7706 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Ali et al.
(t, J = 2.0 Hz, 1H), 8.05 (m, 1H), 7.93-7.90 (m, 2H), 7.60 (t, J =
8.4 Hz, 1H), 7.13 (d, J = 7.2 Hz, 2H), 7.02 (t, J = 7.2 Hz, 2H),
6.85 (t, J = 7.2 Hz, 1H), 6.79 (d, J = 10.0 Hz, 1H), 4.84 (dd, J =
9.6, 5.2 Hz, 1H), 4.29 (m, 1H), 4.09 (t, J = 9.2 Hz, 1H), 3.99 (m,
1H), 3.41 (dd, J = 9.2, 6.0 Hz, 1H), 3.29 (dd, J = 15.6, 9.6 Hz,
1H), 3.14-3.05 (m, 3H), 2.93 (dd, J = 13.2, 6.4 Hz, 1H), 2.78
(dd, J = 13.6, 10.4 Hz, 1H), 1.89 (m, 1H), 0.96 (d, J = 6.8 Hz,
3H), 0.91 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
168.36, 158.38, 155.90, 152.82, 148.86, 138.69, 137.44, 135.68,
134.69, 130.33, 129.61 (2C), 128.62 (2C), 126.67, 125.05, 124.75,
123.92, 122.59, 119.35, 113.01, 72.58, 69.98, 59.12, 53.91, 53.45,
48.10, 35.53, 27.57, 20.38, 20.13. HRMS (ESI) m/z calcd for
C31H34N5O8S [M þ H]þ 668.1849; found 668.1852.
To an ice-cooled solution of compound 29d (0.1 g, 0.157 mmol)
in CH2Cl2 (5 mL) was added an aqueous solution of Na2CO3
(0.026 g, 0.25 mmol) in water (2 mL) followed by the slow
addition of methylchloroformate (12.3 μL, 0.157 mmol). After
15 min, the reaction mixture was warmed to ambient tempera-
ture and stirred until no starting material was detected by TLC
(2 h). Reaction mixture was diluted with CH2Cl2, and layers
were separated. The organic extract was washed with saturated
aqueous NaCl solution (5 mL), dried (Na2SO4), filtered, and
evaporated. The residue was purified by flash chromatography
on silica gel, eluting with a mixture of 2% MeOH in EtOAc to
afford the target compound 31d (0.095 g, 87%) as a white solid.
1H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 8.49 (d, J = 1.6 Hz,
1H), 8.25 (d, J = 8.8 Hz, 1H), 7.91 (dd, J = 8.8, 2.0 Hz, 1H), 7.76
(t, J = 1.6 Hz, 1H), 7.34-7.26 (m, 2H), 7.13 (d, J = 7.2 Hz, 2H),
7.06-6.89 (m, 6H), 4.76 (dd, J = 10.0, 6.0 Hz, 1H), 4.23 (m, 1H),
4.03 (t, J = 10.0 Hz, 2H), 3.82 (br s, 1H), 3.79 (s, 3H), 3.34 (dd,
J = 9.2, 6.0 Hz, 1H), 3.25 (dd, J = 15.6, 9.2 Hz, 1H), 3.19-3.10
(m, 2H), 3.05 (dd, J = 13.6, 8.0 Hz, 1H), 2.94 (dd, J = 13.6,
7.2 Hz, 1H), 2.79 (dd, J = 13.6, 10.8 Hz, 1H), 1.90 (m, 1H), 0.94 (d,
J = 6.8 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ 168.94, 158.31, 155.85, 154.23, 153.21, 139.12, 138.18,
137.57, 135.78, 134.63, 129.94, 129.57 (2C), 128.65 (2C), 126.82,
125.08, 124.69, 122.58, 114.88, 113.11, 108.87, 72.60, 69.94, 59.04,
53.89, 53.75, 52.73, 48.45, 35.88, 27.51, 20.36, 20.12. HRMS (ESI)
m/z calcd for C33H38N5O8S2 [M þ H]þ 696.2162; found 696.2158.
