2,2'-dithiobisbenzamides derived from α-, β- and γ-amino acids possessing anti-HIV activities: Synthesis and structure-activity relationship

Article abstract of DOI:10.1016/S0968-0896(98)00118-7

Nucleocapsid protein (NCp7), which contains highly conserved retroviral zinc fingers, is essential in the early as well as the late phase of human immunodeficiency virus (HIV) life cycle and constitutes a novel target for AIDS therapy. HIV-1 NCp7 is a basic 55 amino acid protein containing two C(X)₂C(X)₄H(X)₄C motif zinc fingers flanked by basic amino acids on each side. 2,2'-dithiobisbenzamides have previously been reported to release zinc from these NCp7 zinc fingers and also to inhibit HIV replication. Specifically, 2,2'-dithiobisbenzamides derived from simple amino acids showed good antiviral activities. The benzisothiazolone 3, the cyclic derivative of 2, was selected for clinical trials as an agent for AIDS therapy. Herein we report the syntheses and antiviral activities, including therapeutic indices, of 2,2'-dithiobisbenzamides derived from α-, β- and γ-amino acids. Electrospray ionization mass spectrometry was used to study the zinc-ejection activity of these compounds. Among the α-amino acid derived 2,2'-dithiobisbenzamides, analogues containing alkyl side chains were found to be antivirally active with good therapeutic indices. 2,2'-Dithiobisbenzamides, derived from β- and γ-amino acids, were found to possess better antiviral and therapeutic efficacies than the α-amino acid analogues. Thus compound 59 was found to possess an EC₅₀ of 1.9μM with a therapeutic index of >50. Interestingly, 2,2'-dithiobisbenzamides derived from α-amino acids containing a protected acid function and polar side chains also exhibited very good antiviral activity. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00118-7

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1707±1730
2,20-Dithiobisbenzamides Derived from ꢀ-, ꢁ- and ꢂ-Amino
Acids Possessing Anti-HIV Activities: Synthesis and
Structure±Activity Relationship
J. V. N. Vara Prasad,a,* Joseph A. Loo,a Frederick E. Boyer,a
Michael A. Stier,a Rocco D. Gogliotti,a William J. Turner,a
Patricia J. Harvey,a Melissa R. Kramer,a David P. Mack,a
Je€erey D. Scholten,b Stephen J. Gracheckc and John M. Domagalaa
aDepartment of Chemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA
bDepartment of Biochemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA
cDepartment of Therapeutics, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA
Received 14 January 1998; accepted 20 April 1998
AbstractÐNucleocapsid protein (NCp7), which contains highly conserved retroviral zinc ®ngers, is essential in the
early as well as the late phase of human immunode®ciency virus (HIV) life cycle and constitutes a novel target for
AIDS therapy. HIV-1 NCp7 is a basic 55 amino acid protein containing two C(X)2C(X)4H(X)4C motif zinc ®ngers
¯anked by basic amino acids on each side. 2,20-dithiobisbenzamides have previously been reported to release zinc from
these NCp7 zinc ®ngers and also to inhibit HIV replication. Speci®cally, 2,20-dithiobisbenzamides derived from simple
amino acids showed good antiviral activities. The benzisothiazolone 3, the cyclic derivative of 2, was selected for clinical
trials as an agent for AIDS therapy. Herein we report the syntheses and antiviral activities, including therapeutic
indices, of 2,20-dithiobisbenzamides derived from a-, b- and g-amino acids. Electrospray ionization mass spectrometry
was used to study the zinc-ejection activity of these compounds. Among the a-amino acid derived 2,20-dithiobisbenz-
amides, analogues containing alkyl side chains were found to be antivirally active with good therapeutic indices. 2,20-
Dithiobisbenzamides, derived from b- and g-amino acids, were found to possess better antiviral and therapeutic e-
cacies than the a-amino acid analogues. Thus compound 59 was found to possess an EC50 of 1.9 mM with a therapeutic
index of >50. Interestingly, 2,20-dithiobisbenzamides derived from a-amino acids containing a protected acid function
and polar side chains also exhibited very good antiviral activity. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
against HIV-1 in cell culture resulted in the identi®ca-
tion of 2,20-dithiobisbenzamides as anti-HIV com-
pounds (i.e. 1 and 2). These compounds displayed low
micromolar activity against laboratory and clinical iso-
lates of HIV, including the strains resistant to both
nucleoside and non-nucleoside reverse transcriptase
inhibitor. These compounds also showed synergistic
activity with reverse transcriptase inhibitors (AZT, ddC)
and the protease inhibitor (KNI-272). Thus, these 2,20-
dithiobisbenzamides could potentially be used in com-
bination with other known anti-HIV drugs. Due to its
novel mechanism of action (vide infra) and its potential
for combination HIV therapy, compound 2 (CI-1013,
DIBA-4) was selected for preclinical evaluation.
Recently we reported a structure-activity relationship on
Targets of antiretroviral therapy for patients infected
with human immunode®ciency virus (HIV), the putative
agent responsible for AIDS, include reverse tran-
scriptase1 and protease2 enzymes. Due to the recent
encouraging results obtained with combination thera-
pies3,4 relative to monotherapies, there is an increased
interest in anti-HIV compounds targeted against novel
viral targets.5,6
Screening of selected compounds, from the Parke-Davis
chemical collection by the National Cancer Institute
*Corresponding author.
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00118-7

1708
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
the basic benzamide structure (1 (DIBA-1) and 2) and
showed that certain (a-amino acid analogues were
among the most active.7 Benzisothiazolone (3, CI-1012),
a cyclic compound derived directly from 2, was selected
for clinical trials as an agent for AIDS therapy. Rice
and co-workers also reported a wide variety of di-
sul®des, for example 4, as anti-HIV agents.8 Another
disul®de containing macrolide (5, SRR-SB3) was also
reported to be an anti-HIV agent9 and is likely to act by
a similar mechanism to these 2,2-dithiobisbenzamides.
Azodicarbonamide (6, ADA),10 introduced into Phase I/
II clinical trials in Europe for advanced AIDS, was also
shown to inhibit HIV-1 replication by targeting the
nucleocapsid protein.11 We wished to expand on the
observation that a-amino acids conferred good activity
to the 2,20-dithiobisbenzamides. Herein we report the
syntheses and a detailed structure±activity relationship
of various 2,2-dithiobisbenzamides derived from a-, b-,
and g-amino acids, including anti-HIV activities and
therapeutic indices.
pared from the commercially available 2,20-dithio-
bisbenzoic acid and thionyl chloride,12 with the
corresponding amino acids as shown in Scheme 1.
Couplings with 7 were performed under a variety of con-
ditions. The amino acid was converted to the corre-
sponding silyl amine by treatment with N-methyl-N-
(trimethylsilyl)acetamide followed by reaction with 7.
Alternately, the amine was converted to its potassium
salt by treatment with potassium t-butoxide in ethanol
and this was coupled with 7. Finally, the amino acid
esters were also coupled with dithiobisbenzoyl chloride
7 in the presence of a base like N-methylmorpholine or
triethylamine.
b-Amino acids, used for the preparation of compounds
57±68, were synthesized from the corresponding pro-
tected a-amino acids via the diazoketone derivatives 8.
Homologation into methyl ester, 9 was accomplished
via treatment with silver benzoate.13 The methyl esters
thus obtained were saponi®ed using potassium carbo-
nate in methanol/water and the Boc group was removed
with a 4 N hydrochloric acid solution in dioxane to
obtain the fully deprotected b-amino acids 10 (Scheme
2). The g-amino acids,14 used for the synthesis of
compounds 69±76, were prepared from the correspond-
ing N-protected g-amino aldehydes 1115 via Wittig
reaction to obtain the a- and b-unsaturated esters 12.
Hydrogenation of 12 gave the saturated N-protected
g-amino esters 13. Carboxyl and amino protective
groups were removed by saponi®cation and acid treat-
ment, respectively, to give the fully deprotected g-amino
acids 14, which were further used to couple to 7
(Scheme 3).
Biological Assays
All the compounds reported in this paper were tested for
anti-HIV activity in an in vitro culture assay with HIV-
IIIB infected human lymphocyte derived CEM cells
using the XTT cytopathic method at Southern Research
Institute.16 The assay measures the protection conferred
by the drug to the lymphocyte cells against the cyto-
pathic e€ects of HIV. The EC50 indicates the con-
centration of the drug that provides 50% protection
against HIV. The TC50 is the concentration of drug that
elicits drug induced cytotoxicity in 50% of uninfected
cells. Some compounds were tested two to three times
and the averages of the runs were reported. Gel shift
assay was performed to measure the ability of selected
compounds to inhibit the binding of NCp7 to its target
cRNA. Selected compounds were also tested for their
ability to extrude zinc from NCp7 using electrospray
mass spectroscopy as described earlier.17 The physical
properties and biological data of the compounds are
shown in Tables 1±6.
Chemistry
The compounds described in this paper were synthesized
via coupling of 2,2-dithiobisbenzoyl chloride 7, pre-

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1709
Scheme 1. Reactions conditions: (a) thionyl chloride re¯ux, overnight; (b) i. 1 equiv of amino acid or its ester, 4.0 equiv of N-methyl-
N-(trimethylsilyl)acetamide, N-methylmorpholine dichloromethane, rt, 30 min, followed by addition of bisacid chloride, rt, 4±12 h;
or ii. Dichloromethane, triethylamine or N-methylmorpholine, rt, overnight, or iii. 1 equiv of amino acid or its ester, potassium
t-butoxide, ethanol, 0 ꢀC, 1 h, followed by addition of bisacid chloride, rt, 4±12 h.
Scheme 2. Reactions conditions: (a) 1 equiv of amino acid, isobutyl chloroformate, triethylamine, 78 ꢀC, 30 min, THF, 1.1 equiv of
diazomethane in ether, rt; (b) silver benzoate, triethylamine, methanol; (c) i. R1=H; potassium carbonate, MeOH,H2O; ii. 4 N HCl in
dioxane, rt, 45 min; (d) bisacid chloride, 6, coupling as in Scheme 1.
ꢀ
ꢀ
.
Scheme 3. Reaction conditions: (a) EDCl, HOBT, N-methylmorpholine, HCl HN(OMe)Me, 0 C to rt; (b) LAH, 0 C; (c) Wittig
reagent, rt; (d) hydrogenation; (e) i. R=H, 3 N NaOH, rt; ii. 4 N HCl in dioxane, rt; (f) bisacid chloride, 7, coupling as in Scheme 1.
Results and Discussion
the a-amino acid portion (21 and 22, respectively)
showed better antiviral activities. 2,20-Dithiobisbenz-
amides 2, 24±30 derived from a-amino acids containing
branching at the b-or g-position of the amino acid
showed good antiviral activities with good therapeutic
indices. However, 2,20-dithiobisbenzamide analogues
31±34, which are disubstituted at the a-position of the
amino acids, either did not exhibit good antiviral activ-
ity or had poor therapeutic indices. Thus aminoiso-
butyric acid analogue 31 and a-methylmethionine
analogue 34 showed EC50s of 25 mM and 46 mM,
respectively, whereas cyclopentyl and cyclohexyl analo-
gues 32 and 33 showed increased cellular toxicities.
Among 2,20-dithiobisbenzamides possessing a phenyl
ring (35±37), compound 37, with a phenyl ring two
2,20-Dithiobisbenzamides derived from ꢀ-amino acids
As reported earlier,7 2,20-dithiobisbenzamides derived
from dl-isoleucine (2), l-isoleucine (15), or d-isoleucine
(16) exhibited similar antiviral activities as well as thera-
peutic indices, a result which suggests that chirality is
not a factor (Table 1). Hence 2,20-dithiobisbenzamides
derived from either dl- or l-amino acids were used in
this current study. The side chain of the amino acid
portion in 2,20-dithiobisbenzamides derived from various
a-amino acids was varied and their antiviral activities
are shown in Table 1. Among 2,20-dithiobisbenzamides
possessing straight chain alkyl groups 18±23, com-
pounds possessing n-butyl and n-pentyl side chains on

1710
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
atoms away from the a-position, exhibited better anti-
viral activity (EC50: 6 mM) relative to their lower homo-
logs 35 and 36 (EC50s: 25 and >100 mM, respectively).
Reduction of the phenyl ring of 37 to give the cyclohexyl
analogue 38 did not a€ect the antiviral activity. Simi-
larly, placement of the hydroxy or amino substituents at
the para position of the phenyl ring of compound 36 to
give 39 and 41 showed no improvement in antiviral
activities over 36. 2,20-Dithiobisbenzamides derived
from a-amino acids possessing polar functional groups
on the side chain, that is, carboxyl, hydroxyl, ether
moieties (compounds 42±47), displayed negligible anti-
viral activities. However, asparagine and glutamine
derived analogues 48 and 49 exhibited EC50s of 51 and
18.3 mM, respectively. Interestingly, the benzamide with
a tertiary amide moiety (compound 50, the N-methyl
Table 1. Dithiobisbenzamides varying amino acid side chains and their physical and antiviral activities tested against HIV in cell
culture
Entry R1
R2
R3
Chirality Mp(ꢀC)
Molecular formula
Elements EC50 TC50
TIc
analyzed (mM) (mM)
.
C26H32N2O6S2 0.26H2O
2
CH(CH3)CH2CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
dl
l
d
-
225±229
195±197
194±196
210±214
226±227
238±240
218±220
218±220
160±163
201±202
231±233
132±135
204±206
264±265
159±161
164±166
230±232
244±245
227±229
254±255
215±217
231±232
CHN
CHN
CHN
CH
9.0 >120 >13.3a
140 20a
113
17.9a
.
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
CH(CH3)CH2CH3
CH(CH3)CH2CH3
H
C26H32N2O6S2 0.7H2O
.
C26H32N2O6S2 0.6H2O
7.0
6.3
C18H16N2O6S2
C20H20N2O6S2
.
C22H24N2O6S2 0.1H2O
.
C24H28N2O6S2 1.4H2O
.
C26H32N2O6S2 0.5H2O
51.3 >100 >1.9a
35.3 >100 >2.8a
25 >100 >4.0
28.5 >80 >2.8
CH3
dl
dl
l
l
dl
l
dl
l
l
l
l
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CH2CH3
CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH2CH2CH3
CH2CH2SCH3
CH(CH3)2
7.4
6.9
51
6.9
9.1
C28H36N2O6S4
.
C24H28N2O6S4 0.6H2O
62.7
43 >100 >2.3
12 >100 >8.3a
18 >100 >5.5
C24H28N2O6S2
C26H32N2O6S2
C26H32N2O6S2
.
C28H36N2O6S2 0.25H2O
.
C26H28N2O6S2 0.61H2O
C(CH3)3
CH2CH(CH3)2
CH2C(CH3)3
8.9
5.7
130 >14.6a
92 16.1
CH2CH(CH3)=CH2
CH2(cyclopentyl)
CH2(cyclohexyl)
CH3
12 >100 >8.3
b
dl
l
C30H36N2O6S2
.
C32H40N2O6S2 0.8H2O
.
C24H24N2O6S2 0.7H2O
.
C26H28N2O6S2 1.9H2O
.
C28H32N2O6S2 1.3H2O
-
8.3
47
23
5.7
9.4
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
2.45
-
25 >100 >4.0
CH2CH2CH2CH2
CH2CH2CH2CH2CH2
CH2CH2SCH3
C6H5
-
>16
6.9
16
1
-
25.6
3.7
.
C26H32N2O6S4 1.0H2O
.
C30H24N2O6S2 1.1H2O
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
46 >100 >2.2
25 >100 >4.0
.
CH2C6H5
143±147 (d) C32H28N2O6S2 0.4H2O
.
>100 >100
1.0
11.3
13.2
1.0
CH2CH2C6H5
CH2CH2C6H11
CH2C6H5(para-OH)
CH2C6H5(para-NHCbz)
CH2C6H5(para-NH2)
CH2COOH
227±229
242±244
196±201
228±230
164±165
177±178
205±206
259±260
195±197
205±206
206±207
208±211
C34H32N2O6S2 0.83H2O
.
C34H44N2O6S2 0.81H2O
6.0
5.7
68
75
C32H28N2O8S2
.
C48H42N4O10S2 1.58H2O
.
C26H32N2O6S2 HBr
65 >165
13 >100
7.7
b
-
>15
15 <1.0
C22H20N2O10S2
.
C24H24N2O10S2 1.8H2O
CHN
CHN
CHN
>100 >100
>100 >100
>100 >100
1.0
1.0
1.0
1.0
1.0
1.0
2.0
5.5
1.0
1.0
CH2CH2COOH
CH2CH2CH2COOH
CH2CH2CH2CH2NH2
CH2OH
C26H28N2O10S2
.
.
C26H34N4O6S2 2.0HCl 3.5H2O CHNSCl >100 >100
.
C20H20N2O8S2 0.3H2O
C28H36N2O8S2
CHN
CHN
CHNS
CHNS
CHN
CHN
>100 >100
>100 >100
51 >100
CH2OtBu
CH2CONH2
C22H22N4O8S2
.
CH2CH2CONH2
CH2CH(CH3)2
CH2CH2CH2CH2 (R1 and R3)
192±194 (d) C24H26N4O8S2 1.7H2O
.
18.3 >100
l
l
120 (d)
88±90
C28H36N2O6S2 0.3H2O
.
C24H24N2O6S2 0.85H2O
>100 >100
>100 >100
aTaken from reference7.
bPurities were checked by HPLC.
cTI=TC50/EC50
.

