Design, synthesis, and X-ray crystallographic analysis of a novel class of Hiv-1 protease inhibitors

Article abstract of DOI:10.1021/jm200778q

In the present paper, design, synthesis, X-ray crystallographic analysis, and HIV-1 protease inhibitory activities of a novel class of compounds are disclosed. Compounds 28-30, 32, 35, and 40 were synthesized and found to be inhibitors of the HIV-1 protease. The crucial step in their synthesis involved an unusual endo radical cyclization process. Absolute stereochemistry of the three asymmetric centers in the above compounds have been established to be (4S,2′R,3′S) for optimal potency. X-ray crystallographic analysis has been used to determine the binding mode of the inhibitors to the HIV-1 protease.

Full text of DOI:10.1021/jm200778q

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and X-ray Crystallographic Analysis of a Novel Class
of HIV-1 Protease Inhibitors†
Ashit K. Ganguly,*,‡ Sesha S. Alluri,‡ Danielle Caroccia,‡ Dipshikha Biswas,‡ Chih-Hung Wang,‡
Eunhee Kang,‡ Yong Zhang,‡ Andrew T. McPhail,§ Steven S. Carroll,|| Christine Burlein,|| Vandna Munshi,||
Peter Orth,^ and Corey Strickland^
‡Department of Chemistry, Chemical Biology and Biomedical Engineering, Stevens Institute of Technology, Hoboken,
New Jersey 07030, United States
§Duke University, Durham, North Carolina 27708, United States
In Vitro Science Department, Merck Research Laboratories, West Point, Pennsylvania 19486, United States
^Structural Chemistry, Merck Research Laboratories, Kenilworth, New Jersey 07033, United States
ABSTRACT: In the present paper, design, synthesis, X-ray crystal-
lographic analysis, and HIV-1 protease inhibitory activities of a
novel class of compounds are disclosed. Compounds 28ꢀ30, 32,
35, and 40 were synthesized and found to be inhibitors of the HIV-1
protease. The crucial step in their synthesis involved an unusual
endo radical cyclization process. Absolute stereochemistry of the
three asymmetric centers in the above compounds have been
established to be (4S,20R,30S) for optimal potency. X-ray crystal-
lographic analysis has been used to determine the binding mode of
the inhibitors to the HIV-1 protease.
’ INTRODUCTION
Using similar approach, we synthesized compounds 15ꢀ20
from 9ꢀ14, respectively (Scheme 2). This allowed us to
synthesize compounds represented by structure 2 by derivitiza-
tion of the free ꢀNH function.
In a recent publication1 we have described the synthesis of
conformationally restricted sulfonamides represented by formula
1 wherein the R0 substituents are alkyl, aryl, and arylalkyl groups
(Figure 1). The ring size could be six, seven, or eight atoms. Our
expectation is that these cyclic sulfonamides will assume fewer
conformations compared to open chain sulfonamides and thus
serve as valuable pharmacophores in drug discovery. In this
communication we disclose the design and synthesis of com-
pounds, represented by the general structure 2, that have been
found to be inhibitors of HIV-1 protease. Several drugs such as 3,
4, and 5, which are HIV-1 protease inhibitors,2ꢀ5 are widely and
successfully used in AIDS patients. In general our compounds
represent novel pharmacophores and are less potent inhibitors of
HIV-1 protease, when compared with the compounds men-
tioned above. Our hope is that with further changes in structures
2, these compounds might show improvements in the spectrum
of activity against resistant organisms and also show improve-
ments in pharmacokinetic properties.
Thus, treatment of 15 with (2S,3S)-1,2-epoxy-3- (bocamino)-
4-phenylbutane (21) in DMF solution and in the presence of
cesium carbonate yielded 22 (Scheme 3). Compound 22, when
treated with 30% trifluoroacetic acid in DCM, yielded inter-
mediate 23a. The crude intermediate 23a was further reacted
with isopropyl 1H-imidazole-1-carboxylate, which was generated
by reacting carbonyldiimidazole with isopropanol, in the pre-
sence of N,N0-diisopropylethylamine to yield compound 23.
Compound 22, when tested in HIV-protease assay, showed a
Ki of 470 nM, and 23 was inactive. As expected, these results
suggest the importance of the free hydroxyl group in 22 for HIV-1
protease inhibitory activity.
Furthermore, we decided to study the effect of substitution on
the cyclic sulfonamide moiety in both the aromatic and alicyclic
portion of 22, as well as study the nature of substituted
carbamates and amides required for optimizing antiviral potency.
The results of these studies are summarized below.
’ RESULTS AND DISCUSSION
Treatment of 16 with 21 in DMF solution and in the presence
of cesium carbonate yielded a mixture of diastereoisomers 24,
Compounds represented by structure 1 were synthesized
using a radical cyclization process. Thus, compound 6, when
treated with tributyltin hydride (TBTH) in refluxing toluene
solution and in the presence of AIBN, yielded 1 via 7 and 8
(Scheme 1).
Received: June 15, 2011
Published: September 14, 2011
r
2011 American Chemical Society
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Figure 1. HIV-1 protease inhibitors.
Scheme 1. Synthesis of Seven-Membered Cyclic Sulfonamide Using Radical Cyclization
Scheme 2. Synthesis of Conformationally Restricted Sulfonamides 15ꢀ20
which could not be separated. However, on treatment with
trifluoroacetic acid in dichloromethane solution, 24 yielded
25 and 26, which could be separated using preparative TLC
(Scheme 4). X-ray crystallographic analysis of 25 (Figure 2)
established its absolute stereochemistry to be (4R,20R,
30S), and therefore, the absolute stereochemistry of 26 is
(4S,20R,30S).
