Squaric Acid-Based Peptidic Inhibitors of Matrix Metalloprotease-1 (MMP-1)

Abstract

A series of squaric acid-peptide conjugates were synthesized and evaluated as inhibitors of MMP-1. The cyclobut-3-enedione core was substituted at the 3-position with several functional groups, such as -N(alkyl)OH, -NHOH and –OH, that are designed to bind to the zinc atom in the active site of the metalloprotease. The 4-position of the cyclobut-3-enedione was derivatized with mono- or dipeptides that are designed to bind in the S1′ and S2′ subsites of the enzyme, and position the metal chelating group appropriately in the active site for binding to zinc. Positional scanning revealed that -N(Me)OH provided the highest level of inhibition among the chelating groups that were tested, and Leu-Tle-NHMe was the preferred amino acid sequence. A combination of these groups yielded an inhibitor with an IC₅₀ value of 95 μM. For one inhibitor, conversion of one of the carbonyl groups on the cyclobut-3-enedione core to a thiocarbonyl group resulted in a 18-fold increase in potency, and yielded a compound with an IC₅₀ value of 15 μM.

Introduction

Matrix Metalloproteases (MMPs)

Matrix metalloproteases are a family of structurally related endopeptidases that degrade and remodel components of the extracellular matrix (ECM).¹ These enzymes regulate structure and sustain a balanced composition of the ECM, two processes that are important for maintaining normal physiology in a number of tissues. For example, MMPs play a crucial role in embryonic development, healing and reproduction.

The activity of MMPs is normally regulated at three levels: 1) gene transcription, 2) activation of MMP propeptides, and 3) inhibition of MMPs by tissue inhibitors of metalloproteases (TIMPs).^2,3 Overexpression of MMPs and deregulation of their activity is associated with a variety pathological conditions including tumor growth and metastasis,⁴ angiogenesis,⁵ destruction of joints that causes osteoarthritis⁶ and rheumatoid arthritis,⁷ periodontal disease⁸ and multiple sclerosis.⁹ Therefore, there is significant interest in developing MMP inhibitors for therapeutic applications.

Biological Applications of Squaric Acids

Squaric acid is a molecule that has significant aromatic character. In one resonance form it has two π electrons and a negative charge on each of the carbonyl oxygen atoms (Figure 1). The conjugate base of squaric acid can serve as an electrostatic mimic of negatively charged groups that are common in biology including carboxylates and phosphate mono- and diesters. As a result, derivatives of squaric acid have been used as a replacement for these groups in a number of medicinal applications.

FIGURE 1.

Resonance structures of squaric acid.

Our research group has used squaric acid derivatives to mimic the phosphate group in phosphotyrosine residues. We prepared a number of 3-hydroxy-4-aryl-cyclobut-3-enediones as non-hydrolyzable isosteres of aryl phosphate esters and found that these compounds are effective inhibitors of protein tyrosine phosphatases.¹⁰ Kim and coworkers have used phosphonocyclobutenedione as a mimic of pyrophosphate and found that it is a selective inhibitor of DNA polymerases from several viruses.¹¹ Sekine has used a diamide of squaric acid to replace a phosphate diester linkage in an oligodeoxynucleotide.¹²

Derivatives of squaric acid have also been used to mimic carboxylates by a number of investigators. Shinada replaced the γ-carboxylic acid of a glutamate residue within a polyamine toxin with a squaric acid derivative. The resulting compound was a selective agonist of ionotropic glutamate receptors.¹³ Sun and coworkers used a derivative of squaric acid in their investigations of an NMDA antagonist that regulates the activation of glutamate receptors.¹⁴ In this example, squaric acid mimics the natural glutamate agonist of the neuronal receptors. In its application as a guanidinium isotere, Butera used diaminocyclobutenedione as a replacement for the N-cyanoguanidine group in a bladder-selective potassium channel opener that is used as a treatment for urge urinary incontinence.¹⁵

Hydroxamic acids are potent inhibitors of zinc metalloproteases. X-ray crystal structures show that hydroxamic acids chelate to the active site zinc atom as shown in structure I (Figure 2).¹⁶ In addition, Bruckner and coworkers have demonstrated that vinylogous hydroxamic acids that are derived from squaric acid are good metal chelators (see structure II in Figure 2).¹⁷ These two observations prompted us to investigate the potential of vinylogous hydroxamic acids that are based upon squaric acid as inhibitors of MMPs. We have derivatized the vinylogous hydroxamic acids with peptides in order to target them to the active site of the proteases.

FIGURE 2.

Known binding mode of hydroxamic acid I versus proposed binding mode by squaric acid derivative II.

Results and Discussion

Synthesis of Compounds 4a–4f

We began our studies by making a series of simple derivatives to determine what functional group is preferred at the R¹ position of the inhibitors (Scheme 1). Squaric acid 1 was converted to its dimethyl ester 2 by treating it with trimethyl orthoformate. Reaction of compound 2 with a series of hydroxylamines gave vinylogous hydroxamic acids 3a–e. Substitution of the remaining methyl ester with several primary amines gave inhibitors 4a–f.

SCHEME 1^a.

^aReagents: (a) CH(OCH₃)₃, MeOH, Δ; (b) HONHR¹·HCl, KOH, MeOH; (c) H₂NR², MeOH.

These compounds were screened for activity against MMP-1. Assays were performed in a buffer of 200 mM NaCl, 50 mM Tris, 5 mM CaCl₂, 20 μM ZnSO₄, 0.05% Brij 35 at pH 7.6 using the fluorogenic substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Nma)-NH₂. In this substrate, N-methylanthranilic acid (Nma) is a fluorophore and the dinitrophenyl (Dnp) group is a quencher.^18,19 The progress of the reactions was monitored by fluorescence spectroscopy with excitation at 340 nm and emission at 460 nm. For inhibitors 4a–b, R¹ = H while the R² position was varied between a branched and a straight-chain alkyl group. Between these two compounds, inhibitor 4b, which incorporates a simple n-hexyl chain, had the better activity. We next screened Me, cyclohexyl and benzyl groups at the R¹ position, and found that large groups are unfavorable at this site. Compounds 4e and 4f, where R¹ is cyclohexyl or benzyl, were poor inhibitors that showed no activity up to 10 mM concentration. Compound 4c, which incorporates a smaller methyl group at the R¹ position, was approximately three times more active than compound 4b, where R¹ = H. Both inhibitors 4c and 4d had IC₅₀ values of 310 μM.

Monopeptide Inhibitors

We next turned our attention to inhibitors that incorporated a single amino acid in order to determine which amino acid side chain is preferred in the S1′ subsite within the context of these inhibitors. As shown in Scheme 2, inhibitors 6a–g were synthesized by reaction of vinylogous hydroxamic acids 3b or 3e with a variety of amino acid methyl esters. We chose amino acids with hydrophobic side chains since data from the literature indicated that such structures are preferred in the S1′ subsite.¹

SCHEME 2^a.

^aReagents: (a) MeOH, KOH. ^bRacemic Nle was used to prepare 5b and 6c.

Comparison of inhibitors 6a and 6b shows that both methyl and isopropyl groups at the R¹ position are small enough to be tolerated by the enzyme (Table 2). In addition, the enzyme appears to be fairly insensitive to the identity of the R³ side chain, since all of the inhibitors in this series gave IC₅₀ values that range from 190 to 380 μM. Among these compounds inhibitor 6a had the best activity with an IC₅₀ value of 190 μM.

TABLE 2.

Inhibition of MMP-1 by Compounds 6a–g

compound	R1	R3 = side chain of amino acid	IC50 (μM)a
6a	Me	Ile	190 ± 30
6b	-CH(CH3)2	Ile	210 ± 10
6c	Me	Nleb	280 ± 20
6d	Me	Leu	320 ± 20
6e	Me	Phe	330 ± 20
6f	Me	Trp	380 ± 50
6g	Me	Met	300 ± 50

^aAll experiments were performed in duplicate.

^bRacemic Nle was used to prepare compound 6c.

Dipeptide Inhibitors

To improve the potency of the inhibitors, we extended their structure by incorporating a second amino acid that is designed to bind in the S2′ subsite. X-ray crystallographic studies have shown that peptide-based hydroxamic acid inhibitors bind in the active site of MMPs by occupying the primed subsites.²⁰ The synthesis of the inhibitors in this series is shown in Scheme 3. For these studies, we used the dibutyl ester of squaric acid 7 as the starting point. We found that this compound is more convenient to work with than the corresponding dimethyl ester, since the dibutyl ester has increased solubility in most common organic solvents.

SCHEME 3^a.

^aReagents: (a) (BuO)₃CH, BuOH, reflux; (b) HONHMe·HCl, KOH, MeOH; (c) NH₂Me, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), DMF; (d) 50% TFA, CH₂Cl₂; (e) NHS, N-Boc-Ile or N-Boc-Leu, EDC, DMF; (f) 50% TFA, CH₂Cl₂; (g) MeOH. Tle = tert-leucine, Chg = cyclohexylglycine, Phg = phenylglycine.

^bSee Table 3 for specific structures.

Squaric acid was treated with tributyl orthoformate to give dibutyl ester 7. Subsequent reaction of 7 with N-methylhydroxylamine yielded compound 8. The dipeptide building blocks were prepared by coupling N-Boc amino acids 9a–g with methylamine to give the corresponding N-methyl amides. These were treated with trifluoroacetic acid to give compounds 10a–g. Coupling of 10a–g with N-Boc-Ile or N-Boc-Leu, followed by Boc deprotection gave dipeptides 11a–h. Finally, reaction of the dipeptides with compound 8 in methanol at room temperature provided the desired inhibitors 12a–h.

Most of the inhibitors in this series incorporate the side chain of Ile at the R³ position because the data presented in Table 2 indicated that Ile is preferred in the S1′ subsite. The side chains at the R⁴ position were chosen based upon literature precedent, which suggest that aromatic and hydrophobic amino acids are preferred at the S2′ subsite.¹ For inhibitors 12a–g, which incorporate an Ile side chain at R³, we did not observe any improvement in activity over the related monopeptide inhibitor 6a. In addition, the activity did not depend strongly on the structure of R⁴, since we observe a difference in IC₅₀ values of only 2.5-fold among these seven compounds.