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1711
derivative of 26) did not show any antiviral activity,
indicating the importance of the NH moiety in the
molecule. For this reason, the proline analogue 51 did
not show any antiviral activity. This observation is
consistent with our previous results.7
erally, 2,20-dithiobisbenzamides derived from b-amino
acids (57±68) showed an increase in anti-HIV activities
relative to 2,20-dithiobisbenzamides derived from a-
amino acids (Table 3). Since the cellular toxicities are
similar, 2,20-dithiobisbenzamides derived from b-amino
acids showed better overall therapeutic indices. Among
these derivatives, compound 59 containing a n-butyl
side chain was the most potent, with a higher thera-
peutic index relative to the lead compound 2. Although,
2,20-dithiobisbenzamides derived from g-amino acids
(69±79) with or without unsaturation exhibited good
anti-HIV activities, their cellular toxicities were also
increased relative to the 2,20-dithiobisbenzamides
derived from a- and b-amino acids leading to lower
therapeutic indices. Interestingly, analogues 67 and 75
containing the benzyl group in the side chain and
derived from b- and g-amino acids, respectively, show
much better antiviral activity and therapeutic index
compared to the a-amino acid analogue 36, which was
inactive.
Variation of spacer in the amino acids
Next, the spacer between the amine and carboxylic acid
was varied in these 2,20-dithiobisbenzamides to give 17,
52±56 and are shown in Table 2. Increases in antiviral
activities were observed with the increase in spacer from
n=1 to n=4. Thus increasing the spacer length from
n=1 (17) to n=2 (52) resulted in ꢁsevenfold increase in
antiviral activity. A further twofold increase in anti-HIV
activity was engendered by increasing the spacer from
n=2 (52) to n=3 (53), and another twofold increase in
potency was observed from n=3 (53) to n=4 (54) or 5
(55). Though the cellular toxicities also increased mod-
estly, compounds 54 and 55 exhibited a therapeutic
indices of 38 and 44, respectively. However, the analo-
gue containing a 6-carbon atom spacer 56 showed anti-
viral activity similar to compound 53 (n=3), and a
further increase in toxicity. This is very interesting
observation because among the 2,20-dithiobisbenamides
54 and 55 represent the most potent of the compounds
described to date with EC50s of 1.7 and 1.8 mM, respec-
tively.
2,20-Dithiobisbenzamides derived from amino derivatives
To explore the importance of the carboxylic group on
the antiviral activities, various analogues derived from
amino acid esters, amides, or amino alcohols were syn-
thesized and are shown in Table 5.
The a-amino acid ester analogues (80±86) containing
simple alkyl side chains exhibited little antiviral activity,
in direct contrast to their acid analogues. Moreover,
2,20-dithiobisbenzamides derived from amino alcohols
(containing alkyl or phenyl side chains) 87±89 were
toxic. However, a-amino acid ester analogues (90±94),
which contain the polar functional groups in the side
chain, amino (90 and 91), hydroxy (92 and 93) and
cyano (94), exhibited better anti-HIV activities relative
2,20-Dithiobisbenzamides derived from ꢁ- and ꢂ-amino
acids
Since 2,20-dithiobisbenzamides showed improved
potencies with increased methylene spacing between the
acid and the amide nitrogen, various 2,20-dithio-
bisbenzamides containing b- and g-amino acids were
synthesized (57±76) and are shown in Table 3. Gen-
Table 2. Dithiobisbenzamides varying spacer between amine and carboxylic groups and their physical and antiviral activities tested
against HIV in cell culture
Entry
n
mp (ꢀC)
Molecular formula
Elements
analyzed
EC50
(mM)
TC50
(mM)
TIb
17
52
53
54
55
56
1
2
3
4
5
6
210±214
201±203
165±167
172±173
137±139
190±192
C18H16N2O6S2
C20H20N2O6S2
C22H24N2O6S2
C24H28N2O6S2
CH
51.3
7.8
3.7
1.7
1.8
3.4
>100
120
>100
64
>1.9a
15.4a
27.0
CHN
CHN
CHN
CHN
CHN
37.7
44.4
C26H32N2O6S2
80
44
.
C28H36N2O6S2 0.4H2O
12.9
aTaken from ref7.
bTI=TC50/EC50
.

1712
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
to the free acid analogues (45 and 46) shown in Table 1.
When a side chain amine group of 90 was protected
using Boc- or Cbz-group (95 and 96) the antiviral
activity is reduced or the toxicity is increased. Interest-
ingly, the analogues containing amide functionalities in
either the backbone or side chain (97±100) exhibited
very good anti-HIV activities usually with good thera-
peutic ecacies. Thus, compound 100 showed EC50:
3.3 mM with a therapeutic index of 27.5. It appears that
hydrogens on the amide nitrogen are important for
antiviral activity, since 101, which is an N-methoxy-N-
methyl amide showed no viral activity. 2,20-Dithio-
bisbenzamide analogues containing two amide groups
(102 and 103) also did not exhibit any antiviral activities.
Table 3. Dithiobisbenzamides derived from b-and g-amino acids possessing various side chains and their physical and antiviral
activities tested against HIV in cell culture
Entry
R
n Chirality Mp (ꢀC)
Molecular formula
Elements EC50 TC50
analyzed (mM) (mM)
TIa
.
C22H24N2O6S2 0.6H2O
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
CH3
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
dl
l
l
l
l
l
l
l
l
dl
l
l
l
l
l
l
l
l
137±138
219±224
231±232
228±229
220±224
271±272
231±232
208±210
197±200
236±237
211±214
146±157
256±258
225±226
254±255
253±256
284±286(d)
231±233
223±226
251±252
CHN
CHN
CHN
CHNS
CHNS
CHNS
CHNS
CHNS
CHNS
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
29.9
59.5
2.0
CH2CH2CH3
CH2CH2CH2CH3
CH(CH3)2
C28H36N2O6S2
C30H40N2O6S2
C26H32N2O6S2
3.2 >100 >31.55
1.9 >100 >52.6
5.1 >100 >19.6
3.2 >100 >31.2
.
CH(CH3)CH2CH3
Cyclohexyl
C28H36N2O6S2 0.4H2O
.
C
C28H36N2O6S2 0.5H2O
32H40N2O6S2 0.3H2O
.
4.3
3.4
76
92
17.7
27
CH2CH(CH3)2
CH2C(CH3)3
CH2(cyclohexyl)
C6H5
.
C30H40N2O6S2 0.4H2O
5.2 >100
6.4 42
6.9 >100
19.2
6.6
.
C34H44N2O6S2 1.2H2O
.
C32H28N2O6S2 0.6H2O
14.5
31.9
13.7
20
.
C34H32N2O6S2 0.53H2O
.
C44H50N4O10S2 0.38H2O
CH2C6H5
2.1
7.1
67
97
50
25
74
60
24
CH2CH2CH2CH2NH(Cbz)
CH2CH2CH3
CH2CH2CH2CH3
CH(CH3)2
C28H36N2O6S2
C30H40N2O6S2 0.3H2O
2.5
4.1
.
6.1
C28H36N2O6S2
C30H40N2O6S2
.
4.3
4.9
17.2
12.2
3.1
CH(CH3)CH2CH3
Cyclohexyl
C
C36H48N2O6S2
34H44N2O6S2 1.1H2O
7.8
>8.5
2.1
CH2(cyclohexyl)
CH2C6H5
CH2CH(CH3)2
8.5
67
1.0
31.7
.
l
l
C36H36N2O6S2 0.42H2O
.
C30H40N2O6S2 0.9H2O
4.0 >100 >25
aTI=TC50/EC50
.
Table 4. Dithiobisbenzamides derived from a,b-unsaturated amino acids possessing various side chains and their physical and
antiviral activities tested against HIV in cell culture
Entry
R
Mp (ꢀC)
Molecular formula
Elements
analyzed
EC50 TC50 TIa
(mM) (mM)
.
77
78
79
CH(CH3)2
CH(CH3)CH2CH3
CH2CH(CH3)3
223±225
238±241
238±240
C28H32N2O6S2 0.7H2O
CHN
CHN
Ðb
4.8
5.0
3.7
74
70
53
15.4
14
.
C30H36N2O6S2 0.77H2O
C30H36N2O6S2
14.3
aTI=TC50/EC50
.
bPurity was checked by HPLC.

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1713

1714
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
Conclusions from SAR
biological roles of the nucleocapsid zinc ®ngers include
RNA packaging, dimerization, assembly of viral struc-
ture and reverse transcription of viral RNA genome.20
These highly conserved nucleocapsid proteins are essen-
tial in multiple phases of the retroviral replication
cycle.21±23 The HIV-1 NCp7 (Fig. 1) is a basic 55 amino
acid protein containing two C(X)2C(X)4H(X)4C motif
zinc ®ngers ¯anked by basic amino acids on each
side.18,19 These motifs are known to bind Zn+2 with
subpicomolar anities.24 The NMR solution structure
of NCp7 as well as NCp7 bound to cRNA have been
reported.25
In summary, SAR of these 2,20-dithiobisbenzamides
shows several important features: (1) Analogues con-
taining straight-chain or a- or b-branched alkyl groups
in the side chain of amino acid portion, were preferred
as they demonstrate excellent antiviral activities; (2)
Variation of spacer between the amine and carboxylic
acid is fruitful, yielding 2,20-dithiobisbenzamides pos-
sessing excellent antiviral activities; (3) Analogues con-
taining either a longer straight alkyl side chain (21 and
22) in the a-amino acid analogues or a longer spacer
between amine and acid portion (54±56) gave more
cytotoxicity, which may be due to less speci®city or
other cellular e€ects; (4) 2,20-Dithiobisbenzamides con-
taining acid functionality exhibited better antiviral
activities than other polar functional group analogues;
(5) It is also apparent that the presence of one polar
group (preferably acid functionality) is necessary for the
anti-HIV activity of 2,20-dithiobisbenzamides, whereas
two polar groups are detrimental for anti-HIV activity;
(6) 2,20-Dithiobisbenzamides containing a tertiary amide
did not exhibit good antiviral activities.
Rice et al. and later Tummino et al. demonstrated that
the 2,2-dithiobisbenzamides cause extrusion of zinc
from the zinc ®ngers of NCp7 resulting in a functionally
ine€ective form of NCp7.5,26 Turpin et al.27 have
observed the direct consequences of NCp7 zinc extru-
sion in HIV infected cells in cell culture with similar
aromatic disul®des. Thus they have shown that 2,20-
dithiobisbenzamides chemically modify the mutationally
intolerant retroviral zinc ®ngers in infected cells, inter-
rupting protease-mediated maturation of virions leading
ultimately to the production of compromised virions. It
was also found that the relative rates of HIV inactiva-
tion by various dithiobenzamides correlate with their
relative kinetic rates of NCp7 zinc ejection.28 Turpin et
al. also showed that this class of disul®des (example 1)
impairs the ability of HIV-1 virions to initiate reverse
transcription through their action on the retroviral zinc
®nger, thereby blocking further rounds of replication.29
Spectrometric studies of 2,2-dithiobisbenzamides using
electrospray ionization mass spectrometry (ESI-MS),
500 MHz one- and two-dimensional nuclear magnetic
resonance spectroscopy and circular dichroism spectro-
scopy demonstrated that Zn ejection is accompanied by
formation of covalent complexes between 2,20-dithio-
bisbenzamide monomers and Cys residues of Zn-deple-
ted NCp7 protein.17 Zinc is ejected sequentially from the
less stable C-terminal zinc ®nger, and then the more
stable N-terminal zinc ®nger.17 Characterization of var-
ious intermediates during the formation of ®nal pro-
ducts in the reaction of NCp7 protein with 2,20-
dithiodipyridine and disul®ram was also reported using
electrospray-mass spectrometry studies.30 It has also
been shown that similar dithiobenzamides with the
amide substituent para instead of ortho do not extrude
zinc or exhibit antiviral activity.6,8,18b
It is not surprising to note that analogues containing
carboxylic functionality are preferred compounds con-
sidering the fact that the zinc ®nger domains and linker
region (Fig. 1) of NCp7 contains basic amino acid resi-
dues.18,19 Molecular modeling studies also showed that
these aromatic disul®des could be docked eciently into
the zinc ®nger domains.8 Our SAR also supports the
overall conclusion from molecular modeling studies
performed by Rice and co-workers.8 In conclusion, an
e€ective ligand should possess a hydrophobic (pre-
ferably alkyl side chains) and hydrophilic (preferably
carboxyl functionality) components for interaction with
NCp7 protein.
Mechanism of action
Rice et al.6 demonstrated in their initial report that HIV
nucleocapsid protein (NCp7) was the target for these
2,20-dithiobisbenzamides e€ecting antiviral activity. The
Characterization of zinc ejection by electrospray
ionization mass spectrometry (ESI-MS)
The time course study of zinc ejection from NCp7 pro-
tein, as measured by changes in mass of Zn-loaded
NCp7 using ESI mass spectrometry, was performed
with selected 2,20-dithiobisbenzamides (2, 33, 51, 59, 72,
Figure 1. Amino acid sequence of nucleocapsid protein, NCp7,
and the peptide fragments used for this study.

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1715
and 79) derived from various amino acids (Fig. 2). After
addition of ®ve molar equivalents of the compound to
the protein, the major components, after 1 h, were the
starting material (NCp7+2Zn) and mono-modi®ed
(NCp7+X+Zn) molecules, where X represents half of
the disul®de drug molecule. At later time periods, the
apo-protein and its various covalently-modi®ed forms
(e.g. NCp7+2X) were the major products. The data
shows that up to two zinc atoms are being ejected upon
the covalent addition of these compounds to NCp7-Zn2
protein during a 1 h reaction time, similar to that pre-
viously observed with compound 2. Figure 3 shows
mass spectra from two time periods after addition of the
compounds to NCp7-Zn2. Although all of the com-
pounds studied by ESI-MS showed some zinc-ejection
activity, the nature with which they acted was somewhat
variable. For example, compounds 33 and 51 appeared
to form more abundant covalent adducts upon zinc-
ejection relative to 59 (Fig. 3). However, a direct corre-
lation between the zinc ejection data and the antiviral
activities of the present set of compound, was not
observed. More surprising is that compound 51 (proline
amide) also ejects zinc, though it did not show any
antiviral activity. This result con®rm that disul®des
themselves eject zinc and that they need not be ®rst
converted to their cyclic analogues, benzisothiazolones,
Figure 2. Time course study of total zinc ejection from NCp7
measured by ESI-MS. The absolute abundances (peak intensity)
for each protein±zinc species were summed in each mass spec-
trum acquired at each time point and normalized to the total
protein ion signal. For example, all of the ion signals for the
NCp7-Zn1 species were counted together (i.e. [NCp7+Zn]+
[NCp7+Zn+drug]+[NCp7+Zn+2drug]+ etc). The follow-
ing symbols were used to represent the data for the various
compounds studied:&, 72; +, 51; O, 2; *, 79; &, 59; Á, 33.
Figure 3. Time course study of zinc ejection from NCp7 by compounds (a) 59, (b) 51, and (c) 33 as monitored by ESI-MS. Decon-
voluted mass spectra acquired 13±15>min (top) and 43±45 min (bottom) after 150 mM of the compound was added to a solution
containing 30 mM NCp7, 60 mM ZnCl2, and 10 mM ammonium acetate pH 6.9. The square symbol represents the amount of zinc
bound to NCp7: a completely ®lled square (&) designates 2 bound-zinc, a half-®lled square (l) designates 1 bound-zinc, and an open
square (&) designates zero bound-zinc (apo-protein). The circle (*) symbol represents covalently-bound compound to NCp7, with
the number of circles equal to the number of molecules binding.

