Modification and structure-activity relationship of a small molecule HIV-1 inhibitor targeting the viral envelope glycoprotein gp120

Article abstract of DOI:10.1039/b415159c

This paper describes selected modification and structure-activity relationship of the small molecule HIV-1 inhibitor, 4-benzoyl-1 -[(4-methoxy-1-H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806). The results revealed: i) that both the presence and configuration (R vs. S) of the 3-methyl group on the piperazine moiety are important for the antiviral activity, with the 3-(R)-methyl derivatives showing the highest activity; ii) that the electronegativity of the C-4 substituent on the indole or azaindole ring seems to be important for the activity, with a small, electron-donating group such as a fluoro or a methoxy group showing enhanced activity, while a nitro group diminishes the activity; iii) that the N-1 position of the indole ring is not eligible for modification without losing activity; and iv) that bulky groups around the C-4 position of the indole or azaindole ring diminish the activity, probably due to steric hindrance in the binding. We found that a synthetic bivalent compound with two BMS-378806 moieties being tethered by a spacer demonstrated about 5-fold enhanced activity in an nM range against HIV-1 infection than the corresponding monomeric inhibitor. But the polyacrylamide-based polyvalent compounds did not show inhibitory activity at up to 200 nM. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b415159c

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
Modification and structure–activity relationship of a small molecule
HIV-1 inhibitor targeting the viral envelope glycoprotein gp120
Jingsong Wang, Nhut Le, Alonso Heredia, Haijing Song, Robert Redfield and Lai-Xi Wang*
Institute of Human Virology, University of Maryland Biotechnology Institute, University of
Maryland, 725 W. Lombard Street, Baltimore, MD, 21201, USA.
E-mail: wangx@umbi.umd.edu; Fax: 410-706-5068; Tel: 410-706-4982
Received (Pittsburgh, PA, USA) 1st October 2004, Accepted 29th March 2005
First published as an Advance Article on the web 12th April 2005
This paper describes selected modification and structure–activity relationship of the small molecule HIV-1 inhibitor,
4-benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806). The
results revealed: i) that both the presence and configuration (R vs. S) of the 3-methyl group on the piperazine moiety
are important for the antiviral activity, with the 3-(R)-methyl derivatives showing the highest activity; ii) that the
electronegativity of the C-4 substituent on the indole or azaindole ring seems to be important for the activity, with a
small, electron-donating group such as a fluoro or a methoxy group showing enhanced activity, while a nitro group
diminishes the activity; iii) that the N-1 position of the indole ring is not eligible for modification without losing
activity; and iv) that bulky groups around the C-4 position of the indole or azaindole ring diminish the activity,
probably due to steric hindrance in the binding. We found that a synthetic bivalent compound with two BMS-378806
moieties being tethered by a spacer demonstrated about 5-fold enhanced activity in an nM range against HIV-1
infection than the corresponding monomeric inhibitor. But the polyacrylamide-based polyvalent compounds did not
show inhibitory activity at up to 200 nM.
throughput screening of a compound library.13 Compound 3,
Introduction
denoted BMS-378806, showed excellent potency against many
The disease AIDS is caused by the infection of the human
HIV type 1 (HIV-1) laboratory and clinical isolates. Notably,
immunodeficiency virus (HIV).1 In the absence of a preventive
BMS-378806 displays many attractive pharmacological prop-
vaccine, effective anti-HIV drugs are urged to combat the
erties such as minimal serum effect on anti-HIV-1 potency,
expanding global epidemic.2–5 Almost all the FDA-approved
excellent oral bioavailability, and a clean safety profile in animal
anti-HIV drugs belong to two classes: the protease inhibitor
toxicology studies.14 Preliminary mechanistic studies suggested
and the reverse transcriptase inhibitor,2,3 with the fusion peptide
that BMS-378806 targets the HIV-1 envelope glycoprotein gp120
inhibitor T-20 (enfuvirtide) that blocks HIV entry as the only
and blocks the binding of gp120 to CD4 receptor.15,16 More
exception.6,7 The highly active antiretroviral therapy (HAART)
recent studies indicated that the inhibitor exhibits its anti-
that combines three or more reverse transcriptase/protease
HIV activity by interrupting the essential CD4 binding-induced
inhibitors has prolonged the survival of AIDS patients and
conformational changes in gp120.17,18
significantly reduced plasma viral loads in HIV infected patients.
As part of our project on developing multivalent HIV-1 entry
However, the emerging of drug-resistant strains as well as the
inhibitors targeting the envelope glycoprotein oligomers,19 we
toxicity associated with prolonged HAART therapy has limited
are particularly interested in modifying BMS-378806 and related
the efficacy of the current combination regimen. New drugs
inhibitors as lead compounds. It is hitherto not clear which part
with distinct mechanism of action and improved anti-HIV
of the molecule can be modified while preserving its antiviral
potency are highly desirable. In contrast to drugs targeting viral
activity. We describe in this paper selected modifications and
protease and reverse transcriptase, inhibitors that block HIV
structure–activity relationship studies of this type of HIV-1
infection at the early stages of HIV entry have the advantage
inhibitor to address the following questions: i) how do the (R)-
of preventing the establishment of the provirus.8–10 A number
methyl group on the piperazine moiety and its configuration
of entry inhibitors have been discovered in recent years. Typical
affect the anti-HIV activity? ii) how does the electronegativity
examples include: inhibition of the binding of HIV-1 gp120 to
of the substituent at C-4 of the indole or azaindole ring influence
receptor CD4;11 blockade of the interaction between gp120 and
the activity? and iii) whether can we improve its activity through
chemokine co-receptors;12 and interference with the function
multivalent assembly?