Treatment of 25 in THF solution with di-tert-butyl dicar-
bonate and diisopropylethylamine yielded 27, and simi-
larly 26 yielded 28. In HIV protease assay, 28 with Ki of
29 nM, was considerably more potent than 22 and it was
also more potent than the diastereoisomer 27 which showed
a Ki of 1000 nM. These results demonstrated the impor-
tance of the methyl substituent at C4 and also the absolute
stereochemistry at C4 to be (S) for optimum HIV protease
inhibitory activity. Thus, 28 with (4S,20R,30S) geometry
was considerably more potent than 27 with (4R,20R,30S)
geometry.
Using the above procedure for the synthesis of 28, we
prepared a number of carbamates maintaining (4S,20R,30S)
geometry and then tested them in the HIV-1 protease inhibitor
assay. The results are summarized in Table 1. We prepared the
corresponding (4R,20R,30S) diastereoisomers and found them to
be considerably less active (results not shown).
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Scheme 3. Synthesis of Compounds 22 and 23
Scheme 4. Synthesis of Diastereoisomers 27 and 28
Table 1. Novel HIV Protease Inhibitors
compd
substitution
Ki (nM)
29
30
31
32
33
34
35
R = R0 = H; R00 = allyl
R = R0 = H; R00 = Et
R = R0 = H; R00 = Ph
R = F; R0 = H; R00 = tert-butyl
R = CF3; R0 = H; R00 = tert-butyl
R = H; R0 = CF3; R00 = tert-butyl
R = OCH3; R0 = H; R00 = tert-butyl
27
34
350
20
186
100
19
Figure 2. X-ray crystallographic structure of compound 25.
Figure 3. X-ray crystallographic structure of 32 bound to HIV-1
protease.
The X-ray crystallographic structure of compound 32 was
solved bound to the HIV-1 protease. Compound 32 occupies
the active site cavity and makes hydrogen bonds to the cata-
lytic aspartic acid residues Asp25 and Asp250 through the
hydroxyl group (Figure 3). The sulfonamide and the carba-
mate carbonyl form hydrogen bonds with a structural water
molecule, which in turn hydrogen-bonds with Ile50 and Ile500
of the protease.
In addition to these favorable hydrogen bonding stabilization
effects, the C4-Me group of compound 32 has extensive hydro-
phobic interactions with side chains of Leu23, Val82, and Ile84.
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’ CONCLUSION
On the basis of a novel pharmacophore structure containing a
conformationally restricted sulfonamide, a class of HIV-1 pro-
tease inhibitors was designed and synthesized. An unusual endo
radical cyclization process was used as a key step in their
synthesis. Several analogues were synthesized to establish a
preliminary structureꢀactivity relationship, and the importance
of the absolute stereochemistry of the three asymmetric centers
for optimum activity was established. These compounds were
shown to possess HIV-1 protease inhibitory activity. X-ray
crystallographic analysis of compound 32, when bound to the
HIV-1 protease, demonstrated that it binds to a similar pocket as
known inhibitors, such as darunavir. The 2.9 Å distance and the
geometry between C4-Me and the carbonyl oxygen of Gly270
explain the strong preference for the (S) stereochemistry of the
C4 position.
Figure 4. Overlay of 32 with darunavir (PDB code 2hs1). Rotation of
cyclized fluorophenyl with respect to noncyclized aromatic plane is
shown with arrow.
Scheme 5. Synthesis of Amide Inhibitor 40
Further work is in progress to improve the potency based on
the X-ray results described above. We are exploring the possibi-
lity of optimizing the C4-methyl binding site and also incorpor-
ating different substitutions on the aromatic ring of the cyclic
sulfonamide moiety. Modification of the carbamate functionality
to capture other interactions with the protein backbone also will
be explored. These cyclic sulfonamide inhibitors offer an exciting
opportunity to expand the scope of compounds active against
HIV-1 protease.
’ EXPERIMENTAL SECTION
General Procedures. All the reactions were performed under a
nitrogen atmosphere with magnetic stirring. Air- and moisture-sensitive
reagents were transferred via disposable syringes. Anhydrous solvents
and commercially available chemicals were used without further drying.
TLC was performed on Analtech silica gel glass plates (250 μm) and
visualized with a UV lamp. NMR spectra were recorded with Varian 400
MHz spectrometer using CDCl3 as solvent unless otherwise noted. The
purity of the compounds was established using various chromatographic
techniques including HPLC and was judged to be >95%.
General Procedure for Sulfonamide Formation, 9ꢀ14. To
a solution of allylamine/2-methylallylamine (1.1 equiv) in pyridine was
added 2-bromobenzenesulfonyl chloride (1 equiv) dissolved in pyridine.
The mixture was stirred at room temperature for 8ꢀ10 h under nitrogen.
TLC was used to monitor the progress of the reaction. After the reaction
was complete, the reaction mixture was neutralized with 30% HCl and
then the aqueous portion was extracted with dichloromethane (DCM).
The combined organic layer was dried over anhydrous sodium sulfate
and evaporated to dryness to yield the crude compound which was
purified by column chromatography using 15% ethyl acetate in hexane to
yield the pure compound.
Furthermore, C4-Me (S configuration) in compound 32 is 2.9 Å
away from the carbonyl oxygen of Gly270. If the C4-Me config-
uration is changed to (R) instead of (S), an unfavorable repulsion
would be created between the C4-Me and the Gly270 carbonyl
group and the interactions to the hydrophobic pocket would be
lost. These results support the experimental observation that
compounds with the C4 methyl group in the (S) configuration
possess optimum activity.
