoles are appealing targets for their antioxidant properties. This moiety is present in natural compounds such as ergothioneine or ovothiol.⁷ In this paper, we wish to report a validation of a general method for the conversion of aminoacids into their mercaptoimidazole analogs.

Results and discussion

As far as the synthesis of 4-mercaptoimidazoles is concerned, few procedures exist. Condensation can be achieved either from an alkylsulfanyl enamine using propane phosphonic anhydride⁸ or from N-acyl, N-alkylaminothioacetamide, readily available from aminoacetonitrile precursors (Fig. 2). The cyclization step requires a selective electrophilic activation of the carboxamide, over the thioamide. This can be achieved if the dehydrating reagent has a better affinity for oxygen than nitrogen or sulfur. Previously, Hopkins et al.⁹ used trimethylsilyl triflate. This procedure, which was successfully employed later,¹⁰ and is amenable to selenoimidazoles,¹¹ is very efficient and allows smooth formation of 4-mercaptoimidazoles. Further workup of the reaction requires the use of NaBH₄ to avoid obtaining disulfides.

Fig. 2 Strategies described in the literature.

In the case of aminoacids and peptides, we chose (Fig. 3) the method described in ref. 9, believing that those mild conditions would be suitable with highly-functionalized aminoacid derivatives. Preliminary results, however, called for further optimisation of the cyclisation step and the introduction of a subsequent protection step.

Fig. 3 Retrosynthetic analysis.

Unraveling the reactivity of this system was initially conducted with N-acetyl-N-benzylaminothioacetamide 1 as a model substrate before being applied to aminoacids. As shown in Scheme 1, several parameters were examined to reach an efficient one-pot process. Preparation of the reagent was accomplished by treating N-benzyl aminoacetonitrile with excess thioacetic acid in pyridine¹² followed by acetylation. Dehydration of this compound with TMSOTf/Et₃N following the literature conditions led to a modest yield of 4-mercaptoimidazole.

Scheme 1 Reagents and conditions: i, TMSOTf, Et₃N, CH₂Cl₂, −78 °C to RT, 6.5 h; ii, see conditions in Table 1.

In addition, the imidazole thiol may be isolated as a thiol or disulfide but characterisation of this compound is facilitated by protecting the thiol moiety. On the one hand, pK_a values¹³ indicate that 4-mercaptoimidazoles predominantly exist under a zwitterionic form, since 1,5-dimethyl-4-mercaptoimidazole exhibited a pK_a of 2.3 for the thiol and 10.3 for the imidazole itself. On the other hand, as free thiols, mercaptoimidazoles in particular are very potent anti-oxidants, and they have a high propensity to undergo oxidative dimerisation. Their corresponding disulfides were more commonly isolated. Thus, in situ electrophilic trapping was used as a mean to facilitate the isolation of the products. As listed in Table 1, in situ protection of the thiol was performed with benzylic halides or di-tert-butyl dicarbonate Boc₂O, though the reaction seemed rather slow in this case. We also noticed that treatment with NaBH₄ prior to S-protection was unnecessary if the reaction is conducted under inert atmosphere (entries 3 and 4). The optimal conditions consisted in treating the crude reaction medium with methanol before protection of the thiol moiety with Boc₂O (Table 1, entry 6). We suggest that S-desilylation occurs with methanol, the free thiol being further protected. Having this result in hand, we examined whether the method was applicable to aminoacid substrates. Aminoacids in which the carboxyl group was replaced with the heterocycle were obtained according to Scheme 2.

Table 1 Optimisation study

Entry	Conditions	Yield (%)
1	NaBH4 then PhCH2Cl	R = Bn (2a), 24%
2	NaBH4 then PMBCl	R = PMB (2b), 32%
3	NaBH4 then Boc2O, 3 h	R = Boc (2c), 31%
4	Boc2O, 3 h	2c, 30%
5	Boc2O, DMAP, 12 h	2c, 50%
6	MeOH , then Boc 2 O , DMAP , 16 h	2c , 60%

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Scheme 2 Reagents and conditions: i, CH₃–NH–CH₂CN, dioxane; ii, EDCI, DMAP in dry MeOH, 56% overall; iii, 8 equiv CH₃COSH, pyridine, 65%; iv, TMSOTf, Et₃N, CH₂Cl₂, −78 °C to RT; v, MeOH, 15 min; vi, Boc₂O, Et₃N, DMAP, CH₂Cl₂, 53%; vii, 1.5 M HCl, 50% aq dioxane, 40 °C, 100%

For instance, with glutamic acid, opening of the anhydride with a N-cyanomethyl amine such as sarcosine nitrile afforded the amide 4 with a good regioselectivity in favour of the α-carbonyl.¹⁴ To facilitate the purification step, the crude reaction mixture was treated with EDCI/DMAP in anhydrous methanol, leading to the amidoester 4. Thioacetic acid-mediated addition of H₂S on the nitrile afforded thioamide 5 in a 65% yield. NMR spectra of most cyanomethyl amides and aminothiocarbonyl amides in CDCl₃ revealed two rotamers (see experimental section).

