Design and synthesis of sulfur based inhibitors of matrix metalloproteinase-1.

Article abstract of DOI:10.1248/cpb.50.239

Fibroblast collagenase (MMP-1), a member of the matrix metalloproteinases family, is believed to be a pathogenesis of arthritis, by cleaving triple-helical type II collagen in cartilage. From the similarity of the active site zinc binding mode with hydroxamate, we designed and synthesized alpha-mercaptocarbonyl possessing compounds (3-5), which incorporated various peptide sequences as enzyme recognition sites. The P4-P1 peptide incorporating compound (3) exhibited as potent inhibition as the hydroxamate (1) and the carboxylate (2) type inhibitors, with an IC50 of 10(-6) M order against MMP-1. But the inhibitor (3) related compounds (6-8) displayed decreased or no inhibitory potencies. These results suggest that the existence of both the carbonyl and thiol groups might be critical for the inhibition, and the distance between the two functional groups is important for inhibitory potency. For Pn' peptide incorporating compounds (4a-k), except for 4h and 4k, all compounds showed IC50 values under sub-nanomolar. Among them, for potent inhibition, Leu was better than Phe and Val as the P1' amino acid, and the P2' position amino acid was necessary, and preferentially Phe. Insertion of the Pn peptide into 4d or 4k, giving compounds 5a-c, did not increase the activities of 4d and 4k. Substitution of the mercapto group with other functional groups lost the activity of compound 4a. The stereochemical preference at the thiol-attached position was also determined by preparation of both isomers of 4a. It was found that the S configuration compound (36b) is approximately 100 times more potent than the corresponding R-isomer (36a).

Full text of DOI:10.1248/cpb.50.239

February 2002
Chem. Pharm. Bull. 50(2) 239—252 (2002)
239
Design and Synthesis of Sulfur Based Inhibitors of Matrix
Metalloproteinase-1
Tetsunori FUJISAWA, Shinjiro ODAKE, Yuji OGAWA, Junko YASUDA, Yasuo MORITA, and
Tadanori MORIKAWA*
Research Institute, Fuji Chemical Industries, Ltd., 530 Chokeiji, Takaoka, Toyama 933–8511, Japan.
Received September 13, 2001; accepted November 16, 2001
Fibroblast collagenase (MMP-1), a member of the matrix metalloproteinases family, is believed to be a
pathogenesis of arthritis, by cleaving triple-helical type II collagen in cartilage. From the similarity of the active
site zinc binding mode with hydroxamate, we designed and synthesized a-mercaptocarbonyl possessing com-
pounds (3—5), which incorporated various peptide sequences as enzyme recognition sites. The P4–P1 peptide in-
corporating compound (3) exhibited as potent inhibition as the hydroxamate (1) and the carboxylate (2) type in-
hibitors, with an IC50 of 10؊6 M order against MMP-1. But the inhibitor (3) related compounds (6—8) displayed
decreased or no inhibitory potencies. These results suggest that the existence of both the carbonyl and thiol
groups might be critical for the inhibition, and the distance between the two functional groups is important for
inhibitory potency. For Pn؅ peptide incorporating compounds (4a—k), except for 4h and 4k, all compounds
showed IC50 values under sub-nanomolar. Among them, for potent inhibition, Leu was better than Phe and Val
as the P1؅ amino acid, and the P2؅ position amino acid was necessary, and preferentially Phe. Insertion of the Pn
peptide into 4d or 4k, giving compounds 5a—c, did not increase the activities of 4d and 4k. Substitution of the
mercapto group with other functional groups lost the activity of compound 4a. The stereochemical preference at
the thiol-attached position was also determined by preparation of both isomers of 4a. It was found that the S con-
figuration compound (36b) is approximately 100 times more potent than the corresponding R-isomer (36a).
Key words matrix metalloproteinase; inhibitor; a-mercaptocarbonyl; isoserine; in vitro activity
Matrix metalloproteinases (MMPs) are zinc endometal-
A number of MMP inhibitors have progressed into clinical
lopeptidases that are involved in the degradation and remod- trials mainly for cancer and rheumatoid arthritis. In the struc-
eling of connective tissues. This family of enzymes shows tures of those MMPs inhibitors, a hydroxamate group is uti-
proteolytic activity towards virtually all of the constituents of lized as a zinc-chelating group (ZCG) predominantly be-
the extracellular matrix. The members of this family, cur- cause of its superior zinc-chelating potency. Previously, we
rently numbering 20, can be classified into four groups, explored MMP-1 inhibitors that had a hydroxamate group
which are: the collagenases that cleave triple-helical intersti- (1)11) and a carboxylate group (2).12) These compounds con-
tial collagen; the gelatinases that cleave denatured collagen, tain a P4–P1 peptide sequence and exhibited inhibitory activ-
elastin, and type IV and V collagen; the stromelysins that ity for MMP-1 in the order of 10Ϫ6 M for IC50 (Fig. 1). Our
mainly cleave proteoglycans; and the membrane-type MMPs objective is to explore the usefulness of novel ZCGs as in-
that have a C-terminal transmembrane domain for anchoring hibitors of MMPs, and in particular of MMP-1. A further
to the cell membrane. The MMPs are involved in crucial aim is to compare the MMP-1 inhibitory potencies of com-
physiological and physiopathological events, such as wound pounds that incorporate various enzyme recognition moi-
healing, nerve growth, angiogenesis, and pregnancy. In these eties.
physiological processes, MMP activity is tightly regulat-
Mercapto groups are good ZCGs, as found in Captopril,
ed.1—5) However, excessive MMP synthesis and release can which is a well-known angiotensin converting enzyme (ACE)
lead to connective tissue degradation and destruction, which inhibitor. In this study, we explored mercapto group based
occurs in tumor invasion, metastasis,6) corneal ulceration,7) compounds that incorporated various peptide sequences as
arthritis,8a) periodontal disease,9) and multiple sclerosis.10)
enzyme recognition functionalities.
Fig. 1. Structures of the Pn Peptide Based Inhibitors that Possess a Hydroxamate or a Carboxyate as ZCG and of Its MMP-1 Inhibitory Potencies
To whom correspondence should be addressed. e-mail: morikawa@daiichi-fcj.co.jp © 2002 Pharmaceutical Society of Japan

240
Vol. 50, No. 2
Fig. 2. The Design of an a-Mercaptocarbonyl Moiety Containing Compounds (3—5) and Related Compounds (6—8)
Design The X-ray crystal structures of MMP-1 with tert-butyloxycarbonyl alanine (Boc–Ala–OH) (9). A mixed
bound inhibitors reveal that the hydroxamate acts as a biden- anhydride, which was prepared from the reaction of 9 with
tate ligand with each oxygen at an optimal distance (2.1— ethyl chloroformate (ClCO2Et)/Et3N, was reacted with excess
2.2 Å) from the active-site zinc ion (structure A, Fig. 2).13) diazomethane to give a diazomethyl ketone (10). This was
From this structural analysis data, we reasoned that the a- converted to the bromomethyl ketone (11) by reaction with
mercaptocarbonyl group (structure B) at the cleavage site HBr in ether.16) The acetylsulfanylmethylketone (12) was ob-
would chelate at the active-site zinc ion by both carbonyl and tained by reaction of 11 with potassium thioacetate followed
mercapto groups in a similar manner as the hydroxamate by purification by silica gel column chromatography.
(Fig. 2). In comparison with the hydroxamate possessing
The deoxygenated compound of 12 depicted as 16 was
MMPs inhibitors, little is known about thiol group including synthesized as follows. The aldehyde (14), obtained via the
compounds. But, previously Donald and colleagues devel- aminoalcohol (13)17) from 9, was treated with the ylide de-
oped a b-mercapto carbonyl group possessing compound rived from potassium bis(trimethylsilyl)amide [[(CH3)3Si]2NK]
that exhibited 10Ϫ7 M order of IC50 against MMP-1.14) The and methyltriphenylphosphonium bromide (CH3PPh3Br), to
compound differs in the distance between the carbonyl and give the olefin (15).18) Then an addition of thioacetic acid to
mercapto groups from the structure B. In addition, Baxter compound 15 was carried out under ultraviolet light cat-
and colleagues have prepared the thiol containing inhibitors alyzed conditions19) to afford 16. The acetylsulfanylethylke-
utilizing computer-aided drug design, expecting new interac- tone (19) that differed from intermediate (12) by the distance
tions with MMP-8 by hydrogen bonds, e.g. Ser172 of MMP- between the carbonyl and mercapto groups, was prepared via
8.15) These compounds exhibited potent inhibitions with an the a,b-unsaturated compound (18).20) Thus the aminoalde-
IC50 of 10Ϫ7—10Ϫ9 M against MMP-3, -8, -9, but were not hyde (14) was transformed into the vinyl alcohol (17), which
tested on MMP-1. We designed three different compounds on Swern oxidation gave the corresponding a,b-unsaturated
containing an a-mercaptocarbonyl group, based on the en- ketone (18). Compound 19 was obtained by an 1,4-addition
zyme recognition sites. The P4–P1 peptide incorporating of thioacetic acid to 18.21) The alcohol derivative (21) was
compound (3), the P1Ј–P2Ј peptide incorporating compound prepared by an epoxide ring opening with thioacetic acid.22)
(4), and both Pn and PnЈ peptides incorporating compound (5) Thus the olefin (15) was treated with m-chloroperbenzoic
were designed. Also we designed three different thiol com- acid (MCPBA), which was allowed to react with thioacetic
pounds based on (3) to confirm the necessity of the carbonyl acid to give the desired alcohol (21). After deprotection of
group and the propriety of the distance between the carbonyl the Boc group from the intermediates (12), we initially uti-
and thiol group: one lacking the carbonyl group (6); insertion lized N-ethyl NЈ-(3-dimethylaminopropyl)carbodiimide (EDC)/
of a methylene spacer between the carbonyl and the mercapto 1-hydroxybenzotriazol (HOBt) as a coupling reagent for
group (7); and replacement of the carbonyl by a hydroxy the condensation with N-benzoyl–glycyl–prolyl–leucine (Bz–
group (8).
Gly–Pro–Leu–OH, 22). But unexpectedly, the desired com-
To verify the usefulness of the a-mercaptocarbonyl group pound (23a) was not obtained owing to the instability of the
for MMP-1 inhibitory potency, substitutions of the mercapto free amino compound. So we selected a mixed anhydride pro-
group with other functional groups (alkylsulfanyl, alkyl- cedure [iso-butylchloroformate/N-ethylmorpholine (NEM)],
sulfinyl, alkyldisulfanyl, hydroxyl, amino, carboxyl, or hy- by which an amide bond could be formed before degradation
droxamate) were conducted and compared with the mercapto of the free amine compound and succeeded in obtaining
group substituted compound against MMP-1 inhibitory po- compound 23a. Compounds 23b and 23c were also prepared
tencies.
by the same method. In the case of compound 23d, BOP
Synthesis The preparations of Pn peptide possessing Reagent [benzotriazol-1-yloxytris(dimethylamino)phospho-
compounds (3, 6—8) are summarized in Chart 1. The key in- nium hexafluorophosphate] was utilized as the coupling
termediates (12), (16), (19) and (21) were prepared from N- reagent to avoid the side reaction caused by the free hydroxy

February 2002
241
Reagents: (a) (1) ClCO2Et/Et3N (2) CH2N2; (b) 0.65 M HBr; (c) AcSK; (d) (1) HOSu/EDC (2) NaBH4; (e) (COCl)2/DMSO; (f) Ph3Pϩ-MeBrϪ/(TMS)2NK; (g) AcSH/UV; (h)
vinylmagnesium bromide; (i) MCPBA; (j) AcSH/Bu4NϩFϪ; (k) AcSH; (l) TFA or 4 N HCl/AcOEt; (m) ClCO2i-Bu/Et3N or BOP reagent; (n) conc.NH4OH/MeOH.
Chart 1. Syntheses of the P4–P1 Peptide Based Compounds
group. Finally, hydrolysis of the thioester (23a—d) gave the
free thiol compounds (3, 6—8).
PnЈ peptide incorporating compounds 4a—k were synthe-
sized by the route shown in Chart 2. The 3-amino-2-hydroxy-
propanoic acid (DL-isoserine), which was prepared from 2-
aminoethanol by Nishizawa and Saino23) was a suitable pre-
cursor for the construction of an a-mercaptocarbonyl moiety,
and the amino group was a good scaffold for the Pn peptide
sequence. The amino group in isoserine was protected by a
Boc group, followed by condensation with various dipeptides
or amino acid N-methylamides (25a—k) using EDC/HOBt
as a coupling reagent. Replacement of the hydroxy group
with an acetylsulfanyl group (27a—k) was achieved via a
mesylate intermediate. The free thiol (4a—k) was obtained
by an alkaline hydrolysis according to a standard procedure.
The physicochemical properties of 26a—k, 27a—k, and
4a—k are shown in Tables 1—6.
Reagents: (a) Et3N/HOBt/EDC; (b) (1) methanesulfonyl chloride/Et3N (2) AcSK; (c)
aq. NaOH/MeOH.
Syntheses of both Pn and PnЈ peptides possessing com-
pounds (5a—c) are summarized in Chart 3. The N-Boc
group of compounds 27d and 27k were removed by treat-
Chart 2. Syntheses of the P1Ј–P2Ј Peptide Incorporating Compounds
ment with 4 N HCl in EtOAc followed by condensation with
N-tert-butyloxycarbonyl–prolyl–leucine (Boc–Pro–Leu–OH,
28a) or N-tert-butyloxycarbonyl–leucine (Boc–Leu–OH,
28b) using a mixed anhydride procedure (ClCO2i-Bu/NEM)

