Synthesis and antiviral activity of P1′ arylsulfonamide azacyclic urea HIV protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.05.036

A series of novel azacyclic urea HIV protease inhibitors bearing a benzenesulfonamide group at P1′ were synthesized utilizing a parallel synthesis method. Structural studies of early analogs bound in the enzyme active site were used to design more potent

Full text of DOI:10.1016/j.bmcl.2004.05.036

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4075–4078
Synthesis and antiviral activity of P10 arylsulfonamide
azacyclic urea HIV protease inhibitors
Peggy P. Huang,* John T. Randolph, Larry L. Klein, Sudthida Vasavanonda,
Tatyana Dekhtyar, Vincent S. Stoll and Dale J. Kempf
Abbott Laboratories, Infectious Disease Research, Dept. R47D, Building AP52N,
200 Abbott Park Road, Abbott Park, IL 60064-6217, USA
Received 25 March 2004; accepted 12 May 2004
Available online 17 June 2004
Abstract—A series of novel azacyclic urea HIV protease inhibitors bearing a benzenesulfonamide group at P10 were synthesized
utilizing a parallel synthesis method. Structural studies of early analogs bound in the enzyme active site were used to design more
potent inhibitors. The effects of substituting the P10 benzenesulfonyl group on antiviral activity and protein binding are described.
Ó 2004 Elsevier Ltd. All rights reserved.
Inhibitors of the human immunodeficiency virus (HIV)
protease are important therapeutic agents used in the
treatment of AIDS infection.1 We have previously
reported azacyclic urea 1a as a potent inhibitor of HIV
protease.2;3 Recent efforts to replace the benzyl sub-
stituent at P10 with a variety of acyl groups resulted in
compounds, which were found to exhibit similar or
greater potency as HIV protease inhibitors.4 For
example, the benzoyl analog 1b had excellent activity
when compared to that of 1a, with a Ki of 0.01 nM (Fig. 1).
However, the benzenesulfonyl analog 1c, was found
to be >10-fold less active than 1a, suggesting that the
sulfonyl analog 1c might have a significantly different
binding mode than 1a and 1b. Molecular modeling
studies encouraged us to further investigate the P10
benzenesulfonyl series in an effort to take advantage of
novel vectors available for substituents on the benzene
ring, thereby obtaining new interactions with the
enzyme that might improve potency in the series. Taking
advantage of the wide variety of substituted benz-
enesulfonyl chlorides that are commercially available,
we were able to develop a parallel synthesis method to
quickly prepare these compounds for testing.
P2
P2'
O
The synthesis of P10 benzenesulfonyl azacyclic ureas is
outlined in Scheme 1. Compound 2 was prepared as
previously reported.2 Debenzylation of 2 by hydrogen-
olysis using Pearlman’s catalyst, followed by reprotec-
tion of the exposed nitrogen with a Cbz group, gave 3.
Bis-alkylation of the urea nitrogens was accomplished
by deprotonation with sodiumhydride in DMF fol-
lowed by the addition of trimethylsilylethoxymethyl
(SEM) protected 4-hydroxybenzyl chloride (4). Sub-
sequent removal of the Cbz protecting group by
hydrogenolysis gave 5, a common intermediate for the
synthesis of P10 acyl and sulfonyl derivatives. P10 sul-
fonyl analogs were synthesized using a simple, rapid
parallel approach, involving sulfonylation and sub-
sequent removal of the SEM protecting groups. First,
the appropriate benzenesulfonyl chloride was added to
HO
OH
N
N
N
R
HO
P1'
P1
Ki (nM)
EC50 (µM)
0.01
1a R = CH2Ph
1b R = COPh
0.07
---
0.01
1.37
1c R = SO2Ph
0.459
Figure 1. Azacyclic urea HIV protease inhibitors.
* Corresponding author. Tel.: +1-847-935-1545; fax: +1-847-938-3403;
e-mail: peggy.huang@abbott.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.036