HIV-1 Protease Inhibition Assays. The HIV-1 protease inhibitory
potencies of all compounds were determined by a fluorescence
resonance energy transfer (FRET) method.20,25,28 Protease sub-
strate (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln Lys-
(DABCYL)-Arg) was purchased from Molecular Probes. The
energy transfer donor (EDANS) and acceptor (DABCYL)
dyes were labeled at two ends of the peptide, respectively, to
perform FRET. Fluorescence measurements were carried
out on a fluorescence spectrophotometer (Photon Technology
International) at 30 °C. Excitation and emission wavelengths
were set at 340 and 490 nm, respectively. Each reaction was
recorded for about 10 min. The expression, isolation, and purifi-
cation of wild-type and mutant HIV-1 protease used for
binding experiments were carried out as previously described.29
Wild-type HIV-1 protease (Q7K) and its MDR variant (L10I,
L63P, A71V, G73S, I84V, L90M) were desalted through PD-
10 columns (Amersham Biosciences). Sodium acetate (20 mM,
pH 5) was used as the elution buffer. Apparent protease con-
centrations were around ∼50 nM as estimated by UV spectro-
photometry at 280 nm. All inhibitors were dissolved in
dimethylsulfoxide (DMSO) and diluted to appropriate con-
centrations. Protease (2 μL) and inhibitor (2 μL) or DMSO
were mixed and incubated for 20-30 min at room temperature
before initializing the substrate cleavage reaction. For all experi-
ments, 150 μL of a 1 μM substrate were used in substrate buffer
[0.1 M sodium acetate, 1 M sodium chloride, 1 mM ethylene-
diaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT),
2% DMSO, and 1 mg/mL bovine serum albumin (BSA) with
an adjusted pH of 4.7]. Inhibitor binding dissociation constant
(Ki) values were obtained by nonlinear regression fitting
(GraFit 5, Erithacus software) of the plot of initial velocity
as a function of inhibitor concentrations based on the Morrison
equation.30 The initial velocities were derived from the linear
range of reaction curves.
(5S)-3-(3-Aminophenyl)-N-[(1S,2R)-3-[(6-benzothiazolylsulfonyl)-
(2-methylpropyl)amino]-2-hydroxy-1-(phenylmethyl)propyl]-
2-oxooxazolidine-5-carboxamide (29d). A mixture of the above
nitro compound 28d (0.335 g, 0.5 mmol) and SnCl2.2H2O (0.565 g,
2.5 mmol) in EtOAc (20 mL) was heated at 80 °C for 3 h. The
reaction mixture was allowed to cool to ambient temperature
and treated with saturated aqueous NaHCO3 solution (15 mL).
The mixture was diluted with EtOAc (50 mL), and layers were
separated. The aqueous layer was further extracted with EtOAc
(2ꢀ 50 mL). The combined organic extract was washed with saturated
aqueous NaCl solution (2 ꢀ 40 mL), dried (Na2SO4), filtered,
and evaporated. The residue was purified by flash chromatog-
raphy on silica gel, eluting with a mixture of 1% MeOH in
EtOAc to provide the target compound 29d (0.29 g, 91%) as
a white solid. 1H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 8.49
(d, J = 2.0 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H), 7.92 (dd, J = 8.8,
1.6 Hz, 1H), 7.17-7.12 (m, 3H), 7.09-7.04 (m, 3H), 6.96 (t, J =
7.6 Hz, 1H), 6.90 (d, J = 9.6 Hz, 1H), 6.55 (dd, J = 8.0, 1.6 Hz,
1H), 6.51 (dd, J = 8.0, 2.0 Hz, 1H), 4.73 (dd, J = 10.0, 6.4 Hz,
1H), 4.21 (m, 1H), 4.0 (t, J = 9.6 Hz, 2H), 3.85 (br s, 1H), 3.75
(br s, 1H), 3.32 (dd, J = 9.6, 6.4 Hz, 1H), 3.25 (dd, J = 15.2, 9.2
Hz, 1H), 3.14 (dd, J = 14.0, 4.8 Hz, 1H), 3.09-3.03 (m, 2H),
2.93 (dd, J = 13.6, 6.8 Hz, 1H), 2.73 (dd, J = 13.6, 10.8 Hz, 1H),
1.89 (m, 1H), 0.95 (d, J = 6.4 Hz, 3H), 0.91 (d, J = 6.4 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 169.07, 158.34, 155.87, 153.14,
147.55, 138.46, 137.47, 135.69, 134.66, 130.10, 129.53 (2C),
128.70 (2C), 126.85, 125.09, 124.72, 122.60, 111.81, 108.37,
105.48, 72.49, 68.87, 59.11, 53.99, 53.74, 48.47, 35.84, 27.55,
20.38, 20.12. HRMS (ESI) m/z calcd for C31H36N5O6S2 [M þ
H]þ 638.2107; found 638.2098.