Compounds 12a and 12h provide an interesting comparison. Both incorporate at bulky t-butyl group at R⁴. Compound 12a incorporates the side chain of Ile at R³, and was the least active among 12a–g. By contrast, 12h has a Leu side chain at R³ and it is 2–5-fold more active than any of the other mono- or dipeptide-based inhibitors. These data suggest that binding interactions in the S1′ and S2′ subsites are dependent on one another. For the monopeptide inhibitors that leave the S2′ subsite empty (Table 2, compare inhibitors 6a and 6d), Ile is preferred over Leu at S1′. This selectivity is reversed in the dipeptide inhibitors. With Tle in the S2′ subsite, Leu is preferred over Ile at S1′ (compare 12a and 12h).

Squaric Acid Monoamides

We also made a cursory examination of a simple monoamide of squaric acid as a potential MMP-1 inhibitor. Scheme 4 shows the synthesis of compound 14, which is the squaric acid analog of the vinylogous hydroxamic acid inhibitor 12h. Reaction of H-Leu-Tle-NHMe (11h) with dimethyl squarate 2 gave compound 13, which was subsequently hydrolyzed under acidic conditions to give compound 14.

SCHEME 4^a.

^aReagents: (a) H-Leu-Tle-NHMe, MeOH, reflux; (b) 0.15 N HCl, MeOH, reflux.

When compound 14 was assayed against MMP-1, we observed no inhibition up to a concentration of 200 μM. In retrospect, this result is not surprising since the bite angle between the two oxygen atoms in squaric acid is known to be too large to form a strong five-membered chelate with Zn⁺² (right side of Figure 3).²¹ By comparison, the vinylogous hydroxamic acid inhibitors have the potential to form a six-membered chelate with zinc (left side of Figure 3). This structure has a reduced bite angle and a shorter distance between the two chelating oxygen atoms. Both of these factors favor binding to the metal center.²²

FIGURE 3.

6-membered vs. 5-membered zinc chelation models.

Thiocarbonyl- Vs. Carbonyl-Containing Inhibitors

Despite the fact that hydroxamic acids are potent inhibitors of metalloproteases, they have met with limited success in the clinic because of unfavorable oral bioavailability, stability in vivo and side effects.²³ As a result, investigators have been working to discover alternate zinc-binding motifs as a substitute for hydroxamic acids. One such example has been published by Cohen and coworkers, who reported hydroxypyridinone and pyrone ligands as promising alternatives to hydroxamic acids.²⁴ Among the compounds assayed, the zinc chelators that incorporated a thiocarbonyl group had lower IC₅₀ values when compared to their non-sulfur containing analogues.

To apply this strategy to the squaric/hydroxamic acid hybrids, we first needed to develop a reliable method for incorporating sulfur into the inhibitors. Squaric acid monoester 3b (Scheme 2) did not react with a variety of thionating agents including P₂S₅/hexamethyldisiloxane and Lawesson’s reagent. By contrast, the squaric acid monoamide 6a reacted with both P₂S₅ and Lawesson’s reagent to give a product that incorporated a single sulfur atom in place of oxygen, as determined by mass spectrometry. Since Lawesson’s reagent provided the cleaner of the two reactions, this method was used to convert 6a, 12d, 12g and 12h into the corresponding thiocarbonyl compounds (Scheme 5).

SCHEME 5.

Thionation of Cyclobutenediones

Determining the Position of Sulfur Incorporation

The inhibitors in Scheme 5 contain three or four different carbonyl groups, any of which could be the site of reaction with Lawesson’s reagent. In order to determine the specific site that sulfur was incorporated into the molecules, we prepared two model compounds to aid us with this analysis (Scheme 6). Dibutyl squarate 7 was treated with two equivalents of NH₂-Phe-OMe to give compound 19. When compound 19 was treated with less than one equivalent of Lawesson’s reagent, the reaction yielded compound 20 in which one carbonyl group on the cyclobutenedione core had been replaced by a thiocarbonyl as determined by mass spectrometry. Ester carbonyl groups do not react with Lawesson’s reagent under the reaction conditions that we employed (25 °C). In a similar manner, compound 6e was converted to 21.

SCHEME 6^a.

^aReagents: (a) L-Phe-OMe·HCl, KOH, MeOH; (b) Lawesson’s reagent, CH₂Cl₂, 25 °C.

The ¹H NMR spectrum of compound 19 (Figure 4) has a resonance at 5.0 PPM that corresponds to the α-protons of the two Phe residues (H^a). When this compound is treated with Lawesson’s reagent to give compound 20, one of the α-protons remains at 5.0 PPM (H^c) while the other is shifted to 6.1 PPM (H^b shown in red). H^b shifts downfield since the neighboring nitrogen atom of this Phe residue is in conjugation with the thiocarbonyl group, which is a better electron acceptor than a standard carbonyl. In a similar manner, compound 6e has a resonance at 5.0 PPM that corresponds the Phe α-proton (H^d). When this compound is converted to the monothiocarbonyl adduct 21, this proton shifts downfield to 6.2 PPM (H^e shown in red). This observation, along with the fact that compound 3b (Scheme 2) that also incorporates a – N(OH)Me group does not react with Lawesson’s reagent, suggests that the carbonyl group opposite the Phe residue in 6e was the site of reaction with Lawesson’s reagent. Since we observe a similar shift for the α-proton of the amino acid that is attached directly to the cyclobutenedione ring in compounds 6a, 12d, 12g and 12h, we infer that all of these compounds react with Lawesson’s reagent on the opposite side of the ring from the peptide chain (see the Supporting Information for the spectra).

FIGURE 4.

Changes that occur in the ¹H NMR spectra of compounds 19 and 6e upon monothionation of the cyclobutenedione core.

Additional evidence for the site of sulfur incorporation comes from the chemical shift of the –OH proton of the vinylogous hydroxamic acid in compounds 6e and 21. This proton appears at 10.7 ppm in compound 6e, since it participates in a strong hydrogen bond with the neighboring carbonyl group on the cyclobutenedione ring. However, the analogous proton in compound 21 appears at 8.5–9.0 ppm. This change in chemical shift is partly due to the fact that the thiocarbonyl group is a weaker hydrogen bond acceptor than the carbonyl group.

Effect of the Thiocarbonyl Group on Inhibition

As shown in Table 4, two of the compounds show improved activity against MMP-1 on conversion to their thiocarbonyl analogs, while the activity of the other two remain unchanged. Compound 15 is 2–3 times more potent than 6a, while the potency of 16 is increased by 18-fold compared to 12d. By contrast, compounds 12g and h have similar IC₅₀ values when compared to their thiocarbonyl analogs 17 and 18. These results suggest that conversion of a carbonyl group on the cyclobutenedione core of the inhibitors to a thiocarbonyl can be an effective method for improving inhibition. However, this improvement is dependent on the specific structure of the inhibitor. One plausible explanation for the observation that the potencies of compounds 12g and h do not change upon thionation is that the improvement in chelation between the cyclobutene core and the active site zinc atom changes the position of the bound inhibitor in the active site. This geometry change could decrease binding interactions between the peptide portion of the inhibitors and the S1′ and S2′ subsites, and offset the improved binding to zinc. By contrast, conversion of compounds 6a and 12d to their thiocarbonyl analogs could lead to improved binding with the zinc atom, and also may reposition the inhibitor in the active site so that it makes more favorable interactions with the distal enzyme subsites.

TABLE 4.

Inhibition of MMP-1 by Thiocarbonyl-Containing Inhibitors

R	compound number for X = S	IC50 (μM)a	compound number for X = O	IC50 (μM)
HN-Ile-OMe	15	70 ± 8	6a	190 ± 30
HN-Ile-Phe-NHMe	16	15 ± 1	12d	270 ± 30
HN-Ile-Leu-NHMe	17	170 ± 40	12g	200 ± 20
HN-Leu-Tle-NHMe	18	96 ± 30	12h	95 ± 7

^aAll experiments performed in duplicate.

Conclusions

We have investigated the potential of a hybrid between squaric and hydroxamic acids to serve as a metal binding motif for the design of metalloprotease inhibitors. While hydroxamic acids are commonly used as MMP inhibitors, to the best of our knowledge this report represents the first use of a squaric acid derivative as a warhead for the design of inhibitors of metalloproteases. The squaric/hydroxamic acid hybrids are generally not as potent as hydroxamic acid-based inhibitors, many of which have inhibition constants in the nM range. However, since hydroxamic acids have not met with much success in the clinic, these hybrids could serve as an alternate starting point for the design of inhibitors with perhaps improved pharmacological properties. The structure of the peptidic portion of the inhibitors helps target them to the active site of zinc proteases, rather than to other classes of enzymes for which squaric acid derivatives can serve as inhibitors.

Among the alkyl groups that we have examined at the –N(OH)alkyl position of the inhibitors, small substituents such as Me and i-Pr are well accommodated in the active site of MMP-1, while sterically demanding groups such as Bn and cyclohexyl are too large and lead to poor activity. Among the inhibitors that do not contain a sulfur atom, we found that the dipeptide Leu-Tle-NHMe provided the highest level of activity when attached to the cyclobutenedione core.

We also developed a regioselective method for converting one of the carbonyl groups on the core into the corresponding thiocarbonyl compound. This reaction occurs specifically at the position on the opposite side of the cyclobutene ring from the peptide substituent. Thionation of the inhibitors leads to improved activity in some cases, but not in others. Compound 12d gave the largest increase in potency upon thionation. The 18-fold improvement resulted in an inhibitor with an IC₅₀ value of 15 μM against MMP-1.