242
Vol. 50, No. 2
Table 1. Physical Data of Compounds 26a—k
c were oxidized by treatment with equimolar amounts of
MCPBA to give the sulfoxides (38a—c). The unsymmetrical
disulfide (39) was synthesized from 4a using diethyl N-ethyl-
sulfenylhydrazodicarboxylate.26) The amino compound (41)
was also prepared via the mesylate followed by treating with
sodium azide, and a hydrogenation.27) The malonyl com-
pound 44 was synthesized from the 3-tert-butyloxycar-
bonylamino-2(R,S)-benzyloxycarbonylpropanoic acid (42),28)
which was coupled to H–Leu–Tyr(Me)–NHMe (25a) using
EDC/HOBt followed by catalytic hydrogenation in the pres-
ence of palladium on charcoal to give the malonyl compound
(44). We also prepared the hydroxamate compound 46, which
was constructed by the condensation of 44 and O-benzylhy-
droxylamine using EDC/HOBt as the coupling reagent and
subsequent removal of the benzyl group by means of a cat-
alytic hydrogenation.
Formula (MϩH),
HR-MS m/z
Calcd (Found)
Yield
(%)
mp
(°C)
[a]D25
Compd. A.A1Ј A.A2Ј
(c, MeOH)
26a
26b
26c
26d
26e
26f
26g
26h
26i
Leu Tyr(Me) 70 172—174
Ϫ28.0
(1.15)
C25H41N4O7
509.2975
(509.2937)
C20H39N4O6
431.2870
(431.2855)
C21H41N4O6
445.3026
(445.3029)
C24H39N4O6
479.2870
(479.2856)
C23H37N4O6
465.2713
(465.2712)
C27H37N4O6
513.2713
(513.2740)
C24H39N4O6
479.2870
(479.2877)
C19H37N4O6
417.2713
Leu Val
Leu Leu
Leu Phe
Phe Val
Phe Phe
Phe Leu
Val Val
Val Phe
Val Leu
69 143—145
93 137—141
86 119—121
75 194—197
82 160—163
77 152—154
62 185—187
83 189—193
81 171—174
68 142—146
Ϫ45.3
(1.03)
Ϫ47.6
(1.01)
Ϫ41.1
(1.03)
Results and Discussion
Ϫ29.2
(1.03)
Pn Peptide Incorporating Compounds As expected,
compound 3 exhibited strong MMP-1 inhibitory activity
(3.5 mM IC50), as potent as the hydroxamate (1) and the car-
boxylate (2). But modified compounds (6) and (7) were two
magnitudes less potent than 3 (530 and 220 mM IC50, respec-
tively). The alcohol compound (8) had no inhibitory potency
(0% inhibition at 10Ϫ3 M). These results clearly suggest that
the existence of both the carbonyl and the thiol groups might
be critical for the MMP-1 inhibition, and the distance be-
tween the two functional groups is important for inhibitory
potency. We aimed to find new mercapto groups in the form
of a-mercaptocarbonyl moiety as a ZCG, which showed no
decrease in inhibitory activity, compared with the hydroxam-
ate (1) or the carboxylate (2).
P1؅–P2؅ Peptide Incorporating Compounds Regarding
the P1Ј and P2Ј amino acids, Van Walt reported the substrate
specificity of MMP-1 by measuring the rate of hydrolysis of
various synthetic oligopeptide substrates.29) Of those studied,
Leu or Met are preferred at A.A1Ј and the aromatic amino
acids Trp or Phe are preferred at A.A2Ј. Based on this infor-
Ϫ13.5
(1.0)
Ϫ15.1
(0.98)
Ϫ46.6
(0.50)
(417.2712)
C23H37N4O6
465.2713
(465.2731)
C20H39N4O6
431.2870
(431.2848)
C15H30N3O5
332.2185
Ϫ31.7
(1.01)
26j
26k
Ϫ33.7
(1.0)
Leu
—
Ϫ22.4
(1.0)
(332.2198)
to give the compounds (29a—c). The free thiol groups (5a— mation, various P1Ј–P2Ј site peptides were incorporated into
c) were also obtained by an alkaline hydrolysis. a-mercaptocarbonyl moiety and their MMP-1 inhibitory ac-
Preparation of the single isomers of 4a described as com- tivities are summarized in Table 7. Although compounds that
pounds 36a, b are shown in Chart 4 and were achieved by have Val at A.A1Ј (4i, 4j) exhibited sub-nanomolar potencies
utilizing chiral isoserines (32a, b) reported by Maeda et al.24) against MMP-1, substitution of Val at A.A2Ј (4h) led to a
Formation of the 2-oxooxazolidine isomers (31a, b) were loss of inhibitory potencies. The decreased affinity for MMP-
conducted by the treatment of malic acid half esters 1 is probably due to the repulsion induced by adjacent iso-
(30a, b)25) with diphenylphosphoryl azide (DPPA) and Et3N. propyl groups in 4h, which makes the molecule distort. In
Alkaline hydrolysis of the 2-oxooxazolidine (31a, b) was fol- the case of A.A1ЈϭPhe (4e—g), the nature of A.A2Ј does af-
lowed by protection of the amino group by treating with di- fect the inhibitory potencies and these compounds all showed
tert-butyl dicarbonate giving N-protected isoserine (33a, b). sub-nanomolar potencies. In a limited number of these com-
After condensations of the isoserine (33a, b) with leucyl–O- pounds, A.A1ЈϭLeu (4a—d) provided the best result with re-
methyl–tyrosine–N-methylamide (H–Leu–Tyr(Me)–NHMe, spect to the MMP-1 inhibitory potencies. That is, the combi-
25a) utilizing EDC/HOBt as a coupling reagent, subsequent nations of Leu at P1Ј and hydrophobic amino acid residues at
conversions of the hydroxy group to the mercapto group P2Ј, such as branched alkyls or an aromatic ring, showed
were carried out by the same method as that described in the nanomolar inhibitory potencies and these results are identical
preparation of 4a.
to the substrate specificity study by Van Walt and coworker29)
The preparations of compounds where the mercapto group A comparison of compound 4 with well-explored P1–P2Ј
was replaced with other functional groups are summarized in mimic hydroxamate inhibitors, such as batimastat,30) reveals
Chart 5. The alkylsulfanyl compounds (37a—c) were ob- that the preference at P2Ј is a little different. In succinyl hy-
tained by the same method described in the conversion of 26 droxamate type inhibitors, the P2Ј site preferred aromatic
to 27 utilizing the corresponding potassium salt of alkylmer- substituents to an alkyl group for in vitro activity,5) but for
captane instead of potassium thioacetate. Sequentially, 37a— the a-mercaptocarbonyl series (4), branched alkyl residues

February 2002
243
Table 2. 1H-NMR Data for Compounds 26a—k
Compd.
A.A1Ј
A.A2Ј
1H-NMR, (CDCl3, d, ppm)
26a
Leu
Tyr(Me)
0.80—1.00 (6H, m), 1.34—1.78 (13H, m), 2.68 (1.5H, d, Jϭ5.0 Hz), 2.71 (1.5H, d, Jϭ4.8 Hz), 2.88—3.09
(2H, m), 3.32—3.51 (1H, m), 3.56—3.74 (1H, m), 3.77 (1.5H, s), 3.78 (1.5H, s), 4.03—4.12 (0.5H, m),
4.18—4.34 (1.5H, m), 4.46—4.60 (1H, m), 5.42 (0.5H, d, Jϭ4.2 Hz), 5.53 (0.5H, d, Jϭ4.6 Hz), 5.87 (0.5H,
d, Jϭ7.9 Hz), 5.98 (0.5H, d, Jϭ8.2 Hz), 6.55—6.66 (0.5H, m), 6.70—6.87 (2.5H, m), 7.02—7.15 (2H, m),
7.20—7.24 (1H, m)
26b
Leu
Val
0.81—1.02 (12H, m), 1.35—1.80 (13H, m), 2.08—2.20 (1H, m), 2.82 (3H, d, Jϭ4.9 Hz), 3.40—3.78 (2H,
m), 4.07—4.28 (2H, m), 4.28—4.35 (1H, m), 5.40—5.56 (0.5H, m), 5.60—5.68 (0.5H, m), 6.06—6.18
(0.5H, m), 6.20—6.31 (0.5H, m), 6.71—6.81 (0.5H, m), 6.81—6.95 (0.5H, m), 7.21—7.34 (0.5H, m),
7.45—7.53 (0.5H, m)
26c
26d
26e
Leu
Leu
Phe
Leu
Phe
Val
0.80—1.01 (12H, m), 1.30—1.75 (16H, m), 2.74 (1.5H, d, Jϭ5.0 Hz), 2.76 (1.5H, d, Jϭ4.7 Hz), 3.41—3.62
(2H, m), 4.04—4.12 (0.5H, m), 4.23—4.35 (2.5H, m), 5.40—5.47 (0.5H, m), 5.51—5.62 (0.5H, m), 6.05—
6.16 (1H, m), 6.62—6.71 (0.5H, m), 6.72—6.80 (0.5H, m), 7.31—7.48 (1H, m)
0.78—0.92 (6H, m), 1.37—1.80 (13H, m), 2.67 (1.5H, d, Jϭ4.9 Hz), 2.70 (1.5H, d, Jϭ4.8 Hz), 2.94—3.05
(2H, m), 3.32—3.54 (2H, m), 4.01 (0.5H, m), 4.10—4.21 (1.5H, m), 4.50—4.60 (1H, m), 5.60—5.72
(0.5H, m), 5.70—5.81 (0.5H, m), 6.20—6.34 (0.5H, m), 6.45—6.53 (0.5H, m), 7.00—7.42 (7H, m)
0.71—0.93 (6H, m), 1.36 (4.5H, s), 1.44 (4.5H, s), 1.59—1.84 (1H, m), 1.95—2.23 (1H, m), 2.74 (1.5H, d,
Jϭ4.8 Hz), 2.75 (1.5H, d, Jϭ5.0 Hz), 3.03—3.28 (2H, m), 3.32—3.75 (2H, m), 4.07—4.21 (2H, m), 4.51—
4.60 (0.5H, m), 4.60—4.72 (0.5H, m), 5.18—5.33 (0.5H, m), 5.92—6.06 (0.5H, m), 6.11—6.21 (0.5H, m),
6.50 (0.5H, d, Jϭ8.1 Hz), 6.70—6.80 (0.5H, m), 6.84 (0.5H, d, Jϭ8.4 Hz), 7.12—7.34 (5H, m), 7.48 (0.5H,
d, Jϭ6.8 Hz), 7.54 (0.5H, d, Jϭ7.5 Hz)
26f
Phe
Phe
1.40 (4.5H, s), 1.44 (4.5H, s), 1.72—2.21 (1H, m), 2.60 (1.5H, d, Jϭ4.8 Hz), 2.65 (1.5H, d, Jϭ4.8 Hz),
2.85—3.18 (4H, m), 3.18—3.38 (1H, m), 3.42—3.67 (1H, m), 3.97—4.05 (0.5H, m), 4.10—4.18 (0.5H, m),
4.47—4.65 (2H, m), 5.11 (0.5H, t, Jϭ6.1 Hz), 5.59—5.68 (0.5H, m), 5.74—5.84 (0.5H, m), 6.31—6.42
(0.5H, m), 6.52 (0.5H, d, Jϭ8.0 Hz), 6.55 (0.5H, d, Jϭ8.3 Hz), 7.02—7.48 (10H, m), 7.72 (0.5H, d,
Jϭ7.7 Hz), 7.84 (0.5H, d, Jϭ8.4 Hz)
26g
26h
26i
Phe
Val
Val
Leu
Val
Phe
0.82—0.97 (6H, m), 1.41—1.84 (13H, m), 2.71 (1.5H, d, Jϭ4.9 Hz), 2.75 (1.5H, d, Jϭ4.9 Hz), 2.94—3.13
(2H, m), 3.33—3.76 (2H, m), 3.98—4.14 (0.5H, m), 4.14—4.26 (1.5H, m), 4.41—4.56 (1H, m), 5.21—5.34
(0.5H, m), 5.40—5.47 (0.5H, m), 5.47—5.56 (0.5H, m), 5.81—5.93 (0.5H, m), 5.93—6.06 (0.5H, m),
6.60—6.78 (0.5H, m), 7.01—7.42 (5H, m), 7.49—7.51 (0.5H, m), 7.59 (0.5H, d, Jϭ7.9 Hz)
0.82—1.08 (12H, m), 1.43 (4.5H, s), 1.45 (4.5H, s), 1.52—1.71 (1H, m), 2.07—2.21 (1H, m), 2.25—2.42
(1H, m), 2.80 (3H, d, Jϭ4.8 Hz), 3.39—3.57 (1H, m), 3.60—3.82 (1H, m), 4.08—4.20 (2H, m), 4.20—4.29
(1H, m), 5.28—5.39 (0.5H, m), 5.47—5.57 (0.5H, m), 5.64—5.71 (0.5H, m), 6.04—6.19 (0.5H, m), 6.83
(0.5H, d, Jϭ9.0 Hz), 6.89—6.94 (0.5H, m), 7.37 (0.5H, d, Jϭ7.7 Hz), 7.52 (0.5H, d, Jϭ5.9 Hz)
0.78 (1.5H, d, Jϭ6.8 Hz), 0.81—0.92 (3H, m), 0.94 (1.5H, d, Jϭ7.0 Hz), 1.43 (4.5H, s), 1.48 (4.5H, s),
1.52—1.72 (1H, m), 2.16—2.36 (1H, m), 2.67 (1.5H, d, Jϭ5.0 Hz), 2.71 (1.5H, d, Jϭ4.8 Hz), 2.98—3.13
(2H, m), 3.31—3.53 (1H, m), 3.53—3.76 (1H, m), 4.04—4.18 (1.5H, m), 4.21—4.28 (0.5H, m), 4.52—4.68
(1H, m), 5.14—5.25 (0.5H, m), 5.46—5.62 (0.5H, m), 5.68—5.93 (0.5H, m), 6.46—6.54 (0.5H, m), 6.56—
6.69 (1H, m), 7.13—7.41 (6H, m)
26j
Val
Leu
—
0.82—1.04 (12H, m), 1.41—1.83 (13H, m), 2.09—2.21 (1H, m), 2.76 (1.5H, d, Jϭ5.0 Hz), 2.78 (1.5H, d,
Jϭ4.8 Hz), 3.38—3.68 (2H, m), 4.06—4.25 (2H, m), 4.30—4.47 (1H, m), 5.42—5.56 (0.5H, m), 5.64—
5.78 (0.5H, m), 6.02—6.23 (1H, m), 6.56—6.72 (0.5H, m), 6.71—6.84 (0.5H, m), 7.30—7.34 (0.5H, m),
7.48—7.59 (0.5H, m)
0.77—0.98 (6H, m), 1.36—1.86 (13H, m), 2.71 (1.5H, d, Jϭ4.9 Hz), 2.74 (1.5H, d, Jϭ5.0 Hz), 3.34—3.51
(2H, m), 4.03 (0.5H, m), 4.12—4.28 (1.5H, m), 4.34—4.47 (1H, m), 6.18—6.32 (1H, m), 6.61—6.83 (1H, m)
26k
Leu
also gave rise to inhibitory potency. Deletion of the P2Ј site zyme recognition site. But insertion of the Pn peptide into 4d
as shown for 4k abolished the MMP-1 inhibitory activity. All or 4k led to loss of activities. By comparing the backbones of
of these compounds (4a—k) had been prepared in a racemic collagen substrate and inhibitors 5a—c, it was found that the
form and thus 1 : 1 mixtures of diastereoisomers because we b-alanine structure in the a-mercaptocarbonyl compounds is
used racemic isoserine as the starting material. To determine longer than the natural peptide sequence, which might intro-
the stereochemical preference at the thiol attached carbon, duce a gap between enzyme subsites and side chains of in-
we prepared both single isomers of 4a described as 36a, b31) hibitors 5a, b, and caused the decreasing affinity for MMP-1.
starting from chiral malic acids. As a result, the S-isomer
(36b) was found to be approximately 100 times more potent tional Groups Using compound 4 as the basic structure,
than the corresponding R-isomer (Table 7).
neither the hydroxy intermediate (26a) nor sulfur atom con-
Substitution of the Mercapto Group with Other Func-
Compounds Incorporating Both Pn and Pn؅ Peptides taining and relatively stable derivatives such as the alkylsul-
The MMP-1 inhibitory activities of compounds incorporat- fanyl compounds (37a—c), nor the oxidized compounds
ing both Pn and PnЈ peptides (5a—c) are also summarized in (38a—c) showed inhibitory potencies (0% at 10Ϫ6 M). The
Table 7. Deletion of the P2Ј residue described for 5c also in- unsymmetrical disulfide of the ethyldisulfanyl compound
duced a significant loss of MMP-1 inhibitory activity. This (39) and the amino compound (41) exhibited little inhibitory
result indicated that the P2Ј site amino acid plays an impor- potency (18 and 11% at 10Ϫ6 M respectively). Then we intro-
tant role in recognition for MMP-1. Compounds 5a and 5b duced a carboxylate and a hydroxamate that have the poten-
were expected to have stronger affinity against the enzyme tial to chelate a zinc ion. But both the carboxylate (44) and
than 4d or 4k, which have only the P1Ј–P2Ј peptide as an en- hydroxamate (46) also showed no inhibitory potencies (0% at