1716
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
Table 6. Binding and antiviral data of selected 2,20-bisdithio-
benzamides as measured by gel shift assay
Entry
Gel shift IC50 (mM)
HIV EC50 (mM)
36
59
76
79
66.2
19.1
12.9
5.89
>100
1.9
4.0
3.7
(Fig. 4). The rate of zinc ejection for either of the ®nger/
linker peptides or the combined sum is not as fast as full
length 1±55 NCp7, indicating the importance of the
total conformation of NCp7 on its reactivity and stabi-
lity. The Arg29-Gly35 region is highly conserved in
human and simian immunode®ciency virus strains. The
linker region appears to a€ect the reactivity of disul®de
Figure 4. Time course study of total zinc ejection by 2 from
zinc-bound peptides derived from the NCp7 amino acid
sequence (see Fig. 1) measured by ESI-MS. The following
symbols were used to represent the data for the various pep-
tides studied: &, peptide 29-55; x, peptides 7±34; O, 1±55
(NCp7). Peptides 13±30 and 34±51 showed no ejection of zinc
in the presence of 2 over a 1 h period. The open square (&)
symbol designate points resulting from the addition of data
from peptides 7±34 (x) and 29±55 (&).
compounds towards zinc-bound NCp7 in
unknown manner.
a still
Gel shift assay of selected 2,20-dithiobisbenzamides
To further address the mechanism of action of these
compounds,
representative group of 2,20-dithio-
a
bisbenzamides was chosen to test their ability to inhibit
cRNA±NCp7 complex formation as measured using a
gel shift assay. This represents a functional assay for
NCp7 and the results are shown in Table 6. Antivirally
inactive compound 36 has the highest IC50 value in this
assay, while the active compounds versus HIV showed
5±19 mM inhibition of cRNA±NCp7 binding. Although
the correlation is not tight, it seems reasonable in quali-
tative terms given the error in these biological measure-
ments.
Figure 5. Amino acid sequence of SP1 protein.
Conclusions
in situ. Since these experiments were conducted on
naked NCp7 in the absence of cRNA, it is possible that
recognition could be a factor in the NCp7±cRNA±drug
ternary complex.
Through a systematic variation of the amino acid por-
tion of these 2,20-dithiobisbenzamide analogues a com-
pound 59, with improved antiviral activity (fourfold)
and improved therapeutic index relative to the lead
compound 2, was obtained. Structural features that
improve antiviral activities were studied and found to be
quite consistent. More importantly, structural features
that increase cellular toxicities were identi®ed, which
may shed some light on the selectivity towards viral zinc
®ngers relative to mammalian zinc ®ngers. Gel shift
studies in our laboratories using SP1 zinc ®nger protein
(a mammalian zinc ®nger containing the CCHH motif,
Figure 5)31 have shown no binding with disul®de 2
(>100 mM at 4 h and >50 mM at 24 h), indicating
selectivity towards NCp7 protein. Moreover, the fact
that these compounds exert in vitro antiviral activity
without excessive cellular toxicity on either primary cell
The mechanism of action of 2 was further investigated
by ESI-MS using peptide fragments derived from the
NCp7 structure. Peptides composed of the amino acid
sequence for the N-terminal zinc ®nger motif (residues
13±30) and the C-terminal zinc ®nger structure (residues
34±51) (see Fig. 1) showed very little zinc-ejection
activity or covalent drug-binding over a 1 h period.
However, peptides containing either the N- or C-term-
inal zinc ®ngers with the highly basic central linker
region attached (residues 7±34 and 29±55, respectively)
showed moderate zinc-ejection and drug-binding activ-
ity over the same time period, with the C-terminal ®n-
ger/linker peptide exhibiting much more reactivity

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1717
lines indicates that there exists a degree of speci®city
toward the retroviral zinc ®ngers.18b
dephosphorylated. The ligation product was trans-
formed into the INVF' cells from the TA cloning kit,
colonies again chosen by restriction digest, and veri®ed
by sequencing. Isolated plasmid containing the correct
DNA was then transformed into BL21(DE3) cells for
expression. The cells were then grown to an OD600 of
0.6 in 300 mL of LB media supplemented with 100 mM
zinc chloride and 100 mg/mL Ampicillin. Isopropyl-
galactosidase (1 mM) was added and after 2 h of further
growth, the cells were harvested. The cells were ali-
quotted and centrifuged for 30 min, 4 ꢀC, 2500 rpm. The
supernatant was then discarded and the pellets were
stored at 80 ꢀC. Partial puri®cation of the peptide was
achieved via a scaled-up version of Desjarlais and
Berg.32 The pellet weight was multiplied by a factor of
0.0177 to give the amount of bu€er to be added (25 mM
Tris±HCl pH 8.0, 100 mM zinc chloride, 5 mM DTT).
The pellets were lique®ed and mixed until uniform. The
cell paste was then aliquotted into 1.5 mL sterile
Eppendorf tubes. The tubes were then placed in a boil-
ing water bath for 15 min. 1 M KCl was added to a
concentration of 150 mM/tube. The tubes were then
vortexed and spun in an Eppendorf centrifuge at
14,000 rpm for 30 min at 4 ꢀC. The supernatant was then
removed to a fresh tube. After pooling the protein, it
was aliquotted and stored at 80 ꢀC. The protein
obtained was then pooled together, aliquotted, and
stored at 80 ꢀC. Protein sequence was veri®ed by
amino acid analysis and the protein concentration was
estimated to be 580 nM. The DNA binding experiments
were carried out at room temperature in a binding buf-
fer consisting of 50 mM Tris±HCl, pH 7.5, 100 mM
KCl, 20 mM MgCl2, and 10% glycerol. Partially pur-
i®ed SP1 zinc ®nger protein (37.7 ng) was incubated with
each compound at two concentrations (50 and 5 mM).
At 1, 4, and 24 h, a 19 mL aliquot from each was added
to 6 mL of 32P labeled SP1 DNA consensus sequence
(<100 pM). After mixing, the solutions were incubated
at room temperature for 15 min. Separation of DNA
and the DNA±protein complex was achieved by gel
electrophoresis on an 8% nondenaturing polyacryl-
amide gel (39/1 crosslinking) using a running bu€er of
50 mM Tris, 45 mM boric acid, 0.5 mM EDTA, pH 8.4.
The gels were pre-run at 4 ꢀC for 30 min at 150V and
continued for 60 min at 250V at 4 ꢀC after loading the
entire reaction with the voltage on. The gels were
dried and then imaged using a Molecular Dynamics
Phosphorimager. After quantitation of the images using
the ImageQuant software package, percentage shift
values were calculated based on the volume of the
DNA±protein complex band compared to the total
volume of all bands present.
The exact mechanism of action of these compounds
with the NCp7 and any cellular targets is uncertain, as
these highly reactive disul®des are capable of multiple
reaction in vivo, to form many potentially active species
(e.g. free thiols, mixed disul®des with other cellular
proteins, etc). Nevertheless, these structurally simple
and easily synthesized compounds appear to possess
good anti-HIV activity, a reasonable therapeutic index,
and importantly have a di€erent mode of action from
known anti-HIV agents, which are approved or in clin-
ical trials.
Experimental
Biological assays. The expression and isolation of NCp7
protein were reported previously.26,27 Peptide fragments
of NCp7 were synthesized by the University of Michi-
gan Protein and Carbohydrate Structure Facility (Ann
Arbor, MI, USA).
NCp7 gel shift assay. The RNA binding experiments
were carried out at room temperature in a binding buf-
fer containing 50 mM Tris±HCl pH 7.5, 100 mM KCl,
20 mM MgCl2, 10% glycerol. The NCp7 at 0.5 mM was
incubated with each compound at six concentrations
(200±0 mM). At 24 h, a 50 mL aliquot from each was
added to an equal volume of 32P-labeled cRNA
(<100 pM) containing 0.01 mg/mL calf thymus DNA.
After mixing, the solutions were incubated for 10 min at
room temperature. Separation of RNA and RNA±pro-
tein complexes was accomplished by gel electrophoresis
on a 8% nondenaturing polyacrylamide gel (39/1 cross-
linking) using a running bu€er of 50 mM Tris, 45 mM
boric acid, 0.5 mM EDTA, pH 8.4. The gels were pre-run
at 4 ꢀC after loading with voltage on. The gels were
dried and then imaged using a Molecular Dynamics
Phosphor Imager. After quantitation of the images, IC50
values were calculated based on the concentration of
compound incubated with protein prior to the addition
of RNA.
SP1 Gel shift assay. The DNA corresponding to the 99
amino acid DNA binding domain of SP1 was obtained
by PCR from a plasmid containing the human coding
sequence for SP1. The primers utilized in the PCR
reaction added a Nde I and Hind III restriction site to
the sequence for cloning purposes. The PCR reaction
was carried out using Vent Polymerase followed by A-
tailing and was ligated into the Invitogen TA cloning kit
plasmid. Colonies were initially chosen by restriction
digest and veri®ed by sequencing. The Nde I/Hind III
fragment was then cloned into pET21a(+) which had
been digested with the same restriction enzymes and
Chemical synthesis. Melting points were determined in
open capillary tubes on a Hoover melting point appa-
ratus and are uncorrected. Infrared (IR) spectra were

1718
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
determined in KBr pellet on a Nicolet FT IR SX-20
spectrophotometer. Proton magnetic resonance (NMR)
spectra were recorded on a 300 or 400 MHz Varian
Unity spectrometer. Chemical shifts are reported in d
values relative to tetramethylsilane. Mass spectra were
recorded on a Finnigan TSQ-70 spectrometer. Ele-
mental analyses were performed on a Perkin±Elmer 240
elemental analyzer or at Robertson Microlit Labora-
tories (Madison, NJ, USA). Flash or medium pressure
chromatography was performed using silica gel 230±400
mesh. All starting materials were commercially available
unless otherwise noted.
was taken in water and ethyl acetate. Aqueous layer was
acidi®ed with 3 N hydrochloric acid to obtain pre-
cipitate. Then, it was extracted into saturated sodium
bicarbonate solution and the free acid was liberated by
added 6 N hydrochloric acid to get the ®nal product.
The ®nal product was puri®ed by washing with either
ether or 10% ethanol in ether, ®ltered and dried.
General method D. Amino acid (1 equiv) was taken in
absolute ethanol and treated with a solution of sodium
(1 equiv) in absolute ethanol, keeping the temperature
between 30 to 50 ꢀC. After 2 h, 2,20-dithiobisbenzoyl
chloride was added portionwise over 1 h. After stirring
overnight at room temperature, the mixture was con-
centrated and the residue was suspended in water. After
®ltering the insoluble material, the ®ltrate was adjusted
to pH 3.0 with 1 N hydrochloric acid and the solid
obtained was collected. The product was again taken in
aqueous sodium bicarbonate solution and acidi®ed with
hydrochloric acid to obtain pure material, which was
dried.
General procedures for the synthesis of 2,20-dithio-
bisbenzamides. The general procedures used in synthesis
of the compounds described above are very much simi-
lar to the ones described previously.7 The synthesis and
spectral data of compounds reported earlier were not
given.
General method A. Initially amino acid hydrochloride
(2±2.5 equiv) was taken in 10 mL of dichloromethane
and treated with N-methylmorpholine (2±2.5 equiv) to
liberate free base. To it N-methyl-N-(trimethylsilyl)acet-
amide (2±4.5 equiv) was added. The reaction mixture
was stirred at room temperature for 1.5 h. To it bisacid
chloride (1 equiv) was added. The reaction mixture was
stirred at room temperature 4±16 h. Solvents were eva-
porated. The residue was stirred with 3 N hydrochloric
acid for 1 h, diluted with ethyl acetate and washed with
3 N hydrochloric acid. The product was extracted into
saturated sodium bicarbonate solution and the free acid
was liberated by added 6 N hydrochloric acid to get the
®nal product. The ®nal product was puri®ed by washing
with either ether or 10% ethanol in ether, ®ltered and
dried.
Examples
[S-(R*,R*)-2-{2-[2-(1-Carboxy-2-methyl-butylcarbamoyl)-
phenyldisulfanyl]-benzoyl-amino}-3-methyl-pentanoic acid
(15). This compound was synthesized according to the
general procedure B using isoleucine t-butyl ester
(5.70 g, 30.4 mmol), pyridine (50 mL), 2,20-dithio-
bisbenzoyl chloride (4.80 g,14.0 mmol) and 70 mL of
CH2Cl2. Isolated yield: 38%; This material was dis-
solved in 50 mL CH2Cl2 and reacted with 50 mL tri-
¯uoroacetic acid at 35 ꢀC. After 2 h, the mixture was
concentrated and the residue triturated with ether. The
solids were suspended in EtOAc/CHCl3 and ®ltered.
1
Isolated yield: 42%; H NMR (400 MHz, DMSO-d6) d
12.70 (bs, 2H), 8.73 (d, 2H), 7.65 (d, 4H), 7.45 (m, 2H),
7.31 (t , 2H), 4.35 (t, 2H ), 1.96 (m, 2H), 1.53 (m, 2H),
1.33 (m, 2H), 0.96 (d, 6H), 0.89 (t, 6H ); IR (KBr) 1722,
1641 cm 1; MS (ESI) m/z 266.
General method B. To the amino acid ester (1 equiv)
taken in dichloromethane was added N-methylmor-
pholine or triethylamine (1.2±10 equiv). To it bisacid
chloride (0.5 equiv) in dichloromethane was added
dropwise at 0±10 ꢀC. The reaction mixture was stirred at
room temperature for 18 h. The mixture was con-
centrated, suspended in water and ®ltered. The solids
were dissolved in chloroform, washed with water, dried
and concentrated. The residue was triturated with ether/
hexanes (1/1) to obtain the ®nal product, which was
puri®ed by ¯ash silica gel chromatography, if needed.
(+, )-2-{2-[2-(1-Carboxy-propylcarbamoyl)-phenyldi-
sulfanyl]benzoylamino}-butyric acid (19). This compound
was synthesized according to the general procedure B
using bisacid chloride (1.64 g, 4.77 mmol), potassium
t-butoxide (1.29 g, 11.45 mmol) and corresponding dl-
amino acid hydrochloride (2.64 g, 19.08 mmol). Isolated
1
yield: 62%; H NMR (400 MHz, DMSO-d6) d 0.97 (t,
6H), 1.66 (m, 2H), 1.86 (m, 2H), 4.31 (m, 2H), 7.33 (t,
2H), 7.47 (t, 2H), 7.66 (d, 2H), 7.72 (d, 2H), 8.83 (d, 2H);
IR (KBr) 3330, 2973, 1716, 1639, 1529, 1288, 1249, 1233,
748 cm 1; MS (ESI) m/z 238, 323, 463, 475.
General method C. Potassium t-butoxide (0.6 equiv) was
taken in 20 mL of ethanol and stirred at room tempera-
ture for 5 min. To it amino acid (1 equiv) was added and
kept under stirring for 15±20 min until it is homo-
geneous. To it bisacid chloride (0.25 equiv) in dichlor-
omethane (5 mL) was added and kept under stirring for
3 h. After 3 h, ethanol was evaporated and the residue
[S-(R*,R*)-2-{2-[2-(1-Carboxy-butylcarbamoyl)-phenyldi-
sulfanyl]benzoylamino}-pentanoic acid (20). This com-
pound was synthesized according to the general method