Experimental Section

Full characterization for compounds 3b, 3c and 3e has been reported in the literature.¹⁷

3-(Hydroxyamino)-4-methoxy-3-cyclobutene-1,2-dione (3a)

3,4-Dimethoxy-3-cyclobutene-1,2-dione 1 (200 mg, 1.4 mmol) was added to 10 mL of MeOH and stirred until all of the solids had dissolved. To this mixture was added hydroxylamine hydrochloride (104 mg, 1.5 mmol), the reaction mixture was cooled to 0 °C, and KOH (94.3 mg, 1.7 mmol) dissolved in 5 mL of MeOH was added. At this point the reaction contained a white precipitate. The reaction was monitored by TLC and upon disappearance of the starting material the mixture was filtered and the solid that was collected was washed with cold MeOH. The filtrate and washes were combined and solvent was evaporated. The crude material was purified by column chromatography (1:9 MeOH/CH₂Cl₂) to give compound 3a as a yellow solid (0.093 g, 0.650 mmol, 47%). ¹H NMR (300 MHz, CD₃OD) δ 4.37 (s, 3 H); ¹³C NMR (100 MHz, CD₃OD) δ 184.9, 181.5, 174.4, 170.1, 60.1; HRMS-ESI (M + H⁺) calcd for C₅H₆NO₄ 144.0297, found 144.0292.

3-[(Benzyl)hydroxyamino]-4-methoxy-3-cyclobutene-1,2-dione (3d)

This compound was prepared using N-benzylhydroxylamine hydrochloride (0.367 g, 2.3 mmol) and 1 (0.30 g, 2.1 mmol) according to the procedure described above for the preparation of compound 3a. Purification of 3d was performed by column chromatography (5:95 MeOH/CH₂Cl₂) to obtain a white solid (0.318 g, 1.37 mmol, 65%). ¹H NMR (300 MHz, CD₃OD) δ 7.39 (m, 5 H), 4.88 (s, 3 H), 4.38 (s, 3 H); ¹³C NMR (75 MHz, CD₃OD) δ 185.0, 181.3, 175.0, 169.0, 135.0, 129.0, 128.8, 128.5, 60.3, 57.3; HRMS-ESI (M + Na⁺) calcd for C₁₂H₁₁NO₄Na 256.0586, found 256.0590.

3-(Hydroxyamino)-4-[(2-methylpropyl)amino]-3-cyclobutene-1,2-dione (4a)

To a solution of compound 3a (0.055 g, 0.383 mmol) dissolved in MeOH (1 mL), isobutylamine (0.062 g, 0.844 mmol) was added and the mixture was stirred at room temperature for 25 h. The solvent was evaporated and the product was purified by column chromatography (1:9:90 30% aqueous NH₄OH/MeOH/CH₂Cl₂) giving 4a (0.009 g, 0.049 mmol, 13%) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 3.39 (d, J = 6.8 Hz, 2 H), 1.85 (m, 1 H), 0.96 (d, J = 5.2 Hz, 6 H); ¹³C NMR (100 MHz, CD₃OD) δ 181.1, 180.3, 168.9, 167.8, 52.8, 31.4, 20.0; HRMS-ESI (M + H⁺) calcd for C₈H₁₂N₂O₃ 185.0926, found 185.0932.

3-(Hexylamino)-4-(hydroxyamino)-3-cyclobutene-1,2-dione (4b)

Compound 3b (100 mg, 0.70 mmol) was dissolved in MeOH (10 mL) and hexylamine (84.9 mg, 0.84 mmol) was added to the solution. The reaction was stirred for 12 h at room temperature, after which time TLC showed that all the starting material was consumed. The solvent was then removed by rotary evaporation. The crude material was purified by column chromatography (1:9:90 30% aqueous NH₄OH/MeOH/CH₂Cl₂) to yield 4b as a yellow oil (17.8 mg, 0.084 mmol, 12%). ¹H NMR (300 MHz, DMSO-d₆) δ 3.35 (m, 2 H), 1.50 (m, 2 H), 1.27 (m, 6 H), 0.86 (m, 3 H); ¹³C NMR (75 MHz, DMSO-d₆) δ 184.2, 183.6, 170.2, 169.6, 44.1, 31.7, 31.6, 26.4, 23.0, 14.8; HRMS-ESI (M + H⁺) calcd for C₁₀H₁₇N₂O₃ 213.1239, found 213.1250.

3-(Hexylamino)-4-(hydroxymethylamino)-3-cyclobutene-1,2-dione (4c)

To a solution of 3b (0.157 g, 1.0 mmol) in MeOH (1 mL), hexylamine (0.111 g, 1.1 mmol) was added and the mixture was stirred at room temperature for 4 h. The solvent was evaporated and the crude product was purified by column chromatography (1:9:90 30% aqueous NH₄OH/MeOH/CH₂Cl₂) giving 4c (0.19 g, 0.84 mmol, 84%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 11.19 (s, 1 H), 7.50 (s, 1 H), 3.59 (m, 5 H), 1.64 (m, 2 H), 1.32 (m, 6 H), 0.88 (t, J = 6.5 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 178.9, 177.5, 166.8, 164.9, 45.0, 41.4, 31.4, 30.9, 26.1, 22.6, 14.0; HRMS-FAB (M + Na⁺) calcd for C₁₁H₁₈N₂O₃Na 249.1215, found 249.1220.

3-(Hydroxymethylamino)-4-(pentylamino)-3-cyclobutene-1,2-dione (4d)

Compound 4d (0.058 g, 0.27 mmol, 37%) was prepared from 3b (0.118 g, 0.75 mmol) and pentylamine (0.65 g, 0.75 mmol) according to the procedure used for preparing 4c. ¹H NMR (400 MHz, CDCl₃) δ 7.16 (s, 1 H), 3.56 (m, 5 H), 1.64 (m, 2 H), 1.33 (m, 4 H), 0.89 (t, J = 6.8 Hz, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 178.9, 177.4, 166.7, 165.1, 45.0, 41.4, 30.7, 28.6, 22.3, 14.0; HRMS-FAB (M + Na⁺) calcd for C₁₀H₁₆N₂O₃Na 235.1059, found 235.1066.

3-[(Cyclohexyl)hydroxyamino]-4-(hexylamino)-3-cyclobutene-1,2-dione (4e)

Compound 4e (0.0047 g, 0.016 mmol, 13%) was prepared as a white solid from 3c (0.027 g, 0.12 mmol) and hexylamine (0.015 g, 0.144 mmol) according to the procedure used for preparation of 4b. ¹H NMR (400 MHz, DMSO-d₆) δ 7.34 (br s, 2 H), 3.76 (br s, 1 H), 3.32 (s, 4 H), 3.52 (s, 2 H), 1.88 (m, 2 H), 1.70 (m, 2 H), 1.57 (s, 3 H), 1.27 (m, 10 H), 1.53 (m, 3 H), 0.86 (m, 3 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 181.7, 181.4, 167.3, 166.4, 51.5, 42.7, 33.2, 30.3, 30.2, 25.0, 24.3, 23.5, 21.5, 13.4; HRMS-ESI (M + H⁺) calcd for C₁₆H₂₇N₂O₃ 295.2022, found 295.2018.

3-[(Benzyl)hydroxyamino]-4-(hexylamino)-3-cyclobutene-1,2-dione (4f)

Compound 4f (0.232 g, 0.77 mmol, 89%) was prepared as a white solid from 3d (0.200 g, 0.86 mmol) and hexylamine (0.101 g, 1.00 mmol) according to the procedure used for the preparation of 4b. ¹H NMR (300 MHz, CD₃OD) δ 7.36 (m, 5 H), 4.94 (s, 2 H), 3.58 (t, J = 7.0 Hz, 2 H), 1.60 (dd, J = 13.9, 6.9 Hz, 2 H), 1.34 (m, 6 H), 0.91 (t, J = 6.7 Hz, 3 H); ¹³C NMR (75 MHz, CD₃OD) δ 180.4, 178.6, 168.2, 166.1, 135.6, 129.1 128.7, 128.3 57.5, 44.4, 31.6, 26.1, 22.7, 13.4; HRMS-ESI (M + H⁺) 303.1709, found 303.1711.

3-(Hydroxymethylamino)-4-(L-isoleucine methyl ester)-3-cyclobutene-1,2-dione (6a)

To a solution of 3b (0.219 g, 1.39 mmol) dissolved in MeOH (5 mL), the hydrochloride salt of L-isoleucine methyl ester (5a) (0.229 g, 1.26 mmol) was added. To this stirred solution KOH (0.0713 g, 1.27 mmol) was added, and immediately a white precipitate formed. The reaction was stirred at room temperature for 12 h and solvent was evaporated. Purification was performed by column chromatography (1:9:90 30% aqueous NH₄OH/MeOH/CH₂Cl₂) to yield a yellowish flaky solid (0.238 g, 0.88 mmol, 70%). ¹H NMR (300 MHz, CD₃OD) δ 5.04 (br s, 2 H), 4.81 (d, J = 7 Hz, 2 H), 3.78 (s, 3 H), 3.50 (s, 3 H), 2.03 (m, 1 H), 1.54 (m, 1 H), 1.30 (m, 1 H), 0.97 (m, 6 H); ¹³C NMR (75 MHz, CD₃OD) δ 180.4, 180.0, 173.0, 167.6, 167.0, 62.4, 52.8, 41.4, 39.4, 25.8, 15.6, 11.6; HRMS-ESI (M + H⁺) calcd for C₁₂H₁₉N₂O₅ 271.1294, found 271.1287.

3-[Hydroxy(1-methylethyl)amino]-4-(L-isoleucine methyl ester)-3-cyclobutene-1,2-dione (6b)

Compound 6b (0.034 g, 0.11 mmol, 26%) was prepared as a colorless solid starting from 3e (0.080 g, 0.432 mmol) and 5a (0.120 g, 0.56 mmol) according to the procedure used for the preparation of 4b. ¹H NMR (300 MHz, CD₃OD) δ 7.28 (br s, 1 H), 4.83 (br s, 1 H), 4.52 (br s, 1 H), 3.73 (s, 3 H), 2.01 (s, 1 H), 1.49 (m, 1 H), 1.31 (m, 7 H), 0.93 (m, 6 H); ¹³C NMR (75 MHz, CDCl₃) δ 179.1, 179.0, 172.0, 166.7, 165.5, 61.8, 55.5, 52.7, 38.7, 25.1, 19.8, 15.5, 11.8; HRMS-ESI (M + H⁺) calcd for C₁₄H₂₃N₂O₅ 299.1607, found 299.1602.