When superimposing the X-ray structure of 32 with darunavir
(3), a similar acyclic inhibitor, there is a nice overlay with the
exception of the aromatic sulfonamide plane, which is rotated by
42ꢀ (Figure 4). The cyclizing linker, including C4, bridges the
hydrophobic area of the active site occupied by the isobutyl of
darunavir (3).
To investigate whether the carbamates in this series of
compounds could be replaced by amides, we treated com-
pound 17 with 21 and obtained a mixture of diastereoisomers
36 that again could not be separated (Scheme 5). Compound
36 on treatment with trifluoro acetic acid yielded 37 and 38,
which were separated using preparative TLC. Diastereoi-
somer 37 with (4S,20R,30S) geometry was treated with 396
in dichloromethane solution and dicyclohexyl carbodiimide to
yield 40 which when tested in HIV-1 protease assay had a Ki
of 30 nM.
N-Allyl-2-bromobenzenesulfonamide (9). The title com-
pound was obtained from 2-bromobenzenesulfonyl chloride and allyl-
amine following the general procedure for sulfonamide formation.
Crystals were obtained from DCM and hexane (1:4). Yield: 70%. Mp
68ꢀ70 ꢀC. 1H NMR (400 MHz, CDCl3) δ ppm 8.14 (dd, J = 7.69, 1.88
Hz, 1H), 7.74 (dd, J = 7.74, 1.36 Hz, 1H), 7.45ꢀ7.40 (m, 2H),
5.72ꢀ5.62 (m, 1H), 5.25ꢀ5.05 (m, 3H), 3.57 (t, J = 6.11 Hz, 2H).
2-Bromo-N-(2-methylallyl)benzenesulfonamide (10). The
title compound was prepared following the general procedure for
sulfonamide formation by using 2-bromobenzenesulfonyl chloride and
2-methylallylamine. Crystals were obtained from DCM and hexane
1
(1:4). Yield: 53%. Mp 83ꢀ85 ꢀC. H NMR (400 MHz, CDCl3) δ
ppm 8.15 (dd, J = 7.73, 1.85 Hz, 1H), 7.76 (dd, J = 7.72, 1.40 Hz, 1H),
7.45ꢀ7.40 (m, 2H), 5.23 (b, 1H), 4.91 (s, 1H), 4.85 (s, 1H), 3.47 (d, J =
6.42 Hz, 2H), 1.71 (s, 3H).
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2-Bromo-4-fluoro-N-(2-methylallyl)benzenesulfonamide
(11). The title compound was prepared following the general procedure
for sulfonamide formation by using 2-bromo-4-fluorobenzenesulfonyl
chloride and 2-methylallylamine. Crystals were obtained from DCM and
hexane (1:4). Yield: 83%. Mp 51ꢀ53 ꢀC. 1H NMR (400 MHz, CDCl3)
δ ppm 8.15 (dd, J = 8.84, 5.80 Hz, 1H), 7.48 (dd, J = 7.90, 2.51 Hz, 1H),
7.19ꢀ7.16 (m, 1H), 5.16 (b, 1H), 4.89 (s, 1H), 4.84 (s, 1H), 3.46 (d, J =
6.37 Hz, 2H), 1.70 (s, 3H). MS (ESI): m/z 307.94 [M + H]+
2-Bromo-N-(2-methylallyl)-4-(trifluoromethyl)benzenes-
ulfonamide (12). The title compound was prepared following the
general procedure for sulfonamide formation, from 2-bromo-4-trifluoro-
methylbenzenesulfonyl chloride and 2-methylallylamine. Crystals were ob-
tained from DCM and hexane (1:5). Yield: 72%. Mp 102ꢀ103 ꢀC. 1H NMR
(400 MHz, CDCl3) δppm 8.26 (d, J=8.23 Hz, 1H), 8.01 (s, 1H), 7.74 (d, J=
8.23 Hz, 1H), 5.2 (t, J= 6.4, 1H), 4.88 (dd, J=18.27, 0.71 Hz, 2H), 3.51 (d, J=
6.35 Hz, 2H), 1.71 (s, 3H). MS (FAB): m/z 358.1 [M + H]+.
2-Bromo-N-(2-methylallyl)-5-(trifluoromethyl)benzene-
sulfonamide (13). The title compound was prepared following the
general procedure for sulfonamide formation, from 2-bromo-5-trifluoro-
methylbenzenesulfonyl chloride and 2-methylallylamine. Crystals were ob-
tained from DCM and hexane (1:5). Yield: 71%. Mp 85ꢀ86 ꢀC. 1H NMR
(400 MHz, CDCl3) δ ppm 8.38 (s, 1H), 7.89 (d, J = 8.32 Hz, 1H), 7.66
(d, J= 8.23 Hz, 1H), 5.22 (t, J=5.90Hz,1H),4.87(d,J= 21.55 Hz, 2H), 3.52
(d, J = 6.36 Hz, 2H), 1.69 (s, 3H). MS (FAB): m/z 358.1 [M + H]+.
