Squaric acid-based peptidic inhibitors of matrix metalloprotease-1

Article abstract of DOI:10.1021/jo0517848

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.

Full text of DOI:10.1021/jo0517848

Squaric Acid-Based Peptidic Inhibitors of Matrix
Metalloprotease-1
M. Burak Onaran,† Anthony B. Comeau,† and Christopher T. Seto*
Department of Chemistry, Brown University, Providence, Rhode Island 02912
christopher_seto@brown.edu
Received August 24, 2005
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
joints that causes osteoarthritis6 and rheumatoid arthri-
tis,7 periodontal disease,8 and multiple sclerosis.9 There-
fore, 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.
Matrix Metalloproteases (MMPs). Matrix metallo-
proteases are a family of structurally related endopep-
tidases 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 inhibi-
tors of metalloproteases (TIMPs).2,3 Overexpression of
MMPs and deregulation of their activity are associated
with a variety of pathological conditions including tumor
growth and metastasis,4 angiogenesis,5 destruction of
(5) Fisher, C.; Gilbertson-Beadling, S.; Powers, E. A.; Petzold, G.;
Poorman, R.; Mitchell, M. A. Dev. Biol. 1994, 162, 499-510.
(6) Billinghurst, R. C.; Dahlberg, L.; Ionescu, M.; Reiner, A.; Bourne,
R.; Rorabeck, C.; Mitchell, P.; Hambor, J.; Diekmann, O.; Tschesche,
H.; Chen, J.; Van Wart, H.; Poole, A. R. J. Clin. Invest. 1997, 7, 1534-
1545.
† These two authors contributed equally to this work.
(1) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. H. Chem.
Rev. 1999, 99, 2735-2776.
(7) Jackson, C.; Nguyen, M.; Arkell, J.; Sambrook, P. Inflammation
Res. 2001, 50, 183-186.
(2) Jones, C. B.; Sane, D. C.; Herrington, D. M. Cardiovasc. Res.
2003, 59, 812-823.
(8) Lee, W.; Aitken, S.; Sodek, J.; McCulloch, C. A. J. Periodontal
Res. 1995, 30, 23-33.
(3) Chakraborti, S.; Mandal, M.; Das, S.; Mandal, A.; Chakraborti,
T. Mol. Cell. Biochem. 2003, 253, 269-285.
(9) Liedtke, W.; Cannella, B.; Mazzaccaro, R. J.; Clements, J. M.;
Miller, K. M.; Wucherpfennig, K. W.; Gearing, A. J. H.; Raine, C. S.
Ann. Neurol. 1998, 44, 35-46.
(4) Westermarck, J.; Kahari, V.-M. FASEB J. 1999, 13, 781-792.
10.1021/jo0517848 CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/17/2005
10792
J. Org. Chem. 2005, 70, 10792-10802