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1719
A using bisacid chloride (2.0 g, 5.85 mmol), N-methyl-N-
(trimethylsilyl)acetamide (6.81 g, 46.84 mmol), corre-
sponding amino acid hydrochloride (1.8 g, 11.71 mmol),
N-methylmorpholine (4.74 g, 46.84 mmol) and 40 mL of
dichloromethane. Isolated yield: 70%; 1H NMR
(400 MHz, DMSO-d6) d 0.92 (t, 6H), 1.47 (m, 4H), 1.81
(m, 4H), 4.4 (m, 2H), 7.33 (t, 2H), 7.47 (t, 2H), 7.66 (t,
2H), 7.72 (d, 2H), 8.83 (d, 2H); IR (KBr) 3279, 2961,
2873, 1723, 1640, 1534, 1260, 746 cm 1; MS (ESI) m/z
252, 286, 503.
(s, 18H), 4.29 (m, 2H), 7.25 (t, 2H), 7.39 (t, 2H), 7.54
(d, 2H), 7.59 (d, 2H), 8.56 (d, 2H); MS (ESI) m/z 532.
[S-(R*,R*)]-2-{2-[2-(1-Carboxy-3,3-dimethyl-butylcar-
bamoyl)-phenyldisulfanyl]-benzoylamino}-4,4-dimethyl-
pentanoic acid (27). This compound was synthesized
according to the general method A using bisacid chlor-
ide (0.59 g, 1.72 mmol), N-methyl-N-(trimethylsilyl)-
acetamide (8.6 g, 59.17 mmol), corresponding amino
acid (1.0 g, 6.88 mmol), N-methylmorpholine (1.39 g,
13.76 mmol) and 20 mL of dichloromethane. Isolated
yield: 82%; 1H NMR (400 MHz, DMSO-d6) d 0.94
(s, 18H), 1.61 (m, 4H), 4.36 (m, 2H), 7.29 (t, 2H), 7.42
[S-(R*,R*)-2-{2-[2-(1-Carboxy-pentylcarbamoyl)-phenyl-
disulfanyl]benzoylamino}-hexanoic acid (21). This com-
pound was synthesized according to the general procedure
C using bisacid chloride (1.64 g, 4.77 mmol), potassium
t-butoxide (1.29 g, 11.45 mmol) and corresponding dl-
amino acid hydrochloride (2.50 g, 19.08 mmol). Isolated
yield: 68%; 1H NMR (400 MHz, DMSO-d6) d 0.83
(t, 6H), 1.28 (m, 8H), 1.75 (m, 4H), 4.31 (m, 2H), 7.28
(t, 2H), 7.42 (t, 2H), 7.61 (d, 2H), 7.66 (d, 2H), 8.77
(d, 2H); IR (KBr) 3282, 2957, 2870, 1718, 1643, 1538,
1458, 1251, 755 cm 1; MS (ESI) m/z 266.
(t, 2H), 7.62 (t, 2H), 7.67 (t, 2H), 8.86 (d, 2H); IR (KBr)
1
3313, 2956, 1711, 1637, 1544, 1434, 1251, 867, 747 cm
MS (ESI) m/z 280, 561.
;
[S-(R*,R*)-2-{2-[2-(1-Carboxy-3-methyl-but-3-enylcar-
bamoyl)-phenyldisulfanyl]-benzoylamino}-4-methyl-pent-4-
enoic acid (28). This compound was synthesized accord-
ing to the general method A using bisacid chloride
(1.32 g, 3.87 mmol), N-methyl-N-(trimethylsilyl)acet-
amide (2.25 g, 15.48 mmol), corresponding amino acid
hydrochloride (1.0 g, 7.75 mmol), N-methylmorpholine
(1.56 g, 15.48 mmol) and 40 mL of dichloromethane.
Isolated yield: 49%; 1H NMR (400 MHz, DMSO-d6)
d 1.75 (s, 6H), 2.55 (m, 4H), 4.61 (m, 2H), 4.83
(brs, 4H), 7.31 (t, 2H), 7.44 (t, 2H), 7.64 (t, 2H), 7.66
(t, 2H), 8.83 (d, 2H); IR (KBr) 3386, 3293, 2923, 1729,
1639, 1527, 1434, 1258, 898, 747 cm 1; MS (ESI) m/z
264.
[S-(R*,R*)-2-{2-[2-(1-Carboxy-hexylcarbamoyl)-phenyl-
disulfanyl]benzoylamino}-heptanoic acid (22). This com-
pound was synthesized according to the general proce-
dure C using bisacid chloride (1.64 g, 4.77 mmol),
potassium t-butoxide (1.29 g, 11.45 mmol) and corres-
ponding dl-amino acid (2.76 g, 19.08 mmol). Isolated
1
yield: 61%; H NMR (400 MHz, DMSO-d6) d 0.86 (t,
6H), 1.31 (m, 8H), 1.44 (m, 4H), 1.77 (m,4H), 4.36 (m,
2H), 7.31 (t, 2H), 7.45 (t, 2H), 7.64 (d, 2H), 7.67 (d, 2H),
8.83 (d, 2H); IR (KBr) 3286, 2955, 2860, 1729, 1634,
1532, 1461, 1247, 745 cm 1; MS (ESI) m/z 280, 559.
(+, )-2-{2-[2-(1-Carboxy-2-cyclopentyl-ethylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-3-cyclopentyl-propionic
acid (29). This compound was synthesized according
[S-(R*,R*)-2-{2-[2-(1-Carboxy-3-methylsulfanyl-propyl-
carbamoyl)-phenyldisulfanyl]-benzoylamino}-4-methyl-
sulfanyl-butyric acid (23). This compound was synthe-
sized according to the general procedure C using bisacid
chloride (1.64 g, 4.77 mmol), potassium t-butoxide
(1.29 g, 11.45 mmol) and corresponding dl-amino acid
hydrochloride (2.84 g, 19.08 mmol). Isolated yield: 52%;
1H NMR (400 MHz, DMSO-d6) d 2.06 (s, 6H), 2.61 (m,
8H), 4.53 (m, 2H), 7.31 (t, 2H), 7.47 (t, 2H), 7.64 (d,
2H), 7.72 (t, 2H), 8.89 (d, 2H); IR (KBr) 3337, 2917,
1727, 1639, 1530, 1433, 1224, 748 cm 1; MS (ESI) m/z
284, 567.
to the general method A using bisacid chloride
(0.54 g, 1.59 mmol), N-methyl-N-(trimethylsilyl)acetamide
(1.85 g, 12.72 mmol), dl-cyclopentylalanine (0.5 g,
3.18 mmol), N-methylmorpholine (1.29 g, 12.72 mmol)
1
and 20 mL of dichloromethane. Isolated yield: 42%; H
NMR (400 MHz, DMSO-d6) d 1.11 (m, 4H), 1.31±2.03
(m, 18H), 4.36 (m, 1H), 5.06 (dd, 1H), 7.39 (t, 2H), 7.62
(t, 2H), 7.83 (d, 2H), 7.92 (d, 2H), 8.8 (d, 2H) IR (KBr)
3411, 2950, 2864, 1737, 1599, 1530, 1448, 1222,
747 cm 1; MS (ESI) m/z 290, 292.
[S-(R*,R*)]-2-{2-[2-(1-Carboxy-2-cyclohexyl-ethylcar-
bamoyl)-phenyldisulfanyl]-benzoylamino}-3-cyclohexyl-
propionic acid (30). This compound was synthesized
according to the general method A using bisacid chloride
(1.0g, 2.92 mmol), N-methyl-N-(trimethylsilyl)acetamide
(1.7 g, 11.68 mmol), l-cyclohexylalanine hydrochloride
(1.33 g, 6.41 mmol), triethylamine (1.3 g, 12.82 mmol)
[S-(R*,R*)]-2-{2-[2-(1-Carboxy-2,2-dimethyl-propylcar-
bamoyl)-phenyldisulfanyl]-benzoylamino}-3,3-dimethyl-
butyric acid (25). This compound was synthesized
according to the general method A using bisacid chlor-
ide (1.57 g, 4.5 mmol), N-methyl-N-(trimethylsilyl)acet-
amide (11.1 mL, 36 mmol), corresponding amino acid
(1.50 g, 11 mmol) and 120 mL of dichloromethane. Iso-
lated yield: 15%; 1H NMR (400 MHz, DMSO-d6) d 1.03
1
and 20 mL of dichloromethane. Isolated yield: 51%; H
NMR (400 MHz, DMSO-d6) d 0.77±1.25 (m, 11H),
1.39±1.86 (m, 15H), 4.36 (m, 2H), 7.33 (t, 2H), 7.44

1720
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
(t, 2H), 7.66 (d, 4H), 8.83 (d, 2H), 12.61 (brs,
2H); IR (KBr) 3287, 2923, 1727, 1631 cm 1; MS (ESI)
m/z 306.
[R-(R*,R*)] (2-{2-[Carboxy-phenyl-methyl)-carbamoyl]-
phenyldisulfanyl}-benzoylamino)-phenyl-acetic acid (35).
t-Butyl ester of the title compound was prepared
according to the general method B using bisacid
chloride (0.43 g, 1.25 mmol), corresponding amino acid
t-butyl ester hydrochloride (0.60 g, 2.89 mmol), triethyl
amine (1.2 g) in 30 mL of dichloromethane. Resulting
compound was taken in 10 mL of dichloromethane and
stirred with 0.73 g of tri¯uoroacetic acid at room tem-
2-{2-[2-(1-Carboxy-1-methyl-ethylcarbamoyl)-phenyl-
disulfanyl]-benzoylamino}-2-methyl-propionic acid (31).
The title compound was synthesized according to the
general method A using bisacid chloride (1.0 g, 2.92 mmol),
N-methyl-N-(trimethylsilyl)acetamide (1.70 g, 11.68 mmol),
corresponding amino acid (1.5 g, 7.4 mmol), N-methyl-
morpholine (1.20 g, 11.68 mmol) and 20 mL of
dichloromethane. Isolated yield: 80%; 1H NMR
(400 MHz, DMSO-d6) d 1.47 (s, 12H), 7.17 (t, 2H), 7.44
(t, 2H), 7.66 (d, 4H), 8.72 (s, 2H), 12.31 (brs, 2H); IR
(KBr) 3329, 2988, 1712, 1633, 1528, 748 cm 1; MS (ESI)
m/z 238.
1
perature to a€ord the product. Isolated yield: 10%; H
NMR (400 MHz, DMSO-d6) d 6.08 (d, 2H), 7.43
(m, 14H), 7.60 (t, 2H), 7.82 (m, 2H); MS (ESI) m/z 286.
[S-(R*,R*)]-2-{2-[2-(1-Carboxy-2-phenyl-ethylcarabamoyl)-
phenyldisulfanyl]-benzoylamino}-3-phenyl-propionic acid
(36). (a) t-Butyl ester of the title compound was pre-
pared according to the general method B using bisacid
chloride (0.87 g, 2.9 mmol), corresponding amino acid t-
butyl ester hydrochloride (1.30 g, 5.05 mmol), triethyl
amine (1.2 g) in 20 mL of dichloromethane. Isolated
Cyclopentanecarboxylic acid, 1,10-[dithiobis[(2,1-phenyl-
enecarbonyl)imino]bis[-, (32). The title compound was
synthesized according to the general method A using
bisacid chloride (1.0 g, 2.92 mmol), N-methyl-N-(tri-
methylsilyl)acetamide (1.70 g, 11.68 mmol), corresponding
amino acid (0.82 g, 6.41 mmol), N-methylmorpholine
(1.20 g, 11.68 mmol) and 20 mL of dichloromethane.
1
yield: 54%; H NMR (400 MHz, DMSO-d6) d 1.35 (s,
18H), 3.03 (dd, 2H), 3.11 (dd, 2H), 4.50 (m, 2H), 7.17
(m, 2H), 7.25 (m, 5H), 7.47 (t, 2H), 7.50 (d, 2H), 7.53 (d,
2H), 8.94 (d, 2H). (b) The above compound was dis-
solved in 10 mL of dichloromethane. To this solution
was added 1.5 mL of tri¯uoroacetic acid and the mixture
was stirred at room temperature for 48 h. The solvents
were evaporated and the residue was triturated with
hexanes to obtain the title compound which was dried in
vacuo. Isolated yield: 99%; 1H NMR (400 MHz,
DMSO-d6) d 3.03 (dd, 2H), 3.22 (dd, 2H), 4.64 (m, 2H),
7.19 (t, 2H), 7.31 (m, 10H), 7.42 (t, 2H), 7.55 (m, 4H),
8.92 (d, 2H); IR (KBr) 3306, 3030, 1721, 1641, 1528,
1260, 749 cm 1; MS (ESI) m/z 300, 601.
1
Isolated yield: 52%; H NMR (400 MHz, DMSO-d6) d
1.69 (m, 8H), 2.09 (m, 8H), 7.3 (t, 2H), 7.43 (t, 2H), 7.61
(d, 2H), 7.64 (d, 2H), 8.65 (s, 2H), 12.28 (brs, 2H); IR
(KBr) 3297, 2959, 1710, 1630, 1530, 1323, 748 cm 1; MS
(ESI) m/z 264.
Cyclohexanecarboxylic acid, 1,10-[dithiobis[(2,1-phenyl-
enecarbonyl)imino]bis[-, (33). The title compound was
synthesized according to the general method A using
bisacid chloride (1.0 g, 2.92 mmol), N-methyl-N-(tri-
methylsilyl)acetamide (1.7 g, 11.68 mmol), corresponding
amino acid (0.92 g, 6.41 mmol), N-methylmorpholine
(1.2 g, 11.68 mmol) and 20 mL of dichloromethane. Iso-
lated yield: 60%; 1H NMR (400 MHz, DMSO-d6) d 1.28
(brm, 2H), 1.55 (brs, 10H), 1.73 (brm, 4H), 2.15
(brd, 4H), 7.31 (t, 2H), 7.44 (t, 2H), 7.61 (d, 2H), 7.66
(d, 2H), 8.53 (s, 2H), 12.28 (brs, 2H); IR (KBr) 3288,
2935, 1711, 1632, 1529, 748 cm 1; MS (ESI) m/z 278.
[R-(R*,R*)]-2-{2-[2-(1-Carboxy-3-phenyl-propylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-4-phenyl-butyric acid (37).
The title compound was synthesized according to the
general method
A using bisacid chloride (1.53 g,
4.46mmol), N-methyl-N-(trimethylsilyl)acetamide (3.24 g,
11.68 mmol), corresponding amino acid (2.0 g, 11.16 mmol),
N-methylmorpholine (2.26 g, 22.32 mmol) and 40 mL of
dichloromethane. Isolated yield: 42%; 1H NMR
(400 MHz, DMSO-d6) d 2.11 (m, 4H), 2.64±2.83 (m,
4H), 4.33 (m, 2H), 7.17±7.4 (m, 12H), 7.5 (t, 2H), 7.69
(d, 2H), 7.75 (d, 2H), 8.95 (d, 2H), 12.69 (brs, 2H); IR
(KBr) 3284, 2926, 1717, 1639, 1532, 747, 699 cm 1; MS
(ESI) m/z 314.
(+, )-2-{2-[2-(1-Carboxy-1-methyl-3-methylsulfanyl-
propylcarbamoyl)-phenyldisulfanyl]-benzoylamino}-2-methyl-
4-methylsfulfanyl-butyric acid (34). This compound was
synthesized according to the general procedure C using
bisacid chloride (1.64 g, 4.77 mmol), potassium t-but-
oxide (1.29 g, 11.45 mmol) and corresponding dl-a-
methylmethionine (3.11 g, 19.08 mmol). Isolated yield:
76%; 1H NMR (400 MHz, DMSO-d6) d 1.32 (s, 6H),
2.03 (s and m, 8H), 2.47 (m, 4H, partially obscured
by dmso peak), 2.33 (m, 2H), 7.31 (t, 2H), 7.44 (t, 2H),
7.61 (d, 2H), 7.66 (d, 2H), 8.66 (d, 2H); IR (KBr) 3325,
2918, 1709, 1629, 1520, 1448, 751 cm 1; MS (ESI) m/z
298.
[S-(R*,R*)]-2-{2-[2-(1-Carboxy-3-cyclohexyl-propylcar-
bamoyl)-phenyldisulfanyl]-benzoylamino}-4-cyclohexyl-
butyric acid (38). The title compound was synthesized
according to the general method A using bisacid chlor-
ide (1.48 g, 4.34 mmol), N-methyl-N-(trimethylsilyl)-
acetamide (4.27 g, 43.24 mmol), corresponding amino
acid (2.0 g, 10.81 mmol), N-methylmorpholine (4.37 g,