of gp41 such as viral membrane fusion.6,7 Recently, a novel
class of small-molecule inhibitors, compounds 1–3 (Fig. 1), was
discovered by a team at Bristol-Myers Squibb through high-
Results and discussion
Synthesis
A facile synthesis of the BMS-378806 (compound 3) was
previously described by Wang and co-workers.13 Therefore,
the synthesis of the related inhibitors for the present studies
was based on the reported procedure with corresponding
modifications. The preparation of the indole derivatives is shown
in Scheme 1. Starting with indole (4) or 4-fluoro-indole (5), the
acyl chloride 6 or 7 was synthesized in one step by reacting
with oxalyl chloride. Coupling of the acyl chloride 6 with the
N-benzoylpiperazine (8), N-benzoyl-2-R-methyl-piperazine (9),
and N-benzoyl-2-S-methyl-piperazine (10) gave compounds 1,
Fig. 1 Structures of small molecule HIV-1 attachment inhibitors.
T h i s j o u r n a l i s
11, 12, respectively. Similarly, coupling of the acyl chloride 7
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 8 1 – 1 7 8 6
1 7 8 1
©

View Article Online
Scheme 1 a) Oxalyl chloride–Et2O; b) N-benzoylpiperazine derivative (8 or 9 or 10), THF, DIPEA; c) N-benzoylpiperazine derivative (8 or 9), THF,
DIPEA; d) NaH–THF–allyl bromide (for 14) or NaH–THF–BrCH2CH2CH2NHCBz (for 15).
Scheme 2 a) PCl3, EtOAc; b) (1) CH3ONa, CH3OH; (2) PCl3, EtOAc; c) (1) HOCH2CH2CH2N3, NaH, ethylene glycol dimethyl ether; (2) PCl3,
EtOAc; d) Pd/C, H2, MeOH; e) acetic anhydride, MeOH, aqueous NaHCO3.
with the respective piperazine derivatives (8 and 9) afforded
the fluoro-containing derivatives 2 and 13, respectively. To
functionalize the derivative, compound 13 was treated with allyl
bromide in the presence of NaH to give the N-allyl derivative
14. On the other hand, reaction of 13 with Br-(CH2)3-NHCBz
gave the derivative 15, which will provide a free amino group
for further manipulation upon removal of the CBz protecting
group.
To address the importance of the substituent at C-4 with
concurrent functionalization of the molecule, we chose to
modify the azaindole derivative (Scheme 2). The 4-nitro N-
oxide intermediate 16 was prepared following the reported
procedure.13 Treatment of 16 with MeONa in MeOH at elevated
temperature, followed by reduction of the N-oxide with PCl3
in EtOAc, gave the known inhibitor 3.13 Removal of the N-
oxide directly from 16 gave the 4-nitro compound 17. On the
other hand, a 3-azido-propoxyl group was introduced at the C-
4 position by treatment of the 4-nitro-N-oxide derivative 16
with 3-azido-propanol and NaH in ethylene glycol dimethyl
ether with subsequent removal of the N-oxide using PCl3
to give the 4-(3-azido-propoxyl) derivative 18. Reduction of
the azido functionality by hydrogenation yielded the amino
derivative 19. Treatment of 19 with acetic anhydride gave the
acetamido derivative 20 (Scheme 2). A bivalent compound
22 was synthesized by tethering two BMS-378806 moieties
through a spacer. Thus, coupling of the amino compound
19 and 3,6,9,12,15,18-hexaoxaeicosane-1,20-dioic acid (21)20
with EDCI gave the bivalent compound 22 (Scheme 3). The
polyacrylamide-based polyvalent inhibitors were synthesized
by reaction of the amino compound 19 with a pre-activated
polymer poly[N-(acryloyloxy)succinimide].21,22 The density of
ligands in the polymer was controlled by altering the ratio of the
amino compound 19 and the poly[N-(acryloyloxy)-succinimide]
in the reaction, giving the polyvalent inhibitors 23a (10% ligand
substitution) and 23b (1% ligand substitution), respectively.
Antiviral activity
The antiviral activities of the inhibitors were evaluated in
the MT-2 cell line infected with HIV-1 IIIB. The results are
summarized in Table 1. Comparison of the antiviral activities
of compounds 1, 11, and 12 revealed the importance of the
(R)-methyl group on the piperazine ring. Compound 11, with
an (R)-methyl group, is 26-fold more active than compound
1 without the methyl group (see Table 1). Reversion of the
configuration of the methyl substituent from (R) (compound 11)
1 7 8 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 8 1 – 1 7 8 6

View Article Online
to the (S)-configuration (compound 12) led to a 10-fold
decrease in antiviral activity. The positive effect of the (R)-
methyl group was also demonstrated for the 4-fluoro derivatives.
Thus, the compound with the (R)-methyl group (13, IC50
=
1.6 nM) is about 24-fold more potent than the one without the
methyl group (2, IC50 = 38 nM). It was shown previously that
introduction of a fluoro group at the C-4 of the indole ring
(1 vs. 2) resulted in a significant enhancement of the antiviral
activity against several types of HIV-1 strains including LAI,
SF-2, and Bal.13 Here, evaluation of the inhibitory activity of
1 and 2 against HIV-1 IIIB also revealed this tendency. Thus,
compound 2 was found to be 26-fold more active than 1 against
HIV-1 IIIB in the cell culture assay. Moreover, the fluoro type
compound 13 (IC50 = 1.6 nM) showed 22-fold enhancement in
antiviral activity against the proto type compound 11 (IC50
=
36 nM). On the other hand, the 1-N-alkylated compounds
14 and 15 showed 600-fold decrease in antiviral activity in
comparison with the parent compound 13. The results suggest
that the N-7 position of the indole ring cannot be modified for
functionalization.