It is worth noticing that the use of TMSOTf under our optimized conditions successfully led to compound 6 (53% yield) from a more sensitive functionalized substrate such as 5. Final deprotection under standard acidic hydrolysis conditions of the thiol, side-chain acid and amine afforded the pure compound 7 as its hydrochloride salt. ¹H NMR in D₂O exhibited a rapid exchange of the proton at C5 of the imidazole ring, due to an easy thiol/thione tautomerism.

As far as the “N-terminus” mimetic was concerned, the cyanomethylamine was prepared directly from the amino group of the aminoacid, as depicted in Scheme 3.

Scheme 3 Reagents and conditions: i, RCOOAc, 50% (9a), 95% (9b); ii, 8 equiv CH₃COSH, pyridine, 78% (10a), 71% (10b); iii, TMSOTf, Et₃N, CH₂Cl₂, −78 °C to RT; iv, MeOH, 15 min; v, Boc₂O, Et₃N, DMAP, CH₂Cl₂, 60% (11a), 66% (11b); vi, 1.5 M HCl, 50% aq dioxane, 40 °C, 100%

Cyanomethylation of aminoacid methyl esters is a known reaction that can be performed smoothly either by direct alkylation with chloroacetonitrile,¹⁵ or via aminomethylation in presence of benzotriazole derivatives.¹⁶ The free cyanomethylamine 8 can be readily purified by flash chromatography on silica gel without noticeable degradation. Subsequent acetylation or formylation was carried out with acetic anhydride or mixed formic acetic anhydride. Compounds 10 were cyclized in good yields with TMSOTf and S-Boc protected as described above, to produce the 4-mercaptoimidazole 12 after final acidic hydrolysis. This new isoleucine derivative 12 bears a potential zinc ligand as a surrogate of the amine moiety.

Further applications of this work were devoted to the evaluation of a dipeptidic substrate. We were interested in knowing whether the formation of the imidazole ring was compatible with the presence of the secondary amide bond. Examination of the reactivity of the dipeptide-derived compound 13 proved that our conditions are amenable to starting materials which contain peptidic linkages. In addition, quenching with methyl iodide instead of Boc₂O at the end of the cyclisation process afforded the S-methyl derivative 15b. This allowed an efficient synthesis of both dipeptides 15b and 16a (Scheme 4).

Scheme 4 Reagents and conditions: i, HCOOH, Ac₂O, 57%; ii, 8 equiv CH₃COSH, pyridine; iii, TMSOTf, Et₃N, CH₂Cl₂, −78 °C to RT; iv, MeOH, 15 min; v, Boc₂O, Et₃N, DMAP, CH₂Cl₂, 42%; vi, CH₃I, Et₃N, 26%; vii, 1.5 M HCl, 50% aq dioxane, 40 °C, 100%.

Recently, 2-substituted 1-benzyl-4-methyl imidazoles were described as acid mimics in aminoacid series. Their preparations used palladium-catalyzed cyclisation of N-allyl oxime aminoacid derivatives¹⁷ or electrocyclization of azomethine ylides.¹⁸ We were interested in examining whether our mercaptoimidazoles could serve as precursors of similar, unsubstituted imidazoles. Initial attempts at desulfurisation of S-Boc derivatives with Raney nickel were unsuccessful. We could, however, overcome this poor reactivity by using the methylsulfanyl imidazole 17. The latter was readily converted to the unsubstituted imidazole by treatment with Raney nickel¹⁹ in refluxing ethanol overnight as shown in Scheme 5.

Scheme 5 Reagents and conditions: i, Raney nickel, EtOH, reflux, 95%

Summary and conclusion

This work allowed an efficient and concise synthesis of optically active aminoacid-derived mercaptoimidazoles and imidazoles. These compounds constitute a new series of heterocyclic mimics of aminoacid and peptides, which opens the way towards new enzyme inhibitors or antioxidants. In addition, as the conditions used for the formation of the heterocycle are compatible with peptides, wide screening of compounds with different aminoacid scaffolds is now envisageable.

Experimental

Unless otherwise stated, reactions were performed under a nitrogen atmosphere using freshly distilled solvents. All reactions were monitored by thin-layer chromatography with Merck silica gel 60 F254 pre-coated aluminum plates (0.25 mm). Flash chromatography was performed with indicated solvents using silica gel (particle size 30–63 μm) purchased from Merck. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance at 300 MHz for ¹H and 75 MHz for ¹³C. Chemical shifts are reported relative to TMS, calibrated with chloroform or deuterium oxide. Coupling constants J are given in Hz.

N-(2-Amino-2-thioxoethyl)-N-benzylacetamide 1

To a solution of N-benzyl-N-cyanomethyl acetamide (2.34 g, 12.4 mmol) in CH₂Cl₂ (23 cm³) was added pyridine (23 cm³), followed by thioacetic acid (7.57 g, 99.6 mmol). Stirring was kept 16 hr at RT. After concentration in vacuo, the residue was purified by flash chromatography through silica gel, using [EtOAc:cyclohexane = 3:2], (Rf 0.28) as an eluent.