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1721
43.24 mmol) and 40 mL of dichloromethane. Isolated
1
[S-(R*,R*)]-2-{2-[2-(1,2-Dicarboxy-ethylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-succinic acid (42). This
compound was synthesized according to the general
procedure C using bisacid chloride (1.1 g, 3.2 mmol),
corresponding amino acid t-butyl ester hydrochloride
(2.0 g ), N-methylmorpholine (1.06 g) in 45 mL of
dichloromethane; The crude compound obtained was
dissolved in 10 mL of dichloromethane. To it 0.41 g of
tri¯uoroacetic acid was added and stirred at room tem-
perature for 24 h. The solvents were evaporated and the
residue was triturated with hexanes to obtain the title
compound, which was dried in vacuo. Isolated yield:
yield: 42%; H NMR (400 MHz, DMSO-d6) d 0.88 (m,
4H), 1.03±1.26 (m, 12H), 2.55±2.92 (m, 14H), 7.31
(t, 2H), 7.44 (t, 2H), 7.64 (d, 2H), 7.69 (d, 2H), 8.83
(d, 2H), 12.64 (brs, 2H); IR (KBr) 3290, 2922, 2851,
1715, 1642, 1532, 742 cm 1; MS (ESI) m/z 320.
[S-(R*,R*)]-2-(2-{2-[1-Carboxy-2-(4-hydroxy-phenyl)-
ethylcarbamoyl]-phenyldisulfanyl}-benzoylamino)-3-(4-
hydroxy-phenyl)-propionic acid (39). (a) To the bisacid
chloride (0.57 g, 0.167 mmol) dissolved in 20 mL of
dichloromethane was added tyrosine (t-butyl) t-butyl
ester hydrochloride (1.10 g, 3.34 mmol) and triethyl-
amine (0.7 g) as described in the general method B, iso-
lated yield: 52%; 1H NMR (400 MHz, DMSO-d6) d 1.25
(s, 18H), 1.37 (s, 18H), 3.08 (m, 4H), 4.5 (m, 2H), 6.89
(d, 4H), 7.22 (d, 4H), 7.28 (t, 2H), 7.42 (t, 2H), 7.53
(d, 2H), 7.58 (d, 2H), 8.94 (d, 2H). (b) The above com-
pound (0.57 g) was dissolved in 10 mL of dichloro-
methane. To this, 1.0 mL of tri¯uoroacetic acid was
added and stirred at room temperature for 24 h. The
solvents were evaporated and the residue on triturating
with hexanes a€orded the title compound. Isolated
yield: 50%; 1H NMR (400 MHz, DMSO-d6) d 2.92
(dd, 2H), 3.08 (dd, 2H), 4.53 (m, 2H), 6.66 (d, 4H), 7.14
(d, 4H), 7.28, (t, 2H), 7.42 (t, 2H), 7.58 (m, 4H), 8.86
(d, 2H), 9.22 (s, 2H); MS (ESI) m/z 316.
1
25%; H NMR (400 MHz, DMSO-d6) d 2.88 (m, 4H),
4.78 (m, 2H), 7.36 (m, 2H), 7.52 (m, 2H), 7.71 (m, 4H),
8.98 (d, 2H); MS (ESI) m/z 536.
[S-(R*,R*)]-2-{2-[2-(1,3-Dicarboxy-propylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-pentanedioic acid (43).
t-Butyl ester of the title compound was prepared
according to general method B using bisacid chloride
(1.05 g, 3.0 mmol), corresponding amino acid t-butyl
ester hydrochloride (2.0 g, 6.7 mmol), triethylamine
(1.2 g) in 45 mL of dichloromethane; The crude com-
pound obtained was dissolved in 10 mL of dichloro-
methane. To this, 2.86 g of tri¯uoroacetic acid was
added and stirred at room temperature for 24 h. The
solvents were evaporated and the residue was triturated
with hexanes to obtain the title compound which was
dried in vacuo. Isolated yield: 16%; 1H NMR
(400 MHz, DMSO-d6) d 2.03 (m, 2H), 2.50 (m, 4H), 4.42
(m, 2H), 7.32 (t, 2H), 7.45 (t, 2H), 7.67 (d, 2H), 7.72 (d,
2H), 8.85 (d, 2H).
[S-(R*,R*)]-3-(4-Benzyloxycarbonylamino-phenyl)-2-(2-
{2-[2-(4-benzyloxycarbonylamino-phenyl)-1-carboxy-ethyl-
carbamoyl] - phenyldisulfanyl} - benzoylamino) - propionic
acid (40). This compound was synthesized according to
the general procedure C using bisacid chloride (1.0 g,
2.41 mmol), potassium t-butoxide (0.43 g, 1.21 mmol)
and corresponding amino acid hydrochloride (obtained
via deprotection of the corresponding amino acid
(1.0 g), where a-amine was protected as Boc derivative
[S-(R*,R*)]-2-{2-[2-((1,4-Dicarboxy-butylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-hexanedioic acid (44).
t-Butyl ester of the title compound was prepared
according to the general method B using bisacid chlor-
ide (1.12 g, 3.2 mmol), corresponding amino acid t-butyl
ester hydrochloride (2.0 g, 7.3 mmol), triethylamine
(1.2 g) in 55 mL of dichloromethane. The crude com-
pound obtained was dissolved in 10 mL of dichloro-
methane. To it tri¯uoroacetic acid was added and
stirred at room temperature for 24 h. The solvents were
evaporated and the residue was triturated with hexanes
to obtain the title compound which was dried in vacuo.
1
(2.41 mmol)]. Isolated yield: 48%; H NMR (400 MHz,
DMSO-d6) d 3.14 (dd, 4H), 4.58 (m, 2H), 5.11 (s, 4H),
7.22 (m, 6H), 7.39 (m, 16H), 7.58 (d, 2H), 7.55 (d, 2H),
8.89 (d, 2H), 9.7 (s, 2H), 12.82 (brs, 2H); IR (KBr) 3310,
1716, 1528, 1223, 745 cm 1; MS (ESI) m/z 451.
[S-(R*,R*)]-3-4-(Amino-phenyl)-2-(2-{2-[2-(4-amino-phenyl)-
1-carboxy-ethylcarbamoyl]-phenyldisulfanyl}-benzoylamino)-
propionic acid (41). The title compound was synthesized
by treating 40 (0.15 g) with 31% HBr in acetic acid
solution (4 mL) at room temperature for 20 min. The
solvents were evaporated and the residue on triturating
with ether a€orded solid, which is hygroscopic. Isolated
yield: 92%; 1H NMR (400 MHz, DMSO-d6) d 3.28
(dd, 2H), 3.36 (dd, 2H), 5.40 (m, 2H), 7.20 (d, 4H), 7.28
(d, 2H), 7.20±7.6 (m, 10H), 7.36 (d, 2H), 7.66 (t, 2H),
7.79 (d, 2H), 7.94 (d, 1H), 8.31 (brs, 1H), 8.97 (d, 2H),
9.8 (brs, 4H); IR (KBr) 3422, 2920, 1732, 1630, 1511,
741 cm 1; MS (ESI) m/z 315, 329.
1
Isolated yield: 30%; H NMR (400 MHz, DMSO-d6) d
1.64 (m, 8H), 2.54 (t, 4H), 4.36 (m, 2H), 7.30 (t, 2H),
7.45 (t, 2H), 7.67 (m, 4H), 8.84 (d, 2H); MS (ESI) m/z
296.
6-Amino-2-{2-[2-(5-amino-1-carboxy-pentylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-hexanoic acid (45). A
solution of 90 (0.51 g, 0.86 mmol) in 100 mL of 1 N HCl
was heated to 50 ꢀC for 7 h. The solution was cooled and
concentrated to give after drying 0.41 g of compound 45
as a solid. 1H NMR (300 MHz, DMSO-d6) d 12.75 (brs,

1722
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
2H), 8,86 (d, 2H), 7.88 (brs, 6H), 7.76 (d, 2H), 7.65 (d,
2H), 7.48 (t, 2H), 7.33 (t, 2H), 4.37 (m, 2H), 2.78 (m,
4H), 1.83 (m, 4H), 1.60 (m, 2H), 1.48 (m, 2H), IR (KBr)
3426, 2926, 1630 cm 1; MS (ESI) m/z 563.
2.10±1.85 (m, 4H); IR (KBr) 2976, 1740, 1601, 1426,
1179, 751 cm 1; MS (ESI) m/z 499.
4-{2-(3-Carboxy-propylcarbamoyl)-phenyldilsulfanyl]-
benzoylamino}-butyric acid (53). t-Butyl ester of the title
compound was prepared according to the general
method B using bisacid chloride (1.96 g, 5.7 mmol), cor-
responding amino acid t-butyl ester hydrochloride
(2.0 g, 12.5 mmol), triethylamine (1.25 mL, 12.0 mmol)
in 85 mL of dichloromethane. The crude compound
obtained was dissolved in 10 mL of dichloromethane.
To it tri¯uoroacetic acid was added and stirred at room
temperature for 24 h. The solvents were evaporated and
the residue was triturated with hexanes to obtain the
title compound which was dried in vacuo. Isolated
yield: 23%; 1H NMR (300 MHz, DMSO-d6) d 1.75
(m, 4H), 2.30 (m, 4H), 3.26 (m, 4H), 7.27 (t, 2H), 7.42
(t, 2H), 7.59 (m, 4H), 8.61 (m, 2H); MS (ESI) m/z
238.
(S-R*,R*)]-2-{2-[2-(2-Carbamoyl-1-carboxy-ethylcar-
bamoyl)-phenyldidsulfanyl]-benzoylamino}-succinamic acid
(48). This compound was prepared from 99 (1.31 g,
2.0 mmol) using 15 mL of dichloromethane and 15 mL
of tri¯uoroacetic acid. The sample was concentrated
and the residue was triturated with ethanol and 1 N HCl
to yield 0.74 g of ®nal product as a solid, mp 208±
211 ꢀC. 1H NMR (DMSO-d6) d 12.74 (brs, 2H), 8.85
(d, 2H), 7.67 (dd, 4H), 7.48 (t, 2H), 7.43 (brs, 2H), 7.31
(t, 2H), 6.98 (brs, 2H), 4.72 (q, 2H), 2.72 (dd, 2H), 2.60
(dd, 2H); IR (KBr) 3270, 1724, 1645, 748 cm 1; MS
(ESI) m/z 533.
(S-R*,R*)]-4-Carbamoyl-2-{2-[2-(3-carbamoyl-1-carboxy-
propylcarbamoyl)-phenyldisulfanyl]-benzoylamino}-butyric
acid (49). This compound was prepared from 100
(1.05 g, 1.6 mmol) using 15 mL of dichloromethane and
15 mL of tri¯uoroacetic acid. The residue was triturated
with ethanol and 1 N HCl to yield 0.74 g of ®nal
product as a solid. 1H NMR (DMSO-d6) d 12.65 (bs,
2H), 8.88 (d, 2H), 7.69 (d, 2H), 7.60 (d, 2H), 7.42 (t,
2H), 7.31 (bs, 2H), 7.26 (t, 2H), 6.80 (brs, 2H), 4.31
(m, 2H), 2.20 (t, 4H), 2.02 (m, 2H), 1.88 (m, 2H); IR
(KBr) 3264, 1718, 1643, 1538, 747 cm 1; MS (APCI) m/z
281.
6-{2-[2-(5-Carboxy-pentylcarbamoyl)-phenyldisulfanyl]-
benzoylamino}-hexanoic acid (56). This compound was
synthesized according to the general procedure C using
bisacid chloride (1.10 g, 3.0 mmol), sodium ethoxide
(prepared from 0.30 g of sodium and 40 mL of ethanol)
and corresponding amino acid (3.0 g, 20 mmol). Isolated
yield: 43%; 1H NMR (300 MHz, DMSO-d6) d 1.30
(m, 8H), 1.48 (m, 8H), 2.18 (t, 4H), 3.24 (m, 4H), 7.27
(t, 2H), 7.39 (t, 2H), 7.60 (m, 4H), 8.57 (m, 2H); MS
(ESI) m/z 280.
[S-(R*,R*)]-2-{2-[2-(1-Carboxy-3-methyl-butyl)-methyl-
carbamoyl]-phenyldisulfanyl}-benzoyl)-methyl-amino]-4-
methyl-pentanoic acid (50). t-Butyl ester of the title
compound was prepared according to the general
method B using bisacid chloride (1.02 g, 2.98 mmol),
corresponding amino acid t-butyl ester hydrochloride
(1.5 g, 7.5 mmol), triethylamine (1.15 mL, 8.25 mmol) in
40 mL of dichloromethane. The crude compound
obtained was dissolved in 10 mL of dichloromethane.
To it tri¯uoroacetic acid was added and stirred at room
temperature for 24 h. The solvents were evaporated and
the residue was triturated with hexanes to obtain the
title compound, which was dried in vacuo. Isolated
(‹)3-{2-(2-Carboxy-1-methyl-ethylcarbamoyl)-phenyldi-
sulfanyl]-benzoylamino}-butyric acid (57). This com-
pound was synthesized according to the general
procedure C using bisacid chloride (1.1 g, 3.0 mmol),
potassium t-butoxide (1.35 g, 1.2 mmol) and corre-
sponding amino acid (2.0 g, 19.3 mmol) and 20 mL of
ethanol. Isolated yield: 37%; 1H NMR (300 MHz,
DMSO-d6) d 1.19 (d, 6H), 2.43 (m, 4H), 4.29 (m, 2H),
7.26 (m, 2H), 7.35 (m, 2H), 7.59 (m, 4H), 8.52 (m, 2H);
MS (ESI) m/z 487.
[S-(R*,R*)]-(3-{2-[2-(1-Carboxymethyl-butylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-hexanoic acid) (58).
The title compound was synthesized according to the
general method A using corresponding b-amino acid
hydrochloride (0.79 g, 2.75 mmol), N-methylmorpholine
(1.0 g (1.08 mL), 9.85 mmol), bisacid chloride (0.70 g,
2.04mmol), N-methyl-N-(trimethylsilyl)acetamide (1.43 g
(1.58 mL), 9.85 mmol), isolated yield: 46%; 1H NMR
(400 MHz, DMSO-d6) d 0.90 (t, 6H), 1.39 (m, 4H), 1.51
(m, 4H), 2.49 (m, 4H), 4.29 (m, 2H), 7.30 (t, 2H), 7.43
(t, 2H), 7.56 (d, 2H), 7.64 ( d, 2H), 8.46 (d, 2H), 11.4 ( br
s, 2H); IR (KBr) 3280, 3060, 2958, 2932, 2872, 1700,
1637, 1587, 1538, 1462, 1432, 1321, 1227, 1185, 1037,
915, 873, 744, 696. 652 cm 1; MS (ESI) m/z 266.3.
1
yield: 23%; H NMR (300 MHz, DMSO-d6) d 0.8 (m,
12H), 1.48 (m, 4H), 1.73 (m, 2H), 2.36 (s, 6H), 7.45 (t,
2H), 5.02 (m, 2H), 7.20 (m, 6H), 7.75 (m, 2H); MS (ESI)
m/z 282.
[S-(R*,R*)]-(1H-Pyrrolidine-2-carboxylic acid, 1,10-[dithio-
bis(2,1-phenylenecarbonyl)]bis-, (51). The title com-
pound was synthesized from 83 (1.36 g, 2.2 mmol) using
15 mL tri¯uoroacetic acid in 15 mL dichloromethane to
1
a€ord 0.78 g of the title compound as a solid. H NMR
(300 MHz, CDCl3) d 7.71 (d, 2H), 7.43±7.20 (m, 6H),
4.76 (m, 2H), 3.28 (m, 4H), 2.39 (m, 2H), 2.18 (m, 2H),