The effects of electronegativity of the C-4 substituent on
the azaindole ring were demonstrated by comparison of the
4-nitro derivative 17 (IC50 = 1000 nM) and the 4-methoxy
derivative 3 (IC50 = 2.3 nM). Attachment of an electron-
withdrawing group such as the nitro group at the C-4 position
led to a dramatic decrease in the antiviral activity. Interestingly,
changing the methoxy group to other alkoxy derivatives with
a terminal functionality such as 18 (IC50 = 35 nM) and 20
(IC50 = 22 nM) resulted in a moderate decrease in activity,
but the compounds are still potent in the nM range. However,
the related alkoxy derivative 19 with a free amino group at the
terminus did not show antiviral activity at up to 1000 nM. The
results suggest that a positive charge around the C-4 position
might cause unfavorable ionic interactions in its binding to
the HIV-1 envelope glycoprotein gp120. As to the multivalent
compounds, it was found that the bivalent compound 22,
which has two BMS-378806 moieties being tethered through a
spacer at the C-4 position, demonstrated about 5-fold enhanced
activity in an nM range against HIV-1 infection than the
corresponding monomeric inhibitor 20. The enhancement of
the inhibitory activity of the bivalent compound over the
monomeric compound might reflect the simultaneous binding
of the bivalent compounds to the two interaction sites in
the trimeric gp120 complex, as exemplified by the bivalent
CD4-mimic miniproteins.19 However, the loss of activity of
the polyvalent compounds (23a and 23b) suggest that steric
hindrance around the C-4 position of the unit might result
in the abortion of the binding for the polyvalent compounds.
No compounds show any cytotoxicity at up to 1000 nM
concentration (Table 1).
In summary, selected modification of the small molecule HIV-
1 inhibitor, 4-benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-
3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806) was per-
formed for evaluating its structure–activity relationship. The
results indicate: i) that both the presence and configuration (R vs.
S) of the 3-methyl group on the piperazine moiety are important
for the antiviral activity, with the 3-(R)-methyl derivatives show-
ing the highest activity; ii) that the electronegativity of the C-4
substituent on the indole or azaindole ring has great influence
on the activity, with a small, electron-donating group such as
a fluoro or a methoxy group showing enhanced activity, while
the electron-withdrawing nitro group diminished the activity;
iii) that the N-1 position is not eligible for modification (e.g., N-
alkylation); and iv) that bulky groups around the C-4 position
of the indole or azaindole ring diminished the activity, probably
due to steric hindrance in the binding. In order to develop an
effective anti-HIV drug based on the lead compound, further
structural modifications should be performed in connection with
mechanistic studies and molecular modeling of the interactions
between the inhibitor and HIV-1 gp120.
Scheme 3 a) EDCI, HOBt, DIPEA, DMF, rt; b) DMF, 60 ◦C, 24 h,
then NH3·H2O, rt, 24 h.
Table 1 HIV-1 inhibitory activity and cytotoxicity of synthetic
compoundsa
Compound
IC50/nMb
CC50/nMc
1
990 150
36 8.0
400 140d
38 6.0
1.6 0.40
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
11
12
2
13
14
15
3
17
18
19
20
22
23a
23b
>1000
2.3 0.55
>1000
35 3.0d
>1000
22 2.5d
4.8 0.7
>200
>200
a MT-2 cells were infected with HIV-1IIIB in the presence or absence
of the synthetic inhibitors at varied concentrations. Viral replication
was assessed by measuring HIV-1 p24 antigen in the culture super-
natants using a commercial p24 ELISA. b The IC50 was defined as
the concentration of the inhibitor that reaches 50% inhibition of viral
replication. c Cytotoxcity assays were performed in the presence of the
inhibitors at varied concentrations for 7 days, and the cell viability was
quantitated by using a MTT assay. CC50 was defined as the concentration
of inhibitor causing 50% decrease in cell viability. d These compounds
showed a normal dose response at 0–100 nM, but seemed to have reduced
inhibitory activities at the high end of the concentrations (500–1000 nM).
The reason is not clear.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 8 1 – 1 7 8 6
1 7 8 3

View Article Online
7.2 Hz), 7.40 (s, 5H), 7.20 (m, 4H), 5.02–2.80 (m, 7H), 1.30 (br s,
3H); 13C NMR (75 MHz, CD3OD): d 185.4, 171.5, 166.5, 136.7,
136.2, 134.4, 129.3, 127.8, 126.1, 124.5, 123.2, 122.1, 120.5,
113.0, 111.4, 49.7, 46.2, 14.4, 13.3. ESI-MS: 376.2 (M + H)+.
Experimental
General
All reagents were purchased from Aldrich Chemical Co. and
used as received. Thin layer chromatography (TLC) was con-
ducted on precoated plates (Merck 60F250). Flash column
chromatography was performed with silica gel 60 (EM science,
particle size 0.040–0.063 mm, 230–240 mesh). 1H NMR spectra
were measured on a QE-300/Tecmag AQuErius Spectrometer
(300 MHz) or a Varian INOVA 500 spectrometer (500 MHz). 13C
NMR spectra were recorded on the QE-300/Tecmag AQuErius
Spectrometer at 75 MHz. The ESI-MS spectra were measured
on a micromass ZQ-4000 single quadruple mass spectrometer.