3-(Hydroxymethylamino)-4-(D,L-norleucine methyl ester)-3-cyclobutene-1,2-dione (6c)

Compound 6c (0.09 g, 0.33 mmol, 45%) was prepared from 3b (0.118 g, 0.75 mmol), D,L-norleucine methyl ester HCl salt (0.136 g, 0.75 mmol) and KOH (0.042 g, 0.75 mmol) according to the procedure used for preparing 6a. ¹H NMR (400 MHz, CDCl₃) δ 7.15 (d, J = 8.4 Hz, 1 H), 4.79 (dt, J = 9.1, 4.8 Hz, 1 H), 3.73 (s, 3 H), 3.55 (s, 3 H), 1.95 (m, 1 H), 1.82 (m, 1 H), 1.36 (m, 4 H), 0.89 (t, J = 7.1 Hz, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 178.8, 178.2, 172.0, 165.9, 165.2, 57.0, 52.5, 41.3, 32.5, 27.6, 22.2, 13.8; HRMS-ESI (M + H⁺) calcd for C₁₂H₁₉N₂O₅ 271.1294, found 271.1285.

3-(Hydroxymethylamino)-4-(L-leucine methyl ester)-3-cyclobutene-1,2-dione (6d)

Compound 6d was prepared as a white solid (0.023 g, 0.076 mmol, 38%) starting from 3b (0.031 g, 0.20 mmol) and 5c (0.047 g, 0.26 mmol) according to the procedure used to prepare 6a. ¹H NMR (300 MHz, CDCl₃) δ 7.14 (br s, 1 H), 4.87 (br s, 1 H), 3.72 (s, 3 H) 3.55 (s, 3 H), 1.75 (m, 1 H), 0.96 (d, J = 4.8 Hz, 6 H); ¹³C NMR (100 MHz, CD₃OD) δ 184.5, 183.5, 174.1, 170.3, 168.7, 54.8, 53.0, 31.3, 25.9, 23.4, 21.7; HRMS-ESI (M + Na⁺) calcd for C₁₂H₁₈N₂O₅Na 293.1113, found 293.1118.

3-(Hydroxymethylamino)-4-(L-phenylalanine methyl ester)-3-cyclobutene-1,2-dione (6e)

Compound 6e (0.135 g, 0.44 mmol, 59%) was prepared from 3b (0.118 g, 0.75 mmol), L-phenylalanine methyl ester HCl salt (0.216 g, 1.0 mmol) and KOH (0.056 g, 1.0 mmol) according to the procedure used for preparing 6a. ¹H NMR (400 MHz, DMSO-d₆) δ 10.75 (s, 1 H), 7.79 (s, 1 H), 7.25 (m, 5 H), 5.06 (s, 1 H), 3.70 (s, 3 H), 3.35 (s, 3 H), 3.28 (dd, J = 14.0, 3.9 Hz, 1 H), 3.18 (m, 1 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.0, 179.2, 171.9, 166.9, 166.0, 137.7, 129.6, 128.8, 127.0, 57.6, 52.8, 41.2, 37.7; HRMS-FAB (M + Na⁺) calcd for C₁₅H₁₆N₂O₅Na 327.0957, found 327.0948.

3-(Hydroxymethylamino)-4-(L-tryptophan methyl ester)-3-cyclobutene-1,2-dione (6f)

Compound 6f (0.16 g, 0.47 mmol, 62%) was prepared from 3b (0.118 g, 0.75 mmol), L-tryptophan methyl ester HCl salt (0.255 g, 1.0 mmol) and KOH (0.056 g, 1.0 mmol) according to the procedure used for preparing 6a. ¹H NMR (300 MHz, CD₃OD) δ 7.56 (d, J = 7.7 Hz, 1 H), 7.34 (d, J = 8.0 Hz, 1 H), 7.07 (m, 3 H), 5.13 (dd, J = 7.7, 5.1 Hz, 1 H), 3.76 (s, 3 H), 3.43 (m, 5 H); ¹³C NMR (75 MHz, CD₃OD) δ 179.6, 179.0, 172.4, 166.9, 166.2, 137.0, 127.7, 123.7, 121.5, 119.0, 118.2, 111.3, 109.1, 57.7, 52.1, 40.3, 28.7; HRMS-ESI (M + Na⁺) calcd for C₁₇H₁₇N₃O₅Na 366.1066, found 366.1075.

3-(Hydroxymethylamino)-4-(L-methionine methyl ester)-3-cyclobutene-1,2-dione (6g)

Compound 6g (0.082 g, 0.28 mmol, 38%) was prepared from 3b (0.118 g, 0.75 mmol), L-methionine methyl ester HCl salt (0.15 g, 0.75 mmol) and KOH (0.042 g, 0.75 mmol) according to the procedure used for preparing 6a. ¹H NMR (400 MHz, CDCl₃) δ 7.30 (d, J = 7.8 Hz, 1 H), 4.93 (dd, J = 12.7, 8.5 Hz, 1 H), 3.75 (s, 3 H), 3.55 (s, 3 H), 2.60 (m, 2 H), 2.27 (m, 1 H), 2.17 (td, J = 14.7, 7.2 Hz, 1 H), 2.10 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 178.8, 178.3, 171.6, 165.9, 165.3, 56.0, 52.8, 41.4, 31.8, 30.1, 15.4; HRMS-FAB (M + Na⁺) calcd for C₁₁H₁₆N₂O₅SNa 311.0678, found 311.0685.

3-Butoxy-4-(hydroxymethylamino)-3-cyclobutene-1,2-dione (8)

To a stirred solution of HONHMe HCl salt (2.631 g, 31.5 mmol) in MeOH (30 ml), KOH (1.767 g, 31.5 mmol) and compound 7 (4.766 g, 21.0 mmol) were added. The mixture was stirred overnight at room temperature, the solvent was evaporated, and the crude product was washed with H₂O, and then dissolved in EtOAc. The aqueous layer was extracted with EtOAc. The organic extracts were combined, dried over MgSO₄, and the solvents were evaporated under reduced pressure. The crude product was purified by column chromatography (3% MeOH/CH₂Cl₂) to yield compound 8 as a pale yellow solid (3.416 g, 17.2 mmol, 82%). ¹H NMR (400 MHz, CDCl₃) δ 4.70 (t, J = 6.6 Hz, 2 H), 3.52 (s, 3 H), 1.77 (m, 2 H), 1.44 (m, 2 H), 0.97 (t, J = 7.4 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 184.0, 180.7, 174.3, 169.1, 74.2, 41.6, 32.3, 18.9, 14.0; HRMS-FAB (M + Na⁺) calcd for C₉H₁₃NO₄Na 222.0742, found 222.0740.

L-tert-Leucine methyl amide (10a)

Compound 10a was synthesized starting from N-Boc- protected amino acid 9a. To a stirred solution of N-hydroxysuccinimide (NHS, 0.925 g, 8.04 mmol) in DMF (20 mL) under N₂, NH₂Me (4.02 mL, 2.0 M in MeOH) was added via syringe and the mixture was stirred for 30 min at room temperature. Formation of a white precipitate was observed. Addition of N-Boc-protected amino acid 9a (1.55 g, 6.7 mmol) and cooling the mixture to 0 °C was followed by addition of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, 1.54 g, 8.04 mmol). The mixture was stirred for 1 h at 0 °C and 2 h at room temperature and then diluted with EtOAc. The solution was washed with H₂O, the organic layer was separated, and the aqueous layer was extracted with EtOAc. The combined organic extracts were washed with saturated NaHCO₃, H₂O, and brine, dried over MgSO₄, and the solvents were evaporated under reduced pressure. The crude product was purified by column chromatography (3% MeOH/CH₂Cl₂), washed with heptane, dried in vacuo and dissolved in CH₂Cl₂ (15 mL). To this stirred solution, TFA (15 mL) was added and the mixture was stirred for 40 min at room temperature. The solvents were removed in vacuo, and the residue was partitioned between CH₂Cl₂ (90 mL) and saturated aqueous NaHCO₃ (90 mL). The organic phase was separated and the aqueous phase was extracted with CH₂Cl₂. The organic extracts were combined and dried over Na₂SO₄, and the solvents were evaporated. Purification was performed by column chromatography (5:95 MeOH/CH₂Cl₂) yielding 10a as a colorless oil (0.637 g, 4.42 mmol, 66%). ¹H NMR (300 MHz, CD₃OD) δ 2.99 (s, 1 H), 2.75 (s, 3 H), 0.98 (s, 9 H); ¹³C NMR (75 MHz, CD₃OD) δ 175.2, 63.9, 34.0, 25.9, 24.9; HRMS-FAB (M + Na⁺) calcd for C₇H₁₆N₂ONa 167.1160, found 167.1163.

L-Cyclohexylglycine methyl amide (10b)

Compound 10b was synthesized starting from N-Boc-protected amino acid 9b. To a stirred solution of N-Boc-protected amino acid 9b (1.16 g, 4.52 mmol) in CH₂Cl₂ (20 mL), HOBt (760 mg, 4.97 mmol) was added and then EDC (911 mg, 4.75 mmol) and mixture was stirred at 0 °C for 30 min. Formation of a white precipitate was observed. To this solution NH₂Me (2.49 mL, 2.0 M in MeOH) was added via syringe and reaction was allowed to stir overnight. The solution was diluted with CH₂Cl₂ (50 mL) and washed once with 1M citric acid (30 mL), once with saturated NaHCO₃ (30 mL) and then with brine (30 mL). The organic layer was then dried over NaSO₄ and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (3% MeOH/CH₂Cl₂) and the product was dissolved in CH₂Cl₂ (15 mL). To this stirred solution TFA (15 mL) was added and the mixture was stirred at room temperature for 40 min. The solvents were removed in vacuo, and the residue was partitioned between 1:1 CH₂Cl₂/saturated aqueous NaHCO₃. The organic phase was separated and the aqueous phase was extracted with CH₂Cl₂. The organic extracts were combined and dried over Na₂SO₄, and solvents were evaporated. Purification was performed by flash chromatography (5% MeOH/CH₂Cl₂) yielding 10b as a white solid (440 mg, 2.80 mmol, 62%). ¹H NMR (CDCl₃, 300 MHz) δ 7.34 (br s, 1 H), 3.22 (d, J = 3.8 Hz, 1 H), 2.80 (d, J = 5.0 Hz, 3 H), 1.27 (m, 14 H); ¹³C NMR (75 MHz, CDCl₃) δ 175.5, 60.5, 41.5, 30.6, 27.0, 26.7, 26.6, 26.5, 26.0; HRMS-FAB (M + Na⁺) calcd for C₉H₁₈N₂NaO 193.1317, found 193.1320.