Compound 1 (2.14 g, 77%) was obtained as a stench, pale yellow oil. (Found: C, 57.29; H 8.02; N 12.38. C₁₁H₁₄N₂OS requires C, 57.39; H, 8.21; N, 12.41); ¹H NMR ratio of rotamers 4:1, major compound : δ_H(300 MHz; CDCl₃) 2.15 (3H, s), 4.29 (2H, s), 4.64 (2H, s), 7.11–7.33 (5H, m), 7.77 (1H, br s), 8.27 (1H, br s); δ_C(75 MHz; CDCl₃) 21.9, 53.4, 57.4, 127.0, 128.4, 129.5, 135.8, 173.2, 204.2

1-Benzyl-2-methyl-4-(Bocsulfanyl)-1H-imidazole 2c

General procedure for the preparation of protected mercaptoimidazoles, e.g.2c:

A solution of N-(2-amino-2-thioxoethyl)-N-benzylacetamide 1 (489 mg, 2.2 mmol) and Et₃N (1.26 cm³, 9.0 mmol) in anhydrous CH₂Cl₂ (15 cm³) was cooled to −78 °C and trimethylsilyl triflate (1.27 cm³, 6.6 mmol) was added dropwise. After stirring 15 min at −78 °C and 6.5 h at RT, methanol was added and stirring continued during 15 min. The solvents were evaporated and a solution of DMAP (27 mg, 0.22 mmol) and anhydrous Et₃N (2 cm³, 14.4 mmol) in CH₂Cl₂ (15 cm³) was added to the residue. To this solution was added dropwise a solution of Boc₂O (1.9 g, 8.8 mmol) in anhydrous CH₂Cl₂ (5 cm³). After stirring overnight and concentration in vacuo, the residue was purified by flash chromatography through silica gel, eluent [EtOAc:cyclohexane:Et₃N= 6:4:0.01], Rf 0.35, yielding 2c (400 mg, 60%) as a colourless oil.

(Found: C, 62.93; H, 6.61; N, 9.19; S, 10.69. C₁₆H₂₀N₂O₂S requires C, 63.13; H, 6.62; N, 9.20; S, 10.53); δ_H(300 MHz; CDCl₃) 1.40 (9H, s), 2.26 (3H, s), 4.95 (2H, s), 6.99–7.02 (2H, m), 7.22–7.29 (3H, m); δ_C(75 MHz; CDCl₃) 13.7, 28.6, 50.5, 85.6, 125.1, 127.1, 127.2, 128.6, 129.5, 135.8, 146.9, 168.3

1-Benzyl-4-(benzylsulfanyl)-2-methyl-1H-imidazole 2a

Compounds 2a and 2b were obtained by the same procedure than for 2c, except that 1.3 molar equiv of the alkylating agent (PhCH₂Cl or 4-methoxybenzyl chloride) was used instead of Boc₂O.

(Found: C, 73.28, H, 6.14, N, 9.25, S, 10.7. C₁₈H₁₈N₂S requires C, 73.43; H, 6.16; N, 9.51; S, 10.89); δ_H(300 MHz; CDCl₃) 2.22 (3H, s), 3.90 (2H, s), 4.83 (2H, s), 6.53 (1H, s), 6.86–6.88 (2H, m), 7.08–7.25 (8H, m); δ_C(75 MHz; CDCl₃) 13.6, 40.3, 50.1, 124.3, 127.0, 127.1, 128.4, 128.6, 129.36, 129.39, 130.3, 136.2, 138.9, 146.3; [EtOAc:cyclohexane= 3:2, 1% Et₃N]: Rf 0.38; m/z (DCI) 295 (100%), 189 (90%).

1-Benzyl-4-(4-methoxybenzylsulfanyl)-2-methyl-1H-imidazole 2b

δ _H(300 MHz; CDCl₃) 2.32 (3H, s), 3.76 (3H, s), 3.95 (2H, s), 4.94 (2H, s), 6.64 (1H, s), 6.76 (2H, d, J 8.7), 6.96–6.99 (2H, m), 7.11 (2H, d, J 8.7), 7.29–7.34 (3H, m); δ_C(75 MHz; CDCl₃) 13.6, 39.8, 50.1, 55.6, 114.0, 124.2, 127.0, 128.4, 129.4, 130.5, 130.6, 131.0, 136.3, 146.3, 158.8; m/z (DCI) 325 (100%), 121 (30%).

N-Boc-Glutamic anhydride 3

Commercially available N-Boc glutamic acid (8 g, 32 mmol) was stirred for 15 min with 80 cm³acetic anhydride at 55 °C. Toluene (100 cm³) was added and the solution was concentrated to dryness. Traces of acetic acid were removed by drying under vacuum over KOH to afford 7.41 g (100%) of anhydride 3.