244
Vol. 50, No. 2
Table 3. Physical Data for Compounds 27a—k
loenzymes (e.g., angiotensin converting enzyme, tissue
necrosis factor-a converting enzyme, aminopeptidase, and
carboxypeptidase) to investigate the selectivity for MMP-1.
Experimental
Melting points were determined on a Yanagimoto melting point apparatus
without correction. Column chromatography was performed on Silica gel
BW-200 (Fuji Silysia Chemical Ltd.). TLC was performed on silica gel
(Kiesel-gel 60 F254, Merck). Rf values refer to the following v/v solvent sys-
tem: Rf1, CHCl3–MeOH–AcOH (5 : 2 : 1); Rf2, CHCl3–MeOH–AcOH (80 :
10 : 5); Rf3, CHCl3–MeOH (10 : 1); Rf4, CHCl3–MeOH (20 : 1). 1H-NMR
was recorded on a JEOL JMN-AL300 spectrometer, and chemical shifts are
given in ppm (d) from tetramethylsilane (TMS), which was used as the in-
ternal standard. Mass spectra were obtained on a JEOL JMS 700 spectrome-
ter by fast atom bombardment (FAB) ionization techniques. Optical rota-
tions were measured in a JASCO DIP-140 apparatus.
Formula (MϩH),
HR-MS m/z
Calcd (Found)
Yield
(%)
mp
(°C)
[a]D25
Compd. A.A1Ј A.A2Ј
(c) solvent
27a
27b
27c
27d
27e
27f
27g
27h
27i
Leu Tyr(Me) 31 148—150
Ϫ39.2
(1.08)
MeOH
Ϫ78.5
(0.45)
CHCl3
Ϫ69.5
(1.07)
CHCl3
Ϫ45.7
(1.08)
CHCl3
Ϫ39.7
(0.74)
CHCl3
Ϫ35.8
(1.04)
CHCl3
Ϫ40.6
(1.06)
CHCl3
Ϫ37.6
(0.98)
CHCl3
Ϫ42.2
(1.02)
CHCl3
Ϫ45.3
(1.06)
CHCl3
Ϫ35.2
(1.03)
C27H43N4O7S
567.2852
(567.2845)
C22H41N4O6S
489.2747
(489.2748)
C23H43N4NaO6S
527.2879
(527.2884)
C26H41N4O6S
537.2747
(537.2741)
C25H39N4O6S
523.2590
(523.2606)
C29H39N4O6S
571.2590
(571.2613)
C26H41N4O6S
537.2747
(537.2756)
C21H39N4O6S
475.2590
Leu Val
Leu Leu
Leu Phe
Phe Val
Phe Phe
Phe Leu
Val Val
Val Phe
Val Leu
46 179—181
50 174—177
50 145—148
49 178—179
52 161—163
37 159—163
42 201—203
47 211—213
45 183—185
34 153—156
1-Bromo-3(S)-tert-butyloxycarbonylamino-2-oxobutane (11) A mixed
anhydride [prepared from Boc–Ala–OH (8.84 g, 46.7 mmol), ethyl chloro-
formate (5.58 g, 51.4 mmol) and Et3N (7.41 ml, 51.4 mmol) as usual] in THF
(150 ml) was added to an ice-cold solution of 0.47 N diazomethane in Et2O
(150 ml, 70 mmol). Then the reaction mixture was stirred for 2 h at the same
temperature, after which it was evaporated in vacuo and the residue was par-
titioned between EtOAc and H2O. The organic layer was washed succes-
sively with H2O, sat. aq. NaHCO3, and sat. aq. NaCl, and then was dried
over MgSO4. The EtOAc solution was concentrated to give crude Boc–Ala–
CHN2 (10) (11.6 g). A HBr solution [20% HBr in AcOH (15.9 ml, 49.0
mmol) diluted with Et2O (30 ml)] was added dropwise to a solution of crude
(10) in anhydrous THF (30 ml) at Ϫ15 °C over a period of 30 min. under a
nitrogen atmosphere, and was stirred for a further 10 min at the same tem-
perature (the final HBr concentration was 0.65 M). The reaction mixture was
diluted with EtOAc, washed with successively H2O, sat. aq. NaHCO3, and
sat. aq. NaCl. Then it was dried over MgSO4, and concentrated to give 11 as
a yellow solid (10.9 g, 88% yield): mp 52—56 °C, [a]D23 Ϫ45.2° (cϭ0.522,
(475.2636)
C25H39N4O6S
523.2590
(523.2583)
C22H41N4O6S
489.2747
(489.2706)
C17H31N3O5S
390.2063
1
MeOH), Rf4 0.69. H-NMR (CDCl3, TMS) d: 1.36 (3H, d, Jϭ6.7 Hz), 1.41
(9H, s), 4.08 (2H, s), 4.53 (1H, m), 5.08 (1H, m). FAB-MS m/z: 268 (MϩH
for 81Br)ϩ, 266 (MϩH for 79Br)ϩ.
1-Acetylsulfanyl-3(S)-tert-butyloxycarbonylamino-2-oxobutane (12)
Potassium thioacetate (90% min, 97.2 mg, 0.767 mmol) was added to a solu-
tion of 11 (200 mg, 0.693 mmol) in anhydrous acetone (15 ml), after which
the mixture was stirred for 30 min and then concentrated. After dilution with
EtOAc and H2O, the organic layer was washed with H2O and sat. aq. NaCl.
Then it was dried over MgSO4 and concentrated to give a yellow oil, which
was purified by silica gel column chromatography (eluent; n-hexane : Et2Oϭ
2 : 1) to give 12 as a colorless solid (164 mg, 98%). mp 65.5—69.0 °C, Rf4
27j
27k
Leu
—
(390.2070)
1
0.66. H-NMR (CDCl3, TMS) d: 1.38 (3H, d, Jϭ6.7 Hz), 1.43 (9H, s), 2.36
10Ϫ6 M). On the basis of these facts, it is speculated that only
the mercapto group incorporating compound (4) provided a
sufficient three dimensional distance between the active site
zinc ion and P1Ј–P2Ј subsites.
(3H, s), 3.85 (2H, s), 4.41 (1H, m), 5.10 (1H, m). FAB-MS m/z: 262
(MϩH)ϩ.
2(S)-tert-Butyloxycarbonylamino-propanol (13) To a solution of Boc–
Ala–OH (9) (20.0 g, 106 mmol) and N-hydroxysuccinimide (13.4 g, 112
mmol) in CH2Cl2 (150 ml), EDC (24.3 g, 127 mmol) was added at Ϫ8 °C,
followed by stirring for 1 h at the same temperature and then at 4 °C for 15 h.
The reaction mixture was washed with H2O and sat. aq. NaCl, and then
dried over MgSO4. The CH2Cl2 solution was concentrated under reduced
pressure, precipitated from 1-propanol, and collected by filtration to give an
activated ester (23.3 g 77% yield). To a solution of this activated ester (23.3
g, 81.4 mmol) in THF (600 ml), NaBH4 (8.01 g, 212 mmol) was added, fol-
lowed by stirring at room temperature for 1 h. Additional NaBH4 (4.00 g,
106 mmol) was added to the reaction mixture and stirring was continued for
1.5 h. Then the reaction mixture was poured into cold 10% aq. citric acid
and the organic material was extracted with EtOAc. Next the organic layer
was dried over MgSO4 and evaporated in vacuo to give crude 13 (9.89 g,
78%) as a colorless oil. 1H-NMR (CDCl3, TMS) d: 1.13 (3H, d, Jϭ6.4 Hz),
1.42 (9H, s), 2.94 (1H, m), 3.58 (2H, m), 3.72 (1H, m), 4.80 (1H, br d, Jϭ
6.7 Hz). FAB-MS m/z: 176 (MϩH)ϩ.
2(S)-tert-Butyloxycarbonylamino-propanal (14) To a solution of oxa-
lyl chloride (5.77 ml, 67.7 mmol) in CH2Cl2 (50 ml), a solution of dimethyl-
sulfoxide (10.6 ml, 149 mmol) in CH2Cl2 (60 ml) was added dropwise at a
temperature below Ϫ68 °C. To the reaction mixture, a solution of the crude
alcohol (13) (9.89 g, 56.4 mmol) in CH2Cl2 (400 ml) was added dropwise
over a period of 15 min. After stirring for 20 min at the same temperature,
Et3N (41.4 ml, 298 mmol) was added to the mixture dropwise over a period
of 30 min. Then the reaction mixture was poured into water and extracted
Conclusion
From the evaluation of the P4–P1 peptide incorporating
compounds 3, 6—8 against MMP-1 inhibitory potency, we
confirmed that the a-mercaptocarbonyl moiety plays a cru-
cial role for chelation of the zinc ion in the MMP-1 active
site. For the PnЈ peptide incorporating compounds (4a—j),
the a-mercaptocarbonyl unit was also effective against
MMP-1 inhibition and Leu at the P1Ј position exhibited the
best results (4a—d) by which we achieved MMP-1 in-
hibitory potency at the nanomolar level. This is as potent as
the inhibition of batimastat. But the intrinsic good chelation
property of the a-mercaptocarbonyl unit was reduced by the
existence of the Pn peptide in 5a, b. Syntheses of both iso-
mers of 4a revealed that the S configuration at the thiol at-
tached carbon was preferred for MMP-1 inhibition and only
the thiol group provided a satisfactory inhibitory result. Fur-
ther evaluation of the a-mercaptocarbonyl moiety will pro-
ceed against not only other MMPs but also general metal- with n-hexane. The organic layer was washed with 0.5 N aq. HCl and sat. aq.

