K. Krajewski et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3034–3038
3037
Table 2. Ex vivo sensitivity of wild-type and NNRTI-resistant HIV-1
RTs to nevirapine, efavirenz, and NSC-380292
1i
40000
30000
20000
10000
0
1ii
6i
No.
HIV-RT
IC50 (lM)
6ii
7i
Nevirapine
Efavirenz
NSC-380292
1
2
3
4
Wild-type
K103N
Y181C
0.060
5.750
6.650
0.860
0.0008
0.0540
0.0060
0.0095
1.2
>30
>30
>30
-10000
-20000
-30000
-40000
-50000
-60000
-70000
Y188C
these inhibitors take up a butterfly-like overall shape in
14
their structure.
In this respect, this family of reverse
transcriptase inhibitors resembles the other NNRTIs.15
Our observations that mutations in HIV-1 RT associat-
ed with resistance to other clinically available NNRTIs
strongly support the view that the NSC-380292 com-
pound is an HIV-1 RT inhibitor and that it binds to
the enzyme in a manner that is similar to other NNRTIs
used to treat HIV-1 infected patients. The results of
HIV-1 reverse transcriptase inhibitory assays for NSC-
380292 (1) demonstrated that only one enantiomer
(R,R; 1i) is responsible for the sample inhibitory activity.
This is also true for all analogues we tested in the homo-
chiral form. The lack of activity of the analogs with R2
other than Me (2 and 3) and a low tolerance for the
changes of R1 suggest that the lead compound (1) is a
unique reverse transcriptase inhibitor. In this regard, it
is found that a number of NNRTIs have been reported
to directly inhibit the reverse transcriptase enzyme in an
allosteric fashion by binding to a pocket near the poly-
merase active site.
195
215
235
255
275
295
315
Wavelength / nm
Figure 4. The CD spectra of compounds 1i (solid gray line), 1ii (solid
black line), 6i (dashed gray line), 6ii (dashed black line) and 7i (dotted
gray line). The CD spectra first fractions from chiral HPLC (1i, 6i, and
7i) (gray lines) or S,S (black lines) reflect the absolute configurations at
the bridgehead carbon atoms of the stereoisomers. Spectra were
collected at 23 ꢁC (0.2 mM in MeOH).
Table 1. Results of ex vivo HIV-1 RT inhibitory assay for compounds
1–10, the R,R or S,S represent absolute configurations of chiral centers
at 2a and 7b carbon atoms
Compounds
IC50 (lM)
No. R1
R2
R,R/S,S R,R S,S
1
2
3
4
5
6
1-Pyrrolidinyl-
1-Pyrrolidinyl-
1-Pyrrolidinyl-
Me
Et
1.2
>20
0.8 >20
>20 >20
The current structure/activity study provides an opti-
mized structure, in compounds 1i and 4i, with R config-
urations at the chiral centers 2a and 7b.
t-Bu >20
—
—
11
R-2-Methyl-1-pyrrolidinyl- Me
S-2-Methyl-1-pyrrolidinyl- Me
—
—
48
0.5
2.3
38
>25
>50
S-2-Methoxymethyl-1-
pyrrolidinyl-
1-Piperidinyl-
Me
7
8
Me
Me
Me
Me
8.9
>10
5.1
50
7.7
—
—
—
>40
—
Acknowledgments
1-Azetidinyl-
2,5-Dihydro-1-pyrrolyl-
Dimethylamino-a
9
10
—
—
We are grateful to Dr. Sheng Jiang for advice, helpful
discussions, and a critical reading of the manuscript.
This research was supported by the Intramural Research
Program of the NIH, National Cancer Institute, Center
for Cancer Research.
a 5-Methoxy-, see Scheme 2.
HIV-1 RT mutations that confer resistance to NNRTIs
also confer resistance to NSC-380292. The sensitivity
of NSC-380292 to HIV-1 RT variants containing
mutations associated with clinical resistance to NNR-
TIs was determined. Mutations K103N, Y181C, and
Y188C are associated with high-level resistance in
treated patients to NNRTIs nevirapine and efavi-
renz.13 HIV-1 vectors that expressed the firefly lucifer-
ase gene and contained wild-type RT or mutant RTs
containing the K103N, Y181C, and Y188C substitu-
tions were cotransfected into 293 T-cells with
pHCMV-G; viruses harvested from the transfected
cells were used to infect target cells in the presence
of increasing concentrations of nevirapine, efavirenz,
or NSC-380292 and the IC50 concentrations were
determined 48–72 h after infection (Table 2).
References and notes
1. Imamichi, T. Curr. Pharm. Des. 2004, 10, 4039.
2. Tronchet, J. M.; Seman, M. Curr. Top. Med. Chem. 2003,
3, 1496.
3. Tarby, C. M. Curr. Top. Med. Chem. 2004, 4, 1045.
4. Balzarini, J. Curr. Top. Med. Chem. 2004, 4, 921.
5. Darr, E. S.; Richman, D. D. AIDS Res. Hum. Retroviruses
2005, 21, 343.
6. 1.1 equiv of n-BuLi (1.6 M solution in hexane,) was added
into thionaphthene solution in dry Et2O, under argon,
after 3 h at room temperature 1.1 equiv of Br2 was added
dropwise within 10 min, at 0 ꢁC. The resulting solution
was stirred at 0 ꢁC under argon, for 2 h, washed with 5%
KOH, brine, dried with MgSO4, and the solvent evapo-
rated. Product was purified by flash chromatography (Isco
CombiFlash system, silica gel, linear gradient from 100%
hexanes to 100% EtOAc within 40 min).
It is clear from the study of the X-ray structures of the
active reverse transcriptase inhibitors (4, 6, and 7), that