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1723
[S-(R*,R*)]-(3-{2-[2-(1-Carboxymethyl-pentylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-heptanoic acid) (59).
The title compound was synthesized according to the
general method A using corresponding b-amino acid
hydrochloride (0.95 g, 5.23 mmol), N-methylmorpholine
(1.11 g (1.21 mL), 10.98 mmol), bisacid chloride (0.78 g,
2.27 mmol), N-methyl-N-(trimethylsilyl)acetamide (1.60 g
2H), 1.73 (m, 8H), 2.41±2.58 (m, 4H), 4.19 (m, 2H), 7.29
(t, 2H), 7.41 (t, 2H), 7.55 (d, 2H), 7.64 (d, 2H), 8.41
(d, 2H), 12.17 (brs, 2H); IR (KBr) 3281, 3061, 2927,
2852, 1670, 1635, 1587, 1538, 1447, 1432, 1416, 1323,
1284, 743 cm 1; MS (ESI) m/z 306.5.
[S-(R*,R*)]-(3-{2-[2-(1-Carboxymethyl-3-methyl-butyl-
carbamoyl)-phenyldisulfanyl]-benzoylamino}-5-methyl-
hexanoic acid) (63). The title compound was synthesized
according to the general method A using corresponding
b-amino acid hydrochloride (0.50 g, 2.75 mmol),
N-methylmorpholine (0.58 g (0.64 mL), 5.78 mmol) bis-
acid chloride (0.45 g, 1.31 mmol), N-methyl-N-tri-
methylsilyl)acetamide (0.84 g (0.93 mL), 5.78 mmol).
Isolated yield: 29.9%; 1H NMR (400 MHz, DMSO-d6) d
0.91 (m, 12H), 1.29 (m, 2H), 1.55 (m, 2H), 1.70 (m, 2H),
2.48 (m, 4H), 4.37 (m, 2H), 7.30 (t, 2H), 7.42 (t, 2H), 7.55
(d, 2H), 7.64 (d, 2H), 8.43 (d, 2H), 12.21 (brs, 2H); IR
(KBr) 3283, 3060, 2956, 2932, 2870, 1712, 1633, 1536,
1463, 1434, 1313, 1283 cm 1; MS (ESI) m/z 280.4.
1
(1.76 mL), 10.98 mmol). Isolated yield: 67%; H NMR
(400 MHz, DMSO-d6) d 0.86 (t, 6H), 1.34 (m, 8H), 1.55
(m, 4H), 2.47 (m, 4H), 4.28 (m, 2H), 7.30 (t, 2H), 7.43
(t, 2H), 7.56 (d, 2H), 7.64 ( d, 2H), 8.45 (d, 2H), 12.22
(s, 2H); IR (KBr) 3278, 3060, 3028, 2957, 2930, 2860,
1701, 1636, 1587, 1537, 1459, 1433, 1413, 1321, 1288,
1251, 1227, 1183, 1138, 1037, 913, 872, 747, 697,
652 cm 1; MS (ESI) m/z 280.3.
[S-(R*,R*)]-(3-{2-[2-(1-Carboxymethyl-2-methyl-propyl-
carbamoyl)-phenyldisulfanyl] - benzoylamino} -4 -methyl-
pentanoic acid) (60). The title compound was synthe-
sized according to the general method
A using
corresponding b-amino acid hydrochloride (0.72 g,
4.29 mmol), N-methylmorpholine (0.91 g (0.99 mL),
9.02 mmol), bisacid chloride (0.64 g, 1.87 mmol),
N-methyl-N-(trimethylsilyl)acetamide (1.31 g (1.45 mL),
9.02 mmol). Isolated yield: 27%; 1H NMR (400 MHz,
DMSO-d6) d 0.91 (m, 12H), 1.85 (m, 2H), 2.41±2.56 (m,
4H), 4.20 (m, 2H), 7.30 (t, 2H), 7.43 (t, 2H), 7.55 (d, 2H),
7.64 ( d, 2H), 8.42 (d, 2H), 12.19 (s, 2H); IR (KBr) 3286,
3062, 2963, 2935, 2874, 1705, 1635, 1587, 1536, 1462, 1432,
1409, 1322, 1284, 1184, 745 cm 1; MS (ESI) m/z 266.4.
[S-(R*,R*)]-(3-{2-[2-(1-Carboxymethyl-3,3-dimethyl-butyl-
carbamoyl)phenyldisulfanyl]-benzoylamino}-5,5-dimethyl-
hexanoic acid) (64). The title compound was synthesized
according to the general method A using correspond-
ing b-amino acid hydrochloride (0.88 g, 4.50 mmol),
N-methylmorpholine (0.96 g (1.04 mL), 9.44 mmol),
bisacid chloride (0.67 g, 1.96 mmol), N-methyl-(N-tri-
methylsilyl) acetamide (1.37 g (1.52 mL), 9.44 mmol) to
yield 0.95 g of beige solid. Isolated yield: 82%; 1H NMR
(400 MHz, DMSO-d6) g 0.95 (s, 18H), 1.35 (d, 2H), 1.66
(dd, 2H), 2.37±2.52 (m, 4H), 4.43 (m, 2H), 7.29 (t, 2H),
7.40 (t, 2H), 7.54 (d, 2H), 7.62 (d, 2H), 8.48 (d, 2H),
12.22 (s, 2H); IR (KBr) 3299, 3062, 2955, 2867, 1712,
1632, 1530, 1465, 1433, 1366, 1322, 1285, 1252, 1202,
747 cm 1; MS (ESI) m/z 294.5.
[S-(R*,R*)]-(3-{2-[2-(1-Carboxymethyl-2-methyl-butyl-
carbamoyl)-phenyldisulfanyl] - benzoylamino} -4 -methyl-
hexanoic acid) (61). The title compound was synthe-
sized according to the general method A using corre-
sponding b-amino acid hydrochloride (0.71 g,
3.91 mmol), N-methylmorpholine (0.83 g (0.9 mL),
8.19 mmol), bisacid chloride (0.58 g, 1.70 mmol) and
N-methyl-N-(trimethylsilyl)acetamide (1.19 g (1.31 mL),
8.19 mmol). Isolated yield: 98%; 1H NMR (300 MHz,
DMSO-d6) d 0.89 (m, 12H), 1.18 (m, 2H), 1.48 (m, 2H),
1.64 (m, 2H), 2.46 (m, 4H), 4.26 (m, 2H), 7.29 (t, 2H),
7.42 (t, 2H), 7.54 (d, 2H), 7.64 (d, 2H), 8.46 (d, 2H),
12.13 (br s, 2H); IR (KBr) 3286, 2965, 1702, 1635, 1587,
1538, 1432 cm 1; MS (APCI) m/z 280.5.
[S-(R*,R*)]-(3-{2-[2-(1-Carboxymethyl-2-cyclohexyl-ethyl-
carbamoyl)-phenyldisulfanyl]-benzoylamino}-4-cyclohexyl-
butyric acid) (65). The title compound was synthesized
according to the general method A using correspond-
ing b-amino acid hydrochloride (0.87 g, 3.92 mmol),
N-methylmorpholine (0.83 g (0.91 mL), 8.24 mmol)
bisacid chloride (0.59 g, 1.71 mmol), N-methyl-N-(tri-
methylsilyl)acetamide (1.20 g (1.32 mL), 8.24 mmol).
1
Isolated yield 40%; H NMR (400 MHz, DMSO-d6) d
[S-(R*,R*)]-(3-{2-[2-(2-Carboxy-2-cyclohexyl-ethylcar-
bamoyl)-phenyldisulfanyl]-benzoylamino}-3-cyclohexyl-
propionic acid) (62). The title compound was synthe-
sized according to the general method A using corre-
sponding b-amino acid hydrochloride (0.55 g,
2.65 mmol), N-methylmorpholine (0.56 g (0.61 mL),
5.56 mmol), bisacid chloride (0.45 g, 1.15 mmol),
N-methyl-N-(trimethylsilyl)acetamide (0.81 g (0.89 mL),
5.56 mmol). Isolated yield: 80%; 1H NMR (400 MHz,
DMSO-d6) (0.99±1.16 (m, 10H), 1.49 (m, 2H), 1.61 (m,
0.83 (m, 2H), 0.95 (m, 2H), 1.14 (m, 6H), 1.41 (m, 6H),
1.60 (m, 8H), 1.95 (br d, 2H), 2.46 (m, 4H), 4.42 (m, 2H),
7.30 (t, 2H), 7.42 (t, 2H), 7.52 (d, 2H), 7.63 (d, 2H), 8.42
(d, 2H), 12.21 (br s, 2H); IR (KBr) 3422, 3368, 3339,
3312, 2922, 2850, 1712, 1631, 1587, 1529, 1447, 1434,
1319, 1283 cm 1; MS (ESI) m/z 320.5.
(‹)-3-{2-[2-(2-Carboxy-1-phenyl-ethylcarbamoyl)-phenyl-
disulfanyl]-benzoylamino}-3-phenyl-propionic acid (66).
This compound was synthesized according to the general

1724
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
procedure C using bisacid chloride (0.7 g, 2.0 mmol),
potassium t-butoxide (0.7g, 8.5 mmol) and corresponding
amino acid (2.0 g, 12.1 mmol) and 20 mL of ethanol.
Isolated yield: 37%; H NMR (300 MHz, DMSO-d6) d
2.77 (m, 4H), 5.39 (m, 2H), 7.16±7.93 (m, 18H), 9.10 (d,
2H), 12.32 (s, 2H); MS (ESI) m/z 599.
method A using corresponding g-amino acid hydro-
chloride (0.96 g, 4.90 mmol), N-methylmorpholine
(1.04 g (1.14 mL), 10.29 mmol) bisacid chloride (0.73 g,
2.13 mmol), N-methyl-N-trimethylsilyl)acetamide (1.49 g
1
1
(1.65 mL), 10.29 mmol). Isolated yield: 32%; H NMR
(400 MHz, DMSO-d6) d 0.87 (t, 6H), 1.33 (m, 8H), 1.50
(m, 4H), 1.67 (m, 2H), 1.79 (m, 2H), 2.31 (t, 4H), 3.94
(m, 2H), 7.30 (t, 2H), 7.42 (t, 2H), 7.57 (d, 2H), 7.65 (d,
2H), 8.31 (d, 2H), 12.05 (s, 2H); IR (KBr) 3281, 3064,
2956, 2930, 2839, 1706, 1631, 1587, 1537, 1450, 1434,
1316, 1288, 1258, 1173, 1091, 745, 698, 652 cm 1; MS
(ESI) m/z 294.4.
[S-(R*,R*)]-(3-{2-[2-(1-Carboxymethyl-2-phenyl-ethyl-
carbamoyl)-phenyldisulfanyl]-benzoylamino} -4-phenyl-
butyric acid) (67). The title compound was synthesized
according to the general method A using correspond-
ing b-amino acid hydrochloride (1.00 g, 4.64 mmol),
N-methylmorpholine (0.99 g (1.07 mL), 9.74 mmol)
bisacid chloride (0.70 g, 2.02 mmol), N-methyl-N-(tri-
methylsilyl)acetamide (1.42 g (1.56 mL), 9.74 mmol).
[S-(R*,R*)]-(4-(2-{2-[1-(2-Carboxy-ethyl)-2-methyl-propyl-
carbamoyl]-phenyldisulfanyl}-benzoylamino)-5-methyl-
hexanoic acid) (71). The title compound was synthesized
according to the general method A using correspond-
ing g-amino acid hydrochloride (1.68 g, 9.25 mmol), N-
methylmorpholine (1.96 g (2.14 mL), 19.42 mmol), bis-
acid chloride (1.38 g, 4.02 mmol), N-methyl-N-(tri-
methylsilyl)acetamide (2.82 g (3.12 mL), 19.42 mmol).
1
Isolated yield: 59%; H NMR (400 MHz, DMSO-d6) d
2.53 (m, 4H), 2.89 (d, 2H), 4.47 (m, 2H), 7.19 (m, 2H),
7.27 (m, 10H), 7.40 (t, 2H), 7.47 (d, 2H), 7.55 (d, 2H), 8.57
(d, 2H), 12.29 (s, 2H); IR (KBr) 3293, 3091, 3028, 2923,
1710, 1637, 1586, 1528, 1433, 1404, 1286, 1258, 1204, 1162,
1082, 1035, 907, 869, 747, 701 cm 1; MS (ESI) m/z 314.4.
1
Isolated yield: 61%; H NMR (400 MHz, DMSO-d6) d
[S-(R*,R*)]-(7-Benzyloxycarbonylamino-3-{2-[2-(5-benzyl-
oxycarbonylamino - 1 - carboxymethyl - pentylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-heptanoic acid) (68).
The title compound was synthesized according to the
general method A using corresponding b-amino acid
hydrochloride (1.31 g, 3.96 mmol), N-methylmorpholine
(0.84 g (0.91 mL), 8.32 mmol) bisacid chloride (0.59 g,
1.72 mmol), N-methyl-N-(trimethylsilyl)acetamide (1.21 g
(1.34 mL), 8.32 mmol). Isolated yield: 74%; 1H NMR
(400 MHz, DMSO-d6) d 1.41 (m, 10H), 1.54 (m, 2H),
2.46 (m, 4H), 2.98 (m, 2H), 4.25 (m, 2H), 4.99 (s, 4H),
7.24±7.37 (m, 12H), 7.43 (t, 2H), 7.57 (d, 2H), 7.64
(d, 2H), 8.44 (d, 2H), 12.24 (s, 2H); IR (KBr) 3332,
3063, 2931, 2861, 1705, 1637, 1534, 1455, 1434, 1311,
1258, 1140, 1029, 746, 698 cm 1; MS (ESI) m/z 429.4.
0.93 (t, 12H), 1.65 (m, 2H), 1.81 (m, 4H), 2.32 (m, 4H),
3.78 (m, 2H), 7.30 (t, 2H), 7.42 (t, 2H), 7.57 (d, 2H),
7.67 (d, 2H), 8.28 (d, 2H), 12.08 (s, 2H); IR (KBr) 3285,
3062, 2963, 2932, 2874, 1702, 1632, 1587, 1537, 1446,
1434, 1318, 1304, 1262, 742, 696 cm 1; MS (ESI) m/z
280.4.
[S-(R*,R*)]-(4-(2-{2-[1-(2-Carboxy-ethyl)-2-methyl-butyl-
carbamoyl]-phenyldisulfanyl}-benzoylamino)-5-methyl-
heptanoic acid) (72). The title compound was synthe-
sized using general method A using corresponding
g-amino acid hydrochloride (1.40 g, 7.15 mmol),
N-methylmorpholine (1.52 g (1.65 mL), 15.02 mmol),
bisacid chloride (1.07 g, 3.11 mmol), N-methyl-N-(tri-
methylsilyl)acetamide (2.18 g (2.42 mL), 15.02 mmol).
1
Isolated yield: 77%; H NMR (400 MHz, DMSO-d6) d
[S-(R*,R*)]-(4-(2-{2-[1-(2-Carboxy-ethyl)-butylcarbamoyl]-
phenyldisulfanyl}-benzoylamino)-heptanoic acid) (69).
The title compound was synthesized according to the
general method A using corresponding g-amino acid
hydrochloride (0.89 g, 4.90 mmol), N-methylmorpholine
(1.04 g (1.14 mL), 10.29 mmol), bisacid chloride (0.73 g,
2.13 mmol), N-methyl-N-(trimethylsilyl)acetamide (1.49 g
0.91 (s, 12H), 1.18 (m, 2H), 1.49 (m, 2H), 1.61 (m, 4H),
1.81 (m, 2H), 2.29 (m, 4H), 3.84 (m, 2H), 7.30 (t, 2H),
7.42 (t, 2H), 7.56 (d, 2H), 7.66 (d, 2H), 8.32 (d, 2H),
12.06 (s, 2H); IR (KBr) 3277, 3062, 2964, 2932, 2876,
1702, 1632, 1538, 1457, 1434, 1302, 746, 697 cm 1; MS
(ESI) m/z 294.4.
1
(1.65 mL), 10.29 mmol). Isolated yield: 59%; H NMR
[R-(R*,R*)]-(4-{2-[2-(3-Carboxy-1-cyclohexyl-propylcar-
bamoyl)-phenyldisulfanyl]-benzoylamino}-4-cyclohexyl-
butyric acid) (73). The title compound was synthesized
according to the general method A using corresponding
g-amino acid hydrochloride (0.38 g, 1.71 mmol), N-
methylmorpholine (0.36 g (0.40 mL), 3.60 mmol), bisacid
chloride (0.26 g, 0.75 mmol), N-methyl-N-(trimethylsilyl)-
acetamide (0.52 g (0.58 mL), 3.60 mmol). Isolated yield:
(400 MHz, DMSO-d6) d 0.90 (t, 6H), 1.38 (m, 4H), 1.48
(m, 4H), 1.67 (m, 2H), 1.81 (m, 2H), 2.31 (t, 4H), 3.96
(m,2H), 7.31 (t, 2H), 7.42 (t, 2H), 7.57 (d, 2H), 7.67
(d, 2H), 8.31 (d, 2H), 12.05 (s, 2H); IR (KBr) 3277, 3172,
2957, 2932, 2871, 1708, 1632, 1536, 1450, 1433, 1317, 1259,
1181, 1092, 1037, 744, 699 cm 1; MS (ESI) m/z 280.3.
1
[S-(R*,R*)]-(4-(2-{2-[1-(2-Carboxy-ethyl)-pentylcarbamoyl]-
phenyldisulfanyl}-benzoylamino)-octanoic acid) (70). The
title compound was synthesized according to the general
23%; H NMR (400 MHz, DMSO-d6) d 1.00±1.28 (m,
10H), 1.44 (m, 2H), 1.60±1.89 (m, 14H), 2.28 (m, 4H),
3.79 (m, 2H), 7.29 (t, 2H), 7.41 (t, 2H), 7.56 (d, 2H),

