Design and synthesis of carboxylate inhibitors for matrix metalloproteinases

Article abstract of DOI:10.1248/cpb.49.1272

A series of carboxylate compounds were prepared from N^α-substituted 2,3-diaminopropionic acid and were tested for efficacy as matrix metalloproteinase (MMP) inhibitors. During modeling of the initial compound 10a, we utilized three-dimensional

Full text of DOI:10.1248/cpb.49.1272

1272
Chem. Pharm. Bull. 49(10) 1272—1279 (2001)
Vol. 49, No. 10
Design and Synthesis of Carboxylate Inhibitors for Matrix
Metalloproteinases
Tetsunori FUJISAWA,a Shin-ichi KATAKURA,b Shinjiro ODAKE,a Yasuo MORITA,a Junko YASUDA,a
Isao YASUMATSU,a and Tadanori MORIKAWA*,a
Research Institute, Fuji Chemical Industries, Ltd.,a 530 Chokeiji, Takaoka, Toyama 933–8511, Japan and Discovery
Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo R&D Center,b 1–16–13 Kita-Kasai, Edogawa-ku, Tokyo
134–8630, Japan. Received April 16, 2001; accepted July 4, 2001
A series of carboxylate compounds were prepared from Na-substituted 2,3-diaminopropionic acid and were
tested for efficacy as matrix metalloproteinase (MMP) inhibitors. During modeling of the initial compound 10a,
we utilized three-dimensional structure modeling software (InsightII/Discover Ver. 2.98). Some of the prepared
carboxylate derivatives, such as carbamate compounds (12c, d, 22) and sulfonamide compounds (14b, c), proved
to be effective MMP-1 inhibitors (with IC50 values of a 10؊6 M order), depending on the substituent at the Na-po-
sition of 2,3-diaminopropionic acid. Some of them were also evaluated for inhibition of stromelysin-1 (MMP-3),
and the sulfonamide compound 14c exceeded the lead compound 5b in its MMP-3 inhibitory potency. For the
carbamate compounds, we investigated the minimum molecular size at which the MMP-1 inhibitory potency was
maintained, and found that this was P3–P1؅ compound 10b.
Key words human fibroblast collagenase; computerized drug design; inhibitor; carboxylate; stromelysin-1
Matrix metalloproteinases (MMPs) are zinc endometal- pounds because of their only modest inhibitory potency (IC50
lopeptidases that are involved in the degradation and remod- values in the order of 10Ϫ6 M for MMP-1). However, we
eling of connective tissues. This family of enzymes shows thought that this type of inhibitor could show further im-
proteolytic activity towards virtually all of the constituents provement of its inhibitory activity, since hydroxamate15) and
of the extracellular matrix. The members of this family, phosphorous16) have been examined as ZCGs.
currently numbering 20, can be classified into four groups,
Design Beside hydroxamate and phosphorous, the ZCGs
which are the collagenases that cleave triple-helical intersti- utilized most frequently are thiol or carboxylate. The free thi-
tial collagen; the gelatinases that cleave denatured collagen, ols are unstable to oxidation in air at neutral pH during
elastin, and type IV and V collagen; the stromelysins that preparation and storage.17) To avoid the disadvantage, we se-
mainly cleave proteoglycans; and the membrane-type MMPs lected a carboxylate, which was expected to be relatively sta-
that have a C-terminal transmembrane domain for anchoring ble and to show good pharmacokinetics, as the ZCG. Al-
to the cell membrane. The MMPs are involved in crucial phys- though the carboxylate is a weaker ZCG than the others for
iological and physiopathological events, such as wound heal- MMP-1,5) we designed the 2,3-diaminopropionic acid deriva-
ing, nerve growth, angiogenesis, and pregnancy. In these phys- tive (6) to compensate for this weakness. The amino acid
iological processes, MMP activity is tightly regulated.1—5) moiety was expected to chelate the active site zinc ion by
However, excessive MMP synthesis and release can lead to
connective tissue degradation and destruction, which occurs
in tumor invasion, metastasis,6) corneal ulceration,7) arthritis
disease,8a) periodontal disease,9) and multiple sclerosis.10) For
example, increased levels of fibroblast collagenase (MMP-1)
and stromelysin-1 (MMP-3) have been observed in the carti-
lage and synovium of patients with rheumatoid arthritis and
osteoarthritis, and are correlated with the severity of the dis-
ease.8b) Therefore, MMPs inhibitors may have a potent thera-
peutic effect on various proteolytic diseases.
Many MMP inhibitors have been synthesized and devel-
oped. Several compounds are currently undergoing advanced
clinical trials, such as batimastat (1),11) marimastat (2),12) tro-
cade (3),13) and AG3340 (4)14) (Fig. 1). These inhibitors
mimic the sequence of the substrate–cleavage site of MMPs
and have hydroxamate as a zinc-chelating group (ZCG). This
kind of design has been widely applied for other MMPs in-
hibitors.
Fig. 1. Selected MMP Inhibitors
Previously, we also developed a series of peptidyl hydroxa-
mates as MMP-1 inhibitors (Fig. 2),15) our compounds mim-
icked the P4–P1 substrate rather than the P1–P1Ј or P1–P2Ј sites
in the above-mentioned MMP inhibitors (1)—(4). Inhibitors
that mimic the PnЈ site have been studied extensively but little
is known about Pn site-mimicking inhibitors like our com-
Fig. 2. Structures of P4–P1 Substrate Mimicked Inhibitors That Possess
Hydroxamate as ZCG
To whom correspondence should be addressed. e-mail: morikawa@fuji-chemi.co.jp
© 2001 Pharmaceutical Society of Japan

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both amino and carboxylate groups in a similar manner as the hydroxamate was bound in a bidentate manner to the
the copper complex of amino acid (Fig. 3: in peptide chem- active site zinc ion on this software. Before generation of
istry, the complex is used to selectively protect the lysine MMP-1 : compound 6 complex, we checked binding feat-
residue). However, the 2,3-diaminopropionic acid derivative ure of MMP-1 : compound 5a complex. Each amino acid
(6) showed no inhibitory activity against MMP-1. This unex- residues were well accommodated to corresponding subsites
pected result led us to perform molecular modeling of com- in a manner similar to the case of Pro–Leu–Gly–NHOH. And
pound 6 after it was docked with MMP-1. No report of X-ray N-terminal Bz–Gly–moiety was fit into a shallow hydropho-
crystallographic studies have yet been reported on a complex bic pocket consisting of Phe185, Pro189, Ile191, and
between the Pn site of mimicking inhibitors and MMP-1. Gly192. Binding features observed in the model of the com-
There is only one report about X-ray crystallography be- pound 6 docked with MMP-1 are represented in Fig. 4 with
tween neutrophil collagenase (MMP-8) and Pro–Leu–Gly– the interatomic distances for important polar interactions. As
NHOH18) which also mimicked Pn site of collagen cleavage a result, the only carboxylate of compound 6 was coordinated
site, but exhibited weaker MMP-1 inhibitory potency than with the active site zinc ion in a bidentate manner, while the
5a.15) However, the sequence homology of the catalytic do- amino group formed a hydrogen bond with Ala 182 and did
main is high between MMP-1 and MMP-8, so both three- not interact with the zinc ion. Comparison with the model of
dimensional structures are very similar. Therefore, we gen- a potent initial inhibitor (5a) docked with MMP-1 showed
erated a three-dimensional structured model of a MMP- that the hydrogen bond between the carbonyl at the leucine in
1 : compound 6 complex based on the crystal structure of compound 6 and Ala 184 in MMP-1 was completely absent.