1-(Benzoyl)-3-S-methyl-4[(1H-indole-3-yl)oxoacetyl]piperazine
(12)
The reaction of (S)-N-benzoyl-3-methylpiperazine (10) (180 mg,
0.882 mmol) and a-oxo-1H-indole-3-acetyl chloride (6) (182 mg,
0.882 mmol) was carried out in the same way as described for
the preparation of 11 to afford 12 (280 mg, 85%) as a white
solid. The 1H NMR, 13C NMR, and mass spectra are identical
to those of compound 11.
N-Benzoylpiperazine (8)
1-(Benzoyl)-4[(1H-indol-3-yl)oxoacetyl]piperazine (1)
To a stirred solution of piperazine (1.72 g, 20 mmol) in
dry CH2Cl2 (100 ml) under an argon atmosphere at room
temperature were added methyl benzoate (2.72 g, 2.5 ml,
20 mmol) and Et2AlCl3 (1.0 M in hexanes, 20 ml, 20 mmol).
The reaction mixture was stirred for 2 days and the reaction
was then quenched by adding 2 M NaOH. The aqueous layer
was extracted with EtOAc. The organic layer was dried over
MgSO4 and concentrated. The residue was purified by flash
chromatography (EtOAc–MeOH, 10 : 1) to give 8 (2.50 g, 66%)
as a white solid. 1H NMR (300 MHz, CDCl3): d 7.37 (m, 5H),
3.73 (m, 2H), 3.37 (m, 2H), 2.92 (m, 2H), 2.82 (m, 2H); 13C NMR
(75 MHz, CDCl3): d 169.9, 135.4, 129.0, 127.9, 126.4, 45.6, 45.2.
The reaction of N-benzoylpiperazine (8) (130 mg, 0.69 mmol)
and a-oxo-1H-indole-3-acetyl chloride (6) (243 mg, 0.69 mmol)
was performed in the same way as described for the preparation
1
of 11 to give compound 1 (213 mg, 85%) as a white solid. H
NMR (300 MHz, CD3OD): d 8.22 (d, 1H, J = 2.2 Hz), 8.11
(d, 1H, J = 6.9 Hz), 7.53 (d, 1H, J = 8.7 Hz), 7.45 (s, 5H),
7.27 (m, 2H), 4.8–3.3 (m, 8H); 13C NMR (75 MHz, DMSO-d6):
d 186.4, 169.8, 166.6, 137.8, 137.4, 136.0, 130.2, 129.0, 127.5,
125.4, 124.2, 123.1, 121.4, 113.6, 113.2, 46.0, 41.1. ESI-MS:
362.1 (M + H)+, 394.2 (M + Na)+.
1-(Benzoyl)-4[(1H-4-fluoroindol-3-yl)oxoacetyl]piperazine (2)
The reaction of 7 (312 mg, 1.38 mmol), prepared from 4-
fluoroindole in the same way as for the preparation of 6, and 8
(263 mg, 1.38 mmol) was performed in the same way as described
for the preparation of 11 to give compound 2 (390 mg, 75%) as
a white solid. 1H NMR (300 MHz, CD3OD): d 8.23 (s, 1H), 7.41
(m, 5H), 7.33 (d, 1H, J = 6.9 Hz), 7.25 (m, 1H), 6.96 (dd, 1H, J =
8.0, 10.6 Hz), 4.8–3.3 (m, 8H); 13C NMR (75 MHz, DMSO-d6):
d 184.9, 169.9, 167.0, 140.5, 138.7, 136.0, 130.2, 129.0, 127.5,
125.1, 113.0, 109.6, 108.6, 108.5, 45.8, 42.0. ESI-MS: 380.2
(M + H)+.
(R)-N-(Benzoyl)-3-methylpiperazine (9)
The reaction of R-2-methyl-piperazine (1.0 g, 10 mmol),
Et2AlCl3 (1.0 M in hexanes, 10 ml, 10 mmol) and methyl
benzoate (1.36 g, 1.24 ml, 10 mmol) was performed as described
for the preparation of 8 to give compound 9 (1.50 g, 74%) as a
white solid. 1H NMR (300 MHz, CDCl3): d 7.45 (m, 5H), 4.50
(m, 1H, J = 9.9 Hz), 3.60 (m, 1H), 3.3–2.6 (m, 5H), 1.05 (broad
peak, 3H); 13C NMR (75 MHz, CDCl3): d 169.7, 135.5, 129.0,
127.9, 126.4, 50.4, 48.6, 47.5, 45.7, 18.8.
(S)-N-(Benzoyl)-3-methylpiperazine (10)
1-(Benzoyl)-3-R-methyl-4[(1H-4-fluoroindol-
3-yl)oxoacetyl]piperazine (13)
The reaction of S-2-methyl-piperazine (1.0 g, 10 mmol),
Et2AlCl3 (1.0 M in hexanes, 10 ml, 10 mmol) and methyl
benzoate (1.35 g, 10 mmol) was performed as described for the
preparation of 8 to give compound 10 (1.53 g, 75%) as a white
solid. 1H NMR (300 MHz, CDCl3): d 7.45 (m, 5H), 4.50 (m, 1H,
J = 9.9 Hz), 3.60 (m, 1H), 3.3–2.6 (m, 5H), 1.10 (m, 1.5H), 0.98
(m, 1.5H); 13C NMR (75 MHz, CDCl3): d 169.7, 135.5, 129.0,
127.9, 126.4, 50.4, 48.6, 47.5, 45.7, 18.8.