(5S)-3-[3-(Acetylamino)phenyl]-N-[(1S,2R)-3-[(6-benzothiazolyl-
sulfonyl)(2-methylpropyl)amino]-2-hydroxy-1-(phenylmethyl)-
propyl]-2-oxooxazolidine-5-carboxamide (30d). To a solution of
compound 29d (0.1 g, 0.157 mmol) in dry CH2Cl2 was added
Ac2O (30 μL), and the resulting solution was stirred at room
temperature for 2 h. The solvents were evaporated under
reduced pressure and the residue was purified by flash chroma-
tography, eluting with 1% MeOH in EtOAc to afford the title
1
compound 30d (0.1 g, 94%) as a white solid. H NMR (400
MHz, CDCl3) δ 9.20 (s, 1H), 8.49 (d, J = 2.0 Hz, 1H), 8.23 (d,
J = 8.8 Hz, 1H), 7.95 (s, 1H), 7.91 (dd, J = 8.8, 1.6 Hz, 1H), 7.72
(s, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.35-7.26 (m, 2H), 7.14 (d, J =
7.2 Hz, 2H), 7.02 (t, J = 7.6 Hz, 2H), 6.90 (t, J = 7.6 Hz, 1H),
6.84 (d, J = 8.0 Hz, 1H), 4.76 (dd, J = 9.6, 6.0 Hz, 1H), 4.22 (m,
1H), 4.11 (m, 2H), 4.0 (t, J = 10.0 Hz, 1H), 3.31-3.15 (m, 4H),
3.05 (dd, J = 13.2, 7.6 Hz, 1H), 2.96 (dd, J = 13.6, 7.2 Hz, 1H),
2.83 (dd, J = 14.0, 10.8 Hz, 1H), 2.19 (s, 3H), 1.92 (m, 1H),
0.92 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H); 13C NMR
(100 MHz, CDCl3) δ 168.97, 158.34, 155.83, 153.43, 139.14,
137.98, 137.81, 135.84, 134.63, 129.87, 129.61 (2C), 128.58 (2C),
126.75, 125.08, 124.66, 122.56, 116.25, 113.81, 110.08, 72.63,
70.02, 58.96, 53.89, 53.80, 48.48, 36.02, 27.44, 24.89, 20.36,
20.11; HRMS (ESI) m/z calcd for C33H38N5O7S2 [M þ H]þ
680.2213; found 680.2225.
Antiviral Assays. Drug susceptibility assays were carried out
by Monogram Biosciences against three patient-derived strains
of wild-type HIV-1 from clades A, B, and C and against two
multidrug-resistant HIV-1 variants. Assays were carried out
according to protocols detailed by Petropoulos et al.31 and on
sion numbers for the PR/RT regions of the six HIV-1 strains
used in antiviral assays are as follows: WT-control, HQ179654;
WT-A, HQ179655; WT-B, HQ179656; WT-C, HQ179657;
MDR, HQ179658; MDR1, HQ179659.
(5S)-N-[(1S,2R)-3-[(6-Benzothiazolylsulfonyl)(2-methylpropyl)-
amino]-2-hydroxy-1-(phenylmethyl)propyl]amino]carbonyl]-
2-oxo-3-oxazolidinyl]phenyl]carbamic acid methyl ester (31d).