2-Bromo-4-methoxy-N-(2-methylallyl)benzenesulfonam-
ide (14). The title compound was prepared following the general
555procedure for sulfonamide formation, from 2-bromo-4-methoxybenze-
nesulfonyl chloride and 2-methylallylamine. Crystals were obtained from
DCM and hexane (1:4). Yield: 31%. Mp 69ꢀ71 ꢀC. 1H NMR (400 MHz,
CDCl3) δppm 8.01 (d, J= 8.84 Hz, 1H), 7.22 (d, J= 2.31 Hz, 1H), 6.90 (dd,
J = 8.81, 2.28 Hz, 1H), 5.13 (t, J = 5.99 Hz, 1H), 4.84 (d, J = 26.57 Hz, 2H),
3.84 (s, 3H), 3.39 (d, J = 6.34 Hz, 2H), 1.68 (s, 3H).
General Procedure for Radical Reaction, 15ꢀ20. Compound
9 (1 equiv) was dissolved in toluene, and to the mixture was added
azobisisobutyronitrile (AIBN) (0.2 equiv). The above solution was
heated to about 60ꢀ70 ꢀC, and then tributyltin hydride (TBTH) (1.1
equiv) was added slowly under nitrogen. After the addition was
complete, the reaction mixture was refluxed for 4ꢀ6 h. Upon comple-
tion (followed by TLC), the reaction mixture was evaporated to dryness.
The residue was extracted with DCM and washed with water. The
organic layers were combined, dried over sodium sulfate, and concen-
trated to yield the crude compound which was further purified by
column chromatography using ethyl acetate and hexane to yield the pure
compound.
6.23 Hz, 1H), 3.6ꢀ3.0 (m, 4H), 1.94 (b, 1H), 0.94 (d, J = 6.84 Hz, 3H).
MS: m/z 230.05 [M + H]+.
4-Methyl-7-(trifluoromethyl)-2,3,4,5-tetrahydro-1H-1λ6,
2-benzothiazepine-1,1-dione (18). The title compound was
prepared from compound 12 following the general procedure for
radical reaction, giving a white solid. Crystals were obtained from
DCM and hexane (1:4). Yield: 71%. Mp 154ꢀ155 ꢀC. 1H NMR (400
MHz, CDCl3) δ ppm 8.03 (d, J = 8.12 Hz,1H), 7.59 (d, J = 8.12 Hz,
1H), 7.52 (s, 1H), 4.61 (t, J = 5.89 Hz, 1H), 3.61ꢀ3.15 (m, 4H), 1.97
(b, 1H), 0.97 (d, J = 6.46 Hz, 3H). MS: m/z 280.06 [M + H]+.
4-Methyl-8-(trifluoromethyl)-2,3,4,5-tetrahydro-1H-1λ6,
2-benzothiazepine-1,1-dione (19). The title compound was
prepared from compound 13 following the general procedure for
radical reaction, giving a white solid. Crystals were obtained from
DCM and hexane (1:4). Yield: 73%. Mp 99ꢀ101 ꢀC. 1H NMR (400
MHz, CDCl3) δ ppm 8.15 (s,1H), 7.67 (d, J = 7.74 Hz,1H), 7.39 (d, J =
7.89 Hz,1H), 4.76 (b, 1H), 3.60ꢀ3.10 (m, 4H), 1.97 (b, 1H), 0.96 (d,
J = 6.73 Hz, 3H). MS: m/z 280.06 [M + H]+.
7-Methoxy-4-methyl-2,3,4,5-tetrahydro-1H-1λ6,2-benzo-
thiazepine-1,1-dione (20). The title compound was prepared from
compound 14 following the general procedure for radical reaction,
1
giving an oil. Yield: 95%. H NMR (400 MHz, CDCl3) δ ppm 7.71
(d, J = 8.42 Hz, 1H), 6.66ꢀ6.1 (m, 2H), 4.56 (t, J = 6.78 Hz, 1H), 3.76
(s, 3H), 3.50ꢀ2.90 (m, 4H), 1.85 (b, 1H), 0.86 (d, J = 7.32 Hz, 3H).
HRMS calcd for C11H16NO3S [M + H]+ 242.0845; found 242.0852.
tert-Butyl N-[(1S,2R)-1-Benzyl-3-(1,1-dioxo-1,3,4,5-tetra-
hydro-2H-1λ6,2-benzothiazepin-2-yl)-2-hydroxypropyl]car-
bamate (22). To a solution of compound 15 (75 mg, 0.37 mmol) in N,
N-dimethylformamide (DMF) (3 mL) was added (2S,3S)-1,2-epoxy-
3-(bocamino)-4-phenylbutane (21) (100 mg, 0.37 mmol), followed by
the addition of cesium carbonate (247 mg, 0.75 mmol). The reaction
mixture was stirred for 10ꢀ12 h at room temperature under nitrogen.
TLC was used to monitor the progress of the reaction. The reaction
mixture was filtered, diluted with ethyl acetate, and then washed with
water. The combined organic layer was dried over anhydrous sodium
sulfate, filtered, and evaporated to yield the crude compound which was
purified by column chromatography using 25% ethyl acetate in hexane to
yield the title compound. Yield: 57%. HPLC: >98% pure. NMR data were
consistent with the proposed structure. MS (ESI): m/z 405.05 [M + H]+.
2-{[(4S,5R)-4-Benzyl-2-oxo-1,3-oxazolidin-5-yl]methyl}-
2,3,4,5-tetrahydro-1H-1 λ6,2-benzothiazepine-1,1-dione
(23). To a solution of compound 22 (50 mg, 0.13 mmol) in DCM
was added 30% trifluoroacetic acid in DCM solution, and the mixture
was stirred for 1 h to obtain compound 23a. In flask 2, isopropyl alcohol
(1 equiv) and carbonyl diimidazole (1 equiv) were allowed to react.