February 2002
245
Table 4. 1H-NMR Data for Compounds 27a—k
Compd.
A.A1Ј
A.A2Ј
1H-NMR, (CD3OD, d, ppm)
27a
Leu
Tyr(Me)
0.81—0.97 (6H, m), 1.33—1.74 (12H, m), 2.35 (1.5H, s), 2.36 (1.5H, s), 2.66 (3H, s), 2.82—3.01 (1H, m),
3.01—3.18 (1H, m), 3.32—3.44 (m), 3.44—3.58 (1H, m), 3.75 (3H, s), 4.10—4.32 (2H, m), 4.48—4.64
(1H, m), 6.89—7.43 (1H, m)
27b
27c
27d
Leu
Leu
Leu
Val
Leu
Phe
0.81—1.01 (12H, m), 1.33—1.78 (12H, m), 1.90—2.08 (1H, m), 2.30 (1.5H, s), 2.34 (1.5H, s), 2.72 (3H, s),
3.20—3.46 (m), 3.46—3.58 (1H, m), 4.11—4.24 (1H, m), 4.30—4.48 (2H, m)
0.79—1.04 (12H, m), 1.43 (9H, m), 1.52—1.80 (6H, m), 2.34 (1.5H, s), 2.35 (1.5H, s), 2.70 (3H, s), 3.20—
3.60 (m), 4.22—4.41 (1H, m)
0.83 (1.5H, d, Jϭ4.6 Hz), 0.85 (1.5H, d, Jϭ4.6 Hz), 0.89 (3H, d, Jϭ6.4 Hz), 1.34—1.74 (12H, m), 2.34
(1.5H, s), 2.37 (1.5H, s), 2.66 (3H, s), 2.81—3.00 (1H, m), 3.03—3.24 (1H, m), 3.24—3.55 (m), 4.14—4.35
(2H, m), 4.43—4.58 (1H, m), 7.12—7.33 (5H, m)
27e
Phe
Val
0.78—0.97 (6H, m), 1.41 (9H, s), 1.93—2.12 (1H, m), 2.28 (1.5H, s), 2.31 (1.5H, s), 2.70 (3H, s), 2.82—
2.98 (1H, m), 3.04—3.20 (1H, m), 3.20—3.51 (m), 3.99—4.12 (1H, m), 4.23 (1H, t, Jϭ7.1 Hz), 4.56—4.73
(1H, m), 7.08—7.31 (5H, m)
27f
Phe
Phe
Phe
Leu
1.42 (9H, s), 2.28 (1.5H, s), 2.33 (1.5H, s), 2.63 (3H, s), 2.75—2.96 (2H, m), 2.96—3.19 (2H, m), 3.25—
3.58 (m), 4.14—4.28 (1H, m), 4.42—4.63 (2H, m), 7.07—7.33 (10H, m)
27g
0.79 (1.5H, d, Jϭ4.6 Hz), 0.81 (1.5H, d, Jϭ5.0 Hz), 0.84 (3H, d, Jϭ6.2 Hz), 1.43 (9H, s), 1.52—1.79 (3H,
m), 2.30 (1.5H, s), 2.32 (1.5H, s), 2.68 (3H, s), 2.86—3.01 (1H, m), 3.17—3.23 (1H, m), 3.25—3.57 (m),
4.24 (1H, t, Jϭ6.9 Hz), 4.28—4.40 (1H, m), 4.52—4.68 (1H, m), 7.15—7.35 (5H, m)
0.81—1.02 (12H, m), 1.42 (9H, s), 1.93—2.19 (2H, m), 2.34 (1.5H, s), 2.36 (1.5H, s), 2.71 (3H, s), 3.23—
3.40 (m), 3.40—3.55 (1H, m), 4.02—4.13 (1H, m), 4.17 (1H, d, Jϭ7.3 Hz), 4.35 (1H, dd, Jϭ6.9 and
13.9 Hz), 4.30—4.41 (1H, m)
0.71—0.93 (6H, m), 1.43 (9H, s), 1.90—2.09 (1H, m), 2.37 (3H, s), 2.66 (3H, s), 2.81—3.00 (1H, m),
3.04—3.23 (1H, m), 3.23—3.41 (m), 3.41—3.58 (1H, m), 4.03 (0.5H, d, Jϭ6.2 Hz), 4.09 (0.5H, d,
Jϭ6.6 Hz), 4.26—4.41 (1H, m), 4.48—4.63 (1H, m), 7.12—7.33 (5H, m)
0.82—1.05 (12H, m), 1.32—1.73 (12H, m), 1.98—2.21 (1H, m), 2.34 (1.5H, s), 2.36 (1.5H, s), 2.71 (3H, s),
3.21—3.98 (m), 3.40—3.59 (1H, m), 4.13 (0.5H, d, Jϭ6.8 Hz), 4.14 (0.5H, d, Jϭ6.6 Hz), 4.27—4.44 (2H,
m)
0.81—1.02 (6H, m), 1.31—1.72 (12H, m), 2.34 (3H, s), 2.68 (3H, s), 3.32—3.54 (2H, m), 4.12—4.35 (2H,
m)
27h
27i
Val
Val
Val
Leu
Val
Phe
Leu
—
27j
27k
as a colorless oil (1.47 g, 63%). 1H-NMR (CDCl3, TMS) d: 1.15 (3H, d, Jϭ
7.1 Hz), 1.42 (9H, s), 2.86 (1H, m), 3.66 (1H, m), 4.01 (1H, m), 4.72 (1H,
br d, Jϭ8.5 Hz), 5.12 (1H, m), 5.31 (1H, m), 5.60—5.98 (1H, m). FAB-MS
m/z: 202 (MϩH)ϩ.
NaCl, dried over MgSO4, and then concentrated to give 14 (8.00 g, 82%) as
a colorless solid. H-NMR (CDCl3, TMS) d: 1.32 (3H, d, Jϭ6.9 Hz), 1.47
1
(9H, s), 4.18 (1H, m), 5.09 (1H, m), 9.50 (1H, s). FAB-MS m/z: 174 (Mϩ
H)ϩ. HR-MS Calcd for C8H16NO3: 174.1130 (MϩH)ϩ. Found: 174.1145.
2(S)-tert-Butyloxycarbonylaminobut-3-ene (15) To a suspension of
methyltriphenylphosphonium bromide (6.94 g, 19.1 mmol) in anhydrous
THF (50 ml), bis(trimethylsilyl)potassium amide (0.5 M solution in toluene,
38 ml, 19.0 mmol) was added dropwise at Ϫ69 °C under a nitrogen atmos-
phere, and the mixture was stirred at the same temperature for 11 h. To this
mixture, a solution of aldehyde (14) (1.57 g, 9.07 mmol) in anhydrous THF
(30 ml) was added and stirred at Ϫ65 °C for 15 min. Then the reaction mix-
ture was warmed to room temperature and stirred for 1 h, after which it was
stirred at 40 °C for 11 h. The reaction was quenched with MeOH, after
which the mixture was washed with aq. Rochelle salt solution, dried over
MgSO4 and evaporated to give a brown residue, which was purified by silica
gel column chromatography (eluent; n-hexane : Et2Oϭ6 : 1) to give 15 as a
colorless oil (1.08 g, 70%). [a]D25 Ϫ0.89° (cϭ1.0, MeOH), Rf4 0.78. 1H-
NMR (CDCl3, TMS) d: 1.19 (3H, d, Jϭ6.7 Hz), 1.45 (9H, s), 4.03—4.61
(2H, m), 4.95—5.30 (2H, m), 5.66—6.07 (1H, m). FAB-MS m/z: 172 (Mϩ
H)ϩ.
2(S)-tert-Butyloxycarbonylamino-3-oxopent-4-ene (18) To a solution
of oxalyl chloride (0.71 ml, 8.32 mmol) and dimethylsulfoxide (1.10 ml,
15.5 mmol) in CH2Cl2 (10 ml), a solution of 17 (1.47 g, 7.30 mmol) in
CH2Cl2 (20 ml) was added dropwise over a period of 15 min. at Ϫ40 °C, fol-
lowed by stirring at Ϫ40 °C for 1 h. After adding Et3N (5.13 ml, 36.9 mmol),
stirring was continued for 6 h at room temperature and then the reaction was
quenched with water. The organic layer was separated and the aqueous layer
was co-extracted with CH2Cl2. Subsequently, the combined organic layer
was washed with sat. aq. NaCl, dried over MgSO4, and then evaporated in
vacuo to give a brown oil, which was purified by silica gel column chro-
matography (eluent; n-hexane : EtOAcϭ10 : 1) to give 18 as a colorless solid
(617 mg, 42%). mp 68.5—72.0 °C, [a]D25 ϩ4.16° (cϭ1.0, MeOH), Rf4 0.72.
1H-NMR (CDCl3, TMS) d: 1.32 (3H, d, Jϭ6.7 Hz), 1.42 (9H, s), 4.55 (1H,
m), 5.45 (1H, m), 5.80 (1H, dd, Jϭ3.7, 7.4 Hz), 6.34 (2H, dd, Jϭ3.7, 7.4
Hz). FAB-MS m/z : 200 (MϩH)ϩ.
1-Acetylsulfanyl-4(S)-tert-butyloxycarbonylamino-3-oxopentane (19)
To a mixture of thioacetic acid (37.3 ml, 0.527 mmol), 18 (100 mg, 0.502
mmol) and anhydrous THF (0.5 ml), tetrabutylammonium fluoride (50 mg)
were added under a nitrogen atmosphere and the mixture was stirred at room
temperature for 2 d. Then the reaction mixture was evaporated and purified
by silica gel column chromatography (eluent; n-hexane : EtOAcϭ6 : 1) to
give 19 as slightly yellow oil (80 mg, 58%). 1H-NMR (CDCl3, TMS) d: 1.28
(3H, d, Jϭ6.7 Hz), 1.42 (9H, s), 2.27 (3H, s), 2.80 (2H, m), 2.99 (2H, m),
4.18 (1H, m), 5.07 (1H, m). FAB-MS m/z: 276 (MϩH)ϩ.
1-Acetylsulfanyl-3(S)-tert-butyloxycarbonylaminobutane (16)
A
mixture of 15 (200 mg, 1.17 mmol) and thiolacetic acid (AcSH, 99 ml,
1.40 mmol) was irradiated with ultraviolet light (below 3000 Å) for 20 min.
Then the reaction mixture was purified by silica gel column chromatography
(eluent; n-hexane : Et2Oϭ4 : 1) to give 16 as colorless solid (253 mg, 87%).
mp 54.5—55.0 °C, [a]D25 Ϫ110° (cϭ1.0, MeOH), Rf4 0.23. 1H-NMR (CDCl3,
TMS) d: 1.11 (3H, d, Jϭ6.7 Hz), 1.42 (9H, s), 1.66 (2H, t, Jϭ7.3 Hz), 2.27
(3H, s), 2.82 (2H, dt, Jϭ3.1, 7.3 Hz), 3.64 (1H, m), 4.09 (1H, br d, Jϭ7.8
Hz). FAB-MS m/z: 248 (MϩH)ϩ.
2(S)-tert-Butyloxycarbonylamino-3(R,S)-hydroxypent-4-ene (17) To
a solution of vinylmagnesium bromide (1.0 M in THF, 36 ml), a solution of
14 (2.00 g, 11.5 mmol) in anhydrous THF (20 ml) was added at 0 °C and
stirred for 2 h, followed by stirring at room temperature for 1 h. The reaction
was quenched with sat. aq. ammonium chloride and the mixture was ex-
tracted with EtOAc. Then the organic layer was washed with sat. aq. NaCl,
dried over MgSO4, and evaporated to give an oil, which was purified by sil-
ica gel column chromatography (eluent; n-hexane : EtOAcϭ4 : 1) to give 17
1(R,S)-[1؅(S)-tert-Butyloxycarbonylaminoethyl]oxirane (20) To a so-
lution of 15 (500 mg, 2.92 mmol) in CH2Cl2 (25 ml), MCPBA (3.61 g, 14.6
mmol) was added and the mixture was stirred at room temperature for 1 h.
Then the mixture was diluted with Et2O, and washed successively with cold
10% aq. Na2CO3, sat. NaHCO3, and sat. aq. NaCl. Subsequently, the mixture
was dried over MgSO4, and then evaporated in vacuo to give crude 20,
which was purified by silica gel column chromatography (eluent; n-hexane :
Et2Oϭ2 : 1) to give 20 as a colorless oil (388 mg, 71%). [a]D25 Ϫ10.5°
1
(cϭ1.0, MeOH), Rf4 0.71. H-NMR (CDCl3, TMS) d: 1.13 (1.1H, d, Jϭ6.7

246
Vol. 50, No. 2
Table 5. Physical Data for Compounds 4a—k
NMR (CDCl3, TMS) d: 0.77—1.16 (9H, m, Jϭ6.7 Hz), 1.42—1.91 (7H, m),
1.93—2.43 (5H, m), 2.75 (2H, dt, Jϭ3.0, 7.7 Hz), 3.41—4.20 (4H, m),
4.20—4.71 (3H, m), 6.67 (1H, d, Jϭ8.1 Hz), 7.32—7.70 (4H, m), 7.82 (2H,
m). FAB-MS m/z: 519 (MϩH)ϩ. HR-MS Calcd for C26H38N4O5S:
519.2641(MϩH)ϩ. Found: 519.2621.
1-Acetylsulfanyl-4(S)-(benzoyl-glycyl-prolyl-leucyl)amino-3-oxopentane
(23c): Yield: 58%. mp 100—102 °C, [a]D25 Ϫ172° (cϭ1.0, MeOH), Rf2 0.57.
1H-NMR (CDCl3, TMS) d: 0.82—1.14 (9H, m, Jϭ6.7 Hz), 1.41—1.92 (3H,
m), 1.93—2.41 (7H, m), 2.61—3.24 (4H, m), 3.43—4.62 (7H, m), 7.11 (2H,
m), 7.30—7.72 (4H, m), 7.80 (2H, m). FAB-MS m/z: 547 (MϩH)ϩ. HR-MS
Calcd for C27H39N4O6S: 547.2590 (MϩH)ϩ. Found: 547.2608.
Formula (MϩH),
HR-MS m/z
Calcd (Found)
Yield
(%)
mp
(°C)
[a]D25
Compd. A.A1Ј A.A2Ј
(c) solvent
4a
4b
4c
4d
4e
4f
Leu Tyr(Me) 81 185—191
Ϫ31.0
(0.92)
CHCl3
Ϫ50.7
(0.98)
CHCl3
Ϫ52.4
(1.10)
CHCl3
Ϫ48.5
(1.03)
MeOH
Ϫ15.2
(1.06)
CHCl3
Ϫ18.1
(0.99)
CHCl3
Ϫ19.4
(1.02)
CHCl3
Ϫ10.4
(0.36)
DMF
C25H41N4O6S
525.2747
(525.2750)
C20H39N4O5S
447.2641
(447.2606)
C21H41N4O5S
461.2798
(461.2829)
C24H39N4O5S
495.2641
(495.2625)
C23H37N4O5S
481.2485
(481.2462)
C27H37N4O5S
529.2485
(529.2496)
C24H39N4O5S
495.2641
(495.2647)
C19H37N4O5S
433.2485
1-Acetylsulfanyl-3(S)-(benzoyl-glycyl-prolyl-leucyl)amino-2(R,S)-hy-
droxybutane (23d): To a solution of 1-acetylsulfanyl-3(S)-amino-2(R,S)-
hydroxybutane hydrochloride [prepared from 21 (497 mg, 1.28 mmol) and
4 N HCl in EtOAc (15 ml)] in DMF (20 ml), benzotriazole-1-yl-oxy-tris(di-
methylamino)phosphonium hexafluorophosphate (BOP reagent, 594 mg,
1.34 mmol), Bz-Gly-Pro-Leu-OH (22) and Et3N (406 ml, 2.92 mmol) were
added successively. The mixture was stirred at room temperature for 15 h,
then evaporated under reduced pressure. The residue was diluted with
EtOAc, washed successively with 1 N aq. HCl, 10% aq. Na2CO3, and sat. aq.
NaCl, dried over MgSO4, and then evaporated in vacuo. The crude product
was purified by silica gel column chromatography (eluent; CHCl3 : MeOHϭ
20 : 1) to give 23d as colorless powder (385 mg, 56%). mp 110—114 °C,
Leu
Leu
Leu
Phe
Phe
Phe
Val
Val
Leu
Phe
Val
Phe
Leu
Val
Phe
Leu
—
72 192—194
60 176—178
84 183—185
79 223—225
82 179—181
56 184—187
69 201—205
74 200—202
66 228—230
92 165—168
1
[a]D25 Ϫ206° (cϭ1.0, MeOH), Rf2 0.46. H-NMR (CDCl3, TMS) d: 0.85—
1.32 (9H, m), 1.41—1.89 (3H, m), 1.90—2.56 (7H, m), 2.98 (2H, m),
3.22—4.87 (9H, m), 6.72 (1H, m), 7.03—7.65 (5H, m), 7.87 (2H, m). FAB-
MS m/z: 535 (MϩH)ϩ. HR-MS Calcd for C26H39N4O6S: 535.2590 (MϩH)ϩ.
Found: 535.2582.
4g
4h
4i
General Procedure for the Preparation of Compounds 3, 6—8 To a
solution of 23a—d in MeOH–THF (1 : 2, 2% w/v), 0.2 N aq. NaOH (1.2 eq
of 23a—d) was added and the mixture was stirred at room temperature for
1 h. Then the reaction mixture was acidified by adding 1 N aq. HCl (pHϭ2—
3) and was evaporated in vacuo, followed by extraction with EtOAc, drying
over MgSO4, and evaporation in vacuo. The residue was precipitated from
Et2O to give 3, 6, 7, or 8 as a colorless solid (the thiol compound was stored
under a nitrogen atmosphere to prevent formation of the disulfide).
3(S)-(Benzoyl-glycyl-prolyl-leucyl)amino-1-mercapto-2-oxobutane (3):
(433.2473)
C23H37N4O5S
481.2485
(481.2508)
C20H39N4O5S
447.2641
(447.2628)
C15H30N3O4S
348.1957
Val
Ϫ14.1
(0.96)
DMF
Ϫ13.8
(0.43)
DMF
Ϫ46.6
(0.42)
MeOH
1
Yield: 83%. mp 121—125 °C, [a]D25 Ϫ156° (cϭ1.0, MeOH), Rf3 0.31. H-
4j
Val
NMR (CDCl3, TMS) d: 0.89 (9H, m), 1.12—1.51 (3H, m), 1.63 (2H, m),
1.83—2.17 (3H, m), 3.10 (2H, m), 3.34—4.02 (4H, m), 4.21—4.76 (3H, m),
5.41 (1H, br t, Jϭ5.1 Hz), 6.93—7.80 (7H, m). FAB-MS m/z: 491 (MϩH)ϩ.
HR-MS Calcd for C24H35N4O5S: 491.2328 (MϩH)ϩ. Found: 491.2312.
3(S)-(Benzoyl-glycyl-prolyl-leucyl)amino-1-mercaptobutane (6): Yield:
86%. mp 164—167 °C, [a]D25 Ϫ133° (cϭ1.0, MeOH), Rf3 0.57. 1H-NMR
(CDCl3, TMS) d: 0.74 (3H, d, Jϭ6.4 Hz), 0.89 (6H, m), 1.32—1.90 (5H,
m), 1.90—2.67 (7H, m), 3.42—4.10 (4H, m), 4.10—4.62 (3H, m), 6.52 (1H,
br d, Jϭ7.2 Hz), 7.08 (1H, br d, Jϭ7.2 Hz), 7.33—7.69 (4H, m), 7.80 (2H,
m). FAB-MS m/z: 477 (MϩH)ϩ. HR-MS Calcd for C24H37N4O4S: 477.2536
4k
Leu
(348.1992)
Hz), 1.24 (1.9H, d, Jϭ6.4 Hz), 1.42, 1.43 (9H, s each), 2.52—3.05 (3H, m),
3.94 (1H, m), 4.40 (1H, m). FAB-MS m/z: 188 (MϩH)ϩ.
1-Acetylsulfanyl-3(S)-tert-butyloxycabonylamino-2(R,S)-hydroxybu- (MϩH)ϩ. Found: 477.2558.
tane (21) Compound 20 (649 mg, 3.47 mmol) was dissolved in thioacetic
4(S)-(Benzoyl-glycyl-prolyl-leucyl)amino-1-mercapto-3-oxopentane (7):
1
acid (2.0 ml) and stirred at room temperature for 15 h. The excess thioacetic Yield: 84%. mp 100—102 °C, [a]D25 Ϫ181° (cϭ1.0, MeOH), Rf2 0.55. H-
acid was removed by evaporation, and then the residue was purified by silica
gel column chromatography (eluent; n-hexane : EtOAcϭ4 : 1) to give 21 as a
slightly yellow oil (293 mg, 32%). [a]D25 Ϫ36.2° (cϭ1.0, MeOH) Rf4 0.35.
NMR (CDCl3, TMS) d: 0.90 (9H, m), 1.16 (1H, d, Jϭ7.1 Hz), 1.41—1.90
(2H, m), 1.90—2.40 (5H, m), 2.40—3.02 (4H, m), 3.45—4.72 (7H, m),
7.00—7.60 (5H, m), 7.60—8.11 (3H, m). FAB-MS m/z: 505 (MϩH)ϩ. HR-
1H-NMR (CDCl3, TMS) d: 1.19 (3H, d, Jϭ6.4 Hz), 1.44 (9H, s), 2.05 (1H, MS Calcd for C25H37N4O5S: 505.2485 (MϩH)ϩ. Found: 505.2494.
d, Jϭ6.4 Hz), 2.36 (3H, s), 3.02 (2H, m), 3.44—4.27 (2H, m), 4.85 (1H, br d,
3(S)-(Benzoyl-glycyl-prolyl-leucyl)amino-2(RS)-hydroxy-1-mercaptobu-
Jϭ8.5 Hz). FAB-MS m/z: 264 (MϩH)ϩ.
tane (8): Yield: 83%. mp 110—114 °C, [a]D25 Ϫ169° (cϭ1.0, MeOH), Rf3
1
General Procedure for the Preparation of Compounds 23a—c
A
0.27. H-NMR (CDCl3, TMS) d: 0.69—1.32 (9H, m), 1.32—1.90 (3H, m),
mixed anhydride [prepared from Bz–Gly–Pro–Leu–OH (22), ethyl chloro- 1.90—2.44 (8H, m), 3.31—4.68 (8H, m), 6.78 (1H, m), 6.90—7.68 (4H, m),
formate (1.1 eq of 22), and N-methylmorpholine (NMM, 1.1 eq of 22) as 7.68—8.13 (3H, m). FAB-MS m/z: 493 (MϩH)ϩ. HR-MS Calcd for
usual] in N,N-dimethylformamide (DMF) (4% w/v) was successively added C24H37N4O5S: 493.2485 (MϩH)ϩ. Found: 493.2476.
to an ice-cold solution of N-deprotected 12, 16, or 19 [prepared by treatment
with TFA] in DMF (4% w/v) and NMM (1 eq of N-deprotected 12, 16, or a solution of 2-tert-butyloxycarbonylaminoethanol (43.9 g, 272 mmol) and
19). The reaction mixture was stirred at Ϫ15 °C for 1 h, and then was evapo- Et3N (114 ml, 817 mmol) in CH2Cl2 (600 ml) cooled to Ϫ10 °C, sulfur triox-
rated and the residue was purified by silica gel column chromatography (elu- ide-pyridine complex (130 g, 817 mmol) in DMSO (400 ml) was added as a
ent; CHCl3 : MeOHϭ40 : 1) to give 23a—c as a colorless solid. bolus. The mixture was stirred vigorously for 10 min at 35 °C, poured into
3-tert-Butyloxycarbonylamino-2(R,S)-hydroxypropionic Acid (24) To
1-Acetylsulfanyl-3(S)-(benzoyl-glycyl-prolyl-leucyl)amino-2-oxobutane cold sat. aq. NaCl (1500 ml), and extracted with cooled Et2O. The organic
(23a): Yield: 76%. 1H-NMR (CDCl3, TMS) d: 0.91 (6H, d, Jϭ6.8 Hz), extracts were washed with cooled 10% aq. citric acid and cooled water, dried
1.10—1.53 (6H, m), 1.62 (2H, m), 2.08 (2H, m), 2.33 (3H, s), 3.25—4.05
over MgSO4, and then evaporated in vacuo to give the corresponding alde-
(6H, m), 4.21—4.89 (3H, m), 5.40 (1H, br t, Jϭ5.1 Hz), 6.92—7.81 (7H,
hyde, which was purified by silica gel column chromatography (eluent;
m). FAB-MS m/z: 533 (MϩH)ϩ. HR-MS Calcd for C26H37N4O6S: 533.2434 CHCl3 : MeOHϭ300 : 1—100 : 1) to give the aldehyde as a yellow oil (18.2
(MϩH)ϩ. Found: 533.2449.
g, 42%). To a solution of the aldehyde in acetone (50 ml), an ice-cold solu-
tion of NaHSO3 (11.9 g, 114 mmol) in water (200 ml) was added, and the
1-Acetylsulfanyl-3(S)-(benzoyl-glycyl-prolyl-leucyl)aminobutane (23b):
1
Yield: 50%. mp 121—123 °C, [a]D25 Ϫ115° (cϭ1.0, MeOH), Rf2 0.69. H- mixture was stirred overnight at 5 °C. To the solution of NaHSO3 adduct, a