δ _H(300 MHz; CDCl₃) 1.45 (9H, s), 1.85–2.0 (1H, m), 2.42–2.46 (1H, dd, J 12.8, 6.2), 3.01 (1H, dd, J 5.5, 2.4), 4.39–4.43 (1H, m), 5.34 (1H, br s); δ_C(75 MHz; CDCl₃) 23.6, 28.3, 29.8, 50.9, 81.1, 155.4, 165.3, 167.2; [α]_D²⁰−21 (c 1 in CH₂Cl₂)

(S)-Methyl-4-(tert-butoxycarbonylamino)-5-(N-cyanomethyl, N-methyl)amino)-5-oxopentanoate 4

A solution of sarcosine nitrile (1.36 g, 19 mmol, obtained from its hydrochloride, by ether extraction from an ice-cold basic solution) in dioxane (5 cm³) was added dropwise at 10 °C to the anhydride 3 (2.98 g, 13 mmol) in 25 cm³dioxane. After 14 h stirring at RT, water (50 cm³) and ethyl acetate (30 cm³) were added. The aqueous layer was treated with ice-cold 1M HCl to reach pH 3. The acid was extracted with ethyl acetate and the organic layer dried over MgSO₄. After solvent evaporation and drying under vacuum, the residue (2.74 g, 9.1 mmol) was dissolved in absolute methanol (70 cm³). To this solution were added EDCI (1.92 g, 10.0 mmol) and DMAP (110 mg, 0.91 mmol). Stirring was kept during 5 h at RT. After solvent evaporation, extractive workup with ethyl acetate/satd NH₄Cl, and drying over MgSO₄, the ester 4 (2.3 g, 56%) was obtained as a pale brown oil.

δ _H(300 MHz; CDCl₃), major rotamer: 1.39 (9H, s), 2.31–2.50 (4H, m), 3.30 (3H, s), 3.71 (3H, s), 4.20–4.41 (3H, m), 5.26–5.34 (1H, m); δ_C(75 MHz; CDCl₃) 27.7, 27.9, 28.3, 35.2, 35.3, 35.7, 52.5, 80.1, 115.4, 155.6, 172.3, 172.8

(S)-Methyl 5-(N-(2-amino-2-thioxoethyl), N-(methyl)amino)-4-(tert-butoxycarbonylamino)-5-oxopentanoate 5

Starting from compound 4 (2.0 g, 6.38 mmol), the thioamide 5 (1.44 g, 65%) was obtained following the procedure described for 1.

(Found: C, 48.32, H, 7.39, N, 12.35, S, 9.16. C₁₄H₂₅N₃O₅S requires C, 48.40; H, 7.25; N, 12.09; S, 9.23); δ_H(300 MHz; CDCl₃): 1.38 (9H, s), 1.57–1.65 (1H, m), 2.26–2.61 (3H, m), 2.98 (3H, s), 3.78 (3H, s), 3.96 (1H, d, J 17.1), 4.38–4.45 (1H, m), 5.03 (1H, d, J 17.0), 5.43 (1H, d, J 8.1), 7.98 (1H, br s), 8.63 (1H, br s); δ_C(75 MHz; CDCl₃) 28.2, 28.3, 29.0, 36.0, 52.1, 52.7, 59.0, 80.4, 156.1, 172.4, 173.1, 203.5; [α]_D²⁰ +20.61 (c 0.97 in CH₂Cl₂); m/z (DCI) 348 (100%).

(S)-Methyl 4-(tert-butoxycarbonylamino)-4-(4-(tert-butoxy carbonylthio)-1-methyl-1H-imidazol-2-yl)butanoate 6

Following the procedure given for 2c and starting from 5 (1.21 g, 3.48 mmol), 6 (790 mg, 53%) was obtained as a pale yellow oil. (Found: C, 53.08; H, 7.39; N, 9.86; S, 7.89. C₁₉H₃₁N₃O₆S requires C, 53.13; H, 7.27; N, 9.78; S, 7.47); δ_H(300 MHz; CDCl₃): 1.27 (9H, s), 1.42 (9H, s), 2.11–2.21 (1H, m), 2.30–2.42 (1H, m), 2.72 (2H, d, J 8.0), 3.55 (3H, s), 3.69 (3H, s), 4.30–4.37 (1H, m), 5.34 (1H, d, J 7.6), 7.06 (1H, s); δ_C(75 MHz; CDCl₃) 28.3, 28.4, 29.8, 30.2, 32.9, 52.6, 53.2, 80.0, 85.4, 124.6, 127.6, 148.6, 155.6, 168.2, 172.7; [α]_D²⁰ +9.41 (c 1.2 in CH₂Cl₂)

(S)-4-Amino-4-(4-mercapto-1-methyl-1H-imidazol-2-yl)butanoic acid hydrochloride 7

Protected compound 6 (95 mg, 0.22 mmol) was dissolved in dioxane under argon. A degased solution of 3M hydrochloric acid was added and the reaction mixture stirred 13 h at 40 °C. Evaporation to dryness followed by freeze-drying from water afforded 56 mg (100%) of the free thiol 7.