Compound 23a was dissolved in toluene, and the mixture was main-
tained at 0 ꢀC. To this was added N,N-diisopropylethylamine followed
by the mixture in flask 2. The resulting reaction mixture was stirred at
room temperature for 6 h. The reaction mixture was evaporated to
dryness and was purified by column chromatography using 20% ethyl
acetate in hexane as mobile phase. Yield: 31%. HPLC: >98% pure. 1H
NMR (400 MHz, CDCl3) δ ppm 7.90 (dd, J = 7.75, 1.11 Hz, 1H), 7.47
(dt, J = 7.45, 1.14 Hz, 1H), 7.39ꢀ7.22 (m, 5H), 7.11 (d, J = 6.91 Hz,
2H), 4.95ꢀ5.01 (m, 1H), 4.79 (s, 1H), 4.19ꢀ4.05 (m, 2H), 3.79ꢀ3.42
(m, 2H), 3.38 (dd, J = 14.86, 3.22 Hz, 1H), 3.12ꢀ2.92 (m, 2H), 2.87
(dd, J = 13.29, 3.30 Hz, 1H), 2.52 (dd, J = 12.83, 11.61 Hz, 1H),
1.90ꢀ1.79 (m, 2H).
2,3,4,5-Tetrahydro-1H-1λ6,2-benzothiazepine-1,1-dione
(15). The title compound was prepared starting from 9 following the
general procedure for radical reaction, giving a white solid. Crystals were
obtained from DCM and hexane (1:4). Yield: 44%. Mp 107ꢀ108 ꢀC. 1H
NMR (400 MHz, CDCl3) δ ppm 7.88 (d, J = 7.69 Hz, 1H), 7.41 (dt, J =
7.49, 1.30 Hz, 1H), 7.35ꢀ7.22 (m, 2H), 4.58 (b, 1H), 3.60 (dd, J = 10.71,
6.11 Hz, 2H), 3.30ꢀ3.25 (m, 2H), 1.80ꢀ1.79 (m, 2H).
4-Methyl-2,3,4,5-tetrahydro-1H-1λ6,2-benzothiazepine-
1,1-dione (16). The title compound was prepared starting from 10
following the general procedure for radical reaction, giving a white solid.
Crystals were obtained from DCM and hexane (1:5). Yield: 85%. Mp
121ꢀ123 ꢀC. 1H NMR (400 MHz, CDCl3) δ ppm 7.85 (dd, J = 7.76,
1.23 Hz, 1H), 7.41 (dt, J = 7.53, 7.53, 1.32 Hz, 1H), 7.28ꢀ7.21 (m, 2H),
4.70 (b, 1H), 3.6ꢀ3.0 (m, 4H), 1.93 (b, 1H), 0.93 (d, J = 6.87 Hz, 3H).
7-Fluoro-4-methyl-2,3,4,5-tetrahydro-1H-1λ6,2-benzoth-
iazepine-1,1-dione (17). The title compound was prepared from
compound 11 following the general procedure for radical reaction,
giving a white solid. Crystals were obtained from DCM and hexane
tert-Butyl N-[(1R,2S)-1-Benzyl-2-hydroxy-3-(4-methyl-1,1-
dioxo-1,3,4,5-tetrahydro-2H-1λ6,2-benzothiazepin-2-yl)pr-
opyl]carbamate (24). To a solution of compound 16 (187 mg, 0.71
mmol) in DMF (3 mL) was added compound 21 (150 mg, 0.71 mmol)
followed by cesium carbonate (462 mg, 1.42 mmol). The mixture was
stirred for 10ꢀ12 h at room temperature under nitrogen. TLC was used
to monitor the progress of the reaction. The reaction mixture was
1
(1:5). Yield: 86%. Mp 144ꢀ146 ꢀC. H NMR (400 MHz, CDCl3) δ
ppm 7.86 (dd, J = 9.14, 5.70 Hz,1H), 6.99ꢀ6.91 (m, 2H), 4.70 (t, J =
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filtered. Water was added and then extracted using ethyl acetate. The
combined organic layer was dried over anhydrous sodium sulfate,
filtered, and evaporated yielding the crude compound which was
purified by column chromatography using 25% ethyl acetate in hexane
to yield compound 24 (170 mg, 50%) as an oil. HPLC: >98% pure. 1H
NMR data were consistent with the structure (mixture of dia-
stereoisomers). MS (ESI): m/z 457.05 [M ꢀ 18 + H]+.
7.43 (dt, J = 7.48, 7.46, 1.37 Hz, 1H), 7.36ꢀ7.17 (m, 7H), 4.69 (d, J =
6.73 Hz, 1H), 4.10 (s, 1H), 3.95ꢀ3.73 (m, 3H), 3.55 (s, 1H), 3.24 (s,
1H), 3.10 (dd, J = 14.84, 5.16 Hz, 1H), 3.02ꢀ2.86 (m, 1H), 2.70 (d, J =
11.08 Hz, 2H), 1.96 (s, 1H), 1.63 (s, 1H), 1.33 (s, 9H), 0.99 (d, J = 6.70
Hz, 3H). HRMS calcd for C25H35N2O5S [M + H]+ 475.2261; found
475.2271.