MMP-1 (PDB entry: 1CGL) and the structure of a complex In the case of compound 6, loss of this hydrogen bond might
between MMP-8 and Pro–Leu–Gly–NHOH (PDB entry: decrease its affinity for MMP-1. This model could indicate
1JAN) using InsightII/Discover Ver2.98 (Molecular Simula- that a hydrophobic pocket, consisting of the residues Leu
tions Inc., San Diego, CA, U.S.A.) (The details of the proce- 181, Val 215, and Tyr 240, is located in the vicinity of the N-
dure are described in the experimental section). Certainly, terminal group of compound 6. It may be presumed that sub-
Pro–Leu–Gly–NHOH, well explored Pn site-mimicking in- stitution at the amino group might create a new hydrophobic
hibitor, was fit into the MMP-1 active site and confirmed that interaction with this pocket. This hypothesis prompted us to
measure the MMP-1 inhibitory activity of intermediate (10a)
and it is noteworthy that compound 10a showed potent
MMP-1 inhibitory activity (the IC50 value was 5.0 mM). Dis-
covery of this novel binding mode led us to explore further
N-substitutents with improved inhibitory potency. We pre-
pared compounds with five different bond structures, that is,
compounds with carbamate (12a—e), amide (13a—f), sul-
fonamide (14a—c), alkylamino (15a, b), and urea (16) bonds
(Chart 1). In order to determine the smallest molecule ex-
hibiting potent MMP-1 inhibitory activity, the peptide was
truncated along the sequence from its N-terminal based on
compound 10a. Furthermore, to assess the interaction be-
tween the S1 pocket of the enzyme and the methyl group, a
2,3-diaminobutanoic acid derivative (22) was also designed.
Synthesis Preparation of the carboxylate compounds is
summarized in Charts 1 and 2. The key intermediate 2,3-di-
aminopropionic acid (8) was prepared from Boc–Ser–OMe
under Mitsunobu conditions19) or from Boc–Asn–OMe with
iodosobenzene diacetate.20) Using N-ethyl NЈ-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDC)/1-hydroxy-
Fig. 3. The Design of a-Amino Acid Type Inhibitor
Fig. 4. Schematic Representation of the Docking Simulation of MMP-1 : Compound 6 Complex
Hydrogen bonds are shown with dotted line.

1274
Vol. 49, No. 10
Reagent: (a) EDC/HOBt; (b) aq. NaOH/MeOH; (c) (1) 4 N HCl/EtOAc (2) neutralization; (d) 4 N HCl/EtOAc; (e) (1) R1O–CO–Cl, R2–CO–Cl or
R3–SO2–Cl (2) aq. NaOH/MeOH; (f) (1) R4–CHO (2) H2/Pd–C (3) aq. NaOH/MeOH; (g) (1) CDI/Et3N/Bn–NH2 (2) aq. NaOH/MeOH.
Chart 1. Synthesis of 2,3-Diaminopropionic Acid Derivatives
Reagent: (a) (1) Ph3P/diethyl azodicarboxylate/HN3 (2) H2/Pd-C; (b) EDC/HOBt; (c) (1) 70% aq. AcOH (2) pyridinium dichromate.
Chart 2. Synthesis of 2,3-Diaminobutanoic Acid Derivative
benzotriazol (HOBt) as the coupling reagent, a fully pro- reductive amination with benzaldehyde or 3-phenylpropi-
tected peptide (9a) was obtained from compound 7 and 8 onaldehyde, followed by saponification of the methyl ester to
after easy work-up. The original amino acid compound 6 was give 15a and 15b. The substituted ureido compound 16 was
obtained by saponification of the ester in 9a, followed by prepared as follows. Compound 11 was treated with 1,1Ј-car-
treatment with 4 N hydrogen chloride in ethyl acetate (4 N bonyldiimidazole (CDI)/Et3N, followed by reaction with ben-
HCl in EtOAc). Truncated compounds, 10b and 10c, were zylamine and then saponification to the carboxylate was ac-
also prepared in the same manner as 10a. The amino group complished under the same conditions described above.
at the a position of the carboxylate provided a functional
For the preparation of compound 22, we initially examined
handle onto which various moieties were introduced, as fol- the Mitsunobu reaction with Boc–Thr–OMe (17) to obtain
lows. The Boc group in the fully protected compound 9a was the protected 2,3-diaminobutanoic acid (18), but a,b-unsatu-
removed using 4 N HCl in EtOAc. Then the residue was rated dehydro amino acid (19) was obtained as the sole prod-
treated with various alkyloxycarbonyl chlorides (for 12a—e), uct (Chart 2). In order to avoid dehydration, N2,O-dipro-
acyl chlorides (for 13a—f), or alkylsulfonyl chlorides (for tected 2(S),3(S)-diaminobutanol (20) was prepared from
14a—c) in the presence of Et3N, followed by saponification Boc–Thr–OH according to the procedure described by Naka-
of the methyl ester. N-Alkylation of 11 was performed by mura et al.21) After condensation of 20 and tripeptide (7a)

October 2001
1275
by the EDC–HOBt condensation method, the tert-butyl- residue. The tert-butyl group was accommodated in this
dimethylsilyl group was removed by treatment with 70% pocket, but did not fully occupy it. Since there was further
aqeous AcOH, followed by oxidation with pyridinium space between the pocket floorboard and the tip of the tert-
dichromate to give the a desired 2,3-diaminobutylic acid de- butyl group, additional elongation of the substituent at the tip
rivative (22).
of this group might lead to an additional hydrophobic inter-
action with the S1Ј subsite. Replacement of the carbamate
group with an amide group led to a dramatic decrease of po-
Results and Discussion
The carbamate compounds, 10a and 12a—e, showed vari- tency, and all the amide compounds (13a—f) had no in-
able of MMP-1 inhibitory activity depending on the size of hibitory activity (0% at 10Ϫ3 M). Also, inhibitory activity was
the substituent (Table 1). The ethyl-substituted compound not observed with alkylamino compounds (15a, b). Probably,
12a exhibited no inhibitory activity, but the compounds with the substituents of the amino groups in amide or alkylamino
bulkier groups, such as an iso-butyl group (12b), showed compounds were not positioned in the S1Ј pocket. The differ-
moderate inhibitory activity (an IC50 of 29 mM). The com- ence of potency may be accounted for by the carbamate oxy-
pound with a tert-butyl group (10a) showed nearly equipo- gen atom forming a hydrogen bond with the enzyme and this
tent inhibitory activity compared with the original hydroxam- bond directing the hydrophobic substituent properly the S1Ј
ate (5a, IC50 of 7.7 mM). Likewise, the aromatic ring com- pocket, but a reasonable explanation of the difference was
pounds (12c, d) exhibited inhibitory activity for MMP-1 in not obtained from our computerized docking study. The sul-
the order of 10Ϫ6 M. However, the bulkiest substituent of fonamide compounds (14a—c) showed significant inhibition
2,2,2-trichloroethyl group (12e), caused a small decrease in like that observed with the carbamate compounds (Table 2).
activity. According to these results, the S1Ј pocket of MMP-1 Although a small substituent methyl group (14a) did not gen-
seemed to be well occupied by tert-butyl or p-methoxybenzyl erate any inhibitory potency, an aromatic ring substituent
groups, while the ethyl group was too small. In order to ver- (14b, c) showed inhibitory activity as potent as that of carba-
ify the MMP-1 model, we assessed compound 10a was
docked into MMP-1, and the binding features are shown in
Figs. 5 and 6. Coordination of the carboxylate group on the
inhibitor with the catalytic zinc atom was observed, and the
hydrogen bond between the carbonyl at leucine and Ala 184
was regained. In MMP-1, the lateral wall of the S1Ј subsite
mainly consists of three amino acids (Leu 181,Val 215, and
Tyr 240) and the floor of the pocket is formed by an Arg 214
Table 1. Inhibition of MMP-1 by Carbamate Type Compounds 12a)
Inhibitory activity
Compounds
R1
(IC50, mM)
12a
12b
10a
12c
12d
12e
Ethyl
Iso-butyl
tert-Butyl
4% (10Ϫ4 M)
29
5.0
2.6
2.2
Fig. 5. Expanded View of the MMP-1 : Compound 10a Complex
Compound 10a shown as a liquorice model, binds to the MMP-1 active site shown as
a Connolly molecular surface of MMP-1. The MMP-1 surface color is ranked accord-
ing to the electrostatic potential on the MMP-1 surface. As shown with a stacked color
bar in the figure, the negative/positive electrostatic potential, the deeper it is colored
red/blue. A pink sphere almost buried in the MM-1 surface is a zinc ion coordinated in
the MMP-1 active site. The figure was generated by Insight II.