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1725
7.66 (d, 2H), 8.27 (d, 2H), 12.05 (s, 2H); IR (KBr) 3306,
3060, 2924, 2851, 1706, 1631, 1587, 1536, 1449, 1433,
1326, 1303, 1288, 1260, 955, 889, 744, 697 cm 1; MS
(CI) m/z 320.
according to the general method A using corresponding
unsaturated g-amino acid hydrochloride (0.58 g,
3.12 mmol), N-methylmorpholine (0.66 g (0.72 mL),
6.55 mmol), bisacid chloride (0.47 g, 1.36 mmol),
N-methyl-N-(trimethylsilyl)acetamide (0.95 g (1.05 mL),
6.55 mmol). Isolated yield: 28%; 1H NMR
(400 MHz, DMSO-d6) (0.95 (m, 12H), 1.95 (m, 2H),
4.50 (m, 2H), 5.95 (d, 2H), 6.86 (dd, 2H), 7.33 (t, 2H),
7.46 (t, 2H), 7.62 (d, 2H), 7.65 (d, 2H), 8.73 (d, 2H),
12.36 (s, 2H); IR (KBr) 3415, 3384, 3314, 3298, 3060,
2963, 2928, 1706, 1640, 1587, 1529, 1461, 1316, 1257,
1178, 983, 747, 698 cm 1; MS (ESI) m/z 278.4.
[R-(R*,R*)]-(4-{2-[2-(3-Carboxy-1-cyclohexylmethyl-
propylcarbamoyl)-phenyldisulfanyl]-benzoylamino}-5-cyclo-
hexyl-pentanoic acid) (74). The title compound was
synthesized according to the general method A using
corresponding g-amino acid hydrochloride (1.42 g,
6.02 mmol), N-methylmorpholine (1.27 g (1.4 mL),
12.65 mmol), bisacid chloride (0.90 g, 2.62 mmol),
N-methyl-N-(trimethylsilyl)acetamide (1.84 g (2.03 mL),
12.65 mmol). Isolated yield: 83%; 1H NMR
(400 MHz, DMSO-d6) d 0.84 (m, 2H), 0.95 (m, 2H), 1.14
(m, 6H), 1.30 (m, 2H), 1.42 (m, 4H), 1.63 (m, 10H), 1.75
(m, 2H), 1.91 (m, 2H), 2.30 (t, 4H), 4.08 (m, 2H), 7.30
(t, 2H), 7.41 (t, 2H), 7.54 (d, 2H), 7.65 (d, 2H), 8.30
(d, 2H), 12.04 (s, 2H); IR (KBr) 3306, 3060, 2924, 2851,
1706, 1631, 1587, 1536, 1449, 1433, 1326, 1303, 1288,
1260, 955, 889, 744, 697 cm 1; MS (ESI) m/z 334.6.
[S-(R*,R*)]-(EE)-(4-{2-[2-(1-sec-Butyl-3-carboxy-allyl-
carbamoyl)-phenyldisulfanyl]-benzoylamino}-5-methyl-hept-
2-enoic acid) (78). The title compound was synthesized
according to the general method A using corresponding
unsaturated g-amino acid hydrochloride (0.96 g,
4.96 mmol), N-methylmorpholine (1.05 g (1.14 mL),
10.41 mmol), bisacid chloride (0.74 g, 2.16 mmol), N-
methyl-N-(trimethylsilyl)acetamide (1.51 g (1.67 mL),
10.41 mmol) to yield 0.22 g of beige solid. Isolated yield:
1
[R-(R*,R*)]-(4-{2-[2-(1-Benzyl-3-carboxy-propylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-5-phenyl-pentanoic acid)
(75). The title compound was synthesized according to
the general method A using corresponding g-amino acid
hydrochloride (0.60 g, 2.61 mmol), N-methylmorpholine
(0.55 g (.60 mL), 5.49 mmol) bisacid chloride (0.39 g,
1.14 mmol), N-methyl-N-(trimethylsilyl)acetamide (0.80 g
(0.88 mL), 5.49 mmol). Isolated yield: 33%; 1H NMR
(400 MHz, DMSO-d6) d 1.74 (m, 2H), 1.84 (m, 2H), 2.35
(m, 4H), 2.85 (d, 4H), 4.19 (m, 2H), 7.19 (m, 2H), 7.27
(m, 10H), 7.39 (t, 2H), 7.44 (d, 2H), 7.56 (d, 2H), 8.43
(d, 2H), 12.06 (s, 2H); IR (KBr) 3300, 3062, 3028, 2924,
1710, 1633, 1586, 1534, 1453, 1446, 1434, 1323, 1308,
1299, 1257, 1160, 1086, 1036, 944, 747, 699 cm 1; MS
(CI) m/z 328.4.
18%; H NMR (400 MHz, DMSO-d6) d 0.91 (m, 12H),
1.21 (m, 2H), 1.51 (m, 2H), 1.75 (m, 2H), 4.57 (m, 2H),
5.95 (d, 2H), 6.85 (dd, 2H), 7.31 (t, 2H), 7.46 (t, 2H),
7.62 (d, 2H), 7.66 (d, 2H), 8.75 (d, 2H), 12.34 (s, 2H); IR
(KBr) 3426, 3343, 3314, 3300, 3060, 3026, 2964, 2928,
2877, 1704, 1638, 1587, 1530, 1454, 1383, 1307, 1285,
1259, 1166, 982, 748, 698 cm 1; MS (ESI) m/z 306.5.
[S-(R*,R*)]-(EE)-(4-{2-[2-(3-Carboxy-1-isobutyl-allyl-
carbamoyl)-phenyldisulfanyl]-benzoylamino}-6-methyl-hept-
2-enoic acid (79). The title compound was synthesized
according to the general method A using correspond-
ing b-amino acid hydrochloride (0.72 g, 4.29 mmol),
N-methylmorpholine (0.91 g (0.99 mL), 9.02 mmol),
bisacid chloride (0.64 g, 1.87 mmol), N-methyl-N-(tri-
methylsilyl)acetamide (1.31 g (1.45 mL), 9.02 mmol).
1
[S-(R*,R*)]-4-(2-{2-[1-(2-Carboxy-ethyl)-3-methyl-butyl-
carbamoyl] -phenyldisulfanyl}-benzoylamino)-6-methyl-
heptanoic acid (76). The title compound was synthe-
sized according to the general method A using bisacid
chloride (0.22 g, 0.64 mmol), N-methyl-N-(trimethylsilyl)
acetamide (1.5 g, 10.24 mmol), corresponding amino
acid (0.5 g, 2.561 mmol), N-methylmorpholine (1.04 g,
10.24 mmol) and 10 mL of dichloromethane. Isolated
yield: 48%; 1H NMR (400 MHz, DMSO-d6) d 0.89
(d, 6H), 0.92 (d, 6H), 1.26 (m, 2H), 1.5 (m, 2H), 1.58±
1.86 (m, 8H), 2.31 (t, 4H), 4.06 (m, 2H), 7.29 (d, 2H),
7.42 (d, 2H), 7.55 (d, 2H), 7.66 (d, 2H), 8.32 (d, 2H),
12.07 (s, 2H); IR (KBr) 3276, 2956, 1711, 1629, 1538,
744 cm 1; MS (ESI) m/z 282.
Isolated yield : 27%; H NMR (400 MHz, DMSO-d6) d
0.92 (m, 12H), 1.44 (m, 2H) 1.58 (m, 2H), 1.69 (m, 2H),
4.72 (m, 2H), 5.89 (d, 2H), 6.80 (dd, 2H), 7.31 (t, 2H),
7.44 (t, 2H), 7.64 (d, 2H), 7.66 ( d, 2H), 8.75 (d, 2H),
12.19 (s, 2H); IR (KBr) 3274, 2957, 1702, 1632, 1534,
1284, 745 cm 1; MS (ESI) m/z 292, 324.
[S-(R*,R*)]-2-(2-{2-[1-(1-Methoxycarbonyl-3-methyl-butyl-
carbamoyl]-phenyldisulfanyl}-benzoylamino)-4-methyl-
pentanoic acid methyl ester (80). The title compound
was synthesized according to the general method A
using bisacid chloride (2.07 g, 6.04 mmol), N-methyl-N-
(trimethylsilyl)acetamide (7.0 g, 48.34 mmol), corre-
sponding amino acid methyl ester hydrochloride (2.0 g,
12.08 mmol), N-methylmorpholine (4.89 g, 48.34 mmol)
1
[S-(R*,R*)]-(EE)-(4-{2-[2-(3-Carboxy-1-isopropyl-allyl-
carbamoyl)-phenyldisulfanyl]benzoylamino}-5-methyl-hex-
2-enoic acid) (77). The title compound was synthesized
and 10 mL of dichloromethane. Isolated yield: 53%; H
NMR (400 MHz, DMSO-d6) d 0.92 (d, 6H), 0.94 (d, 6H),
1.58 (m, 2H), 1.76 (m, 4H), 3.66 (s, 6H), 4.54 (m, 2H),

1726
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
7.33 (d, 2H), 7.47 (d, 2H), 7.66 (d, 2H), 7.69 (d, 2H),
8.97 (d, 2H); IR (KBr) 3320, 2956, 1744, 1637, 1532,
747 cm 1; MS (ESI) m/z 280.
(27.23 g (29.6 mL), 269.19 mmol) to yield 62.9 g (83.7%)
of white solid (diastereomeric mixture). 1H NMR
(400 MHz, DMSO-d6) d 0.89 (t, 6H), 0.96 (m, 6H), 1.21±
1.38 (m, 2H), 1.44 (s, 18H), 1.40±1.61 (m, 2H), 1.85±2.03
(m, 2H), 4.24 and 4.42 (t and dd, 2H), 7.32 (t, 2H), 7.45
(t, 2H), 7.64 (m, 4H), 8.70 and 8.77 (d and d, 2H); IR
(KBr) 3322, 3060, 2969, 2933, 2876, 1729, 1647, 1587,
1523, 1431, 1368, 1351, 1301, 1282, 1155, 1038, 847,
745 cm 1; MS (APCI) m/z 266.3.
[S-(R*,R*)]-2-(2-{2-[1-(1-t-Butoxycarbonyl-3-methyl-butyl-
carbamoyl]-phenyldisulfanyl}-benzoylamino)-4-methyl-
pentanoic acid t-butyl ester (81). The title compound
was synthesized according to the general method A
using bisacid chloride (1.23 g, 3.59 mmol), N-methyl-N-
(trimethylsilyl)acetamide (2.6 g, 17.94 mmol), corre-
sponding amino acid methyl ester hydrochloride (2.0 g,
8.97 mmol), N-methylmorpholine (1.81 g, 17.94 mmol)
(+, )2-(2-{2-[(Bismethoxycarbopnyl-methyl)-carbamoyl]-
phenyldisulfanyl}-benzoylamino)malonic acid dimethyl
ester (85). The title compound was synthesized accord-
ing to the general method A using bisacid chloride
(1.03 g, 3.09 mmol), N-methyl-N-(trimethylsilyl)acet-
amide (1.8 g, 12.39 mmol), corresponding amine (2.25 g,
12.39 mmol), N-methylmorpholine (1.25 g, 12.39 mmol)
1
and 40 mL of dichloromethane. Isolated yield: 70%; H
NMR (400 MHz, DMSO-d6) d 0.92 (d, 6H), 0.94 (d, 6H),
1.44 (s, 18H), 1.55 (m, 2H), 1.75 (m, 4H), 4.36 (m, 2H),
7.33 (d, 2H), 7.47 (d, 2H), 7.64 (s, 2H), 7.65 (s, 2H), 8.87
(d, 2H); IR (KBr) 3314, 2958, 1729, 1636, 1530, 1156,
745 cm 1; MS (ESI) m/z 266, 645.
1
and 25 mL of dichloromethane. Isolated yield: 45%; H
NMR (400 MHz, DMSO-d6) d 3.75 (s, 12H), 7.36 (t,
2H), 7.42 (d, 2H), 7.5 (t, 2H), 7.97 (d, 2H), 9.31 (s, 2H);
IR (KBr) 3164, 3061, 2890, 1749, 1683, 1445, 1278,
743 cm 1; MS (ESI) m/z 282.3, 563.4.
[S-(R*,R*)]-2-(2-{2-[1-(1-Allyloxycarbonyl-3-methyl-butyl-
carbamoyl]-phenylsidusulfanyl}-benzoylamino)-4-methyl-
pentanoic acid allyl ester (82). The title compound was
synthesized according to the general method A using
bisacid chloride (0.8 g, 2.33 mmol), N-methyl-N-(tri-
methylsilyl)acetamide (1.7 g, 11.66 mmol), correspond-
ing amino acid allyl ester p-toluenesulfonate (2.0 g,
5.83 mmol), N-methylmorpholine (1.18 g, 11.66 mmol)
[S-(R*,R*)])(1H-Pyrrolidine-2-carboxylic acid, 1,1(-[di-
thiobis(2,1-phenylenecarbonyl)]bis,bis(1,1-dimethylethyl)
ester (86). This compound was prepared according to
the general method B using bisacid chloride (1.0 g,
3 mmol) in 15 mL of dichloromethane and proline-t-
butyl ester (1.08 g, 6 mmol), N-methylmorpholine
(0.99 mL, 9 mmol) in 10 mL of dichloromethane. The
compound was puri®ed by chromatography (SiO2,
CHCl3/MeOH, 98/2) to a€ord 1.59 g of the title com-
pound as a solid. 1H NMR (300 MHz, CDCl3) d 7.70
(m, 2H), 7.33 (m, 4H), 7.24 (m, 2H), 4.57 (m, 2H), 3.83±
3.31 (m, 4H), 2.99 (m, 2H), 1.99 (m, 6H), 1.51 (s, 18H);
IR (KBr) 2976, 1736, 1637, 1414, 1152 cm 1; MS
(APCI) m/z 613.
1
and 40 mL of dichloromethane. Isolated yield: 70%; H
NMR (400 MHz, DMSO-d6) d 0.89 (d, 6H), 0.94 (d,
6H), 1.61 (m, 2H), 1.76 (m, 4H), 4.37 (m, 2H), 4.61 (m,
4H), 5.22 (dd, 2H), 5.33 (dd, 2H), 5.94 (m, 2H), 7.33 (t,
2H), 7.47 (t, 2H), 7.64 (d, 2H), 7.68 (d, 2H), 9.0 (d, 2H);
IR (KBr) 3309, 2957, 1742, 1635, 1531, 745 cm 1; MS
(ESI) m/z 306, 613.
[S-(R*,R*)]-2-(2-{2-[1-(1-Benzloxycarbonyl-3-methyl-butyl-
carbamoyl]-phenyldisulfanyl}-benzoylamino)-4-methyl-
pentanoic acid benzyl ester (83). The title compound
was synthesized according to the general method A
using bisacid chloride (0.696 g, 2.03 mmol), N-methyl-N-
(trimethylsilyl)acetamide (1.46 g, 10.16 mmol), corre-
sponding amino acid benzyl ester p-toluenesulfonate (2.0 g,
5.08 mmol), N-methylmorpholine (1.03 g, 10.16 mmol)
[S-(R*,R*)]-Benzamide, 2,2(-dithiobis[N-[1-hydroxymethyl)-
3-methylbutyl] (87). The title compound was synthesized
according to the general method A using corresponding
amino alcohol (1.17 g, 10.8 mmol), N-methylmorpholine
(0.83 g (0.91 mL), 8.24mmol) bisacid chloride (1.59 g,
4.3 mmol), N-methyl-N-(trimethylsilyl)acetamide (3.2 mL,
19.9 mmol). Isolated yield 29%; 1H NMR (300 MHz,
DMSO-d6) d 0.91 (d, 12H), 1.41 (m, 4H), 1.71 (m, 2H),
3.40 (m, 4H), 4.73 (t, 2H), 7.29 (d, 2H), 7.39 (t, 2H),
7.62 (m, 4H), 8.23 (d, 2H); MS (ESI) m/z 252.
1
and 40 mL of dichloromethane. Isolated yield: 60%; H
NMR (400 MHz, DMSO-d6) d 0.84 (d, 6H), 0.89 (d, 6H),
1.54 (m, 2H), 1.72 (m, 4H), 4.5 (m, 2H), 5.12 (s, 4H),
7.31 (m, 12H), 7.39 (t, 2H), 7.58 (d, 2H), 7.61 (d, 2H),
8.97 (d, 2H); IR (KBr) 3298, 2956, 1741, 1634, 1531,
1
745 cm MS (ESI) m/z 356, 713.
[R-(R*,R*)]-Benzamide, 2,20-dithiobis[N-(2-hydroxy-1-phenyl-
ethyl) (88). The title compound was synthesized using
[S-(R*,R*)]-(2-{2-[2-(1-t-Butoxycarbonyl-2-methyl-butyl-
carbamoyl)-phenyldisulfanyl]-benzoylamino}-3-methyl-
pentanoic acid t-butyl ester) (84). This compound was
prepared according to the general method B using iso-
leucine t-butyl ester (48.0 g, 256.37 mmol), bisacid
chloride (40.0 g, 116.53 mmol), N-methylmorpholine
general method
A using bisacid chloride (1.0 g,
2.94 mmol), N-methyl-N-(trimethylsilyl)acetamide (3.4 mL,
11.76 mmol), corresponding amino alcohol (1.0 g,
4.0 mmol) and 70 mL of dichloromethane. Isolated
yield: 74%; 1H NMR (300 MHz, DMSO-d6) d 3.33