δ _H(300 MHz; D₂O) 2.32–2.39 (2H, m), 3.06–3.24 (2H, m), 3.75 (3H, s), 4.11 (1H, t, J 6.0), 7.51 (1H, br s); δ_C(75 MHz; D₂O) 21.4, 26.8, 34.8, 52.8, 123.3, 149.1, 172.1; [α]_D²⁰ +24.3 (c 1 in abs EtOH).

(S)-Methyl 2-(cyanomethylamino)-4-methylpentanoate 8

A 1 M aqueous solution of NaOH (27.6 cm³) was added dropwise to a suspension of valine methyl ester (5 g, 27.6 mmol) and benzotriazol (3.61 g, 30.3 mmol) in methanol (50 cm³). Formaline (2.1 cm3, 5.5 mmol) was added and the reaction medium was stirred 4 hr at RT. Extraction with petroleum ether followed by drying over MgSO₄, filtration and evaporation gave an oil (6.87 g) which was redissolved in DMSO (50 cm³). Sodium cyanide (1.64 g, 33.6 mmol) was added. After stirring 40 hr at RT, ethyl acetate was added, the organic layer was decanted off and washed with Na₂CO₃, brine and dried over MgSO₄. The aqueous residues were treated with bleach before discarding.

(Found: C, 58.36; H 8.81; N 15.94. C₉H₁₆N₂O₂ requires C, 58.67; H, 8.75; N, 15.31); δ_H(300 MHz; CDCl₃) 0.92 (6H, d, J 6.6), 1.41–1.58 (2H, m), 1.67–1.82 (2H, m), 3.39 (1H, dd, J 8.1, 6.2), 3.54 (1H, d, J 17.4), 3.64 (1H, d, J 17.4), 3.75 (3H, s); δ_C(75 MHz; CDCl₃) 21.8, 22.7, 24.6, 35.9, 42.1, 52.0, 58.7, 117.5, 174.8; [α]_D= −32.0 (c 1, CH₂Cl₂).

(S)-Methyl 2-(N-(cyanomethyl)acetamido)-4-methyl pentanoate 9a

Aminonitrile 8 (700 mg, 3.78 mmol) in CH₂Cl₂ (5 cm³) was treated with 640 mg (6.22 mmol) of acetic anhydride. After 2 h stirring at RT, evaporation and flash chromatography through silica gel, eluent [EtOAc:cyclohexane = 6:4], Rf 0.55 gave compound 9a (425 mg, 50%).

(Found: C, 58.43; H 8.29; N 12.55. C₁₁H₁₈N₂O₃ requires C, 58.39; H, 8.02; N, 12.38); δ_H(300 MHz; CDCl₃): two rotamers, ratio 3:2 in this solvent: major: 0.95–1.02 (6H, m), 1.64–1.89 (3H, m), 2.30 (3H, s), 3.72 (3H, s), 4.09 (1H, d, J 15.0), 4.36 (1H, d, J 15.0), 4.45 (1H, dd, J 9.1, 6.0); minor: 0.95–1.02 (6H, m), 1.64–1.89 (3H, m), 2.20 (3H, s), 3.77 (3H, s), 4.28 (2H, s), 5.41 (1H, dd, J 9.9, 5.4); δ_C(75 MHz; CDCl₃) 24.7, 30.4, 38.2, 52.6, 54.0, 115.7, 171.0, 171.1; [α]_D²⁰−28.4 (c 1.09 in CH₂Cl₂).

(S)-Methyl 2-(N-(cyanomethyl)formamido)-4-methyl pentanoate 9b

An equimolar mixture of formic acid (9.7 g, 212 mmol) and acetic anhydride was heated at 65 °C during 10 minutes. After cooling to 0 °C, this solution was added to aminonitrile 8 (1.5 g, 8.14 mmol) in CH₂Cl₂ (5 cm³). After 10 min stirring at RT, treatment with ice-cold water (240 cm³) and extractive work up with CH₂Cl₂ followed by washing with satd NaHCO₃ gave compound 9b (1.6 g, 95%).

(Found: C, 56.51; H, 7.55; N, 13.69. C₁₀H₁₆N₂O₃ requires C, 56.59; H, 7.60; N, 13.20); δ_H(300 MHz; CDCl₃) two rotamers, ratio 4:1 in this solvent: major: 0.95–1.02 (6H, m), 1.59–1.69 (1H, m), 1.76–1.87 (2H, s), 3.78 (3H, s), 4.19–4.39 (3H, m), 8.15 (1H, s); δ_C(75 MHz; CDCl₃) 21.0, 22.7, 29.2, 37.9, 52.8, 58.2, 114.9, 162.8, 170.9; [α]_D²⁰−24.4 (c 0.53 in CH₂Cl₂).

(S)-Methyl 2-(N-(2-amino-2-thioxoethyl)acetamido)-4-methylpentanoate 10a

Following the procedure described for the formation of 1, compound 9a (250 mg, 1.10 mmol) was treated with thioacetic acid, leading to 10a (223 mg, 78%).