Benzyl
p-Methoxybenzyl
2,2,2-Trichloroethyl
28
a) Standard deviations for enzyme assays were typically Ϯ10% of the mean or less.
Fig. 6. Schematic Representation of the Docking Simulation of MMP-1 : Compound 10a Complex
Hydrogen bonds are shown with dotted line.

1276
Vol. 49, No. 10
mate compound 12c. As described for the carbamate and sul- about 25-fold less potency for inhibition of MMP-3 than
fonamide compounds, a benzyl substituent at the Na position MMP-1, 14c exhibited 10 mM potency for MMP-3 (only a 4-
promoted inhibition, so a ureido compound incorporating a fold decrease in potency compared with its inhibition of
benzyl group (16) was prepared. However, 16 only exhibited MMP-1) and was 16-fold more potent than the original hy-
moderate inhibitory activity (IC50ϭ60 mM). Installation of the droxamate compound 5b. Comparison of these carboxylate
methyl group at the P1 position (described as compound 22) compounds and 5b suggested that the P1Ј substituent was im-
did not cause a dramatic shift of potency compared with that portant for inhibition of MMP-3, with the sulfonamide struc-
of 10a (IC50ϭ5.0, 2.4 mM, respectively). This observation as ture being especially appropriate.
supported by our computerized docking study, in which the
installed methyl group was exposed to the solvent and not Conclusion
oriented towards the enzyme.
In summary, we have discovered non-hydroxamate MMP
Comparison between the truncated peptides (10b, c) and inhibitors with a carboxylate ZCG that show no decrease of
the original peptide (10a) showed that loss of the Gly residue inhibitory activity compared with the lead compounds 5.
(10b) represented only caused a 3-fold decrease in inhibition During construction of the carboxylate compounds, we gen-
of MMP-1 compared with 10a, but a shorter peptide with erated a three-dimensional model of an MMP-1: compound 6
deletion of the Gly–Pro sequence (10c) showed complete complex based on the crystal structure of MMP-1 and the
loss of inhibitory activity (Table 3). Based on these results complex of MMP-8 with an inhibitor (Pro–Leu–Gly–NHOH)
and the lack of activity of compound 6, it seems that both the using computerized docking. We found structurally new
Boc group and the pyrrolidine ring of proline could have a MMP inhibitors, such as carbamate compound (10a, 12c,
critical role in recognition of the enzyme. The benzoyl group 12d, 22) and sulfonamide compound (14b, c), which were ef-
also contributed to increasing the affinity for MMP-1.
fective against MMP-1 with IC50 values in the order of
For some of the carboxylate compounds that showed mi- 10Ϫ6 M. Compound 10a showed that important features for
cromolar potencies against MMP-1 i.e., 10a, 12c, 14c, and the inhibitory potency were both the Boc group and the
22, MMP-3 inhibitory activity was also measured (Table 4). pyrrolidine ring of proline. With respect to MMP-3 in-
Although the carbamate compounds (10a, 12c, 22) showed hibitory potency, compound 14c showed stronger inhibitory
activity compared with the lead compounds 5.
Table 2. Inhibition of MMP-1 by Sulfonamide Type Compounds 14a)
Because hydroxamates are often unstable and there is a
risk of chronic toxicity arising from metabolic degradation to
hydroxylamine, our carboxylate compounds may be safe and
have better pharmacokinetics. Further chemical and pharma-
cological evaluation of this series will be reported in due
course and it is hoped that these potent MMPs inhibitors will
aid in the treatment of various enzymatic diseases.
Inhibitory activity
Compounds
R3
(IC50, mM)
Experimental
14a
14b
14c
Methyl
Benzyl
p-Tolyl
0% (10Ϫ5 M)
4.7
2.5
Model Building The atomic coordinates of the crystal structure of
MMP-1 and MMP-8 complexed with an inhibitor (Pro–Leu–Gly–NHOH)
were retrieved from the RCSB Protein Data Bank22) (PDB entries: 1CGL23)
and 1JAN,24) respectively). To form the S2 pocket of MMP-1, Asp186
residue was moved outside of the pocket in a similar manner to Gln165 of
MMP-8. Thus, Pro–Leu–Gly–NHOH could be accommodated in the MMP-
1 active site. Then the three-dimensional structure of compound 5a was con-
a) Standard deviations for enzyme assays were typically Ϯ10% of the mean or less.
Table 3. Inhibition of MMP-1 by Truncated Carbamate Type Compounds
10a)
Table 4. Inhibition of MMP-3 by Carboxylate Compoundsa)
Compounds
Inhibitory activity (IC50, mM)
Inhibitory activity
Compounds
Y
5b
10a
22
160
128
57
(IC50, mM)
10a
10b
10c
Gly–Pro–Leu
Pro–Leu
Leu
5.0
12d
14c
65
10
17
0% (10Ϫ3 M)
a) Standard deviations for enzyme assays were typically Ϯ10% of the mean or less.
a) Standard deviations for enzyme assays were typically Ϯ10% of the mean or less.
Fig. 7. Construction of Compound 6 Structure

October 2001
1277
structed based upon Pro–Leu–Gly–NHOH to check the binding feature and ture was neutralized by adding 1 N aq. HCl and MeOH was removed by
decide the three-dimensional structure of Bz–Gly–Pro–Leu–NH–CH moiety, evaporation. The resultant solution was adjusted to pH 2 by adding 1 N aq.
which is common structure of compound 6. The Bz–Gly– moiety was ini- HCl at 0 °C. The organic material was extracted with EtOAc, washed with
tially constructed from standard values (bond length, bond angle, and torsion sat. aq. NaCl, dried (MgSO4) and evaporated in vacuo. The resultant residue
angle) followed by searching for a pocket that could accommodate an aro- was precipitated from Et2O to give the title compound as colorless powder.
matic ring by altering all of the torsion angles except for the amide bond. As
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(tert-butyloxycarbonyl-
a result, we found that the aromatic ring of the Bz–Gly– moiety could fit into amino)propionic Acid (10a): Yield: 95%. mp; 169—172 °C, [a]D25 Ϫ109°
a hydrophobic pocket consisting of Phe185, Pro189, Ile191, and Gly192. (cϭ1.08, MeOH), R f 1 0.45. R f 2 0.29. IR (neat) cmϪ1: 3327, 3066, 2958,
Eventually, based upon the structure of 5a, three-dimensional structure of 2878, 1719, 1655. H-NMR (CDCl3, TMS) d: 0.8—1.0 (6H, m), 1.0—1.9
1
compound 6 was constructed. The amino acid moiety was also constructed (14H, m), 1.9—2.4 (2H, m), 3.1—3.4 (2H, m), 3.4—3.8 (2H, m), 3.9—4.6
utilizing standard values (bond length, bond angle, and torsion angle). For (5H, m), 6.16 (1H, br d, Jϭ8.1 Hz), 6.44 (1H, br d, Jϭ8.5 Hz), 6.63 (1H,
modeling of the amino acid moiety, two conformations were assessed based br d, Jϭ8.6 Hz), 6.73 (1H, br d, Jϭ9.2 Hz), 7.3—7.6 (3H, m), 7.8—8.0
on Ca–Cb free rotation, which are described as conformers A and B in Fig.