Study of Squaric Acid-Based Peptidic Inhibitors
vinylogous hydroxamic acids that are based upon squaric
acid as inhibitors of MMPs. We have derivatized the
vinylogous hydroxamic acids with peptides to target them
to the active site of the proteases.
FIGURE 1. Resonance structures of squaric acid.
Results and Discussion
Synthesis of Compounds 4a-f. We began our stud-
ies 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.
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, and 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 sub-
strate, 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. Com-
pound 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 atten-
tion to inhibitors that incorporated a single amino acid
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
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.
FIGURE 2. Known binding mode of hydroxamic acid I versus
proposed binding mode by squaric acid derivative II.
Our research group has used squaric acid derivatives
to mimic the phosphate group in phosphotyrosine resi-
dues. We prepared a number of 3-hydroxy-4-arylcyclobut-
3-enediones as nonhydrolyzable isosteres of aryl phos-
phate esters and found that these compounds are effective
inhibitors of protein tyrosine phosphatases.10 Kim and
co-workers 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. Shi-
nada 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 co-
workers 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 re-
placement 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 metal-
loproteases. X-ray crystal structures show that hydrox-
amic acids chelate to the active site zinc atom as shown
in structure I (Figure 2).16 In addition, Bruckner and co-
workers 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
(10) Xie, J.; Comeau, A. B.; Seto, C. T. Org. Lett. 2004, 6, 83-86.
(11) Kim, C. U.; Misco, P. F. Tetrahedron Lett. 1992, 33, 3961-3962.
(12) Sato, K.; Seio, K.; Sekine, M. J. Am. Chem. Soc. 2002, 124,
12715-12724.
(13) Shinada, T.; Nakagawa, Y.; Hayashi, K.; Corzo, G.; Nakajima,
T.; Ohfune, Y. Amino Acids 2003, 24, 293-301.
(14) Sun, L.; Chiu, D.; Kowal, D.; Simon, R.; Smeyne, M.; Zukin, R.
S.; Olney, J.; Baudy, R.; Lin, S. J. Pharmacol. Exp. Ther. 2004, 310,
563-570.
(15) Butera, J. A.; Antane, M. M.; Antane, S. A.; Argentieri, T. M.;
Freeden, C.; Graceffa, R. F.; Hirth, B. H.; Jenkins, D.; Lennox, J. R.;
Matelan, E.; Norton, N. W.; Quagliato, D.; Sheldon, J. H.; Spinelli,
W.; Warga, D.; Wojdan, A.; Woods, M. J. Med. Chem. 2000, 43, 1187-
1202.
(16) Lovejoy, B.; Welch, A. R.; Carr, S.; Luong, C.; Broka, C.;
Hendricks, T.; Campbell, J. A.; Walker, K. A. M.; Martin, R.; Van Wart,
H.; Browner, M. F. Nat. Struct. Biol. 1999, 6, 217-221.
(17) Lim, N. C.; Morton, M. D.; Jenkins, H. A.; Bruckner, C. J. Org.
Chem. 2003, 68, 9233-9241.
Dipeptide Inhibitors. To improve the potency of the
inhibitors, we extended their structure by incorporating
(18) Bickett, D. M.; Green, M. D.; Berman, J.; Dezube, M.; Howe,
A. S.; Brown, P. J.; Roth, J. T.; McGeehan, G. M. Anal. Biochem. 1993,
212, 58-64.
(19) Le Diguarher, T.; Chollet, A.-M.; Bertrand, M.; Hennig, P.;
Raimbaud, E.; Sabatini, M.; Guilbaud, N.; Pierre, A.; Tucker, G. C.;
Casara, P. J. Med. Chem. 2003, 46, 3840-3852.
J. Org. Chem, Vol. 70, No. 26, 2005 10793

Onaran et al.
SCHEME 1a
a Reagents: (a) CH(OCH3)3, MeOH, ∆; (b) HONHR1‚HCl, KOH, MeOH; (c) H2NR2, MeOH.
SCHEME 2a
TABLE 2. Inhibition of MMP-1 by Compounds 6a-g
R3 ) side chain
of amino acid
compd
R1
IC50 (µM)a
6a
6b
6c
6d
6e
6f
Me
Ile
190 ( 30
210 ( 10
280 ( 20
320 ( 20
330 ( 20
380 ( 50
300 ( 50
-CH(CH3)2
Ile
Me
Me
Me
Me
Me
Nleb
Leu
Phe
Trp
Met
6g
a Reagents: (a) MeOH, KOH. bRacemic Nle was used to prepare
5b and 6c.
a All experiments were performed in duplicate. b Racemic Nle
was used to prepare compound 6c.
TABLE 1. Inhibition of MMP-1 by Compounds 4a-f
amino acids 9a-g with methylamine to give the corre-
sponding 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 improve-
ment 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 com-
parison. Both incorporate a bulky tert-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).
compd
R1
R2
IC50 (mM)a
4a
4b
4c
4d
4e
4f
H
-CH2CH(CH3)2
-(CH2)5CH3
-(CH2)5CH3
-(CH2)4CH3
-(CH2)5CH3
-(CH2)5CH3
1.8 ( 0.3
1.0 ( 0.2
0.31 ( 0.03
0.31 ( 0.03
>10
H
Me
Me
cyclohexyl
benzyl
>10
a All experiments were performed in duplicate.
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.
Squaric acid was treated with tributyl orthoformate
to give dibutyl ester 7. Subsequent reaction of 7 with
N-methylhydroxylamine yielded compound 8. The dipep-
tide building blocks were prepared by coupling N-Boc
(20) Borkakoti, N.; Winkler, F. K.; Williams, D. H.; D’Arcy, A.;
Broadhurst, M. J.; Brown, P. A.; Johnson, W. H.; Murray, E. J. Nat.
Struct. Biol. 1994, 1, 106-110.
10794 J. Org. Chem., Vol. 70, No. 26, 2005