February 2002
247
Table 6. 1H-NMR Data for Compounds 4a—k
Compd.
A.A1Ј
A.A2Ј
1H-NMR, (CDCl3, d, ppm)
4a
Leu
Tyr(Me)
0.80—1.01 (6H, m), 1.27—1.70 (12H, m), 2.04 (0.5H, d, Jϭ10.3 Hz), 2.11 (0.5H, d, Jϭ10.1 Hz), 2.71 (3H,
d, Jϭ4.8 Hz), 2.90—3.08 (2H, m), 3.28—3.58 (3H, m), 3.78 (3H, s), 4.21—4.38 (1H, m), 4.33—4.50 (1H,
m), 5.34 (0.5H, t, Jϭ6.5 Hz), 5.60—5.69 (0.5H, m), 6.72—6.93 (3H, m), 7.00—7.18 (3H, m), 7.26—7.34
(0.5H, m), 7.61—7.70 (0.5H, m)
4b
4c
4d
4e
4f
Leu
Leu
Leu
Phe
Phe
Phe
Val
Val
Leu
Phe
Val
Phe
Leu
Val
Phe
0.78—1.01 (12H, m), 1.27—1.78 (12H, m), 1.89—1.98 (1H, m), 2.03 (0.5H, d, Jϭ10.0 Hz), 2.10 (0.5H, d,
Jϭ10.3 Hz), 2.70 (1.5H, d, Jϭ4.7 Hz), 2.72 (1.5H, d, Jϭ4.7 Hz), 3.30—3.61 (3H, m), 4.22—4.50 (2H, m),
5.21 (0.5H, d, Jϭ6.4 Hz), 5.32—5.41 (0.5H, m), 5.56—5.64 (0.5H, m), 5.84—5.94 (0.5H, m), 6.40—6.48
(0.5H, m), 6.48—6.58 (0.5H, m), 6.75 (0.5H, d, Jϭ7.5 Hz), 6.80—6.92 (0.5H, m)
0.78—1.02 (12H, m), 1.41 (4.5H, s), 1.43 (4.5H, s), 1.51—1.82 (6H, m), 2.06 (0.5H, d, Jϭ10.3 Hz), 2.12
(0.5H, d, Jϭ10.5 Hz), 2.80 (3H, d, Jϭ4.8 Hz), 3.26—3.61 (2H, m), 3.61—3.76 (0.5H, m), 3.76—3.91
(0.5H, m), 4.39—4.66 (2H, m), 5.31 (0.5H, t, Jϭ6.1 Hz), 5.51—5.68 (0.5H, m), 6.71—6.85 (0.5H, m), 6.93
(0.5H, d, Jϭ9.2 Hz), 7.02—7.21 (1H, m), 7.21—7.36 (0.5H, m), 7.67 (0.5H, d, Jϭ8.4 Hz)
0.78—1.02 (6H, m), 1.31—1.78 (12H, m), 2.01 (0.5H, d, Jϭ10.5 Hz), 2.08 (0.5H, d, Jϭ10.1 Hz), 2.70
(1.5H, d, Jϭ4.8 Hz), 2.71 (1.5H, d, Jϭ4.8 Hz), 2.99—3.15 (2H, m), 3.27—3.73 (3H, m), 4.28—4.52 (1H,
m), 4.56—4.74 (1H, m), 5.28 (0.5H, t, Jϭ6.1 Hz), 5.49—5.60 (0.5H, m), 6.00 (0.5H, d, Jϭ6.5 Hz), 6.28—
6.40 (0.5H, m), 6.70—6.83 (0.5H, m), 6.93 (0.5H, d, Jϭ9.1 Hz), 7.05—7.38 (6H, m)
0.78—0.97 (6H, m), 1.42 (9H, s), 1.84—1.96 (1H, m), 2.02 (0.5H, d, Jϭ10.2 Hz), 2.10 (0.5H, d,
Jϭ10.4 Hz), 2.70 (1.5H, d, Jϭ4.8 Hz), 2.72 (1.5H, d, Jϭ4.8 Hz), 2.96—3.12 (2H, m), 3.28—3.67 (3H, m),
4.16—4.21 (1H, m), 4.36—4.51 (1H, m), 5.22 (0.5H, d, Jϭ6.1 Hz), 5.37—5.47 (0.5H, m), 5.60—5.74
(0.5H, m), 5.77—5.89 (0.5H, m), 6.41—6.53 (0.5H, m), 6.53—6.65 (0.5H, m), 7.00—7.43 (6H, m)
1.43 (9H, s), 1.87 (0.5H, d, Jϭ9.9 Hz), 1.89 (0.5H, d, Jϭ10.1 Hz), 2.66 (1.5H, d, Jϭ4.8 Hz), 2.68 (1.5H, d,
Jϭ4.8 Hz), 2.86—3.15 (4H, m), 3.26—3.55 (3H, m), 4.47—4.69 (2H, m), 5.07—5.21 (0.5H, m), 5.26—
5.39 (0.5H, m), 5.55—5.70 (0.5H, m), 5.77—5.92 (0.5H, m), 6.44 (0.5H, d, Jϭ8.3 Hz), 6.49 (0.5H, d,
Jϭ8.6 Hz), 6.73 (0.5H, d, Jϭ6.6 Hz), 6.78 (0.5H, d, Jϭ7.0 Hz), 6.97—7.39 (10H, m)
0.78—0.97 (6H, m), 1.32—1.78 (12H, m), 2.02 (0.5H, d, Jϭ10.2 Hz), 2.09 (0.5H, d, Jϭ10.5 Hz), 2.70
(1.5H, d, Jϭ4.8 Hz), 2.71 (1.5H, d, Jϭ4.8 Hz), 2.92—3.21 (2H, m), 3.32—3.56 (2H, m), 3.60—3.72 (1H,
m), 4.32—4.51 (1H, m), 4.51—4.73 (1H, m), 5.30 (0.5H, t, Jϭ6.0 Hz), 5.49—5.57 (0.5H, m), 5.92—6.02
(0.5H, m), 6.31—6.43 (0.5H, m), 6.72—6.82 (0.5H, m), 6.85—6.96 (0.5H, m), 7.01—7.46 (6H, m)
0.77—0.97 (12H, m), 1.41 (9H, s), 1.82—2.01 (2H, m), 2.04 (0.5H, d, Jϭ10.1 Hz), 2.11 (0.5H, d,
Jϭ10.5 Hz), 2.70 (1.5H, d, Jϭ4.8 Hz), 2.72 (1.5H, d, Jϭ4.8 Hz), 3.27—3.81 (3H, m), 4.45—4.78 (2H, m),
5.49—5.56 (0.5H, m), 5.67—5.78 (0.5H, m), 6.16—6.29 (0.5H, m), 6.50—6.60 (0.5H, m), 6.63—6.76
(0.5H, m), 6.80—6.87 (0.5H, m), 6.95—7.08 (0.5H, m), 7.08—7.18 (0.5H, m)
4g
4h
4i
Val
0.72—0.98 (6H, m), 1.41 (9H, s), 1.89—2.01 (1H, m), 2.03 (0.5H, d, Jϭ10.1 Hz), 2.09 (0.5H, d,
Jϭ10.5 Hz), 2.73 (3H, d, Jϭ4.7 Hz), 2.98—3.21 (2H, m), 3.32—3.57 (3H, m), 4.37—4.68 (2H, m), 5.06—
5.20 (0.5H, m), 5.31—5.44 (0.5H, m), 5.61—5.73 (0.5H, m), 5.90—6.12 (0.5H, m), 6.41 (0.5H, d,
Jϭ8.1 Hz), 6.46 (0.5H, d, Jϭ8.6 Hz), 6.76 (0.5H, d, Jϭ8.1 Hz), 6.82 (0.5H, d, Jϭ8.6 Hz), 7.10—7.41 (5H,
m)
4j
Val
Leu
—
0.73—0.97 (12H, m), 1.28—1.62 (12H, m), 1.90—1.98 (1H, m), 2.03 (0.5H, d, Jϭ10.1 Hz), 2.08 (0.5H, d,
Jϭ10.1 Hz), 2.70 (3H, d, Jϭ4.8 Hz), 3.37—3.72 (3H, m), 4.30—4.44 (1H, m), 4.51—4.63 (1H, m), 5.34
(0.5H, t, Jϭ6.3 Hz), 5.43—5.61 (0.5H, m), 6.29—6.41 (0.5H, m), 6.60—6.73 (0.5H, m), 6.81—6.88 (1H,
m), 6.93—7.05 (0.5H, m), 7.12—7.28 (0.5H, m)
0.93 (3H, d, Jϭ6.2 Hz), 0.95 (3H, d, Jϭ6.4 Hz), 1.36—1.84 (12H, m), 2.07 (1H, d, Jϭ9.7 Hz), 2.82 (3H, d,
Jϭ4.8 Hz), 3.34—3.64 (3H, m), 4.32—4.49 (1H, m), 5.08—5.27 (1H, m), 6.06—6.28 (1H, m), 6.65—6.83
(1H, m)
4k
Leu
Reagents: (a) 4 N HCl in EtOAc; (b) NEM/ClCO2i-Bu; (c) aq. NaOH/MeOH.
Chart 3. Syntheses of the Both Pn and PnЈ Peptides Incorporating Compounds