7. After docking of conformer B into MMP-1, strong electrostatic repulsion C28H41N5O8·1/2H2O: C, 57.52; H, 7.24; N, 11.98. Found: C, 57.34; H, 7.28;
(2H, m). FAB-MS m/z: 598 (MϩNa)ϩ, 576 (MϩH)ϩ. Anal. Calcd for
could be expected between both the carboxylate of the amino acid moiety
and the Glu 219 residue, resulting in a failure of docking simulation. For this
N, 12.19.
3-(Na-Benzoyl-prolyl-leucyl-amino)-2(S)-(tert-butyloxycarbonylamino)-
reason, we chose conformer A as the docking model and then carried out en- propionic Acid (10b): Yield: 97%. mp; 110—112 °C, [a]D25 Ϫ90° (cϭ1.24,
ergy minimization using Discover (Ver 2.98, Molecular Simulations Inc., MeOH), R f 1 0.56. IR (neat) cmϪ1: 3280, 3079, 2959, 1727, 1686, 1647,
San Diego, CA, U.S.A.). Minimization was preformed by 300 steps of the 1560. H-NMR (CDCl3, TMS) d: 0.89 (6H, m), 1.40 (9H, s), 1.64 (3H, m),
steepest descent, followed by 3000 steps of the VA09A method to remove 1.86 (1H, m), 2.03 (1H, m), 2.22 (2H, m), 3.4—3.7 (3H, m), 3.79 (1H, m),
1
steric clashes.
4.2—4.6 (3H, m), 4.72 (1H, t, Jϭ6.8 Hz), 6.07 (1H, d, Jϭ7.2 Hz), 7.2—7.5
Chemistry Melting points were determined on a Yanagimoto melting (4H, m), 7.55 (2H, m). Anal. Calcd for C26H38N4O7·1/2H2O: C, 58.20; H,
point apparatus without correction. IR spectra were taken with a PERKIN 7.33; N, 10.44. Found: C, 58.14; H, 7.48; N, 10.35.
ELMER PARAGON 1000 FT-IR spectrometer. Column chromatography
3-(Na-Benzoyl-leucyl-amino)-2(S)-(tert-butyloxycarbonylamino)propi-
was performed on Silica gel BW-200 (Fuji Silysia Chemical Ltd.). TLC was onic Acid (10c): Yield: 82%. mp; 127—130 °C, [a]D25 Ϫ15.8° (cϭ1.0,
1
performed on silica gel (Silica gel 60 F254, Merck). Rf values refer to the MeOH), R f 2 0.60. H-NMR (CD3OD, TMS) d: 0.97 (6H, m), 1.42 (9H, s),
following v/v solvent system: Rf1, CHCl3–MeOH–AcOH (5 : 2 : 1); R f 2,
CHCl3–MeOH–AcOH (80 : 10 : 5); R f 3, CHCl3–MeOH (10 : 1); R f 4,
1.6—1.8 (3H, m), 3.41 (1H, dd, Jϭ8.1, 13.8 Hz), 3.71 (1H, dd, Jϭ4.5,
13.8 Hz), 4.26 (1H, m), 4.61 (1H, m), 7.4—7.6 (3H, m), 7.84 (2H, m). Anal.
Calcd for C21H31N3O6: C, 59.84; H, 7.41; N, 9.97. Found: C, 59.80; H, 7.45;
1
CHCl3–MeOH (20 : 1). H-NMR was recorded on a JEOL FX90A or JEOL
JMN-AL300 spectrometer, and chemical shifts are given in ppm (d) from N, 9.92.
tetramethylsilane (TMS), which was used as the internal standard. Mass
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-aminopropionic Acid
spectra were obtained on a JEOL JMS 700 spectrometer by electrospray ion- (6) Compound 10a (254 mg, 0.441 mmol) was treated with 4 N HCl in
ization (ESI) or fast atom bombardment (FAB) ionization techniques. Opti- EtOAc (10 ml) at 0 °C for 1 h. The reaction mixture was evaporated in vacuo
cal rotations were measured in a JASCO DIP-140 apparatus.
and the residue was dissolved in H2O (10 ml). The solution was adjusted to
pH 5 by adding 1 N aq. NaOH then concentrated to ca. 2 ml to give crystals,
which were collected by filtration, washed with H2O and dried to afford
General Procedure for the Preparation of Compounds (9) EDC
(1.1 eq of 7) was added to a mixture of 3-amino-2(S)-(tert-butyloxycar-
bonyl-amino)propionic acid methyl ester (8) (1.05 eq of 7), HOBt (1.05 eq 140 mg of 6 (96%) as a colorless solid. mp 216—219 °C (dec.), [a]D25
of 7), compound 7, and N,N-dimethylformamide (DMF) (4—7% v/w) at
Ϫ95.6° (cϭ1.0, 0.1 N aq. NaOH), R f 1 0.39. IR (neat) cmϪ1: 3362, 3096,
Ϫ12 °C. The mixture was stirred at Ϫ3 °C for 6 h then 0 °C for 15 h. The 2956, 2869, 2360, 2057, 1680, 1594, 1549. 1H-NMR [D2OϩNaOD, 3-
reaction mixture was evaporated employing a rotary evaporator and a (trimethylsilyl)propionic 2,2,3,3-d4 acid, sodiun salt] d: 0.88 (3H, d, Jϭ
vacuum pump. The residue was dissolved in EtOAc, washed with 1 N aq.
5.1 Hz), 0.93 (3H, d, Jϭ5.3 Hz), 1.6—1.9 (3H, m), 1.9—2.4 (4H, m), 3.2—
HCl, sat. aq. NaCl, 10% aq. Na2CO3 and sat. aq. NaCl successively, dried 3.4 (2H, m), 3.6—3.8 (2H, m), 4.2—4.4 (2H, m), 4.45 (1H, m), 4.7—5.0
(MgSO4), evaporated in vacuo to give a crude product, which was purified (m), 7.5—7.9 (5H, m). Anal. Calcd for C23H33N5O6·H2O: C, 56.01; H, 7.15;
by silica gel column chromatography (eluent; CHCl3 : MeOHϭ50 : 1) and
precipitated from Et2O to give 9 as colorless powder.
N, 14.20. Found: C, 56.26; H, 7.32; N, 13.93.
General Procedure for the Preparation of Compounds (12)—(14)
Compound 9a was treated with cold 4 N HCl in EtOAc (4% v/w) for 0.5—
1.5 h. The reaction mixture was evaporated in vacuo and the residue was
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(tert-butyloxycarbony-
lamino)propionic Acid Methyl Ester (9a): Yield: 67%. mp; 176—181 °C,
[a]D25 Ϫ65.8° (cϭ1.0, MeOH), R f 4 0.17. IR (neat) cmϪ1: 3332, 3070, 2954, precipitated from Et2O, which were collected by filtration, dried under re-
2872, 1747, 1708, 1690, 1654. 1H-NMR (CDCl3, TMS) d: 0.8—1.0 (6H, duced pressure to give hydrochloride salt (11) as quantitative yield. To a so-
m), 1.1—1.9 (14H, m), 1.9—2.4 (2H, m), 3.1—3.4 (2H, m), 3.4—3.8 (5H, lution of 11 in DMF (4—7% v/w), Et3N (2 eq of 11) and appropriate acyl
m), 3.9—4.6 (5H, m), 6.16 (1H, br d, Jϭ8.0 Hz), 6.46 (1H, m), 6.67 (1H, chloride or sulfonyl chloride (1.05—1.1 eq of 11) were added successively.
br d, Jϭ8.0 Hz), 6.94 (1H, br d, Jϭ7.9 Hz), 7.4—7.6 (3H, m), 7.7—8.1 (2H, The mixture was stirred at room temperature overnight. Then the reaction
m). Anal. Calcd for C29H43N5O8: C, 59.07; H, 7.35; N, 11.88. Found: C, mixture was diluted with EtOAc, washed with sat. aq. NaCl, water, 10% aq.