The reaction of a-oxo-4-fluoro-1H-indole-3-acetyl chloride (7)
(60 mg, 0.269 mmol) and (R)-N-(benzoyl)-3-methylpiperazine
(9) (55 mg, 0.269 mmol) was carried out as described for the
preparation of 11 to afford compound 13 (91 mg, 80%) as a
white solid: 1H NMR (300 MHz, CD3OD): d 11.2 (s, 1H), 7.73
(s, 1H), 7.40 (m, 5H), 7.07 (ddd, J = 4.7, 7.7, 8.4 Hz), 6.88 (dd,
J = 7.7, 8.4 Hz), 4.9–2.9 (m, 7H), 1.30 (br s, 3H); 13C NMR
(75 MHz, CD3OD): d 184.3, 171.5, 166.7, 154.0. 139.6, 136.8,
129.3, 127.8, 126.1, 124.0, 112.3, 107.9, 107.8, 107.6, 107.3, 59.5,
49.8, 14.3, 13.1. ESI-MS: 394.2 (M + H)+.
1-(Benzoyl)-3-R-methyl-4[(1H-indol-3-yl)oxoacetyl]piperazine
(11)
To a solution of indole (200 mg, 1.71 mmol) in anhydrous diethyl
ether (10 ml) was added oxalyl chloride (252 mg, 0.173 ml,
1.98 mmol) over 30 min. The reaction mixture was stirred at
1-(Benzoyl)-3-R-methyl-4[(1-allyl-4-fluoroindol-
3-yl)oxoacetyl]piperazine (14)
◦
0 C for 3 h, then allowed to warm to room temperature over
To a stirred suspension of NaH (30 mg, 60% in mineral
oil, 0.75 mmol) in dry THF (10 ml) was added compound
13 (147 mg, 0.374 mmol) in THF (5 ml), and the resulting
mixture was stirred at room temperature for 30 min. Then
allyl bromide (453 mg, 3.75 mmol) was added. The reaction
mixture was refluxed for 30 min and the reaction was quenched
by adding methanol (1 ml). The solvent was evaporated and
the residue was partitioned between water and EtOAc. The
organic layer was dried over MgSO4, filtered, and the filtrate
was concentrated under vacuum. The residue was purified by
flash chromatography (EtOAc–hexanes, 1 : 1) to give compound
14 (136 mg, 84%) as a white solid. 1H NMR (500 MHz, CDCl3):
d 7.96 (d, 1H, J = 2.5 Hz), 7.41 (s, 5H), 7.28 (m, 2H), 7.15 (d, 1H,
J = 8.4 Hz), 6.98 (t, 1H, J = 8.4 Hz), 5.98 (m, 1H), 5.34 (d, 1H,
1 h. The resulting yellow crystals were collected by filtration,
washed with cold anhydrous ether (20 ml), and dried under
vacuum to yield a-oxo-1H-indole-3-acetyl chloride (6) (320 mg,
91%), which was used immediately for the next step. A solution
of (R)-N-benzoyl-3-methylpiperazine (9) (179 mg, 0.877 mmol)
in dry THF (5 ml) was added to a stirred solution of 6 (182 mg,
0.877 mmol) in dry THF (5 ml). The mixture was cooled to 0 ◦C
and diisopropylethylamine (135 mg, 0.104 mmol) was added
dropwise. The mixture was warmed to room temperature and
stirred for 3 h. The reaction mixture was evaporated and the
residue was subject to flash chromatography to give compound
11 (293 mg, 90%) as a white solid. 1H NMR (300 MHz, CD3OD):
d 10.88 (d, 1H, J = 5.4 Hz), 8.21 (s, 1H), 7.70 (dd, 1H, J = 3.0,
1 7 8 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 8 1 – 1 7 8 6

View Article Online
1
J = 10.3 Hz), 5.20 (dd, 1H, J = 4.4, 6.8 Hz), 4.70 (d, 2H,
J = 5.2 Hz), 4.7–2.95 (m, 7H), 1.3 (broad peak, 3H); 13C NMR
(75 MHz, CD3OD): d 183.8, 171.5, 166.6, 139.4, 134.4, 131.5,
129.3, 127.8, 126.1, 124.1, 124.0, 116.9, 116.8, 113.0, 111.5,
108.0, 107.8, 106.8, 106.7, 49.1, 48.8, 14.8, 13.0. ESI-MS: 434.3
(M + H)+, 456.3 (M + Na)+.
afford compound 18 (70 mg, 25%) as a white solid. H NMR
(300 MHz, CD3OD): d 8.40 (d, 1H, J = 5.2 Hz), 8.30 (s, 0.5H),
8.21 (s, 0.5H), 7.40 (s, 5H), 7.28 (d, 1H, J = 5.2 Hz), 4.8–3.1 (m,
11H), 2.15 (m, 2H), 1.3 (broad peak, 3H). 13C NMR (75 MHz,
CD3OD): d 184.9, 176.4, 167.2, 145.5, 136.7, 134.9, 130.0, 128.5,
126.8, 112.5, 65.3, 50.4, 47.7, 28.1, 14.9, 14.3. ESI-MS: 476.23
(M + H)+.