Study of Squaric Acid-Based Peptidic Inhibitors
SCHEME 3a
a Reagents: (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.
TABLE 3. Inhibition of MMP-1 by Dipeptides 12a-ha
atoms. Both of these factors favor binding to the metal
center.22
Thiocarbonyl-Containing vs Carbonyl-Contain-
ing 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 hydrox-
amic acids. One such example has been published by
Cohen and co-workers, who reported hydroxypyridinone
and pyrone ligands as promising alternatives to hydrox-
amic 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.
R3 ) side chain
R4 ) side chain
compd
of amino acid
of amino acid
IC50 (µM)a
12a
12b
12c
12d
12e
12f
Ile
Ile
Ile
Ile
Ile
Ile
Ile
Leu
Tle
500 ( 100
300 ( 100
280 ( 30
270 ( 30
260 ( 25
210 ( 30
200 ( 20
95 ( 7
Chg
Tyr(Me)
Phe
Phg
Trp
Leu
Tle
12g
12h
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 spec-
trometry. 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).
a All experiments performed in duplicate.
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 analogue 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.
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 Zn2+ (right side of Figure 3).21 By compari-
son, 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
Determining the Position of Sulfur Incorpora-
tion. The inhibitors in Scheme 5 contain three or four
(22) An X-ray crystal structure of a hydroxamic acid/zinc complex
shows that the bite angle in this complex is 81.1°. Ruf, M.; Weis, K.;
Brasack, I.; Vahrenkamp, H. Inorg. Chim. Acta 1996, 250, 271-281.
For comparison, we used HyperChem 3D to calculate the bite angle
in a zinc complex of the squaric acid/hydroxamic acid hybrids, which
was calculated to be 117.9°.
(23) Reich, R.; Katz, Y.; Hadar, R.; Breuer, E. Clin. Cancer Res. 2005,
11, 3925-3929.
(24) Puerta, D. T.; Lewis, J. A.; Cohen, S. M. J. Am. Chem. Soc.
2004, 126, 8388-8389.
(21) Solans, X.; Aguilo, M.; Gleizes, A.; Faus, J.; Julve, M.; Ver-
daguer, M. Inorg. Chem. 1990, 29, 775-784.
J. Org. Chem, Vol. 70, No. 26, 2005 10795

Onaran et al.
SCHEME 4a
a Reagents: (a) H-Leu-Tle-NHMe, MeOH, reflux; (b) 0.15 N HCl, MeOH, reflux.
other is shifted to 6.1 ppm (Hb shown in red). Hb 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 R-proton
(Hd). When this compound is converted to the monothio-
carbonyl adduct 21, this proton shifts downfield to 6.2
ppm (He 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 Lawes-
son’s reagent. Since we observe a similar shift for the
R-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 3. Six-membered vs five-membered zinc chelation
models.
SCHEME 5. Thionation of Cyclobutenediones
SCHEME 6a
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 analogues, 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 that of 12d. By contrast, compounds
12g and 12h have similar IC50 values when compared to
their thiocarbonyl analogues 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
12h do not change upon thionation is that the improve-
ment 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 analogues
a Reagents: (a) L-Phe-OMe‚HCl, KOH, MeOH; (b) Lawesson’s
reagent, CH2Cl2, 25 °C.
different carbonyl groups, any of which could be the site
of reaction with Lawesson’s reagent. To determine the
specific site that sulfur was incorporated into the mol-
ecules, we prepared two model compounds to aid us with
this analysis (Scheme 6). Dibutyl squarate 7 was treated
with 2 equiv of NH2-Phe-OMe to give compound 19. When
compound 19 was treated with <1 equiv 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.
The 1H NMR spectrum of compound 19 (Figure 4) has
a resonance at 5.0 ppm that corresponds to the R-protons
of the two Phe residues (Ha). When this compound is
treated with Lawesson’s reagent to give compound 20,
one of the R-protons remains at 5.0 ppm (Hc) while the
10796 J. Org. Chem., Vol. 70, No. 26, 2005