248
Vol. 50, No. 2
Reagents: (a) (1) Trifluoroacetic anhydride, (2) BnOH; (b) diphenylphosphoryl azide/Et3N; (c) aq. NaOH/MeOH; (d) (Boc)2O/aq. NaOH/acetone; (e) Et3N/EDC/HOBt; (f) (1)
methanesulfonyl chloride/Et3N (2) AcSK; (g) aq. NaOH/MeOH.
Chart 4. Preparation of the Single Isomers of Compound 4a
Table 7. Structure and MMP-1 Inhibitory Activities of a-Mercaptocar-
bonyl Compounds (4a—k, 5a—c, and 36a, b)a)
Inhibitory activity
Compd.
A.A3
A.A2
A.A1Ј
A.A2Ј
(IC50, nM)
4a
36ab)
36bc)
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
5a
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Pro
Pro
—
—
—
—
—
—
—
—
—
—
—
—
—
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Val
Val
Val
Leu
Leu
Leu
Leu
Tyr(Me)
Tyr(Me)
Tyr(Me)
Val
Leu
Phe
Val
Phe
Leu
Val
Phe
Leu
—
Phe
Phe
—
6.8
240
2.5
6.7
8.1
1.4
48
23
40
140
15
24
8400
23
170
5b
5c
Ͼ10000
a) Values are averages for 3 experiments. b) (R)-isomer. c) (S)-isomer.
evaporation in vacuo. Then the residue was precipitated from Et2O–n-
hexane to give 8.0 g of 24 (72%) as a colorless solid. mp 92—95 °C, Rf2
0.59. 1H-NMR (CD3OD, TMS) d: 1.43 (9H, s), 3.23—3.35 (m), 3.35—3.47
(1H, m), 4.03—4.27 (1H, dd, Jϭ4.1, 6.8 Hz). FAB-MS m/z : 206 (MϩH)ϩ.
General Procedure for the Preparation of Compounds 26a—k EDC
(1.1 eq of 24) was added to a mixture of H–A.A1Ј–A.A2Ј–NHMe·HCl
(1.05 eq of 24; prepared from the corresponding Boc–A.A1Ј–A.A2Ј–NHMe
and 4 N HCl in EtOAc), Et3N (1.0 eq of H–A.A1Ј–A.A2Ј–NHMe·HCl),
HOBt (1.05 eq of 24), compound 24, and DMF (4—7% w/v) at Ϫ12 °C. The
mixture was stirred at Ϫ3 °C for 6 h and then at 0 °C for 15 h. The reaction
mixture was subsequently diluted with EtOAc, washed successively with sat.
aq. NaCl, 1 N aq. HCl, sat. aq. NaCl, 10% aq. Na2CO3, and sat. aq. NaCl,
dried over MgSO4, and evaporated in vacuo to give a crude product, which
was purified by silica gel column chromatography (eluent; CHCl3 : MeOHϭ
50 : 1—30 : 1) to give 26a—k as a colorless solid. Yield and spectral data for
26a—k are listed in Tables 1 and 2.
General Procedure for the Preparation of Compounds 27a—k To a
solution of 26 in CH2Cl2–pyridine (3 : 1, 4% w/v), methanesulfonyl chloride
(1.5 eq of 26) was added dropwise at 0 °C, and the mixture was stirred at the
same temperature for 2 h. Then the reaction mixture was diluted with EtOAc
(0.6% w/v) and poured into cold 1 N aq. HCl (40 eq of 26). The organic layer
was washed with sat. aq. NaCl, dried over MgSO4, and evaporated to give a
mesylate. To a solution of the mesylate in DMF (5% w/v), potassium thioac-
etate (5 eq of mesylate) was added, and the mixture was stirred at room tem-
perature for 2 d. Then the reaction mixture was diluted with EtOAc, washed
Reagents: (a) (1) methanesulfonyl chloride/Et3N (2) R3-SH/KOH; (b) MCPBA; (c)
(1) methanesulfonyl chloride/Et3N (2) AcSK (3) aq. NaOH/MeOH; (4) EtO2C-N(SEt)-
NH-CO2Et; (d) (1) methanesulfonyl chloride/Et3N (2) NaN3; (e) H2/Pd–C; (f)
Et3N/HOBt/EDC; (g) BnONH2/HOBt/EDC.
Chart 5. Substitution of the Mercapto Group with Other Functional
Groups
solution of KCN (7.61 g, 114 mmol) in water (30 ml) was added. After
stirred for 4 h, the mixture was extracted with EtOAc and evaporated to give
the cyanohydrin as an oil. Then it was hydrolyzed in dioxane–conc. HCl (1 :
1, 100 ml) by reflux for 12 h. The hydrolyzate was evaporated after washing
with Et2O and the residue was dissolved in H2O–acetone (1 : 1, 100 ml),
which was adjusted to pH 5.5 with NH4OH. Crystals were precipitated by
letting the mixture stand overnight at 4 °C and were filtered and washed with
acetone to give 5.66 g of 3-amino-2(RS)-hydroxypropionic acid (47% yield
from the aldehyde) as a colorless solid. To a mixture of 3-amino-2(RS)-hy-
droxypropionic acid (5.66 g, 53.9 mmol), dioxane–H2O (1 : 1, 100 ml), and
1 N aq. NaOH (53.9 ml), a solution of (Boc)2O (12.3 g, 56.6 mmol) in diox-
ane (50 ml) was added and the mixture was stirred at room temperature for
4 h. After washing with Et2O, the reaction mixture was adjusted to pH 2 with
1 N aq. HCl, followed by extraction with EtOAc, drying over MgSO4, and

February 2002
249
with sat. aq. NaCl, dried over MgSO4, and evaporated in vacuo to give a
4.67—4.81 (1H, m), 6.31—6.71 (1H, m), 6.99—7.33 (7H, m), 7.33—7.48
slightly brown oil, which was purified by silica gel column chromatography (1H, m), 7.68—7.91 (1H, m). FAB-MS m/z: 705 (MϩH)ϩ. HR-MS Calcd
(eluent; CHCl3 : MeOHϭ60 : 1—30 : 1) to give 27a—k as a colorless solid. for C35H57N6O7S: 705.4009 (MϩH)ϩ. Found: 705.4031.
Yield and spectral data for 27a—k are listed in Tables 3 and 4.
General Procedure for the Preparation of Compounds 4a—k To a
solution of 27 in MeOH (5% w/v), 1 N aq. NaOH (1.05 eq of 27) was added,
and the mixture was stirred at room temperature for 0.5—1 h. Then the reac- NMR (CDCl3, TMS) d: 0.80—1.01 (12H, m), 1.31—1.78 (15H, m), 1.80—
tion mixture was diluted with EtOAc, washed successively with sat. aq. 2.17 (3H, m), 2.17—2.29 (2H, m), 2.81 (3H, d, Jϭ4.8 Hz), 3.30—3.50 (2H,
Na-[3-(tert-Butyloxycarbonyl-prolyl-leucyl-amino)-2(R,S)-(mercapto)pro-
pionyl]-leucine N-Methylamide (5c, A.A3ϭPro, A.A2ϭLeu, A.A1ЈϭLeu):
Yield: 74%. mp 202—205 °C, [a]D25 Ϫ85.2° (cϭ1.03, MeOH), Rf3 0.28. 1H-
NaCl, 1 N aq. HCl, and sat. aq. NaCl, dried over MgSO4, and evaporated in
m), 3.50—3.78 (3H, m), 4.15—4.41 (3H, m), 6.31—6.50 (1H, m), 6.93—
vacuo to give a colorless residue, which was precipitated from Et2O to give 7.20 (2H, m), 7.39—7.49 (1H, m). FAB-MS m/z: 558 (MϩH)ϩ. HR-MS
4a—k as a colorless solid. Yield and spectral data for 4a—k are listed in Ta- Calcd for C26H48N5O6S: 558.3325 (MϩH)ϩ. Found: 558.3322.
bles 5 and 6.
(S)-Malic Acid 1-Monobenzyl Ester (30a)25) Trifluoroacetic anhydride
(33.6 ml, 238 mmol) was added to L-malic acid (13.4 g, 99.9 mmol) and the
mixture was stirred at 0 °C for 2.5 h. Then the reaction mixture was concen-
trated using a vacuum pump while the flask was kept at 0 °C. To the result-
General Procedure for the Preparation of Compounds 29a—c Com-
pound 27d or 27k was treated with cold 4 N HCl in EtOAc (4% w/v) for
0.5—1.0 h. The reaction mixture was evaporated in vacuo and the residue
was precipitated from Et2O, collected by filtration, and dried under reduced ing residue, benzylalcohol (20.7 ml, 200 mmol) was added, after which the
pressure to give the hydrochloride salt in a quantitative yield. A mixed anhy- mixture was stirred at room temperature for 2 h and then diluted with
dride [prepared from Boc–A.A3–A.A2–OH (28a or b), ethyl chloroformate EtOAc. The reaction mixture was subsequently adjusted to pH 7 by adding
(1.1 eq of 28a or b), and NMM (1.1 eq of 28a or b) as usual] in DMF (4%
w/v) was added to an ice-cold solution of the amine hydrochloride salt
(0.95 eq of 28a or b) in DMF (7% w/v). Then NMM (1 eq of amine hy-
10% aq. Na2CO3 to give a colorless suspension, which was collected by fil-
tration and washed with EtOAc. The sodium salt of 30a was dissolved in 1 N
aq. HCl (160 ml), after which it was extracted with EtOAc, washed with sat.
drochloride salt) was added and the mixture was stirred at 0 °C for 1 h. Sub- aq. NaCl, dried over MgSO4, and evaporated in vacuo to give 30a (13.3 g,
sequently, the reaction mixture was partitioned between EtOAc and H2O. 59%) as a colorless oil. [a]D25 Ϫ13.2° (cϭ1.0, MeOH). 1H-NMR (CDCl3,
The organic layer was washed successively with H2O, sat. aq. NaHCO3, and
TMS) d: 2.76—2.97 (2H, m), 4.54 (1H, dd, Jϭ4.4, 6.1 Hz), 5.12 (2H, s),
sat. aq. NaCl, dried over MgSO4, and evaporated in vacuo to give a crude 6.51 (2H, m), 7.34 (5H, s). FAB-MS m/z: 225 (MϩH)ϩ.
product, which was purified by silica gel column chromatography (eluent;
CHCl3 : MeOHϭ80 : 1—40 : 1) to give 29 as a colorless solid.
(R)-Malic Acid 1-Monobenzyl Ester (30b) Compound 30b was pre-
pared by the same method as that described for 30a. [a]D25 ϩ12.7° (cϭ1.0,
MeOH).
Na-[3-(tert-Butyloxycarbonyl-leucyl-amino)-2(R,S)-(acetylsulfanyl)propi-
onyl]-leucyl-phenylalanine N-Methylamide (29a, A.A2ϭLeu, A.A1ЈϭLeu,
Benzyl (S)-2-Oxooxazolidine-5-carboxylate (31a)25) A mixture of 30a
A.A2ЈϭPhe): Yield: 92%. mp 126—135 °C, [a]D25 Ϫ34.9° (cϭ0.29, CHCl3), (4.06 g, 18.1 mmol), diphenylphosphoryl azide (4.29 ml, 19.9 mmol), Et3N
Rf4 0.19 and 0.26. 1H-NMR (CD3OD, TMS) d: 0.80—1.01 (12H, m), 1.31—
(2.91 ml, 20.9 mmol), and benzene (60 ml) was heated under reflux for 4 h.
1.72 (15H, m), 2.35 (3H, s), 2.67 (3H, s), 2.85—2.98 (1H, m), 2.98—3.15 The reaction mixture was evaporated in vacuo and the residue was dissolved
(1H, m), 3.45—3.65 (2H, m), 4.10—4.41 (3H, m), 4.41—4.56 (1H, m), in H2O (20 ml), which was saturated with NaCl and then extracted with
7.12—7.33 (5H, m). FAB-MS m/z : 650 (MϩH)ϩ. HR-MS Calcd for EtOAc. The combined organic layer was washed with sat. aq. NaHCO3 and
C32H52N5O7S: 650.3587 (MϩH)ϩ. Found: 650.3628.
sat. aq. NaCl, after which it was dried over MgSO4 and evaporated in vacuo
to leave a yellow solid, which was recrystallized from MeOH–diisopropyl
Na-[3-(tert-Butyloxycarbonyl-prolyl-leucyl-amino)-2(R,S)-(acetylsul-
fanyl)propionyl]-leucyl-phenylalanine N-Methylamide (29b, A.A3ϭPro, ether to give 31a (2.50 g, 62%) as slightly yellow crystals. mp 132—134 °C,
A.A2ϭLeu, A.A1ЈϭLeu, A.A2ЈϭPhe): Yield: 40%. mp 187—189 °C, [a]D25
Ϫ53.8° (cϭ1.02, CHCl3), Rf2 0.52. 1H-NMR (CD3OD, TMS) d: 0.78—1.01
[a]D25 Ϫ1.5° (cϭ1.0, MeOH). 1H-NMR (CDCl3, TMS) d: 3.69 (1H, dd,
Jϭ5.5, 9.0 Hz), 3.88 (1H, t, Jϭ9.0 Hz), 5.05 (1H, dd, Jϭ5.5, 9.5 Hz), 5.24—
(12H, m), 1.28—1.77 (15H, m), 1.77—2.08 (3H, m), 2.08—2.29 (1H, m), 5.36 (3H, m), 7.37 (5H, s). FAB-MS m/z: 222 (MϩH)ϩ.
2.35 (3H, s), 2.67 (3H, s), 2.80—2.95 (1H, m), 3.10—3.23 (1H, m), 3.35—
Benzyl (R)-2-Oxooxazolidine-5-carboxylate (31b) Compound 31b
3.68 (4H, m), 4.10—4.42 (4H, m), 4.42—4.57 (1H, m), 7.12—7.31 (5H, m). was prepared by the same method as that described for 31a. [a]D25 ϩ1.4°
FAB-MS m/z: 747 (MϩH)ϩ. HR-MS Calcd for C37H59N6O8S: 747.4115 (cϭ1.0, MeOH).
(MϩH)ϩ. Found: 747.4159.
N-tert-Butyloxycarbonyl-(S)-isoserine (33a)25) A solution of 31a
(4.65 g, 21 mmol) in 3 N aq. NaOH (61.4 ml)–MeOH (15.4 ml) was stirred at
Na-[3-(tert-Butyloxycarbonyl-prolyl-leucyl-amino)-2(R,S)-(acetylsul-
fanyl)propionyl]-leucine N-Methylamide (29c, A.A3ϭPro, A.A2ϭLeu, 60 °C for 4 h. Then MeOH was evaporated in vacuo and washed with Et2O,
A.A1ЈϭLeu): Yield: 65%. mp 187—193 °C, [a]D25 Ϫ41.0° (cϭ0.79, CHCl3), neutralized by adding conc. HCl, and concentrated to dryness. To the
Rf3 0.36. 1H-NMR (CD3OD, TMS) d: 0.80—1.02 (12H, m), 1.30—1.76
residue, 1 N aq. NaOH (42 ml) and di-tert-butyl dicarbonate [4.82 g (22
(15H, m), 1.76—2.07 (3H, m), 2.07—2.29 (1H, m), 2.34 (3H, s), 2.71 (3H, mmol) in acetone (45 ml)] were added, and the mixture was stirred for 14 h
s), 3.35—3.56 (3H, m), 3.56—3.70 (1H, m), 4.16—4.40 (4H, m). FAB-MS at 4 °C. Acetone was evaporated, after which the residue was washed with
m/z: 600 (MϩH)ϩ. HR-MS Calcd for C28H50N5O7S: 600.3431 (MϩH)ϩ.
Found: 600.3435.
Et2O (2ϫ80 ml) and then adjusted to pH 2 by adding 1 N aq. HCl. The de-
sired material was extracted with EtOAc, washed with sat. aq. NaCl, dried
over MgSO4, and evaporated in vacuo to leave a colorless solid, which was
recrystallized from EtOAc–Et2O to give 33a (3.42 g, 79%) as a colorless
General Procedure for the Preparation of Compounds 5a—c To a
solution of 29 in MeOH (5% w/v), 1 N aq. NaOH (1.05 eq of 29) was added
and the mixture was stirred at room temperature for 0.5—1 h. Then the reac- solid. mp 86—88 °C, [a]D25 ϩ6.3° (cϭ1.0, MeOH). 1H-NMR (CD3OD,
tion mixture was diluted with EtOAc, washed successively with sat. aq. TMS) d: 1.43 (9H, s), 3.23—3.35 (m), 3.44 (1H, dd, Jϭ4.2, 13.9 Hz), 4.17
NaCl, 1 N aq. HCl, and sat. aq. NaCl, dried over MgSO4, and evaporated in
vacuo to give a colorless residue, which was precipitated from EtOAc–Et2O
to give 5a—c as a colorless solid.
(1H, dd, Jϭ4.2, 6.7 Hz). FAB-MS m/z: 206 (MϩH)ϩ.
N-tert-Butyloxycarbonyl-(R)-isoserine (33b) Compound 33b was pre-
pared by the same method as that described for 33a. [a]D25 Ϫ6.4° (cϭ1.0,
MeOH).
Na-[3-(tert-Butyloxycarbonyl-leucyl-amino)-2(R,S)-(mercapto)propi-
onyl]-leucyl-phenylalanine N-Methylamide (5a, A.A2ϭLeu, A.A1ЈϭLeu,
Na-[3-(tert-Butyloxycarbonylamino)-2(S)-hydoxypropionyl]-leucyl-O-
A.A2ЈϭPhe): Yield: 33%. mp 198—202 °C, [a]D25 Ϫ40.2° (cϭ0.53, CHCl3), methyl-tyrosine N-Methylamide (34a) Condensation of N-protected
Rf2 0.49. 1H-NMR (CD3OD, TMS) d: 0.80—1.02 (12H, m), 1.37—1.78 isoserine (33a, 1.44 g, 7.01 mmol) and the dipeptide (25a, 2.28 g, 6.37
(15H, m), 2.67 (3H, s), 2.87—3.00 (1H, m), 3.03—3.19 (1H, m), 3.45— mmol) was performed by the same method as that described for the prepara-
3.64 (2H, m), 3.95—4.12 (1H, m), 4.18—4.30 (1H, m), 4.45—4.66 (2H, m), tion of 26. Yield; 75%, mp 196—198 °C, [a]D25 Ϫ48.3° (cϭ1.0, MeOH), 1H-
7.14—7.31 (5H, m). FAB-MS m/z: 608 (MϩH)ϩ. HR-MS Calcd for NMR (CDCl3, TMS) d: 0.87 (3H, d, Jϭ6.2 Hz), 0.92 (3H, d, Jϭ6.2 Hz),
C30H50N5O6S: 608.3482 (MϩH)ϩ. Found: 608.3528.
1.37—1.70 (12H, m), 1.82 (1H, br s), 2.70 (3H, d, Jϭ4.8 Hz), 2.94—3.05
Na-[3-(tert-Butyloxycarbonyl-prolyl-leucyl-amino)-2(R,S)-(mercapto)pro- (2H, m), 3.38—3.51 (1H, m), 3.77 (3H, s), 4.07—4.15 (1H, m), 4.28—4.38
pionyl]-leucyl-phenylalanine N-Methylamide (5b, A.A3ϭPro, A.A2ϭLeu, (1H, m), 4.53 (1H, dd, Jϭ7.0, 14.9 Hz), 5.23—5.32 (1H, m), 5.43 (1H, d,
A.A1ЈϭLeu, A.A2ЈϭPhe): Yield: 58%. mp 201—206 °C, [a]D25 Ϫ71.8° Jϭ4.6 Hz), 6.05—6.14 (1H, m), 6.79—6.84 (2H, m), 6.87 (1H, d,
(cϭ1.08, MeOH), Rf3 0.34. 1H-NMR (CDCl3, TMS) d: 0.62—1.01 (12H,
m), 1.28—1.80 (15H, m), 1.80—2.29 (4H, m), 2.34—2.57 (1H, m), 2.72
(3H, d, Jϭ4.6 Hz), 2.87—3.02 (1H, m), 3.02—3.31 (1H, m), 3.31—3.55
Jϭ8.1 Hz), 7.03—7.12 (2H, m), 7.29 (1H, d, Jϭ7.3 Hz). FAB-MS m/z: 509
(Mϩ1)ϩ.
Na-[3-(tert-Butyloxycarbonylamino)-2(R)-hydoxypropionyl]-leucyl-O-
(2H, m), 3.55—3.87 (3H, m), 4.05—4.21 (1H, m), 4.21—4.50 (2H, m), methyl-tyrosine N-Methylamide (34b) Compound 34b was prepared by