O-Methyl-L-tyrosine methyl amide (10c)

The synthesis and the ¹H NMR spectrum for this compound have been reported previously.^{25 13}C NMR (75 MHz, CD₃OD) δ 175.9, 158.6, 129.9, 129.3, 113.5, 56.5, 54.2, 40.3, 24.7; HRMS-ESI (M + Na⁺) calcd for C₁₁H₁₆N₂O₂Na 231.1109, found 231.1115.

L-Phenylalanine methyl amide (10d)

Compound 10d (105 mg, 0.590 mmol, 52%) was prepared as a white solid from 9d (300 mg, 1.13 mmol), HOBt (190 mg, 1.24 mmol), EDC (228 mg, 1.19 mmol) and NH₂Me (624 μL of a 2.0 M solution in MeOH) according to the procedure used to prepare 10b. ¹H NMR (300 MHz, CDCl₃,) δ 7.26 (m, 5 H), 3.60 (dd, J = 9.5, 4.0 Hz, 1 H), 3.28 (dd, J = 13.7, 4.0 Hz, 1 H), 2.82 (d, J = 5 Hz, 3 H), 2.67 (dd, J = 13.7, 9.5 Hz, 1 H), 1.39 (br s, 2 H); ¹³C NMR (75 MHz, CDCl₃) δ 174.8, 138.0, 129.3, 128.7, 126.8, 56.5, 41.0, 25.8; HRMS-FAB (M + Na⁺) calcd for C₁₀H₁₄N₂NaO 201.1004, found 201.1007.

L-Phenylglycine methyl amide (10e)

Compound 10e (0.836 g, 5.1 mmol, 51%) was prepared from 6e (2.513 g, 10 mmol), NH₂Me (6.0 mL of a 2.0 M solution in MeOH), NHS (1.381 g, 12 mmol), EDC (2.3 g, 12 mmol) and TFA (15 mL) according to the procedure used for preparing 10a. The ¹H NMR and ¹³C NMR spectra, and the HRMS for this compound have been reported previously.²⁶

L-Tryptophan methyl amide (10f)

The synthesis and the ¹H NMR spectrum for this compound have been reported previously.^{24 13}C NMR (75 MHz, CD₃OD) δ 174.8, 135.3, 125.9, 121.8, 119.6, 116.9, 116.5, 109.4, 108.3, 54.0, 29.3, 23.3; HRMS-FAB (M + Na⁺) calcd for C₁₂H₁₅N₃ONa 240.1113, found 240.1117.

L-Leucine methyl amide (10g)

Compound 10g (0.548 g, 3.8 mmol, 42%) was prepared from 9g (2.245 g, 9 mmol), NH₂Me (5.4 mL of a 2.0 M solution in MeOH), NHS (1.243 g, 10.8 mmol), EDC (2.068 g, 10.8 mmol) and TFA (12 mL) according to the procedure used for preparing 10a. ¹H NMR (300 MHz, CD₃OD) δ 3.33 (dd, J = 7.7, 6.5 Hz, 1 H), 2.76 (s, 3 H), 1.68 (qt, J = 12.9, 6.5 Hz, 1 H), 1.54 (ddd, J = 13.8, 7.4, 6.4 Hz, 1 H), 1.39 (m, 1 H), 0.95 (t, J = 6.6 Hz, 6 H); ¹³C NMR (75 MHz, CD₃OD) δ 177.0, 53.2, 44.2, 24.8, 24.5, 22.0, 21.1; HRMS-FAB (M + Na⁺) calcd for C₇H₁₆N₂ONa 167.1160, found 167.1166.

L-Isoleucyl-L-tert-leucine methyl amide (11a)

To a stirred solution of NHS (0.138 g, 1.2 mmol) in DMF (5 mL) under N₂, 10a (0.144 g, 1.0 mmol) was added and the mixture was stirred at room temperature for 30 min. Addition of N-Boc-L-isoleucine (0.288 g, 1.2 mmol) and cooling the mixture to 0 °C was followed by addition of EDC (0.23 g, 1.2 mmol). The mixture was stirred for 1 h at 0 °C and 2 h at room temperature and then diluted with EtOAc. The solution was washed with H₂O, the organic layer was separated, and the aqueous layer was extracted with EtOAc. The combined organic extracts were washed with saturated NaHCO₃, H₂O, and brine, dried over MgSO₄, and the solvents were evaporated under reduced pressure. The crude product was purified by column chromatography (3:97 MeOH/CH₂Cl₂), washed with heptane, dried in vacuo and dissolved in CH₂Cl₂ (2 mL). To this solution TFA (2 mL) was added and the mixture was stirred at room temperature for 40 min. The solvents and TFA were removed, and the residue was partitioned between CH₂Cl₂ (15 mL) and saturated aqueous NaHCO₃ (15 mL). Organic phase was separated and the aqueous phase was extracted with CH₂Cl₂. The organic extracts were combined and dried over Na₂SO₄, and the solvent was evaporated. Purification was performed by flash chromatography (5:95 MeOH/CH₂Cl₂) yielding 11a as a colorless oil (0.162 g, 0.63 mmol, 63%). ¹H NMR (300 MHz, CD₃OD) δ 5.51 (s, 1 H), 4.19 (s, 1 H), 2.73 (s, 3 H), 1.79 (m, 1 H), 1.48 (m, 1 H), 1.16 (m, 1 H), 0.95 (m, 15 H); ¹³C NMR (75 MHz, CD₃OD) δ 175.5, 171.8, 60.6, 59.4, 38.6, 33.8, 25.7, 24.6, 23.8, 14.8, 10.7; HRMS-FAB (M + Na⁺) calcd for C₁₃H₂₇N₃O₂Na 280.2001, found 280.2000.

L-Isoleucyl-L-cyclohexylglycine methyl amide (11b)

N-Boc-L-Isoleucine (140 mg, 0.584 mmol) was dissolved in CH₂Cl₂ (20 mL) and the solution was cooled to 0 °C. To this solution HOBt (89.5 mg, 0.584 mmol) and EDC (201 mg, 0.558 mmol) were added and the mixture was stirred for 30 min. To this mixture compound 10b (83.5 mg, 0.531 mmol) was then added and reaction was stirred overnight. The solution was diluted with CH₂Cl₂ (50 mL) and washed once with 1M citric acid (30 mL), once with saturated NaHCO₃ (30 mL) and then with brine (30 mL). The organic layer was dried over NaSO₄ and the solvents evaporated under reduced pressure. The crude product was purified by column chromatography (3% MeOH/CH₂Cl₂) and then dissolved in CH₂Cl₂ (15 mL). To this solution TFA (15 mL) was added and the mixture was stirred at room temperature for 40 min. The solvents and TFA were evaporated, and the residue was partitioned between a 1:1 mixture of CH₂Cl₂:saturated aqueous NaHCO₃. The organic phase was separated and the aqueous phase was extracted with CH₂Cl₂. The organic extracts were combined and dried over Na₂SO₄, and the solvent was evaporated. Purification was performed by flash chromatography (5% MeOH/CH₂Cl₂) yielding 11b as a white solid (69 mg, 0.244 mmol, 46%). ¹H NMR (400 MHz, CD₃OD) δ 7.94 (d, J = 9.0 Hz, 1 H), 6.98 (m, 1 H), 4.26 (m, 1 H), 3.28 (d, J = 4.0 Hz, 1 H), 2.78 (d, J = 4.8 Hz, 3 H), 1.93 (m, 1 H), 1.75 (m, 7 H), 1.39 (m, 2 H), 1.25 (m, 2 H), 1.13 (m, 4 H), 0.96 (d, J = 6.9 Hz, 3 H), 0.90 (t, J = 7.3 Hz, 3 H); ¹³C NMR (100 MHz, CD₃OD) δ 174.6, 171.9, 59.8, 57.8, 39.7, 38.1, 29.8, 28.8, 26.1, 25.9, 25.8, 25.8, 24.0, 16.1, 11.9 HRMS-FAB (M + Na⁺) calcd for C₁₅H₂₉N₃NaO₂ 306.2157, found 306.2150.

L-Isoleucyl-O-methyl-L-tyrosine methyl amide (11c)

Compound 11c (0.265 g, 0.82 mmol, 58%) was prepared as a white solid from 10c (0.295 g, 1.42 mmol), NHS (0.196 g, 1.7 mmol), N-Boc-L-isoleucine (0.408 g, 1.7 mmol), EDC (0.326 g, 1.7 mmol) and TFA (3 mL) according to the procedure used for preparing 11a. ¹H NMR (300 MHz, CD₃OD) δ 7.16 (d, J = 8.5 Hz, 2 H), 6.84 (d, J = 8.5 Hz, 2 H), 4.55 (dd, J = 8.6, 6.3 Hz, 1 H), 3.76 (s, 3 H), 3.17 (d, J = 5.0 Hz, 1 H), 3.07 (dd, J = 13.8, 6.2 Hz, 1 H), 2.86 (dd, J = 13.8, 8.8 Hz, 1 H), 2.69 (s, 3 H), 1.66 (m, 1 H), 1.18 (m, 1 H), 0.99 (m, 1 H), 0.83 (m, 6 H); ¹³C NMR (75 MHz, CD₃OD) δ 175.6, 172.7, 158.7, 129.9, 128.8, 113.5, 59.6, 54.7, 54.2, 38.5, 37.0, 24.9, 23.5, 14.7, 10.6; HRMS-FAB (M + Na⁺) calcd for C₁₇H₂₇N₃O₃Na 344.1950, found 344.1942.