Allyl N-{(1R,2S)-1-Benzyl-2-hydroxy-3-[(4S)-4-methyl-1,1-
dioxo-1,3,4,5-tetrahydro-2H-1λ6,2-benzothiazepin-2-yl]pr-
(4R)-2-[(2S,3R)-3-Amino-2-hydroxy-4-phenylbutyl]-4-me-
thyl-2,3,4,5-tetrahydro-1H-1λ6,2-benzothiazepine-1,1-dione
(25) and (4S)-2-[(2S,3R)-3-Amino-2-hydroxy-4-phenylbutyl]-
4-methyl-2,3,4,5-tetrahydro-1H-1λ6,2-benzothiazepine-1,
1-dione (26). Compound 24 (125 mg, 0.263 mmol) was dissolved in
a mixture of DCM and trifluoroacetic acid (1.5 mL/1.5 mL), and the
mixture was stirred at room temperature under nitrogen for 3ꢀ4 h.
TLC was used to monitor the progress of the reaction. After the
reaction was complete, the reaction mixture was basified with sodium
hydroxide solution (50%) and the aqueous portion was extracted with
ethyl acetate. The combined organic layer was dried with anhydrous
sodium sulfate and evaporated to yield the crude compound. Com-
pounds 25 (40 mg, 41%) and 26 (50 mg, 51%) were separated by
preparative TLC (6% methanol in chloroform) and were recrystallized
from DCM/hexane (1:4).
1
opyl}carbamate (29). HPLC: >98% pure. H NMR (400 MHz,
CDCl3) δ ppm 7.90 (dd, J = 7.74, 1.31 Hz, 1H), 7.43 (dt, J = 7.54, 7.52,
1.36 Hz, 1H), 7.37ꢀ7.16 (m, 7H), 5.81 (ddd, J = 15.76, 9.78, 5.08 Hz,
1H), 5.30ꢀ5.07 (m, 2H), 4.95 (d, J = 7.23 Hz, 1H), 4.46 (dd, J = 6.56,
3.54 Hz, 2H), 4.00ꢀ3.71 (m, 3H), 3.55 (s, 1H), 3.30ꢀ2.85 (m, 4H),
2.69 (d, J = 10.64 Hz, 2H), 1.95 (s,1H), 1.25 (s, 1H), 0.99 (d, J = 6.69 Hz,
3H). HRMS calcd for C24H31N2O5S [M + H]+ 459.1953; found
459.1946.
Ethyl N-{(1R,2S)-1-Benzyl-2-hydroxy-3-[(4S)-4-methyl-
1,1-dioxo-1,3,4,5-tetrahydro-2H-1λ6,2-benzothiazepin-2-
yl]propyl}carbamate (30). HPLC: >98% pure. 1H NMR (400
MHz, CDCl3) δ ppm 7.91 (dd, J = 7.70, 1.32 Hz, 1H), 7.44 (dt, J =
7.52, 7.52, 1.43 Hz, 1H), 7.35ꢀ7.16 (m, 7H), 4.89 (s, 1H), 4.05ꢀ3.77
(m, 4H), 3.55 (s, 1H), 2.90ꢀ3.21 (m, 7H), 1.96 (s, 2H), 1.2ꢀ1.15 (m,
3H), 1.00 (d, J = 6.75 Hz, 3H). HRMS calcd for C23H30N2O5S [M +
Na]+ 469.1773; found 469.1773.
Phenyl N-{(1R,2S)-1-Benzyl-2-hydroxy-3-[(4S)-4-methyl-
1,1-dioxo-1,3,4,5-tetrahydro-2H-1λ6,2-benzothiazepin-2-
yl]propyl}carbamate (31). HPLC >98% pure. 1H NMR (400 MHz,
CDCl3) δ ppm 7.93 (dd, J = 7.73, 1.31 Hz, 1H), 7.45 (dt, J = 7.50,
7.47, 1.39 Hz, 1H), 7.36ꢀ7.13 (m, 10H), 6.94 (d, J = 7.67 Hz, 2H), 5.31
(d, J = 7.68 Hz, 1H), 4.03ꢀ3.84 (m, 3H), 3.70ꢀ3.41 (m, 2H), 3.24ꢀ3.12
(m, 3H), 2.96 (dd, J = 13.50, 8.88 Hz, 1H), 2.73 (d, J = 12.33 Hz, 2H),
1.97 (s, 1H), 1.00 (d, J = 6.70 Hz, 3H). HRMS calcd for C27H31N2O5S
[M + H]+ 495.1948; found 495.1962.
tert-Butyl N-[(1R,2S)-1-Benzyl-3-[(4S)-7-fluoro-4-methyl-
1,1-dioxo-1,3,4,5-tetrahydro-2H-1λ6,2-benzothiazepin-2-
yl]-2-hydroxypropyl]carbamate (32). HPLC: >98% pure. 1H
NMR (400 MHz, CDCl3) δ ppm 7.95ꢀ7.87 (m, 1H), 7.32ꢀ7.17 (m,
5H), 7.02ꢀ6.96 (m, 2H), 4.67 (d, J = 6.38 Hz, 1H), 4.08 (s, 1H),
3.95ꢀ3.71 (m, 2H), 3.54 (s, 1H), 3.24 (s, 1H), 3.16ꢀ2.83 (m, 4H), 2.69
(d, J = 10.50 Hz, 2H), 1.96 (s, 1H), 1.34 (s, 9H), 0.99 (d, J = 6.74 Hz,
3H). MS (ESI): m/z 493.21 [M + H]+. HRMS calcd for C20H26FN2O3S
[M + H-Boc]+ 393.1648; found 393.1653.
tert-Butyl N-{(1S,2R)-1-Benzyl-2-hydroxy-3-[(4S)-4-meth-
yl-1,1-dioxo-7-(trifluoromethyl)-1,3,4,5-tetrahydro-2H-1λ6,-
2-benzothiazepin-2-yl]propyl}carbamate (33). HPLC: >98%
pure. 1H NMR (400 MHz, CDCl3) δ ppm 8.02 (d, J = 7.98 Hz, 1H),
7.66ꢀ7.45 (m, 2H), 7.39ꢀ7.10 (m, 5H), 4.67 (d, J = 6.91 Hz, 1H),
4.18ꢀ4.02 (m, 1H), 3.99ꢀ3.67 (m, 3H), 3.72ꢀ3.48 (m, 1H),
3.43ꢀ3.19 (m, 1H), 3.09 (dd, J = 14.51, 5.32 Hz, 1H), 3.03ꢀ2.59
(m, 4H), 2.08ꢀ1.98 (m, 1H), 1.32 (s, 9H), 1.02 (d, J = 6.34 Hz, 3H).