(Found: C, 45.66; H, 7.21; N, 8.71; S, 8.20. C₁₁H₂₀N₂O₃S requires C, 50.75; H, 7.74; N, 10.76; S, 12.32); δ_H(300 MHz; CDCl₃): two rotamers, ratio 6.7:1 in this solvent: major: 0.93–0.99 (6H, m), 1.41–1.58 (1H, m), 1.80 (2H, t, J 7.4), 2.08 (3H, s), 3.8 (3H, s), 4.27 (1H, t, J 7.0), 4.36 (1H, d, J 19.4), 4.52 (1H, d, J 19.4), 7.83 (1H, br s), 10.1 (1H, br s); δ_C(75 MHz; CDCl₃) 22.0, 22.1, 23.2, 25.2, 38.0, 53.3, 59.4, 172.2, 175.0, 203.5; [α]_D²⁰−63.8 (c 0.8 in CH₂Cl₂).

(S)-Methyl 2-(N-(2-amino-2-thioxoethyl)formamido)-4-methylpentanoate 10b

Following the procedure described for the formation of 1, compound 9b (1.6 g, 7.54 mmol) was treated with thioacetic acid, leading to 10b (1.31 g, 71%).

(Found: C, 48.68; H, 7.39; N, 11.33; S, 12.67. C₁₁H₁₄N₂OS requires C, 48.76; H, 7.37; N, 11.37; S, 13.02; δ_H(300 MHz; CDCl₃): two rotamers, ratio 1.25:1 in this solvent: major: 0.82–0.92 (6H, m), 1.55–1.65 (3H, m), 3.60 (3H, s), 3.98–4.34 (2H, m), 4.78 (1H, m), 8.14 (1H, s), 9.37 (1H, br s), 9.80 (1H, br s); minor: 0.82–0.92 (6H, m), 1.55–1.65 (3H, m), 3.65 (3H, s), 3.98–4.34 (2H, m), 4.43 (1H, m), 8.24 (1H, s), 8.91 (1H, br s), 9.70 (1H, br s).

(S)-Methyl 2-(4-(tert-butoxycarbonylthio)-2-methyl-1H-imidazol-1-yl)-4-methylpentanoate 11a

Following the procedure described for the formation of 2, compound 10a (160 mg, 0.62 mmol) led to protected mercaptoimidazole 11a (127 mg, 60%).

(Found: C, 55.49; H, 7.78; N, 7.76; S, 9.25. C₁₆H₂₆N₂O₄S requires C, 56.12; H, 7.65; N, 8.18; S, 9.36); δ_H(300 MHz; CDCl₃): 0.92 (6H, m), 1.47 (9H, s), 1.47 (1H, m), 1.84–2.03 (2H, m), 2.39 (3H, s), 3.73 (3H, m), 4.67 (1H, dd, J 9.0, 6.6), 7.23 (1H, s); δ_C(75 MHz; CDCl₃) 13.6, 21.8, 22.7, 24.6, 28.3, 41.2, 53.1, 56.9, 85.3, 123.7, 125.6, 146.3, 167.6, 170.2; [α]_D²⁰−3.27 (c 0.61 in CH₂Cl₂); m/z (DCI) 343 (100%), 299 (10%), 287 (25%).

(S)-Methyl 2-(4-(tert-butoxycarbonylthio)-1H-imidazol-1-yl)-4-methylpentanoate 11b

Following the procedure described for the formation of 2, compound 10b (299 mg, 1.21 mmol) led to protected mercaptoimidazole 11b (262 mg, 66%).

(Found: C, 55.04; H, 7.51; N, 8.57; S, 9.56. C₁₅H₂₄N₂O₄S requires C, 54.86; H, 7.37; N, 8.53; S, 9.76); δ_H(300 MHz; CDCl₃): 0.90–0.94 (6H, m), 1.38–1.47 (1H, m), 1.47 (9H, s), 1.91–1.97 (2H, m), 3.74 (3H, m), 4.74 (1H, dd, J 8.7, 7.2), 7.29 (1H, d, J 1.3), 7.62 (1H, d, J 1.3); δ_C(75 MHz; CDCl₃) 21.6, 22.6, 24.6, 28.2, 41.7, 53.1, 60.5, 85.4, 124.6, 127.6, 137.8, 167.3, 170.1; m/z (DCI) 329 (100%), 273 (90%), 229 (20%).

(S)-2-(4-Mercapto-2-methyl-1H-imidazol-1-yl)-4-methyl pentanoic acid 12a

Hydrolysis was carried out by stirring a solution of compound 11b (86 mg, 0.26 mmol) in 1.5 M HCl in H₂O:dioxane 1:1 at 40 °C overnight. After concentration in vacuo, the residue was taken up in water and freeze-dried. 56 mg (100%) of compound 12b were obtained as a pale yellow foam.

δ _H(300 MHz; DMSO) 0.85 (3H, d, J 6.6), 0.88 (3H, d, J 6.6), 1.17–1.20 (1H, m), 1.81–2.02 (2H, m), 2.40 (3H, s), 4.62 (1H, dd, J 9.8, 6.0), 7.20 (1H, s); δ_C(75 MHz; DMSO) 11.6, 21.3, 22.6, 24.3, 58.0, 126.2, 148.3, 170.0.