250
Vol. 50, No. 2
the same method as that described for 34a. Yield; 79%, mp 187—189 °C, m), 6.73—6.81 (2H, m), 7.04—7.16 (2H, m), 7.18—7.40 (5H, m). FAB-MS
[a]D25 Ϫ11.8° (cϭ1.0, MeOH). 1H-NMR (CDCl3, TMS) d: 0.88 (3H, d, m/z: 615 (MϩH)ϩ. HR-MS Calcd for C32H47N4O6S: 615.3216 (MϩH)ϩ.
Jϭ6.1 Hz), 0.93 (3H, d, Jϭ6.1 Hz), 1.40—1.54 (10H, m), 1.59—1.85 (2H,
m), 2.68 (3H, d, Jϭ5.0 Hz), 2.87—3.06 (2H, m), 3.33—3.46 (1H, m), 3.68
(1H, ddd, Jϭ3.3, 5.2, 14.9 Hz), 3.77 (3H, s), 4.19—4.31 (2H, m), 4.53 (1H,
Found: 615.3251.
General Procedure for the Preparation of Compounds 38a—c To a
solution of compound 37a—c in CH2Cl2–MeOH (6 : 1, 3% w/v), MCPBA
dd, Jϭ8.3, 15.6 Hz), 5.50 (1H, d, Jϭ3.5 Hz), 6.00 (1H, d, Jϭ4.4 Hz), 6.56— (70% min, 1.06 eq of 37a—c) was added and the mixture was stirred at
6.65 (1H, m), 6.71—6.84 (3H, m), 7.05—7.14 (2H, m), 7.20—7.30 (1H, m). room temperature for 1 h. The reaction mixture was diluted with EtOAc,
Na-[3-(tert-Butyloxycarbonylamino)-2(R)-(acetylsulfanyl)propionyl]- washed successively with 5% aq. NaHCO3, and sat. aq. NaCl, dried over
leucyl-O-methyl-tyrosine N-Methylamide (35a) Conversion of the hy- MgSO4, and evaporated in vacuo to yield a colorless syrup, which was re-
droxy group (34a) to the acetylsulfanyl group (35a) was performed by the precipitated from Et2O to give 38a—c as a colorless solid.
same method as that described for the preparation of 27. Yield; 81%, mp
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(ethylsulfinyl)propionyl]-
195—197 °C, [a]D25 Ϫ6.8° (cϭ1.0, MeOH). 1H-NMR (CD3OD, TMS) d: leucyl-O-methyl-tyrosine N-Methylamide (38a, R3ϭEthyl): Yield: 82%. mp
0.85 (3H, d, Jϭ6.4 Hz), 0.90 (3H, d, Jϭ6.6 Hz), 1.34—1.51 (11H, m),
1.51—1.67 (1H, m), 2.36 (3H, s), 2.67 (3H, s), 2.81 (1H, dd, Jϭ9.4, TMS) d: 0.76—0.97 (6H, m), 1.24—1.71 (15H, m), 2.66 (3H, s), 2.70—
13.8 Hz), 3.10 (1H, dd, Jϭ5.6, 13.8 Hz), 3.26—3.48 (2H, m), 3.75 (3H, s), 3.03 (2H, m), 3.03—3.26 (2H, m), 3.48—3.70 (2H, m), 3.74 (3H, s), 4.02—
137—139 °C, [a]D25 Ϫ36.6° (cϭ1.0, MeOH), Rf3 0.44. 1H-NMR (CD3OD,
4.21 (1H, dd, Jϭ6.4, 8.8 Hz), 4.30 (1H, t, Jϭ7.3 Hz), 4.44 (1H, dd, Jϭ5.6, 4.33 (2H, m), 4.38—4.51 (1H, m), 6.74—6.88 (2H, m), 7.04—7.19 (2H, m).
9.4 Hz), 6.78—6.87 (2H, m), 7.09—7.18 (2H, m). FAB-MS m/z: 567
FAB-MS m/z: 569 (MϩH)ϩ. HR-MS Calcd for C27H45N4O7S: 569.3009
(MϩH)ϩ. Found: 569.2977.
(Mϩ1)ϩ.
Na-[3-(tert-Butyloxycarbonylamino)-2(S)-(acetylsulfanyl)propionyl]-
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(iso-butylsulfinyl)propionyl]-
leucyl-O-methyl-tyrosine N-Methylamide (35b) Conversion of the hy- leucyl-O-methyl-tyrosine N-Methylamide (38b, R3ϭIso-butyl): Yield: 80%.
droxy group (34b) to the acetylsulfanyl group (35b) was performed by the mp 138—142 °C, [a]D25 Ϫ39.9° (cϭ1.0, MeOH), Rf3 0.57. 1H-NMR
same method as that described for the preparation of 27. Yield; 77%, mp (CD3OD, TMS) d: 0.79—0.96 (6H, m), 1.03—1.22 (6H, m), 1.28—1.71
161—163 °C, [a]D25 Ϫ67.7° (cϭ1.0, MeOH). 1H-NMR (CD3OD, TMS) d: (12H, m), 2.08—2.48 (1H, m), 2.61—3.25 (7H, m), 3.50—3.70 (2H, m),
0.83 (3H, d, Jϭ6.4 Hz), 0.89 (3H, d, Jϭ6.4 Hz), 1.36—1.63 (12H, m), 2.35 3.74 (3H, s), 3.96—4.50 (3H, m), 6.73—6.89 (2H, m), 7.06—7.20 (2H, m).
(3H, s), 2.66 (3H, s), 2.87 (1H, dd, Jϭ8.4, 13.8 Hz), 3.05 (1H, dd, Jϭ6.4, FAB-MS m/z: 597 (MϩH)ϩ. HR-MS Calcd for C29H49N4O7S: 597.3322
13.8 Hz), 3.28—3.38 (m), 3.41—3.54 (1H, m), 3.74 (3H, s), 4.20—4.31 (MϩH)ϩ. Found: 597.3323.
(2H, m), 4.46 (1H, dd, Jϭ6.4, 8.4 Hz), 6.79—6.85 (2H, m), 7.08—7.15 (2H,
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(benzylsulfinyl)propionyl]-
leucyl-O-methyl-tyrosine N-Methylamide (38c, R3ϭBenzyl): Yield: 82%.
m).
Na-[3-(tert-Butyloxycarbonylamino)-2(R)-mercaptopropionyl]-leucyl- mp 152—154 °C, [a]D25 Ϫ37.4° (cϭ1.0, MeOH), Rf3 0.56. 1H-NMR
O-methyl-tyrosine N-Methylamide (36a) Alkaline hydrolysis of acetyl- (CD3OD, TMS) d: 0.74—1.00 (6H, m), 1.20—1.77 (12H, m), 2.58—2.72
sulfanyl ester (35a) was performed by the same method as that described for
(3H, m), 2.72—2.94 (1H, m), 3.00—3.21 (1H, m), 3.57—3.83 (5H, m),
the preparation of 4. Yield; 78%, mp 193—196 °C, [a]D25 Ϫ21.7° (cϭ0.3, 4.04—4.64 (5H, m), 6.72—6.88 (2H, m), 7.04—7.19 (2H, m), 7.29—7.55
1
MeOH). H-NMR (CD3OD, TMS) d: 0.78 (3H, d, Jϭ6.4 Hz), 0.82 (3H, d, (5H, m). FAB-MS m/z: 631 (MϩH)ϩ. HR-MS Calcd for C32H47N4O7S:
Jϭ6.4 Hz), 1.26—1.60 (12H, m), 2.57 (3H, s), 2.78 (1H, dd, Jϭ7.9, 631.3165 (MϩH)ϩ. Found: 631.3146.
13.9 Hz), 2.93 (1H, dd, Jϭ6.6, 13.9 Hz), 3.14—3.30 (m), 3.36—3.50 (1H,
m), 3.64 (3H, s), 4.18 (1H, dd, Jϭ6.2, 8.8 Hz), 4.25—4.42 (2H, m), 6.67—
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(ethyldisulfanyl)propi-
onyl]-leucyl-O-methyl-tyrosine N-Methylamide (39) A mixture of di-
6.78 (2H, m), 6.96—7.08 (2H, m). HR-MS Calcd for C25H41N4O6S: ethyl azodicarboxylate (129 ml, 0.787 mmol), ethanethiol (56 ml, 0.75 mmol),
525.2747 (MϩH)ϩ. Found: 525.2782.
and benzene (1.5 ml) was gently refluxed for 2 h under nitrogen atmosphere
Na-[3-(tert-Butyloxycarbonylamino)-2(S)-mercaptopropionyl]-leucyl-
to give diethyl N-ethylsulfenylhydrazodicarboxylates27) as a yellow solution.
O-methyl-tyrosine N-Methylamide (36b) Alkaline hydrolysis of acetyl- To the solution, a suspension of 4a (375 mg, 0.715 mmol) in benzene
sulfanyl ester (35b) was performed by the same method as that described for (5.0 ml) was added, and the mixture was stirred for 24 h at room temperature
the preparation of 4. Yield; 92%, mp 190—192 °C, [a]D25 Ϫ24.4° (cϭ1.0,
under a nitrogen atmosphere. Then the reaction mixture was evaporated to
1
MeOH). H-NMR (CD3OD, TMS) d: 0.78 (3H, d, Jϭ6.4 Hz), 0.82 (3H, d, give a yellow residue, which was purified by silica gel column chromatogra-
Jϭ6.4 Hz), 1.27—1.60 (12H, m), 2.56 (3H, s), 2.77 (1H, dd, Jϭ8.3, phy (eluent; CHCl3 : MeOHϭ80 : 1) and precipitated from Et2O–n-hexane to
1
13.8 Hz), 2.94 (1H, dd, Jϭ6.7, 13.8 Hz), 3.17—3.29 (m), 3.34—3.43 (1H, give 39 as a colorless solid (78 mg, 19 %). mp 119—124 °C, Rf3 0.74. H-
m), 3.65 (3H, s), 4.16 (1H, dd, Jϭ6.2, 8.8 Hz), 4.36 (1H, dd, Jϭ6.7, 8.3 Hz), NMR (CDCl3, TMS) d: 0.81—1.01 (6H, m), 1.28 (1.5H, t, Jϭ7.2 Hz), 1.32
4.48—4.54 (1H, m), 6.69—6.77 (2H, m), 6.97—7.05 (2H, m). HR-MS (1.5H, t, Jϭ7.3 Hz), 1.38—1.73 (12H, m), 2.64—2.83 (5H, m), 2.92—3.13
Calcd for C25H41N4O6S: 525.2747 (MϩH)ϩ. Found: 525.2772.
(2H, m), 3.44—3.64 (2H, m), 3.78 (3H, s), 4.16—4.25 (1H, m), 4.25—4.41
General Procedure for the Preparation of Compounds 37a—c These (1H, m), 4.46—4.62 (1H, m), 5.20 (0.5H, m), 5.31 (0.5H, m), 5.82 (0.5H,
compounds were prepared from 26a and the corresponding R3-SK [prepared m), 6.00 (0.5H, m), 6.36—6.51 (1H, m), 6.56—6.68 (1H, d, Jϭ8.3 Hz),
from R3-SH and KOH in MeOH] by the same method as that described for 6.73—6.89 (2H, m), 7.01—7.15 (2H, m). FAB-MS m/z: 585 (MϩH)ϩ. HR-
the preparation of 27.
MS Calcd for C27H45N4O6S2: 585.2781 (MϩH)ϩ. Found: 585.2787.
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-azidopropionyl]-leucyl-O-
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(ethylsulfanyl)propionyl]-
leucyl-O-methyl-tyrosine N-Methylamide (37a, R3ϭEthyl): Yield: 79%. mp methyl-tyrosine N-Methylamide (40) To a solution of the mesylate
160—165 °C, [a]D25 Ϫ33.8° (cϭ1.0, MeOH), Rf3 0.48. 1H-NMR (CD3OD, [1.0 g, 1.71 mmol, derived from 26a in the same manner as that described
TMS) d: 0.80—1.05 (9H, m), 1.26 (3H, t, Jϭ7.8 Hz), 1.36—1.80 (12H, m), for the preparation of 27] in DMF (17 ml), sodium azide (600 mg,
2.31—2.50 (2H, m), 2.67 (3H, s), 2.78—2.96 (1H, m), 2.96—3.12 (1H, m),
9.23 mmol) was added, and the mixture was stirred under a nitrogen atmos-
3.12—3.60 (m), 3.72 (3H, s), 4.14—4.50 (2H, m), 6.74—6.87 (2H, m), phere for 20 h at room temperature. The reaction mixture was diluted with
7.05—7.17 (2H, m). FAB-MS m/z: 553 (MϩH)ϩ. HR-MS Calcd for EtOAc (60 ml), washed successively with sat. aq. NaCl, H2O, and sat. aq.
C27H45N4O6S: 553.3060 (MϩH)ϩ. Found: 553.3049.
NaCl, dried over MgSO4, and evaporated in vacuo to leave a slightly yellow
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(iso-butylsulfanyl)propi- solid, which was purified by flash chromatography (eluent; n-hexane :
onyl]-leucyl-O-methyl-tyrosine N-Methylamide (37b, R3ϭIso-butyl): Yield:
EtOAcϭ1 : 2) to give 40 (803 mg, 88%) as a colorless solid. mp 170—
78%. mp 150—152 °C, [a]D25 Ϫ33.8° (cϭ1.0, MeOH), Rf3 0.48. 1H-NMR 172 °C, [a]D25 Ϫ30.3° (cϭ1.0, MeOH), 1H-NMR (CD3OD, TMS) d: 0.78
(CD3OD, TMS) d: 0.87—1.10 (12H, m), 1.26—1.88 (13H, m), 2.33—2.52 (3H, d, Jϭ6.2 Hz), 0.82 (3H, d, Jϭ6.2 Hz), 1.12—1.26 (1H, m), 1.30—1.57
(2H, m), 2.65 (3H, s), 2.77—2.94 (1H, m), 2.94—3.09 (1H, m), 3.12—3.56
(11H, m), 2.57 (3H, s), 2.73—2.84 (1H, m), 2.88—3.00 (1H, m), 3.15—
(m), 3.74 (3H, s), 4.18—4.56 (2H, m), 6.68—6.89 (2H, m), 6.98—7.17 (2H, 3.28 (m), 3.30—3.45 (1H, m), 3.65 (3H, s), 3.88—3.96 (1H, m), 4.17—4.28
m). FAB-MS m/z: 581 (MϩH)ϩ. HR-MS Calcd for C29H49N4O6S: 581.3373 (1H, m), 4.32—4.41 (1H, m), 6.70—6.78 (2H, m), 6.99—7.08 (2H, m).
(MϩH)ϩ. Found: 581.3395.
FAB-MS m/z: 534 (MϩH)ϩ.
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(benzylsulfanyl)propionyl]-
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-aminopropionyl]-leucyl-
leucyl-O-methyl-tyrosine N-Methylamide (37c, R3ϭBenzyl): Yield: 81%. O-methyl-tyrosine N-Methylamide (41) A solution of 40 (270 mg, 0.506
mp 164—166 °C, [a]D25 Ϫ26.8° (cϭ1.0, MeOH), Rf3 0.49. 1H-NMR mmol) in MeOH (15 ml) was hydrogenated on 10% Pd–C (50% wet, 150
(CD3OD, TMS) d: 0.76—1.03 (6H, m), 1.27—1.69 (12H, m), 2.65 (3H, s), mg) for 2.5 h at room temperature and nomal pressure. After removal of the
2.82—2.94 (1H, m), 2.96—3.08 (1H, m), 3.34—3.60 (3H, m), 3.69 (1.5H, catalyst, the filtrate was evaporated, and the residue was precipitated from
s), 3.73 (1.5H, s), 3.75—3.90 (2H, m), 4.22—4.40 (1H, m), 4.42—4.51 (1H,
Et2O to give 41 as a colorless solid (226 mg, 88 %). mp 169—171 °C, [a]D25