Study of Squaric Acid-Based Peptidic Inhibitors
activity. Among the inhibitors that do not contain a sulfur
atom, we found that the dipeptide Leu-Tle-NHMe pro-
vided the highest level of activity when attached to the
cyclobutenedione core.
We also developed a regioselective method for convert-
ing one of the carbonyl groups on the core into the
corresponding thiocarbonyl compound. This reaction oc-
curs specifically at the position on the opposite side of
the cyclobutene ring from the peptide substituent. Thion-
ation 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 improve-
ment resulted in an inhibitor with an IC50 value of 15
µM against MMP-1.
Experimental Section
FIGURE 4. Changes that occur in the 1H NMR spectra of
compounds 19 and 6e upon monothionation of the cyclobutene-
dione core.
Full characterization for compounds3b, 3c, and 3e has been
reported in the literature.17
3-(Hydroxyamino)-4-methoxy-3-cyclobutene-1,2-di-
one (3a). 3,4-Dimethoxy-3-cyclobutene-1,2-dione (1) (200 mg,
1.4 mmol) was added to 10 mL of MeOH and the solution was
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 mate-
rial was purified by column chromatography (1:9 MeOH/CH2-
Cl2) to give compound 3a as a yellow solid (0.093 g, 0.650
TABLE 4. Inhibition of MMP-1 by
Thiocarbonyl-Containing Inhibitors
compd no.
compd no.
R
for X ) S IC50 (µM)a for X ) O IC50 (µM)
HN-Ile-OMe
15
16
17
18
70 ( 8
15 ( 1
170 ( 40
96 ( 30
6a
190 ( 30
270 ( 30
200 ( 20
95 ( 7
HN-Ile-Phe-NHMe
HN-Ile-Leu-NHMe
HN-Leu-Tle-NHMe
12d
12g
12h
1
mmol, 47%). H NMR (300 MHz, CD3OD) δ 4.37 (s, 3 H); 13C
a All experiments performed in duplicate.
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.
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.
3-[(Benzyl)hydroxyamino]-4-methoxy-3-cyclobutene-
1,2-dione (3d). This compound was prepared with use of
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.
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.
3-(Hydroxyamino)-4-[(2-methylpropyl)amino]-3-cyclo-
butene-1,2-dione (4a). To a solution of compound 3a (0.055
g, 0.383 mmol) dissolved in MeOH (1 mL) was added isobu-
tylamine (0.062 g, 0.844 mmol) 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
1
mmol, 13%) as a white solid. H 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 re-
moved 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
Among the alkyl groups that we have examined at the
-N(OH)alkyl position of the inhibitors, small substitu-
ents 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
1
mmol, 12%). H NMR (300 MHz, DMSO-d6) δ 3.35 (m, 2 H),
J. Org. Chem, Vol. 70, No. 26, 2005 10797

Onaran et al.
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.
11.8; HRMS-ESI (M + H+) calcd for C14H23N2O5 299.1607,
found 299.1602.
3-(Hydroxymethylamino)-4-(D,L-norleucine methyl es-
ter)-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
3-(Hexylamino)-4-(hydroxymethylamino)-3-cyclo-
butene-1,2-dione (4c). To a solution of 3b (0.157 g, 1.0 mmol)
in MeOH (1 mL) was added hexylamine (0.111 g, 1.1 mmol)
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
1
preparing 6a. H 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.
1
solid. H 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-(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 C12H18N2O5-
Na 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-(pentylamino)-3-cyclo-
butene-1,2-dione (4d). Compound 4d (0.058 g, 0.27 mmol,
37%) was prepared from 3b (0.118 g, 0.75 mmol) and pentyl-
amine (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-cyclo-
butene-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 the 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-cyclo-
butene-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) was added the hydro-
chloride salt of L-isoleucine methyl ester (5a) (0.229 g, 1.26
mmol). To this stirred solution was added KOH (0.0713 g, 1.27
mmol), 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 chro-
matography (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-(Hydroxymethylamino)-4-(L-tryptophan methyl es-
ter)-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
1
preparing 6a. H 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 es-
ter)-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
1
preparing 6a. H 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) were added KOH (1.767 g,
31.5 mmol) and compound 7 (4.766 g, 21.0 mmol). 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 and dried over
MgSO4, and the solvents were evaporated under reduced
pressure. The crude product was purified by column chroma-
tography (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,
3-[Hydroxy(1-methylethyl)amino]-4-(L-isoleucine meth-
yl 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,
10798 J. Org. Chem., Vol. 70, No. 26, 2005