(S)-2-(4-Mercapto-1H-imidazol-1-yl)-4-methyl pentanoic acid 12b

Hydrolysis was carried out by stirring a solution of compound 11b (86 mg, 0.26 mmol) in 1.5 M HCl in H₂O:dioxane 1:1 at 40 °C overnight. After concentration in vacuo, the residue was taken up in water and freeze-dried. 56 mg (100%) of compound 12b were obtained as a pale yellow foam.

δ _H(300 MHz; D₂O) 0.76 (3H, d, J 6.6), 0.78 (3H, d, J 6.6), 1.38–1.47 (1H, m), 1.88–2.05 (2H, m), 5.15 (1H, dd, J 10.4, 5.5), 7.51 (1H, d, J 1.3), 8.79 (1H, s); δ_C(75 MHz; D₂O) 20.5, 22.2, 24.7, 39.0, 62.1, 125.5, 127.7, 138.8, 141.4, 147.5, 172.9. [α]_D²⁰ +72.6 (c 1.22 in EtOH).

(S)-Methyl 2-(2-(cyanomethylamino)-3-phenyl propanamido) acetate 13

The dipeptide Phe-Gly-OMe was treated as described for 8.

(Found: C, 60.57; H, 6.17; N, 15.86. C₁₄H₁₇N₃O₃ requires C, 61.08; H, 6.22; N, 15.26); δ_H(300 MHz; DMSO + TFA) 3.11 (2H, d, J 6.6), 3.63 (3H, s), 3.92 (2H, d, J 5.6), 4.17 (2H, s), 7.24–7.31 (5H, m), 7.40 (1H, dd, J 6.4, 3.0), 7.87 (1H, dd, J 6.2, 3.0), 9.11 (1H, t, J 5.6); δ_C(75 MHz; DMSO + TFA) 36.2, 41.0, 52.1, 60.7, 125.6, 128.8, 129.8, 134.3, 167.4, 169.9; [α]_D²⁰−28.9 (c 0.94 in EtOH).

(S)-Methyl 2-(2-(N-(cyanomethyl)formamido)-3-phenyl propanamido)acetate 14

Aminonitrile 13 was treated with HCOOH/Ac₂O, using the same protocol than for 9b.

(Found: C, 59.62; H 5.14; N 13.68. C₁₅H₁₇N₃O₄ requires C, 59.40; H, 5.65; N, 13.85); δ_H(300 MHz; DMSO + TFA) 3.01 (1H, dd, J 14.3, 10.1), 3.21 (1H, dd, J 14.3, 5.5), 3.61 (3H, s), 3.89 (1H, d, J 5.9), 4.19 (1H, d, J 17.5), 4.34 (1H, d, J 17.5), 4.64–4.69 (1H, m), 7.18–7.27 (5H, m), 7.92 (1H, s), 8.79 (1H, t, J 5.7); ¹³C NMR (CDCl₃, 75 MHz) 29.8, 36.2, 41.4, 52.7, 62.6, 115.3, 127.7, 129.1, 129.3, 135.5, 168.6, 168.9, 170.0; [α]_D²⁰−62.6 (c 1 in CH₂Cl₂).

(S)-Methyl 2-(2-(N-(2-amino-2-thioxoethyl)formamido)-3-phenylpropanamido)acetate

Obtained by treatment of the aminonitrile 14 with thioacetic acid (see procedure for 1).

(Found: C, 53.42; H 5.82; N 12.32, S 9.43. C₁₅H₁₉N₃O₄S requires C, 53.40; H, 5.68; N, 12.45; S, 9.50); δ_H(300 MHz; DMSO + TFA) two rotamers, ratio 3:1, major: 3.05–3.26 (2H, m), 3.60 (3H, s), 3.84 (2H, d, J 5.7), 4.00 (1H, d, J 17.5), 4.09 (1H, d, J 17.5), 4.54 (1H, t, J 7.7), 7.19–7.30 (5H, m), 8.07 (1H, s), 8.77 (1H, t, J 5.6), 8.87 (1H, br s), 9.70 (1H, br s); δ_C(75 MHz; CDCl₃) 37.3, 41.9, 52.7, 54.7; 63.6, 127.9, 129.6, 130.1, 137.7, 164.8, 170.8, 171.3, 204.2; [α]_D²⁰−95.1 (c 1 in CH₂Cl₂); m/z (DCI) 338 (100%), 304, 265, 90, 76

(S)-Methyl 2-(2-(4-(tert-butoxycarbonylthio)-1H-imidazol-1-yl)-3-phenylpropanamido)acetate 15a

Thioamide resulting from treatment of 14 (250 mg, 0.74 mmol) was dehydrated to give the S-Boc mercaptoimidazole 15a (131mg, 42%), using the procedure described for 2c.