HRMS calcd for C21H26F3N2O3S [M + H ꢀ Boc]+ 443.161 62; found
443.160 71.
tert-Butyl N-{(1S,2R)-1-Benzyl-2-hydroxy-3-[(4S)-4-meth-
yl-1,1-dioxo-8-(trifluoromethyl)-1,3,4,5-tetrahydro-2H-1λ6,-
2-benzothiazepin-2-yl]propyl}carbamate (34). HPLC: >98%
pure. 1H NMR (400 MHz, CDCl3) δ ppm 8.15 (d, J = 1.18 Hz, 1H),
7.68 (d, J = 7.95 Hz, 1H), 7.41 (d, J = 7.86 Hz, 1H), 7.31ꢀ7.19 (m, 5H),
4.69 (d, J = 7.74 Hz, 1H), 4.11 (dd, J = 14.28, 7.13 Hz, 1H), 3.97ꢀ3.18
(m, 5H), 3.16ꢀ2.71 (m, 5H), 2.03ꢀ1.84 (m, 1H), 1.32 (s, 9H), 1.01
(d, J = 6.56 Hz, 3H). HRMS calcd for C26H34F3N2O5S [M + H]+
543.2135; found 543.2144.
1
Compound 25. Mp 153ꢀ155 ꢀC. H NMR (400 MHz, CDCl3) δ
ppm 7.91 (dd, J = 7.74, 1.24 Hz, 1H), 7.48ꢀ7.41 (m, 1H), 7.36ꢀ7.14
(m, 7H), 3.87 (t, J = 13.48 Hz, 1H), 3.60 (s, 2H), 3.19ꢀ3.03 (m, 3H),
3.00ꢀ2.86 (m, 2H), 2.72 (s, 1H), 2.54 (dd, J = 13.51, 9.38 Hz, 1H),
1.99 (s, 4H), 1.02 (d, J = 6.81 Hz, 3H). HRMS calcd for C20H27N2O3S
[M + H]+ 375.1737; found 375.1737.
1
Compound 26. Mp 117ꢀ119 ꢀC. H NMR (400 MHz, CDCl3) δ
ppm 7.92 (dd, J = 7.69, 1.21 Hz, 1H), 7.45 (dt, J = 7.54, 7.53, 1.34 Hz,
1H), 7.36ꢀ7.17 (m, 7H), 3.86 (ddd, J = 16.10, 11.48, 7.92 Hz, 2H), 3.57
(s, 1H), 3.35 (s, 1H), 3.21ꢀ3.06 (m, 2H), 2.98 (dd, J = 13.29, 3.12 Hz,
1H), 2.90ꢀ2.62 (m, 2H), 2.44 (dd, J = 13.45, 10.22 Hz, 1H), 2.04ꢀ1.95
(m, 3H), 1.28ꢀ1.20 (m, 1H), 1.02 (d, J = 6.80 Hz, 3H). HRMS calcd for
C20H27N2O3S [M + H]+ 375.1737; found 375.1736.
General Procedure for Carbamate Formation, 27, 28,
34ꢀ40. To an ice cold solution of compound 25 (1 equiv) in THF,
diisopropylethylamine (DIPEA) (1 equiv) was added followed by the
addition of di-tert-butyl dicarbonate/alkyl chlorofomate (1 equiv) in
THF. The reaction mixture was slowly allowed to reach room tempera-
ture and stirring was continued for 10ꢀ12 h under nitrogen. The
progress of the reaction was monitored by TLC. When the reaction was
complete, the reaction mixture was diluted with water and extracted with
chloroform. The combined organic layer was dried over anhydrous
sodium sulfate and evaporated to yield the crude compound. The crude
compound was purified by column chromatography using ethyl acetate
and hexane.
tert-Butyl N-{(1R,2S)-1-Benzyl-2-hydroxy-3-[(4R)-4-meth-
yl-1,1-dioxo-1,3,4,5-tetrahydro-2H-1λ6,2-benzothiazepin-
2-yl]propyl}carbamate (27). The title compound was synthesized
from compound 25, diisopropylethylamine (DIPEA), and di-tert-butyl
dicarbonate using the general procedure for carbamate formation. Yield:
89%. HPLC: >98% pure. 1H NMR (400 MHz, CDCl3) δ ppm 7.89 (dd,
J = 7.59, 1.09 Hz, 1H), 7.44 (dt, J = 7.53, 7.50, 1.39 Hz, 1H), 7.33 (dt, J =
7.64, 7.55, 1.09 Hz, 1H), 7.28ꢀ7.14 (m, 6H), 4.71 (b, 1H), 3.94ꢀ3.40
(m, 4H), 3.24ꢀ2.59 (m, 5H), 1.88 (s, 1H), 1.60 (s, 2H), 1.32 (s, 9H),
0.99 (d, J = 6.73 Hz, 3H). HRMS calcd for C25H35N2O5S [M + H]+
475.2261; found 475.2271.
tert-Butyl N-{(1R,2S)-1-Benzyl-2-hydroxy-3-[(4S)-4-meth-
yl-1,1-dioxo-1,3,4,5-tetrahydro-2H-1λ6,2-benzothiazepin-
2-yl]propyl}carbamate (28). The title compound was synthesized
from 26, DIPEA, and di-tert-butyl dicarbonate using the general
procedure for carbamate formation. Yield: 75%. HPLC: >98% pure.