59.23; H, 7.31; N, 11.69.
Na2CO3 and sat. aq. NaCl successively, dried (MgSO4) then evaporated in
vacuo. The residue was dissolved in MeOH (7—10% v/w) and 2 N aq.
NaOH (2 eq of acylated compound) was added at 0 °C, then the mixture was
stirred for 1—2 h. The reaction mixture was acidified by adding 1 N aq. HCl
3-(Na-Benzoyl-prolyl-leucyl-amino)-2(S)-(tert-butyloxycarbonylamino)-
propionic Acid Methyl Ester (9b): Yield: 63%. mp; 77—79 °C, [a]D25
1
Ϫ87.5° (cϭ1.04, MeOH), R f 3 0.31. H-NMR (CDCl3, TMS) d: 0.90 (6H,
m), 1.40 (9H, s), 1.5—1.6 (2H, m), 1.78 (1H, m), 1.92 (1H, m), 2.03 (1H, (pHϭ2—3), MeOH was removed by evaporation, extracted with EtOAc,
m), 2.21 (1H, m), 2.37 (1H, m), 3.4—3.6 (3H, m), 3.70 (3H, s), 3.8—3.9
(1H, m), 4.30 (2H, m), 4.75 (1H, m), 6.03 (1H, d, Jϭ7.9 Hz), 6.89 (1H, d,
Jϭ7.5 Hz), 7.00 (1H, m), 7.4—7.5 (3H, m), 7.56 (2H, m). Anal. Calcd for
C27H40N4O7: C, 60.88; H, 7.57; N, 10.52. Found: C, 60.62; H, 7.73; N,
10.51.
dried (MgSO4) and evaporated in vacuo. The residue was precipitated from
Et2O and reprecipitated from appropriate solvent system to give 12—14.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(ethyloxycarbonyl-
amino)propionic Acid (12a, R1ϭEthyl): Yield: 62% (reprecipitated from
THF–n-hexane). mp 105—107 °C, [a]D25 Ϫ62.6° (cϭ1.03, MeOH), R f 1
0.38. 1H-NMR (CDCl3, TMS) d: 0.75—1.0 (6H, m) 1.13 (3H, t, Jϭ7.1 Hz),
3-(Na-Benzoyl-leucyl-amino)-2(S)-(tert-butyloxycarbonylamino)propi-
onic Acid Methyl Ester (9c): Yield: 39%. mp; 69—71 °C, [a]D25 Ϫ16.9° 1.5—1.8 (3H, m), 1.9—2.3 (4H, m), 3.30 (2H, m), 3.5—3.8 (2H, m), 3.8—
(cϭ1.15, MeOH), R f 4 0.40. IR (neat) cmϪ1: 3304, 3072, 2957, 2870, 1751,
1718, 1647, 1604, 1578, 1540. 1H-NMR (CDCl3, TMS) d: 0.96 (6H, m),
1.42 (9H, s), 1.6—1.8 (3H, m), 3.63 (2H, m), 3.70 (3H, s), 4.40 (1H, m),
4.64 (1H, m), 5.59 (1H, d, Jϭ7.7 Hz), 6.74 (1H, d, Jϭ8.1 Hz), 7.4—7.54
(3H, m), 7.80 (2H, m). Anal. Calcd for C22H33N3O6·3/4H2O: C, 58.85; H,
7.74; N, 9.36. Found: C, 58.78; H, 7.80; N, 9.26.
4.3 (5H, m), 4.4—4.6 (2H, m), 6.79 (1H, d, Jϭ7.0 Hz), 7.18 (2H, m), 7.3—
7.5 (3H, m), 7.82 (2H, m), 8.23 (1H, m). Anal. Calcd for C26H37N5O8·
1/3H2O: C, 56.41; H, 6.86; N, 12.65. Found: C, 56.59; H, 7.00; N, 12.43.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(iso-butyloxycarbonyl-
amino)propionic Acid (12b, R1ϭIso-butyl): Yield: 70% (reprecipitated from
THF–n-hexane). mp 134—137 °C, [a]D25 Ϫ72.6° (cϭ1.01, MeOH), R f 1
General Procedure for the Preparation of Compounds (10) Two nor- 0.42. IR (neat) cmϪ1: 3312, 3072, 2962, 1746, 1700, 1654, 1558. H-NMR
1
mal (2 N) aq. NaOH (3 eq of 9) was added to a solution of 9 in MeOH (5—
7% v/w) at 0 °C and stirred at room temperature for 1 h. The reaction mix- 3.25 (2H, m), 3.6—3.9 (4H, m), 3.9—4.3 (3H, m), 4.4—4.6 (2H, m), 6.51
(CDCl3, TMS) d: 0.8—1.0 (12H, m) 1.4—1.9 (4H, m), 2.0—2.4 (4H, m),

1278
Vol. 49, No. 10
propionic Acid (13f, R2ϭp-Nitrophenyl): Yield: 55% (recrystallized from
EtOAc–Et2O). mp 132—135 °C, [a]D25 Ϫ52.1° (cϭ1.01, MeOH), R f 1 0.39.
IR (neat) cmϪ1: 3322, 3075, 2961, 2874, 1734, 1654, 1541. 1H-NMR
(CDCl3, TMS) d: 0.92 (3H, d, Jϭ5.9 Hz), 1.02 (3H, d, Jϭ6.1 Hz), 1.59 (1H,
m), 1.76 (2H, m), 1.8—2.5 (4H, m), 3.43 (2H, m), 3.73 (2H, m), 4.23 (1H,
m), 4.26 (1H, m), 4.5—4.3 (2H, m), 4.58 (2H, m), 7.01 (1H, m), 7.18 (2H,
m), 7.50 (2H, m), 7.87 (4H, m), 8.22 (2H, m), 8.70 (1H, m), 8.89 (1H, d,
Jϭ4.2 Hz). FAB-MS m/z: 647 (MϩNa)ϩ, 625 (MϩH)ϩ. Anal. Calcd for
C30H36N6O9: C, 57.68; H, 5.81; N, 13.45. Found: C, 57.82; H, 5.88; N,
13.21.