1-(Benzoyl)-3-R-methyl-4[1-(3-benzyloxycarbonyl-
aminopropyl)-4-fluoroindol-3-yl-oxoacetyl]piperazine (15)
4-Benzoyl-1-[4-(3-aminopropoxy)-1H-pyrrolo[2,3-b]pyridin-
3-yl)oxoacetyl]-2-(R)-methyl-piperazine (19)
Compound 13 (90 mg, 0.228 mmol) was reacted with Br-(CH2)3-
NHCBz (350 mg, 1.29 mmol) in the same way as described for
the preparation of 14 to give compound 15 (115 mg, 88%) as a
A solution of 4-benzoyl-1-[4-(3-azidopropoxy)-1H-pyrrolo[2,3-
b]pyridine-3-yl)oxoacetyl]-2-(R)-methyl-piperazine 18 (50 mg,
0.105 mmol) in MeOH (10 ml) containing Pd–C (20 mg) was
hydrogenated under 40 psi for 2 h. The mixture was filtered
through a pad of celite, and the pad was washed with MeOH
(4 ml). The filtrate and washings were combined and evaporated
at a reduced pressure to give compound 19 (42 mg, 90%) as a
white solid. 1H NMR (300 MHz, CD3OD): d 8.18 (s, 0.5H), 8.10
(s, 0.5H), 7.81 (s, 2H), 7.40 (s, 5H), 4.6–3.3 (m, 9H), 2.65 (m,
2H), 2.21 (m, 2H), 1.3 (broad peak, 3H). 13C NMR (75 MHz,
CD3OD): d 185.0, 171.5, 160.0, 146.2, 137.9, 134.3, 129.4, 127.8,
126.1, 118.0, 66.9, 58.4, 50.2, 45.8, 37.5, 28.6, 14.3, 13.9. ESI-
MS: 450 (M + H)+.
1
white solid. H NMR (300 MHz, CDCl3): d 8.08 (d, 1H, J =
2.8 Hz), 7.47 (m, 5H), 7.38 (m, 5H), 7.27 (q, 1H, J = 6.5 Hz), 7.17
(d, 1H, J = 8.4 Hz), 7.02 (t, 1H, J = 8.4 Hz), 5.34 (br s, 1H), 5.15
(s, 2H), 4.9–3.0 (m, 11H), 2.08 (m, 2H), 1.35 (br s, 3H); 13C NMR
(75 MHz, CD3OD): d 183.8, 139.4, 134.4, 127.8, 127.5, 127.0,
126.7, 126.1, 124.2, 124.1, 113.0, 111.2, 108.0, 107.6, 106.4, 65.5,
44.5, 43.9, 36.8, 28.9, 14.3, 13.1. ESI-MS: 585.2 (M + H)+.
4-Benzoyl-1-[4-nitro-1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-
(R)-methyl-piperazine (17)
Benzoyl-2-(R)-methyl-1-[(4-nitro-7-oxido-1H-pyrrolo[2,3-b]pyri-
din-3-yl)oxoacetyl]piperazine 16 (85.1 mg, crude), prepared
from azaindole according to the reported procedure,13 was
suspended in EtOAc (10 ml). To this suspension was added
PCl3 (0.8 ml) and the resulting mixture was stirred at room
temperature for 5 h. The reaction was cooled down to 0 ◦C
and neutralized to pH 6 with 2 M NaOH. The aqueous layer
was extracted by EtOAc, and the combined organic layer was
dried over MgSO4, filtered, and concentrated. The residue was
subject to flash chromatography (EtOAc–MeOH, 50 : 1) to
4-Benzoyl-1-[4-(3-acetamidopropoxy)-1H-pyrrolo[2,3-b]pyridin-
3-yl)oxoacetyl]-2-(R)-methyl-piperazine (20)
To a solution of 19 (12 mg, 26.7 lmol) in aqueous MeOH
containing NaHCO3 (3 ml, pH = 8.2) was added Ac2O (11 mg,
106 lmol). The mixture was stirred at room temperature until
TLC analysis showed the disappearance of the starting material.
MeOH (10 ml) was added to the mixture to quench the reaction.
The mixture was concentrated and the residue was purified by
flash chromatography to give compound 20 (10 mg, 76%) as a
white solid. 1H NMR (300 MHz, CD3OD): d 8.18 (s, 2H), 8.11
(s, 1H), 7.50 (s, 5H), 4.50–3.05 (m, 11H), 2.18 (m, 2H), 1.97
(s, 3H), 1.32 (broad peak, 3H). 13C NMR (75 MHz, CD3OD):
d 182.1, 170.8, 170.2, 159.0, 145.9, 139.6, 135.2, 129.9, 128.4,
127.5, 126.7, 116.5, 65.6, 56.8, 45.7, 30.0, 21.2, 13.0, 11.9. ESI-
MS: 492.2 (M + H)+, 514.2 (M + Na)+.
1
give compound 17 (36 mg, 44%) as a yellow solid. H NMR
(300 MHz, CD3OD): d 8.53 (d, 1H, J = 4.8 Hz), 8.48 (s, 0.5H),
8.44 (s, 0.5H) (these are from the rotamers), 7.59 (s, 1H), 7.41 (s,
5H), 4.7–3.3 (m, 7H), 1.3 (s, 3H); 13C NMR (75 MHz, CD3OD):
d 184.0, 172.1, 166.2, 145.6, 139.9, 134.9, 130.0, 128.5, 126.8,
111.7, 111.4, 50.4, 45.5, 15.2, 13.9; ESI-MS: 422 (M + H)+, 444
(M + Na)+.