1H NMR (400 MHz, CDCl3) δ ppm 7.91 (dd, J = 7.69, 1.32 Hz, 1H),
tert-Butyl N-{(1S,2R)-1-Benzyl-2-hydroxy-3-[(4S)-7-meth-
oxy-4-methyl-1,1-dioxo-1,3,4,5-tetrahydro-2H-1λ6,2-ben-
zothiazepin-2-yl]propyl}carbamate (35). HPLC: >98% pure. 1H
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Journal of Medicinal Chemistry
ARTICLE
NMR (400 MHz, CDCl3) δ ppm 7.84 (d, J = 9.32 Hz, 1H), 7.32ꢀ7.15
(m, 5H), 6.77 (d, J = 5.75 Hz, 2H), 4.69 (d, J = 4.87 Hz, 1H), 4.20ꢀ3.98
(m, 1H), 3.84 (s, 3H), 3.51 (s, 1H), 3.36ꢀ2.83 (m, 5H), 2.80ꢀ2.51
(m, 3H), 2.03ꢀ1.84 (m, 2H), 1.34 (s, 9H), 0.99 (d, J = 6.71 Hz, 3H).
MS (ESI): m/z 505.24 [M + H]+. HRMS calcd for C26H36N2O6S
[M + Na]+ 527.2186; found 527.2175.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +1-201-216-5540. Fax: +1-201-216-8240. E-mail:
akganguly1@aol.com.
(5S)-N-{(1R,2S)-1-Benzyl-3-[(4S)-7-fluoro-4-methyl-1,1-di-
oxo-1,3,4,5-tetrahydro-2H-1λ6,2-benzothiazepin-2-yl]-2-
hydroxypropyl}-2-oxo-3-phenyl-1,3-oxazolidine-5-carbox-
amide (40). To a solution of compound 37 (50 mg, 0.127 mmol) in
DCM was added dicyclohexylcarbodiimide (DCCI) (24.1 mg, 0.191
mmol), and the mixture was stirred. Compound 39 (29.0 mg, 0.14
mmol) was added, and the mixture was stirred for 10ꢀ12 h at room
temperature under nitrogen. The progress of the reaction was mon-
itored by TLC. Upon completion of the reaction, the reaction mixture
was extracted with DCM and washed with water. The combined organic
layer was dried with anhydrous sodium sulfate, filtered, and evaporated
to yield the crude compound which was purified by preparative TLC
(7.5% methanol in chloroform) (15 mg, 20%). HPLC: >98% pure.
NMR data were consistent with the proposed structure. MS (ESI): m/z
582.20 [M + H]+.
In Vitro HIV-1 Protease Assay. Compounds were evaluated for
the ability to inhibit the enzymatic activity of HIV-1 protease in an in
vitro assay.7 Protease activity was assessed in reactions catalyzed by
purified HIV-1 protease at 15 pM in buffer (50 mM sodium acetate, pH
5.5, 100 mM NaCl, 1 mg/mL bovine serum albumin) using a peptide
substrate with sequence Val-Ser-Gln-Asn-(β-naphthylalanine)-Pro-Ile-
Val at 440 μM in a final volume of 80 μL. Compounds in DMSO stock
were added to a final DMSO concentration of 2.5% and preincubated
with enzyme prior to the initiation of the reaction by addition of
substrate. Inhibitor dose titrations were used to determine IC50 values.
Reactions were incubated at 30 ꢀC for 60 min and were then quenched
by the addition of 120 μL of 10% H3PO4. The amount of product
formed was determined using high performance liquid chromatography
with a Vydac C18 column and fluorescence detection of product.
Percent inhibition was determined relative to control samples without
inhibitor, and IC50 values were determined using a standard four-
parameter fit to the inhibition data. Ki values were determined from
eq 1 for competitive inhibitors,
’ ACKNOWLEDGMENT
We thank Stevens Institute of Technology, Hoboken, NJ, for
generous financial support.
’ ABBREVIATIONS USED
HIV, human immunodeficiency virus; AIDS, acquired immuno
deficiency syndrome; Ki, absolute inhibition constant; IC50, half
maximal inhibitory concentration; AIBN, azobisisobutyronitrile;
DCM, dichloromethane; THF, tetrahydrofuran; DMF, N,N-di-
methylformamide; TLC, thin layer chromatography
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ð1Þ
X-ray Crystallography. Crystallographic data for 25 have been
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the whole data set and were 48.9% and 2.8 for the highest resolution
shell, respectively. Data were reduced to space group P21212 with unit
cell dimensions of a = 58.6 Å, b = 85.9 Å, c = 46.2 Å and one dimer per
asymmetric unit. The structure was refined using Buster10 and refitted
using Coot,11 resulting in final Rwork (Rfree) of 18.0% (19.1%) and
excellent geometry. Figures were generated using Pymol.12
Accession Codes
†The PDB code for HIV-1 protease complex with 32 is 3TH9.
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Journal of Medicinal Chemistry
ARTICLE
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