Study of Squaric Acid-Based Peptidic Inhibitors
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.
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-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 was added NH2Me (4.02 mL,
2.0 M in MeOH) 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 and 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 was added
TFA (15 mL) 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
L-Tryptophan Methyl Amide (10f). The synthesis and the
1H NMR spectrum for this compound have been reported
previously.24 13C 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), NH2-
Me (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
1
to the procedure used for preparing 10a. H 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 was added 10a (0.144 g, 1.0 mmol) 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 and 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 CH2-
Cl2 (2 mL). To this solution was added TFA (2 mL) and the
mixture was stirred at room temperature for 40 min. The
solvents and TFA were removed, and the residue was parti-
tioned between CH2Cl2 (15 mL) and saturated aqueous NaH-
CO3 (15 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 solvent
was evaporated. Purification was performed by flash chroma-
tography (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.
1
a colorless oil (0.637 g, 4.42 mmol, 66%). H 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) was added HOBt (760 mg,
4.97 mmol) and then EDC (911 mg, 4.75 mmol) and the
mixture was stirred at 0 °C for 30 min. Formation of a white
precipitate was observed. To this solution was added NH2Me
(2.49 mL, 2.0 M in MeOH) via syringe and the reaction mixture
was stirred overnight. The solution was diluted with CH2Cl2
(50 mL) and washed once with 1 M citric acid (30 mL), once
with saturated NaHCO3 (30 mL), and then once 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 was added TFA (15 mL) 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
1
white solid (440 mg, 2.80 mmol, 62%). H 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 13C 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
L-Isoleucyl-L-cyclohexylglycine Methyl Amide (11b).
N-Boc-L-Isoleucine (140 mg, 0.584 mmol) was dissolved in CH2-
Cl2 (20 mL) and the solution was cooled to 0 °C. To this
solution were added HOBt (89.5 mg, 0.584 mmol) and EDC
(201 mg, 0.558 mmol) and the mixture was stirred for 30 min.
To this mixture was then added compound 10b (83.5 mg, 0.531
(26) Reichard, G. A.; Stengone, C.; Paliwal, S.; Mergelsberg, I.;
Majmundar, S.; Wang, C.; Tiberi, R.; McPhail, A. T.; Piwinski, J. J.;
Shih, N.-Y. Org. Lett. 2003, 5, 4249-4251.
(25) Kortylewicz, Z. P.; Galardy, R. E. J. Med. Chem. 1990, 33, 263-
273.
J. Org. Chem, Vol. 70, No. 26, 2005 10799

Onaran et al.
mmol) and the reaction mixture was stirred overnight. The
solution was diluted with CH2Cl2 (50 mL) and washed once
with 1 M citric acid (30 mL), once with saturated NaHCO3
(30 mL), and then once 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 was added TFA (15 mL) 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 evapo-
rated. Purification was performed by flash chromatography
(5% MeOH/CH2Cl2) yielding 11b as a white solid (69 mg, 0.244
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
1
(2.5 mL) according to the procedure used to prepare 11a. H
NMR (300 MHz, CD3OD) δ 4.4 (t, J ) 7.5 Hz, 1 H), 3.23 (d, J
) 5.5 Hz, 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
1
mmol, 46%). H 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.
1
11a. H NMR (400 MHz, CD3OD) δ 4.22 (s, 1 H), 3.46 (t, J )
5.3 Hz, 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 equiv) dissolved in MeOH
was added 11a-h (1.0 equiv) 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-leu-
cine methyl amide)-3-cyclobutene-1,2-dione (12a). Com-
pound 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
1
to the general procedure. H 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.
L-Isoleucyl-L-phenylalanine Methyl Amide (11d). Com-
pound 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
1
11b. H NMR (400 MHz, CDCl3) δ 8.04 (d, J ) 8.8 Hz, 1 H),
3-(Hydroxymethylamino)-4-(L-isoleucyl-L-cyclohexyl-
glycine 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
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.
1
solid according to the general procedure. H 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.
L-Isoleucyl-L-phenylglycine Methyl Amide (11e). Com-
pound 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
3-(Hydroxymethylamino)-4-(L-isoleucyl-O-methyl-L-ty-
rosine methyl amide)-3-cyclobutene-1,2-dione (12c). Com-
pound 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
1
11a. H 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 C15H23N3O2-
Na 300.1688, found 300.1680.
1
to the general procedure. H 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.
L-Isoleucyl-L-tryptophan Methyl Amide (11f). Com-
pound 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
Hz, 1 H), 3.2 (d, J ) 6.5 Hz, 1 H), 2.62 (d, J ) 1.9 Hz, 3 H),
1.60 (m, 1 H), 1.14 (m, 1 H), 0.9 (m, 1 H), 0.75 (m, 6 H); 13C
3-(Hydroxymethylamino)-4-(L-isoleucyl-L-phenylala-
nine methyl amide)-3-cyclobutene-1,2-dione (12d). Com-
pound 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
10800 J. Org. Chem., Vol. 70, No. 26, 2005

