New HIV-1 reverse transcriptase inhibitors based on a tricyclic benzothiophene scaffold: Synthesis, resolution, and inhibitory activity

Article abstract of DOI:10.1016/j.bmcl.2006.02.049

We synthesized, separated into enantiomers, and tested for the HIV-1 reverse transcriptase inhibitory activity a group of analogs of dimethyl-1-(1-piperidynyl)cyclobuta[b][1]benzothiophene-2,2a(7bH)-dicarboxylate (NSC-380292). Absolute configurations of the enantiomers were determined based on absolute X-ray structures and analysis of CD spectra. Within pairs of enantiomers the (R,R)-enantiomer was always much more potent HIV-1 reverse transcriptase inhibitor.

Full text of DOI:10.1016/j.bmcl.2006.02.049

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3034–3038
New HIV-1 reverse transcriptase inhibitors based on a tricyclic
benzothiophene scaffold: Synthesis, resolution, and
inhibitory activity
Krzysztof Krajewski,a Yijun Zhang,b Damon Parrish,c Jeffrey Deschamps,c
Peter P. Rollera,* and Vinay K. Pathakb,*
aLaboratory of Medicinal Chemistry, CCR, NCI-Frederick, NIH, Frederick, MD 21702, USA
bHIV Drug Resistance Program, NCI-Frederick, NIH, Frederick, MD 21702, USA
cNaval Research Laboratory, Washington, DC 20375, USA
Received 25 January 2006; revised 17 February 2006; accepted 20 February 2006
Available online 9 March 2006
Abstract—We synthesized, separated into enantiomers, and tested for the HIV-1 reverse transcriptase inhibitory activity a group of
analogs of dimethyl-1-(1-piperidynyl)cyclobuta[b][1]benzothiophene-2,2a(7bH)-dicarboxylate (NSC-380292). Absolute configura-
tions of the enantiomers were determined based on absolute X-ray structures and analysis of CD spectra. Within pairs of enanti-
omers the (R,R)-enantiomer was always much more potent HIV-1 reverse transcriptase inhibitor.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The HIV-1 reverse transcriptase (RT) is one of the
enzymes crucial for the HIV virus replication cycle.
Upon entering a host cell, RT converts the single-
stranded viral RNA into double-stranded DNA, which
is then inserted into the genome of the host cell. RT
has a DNA polymerase domain that can copy either
RNA or DNA, and an RNase H domain that cleaves
RNA. RNase H is required for the degradation of the
RNA template after it has been copied into DNA, which
then permits the synthesis of the second DNA strand by
DNA polymerase. These enzymatic activities are essen-
tial for viral replication, therefore RT is an important
target for anti-HIV drug development and its inhibitors
are widely used as anti-HIV drugs.1 Two major classes
of HIV-1 RT inhibitors are currently used as anti-viral
drugs. Nucleoside RT inhibitors (NRTIs) terminate
DNA synthesis after their incorporation into the grow-
ing DNA chain. Non-nucleoside RT inhibitors (NNR-
TIs) bind to RT and inhibit its enzymatic activity.2–4
Despite the presence of many RT inhibitors, a search
for more suitable ones is still very important, because
of the appearance of a large number of drug-resistant
mutants of HIV virus.5
During the screening of compounds from the NCI
chemical repository for anti-HIV activity, we (Y. Zhang
and V. Pathak) found that compound NSC-380292
(dimethyl-1-(1-pyrrolidinyl)cyclobuta[b][1]benzothioph-
ene-2,2a(7bH)-dicarboxylate, Fig. 1) is a potent inhibi-
tor of HIV-1 RT with an IC50 = 1.24 lM and
therapeutic index of over 800 (CC50 > 1 mM).
To explore the relationship between the lead compound
structure and the inhibitory activity, we synthesized
N
1
7b
*
O
2
*2a
O
S
O
O
Keywords: Reverse transcriptase inhibitors; Synthesis; Chiral
separation; CD spectra; X-ray structures; RT inhibitory assay.
*
Figure 1. NSC-380292 (1) is a mixture of 2aS,7bS- and 2aR,7bR-
enantiomers of dimethyl-1-(1-pyrrolidinyl)cyclobuta[b][1]benzothioph-
ene-2,2a(7bH)-dicarboxylate.
Corresponding authors. Tel.: +1 301 846 1710 (V.K.P.); tel.: +1 301
846 5904; fax: +1 301 846 6033 (P.P.R.); e-mail addresses:
proll@helix.nih.gov; vpathak@ncifcrf.gov
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.049