(1H, m), 6.75 (1H, m), 7.04 (1H, m), 7.4—7.55 (3H, m), 7.62 (1H, m),
7.8—7.95 (2H, m). Anal. Calcd for C28H41N5O8·3/4H2O: C, 57.08; H, 7.27;
N, 11.89. Found: C, 56.92; H, 7.28; N, 12.10.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(benzyloxycarbony-
lamino)propionic Acid (12c, R1ϭBenzyl): Yield: 69% (reprecipitated from
MeOH–Et2O). mp 214—216 °C, [a]D25 Ϫ107° (cϭ0.98, MeOH), R f 1 0.51,
1
R f 2 0.29. IR (neat) cmϪ1: 3317, 3066, 2954, 2875, 1735, 1662, 1560. H-
NMR (CDCl3, TMS) d: 0.8—1.0 (6H, m) 1.5—1.85 (3H, m), 1.9—2.4 (4H,
m), 3.4—3.7 (2H, m), 3.7—4.3 (3H, m), 4.48 (2H, m), 4.97 (2H, m), 6.87
(1H, d, Jϭ6.6 Hz), 7.78 (4H, m), 7.1—7.6 (7H, m), 8.15 (1H, m). FAB-MS
m/z 632 (MϩNa)ϩ, 610 (MϩH)ϩ. Anal. Calcd for C31H39N5O8: C, 61.07; H,
6.45; N, 11.49. Found: C, 61.14; H, 6.67; N, 11.43.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(methansulfonylamino)-
propionic Acid (14a, R3ϭMethyl): Yield: 47% (reprecipitated from EtOAc–
Et2O). mp 253—254 °C, R f 1 0.18. IR (neat) cmϪ1: 3439, 3368, 3319, 3280,
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(p-methoxybenzyloxy-
carbonylamino)propionic Acid (12d, R1ϭp-Methoxybenzyl): Yield: 47%
(precipitated from EtOAc–Et2O). mp 194—196 °C, [a]D25 Ϫ54.3° (cϭ1.02,
MeOH), R f 1 0.49, R f 2 0.27. IR (neat) cmϪ1: 3285, 3068, 2960, 1740, 1696,
1654, 1559, 1254, 1031. 1H-NMR (CDCl3, TMS) d: 0.8—1.0 (6H, m)
1.45—1.85 (3H, m), 1.9—2.3 (4H, m), 3.29 (2H, m), 3.61 (2H, m), 3.79
(3H, s), 4.0—4.2 (3H, m), 4.50 (2H, m), 4.88 (2H, m), 6.84 (3H, m), 7.1—
7.5 (7H, m), 7.79 (2H, m), 8.12 (1H, m). Anal. Calcd for C32H41N5O9: C,
59.25; H, 6.53; N, 10.80. Found: C, 59.38; H, 6.51; N, 10.67.
1
2959, 1744, 1696, 1654. H-NMR (DMSO-d6, TMS) d: 0.7—0.9 (6H, m),
1.4—1.7 (3H, m), 1.7—2.1 (4H, m), 2.89 (3H, s), 3.1—3.7 (m), 3.9—4.25
(4H, m), 4.35 (1H, m), 7.4—7.6 (4H, m), 7.78 (1H, m), 7.8—8.0 (2H, m),
8.32 (1H, m), 8.66 (1H, m). Anal. Calcd for C24H35N5O8S: C, 52.07; H,
6.37; N, 12.65. Found: C, 52.24; H, 6.20; N, 12.69.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(benzylsulfonylamino)-
propionic Acid (14b, R3ϭBenzyl): Yield: 51% (reprecipitated from EtOAc–
Et2O). mp 193—194 °C, [a]D25 Ϫ52.7° (cϭ1.02, MeOH), R f 1 0.46, R f 2
0.25. IR (neat) cmϪ1: 3318, 3065, 2958, 2875, 1735, 1654, 1542, 1332,
1157. 1H-NMR (CDCl3, TMS) d: 0.7—1.0 (6H, m), 1.4—1.8 (3H, m), 1.9—
2.4 (4H, m), 3.1—3.4 (2H, m), 3.63 (2H, m), 4.02 (1H, m), 4.19 (2H, m),
4.31 (2H, s), 4.51 (2H, m), 6.71 (1H, d, Jϭ8.8 Hz), 7.02 (1H, m), 7.3—7.6
(9H, m), 7.84 (2H, m), 8.0 (1H, m). FAB-MS m/z: 652 (MϩNa)ϩ, 630
(MϩH)ϩ. Anal. Calcd for C30H39N5O8S·1/2H2O: C, 56.41; H, 6.31; N,
10.96. Found: C, 56.36; H, 6.58; N, 10.69.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(2,2,2-trichloroethyloxy-
carbonyl-amino)propionic Acid (12e, R1ϭ2,2,2-Trichloroethyl): Yield: 61%
(reprecipitated from THF–n-hexane). mp 119—122 °C, [a]D25 Ϫ60.3°
(cϭ1.04, MeOH), R f 1 0.36. IR (neat) cmϪ1: 3302, 3062, 2957, 2877, 1734,
1654, 1542, 818. 1H-NMR (CDCl3, TMS) d: 0.88 (3H, d, Jϭ6.1 Hz) 0.95
(3H, d, Jϭ6.1 Hz), 1.5—1.8 (3H, m), 1.9—2.4 (4H, m), 3.37 (2H, m), 3.5—
3.8 (2H, m), 4.07 (1H, m), 4.26 (2H, m), 4.48 (2H, m), 4.61 (2H, m), 7.10
(1H, m), 7.23 (m), 7.3—7.6 (4H, m), 7.89 (2H, m), 8.04 (1H, m). Anal.
Calcd for C26H34Cl3N5O8·H2O: C, 46.68; H, 5.42; N, 10.47. Found: C,
46.41; H, 5.53; N, 10.54.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(p-toluenesulfonyl-
amino)propionic Acid (14c, R3ϭp-Tolyl): Yield: 29% (reprecipitated from
EtOAc–Et2O). mp 120—122 °C, [a]D25 Ϫ101° (cϭ0.60, MeOH), R f 1 0.47,
R f 2 0.26. IR (neat) cmϪ1: 3310, 3072, 2954, 1735, 1671, 1654, 1542, 1338,
1165. 1H-NMR (CDCl3, TMS) d: 0.8—1.0 (6H, m), 1.5—1.8 (3H, m), 1.9—
2.3 (4H, m), 2.44 (3H, s), 3.58 (2H, m), 3.62 (2H, m), 3.90 (1H, m), 4.15
(1H, m), 4.3—4.45 (2H, m), 4.56 (1H, m), 6.04 (1H, m), 6.71 (1H, m),
7.3—7.5 (6H, m), 7.7—7.8 (4H, m), 8.42 (1H, m). FAB-MS m/z: 653
(MϩNa)ϩ, 631 (MϩH)ϩ. Anal. Calcd for C30H39N5O8S·3/4H2O: C, 56.02;
H, 6.35; N, 10.89. Found: C, 56.17; H, 6.39; N, 10.74.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(acetylamino)propionic
Acid (13a, R2ϭMethyl): Yield: 67% (reprecipitated from EtOAc–n-hexane).