4-Benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-
3-yl)oxoacetyl]-2-(R)-methylpiperazine (3)
Preparation of the bivalent compound 22
A solution of compound 19 (8 mg, 17.8 lmol), the 3,6,9,12,15,
18-hexaoxaeicosane-1,20-dioic acid (21)20 (2.1 mg, 5. 94 lmol),
EDCI (3.4 mg, 17.8 lmol), HOBt (17.8 lmol, 17.8 ll of 1 M
NMP solution), and DIPEA (2 ll) in dry DMF (1 ml) was
stirred at rt under an argon atmosphere for 24 h. The reaction
mixture was concentrated in vacuo and the residue was purified
by preparative silica gel thin-layer chromatography (EtOAc–
MeOH 10 : 1) to give the bivalent compound 22 (3.5 mg, 48%)
as a white solid. 1H NMR (300 MHz, CD3OD): d 8.05 (s, 0.5H),
7.95 (s, 0.5H), 7.84 (s, 2H), 7.60–7.40 (br. s, 5H), 4.6–3.3 (br. s,
31H), 2.21 (m, 2H), 1.3 (br. s, 3H). ESI-MS: 1217.0 (M + H)+.
Compound 16 (230 mg, crude) was treated with MeONa–MeOH
(0.5 M, 16 ml). Work-up of the reaction and purification of the
product according to the reported procedure13 gave 3 (85 mg,
1
40%) as white solid. The H NMR, 13C NMR, mass spectra
were in agreement with the reported data.13
4-Benzoyl-1-[4-(3-azidopropoxy)-1H-pyrrolo[2,3-b]pyridin-
3-yl)oxoacetyl]-2-(R)-methyl-piperazine (18)
To a stirred suspension of NaH (240 mg, 60% in mineral oil,
6 mmol) in dry ethylene glycol dimethyl ether (15 ml) was
added 3-azido-1-propanol (606 mg, 6 mmol, prepared by the
reaction of sodium azide with 3-bromo-1-propanol) under an
argon atmosphere. The mixture was stirred at room temperature
for 2 h, then 4-benzoyl-2(R)-methyl-1-[(4-nitro-7-oxido-1H-
pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]piperazine (16) (263 mg,
crude) in ethylene glycol dimethyl e◦ther (5 ml) was added. The
resulting mixture was stirred at 70 C overnight. The reaction
was then quenched by adding MeOH (2 ml), concentrated and
dried under vacuum to give a brown powder. The residue was
suspended in EtOAc (8 ml), and PCl3 (0.5 ml) was added. The
mixture was stirred at room temperature for 5 h. The mixture
was cooled down to 0 ◦C and neutralized to pH 6 by adding 2 M
NaOH. The aqueous phase was extracted with ethyl acetate.
The combined organic layer was dried over MgSO4 and filtered.
The filtrate was concentrated under vacuum and the residue
was subject to flash chromatography (EtOAc–MeOH, 50 : 1) to
Preparation of the polyacrylamide-based polymers (23a and 23b)
of the 4-benzoyl-1-[4-(3-aminopropoxy)-1H-pyrrolo[2,3-
b]pyridin-3-yl)oxoacetyl]-2-(R)-methyl-piperazine
A solution of 19 (12 mg, 26.7 lmol) in DMF (1 ml) containing
diisopropylethylamine (5 ll) was added to a stirred solution of
poly[N-(acryloyloxy)succinimide] (45 mg), which was prepared
according to the literature21,22 (Mw = 216 kD; Mn = 157 kD;
Mw/Mn = 1.4), in DMF (5 ml) under an argon atmosphere.
◦
The mixture was stirred at 60 C for 24 h. Then concentrated
NH3·H2O (0.5 ml) was added dropwise and the resulting mixture
was stirred at rt for 24 h. The reaction mixture was dialyzed
exhaustively against distilled water and lyophilized to yield a
polymer (23a) containing 4-benzoyl-1-[4-(3-aminopropoxy)-
1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methyl-pipera-
zine as a white powder (28 mg, 93%). The density (molar
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 8 1 – 1 7 8 6
1 7 8 5

View Article Online
ratio m/n) of the small molecule ligand in the polymer 23a
was determined to be about 1 : 10 (cat. 10% subsitution by the
ligand) based on the integration of the aromatic signal (d 7.40)
for the small molecule moiety and the a-proton signal (d 1.80)
for the acrylamide moiety in the H NMR (500 MHz, D2O):
d 8.18 (br. s), 8.10 (br. s), 7.81 (br. s), 7.62 (br. s), 7.40 (br. s),
4.6–3.3 (m), 2.2 (m), 1.8 (m).
Changing the ratio of the amino compound 19 and the
poly[N-(acryloyloxy)-succinimide] in the reaction yielded poly-
mers with varied density of the ligands. Accordingly, the reaction
of 19 (1.5 mg) with the poly[N-(acryloyloxy)-succinimide]
(45 mg), followed by the work-up described above, gave the
polymer 23b with a m/n ratio of about 1 : 100 (cat. 1%
substitution by the ligand).
4 L. X. Wang, Curr. Pharm. Des., 2003, 9, 1771–1787.
5 S. Zolla-Pazner, Nat. Rev. Immunol., 2004, 4, 199–210.
6 J. M. Kilby, S. Hopkins, T. M. Venetta, B. DiMassimo, G. A. Cloud,
J. Y. Lee, L. Alldredge, E. Hunter, D. Lambert, D. Bolognesi, T.
Matthews, M. R. Johnson, M. A. Nowak, G. M. Shaw and M. S.
Saag, Nat. Med., 1998, 4, 1302–1307.
1
7 C. T. Wild, D. C. Shugars, T. K. Greenwell, C. B. McDanal and
T. J. Matthews, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 9770–
9774.
8 T. C. Pierson and R. W. Doms, Immunol. Lett., 2003, 85, 113–118.
9 N. A. Meanwell and J. F. Kadow, Curr. Opin. Drug Discovery Dev.,
2003, 6, 451–461.