L-Isoleucyl-L-phenylalanine methyl amide (11d)

Compound 11d (373 mg, 1.28 mmol, 61%) was prepared as a white solid from 10d (374 mg, 2.1 mmol), HOBt (322 mg, 2.1 mmol), EDC (383 mg, 2.0 mmol) and N-Boc-L-Isoleucine (456 mg, 1.9 mmol) according to the procedure that was used to prepare 11b. ¹H NMR (400 MHz, CDCl₃) δ 8.04 (d, J = 8.8 Hz, 1 H), 7.18 (m, 5 H), 4.78 (dt, J = 8.7, 6.3 Hz, 1 H), 2.98 (dd, J = 13.8, 8.8 Hz, 1 H), 2.68 (d, J = 4.8 Hz, 3 H), 1.77 (m, 1 H), 0.99 (m, 1 H), 0.88 (m, 1 H), 0.81 (d, J = 7.0 Hz, 3 H), 0.75 (t, J = 7.3 Hz, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 175.0, 171.9, 137.1, 129.2, 128.4, 126.7, 59.8, 54.0, 38.4, 26.0, 23.4, 15.9, 11.8; HRMS-FAB (M + Na⁺) calcd for C₁₆H₂₅N₃NaO₂ 314.1844, found 314.1850.

L-Isoleucyl-L-phenylglycine methyl amide (11e)

Compound 11e (0.032 g, 0.115 mmol, 53%) was prepared from 10e (0.036 g, 0.217 mmol), NHS (0.03 g, 0.26 mmol), N-Boc-L-isoleucine (0.062 g, 0.26 mmol), EDC (0.05 g, 0.26 mmol) and TFA (0.35 mL) according to the procedure used for preparing 11a. ¹H NMR (400 MHz, CD₃OD) δ 7.44 (m, 2 H), 7.34 (m, 3 H), 5.42 (s, 1 H), 3.29 (d, J = 5.3 Hz, 1 H), 2.74 (s, 3 H), 1.77 (m, 1 H), 1.47 (m, 1 H), 1.15 (m, 1 H), 0.96 (d, J = 6.9 Hz, 3 H), 0.89 (t, J = 7.4 Hz, 3 H); ¹³C NMR (100 MHz, CD₃OD) δ 175.1, 171.4, 137.7, 128.3, 127.9, 127.1, 59.2, 57.0, 38.8, 25.0, 23.9, 14.6, 10.6; HRMS-FAB (M + Na⁺) calcd for C₁₅H₂₃N₃O₂Na 300.1688, found 300.1680.

L-Isoleucyl-L-tryptophan methyl amide (11f)

Compound 11f (0.103 g, 0.313 mmol, 55%) was prepared from 10f (0.124 g, 0.573 mmol), NHS (0.079 g, 0.688 mmol), N-Boc-L-isoleucine (0.165 g, 0.688 mmol), EDC (0.132 g, 0.688 mmol) and TFA (1 mL) according to the procedure used to prepare 11a. ¹H NMR (300 MHz, CD₃OD) δ 7.57 (d, J = 7.8 Hz, 1 H), 7.29 (dd, J = 8.1, 0.9 Hz, 1 H), 7.02 (m, 3 H), 4.59 (t, J = 6.5, 1 H), 3.2 (d, J = 6.5, 1 H), 2.62 (d, J = 1.9, 3 H), 1.60 (m, 1 H), 1.14 (m, 1 H), 0.9 (m, 1 H), 0.75 (m, 6 H); ¹³C NMR (75 MHz, CD₃OD) δ 174.3, 172.1, 135.6, 126.2, 122.1, 119.9, 117.2, 116.8, 109.7, 108.3, 58.3, 52.9, 37.2, 26.7, 23.8, 22.3, 13.4, 9.4; HRMS-ESI (M + H⁺) calcd for C₁₈H₂₇N₄O₂ 331.2134, found 331.2138.

L-Isoleucyl-L-leucine methyl amide (11g)

Compound 11g (0.219 g, 0.851 mmol, 43%) was prepared from 10g (0.285 g, 1.98 mmol), NHS (0.273 g, 2.375 mmol), N-Boc-L-isoleucine (0.57 g, 2.375 mmol), EDC (0.455 g, 2.375 mmol) and TFA (2.5 mL) according to the procedure used to prepare 11a. ¹H NMR (300 MHz, CD₃OD) δ 4.4 (t, J = 7.5, 1 H), 3.23 (d, J = 5.5, 1 H), 2.73 (s, 3 H), 1.61 (m, 5 H), 1.18 (m, 1 H), 0.94 (m, 12 H); ¹³C NMR (75 MHz, CD₃OD) δ 175.9, 174.2, 59.9, 51.9, 41.2, 39.3, 25.4, 24.9, 24.4, 22.5, 21.1, 15.1, 11.0; HRMS-FAB (M + Na⁺) calcd for C₁₃H₂₇N₃O₂Na 280.2001, found 280.2006.

L-Leucyl-L-tert-leucine methyl amide (11h)

Compound 11h (0.054 g, 0.208 mmol, 65%) was prepared from 10h (0.046 g, 0.322 mmol), NHS (0.045 g, 0.386 mmol), N-Boc-L-leucine (0.096 g, 0.386 mmol), EDC (0.074 g, 0.386 mmol) and TFA (0.75 mL) according to the procedure that was used to prepare 11a. ¹H NMR (400 MHz, CD₃OD) δ 4.22 (s, 1 H), 3.46 (t, J = 5.3, 1 H), 2.74 (s, 3 H), 1.76 (m, 1 H), 1.57 (m, 1 H), 1.41 (m, 1 H), 1.01 (m, 9 H), 0.97 (m, 6 H); ¹³C NMR (100 MHz, CD₃OD) δ 176.8, 172.1, 60.8, 53.6, 44.5, 34.5, 26.2, 25.1, 24.9, 22.7, 21.2; HRMS-FAB (M + Na⁺) calcd for C₁₃H₂₇N₃O₂Na 280.2001, found 280.2010.

General Procedure for the Synthesis of Compounds 12a–h

To a stirred solution of 8 (1.0 eq) dissolved in MeOH, 11a–h (1.0 eq) was added and the mixture was stirred at room temperature overnight. The solvent was evaporated and the product was purified by column chromatography (3:97 MeOH/CH₂Cl₂).

3-(Hydroxymethylamino)-4-(L-isoleucyl-L-tert-leucine methyl amide)-3-cyclobutene-1,2-dione (12a)

Compound 12a (0.03 g, 0.077 mmol, 71%) was prepared from 8 (0.022 g, 0.109 mmol) and 11a (0.028 g, 0.109 mmol) according to the general procedure. ¹H NMR (300 MHz, CD₃OD) δ 4.58 (d, J = 8.7 Hz, 1 H), 4.23 (s, 1 H), 3.49 (s, 3 H), 2.73 (s, 3 H), 1.93 (m, 1 H), 1.62 (m, 1 H), 1.23 (m, 1 H), 0.99 (s, 9 H), 0.94 (m, 6 H); ¹³C NMR (75 MHz, CD₃OD) δ 179.5, 178.9, 172.0, 171.8, 166.5, 166.3, 62.4, 61.3, 40.2, 37.7, 34.2, 26.1, 24.9, 24.4, 14.5, 10.0; HRMS-ESI (M + Na⁺) calcd for C₁₈H₃₀N₄O₅Na 405.2114, found 405.2126.

3-(Hydroxymethylamino)-4-(L-isoleucyl-L-cyclohexylglycine methyl amide)-3-cyclobutene-1,2-dione (12b)

Compound 12b (0.0106 g, 0.026 mmol, 11%) was prepared from 8 (0.038 g, 0.24 mmol) and 11b (0.068 g, 0.24 mmol) as a white solid according to the general procedure. ¹H NMR (300 MHz, CD₃OD) δ 4.56 (d, J = 9 Hz, 1 H), 4.14 (d, J = 6 Hz, 1 H), 3.49 (s, 3 H), 2.74 (s, 3 H), 1.92 (m, 1 H), 1.67 (m, 7 H), 1.22 (m, 5 H), 0.94 (m, 7 H); ¹³C NMR (75 MHz, DMSO-d₆) δ 183.8, 182.7, 171.7, 171.1, 169.1, 168.1, 61.3, 58.4, 40.7, 39.2, 39.1, 31.1, 30.0, 29.2, 26.9, 26.8, 26.7, 24.4, 15.8, 12.0; HRMS-FAB (M + Na⁺) calcd for C₂₀H₃₂N₄NaO₅ 431.2270, found 431.2261.

3-(Hydroxymethylamino)-4-(L-isoleucyl-O-methyl-L-tyrosine methyl amide)-3- cyclobutene-1,2-dione (12c)

Compound 12c (0.054 g, 0.12 mmol, 76%) was prepared from 8 (0.032 g, 0.16 mmol) and 11c (0.051 g, 0.16 mmol) according to the general procedure. ¹H NMR (400 MHz, CD₃OD) δ 7.11 (d, J = 8.5 Hz, 2 H), 6.77 (d, J = 8.6 Hz, 2 H), 4.60 (dd, J = 9.6, 5.5 Hz, 1 H), 4.41 (d, J = 8.7 Hz, 1 H), 3.75 (s, 3 H), 3.49 (s, 3 H), 3.06 (dd, J = 13.8, 5.5 Hz, 1 H), 2.81 (dd, J = 13.9, 9.7 Hz, 1 H), 2.73 (s, 3 H), 1.87 (m, 1 H), 1.53 (m, 1 H), 1.18 (m, 1 H), 0.90 (m, 6 H); ¹³C NMR (100 MHz, CD₃OD) δ 179.1, 178.5, 172.4, 171.3, 165.9, 165.7, 158.6, 129.8, 128.6, 113.4, 62.0, 54.5, 54.2, 40.0, 37.0, 36.7, 24.9, 24.2, 14.1, 9.5; HRMS-FAB (M + Na⁺) calcd for C₂₂H₃₀N₄O₆Na 469.2063, found 469.2078.