1
mp 89—92 °C, [a]D25 Ϫ57.1° (cϭ1.0, MeOH), R f 1 0.16. H-NMR (CDCl3,
TMS) d: 0.8—1.0 (6H, m), 1.1—1.8 (6H, m), 1.9—2.3 (4H, m), 3.30 (2H,
m), 3.61 (2H, m), 4.0—4.2 (3H, m), 4.42 (2H, m), 6.80 (1H, d, Jϭ7.0 Hz),
7.2—7.5 (5H, m), 7.75 (2H, m), 8.06 (1H, m). Anal. Calcd for C25H35N5O7:
C, 58.01; H, 6.82; N, 13.53. Found: C, 58.26; H, 6.79; N, 13.38.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(benzylamino)propi-
onic Acid (15a, R4؍Benzyl) NaBH4 (31 mg, 0.494 mmol) was added to a
mixture of 11 (260 mg, 0.494 mmol), benzaldehyde (50 ml, 0.494 mmol) and
MeOH (3 ml) at 0 °C. After stirring for 16 h at room temperature, the reac-
tion mixture was evaporated in vacuo, diluted with EtOAc, washed with
water and sat. aq. NaCl, dried (MgSO4), then evaporated in vacuo. The
crude product was purified by silica gel column chromatography (eluent;
CHCl3 : MeOHϭ20 : 1) to give methyl ester of the title compound as a white
waxen oil (257 mg, 90%). The methyl ester was saponified by the same
method used for preparation of 12 and precipitated from EtOAc–Et2O to
give 130 mg of 15 (55%) as colorless powder. mp 128—131 °C, [a]D25
Ϫ64.0° (cϭ1.03, MeOH), R f 1 0.39. 1H-NMR (CDCl3, TMS) d: 0.87 (3H, d,
Jϭ6.2 Hz), 0.95 (3H, d, Jϭ6.2 Hz), 1.5—1.8 (3H, m), 2.0—2.3 (4H, m),
3.4—3.75 (6H, m), 3.88 (1H, m), 4.17 (1H, m), 4.3—4.45 (2H, m), 4.54
(1H, m), 7.01 (1H, br d, Jϭ8.6 Hz), 7.20 (1H, br d, Jϭ8.5 Hz), 7.3—7.6 (9H,
m), 7.86 (2H, m), 8.02 (1H, d, Jϭ8.1 Hz). Anal. Calcd for C30H39N5O6·
1/2H2O: C, 62.70; H, 7.02; N, 12.19. Found: C, 62.92; H, 7.09; N, 11.99.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-[(3-phenylpropyl)-
amino]propionic Acid (15b, R4؍3-Phenylpropyl) The title compound
was prepared from 11 and 3-phenylpropionaldehyde by the same method
used for 15 (R4ϭbenzyl). Yield: 43%. mp 182—187 °C (dec.), [a]D25 Ϫ56.9°
(cϭ1.0, MeOH), R f 1 0.44. IR (neat) cmϪ1: 3348, 2959, 2872, 1684, 1654,
1550. 1H-NMR (CDCl3, TMS) d: 0.88 (3H, d, Jϭ6.1 Hz), 0.96 (3H, d,
Jϭ6.1 Hz), 1.5—1.8 (3H, m), 1.8—2.3 (6H, m), 2.7—3.0 (4H, m), 3.33 (2H,
m), 3.59 (2H, m), 4.02 (1H, m), 4.13 (2H, m), 4.38 (2H, m), 7.02 (2H, m),
7.1—7.5 (9H, m), 7.59 (1H, br d, Jϭ8.6 Hz), 7.90 (2H, m). FAB-MS m/z
616 (MϩNa)ϩ, 594 (MϩH)ϩ. Anal. Calcd for C32H43N5O6·3/2H2O: C,
61.92; H, 7.47; N, 11.28. Found: C, 62.26; H, 7.59; N, 11.21.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(tert-butylcarbonyl-
amino)propionic Acid (13b, R2ϭtert-Butyl): Yield: 31% (reprecipitated
from THF–Et2O). mp 142—145 °C, [a]D25 Ϫ80.2° (cϭ1.03, MeOH), R f 1
0.36. 1H-NMR (CDCl3, TMS) d: 0.84 (3H, d, Jϭ5.2 Hz), 0.93 (3H, d,
Jϭ5.4 Hz), 1.23 (9H, s), 1.5—1.8 (3H, m), 2.0—2.3 (4H, m), 3.41 (2H, m),
3.70 (2H, m), 4.10 (1H, m), 4.25 (1H, m), 4.3—4.6 (3H, m), 7.03 (1H, m),
7.20 (1H, m), 7.3—7.6 (4H, m), 7.80 (2H, m), 8.01 (1H, m). Anal. Calcd for
C28H41N5O7·1/4H2O: C, 59.61; H, 7.41; N, 12.41. Found: C, 59.64; H, 7.66;
N, 12.19.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(tert-butylacetylamino)-
propionic Acid (13c, R2ϭ2,2-Dimethylpropyl): Yield: 52% (reprecipitated
from THF–Et2O). mp 119—121 °C, [a]D25 Ϫ57.5° (cϭ1.01, MeOH), R f 1
1
0.37. IR (neat) cmϪ1: 3318, 3064, 2956, 2872, 1734, 1654, 1544. H-NMR
(CDCl3, TMS) d: 0.8—1.1 (15H, m), 1.58 (1H, m), 1.78 (2H, m), 1.9—2.4
(6H, m), 3.43 (2H, m), 3.74 (2H, m), 4.23 (1H, m), 4.26 (1H, m), 4.3—4.6
(3H, m), 7.10 (1H, m), 7.20 (1H, m), 7.3—7.5 (4H, m), 7.84 (2H, m), 7.93
(1H, m). FAB-MS m/z: 596 (MϩNa)ϩ, 574 (MϩH)ϩ. Anal. Calcd for
C29H43N5O7·3/4H2O: C, 59.32; H, 7.64; N, 11.93. Found: C, 59.53; H, 7.59;
N, 11.76.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(benzylcarbonylamino)-
propionic Acid (13d, R2ϭBenzyl): Yield: 52% (recrystallized from EtOAc–
Et2O). mp 156—159 °C, [a]D25 Ϫ92.8° (cϭ1.05, MeOH), R f 1 0.55. 1H-NMR
(CDCl3, TMS) d: 0.8—1.0 (6H, m), 1.5—1.8 (3H, m), 2.0—2.3 (4H, m),
3.4—3.7 (6H, m), 3.88 (1H, m), 4.17 (1H, m), 4.3—4.55 (2H, m), 4.54 (1H,
m), 7.01 (1H, m), 7.20 (1H, m), 7.3—7.6 (9H, m), 7.86 (2H, m), 8.02 (1H,
m). Anal. Calcd for C31H39N5O7: C, 62.72; H, 6.62; N, 11.80. Found: C,
62.84; H, 6.70; N, 11.69.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(benzoylamino)propi-
onic Acid (13e, R2ϭPhenyl): Yield: 51% (reprecipitated from THF–Et2O).
mp 188—190 °C, [a]D25 Ϫ59.0° (cϭ1.15, MeOH), R f 2 0.59. 1H-NMR
(CDCl3, TMS) d: 0.8—1.0 (6H, m), 1.5—1.8 (3H, m), 1.9—2.3 (4H, m),
3.58 (2H, m), 3.64 (2H, m), 3.90 (1H, m), 4.15 (1H, m), 4.3—4.45 (2H, m),
4.56 (1H, m), 6.10 (1H, m), 6.72 (1H, m), 7.3—7.5 (9H, m), 7.7—7.8 (2H,
m), 8.0 (1H, m). Anal. Calcd for C30H37N5O7·1/2H2O: C, 61.25; H, 6.51; N,
11.90. Found: C, 61.14; H, 6.59; N, 12.16.
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-N؅-benzylureidopro-
pionic Acid (16) To a mixture of 11 (260 mg, 0.5 mmol) , Et3N (70 ml,
0.5 mmol), and THF (10 ml), CDI (97 mg, 0.6 mmol) was added at 0 °C, and
the mixture was stirred at room temperature for 2 h. Then benzylamine
(55 ml, 0.5 mmol) was added and stirred overnight at the same temperature.
The reaction mixture was diluted with EtOAc and washed with 1 N aq. HCl,
sat. aq. NaCl, 10% aq. NaHCO3, and sat. aq. NaCl successively, dried
(MgSO4), then evaporated in vacuo. The residue was purified by silica gel
3-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(p-nitrobenzoylamino)-

October 2001
1279
by adding 10 ml of 15 mM aq. diisopropyl fluorophosphate to quench the
plasmin activity. A 15 ml aliquot of inhibitor at varying concentrations of in-
hibitor plus 35 ml of an assay buffer were added to the mixture and incubated
further 15 min at 35 °C. Then 50 ml of 0.1% FITC-labeled casein in an assay
buffer was added, followed by incubation at 37 °C for 2 h. Then the reaction
was terminated by adding 120 ml of 5% aq. TCA and vortexed for 30 s, fol-
lowed by centrifugation for 20 min at 3000 rpm to obtain its supernatant
fluid. Fluoresence intensity of such a supernatant was measured with (lex
495 nm, lem 520 nm). Inhibitory activity was defined as a percentage of con-
trol activity in the absence of inhibitor.
column chromatography (eluent; CHCl3 : MeOHϭ20 : 1). The methyl ester
of the title compound was isolated as a colorless oil (230 mg, 77%). 2 N aq.