K. Krajewski et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3034–3038
3035
several, closely related structural analogs of this com-
pound (see Fig. 2).
ii
i
Br
S
S
There are two chiral centers in this molecule, but be-
cause of the compound structure, only one pair of enan-
tiomers can exist. The tested repository sample was a
mixture of both enantiomers, so we separated them
using a chiral HPLC column and tested for HIV-1 RT
inhibitory activity.
N
O
iii
N
O
O
S
S
O
Most synthesized analogs were also separated into enan-
tiomers and tested in homochiral forms. The absolute
configurations of all compounds were determined using
X-ray crystallography and CD spectroscopy.
iv
N
N
(HPLC separation)
Synthesis and chiral separation. The synthesis of com-
pound NSC-380292 and analogs is presented in
Scheme 1 (except compound 10, see Scheme 2). The
commercially available thionaphthene (benzo[b]thio-
phene) was used as a starting material. The selective
bromination at the position 2 was performed by treat-
ment of thionaphthene with n-BuLi followed by
addition of Br2.6,7
O
O
H
H
(S)
(R)
(R)
(S)
O
O
O
O
S
S
O
O
Rt = 8.5 min
Rt = 14.5 min
Scheme 1. The synthesis of NSC-380292 analogs (compound 7 as an
example). Reagents and conditions: (i) 1—n-BuLi in hexane/Et2O,
2—Br2, (66%); (ii) piperidine, Pd2(dba)3, ( )BINAP, t-BuONa in
toluene, under argon (26 h, 80 ꢁC, 41%); (iii) dimethyl acetylenedi-
carboxylate in acetonitrile (22 h, rt).
R1
O
H
OH
O
*
R2
N
*
H
N
i
O
+
R1 =
S
O
S
N
N
O
S
O
R2
1-pyrrolidinyl-
O
O
N
N
iii
ii
N
OMe
OMe
S
S
O
2-R-methyl-1-pyrrolidinyl-
S-2-methyl-1-pyrrolidinyl-
O
O
N
Scheme 2. The synthesis of compound 10. Reagents and conditions: (i)
i-Pr2NH, n-BuLi in THF/hexane (ꢀ78 ꢁC ! 0 ꢁC, 2.5 h, 30%); (ii)
MsOH in DCM (2.5 h, rt 65%); (iii) dimethyl acetylenedicarboxylate in
acetonitrile (22 h, rt).
N
O
1-piperidinyl-
N
S-2-methoxymethyl-1-pyrrolidinyl-
N
2-Bromobenzo[b]thiophene was used in the next step,
palladium-catalyzed substitution of bromine by cyclic
secondary amines.8
1-azetidinyl-
2,5-dihydro-1-pyrrolyl-
Compounds
R1
1-pyrrolidinyl-
1-pyrrolidinyl-
1-pyrrolidinyl-
No
1
2
3
4
5
6
7
8
R2
Me
Et
The last step of synthesis was the addition reaction be-
tween the benzo[b]-thiophene derivative and dimethyl
acetylenedicarboxylate, and a subsequent rearrange-
ment.9 This reaction was carried out in acetonitrile, at
room temperature, for 22 h.
tBu
Me
R-2-methyl-1-pyrrolidinyl-
S-2-methyl-1-pyrrolidinyl-
S-2-methoxymethyl-1-pyrrolidinyl-
1-piperidinyl-
Me
Me
Me
Me
Me
Me
Compound 10 was synthesized starting from commer-
cially available anisaldehyde and N,N-dimethylthiofor-
mamide.10 The product of condensation of these
compounds was converted into thionaphthene deriva-
tive by treatment with methanesulfonic acid in
DCM.10 The final step was the same as for the other
compounds. The products were purified by a flash
1-azetidinyl-
2,5-dihydro-1-pyrrolyl-
dimethylamino-*
9
10
* 5-methoxy-, see Scheme 2
Figure 2. Modifications of R1 and R2 groups in the synthesized
analogs of NSC-380292.