3-(Hydroxymethylamino)-4-(L-isoleucyl-L-phenylalanine methyl amide)-3- cyclobutene-1,2-dione (12d)

Compound 12d (0.224 g, 0.54 mmol, 75%) was prepared from 8 (0.113 g, 0.72 mmol) and 11d (0.210 g, 0.72 mmol) as a white solid according to the general procedure. ¹H NMR (300 MHz, CD₃OD) δ 7.21 (m, 5 H), 4.63 (dd, J = 9.6, 9.3 Hz, 1 H), 4.43 (d, J = 8.6 Hz, 1H) 3.49 (s, 3 H), 3.12 (dd, J = 6.3, 5.3 Hz, 1 H), 2.88 (dd, J = 9.7, 9.3 Hz, 1 H), 2.71 (s, 3 H), 1.86 (m, 1 H), 1.52 (m, 1 H), 1.16 (m, 1 H), 0.90 (m, 6 H); ¹³C NMR (75 MHz, CD₃OD) δ 179.6, 178.9, 172.7, 171.7, 166.4, 166.2, 137.2, 129.2, 128.4, 126.7, 62.3, 54.9, 40.4, 38.1, 37.3, 25.4, 24.5, 14.5, 9.92; HRMS-FAB (M + Na⁺) calcd for C₂₁H₂₈N₄NaO₅ 439.1957, found 439.1968.

3-(Hydroxymethylamino)-4-(L-isoleucyl-L-phenylglycine methyl amide)-3- cyclobutene-1,2-dione (12e)

Compound 12e (0.021 g, 0.053 mmol, 65%) was prepared from 8 (0.016 g, 0.08 mmol) and 11e (0.023 g, 0.083 mmol) according to the general procedure. ¹H NMR (300 MHz, CD₃OD) δ 7.42 (m, 2 H), 7.35 (m, 3 H), 5.42 (s, 1 H), 4.6 (d, J = 6 Hz, 1 H), 3.47 (s, 3 H), 2.73 (s, 3 H), 1.96 (m, 1 H), 1.63 (m, 1 H), 1.26 (m, 1 H), 1.0 (m, 6 H); ¹³C NMR (75 MHz, CD₃OD) δ 179.1, 178.5, 171.1, 171.0, 166.1, 165.9, 137.3, 128.4, 128.0, 127.2, 61.8, 57.3, 40.0, 37.7, 25.0, 24.2, 14.1, 9.8; HRMS-FAB (M + Na⁺) calcd for C₂₀H₂₆N₄O₅Na 425.1801, found 425.1810.

3-(Hydroxymethylamino)-4-(L-isoleucyl-L-tryptophan methyl amide)-3-cyclobutene- 1,2-dione (12f)

Compound 12f (0.057 g, 0.125 mmol, 78%) was prepared from 8 (0.032 g, 0.16 mmol) and 11f (0.053 g, 0.16 mmol) according to the general procedure. ¹H NMR (400 MHz, CD₃OD) δ 7.57 (d, J = 7.8 Hz, 1 H), 7.31 (d, J = 8.1 Hz, 1 H), 7.07 (m, 2 H), 6.99 (m, 1 H), 4.66 (dd, J = 8.1, 6.4 Hz, 1 H), 4.46 (d, J = 8.2 Hz, 1 H), 3.47 (s, 3 H), 3.25 (dd, J = 14.6, 6.3 Hz, 1 H), 3.11 (dd, J = 14.5, 8.1 Hz, 1 H), 2.67 (s, 3 H), 1.86 (m, 1 H), 1.50 (m, 1 H), 1.13 (m, 1 H), 0.88 (m, 6 H); ¹³C NMR (100 MHz, CD₃OD) δ 179.1, 178.5, 172.8, 171.3, 166.0, 165.8, 136.6, 127.3, 123.3, 120.9, 118.4, 117.9, 110.9, 109.3, 62.0, 54.3, 40.0, 37.3, 27.8, 25.0, 24.1, 14.1, 9.7; HRMS-FAB (M + Na⁺) calcd for C₂₃H₂₉N₅O₅Na 478.2066, found 478.2060.

3-(Hydroxymethylamino)-4-(L-isoleucyl-L-leucine methyl amide)-3-cyclobutene-1,2- dione (12g)

Compound 12g (0.051 g, 0.133 mmol, 82%) was prepared from 8 (0.032 g, 0.16 mmol) and 11g (0.042 g, 0.163 mmol) according to the general procedure. ¹H NMR (400 MHz, CD₃OD) δ 4.57 (d, J = 8.1 Hz, 1 H), 4.41 (dd, J = 9.4, 5.4 Hz, 1 H), 3.50 (s, 3 H), 2.74 (s, 3 H), 1.96 (m, 1 H), 1.63 (m, 3 H), 1.53 (m, 1 H), 1.22 (m, 1 H), 0.94 (m, 12 H); ¹³C NMR (100 MHz, CD₃OD) δ 179.1, 178.6, 173.5, 171.6, 166.3, 165.9, 61.9, 51.9, 40.6, 40.1, 37.6, 25.0, 24.5, 24.3, 22.0, 20.7, 14.3, 9.9; HRMS-FAB (M + H⁺) calcd for C₁₈H₃₁N₄O₅ 383.2295, found 383.2304.

3-(Hydroxymethylamino)-4-(L-leucyl-L-tert-leucine methyl amide)-3-cyclobutene- 1,2-dione (12h)

Compound 12h (0.028 g, 0.074 mmol, 74%) was prepared from 8 (0.02 g, 0.1 mmol) and 11h (0.026 g, 0.1 mmol) according to the general procedure. ¹H NMR (400 MHz, DMSO-d₆) δ 4.76 (br s, 1 H), 4.2 (d, J = 9.6 Hz, 1 H), 3.38 (s, 3 H), 2.57 (d, J = 4.4 Hz, 3 H), 1.76 (m, 1 H), 1.62 (m, 1 H), 1.51 (m, 1 H), 0.87 (s, 15 H); ¹³C NMR (75 MHz, CD₃OD) δ 179.8, 178.9, 172.7, 171.9, 166.5, 166.3, 61.3, 56.6, 41.1, 40.4, 34.4, 26.1, 25.0, 24.9, 22.5, 20.8; HRMS-FAB (M + Na⁺) calcd for C₁₈H₃₀N₄O₅Na 405.2114, found 405.2105.

3-(L-Leucyl-L-tert-leucine methyl amide)-4-methoxy-3-cyclobutene-1,2-dione (13)

To a stirred solution of 2 (0.021 g, 0.15 mmol) in MeOH, 11h (0.042 g, 0.165 mmol) was added and the mixture was heated at reflux overnight. The solvent was evaporated and the crude product was purified by column chromatography (3:97 MeOH/CH₂Cl₂) giving compound 13 (0.052 g, 0.136 mmol, 95%). ¹H NMR (300 MHz, CD₃OD) δ 4.40 (s, 4 H), 4.25 (d, J = 9 Hz, 1 H), 2.74 (s, 3 H), 1.68 (m, 3 H), 0.99 (d, J = 6 Hz, 15 H); ¹³C NMR (75 MHz, CD₃OD) δ 188.7, 187.9, 184.2, 183.7, 178.0, 176.9, 173.1, 172.6, 172.0, 171.9, 171.4, 60.8, 60.7, 59.9, 56.8, 56.1, 25.6, 24.6, 24.5, 22.1, 20.0; HRMS-ESI (M + Na⁺) calcd for C₁₈H₂₉N₃O₅Na 390.2005, found 390.2010.

3-Hydroxy-4-(L-leucyl-L-tert-leucine methyl amide)-3-cyclobutene-1,2-dione (14)

To a stirred solution of 13 (0.05 g, 0.136 mmol) in MeOH (3 mL), 0.15 N HCl (1 mL) was added and the mixture was heated at reflux overnight. The solvent was evaporated and the crude product was purified by column chromatography (3:97 MeOH/CH₂Cl₂) giving 14 (0.032 g, 0.091 mmol, 67%). ¹H NMR (300 MHz, CD₃OD) δ 4.22 (s, 1 H), 3.32 (s, 1 H), 2.74 (s, 3 H), 1.69 (m, 3 H), 0.96 (s, 15 H); ¹³C NMR (75 MHz, CD₃OD) δ 196.3, 187.8, 180.7, 172.5, 171.9, 61.3, 55.8, 40.6, 34.5, 26.1, 25.1, 24.8, 22.5, 20.9; HRMS-ESI (M − H⁺) calcd for C₁₇H₂₆N₃O₅ 352.1872, found 352.1880.

3-(Hydroxymethylamino)-2-(L-isoleucine methylester)-4-thioxo-2-cyclobuten-1-one (15)

Compound 6a (0.100 g, 0.370 mmol) was dissolved in 2 mL of CH₂Cl₂ and allowed to stir while Lawesson’s reagent (0.150 g, 0.370 mmol) was added. The reaction was monitored by TLC until all the starting material had been consumed. The solvent was then evaporated and the crude material was purified by column chromatography (5:95 MeOH/CH₂Cl₂) providing 15 as a yellow solid (0.062 g, 0.23 mmol, 62%). ¹H-NMR (300 MHz, CDCl₃) δ 9.02 (br s, 1 H), 8.19 (br s, 1 H), 5.93 (br s, 1 H), 3.76 (s, 3 H), 3.67 (s, 3 H), 2.20 (m, 1 H), 2.07 (m, 1 H), 1.48 (m, 1 H), 1.26 (m, 1 H), 1.00 (d, J = 6.8 Hz, 3 H), 0.93 (t, J = 7.3 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 205.4, 201.6, 170.9, 170.8, 170.6, 61.2, 52.9, 39.4, 31.6, 25.3, 15.8, 12.2; ESI-MS (M + H⁺) calcd for C₁₂H₁₉N₂O₃S₂ 271.1294, found 271.1287.