NaOH (0.5 ml, 1 mmol) was added to a solution of methyl ester (220 mg,
0.37 mmol) in acetone (3 ml) and stirred at room temperature for 1 h. The re-
action mixture was diluted with EtOAc and washed with 1 N aq. HCl, sat. aq.
NaCl, 10% aq. NaHCO3, and sat. aq. NaCl successively, dried (MgSO4),
then evaporated in vacuo. The residue was precipitated from EtOH–Et2O to
give 16 as a white powder (100 mg, 47%). mp 152—154 °C (dec.), [a]D25
Ϫ59.2° (cϭ1.02, MeOH), R f 1 0.41. IR (neat) cmϪ1: 3280, 2954, 1735,
1663, 1560. 1H-NMR (CDCl3ϩMeOH-d4, TMS) d: 0.8—1.0 (6H, m) 1.4—
1.8 (3H, m), 2.0—2.2 (4H, m), 3.56 (2H, m), 3.6—3.9 (2H, m), 4.0—4.5
(7H, m), 6.07 (1H, m), 6.19 (1H, m), 6.45 (1H, m), 7.1—7.6 (9H, m), 7.79
(2H, m), 8.14 (1H, m). Anal. Calcd for C31H40N6O7·H2O: C, 59.41; H, 6.75;
N, 13.41. Found: C, 59.40; H, 6.83; N, 13.34.
References and Notes
1) Birkedal-Hansen H., Moore W. G. I., Bodden M. K., Windsor L. T.,
Birkedal-Hansen B., DeCarlo A., Engler J. A., Crit. Rev. Oral Biol.
Med., 4, 197—250 (1993).
2) Morphy J. R., Millican T. A., Porter J. R., Curr. Med. Chem., 2, 743—
762 (1995).
3) Hagmann W. K., Lark M. W., Becker J. W., Ann. Rep. Med. Chem., 31,
231—240 (1996).
4) Babin R. E., Bender S. L., Chem. Rev., 97, 1420—1437 (1997).
5) Whittaker M., Floyd C. D., Brown P., Gearing A. J. H., Chem. Rev., 99,
2735—2776 (1999).
3(S)-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-tert-(butyloxycar-
bonylamino)-O-tert-butyldimethylsilylbutanol (21) Compound 21 was
prepared from Bz–Gly–Pro–Leu–OH (7a) and 3(S)-amino-2(S)-(tert-butyl-
oxycarbonylamino)-O-tert-butyldimethylsilylbutanol (20)20) by the same
method used for preparation of 9a to give 950 mg of 21 (69%) as a colorless
solid. mp 120—121 °C, [a]D25 Ϫ61.7° (cϭ0.575, MeOH), R f 4 0.25. IR
1
(neat) cmϪ1: 3296, 2959, 2873, 1696, 1662, 1550. H-NMR (CDCl3, TMS)
d: 0.7—0.9 (19H, m), 1.36 (9H, s), 1.4—1.7 (3H, m), 1.9—2.3 (4H, m),
3.2—3.9 (5H, m), 3.9—4.6 (5H, m), 4.86 (1H, br d, Jϭ8.0 Hz), 6.92 (2H,
m), 7.2—7.5 (4H, m), 7.6—7.8 (2H, m). Anal. Calcd for C35H59N5O7Si·
4/3H2O: C, 58.89; H, 8.71; N, 9.81. Found: C, 58.98; H, 8.60; N, 9.74.
3(S)-(Na-Benzoyl-glycyl-prolyl-leucyl-amino)-2(S)-(tert-butyloxycar-
bonylamino)butanoic Acid (22) A mixture of 21 (950 mg, 1.37 mmol)
and 70% aq. AcOH (20 ml) was stirred at room temperature for 4 h. The re-
action mixture was lyophilized and the residue was precipitated from Et2O to
give 750 mg of primary alcohol intermediate (95%) as a colorless powder.
To a solution of this intermediate (350 mg, 0.594 mmol) in DMF (4 ml),
pyridinium dichromate (1.36 g, 6.31 mmol) was added and stirred at room
temperature for 16 h. The reaction mixture was diluted with EtOAc (30 ml)
and washed with sat. aq. NaCl, 1 N aq. HCl and sat. aq. NaCl successively,
dried (MgSO4), then evaporated in vacuo. The residue was precipitated from
EtOAc–Et2O to give 215 mg of 22 (62%) as a pale green powder. mp 201—
203 °C, [a]D25 Ϫ140° (cϭ0.96, MeOH), R f 1 0.41. IR (neat) cmϪ1: 3400,
3290, 3065, 2976, 1736, 1702, 1656. 1H-NMR (CDCl3ϩMeOH-d4, TMS) d:
0.89 (3H, d, Jϭ6.1 Hz), 0.94 (3H, d, Jϭ6.1 Hz), 1.10 (3H, d, Jϭ6.6 Hz),
1.4—1.7 (12H, m), 1.9—2.3 (4H, m), 3.25 (1H, m), 3.61 (2H, m), 4.0—4.2
(3H, m), 4.3—4.6 (2H, m), 6.21 (1H, br d, Jϭ8.1 Hz), 6.46 (1H, br d,
Jϭ8.5 Hz), 6.63 (1H, br d, Jϭ8.6 Hz), 6.94 (1H, br d, Jϭ8.5 Hz), 7.3—7.6
(3H, m), 7.6—7.8 (2H, m). FAB-MS m/z 612 (MϩNa)ϩ, 590 (MϩH)ϩ.
Anal. Calcd for C29H43N5O8·1/2H2O: C, 58.18; H, 7.41; N, 11.70. Found: C,
58.00; H, 7.52; N, 11.96.
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MMP-1 Inhibition Assay The test was carried out essentially as in
Nagai et al.25) In a microtube, 20 ml of proMMP-1 [15 U/ml 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 an assay buffer. And the mixture was incubated at 35 °C for 2 h
to activate the proenzyme. A 20 ml aliquot of inhibitor at varying concentra-
tions of inhibitor plus 40 ml of an assay buffer were added to the mixture and
incubated further 15 min at 35 °C: In a case of thiol possessing compounds,
to ensure that inhibitors remain unoxidized, a 1ϫ10Ϫ4 M b-mercaptoethanol
supplemented with an assay buffer was used. Then 100 ml of 0.05% FITC-
labeled collagen in an assay buffer was added, followed by incubation at
35 °C for 2 h. Then 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 fragment. To the reac-
tion mixture, 400 ml of 70% ethanol in 0.17 M Tris–HCl (pH 9.5)/0.67 M
NaCl was added and vortexed for 30 s, followed by centrifugation for 20 min
at 3000 rpm to obtain its supernatant fluid. Fluoresence intensity of such a
supernatant was measured with (lex 495 nm, lem 520 nm). Inhibitory activity
was defined as a percentage of control activity in the absence of inhibitor.
MMP-3 Inhibition Assay In a microtube, 20 ml of proMMP-3 [1 U/ml
an assay buffer [50 mM Tris–HCl (pH 7.8), 5 mM CaCl2] containing 0.02%
BSA] was mixed with 20 ml of 2 plasmin (20 mg/ml assay buffer). And the
mixture was incubated at 37 °C for 2 h to activate the proenzyme, followed
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