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1727
1
(m, 2H), 5.05 (m, 2H), 7.36 (m, 14H), 7.62 (d, 2H), 7.79
(d, 2H), 8.94 (d, 2H); MS (ESI) m/z 274.
2.15 g. H NMR (300 MHz, DMSO-d6) d 8.51 (d, 2H),
7.70 (dd, 2H), 7.61 (dd, 2H), 7.44 (t, 2H), 7.29 (t, 2H),
4.90 (d, 2H), 4.48 (m, 2H), 4.16 (m, 2H), 3.63 (s, 6H),
1.13 (d, 6H); IR (KBr) 3315, 1742, 1625, 1539,
1086 cm 1; MS (APCI) m/z 537.
[S-(R*,R*)]-Benzamide,
2,20-dithiobis[N-hydroxy-1-(1-
methylethyl)-3-pentenyl] (89). The title compound was
synthesized according to the general method A using
bisacid chloride (2.74 g, 8.0 mmol), N-methyl-N-(tri-
methylsilyl)acetamide (4.65 g, 32 mmol), corresponding
amino alcohol (0.78 g, 4.0 mmol), N-methylmorpholine
(3.20 g, 32 mmol) and 40 mL of dichloromethane. Iso-
lated yield: 41%; 1H NMR (400 MHz, DMSO-d6) d 0.89
(d, 6H), 0.93 (d, 6H), 1.36 (m, 2H), 1.54 (m, 2H), 1.69
(m, 2H), 3.92 (m, 4H), 4.58 (m, 2H), 4.69 (t, 2H), 5.66
(m, 4H), 7.31 (t, 2H), 7.44 (t, 2H), 7.61 (d, 2H), 7.66 (d,
2H), 8.55 (d, 2H); IR (KBr) 3314, 2955, 1634, 1532,
746 cm 1; MS (ESI) m/z 278, 557.
[S-(R*,R*)]-(N,N0 -[Dithiobis(2,1-phenylenecarbonyl)bis-
L-serine-bis-dimethyl ester) (93). This compound was
prepared according to the general method B using bis-
acid chloride (1.50 g, 4.4 mmol) in 30 mL of dichloro-
methane and serine methyl ester hydrochloride (1.37 g,
8.8 mmol), N-methylmorpholine (1.93 mL, 17.6 mmol)
in 40 mL of dichloromethane. The organic layer was
washed with 1 N HCl, saturated bicarbonate and brine.
The solution was concentrated to give 1.5 g of a foam.
The compound was puri®ed by chromatography (SiO2,
CHCl3/MeOH, 95/5) to a€ord 0.98 g of a solid. The
sample was recrystallized from hot CHCl3 to give 0.87 g
6-Amino-2-{2-[2-(5-amino-1-carboxy-pentylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-hexanoic acid methyl
ester (90). A solution of 96 (1.54 g, 1.75 mmol) in 30 mL
of dichloromethane was treated with HCl gas at 0 ꢀC.
After 18 h the solution was concentrated to a solid and
dissolved in methanol. HCl gas was bubbled in at 0 ꢀC
and it was stirred for 3 h. The solution was concentrated
1
of 93 as a solid. H NMR (300 MHz, DMSO-d6) d 8.78
(d, 2H), 7.71 (d, 2H), 7.60 (d, 2H), 7.44 (t, 2H), 7.28
(t, 2H), 5.03 (t, 2H), 4.48 (m, 2H), 3.75 (t, 4H), 3.62 (s,
6H); IR (KBr) 3434, 3331, 1750, 1738, 1644, 1544,
748 cm 1; MS (APCI) m/z 509.
1
to a€ord 1.38 g of 90 as a foam. H NMR (300 MHz,
[S-(R*,R*)]-(5-Cyano-4-{2-[2-(1-cyanomethyl-3-methoxy-
carbonyl-propylcarbamoyl)-phenyldisulfanyl]-benzoylamino}-
pentanoic acid methyl ester) (94). This compound was
prepared according to the general method A using
bisacid chloride (0.18 g, .52 mmol) in 35 mL of dichloro-
methane and threonine methyl ester hydrochloride (2.0 g,
11.8 mmol), N-methylmorpholine (0.13 g (0.14 mL),
1.30 mmol), N-methyl-N-(trimethylsilyl)acetamide (0.36 g
(0.40 mL), 2.49 mmol) in 40 mL of dichloromethane.
Isolated yield: 19.6%. 1H NMR (300 MHz, DMSO-d6) d
1.88 (m, 4H), 2.46 (m, 4H), 2.83 (m, 4H), 3.58 (s, 6H),
4.21 (m, 2H), 7.33 (t, 2H), 7.43 (t, 2H), 7.62 (d, 2H),
7.68 (d, 2H), 8.78 (d, 2H); IR (KBr) 3326, 3316, 3059,
2952, 2851, 2250, 1734, 1641, 1586, 1532, 1436, 1355,
1307, 1261, 1206, 1172, 1091, 1038, 985, 872, 789, 749,
699 cm 1; MS (APCI) m/z 291.
DMSO-d6) d 9.02 (d, 2H), 7.91 (bs, 4H), 7.76 (dd, 2H),
7.64 (d, 2H), 7.49 (t, 2H), 7.34 (t, 2H), 4.42 (m, 2H),
3.68 (s, 6H), 2.77 (m, 4H), 1.81 (m, 2H), 1.61 (m, 4H),
1.45 (m, 4H); IR (KBr) 2950, 1739, 1638, 1529, 1163,
745 cm 1; MS (ESI) m/z 591.
[S-(R*,R*)]-7-Amino-3-{2-[2-(5-amino-1-methoxycarbonyl-
methyl-pentylcarbamoyl)-phenyldisulfanyl]-benzoylamino}-
heptanoic acid methyl ester (91). Synthesized from 95
(1.0 g, 1.13 mmol) by stirring in 33% HBr/AcOH
(20 mL) for 1 h at room temperature. Diethyl ether was
then added to cause a solid to precipitate. The pre-
cipitate was ®ltered o€ and washed several times with
diethyl ether followed by several washes with ethyl
1
acetate to yield a beige solid. Isolated yield: 76.1%. H
NMR (400 MHz, DMSO-d6) d 1.41 (m, 4H), 1.56 (m,
8H), 2.59 (d, 4H), 2.78 (m, 4H), 3.60 (s, 6H), 4.30
(m, 2H), 7.32 (t, 2H), 7.45 (t, 2H), 7.62 (d, 2H), 7.69
(brs, 6H), 8.52 (d, 2H); IR (KBr) 3229, 2947, 2867, 1733,
1634, 1539, 1457, 1436, 1314, 1161, 1036, 989, 867,
747 cm 1; MS (ESI) m/z 619.4.
[S-(R*,R*)]-(7-Benzyloxycarbonylamino-3-{2-[2-(5-benzyl-
oxycarbonylamino-1-methoxycarbonylmethyl-pentylcar-
bamoyl)-phenyldisulfanyl]-benzoylamino}-heptanoic acid
methyl ester) (95). The title compound was synthesized
according to the general method B using b-amino acid
(3.33 g, 9.66 mmol), bisacid chloride (1.44 g, 4.20 mmol),
N-methylmorpholine (2.05 g (2.23 mL), 20.28 mmol) to
yield 2.75 g of beige solid. Isolated yield: 74%. 1H NMR
(400 MHz, DMSO-d6) d 1.23±1.50 (m, 8H), 1.54 (m,
2H), 2.56 (d, 4H), 2.98 (m, 4H), 3.56 (s, 6H), 4.27
(m, 2H), 4.99 (s, 4H), 7.24±7.37 (m, 12H), 7.43 (t, 2H),
7.56 (d, 2H), 7.64 (d, 2H), 8.48 (d, 2H); IR (KBr) 3325,
3279, 3062, 2944, 2860, 1733, 1690, 1633, 1586, 1537,
1456, 1435, 1313, 1262, 1202, 1158, 1137, 1034, 993, 876,
742, 697, 652 cm 1; MS (ESI) m/z 443.5.
[S-(R*,R*)]-(N,N0-[Dithiobis(2,1-phenylenecarbonyl)bis-
L-threonine-bis-dimethyl ester) (92). This compound was
prepared according to the general method B using bis-
acid chloride (2.01 g, 5.9 mmol) in 35 mL of dichloro-
methane and threonine methyl ester hydrochloride
(2.0 g, 11.8 mmol), N-methylmorpholine (2.72 mL,
24.8 mmol) in 40 mL of dichloromethane. The organic
layer was washed with 1 N HCl and 92 precipitated
from the organic layer. The solid was dried to give

1728
J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
[S-(R*,R*)]-6-t-Butoxycarbonylamino-2-{2-[2-(1-t-butoxy-
carbonyl)-5-t-butoxycarbonylamino -pentylcarbamoyl)-
phenylcarbamoyl)phenydisulfanyl]-benzoylamino}-hexanoic
acid t-butyl ester (96). This compound was prepared
according to the general method B using bisacid chlor-
ide (1.0 g, 3.0 mmol) in 15 mL of dichloromethane and
g-N-Boc-lysine-t-butyl ester hydrochloride (2.24 g,
6.6 mmol), N-methylmorpholine (1.65 mL, 15 mmol) in
20 mL of dichloromethane. The compound was puri®ed
by chromatography (SiO2, CHCl3/MeOH, 95/5) to
a€ord 2.25 g of 96 as a solid. 1H NMR (300 MHz,
CDCl3) d 7.72 (d, 2H), 7.52 (d, 2H), 7.32 (t, 2H), 7.18
(t, 2H), 6.73 (bs, 2H), 4.63 (m, 4H), 3.07 (m, 4H), 1.93
(m, 2H), 1.76 (m, 2H), 1.45 (s, 18H), 1.55±1.30 (m, 8H),
1.34 (s, 18H); IR (KBr) 3351, 2976, 1714, 1694, 1648,
1525, 1159 cm 1, MS (APCI) m/z 876.
2H), 7.63 (d, 4H), 7.46 (m, 4H), 7.31 (t, 2H), 6.98 (bs,
2H), 4.60 (m, 2H), 2.70 (m, 2H), 2.57 (m, 2H), 1.41 (s,
18H); IR (KBr) 3327, 1731, 1669, 1639, 1539,
1154 cm 1; MS (APCI) m/z 646.
[S-(R*,R*)]-2-{2-[2-(1-t-Butoxycarboyl-3-carbamoyl-
propylcarbamoyl)-phenyldisulfanyl]-benzoylamino}-4-car-
bamoyl-butyric acid t-butyl ester (100). This compound
was prepared according to the general method B using
bisacid chloride (1.5 g, 4.5 mmol) in 25 mL of dichloro-
methane and glutamine-t-butyl ester hydrochloride
(2.36 g, 9.9 mmol), N-methylmorpholine (2.47 mL,
22.5 mmol) in 30 mL of dichloromethane. A precipitate
formed which was collected and washed with ether. The
solid was recrystallized from ethanol to a€ord 1.21 g of
1
100 as a solid. H NMR (300 MHz, DMSO-d6) d 8.95
(d, 2H), 7.71 (d, 2H), 7.64 (d, 2H), 7.47 (t, 2H), 7.35
(bs, 2H), 7.33 (t, 2H), 6.85 (brs, 2H), 4.25 (m, 2H), 2.25
(t, 4H), 2.03 (m, 2H), 1.93 (m, 2H), 1.43 (s, 18H); IR (KBr)
3398, 1731, 1651, 1156, 744 cm 1; MS (APCI) m/z 675.
[S-(R*,R*)]Benzamide, 2,20-dithiobis[N-[1-(aminocabonyl)-
3-methylbutyl] (97). The title compound was synthe-
sized according to the general method A using bisacid
chloride (2.5 g, 7.3 mmol), N-methyl-N-(trimethylsilyl)-
acetamide (8.49 g, 58.44 mmol), l-leucine amide (2.2 g,
14.61 mmol), N-methylmorpholine (5.91 g, 58.44 mmol)
[S-(R*,R*)]2,2-Dithiobis[N-[1-(methoxymethylamino)-
carbonyl]-3-methylbutyl]benzamide] (101). The title com-
pound was synthesized according to the general method
A using bisacid chloride (1.59 g, 4.64 mmol), N-methyl-
N-(trimethylsilyl)acetamide (2.7 g, 18.56 mmol), corre-
sponding amino acid amide hydrochloric acid salt
(0.49 g, 2.32 mmol), N-methylmorpholine (1.88 g,
18.56 mmol) and 40 mL of dichloromethane. Isolated
1
and 40 mL of dichloromethane. Isolated yield: 81%. H
NMR (400 MHz, DMSO-d6) d 0.92 (d, 6H), 0.94 (d,
6H), 1.72 (m, 2H), 1.55 (m, 4H), 4.47 (m, 2H), 7.06 (brs,
2H), 7.31 (t, 2H), 7.44 (t, 2H), 7.47 (brs, 2H), 7.64 (d,
2H), 7.75 (d, 2H), 8.64 (d, 2H); IR (KBr) 3373, 3281,
2956, 1670, 1633, 1530, 743 cm 1; MS (ESI) m/z 531.
1
yield: 30%. H NMR (400 MHz, DMSO-d6) d 0.92 (d,
Benzamide, 2,20-dithiobis[N-(3-amino-3-oxopropyl) (98).
This compound was prepared according to the general
method A using bisacid chloride (2.74 g, 8 mmol) in
40 mL of dichloromethane and b-alanine amide (2.0 g,
16 mmol) and N-methyl-N-(trimethylsilyl)acetamide
(7.7 mL, 48 mmol) in 40 mL of dichloromethane. After
18 h the mixture was concentrated and triturated with
1 N HCl. Acetonitrile was added and the precipitate was
®ltered and washed with diethyl ether. The solid was
dried to a€ord 0.83 g of 98. 1H NMR (300 MHz,
DMSO-d6) d 8.67 (t, 2H), 7.60 (d, 4H), 7.43 (m, 4H),
7.27 (t, 2H), 6.86 (brs, 2H), 2.48 (m, 4H), 2.36 (m, 4H);
IR (KBr) 3268, 1663, 1633, 737 cm 1; MS (APCI) m/z
447.
6H), 0.94 (d, 6H), 1.42 (m, 2H), 1.75 (m, 4H), 3.14 (s,
6H) 3.81 (s, 6H), 4.97 (m, 2H), 7.31 (t, 2H), 7.44 (t, 2H),
7.64 (d, 2H), 7.70 (d, 2H), 8.81 (d, 2H); IR (KBr) 3324,
2957, 1638, 1531, 746 cm 1; MS (ESI) m/z 309, 619.
2-{2-[2-(Dicarbamoylmethyl-carbamoyl)-phenyldisulfanyl]-
benzoylamino}-malonamide (102). The title compound
was synthesized using general method A using bisacid
chloride (2.93 g, 8.55 mmol), N-methyl-N-(trimethyl-
silyl)acetamide (4.96 g, 34.2 mmol), corresponding
amine (2.0 g, 17.09 mmol), N-methylmorpholine (3.46 g,
34.2 mmol) and 25 mL of dichloromethane. Isolated
yield: 65%. 1H NMR (400 MHz, DMSO-d6) d 7.33
(t, 2H), 7.39 (d, 2H), 7.5 (t, 2H), 7.72 (s, 4H), 7.89 (s,
2H), 7.94 (d, 2H), 8.17 (s, 2H); IR (KBr) 3342, 3177,
1715, 1644, 1448, 743 cm 1; MS (ESI) m/z 208, 252.
[S-(R*,R*)]-2-{2-[2-(1-t-Butoxycarbamoyl-carbamoyl)-
phenyldisulfanyl]benzoylamino}-succinamic acid t-butyl
ester (99). This compound was prepared according to
the general method B using bisacid chloride (1.0 g,
3.0 mmol) in 15 mL of dichloromethane and arginine-t-
butyl ester hydrochloride (1.48 g, 6.6 mmol), N-methyl-
morpholine (1.65 mL, 15 mmol) in 20 mL of dichloro-
methane. A precipitate formed which was collected and
washed with ether. The solid was treated with boiling
isopropanol and ®ltered hot to yield 1.15 g of the title
[S-(R*,R*)] 2-{2-[2-(1,2-Dicarbamoyl-ethylcarbamoyl)-
phenyldisulfanyl]-benzoylamino}-succinamide (103). This
compound was prepared according to the general
method B using bisacid chloride (0.82 g, 2.39 mmol),
hydrochloric acid salt of asparagine amide (1.0 g,
5.97 mmol), N-methylmorpholine (1.21 g, 11.94 mmol),
1.30 mmol), N-methyl-N-(trimethylsilyl)acetamide (1.74 g,
11.94 mmol) in 40 mL of dichloromethane. Isolated
yield: 42%. 1H NMR (300 MHz, DMSO-d6) d 2.58
1
compound. H NMR (300 MHz, DMSO-d6) d 8.91 (d,

J. V. N. Vara Prasad et al./Bioorg. Med. Chem. 6 (1998) 1707±1730
1729
(m, 4H), 4.69 (m, 2H), 6.94 (brs, 2H), 7.14 (brs, 2H),
7.31 (t, 2H), 7.39 (s, 4H), 7.48 (t, 2H), 7.66 (d, 2H), 7.77
(d, 2H), 8.69 (d, 2H); IR (KBr) 3395, 3312, 3203, 1663,
1530, 1432, 1332, 745 cm 1; MS (APCI) m/z 266.4.
P.; Hupe, D.; Sharmeen, L.; Mack, D.; Scholten, J.; Saunders,
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equipped with a position-and-time-resolved-ion-count-
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net and before quadrupole analyzer.33 Protein solutions
for ESI-MS measurements were prepared at a con-
centration of 10±30 mM in 10 mM ammonium acetate
(pH 6.9) with two times the protein molar concentration
of zinc (ZnCl2) in water. Stock solutions of the disul®de
compounds were prepared at a concentration of ca. 5±
10 mM in acetonitrile/methanol/THF mixtures. Sample
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1
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All the EC50 and TC50 measurements were performed at
Southern Research Institute, Birmingham, AL. All
spectra reported were recorded at the Parke-Davis
Analytical Research Section facility. The authors thank
all those who participated in anti-HIV activity testing
and measurement of the analytical data. We also thank
Ms Susan Hagen, Dr. James Kaltenbronn and Dr. Alex
Bridges for proof reading as well their suggestions.
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