3036
K. Krajewski et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3034–3038
chromatography. The products of the rearrangement
were mixtures of two stereoisomers (with S,S and R,R
absolute configurations at the chiral bridgehead carbon
atoms). We used preparative HPLC on a chiral col-
umn11 to obtain homochiral compounds. Fractions with
shorter retention times were denoted i (e.g., 1i), fractions
with longer retention times ii (e.g., 1ii). We were unable
to separate mixture of 3. The purity of final products
was confirmed by 1H NMR and MALDI-TOF-MS
(Kratos Axima-CFR instrument, matrix: a-cyano-4-hy-
droxycinnamic acid). The observed monoisotopic mass-
es, measured for the mixtures of stereoisomers and for
the homochiral compounds after chiral HPLC, were in
good agreement with theoretical masses.
Determination of absolute configuration. For three of the
homochiral products, the structures and the absolute
configurations were determined by X-ray crystallo-
graphy (see Fig. 3). All three compounds were found in
the early eluting fractions (4i, 6i, and 7i) obtained after
preparative HPLC of 4, 6, and 7, on Chiralcel OD col-
umn. Monocrystals for the X-ray structure determina-
tion were obtained by a slow isothermal crystallization
from cyclopentane/toluene (4i), toluene (7i), or Et2O/
hexane (6i). The absolute configurations of 4i and 7i were
determined using an anomalous dispersion, based on the
presence of sulfur atom in these molecules. The absolute
configurations on bridgehead carbon atoms in 6i were as-
signed based on a known configuration of the third chiral
center (from S-2-methoxymethylpyrrolidine). For all X-
ray structures, the determined absolute configurations
were R on both bridgehead carbons. Full crystallograph-
ic details have been deposited with Cambridge Crystallo-
graphic Data Center (deposition numbers: 286459 for 4i,
286458 for 6i, and 286457 for 7i). A comparison of CD
spectra (see Fig. 4) of 1i, 6i, and 7i with spectra of 1ii,
6ii, and 7ii showed that signs of four strong Cotton ef-
fects around 295, 260, 230, and 205 nm are determined
by the configurations on the chiral bridgehead carbon
atoms (7ii omitted for clarity). For the R,R configura-
tions (1i, 6i, and 7i) the Cotton effect around 230 nm is
positive and the other Cotton effects are negative. For
the S,S configurations (1ii, 6ii, and 7ii) the signs of all
these Cotton effects are opposite. The comparison of
these data with CD spectra of other homochiral products
allowed us to assign the absolute configuration for all
isomers (for clarity we show only a superposition of five
CD spectra). For all compounds, the first fractions from
the chiral HPLC (1i, 4i, 5i, 6i, 7i, 8i, 9i, and 10i) have R,R
configurations on the chiral bridgehead carbon atoms.
The third center of chirality present in 4 and 6 is respon-
sible for the modulation of intensity of the Cotton effect
around 260 nm and an additional Cotton effect at
220 nm.
Figure 3. X-ray structures of (a) 7i-R,R enantiomer of 7; (b) 6i-R,R-
stereoisomer of 6; (c) 4i-R,R-stereoisomer of 4.
Inhibition of HIV-1 replication. The ability of these
compounds to inhibit HIV-1 replication was determined
(Table 1) as previously described.12 Briefly, an HIV-1
vector that expresses the firefly luciferase reporter gene
and all HIV-1 proteins except for the Env and Nef
was used. The vector was cotransfected into 293 T-cells
with pHCMV-G, which expresses the G-glycoprotein of
vesicular stomatitis virus. Virus harvested from the
transfected cells was used to infect target cells in the
presence or absence of the compounds tested. The com-
pounds were added to target cell cultures for 4 h prior to
infection and were present for the next 48–72 h, at which
point the ability of the compounds to inhibit viral repli-
cation was determined by measuring the amount of
luciferase activity in the infected cells.

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).
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It is clear from the study of the X-ray structures of the
active reverse transcriptase inhibitors (4, 6, and 7), that

3038
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