Study of Squaric Acid-Based Peptidic Inhibitors
1
solid according to the general procedure. H NMR (300 MHz,
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.
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)-2-(L-isoleucine methyl ester)-
4-thioxo-2-cyclobuten-1-one (15). Compound 6a (0.100 g,
0.370 mmol) was dissolved in 2 mL of CH2Cl2 and the solution
was 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
3-(Hydroxymethylamino)-4-(L-isoleucyl-L-phenylgly-
cine methyl amide)-3-cyclobutene-1,2-dione (12e). Com-
pound 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
1
to the general procedure. H 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.
1
a yellow solid (0.062 g, 0.23 mmol, 62%). H 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)-4-(L-isoleucyl-L-trypto-
phan methyl amide)-3-cyclobutene-1,2-dione (12f). Com-
pound 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
3-(Hydroxymethylamino)-2-(L-isoleucyl-L-phenylala-
nine methyl amide)-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 meth-
yl 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.
1
to the general procedure. H 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 meth-
yl amide)-3-cyclobutene-1,2-dione (12 g). 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-leucinemeth-
yl 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
1
procedure. H 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 was added 11h (0.042 g, 0.165 mmol)
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-(Hydroxymethylamino)-2-(L-leucyl-L-tert-leucine
methyl amide)-4-thioxo-2-cyclobuten-1-one (18). Com-
pound 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) were
added KOH (0.168 g, 3.0 mmol) and compound 7 (0.256 g, 1.13
mmol). 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
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) was added 0.15 N HCl (1 mL)
J. Org. Chem, Vol. 70, No. 26, 2005 10801

Onaran et al.
washed with cold MeOH and dried in vacuo to give 19 as a
blasts, 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 proce-
dure 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 subse-
quently 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 with use of excitation and emission wavelengths of
340 and 460 nm, respectively. IC50 values were calculated with
the commercial graphing package Grafit (Erithacus Software
Ltd.). Data were obtained for assays with at least five different
concentrations, in duplicate, of each inhibitor.
1
white solid (0.363 g, 0.831 mmol, 74%). H 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-cyclo-
buten-1-one (20). To a stirred solution of 19 (0.112 g, 0.257
mmol) in dry CH2Cl2 (5 mL) was added Lawesson’s reagent
(0.021 g, 0.052 mmol). After TLC analysis indicated that the
reaction was complete, the crude product was purified by
column chromatography (gradient of 0.25-1.0% MeOH/CH2-
Cl2) 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 solu-
tion of 6e (0.171 g, 0.562 mmol) in dry CH2Cl2 (5 mL) was
added Lawesson’s reagent (0.227 g, 0.561 mmol). 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 Hz, 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 fibro-
Acknowledgment. We thank Mr. Jian Xie for
helpful discussions. This research was supported by the
NIH NIGMS (Grant R01 GM057327).
Supporting Information Available: 1H and 13C NMR
spectra for all new compounds; Lineweaver-Burk plot for
compound 16. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO0517848
10802 J. Org. Chem., Vol. 70, No. 26, 2005