February 2002
251
1
4.40 (1H, m), 6.68—6.78 (2H, m), 6.97—7.12 (2H, m). FAB-MS m/z: 552
(MϩH)ϩ. HR-MS Calcd for C26H42N5O8: 552.3034 (MϩH)ϩ. Found:
552.3068.
Ϫ31.0° (cϭ1.0, MeOH). H-NMR (CD3OD, TMS) d: 0.83—0.96 (6H, m),
1.35—1.67 (12H, m), 2.66 (3H, s), 2.79—2.93 (1H, m), 2.98—3.23 (2H,
m), 3.37—3.51 (1H, m), 3.74 (3H, s), 4.08—4.39 (2H, m), 4.42—4.52 (1H,
m), 6.78—6.88 (2H, m), 7.05—7.18 (2H, m). FAB-MS m/z: 508 (MϩH)ϩ.
HR-MS Calcd for C25H42N5O6: 508.3135 (MϩH)ϩ. Found: 508.3109.
Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(benzyloxycarbonyl)pro-
pionyl]-leucyl-O-methyl-tyrosine N-Methylamide (43) To a stirred solu-
tion of diisopropylamine (2.0 ml, 14.3 mmol) in THF (12 ml), 1.6 M n-BuLi
in hexane (8.95 ml, 14.3 mmol) was added at Ϫ10 °C under a nitrogen at-
mosphere. After 20 min, a solution of benzyl 3-(tert-butyloxycarbonyl-
amino)propionate (2.00 g, 7.16 mmol) in THF (8 ml) was added dropwise
over a period of 15 min at Ϫ70 °C, and the mixture was stirred at the same
temperature for 20 min. Then the reaction mixture (a slightly yellow solu-
tion) was poured into dry ice and diluted with 0.1 N aq. H2SO4 (225 ml). The
organic material was extracted with EtOAc, washed with sat. aq. NaCl, dried
over MgSO4, and evaporated in vacuo to give crude 3-(tert-butyloxycar-
bonylamino)-2(R,S)-(benzyloxycarbonyl)propionic acid as a slightly yellow
oil, which was used for the subsequent condensation procedure without fur-
ther purification. EDC (1.51 g, 7.88 mmol) was added to a mixture of H-
Leu-Tyr(Me)-NHMe·EHCl (25a, 2.68 g, 7.52 mmol), Et3N (1.05 ml, 7.52
mmol), HOBt (1.02g, 7.52 mmol), crude 3-(tert-butyloxycarbonylamino)-
2(R,S)-(benzyloxycarbonyl)propionic acid, and DMF (80 ml) at Ϫ12 °C. The
mixture was stirred at Ϫ3 °C for 6 h and then at 0 °C for 15 h. The reaction
mixture was diluted with EtOAc, washed successively with sat. aq. NaCl, 1 N
aq. HCl, sat. aq. NaCl, 10% aq. Na2CO3, and sat. aq. NaCl, dried over
MgSO4, and evaporated in vacuo to give crude product, which was purified
by silica gel column chromatography (eluent; CHCl3 : MeOHϭ80 : 1—30 :
1) to give 43 as a colorless solid. mp 153—156 °C, Rf3 0.49. 1H-NMR
(CD3OD, TMS) d: 0.75 (1.5H, d, Jϭ6.4 Hz), 0.82 (1.5H, d, Jϭ6.4 Hz), 0.85
(1.5H, d, Jϭ6.4 Hz), 0.89 (1.5H, d, Jϭ6.6 Hz), 1.29—1.67 (12H, m), 2.63
(3H, s), 2.73—2.92 (1H, m), 2.98—3.12 (1H, m), 3.40—3.85 (6H, m),
4.19—4.33 (1H, m), 4.39—4.50 (1H, m), 5.14 (1H, m), 5.20 (1H, m),
6.73—6.87 (2H, m), 7.02—7.16 (2H, m), 7.25—7.44 (5H, m). FAB-MS
m/z: 627 (MϩH)ϩ. HR-MS Calcd for C33H47N4O8: 627.3394 (MϩH)ϩ.
Found: 627.3422.
MMP-1 Inhibition Assay The test was carried out essentially as de-
scribed by Nagai et al.32) In a microtube, 20 ml of proMMP-1 [15 U/ml in an
assay buffer [50 mM Tris–HCl (pH 7.5), 0.2 M NaCl, 5 mM CaCl2] containing
0.02% BSA] was mixed with 20 ml of 2 mM 4-aminophenylmercuric acetate
(APMA) in assay buffer. Then the mixture was incubated at 35 °C for 2 h to
activate the proenzyme. A 20 ml aliquot of inhibitor (at various concentra-
tions) plus 40 ml of assay buffer were added to the mixture and further incu-
bation was done for 15 min at 35 °C. In the case of thiol componds, to en-
sure that the inhibitor was not oxidised, 1ϫ10Ϫ4 M b-mercaptoethanol was
added to the assay buffer. Then 100 ml of 0.05% FITC-labeled collagen in
assay buffer was added, followed by incubation at 35 °C for 2 h, after which
the reaction was terminated by adding 10 ml of 80 mM o-phenanthroline in
50% ethanol solution. A 200 ml of aliquot of elastase (25 mg/ml assay buffer)
was added and the mixture was incubated for 10 min at 35 °C to digest the
cleaved FITC-labeled collagen fragments. Next, 400 ml of 70% ethanol in
0.17 M Tris–HCl (pH 9.5)/0.67 M NaCl was added and the mixture was vor-
texed for 30 s, followed by centrifugation for 20 min at 3000 rpm to obtain
the supernatant. The fluoresence intensity of this supernatant was measured
(lex 495 nm, lem 520 nm) and the inhibitory activity was calculated as a per-
centage of the control activity in the absence of any inhibitor.
References and Notes
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Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(hydroxycarbonyl)propi-
onyl]-leucyl-O-methyl-tyrosine N-Methylamide (44) A solution of 43
(300 mg, 0.479 mmol) in MeOH (20 ml) was hydrogenated on 10% Pd–C
(50% wet, 100 mg) for 1 h at room temperature and nomal pressure. After
removal of the catalyst, the filtrate was evaporated and the residue was pre-
cipitated from Et2O–EtOAc to give 44 as a colorless solid (234 mg, 91%).
mp 201—203 °C, Rf2 0.31. 1H-NMR (CD3OD, TMS) d: 0.84 (3H, d,
Jϭ6.6 Hz), 0.87 (1.5H, d, Jϭ6.4 Hz), 0.90 (1.5H, d, Jϭ6.6 Hz), 1.26—1.69
(12H, m), 2.68 (3H, s), 2.73—2.92 (1H, m), 3.01—3.23 (1H, m), 3.41—
3.64 (3H, m), 3.74 (3H, s), 4.15—4.29 (1H, m), 4.38—4.52 (1H, m), 6.74—
6.89 (2H, m), 7.04—7.20 (2H, m). FAB-MS m/z: 537 (MϩH)ϩ. HR-MS
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propionyl]-leucyl-O-methyl-tyrosine N-Methylamide (45) EDC (371
mg, 1.94 mmol) was added to a mixture of O-benzylhydroxylamine hy-
drochloride (337 mg, 2.11 mmol), Et3N (293 ml, 2.11 mmol), HOBt (250 mg,
1.85 mmol), 44 (940 mg, 1.76 mmol), and DMF (25 ml) at Ϫ12 °C. The mix-
ture was stirred at Ϫ3 °C for 6 h and then at 0 °C for 15 h. Then the reaction
mixture was poured into ice water (200 ml) and insoluble material was col-
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water, 5% aq. NaHCO3, and water, after which it was dried over P2O5 under
reduced pressure to give 45 as a colorless solid (1.01 g, 90%). mp 236—
238 °C, Rf2 0.69. 1H-NMR (DMSO-d6, TMS) d: 0.78 (3H, d, Jϭ6.4 Hz),
0.83 (3H, d, Jϭ6.4 Hz), 1.21—1.58 (12H, m), 2.38—2.60 (m), 2.64—2.80
(1H, s), 2.82—2.97 (1H, m), 3.10—3.50 (m), 3.67 (3H, s), 4.13—4.41 (2H,
m), 4.75 (2H, s), 6.67—6.86 (3H, m), 7.00—7.15 (2H, m), 7.36 (5H, m),
7.69—7.86 (2H, m), 7.94 (1H, d, Jϭ7.7 Hz), 8.04 (1H, d, Jϭ8.3 Hz). FAB-
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Na-[3-(tert-Butyloxycarbonylamino)-2(R,S)-(hydroxycarbamoyl)pro-
pionyl]-leucyl-O-methyl-tyrosine N-Methylamide (46) A solution of 45
(710 mg, 1.11 mmol) in MeOH (100 ml) was hydrogenated on 10% Pd–C
(50% wet, 200 mg) for 3 h at room temperature and nomal pressure. After
removal of the catalyst, the filtrate was concentrated to ca. 10 ml and was
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205—209 °C, Rf2 0.32. 1H-NMR (CD3OD, TMS) d: 0.75 (1.5H, d, Jϭ6.4
Hz), 0.76 (1.5H, d, Jϭ6.4 Hz), 0.80 (1.5H, d, Jϭ6.6 Hz), 0.81 (1.5H, d,
Jϭ6.4 Hz), 1.21—1.57 (12H, m), 2.59 (3H, s), 2.72—2.86 (1H, m), 2.92—
3.08 (1H, m), 3.31—3.49 (3H, m), 3.65 (3H, s), 4.08—4.21 (1H, m), 4.30—
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252
Vol. 50, No. 2
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