3-(Hydroxymethylamino)-2-(L-isoleucyl-L-phenylalanine methylamide)-4-thioxo-2- cyclobuten-1-one (16)

Compound 16 (0.093 g, 0.216 mmol, 71%) was prepared as a yellow solid according to the procedure used to prepare 15 starting from 12d (0.127 g, 0.305 mmol) and Lawesson’s reagent (0.123 g, 0.304 mmol). The crude material was purified by column chromatography (3:97 MeOH/CH₂Cl₂). ¹H-NMR (300 MHz, CD₃OD) 7.15 (m, 5 H), 5.56 (d, J = 7.6 Hz, 1 H), 4.90 (s, 3 H), 4.68 (m, 1 H), 3.63 (s, 2 H), 3.10 (m, 1 H), 2.72 (d, J = 0.6 Hz, 3 H), 1.91 (m, 1 H), 1.49 (m, 1 H) 1.17 (m, 1 H) 0.87 (m, 6 H); ¹³C NMR (75 MHz, CD₃OD) δ 206.7, 204.0, 172.7, 171.1, 170.7, 170.4, 137.1, 129.3, 128.4, 126.5, 60.8, 54.9, 38.3, 37.6, 30.4, 25.5, 24.4, 14.5, 10.3; ESI-MS (M + Na⁺) calcd for C₂₁H₂₈N₄O₄SNa 455.1729, found 455.1738.

3-(Hydroxymethylamino)-2-(L-isoleucyl-L-leucine methyl amide)-4-thioxo-2- cyclobuten-1-one (17)

Compound 17 was prepared as a yellow solid (0.013 g, 0.032 mmol, 59%) from 12g (0.021 g, 0.055 mmol) and Lawesson’s reagent (0.022 g, 0.055 mmol) according to the procedure used to prepare compound 15. The crude material was purified by column chromatography (1.3:98.7 MeOH/CH₂Cl₂). ¹H NMR (300 MHz, CD₃OD) δ 5.67 (d, J = 6.8 Hz, 1 H), 4.39 (dd, J = 9.5, 5.2 Hz, 1 H), 3.63 (s, 3 H), 2.73 (s, 3 H), 2.00 (m, 1 H), 1.56 (m, 4 H), 1.24 (m, 1 H), 0.96 (m, 12 H); ¹³C NMR (75 MHz, CD₃OD) δ 206.6, 203.9, 173.5, 171.2, 170.9, 170.2, 60.5, 51.9, 40.6, 37.9, 29.9, 24.9, 24.5, 23.9, 22.0, 20.5, 14.2, 10.2; HRMS-FAB (M + Na⁺) calcd for C₁₈H₃₀N₄O₄SNa 421.1885, found 421.1880.

3-(Hydroxymethylamino)-2-(L-leucyl-L-tert-leucine methyl amide)-4-thioxo-2- cyclobuten-1-one (18)

Compound 18 was prepared as a yellow solid (0.014 g, 0.035 mmol, 41%) from 12h (0.033 g, 0.086 mmol) and Lawesson’s reagent (0.035 g, 0.086 mmol) according to the procedure used to prepare compound 15. The crude material was purified by column chromatography (1.3:98.7 MeOH/CH₂Cl₂). ¹H NMR (400 MHz, CD₃OD) δ 5.92 (s, 1 H), 4.21 (s, 1 H), 3.62 (s, 3 H), 2.74 (s, 3 H), 1.72 (m, 3 H), 0.98 (m, 15 H); ¹³C NMR (100 MHz, CD₃OD) δ 206.1, 203.5, 171.7, 171.4, 170.7, 170.0, 61.2, 54.8, 40.7, 34.2, 30.2, 25.9, 24.8, 24.5, 22.2, 20.6; LRMS-FAB (M + Na⁺) calcd for C₁₈H₃₀N₄O₄SNa 421.0, found 421.1.

3,4-Di-(L-phenylalanine methyl ester)-3-cyclobutene-1,2-dione (19)

To a stirred solution of L-phenylalanine methyl ester HCl salt (0.646 g, 3.0 mmol) in MeOH (10 ml), KOH (0.168 g, 3.0 mmol) and compound 7 (0.256 g, 1.13 mmol) were added. Formation of a white precipitate was observed. The mixture was stirred for 3 h at room temperature, the solvent was evaporated, EtOAc was added and the solution was washed with H₂O. The organic layer was dried over MgSO₄, and the solvent was evaporated. The crude product was recrystallized from MeOH. The resulting white crystals were washed with cold MeOH and dried in vacuo to give 19 as a white solid (0.363 g, 0.831 mmol, 74%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (br s, 1 H), 7.27 (m, 6 H), 7.14 (d, J = 6.4 Hz, 4 H), 5.00 (d, J = 6.1 Hz, 2 H), 3.70 (s, 6 H), 3.17 (dd, J = 13.5, 4.9 Hz, 2 H), 3.06 (m, 2 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 183.3, 171.6, 167.5, 136.4, 129.8, 128.9, 127.3, 57.2, 55.3, 52.8; HRMS-ESI (M + H⁺) calcd for C₂₄H₂₅N₂O₆ 437.1713, found 437.1722.

2,3-Bis(L-phenylalanine methyl ester)-4-thioxo-2-cyclobuten-1-one (20)

To a stirred solution of 19 (0.112 g, 0.257 mmol) in dry CH₂Cl₂ (5 ml), Lawesson’s reagent (0.021 g, 0.052 mmol) was added. After TLC analysis indicated that the reaction was complete, the crude product was purified by column chromatography (gradient of 0.25–1.0% MeOH/CH₂Cl₂) to yield compound 20 as a yellow solid (0.038 g, 0.084 mmol, 40%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.76 (d, J = 7.9 Hz, 1 H), 8.31 (d, J = 9.1 Hz, 1 H), 7.21 (m, 10 H), 6.12 (dd, J = 15.0, 6.3 Hz, 1 H), 5.01 (dd, J = 12.7, 6.7 Hz, 1 H), 3.73 (s, 3 H), 3.70 (s, 3 H), 3.18 (m, 4 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 204.0, 180.4, 172.0, 171.4, 170.8, 168.9, 136.0, 129.8, 129.7, 129.0, 128.9, 127.5, 127.4, 58.1, 55.8, 53.1, 52.9; HRMS-ESI (M + H⁺) calcd for C₂₄H₂₅N₂O₅S 453.1484, found 453.1490.

3-(Hydroxymethylamino)-2-(L-phenylalanine methyl ester)-4-thioxo-2-cyclobuten-1- one (21)

To a stirred solution of 6e (0.171 g, 0.562 mmol) in dry CH₂Cl₂ (5 ml), Lawesson’s reagent (0.227 g, 0.561 mmol) was added. After TLC analysis indicated that the reaction was complete, the solvent was evaporated and the crude product was purified by column chromatography (CH₂Cl₂) to yield compound 21 as a yellow solid (0.104 g, 0.325 mmol, 58%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (br s, 1 H), 8.56 (br s, 1 H), 7.24 (m, 5 H), 6.23 (t, J = 6.0, 1 H), 3.72 (s, 3 H), 3.50 (s, 3 H), 3.21 (m, 2 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 206.5, 203.2, 171.0, 170.7, 170.6, 135.7, 129.9, 129.0, 127.5, 56.3, 53.1, 30.8; HRMS-FAB (M + Na⁺) calcd for C₁₅H₁₆N₂O₄SNa 343.0729, found 343.0740.

Procedure for the Assay of MMP-1

MMP-1, prepared from a culture medium of human rheumatoid synovial fibroblasts, was obtained from Calbiochem. The assays were based on the enzymatic hydrolysis of the peptide substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Nma)-NH₂,¹⁸ using the procedure described by Le Diguarher.¹⁹ This is a fluorescence quenching assay in which N-methylanthranilic acid (Nma) is the fluorophore and dinitrophenyl (Dnp) is the quencher.

Pro-MMP-1 was dissolved in assay buffer (200 mM NaCl, 50 mM Tris, 5 mM CaCl₂, 20 μM ZnSO₄, 0.05% Brij 35, pH 7.6) at a concentration of 1.25 μg/mL. APMA solution (p-aminophenylmercuric acetate, 2 mM in 0.1 N NaOH) was prepared. Proenzyme activation was performed by mixing the proenzyme solution and the APMA solution in a 10:1 ratio. This reaction was incubated at 37 °C for 30 min and subsequently transferred to ice and then to a freezer. The substrate was dissolved in DMSO at a concentration of 2 mM, and then diluted to 0.2 mM with H₂O. Inhibitors were dissolved in a 10:90 DMSO/buffer solution. Fluorescence measurements were performed in 96-well plates by incubating assay buffer (76 μl), activated enzyme (4 μl), and the inhibitor solution (or buffer for the blank) (10 μl) at 37 °C for 30 min and then adding the substrate (10 μl) to the wells. The change in fluorescence was measured using excitation and emission wavelengths of 340 nm and 460 nm, respectively. IC₅₀ values were calculated using the commercial graphing package Grafit (Erithacus Software Ltd.). Data was obtained for assays with at least 5 different concentrations, in duplicate, of each inhibitor.

Supplementary Material

1si20051013_01. Supporting Information Available.

¹H and ¹³C NMR spectra for all new compounds; Lineweaver-Burk plot for compound 16 (50 pages). This information is available free of charge via the internet at http://pubs.acs.org.

TABLE 1.

Inhibition of MMP-1 by Compounds 4a–f

compound	R1	R2	IC50 (mM)a
4a	H	-CH2CH(CH3)2	1.8 ± 0.3
4b	H	-(CH2)5CH3	1.0 ± 0.2
4c	Me	-(CH2)5CH3	0.31 ± 0.03
4d	Me	-(CH2)4CH3	0.31 ± 0.03
4e	cyclohexyl	-(CH2)5CH3	>10
4f	benzyl	-(CH2)5CH3	>10

^aAll experiments were performed in duplicate.

TABLE 3.

Inhibition of MMP-1 by Dipeptides 12 a–h^a

compound	R3 =side chain of amino acid	R4 =side chain of amino acid	IC50 (μM)a
12a	Ile	Tle	500 ± 100
12b	Ile	Chg	300 ± 100
12c	Ile	Tyr(Me)	280 ± 30
12d	Ile	Phe	270 ± 30
12e	Ile	Phg	260 ± 25
12f	Ile	Trp	210 ± 30
12g	Ile	Leu	200 ± 20
12h	Leu	Tle	95 ± 7

^aAll experiments performed in duplicate.