(Found: C, 57.69; H, 5.76; N, 9.89; S, 7.75. C₂₀H₂₅N₃O₅S requires C, 57.26; H, 6.01; N, 10.02; S, 7.64); δ_H(300 MHz; CDCl₃), two rotamers, ratio 9:1, major: 1.48 (9H,s), 3.17 (1H, dd, J 14.0, 9.4), 3.50 (1H, dd, J 14.0, 5.7), 3.71 (3H, s), 3.99 (2H, t, J 5.5), 4.81 (1H, dd, J 9.2, 5.8), 6.99–7.02 (2H, m), 7.16–7.22 (3H, m), 7.29 (1H, s), 7.32 (1H, d, J 1.1); δ_C(75 MHz; CDCl₃) 28.3, 39.3, 41.5, 52.5, 62.9, 85.8, 124.7, 127.4, 127.7, 128.9, 129.0, 135.8, 138.5, 154.8, 168.4, 169.7; [α]_D²⁰−55.3 (c 0.85 in CH₂Cl₂); m/z (DCI) 420, 376, 320, 222, 79

(S)-Methyl 2-(2-(4-(methylthio)-1H-imidazol-1-yl)-3-phenyl propanamido)acetate 15b

The procedure for the formation of 2c was used, except that methyl iodide (2 molar equiv) was used instead of Boc₂O.

δ _H(300 MHz; CDCl₃): 2.35 (3H, s), 3.17 (1H, dd, J 14.1, 9.6), 3.51 (1H, dd, J 14.1, 5.7), 3.70 (3H, s), 4.00 (2H, d, J 5.4), 4.90 (1H, dd, J 9.4, 5.5), 6.97–7.00 (2H, m), 7.06 (1H, s), 7.19–7.21 (3H, m), 7.26 (1H, s), 7.88 (1H, t, J 5.4); δ_C(75 MHz; CDCl₃) 18.1, 39.1, 41.3, 52.4, 62.5, 118.3, 127.3, 128.7, 128.8, 135.9, 136.1, 137.6, 168.9, 169.9; m/z (ESI) 334; [α]_D²⁰−54.5 (c 0.22 in CH₂Cl₂)

(S)-2-(2-(4-Mercapto-1H-imidazol-1-yl)-3-phenyl propanamido)acetic acid 16a

The procedure used for compound 7 was applied.

δ _H(300 MHz; D₂O) 3.32 (1H, dd, J 13.9, 10.0), 3.54 (1H, dd, J 13.9, 5.9), 3.93 (1H, d, J 17.9), 4.02 (1H, d, J 17.9), 5.38 (1H, dd, J 9.0, 6.0), 7.14–7.17 (2H, m), 7.29–7.31 (3H, m), 7.44 (1H, s), 8.65 (1H, s); δ_C(75 MHz; CDCl₃) 38.4, 41.5, 63.7, 127.9, 128.0, 129.0, 129.2, 129.3, 134.9, 138.5, 169.6, 173.0; [α]_D²⁰−96.9 (c 0.8 in CH₂Cl₂); m/z (ESI) 304

(S)-Methyl 4-(tert-butoxycarbonylamino)-4-(1-methyl-4-(methylthio)-1H-imidazol-2-yl)butanoate 17

Following the procedure described for the formation of 15b, thioamide 5 (444 mg, 1.28 mmol) led to protected mercaptoimidazole 17 (198 mg, 45%).

δ _H(300 MHz; CDCl₃): 1.43 (9H, s), 2.16–2.38 (2H, m), 2.39 (3H, s), 2.71 (2H, t, J 7.4), 3.51 (3H, s), 3.72 (3H, s), 4.35 (1H, m), 5.77 (1H, br d, J 7.0), 6.77 (1H, s); δ_C(75 MHz; CDCl₃) 19.0, 23.3, 28.7, 29.9, 32.9, 52.7, 53.9, 80.1, 121.9, 133.2, 148.3, 156.0, 173.1

(S)-Methyl 4-(tert-butoxycarbonylamino)-4-(1-methyl-1H-imidazol-2-yl)butanoate 18

Refluxing sulfide 17 (99 mg, 0.29 mmol) in ethanol (15 cm³), in the presence of 50 mg of Raney nickel (50% in water) during 14 h, followed by filtration through celite, gave 82 mg (95%) of compound 18.

δ _H(300 MHz; CDCl₃): 1.43 (9H, s), 2.11–2.37 (2H, m), 2.72 (2H, t, J 7.7), 3.54 (3H, s), 3.70 (3H, s), 4.35 (1H, m), 5.60 (1H, br d, J 7.7), 6.77 (1H, s), 6.89 (1H, s); δ_C(75 MHz; CDCl₃) 22.8, 28.4, 30.1, 32.6, 52.5, 53.3, 80.2, 120.8, 127.1, 147.0, 155.7, 172.9

Acknowledgements

We are grateful to the FONGECIF, CNAM and the Région Haute Normandie for generous financial support to Alain Crépin and Sylvain Petit, respectively. The Centre Universitaire Normand de Chimie Organique (CRUNCh) is also gratefully acknowledged. Dr Christian A. G. N. Montalbetti (Evotec, U.K.), is gratefully acknowledged for helpful discussions.

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Footnote

† Electronic supplementary information (ESI) available: ¹H and ¹³C NMR spectra. See DOI: 10.1039/b810678a

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