10 S. Jiang, Q. Zhao and A. K. Debnath, Curr. Pharm. Des., 2002, 8,
563–580.
11 J. M. Jacobson, R. J. Israel, I. Lowy, N. A. Ostrow, L. S. Vassilatos,
M. Barish, D. N. Tran, B. M. Sullivan, T. J. Ketas, T. J. O’Neill, K. A.
Nagashima, W. Huang, C. J. Petropoulos, J. P. Moore, P. J. Maddon
and W. C. Olson, Antimicrob. Agents Chemother., 2004, 48, 423–429.
12 J. M. Strizki, S. Xu, N. E. Wagner, L. Wojcik, J. Liu, Y. Hou, M.
Endres, A. Palani, S. Shapiro, J. W. Clader, W. J. Greenlee, J. R.
Tagat, S. McCombie, K. Cox, A. B. Fawzi, C. C. Chou, C. Pugliese-
Sivo, L. Davies, M. E. Moreno, D. D. Ho, A. Trkola, C. A. Stoddart,
J. P. Moore, G. R. Reyes and B. M. Baroudy, Proc. Natl. Acad. Sci.
U. S. A., 2001, 98, 12718–12723.
13 T. Wang, Z. Zhang, O. B. Wallace, M. Deshpande, H. Fang, Z. Yang,
L. M. Zadjura, D. L. Tweedie, S. Huang, F. Zhao, S. Ranadive, B. S.
Robinson, Y. F. Gong, K. Ricarrdi, T. P. Spicer, C. Deminie, R. Rose,
H. G. Wang, W. S. Blair, P. Y. Shi, P. F. Lin, R. J. Colonno and N. A.
Meanwell, J. Med. Chem., 2003, 46, 4236–4239.
14 H. G. Wang, R. E. Williams and P. F. Lin, Curr. Pharm. Des., 2004,
10, 1785–1793.
15 P. F. Lin, W. Blair, T. Wang, T. Spicer, Q. Guo, N. Zhou, Y. F. Gong,
H. G. Wang, R. Rose, G. Yamanaka, B. Robinson, C. B. Li, R.
Fridell, C. Deminie, G. Demers, Z. Yang, L. Zadjura, N. Meanwell
and R. Colonno, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 11013–
11018.
Antiviral assays
The antiviral activities of the inhibitors were tested in the MT-2
cell line infected with HIV-1 IIIB at a multiplicity of infection
(m.o.i) of 0.002. Virus inoculum was preincubated with serial
dilutions of the inhibitors for 1 h at 37 ◦C prior to addition
to the cells. Virus inoculum was then added to cells and the
sample was incubated for 2 h at 37 ◦C. Infected cells were
washed 3 times with PBS to remove non-adsorbed virus, and
then plated in RPMI-10% FBS containing the inhibitors at
the same concentration as in the preincubation step. Cells were
plated in triplicate in a 96-well plate at 1 × 105 cells per well in a
volume of 200 ll. On day 4 after infection, half of the medium
was replenished with fresh medium and inhibitors. On day
7, virus replication was monitored by measuring p24 content
in the culture supernatants using a commercial p24 ELISA
(NCI, Frederick, MD). Cell viability in culture in the presence
or absence of antiviral agents was measured by commercial
MTT assay, as per the manufacturer’s instructions (Boehringer
Mannheim).
16 Q. Guo, H. T. Ho, I. Dicker, L. Fan, N. Zhou, J. Friborg, T. Wang,
B. V. McAuliffe, H. G. Wang, R. E. Rose, H. Fang, H. T. Scarnati,
D. R. Langley, N. A. Meanwell, R. Abraham, R. J. Colonno and P. F.
Lin, J. Virol., 2003, 77, 10528–10536.
17 Z. Si, N. Madani, J. M. Cox, J. J. Chruma, J. C. Klein, A. Schon, N.
Phan, L. Wang, A. C. Biorn, S. Cocklin, I. Chaiken, E. Freire, A. B.
Smith, 3rd and J. G. Sodroski, Proc. Natl. Acad. Sci. U. S. A., 2004,
101, 5036–5041.
18 N. Madani, A. L. Perdigoto, K. Srinivasan, J. M. Cox, J. J. Chruma,
J. LaLonde, M. Head, A. B. Smith, 3rd and J. G. Sodroski, J. Virol.,
2004, 78, 3742–3752.
Acknowledgements
The work was supported in part by the Institute of Human
Virology, University of Maryland Biotechnology Institute, and
the National Institutes of Health (AI51235).
19 H. Li, H. Song, A. Heredia, N. Le, R. Redfield, G. K. Lewis and
L. X. Wang, Bioconjug. Chem., 2004, 15, 783–789.
20 V. Wittmann, S. Takayama, K. W. Gong, G. Weitz-Schmidt and C.-H.
Wong, J. Org. Chem., 1998, 63, 5137–5143.
21 J.-Q. Wang, X. Chen, W. Zhang, S. Zacharek, Y. Chen and P. G.
Wang, J. Am. Chem. Soc., 1999, 121, 8174–8181.
22 M. Mammen, G. Dahmann and G. M. Whitesides, J. Med. Chem.,
1995, 38, 4179–4190.
References
1 R. C. Gallo and L. Montagnier, New Engl. J. Med., 2003, 349, 2283–
2285.
2 D. D. Richman, Nature, 2001, 410, 995–1001.
3 C. F. Pereira and J. T. Paridaen, Curr. Pharm. Des., 2004, 10, 4005–
4037.
1 7 8 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 8 1 – 1 7 8 6