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P. P. Huang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4075–4078
O
O
O
SEMO
OSEM
HN
O
NH
N
HN
O
NH
N
O
N
NH
N
O
a,b
c,d
O
SEM
SEM
SEM
2
3
5
O
HO
OH
N
N
N
e
O
S
SEMO
Cl
O
HO
4
R
6
Scheme 1. Reagents and conditions: (a) Pd(OH)2/C, H2, MeOH, 50 °C, 77%; (b) BnOCOCl, Hunig’s base, CH2Cl2, 77%; (c) 4, NaH, DMF, 75%; (d)
10% Pd/C, H2, EtOAc, 97%; (e) (i) ArSO2Cl, NEt3, DMAP, ClCH2CH2Cl, 40–50 °C, 18–90 h, (ii) polyamine resin, rt, 1 h, (iii) 4 N HCl, MeOH, 1 h,
16–78% (three steps).
5, followed by addition of a polyamine resin5 to scav-
enge excess benzenesulfonyl chloride. Upon filtration of
resin and evaporation of solvent, removal of the SEM
ether protecting groups using anhydrous HCl in meth-
anol provided the target compounds 6.
over 1c. Monosubstitution at the para-position resulted
in a greater than 2-fold loss in activity relative to the
unsubstituted analog (for examples, see 6h–k). Further,
disubstitution resulted in compounds that were less
active than the unsubstituted benzene analog 1c (for
examples, see dichloro derivatives 6l–o). In general,
monosubstitution in either the ortho- or meta-position
was better tolerated. Also, the electron withdrawing/
donating nature of the substituents did not seemto have
a significant effect on antiviral activity.
Table 1 shows a summary of SAR for a series of
substituted benzenesulfonyl azacyclic ureas prepared
using the described method. The ortho-methyl substi-
tuted compound 6a, showed a 2-fold improvement in
EC50 over the parent compound 1c. Of the numerous
compounds prepared with a single substituent in the
meta position (see, as examples, 6b–g), only 6b, the
meta-cyano analog, showed an improvement in activity
An X-ray crystal structure of compound 6b bound in the
HIV protease active site revealed that the P10 meta-
cyano group is projected into a water pocket on the side
of the S10 site and displaces one of two water molecules
typically observed in this pocket (see Fig. 2a).7 An
analogous water pocket is also found in many other
aspartyl proteases.8 However, to date, we are unaware
of a report in which this pocket has been exploited by an
inhibitor for any aspartyl protease. As shown in Figure
2a, a vector into this novel S10 subsite is provided by the
meta-position of the benzenesulfonamide. Although
activity data in Table 1 indicates no significant advan-
tage for initial analogs bearing a meta-substituent over
the unsubstituted analog 1c, further exploration of this
site with novel substituents in the meta-position of the
benzene ring could result in compounds with improved
binding. In order to achieve an improvement in potency,
it was believed that the new meta-substituents would
need to displace water fromthe site and effectively stack
against the planar surface formed by the interaction of
Arg8 with Asp29 (see Fig. 2b).
Table 1. SAR for P10 benzenesulfonyl azacyclic ureas bearing 4-hy-
droxybenzyl substituents at P2 and P20
O
HO
OH
N
N
N
O
S
O
HO
R
Compd
R
H
EC50 (lM)a
1c
6a
6b
6c
6d
6e
6f
0.459
0.238
0.2
o-CH3
m-CN
m-Br
0.428
0.469
0.598
0.817
0.927
0.758
0.879
1.1
m-OCH3
m-F
m-NO2
m-CH3
p-Methoxy
p-CH3
p-Cl
6g
6h
6i
The X-ray crystal structure of 6b was used as a model to
design a new series of inhibitors to explore this novel S10
subsite. For this new series, the alkyl substituents on the
urea nitrogens were modified to give optimal binding at
the P2/P20 positions. The substitution pattern used was
based in part on literature reports, in which the ami-
doxime group9 and the n-butyl group10 were both shown
to give compounds with improved antiviral activity.
Thus, a series of compounds was prepared bearing an
6j
6k
6l
p-CF3
1.1
0.562
0.67
2,3-Dichloro
3,5-Dichloro
3,4-Dichloro
2,4-Dichloro
6m
6n
6o
0.946
3.2
a For assay procedures, see Ref. 6.

P. P. Huang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4075–4078
4077
Figure 2. (a) Crystal structure of 6b bound to HIV protease indicating a cavity on the side of the S10 pocket to be probed with new substituents in this
series. (b) Crystal structure of 6b bound to HIV protease showing a well known aspartyl protease water pocket on the side of the S10 pocket formed
by Arg8 and Asp29. In the presence of 6b, there are two water molecules remaining in this pocket, one of which is H-bonded to the meta-CN group.
amidoxime group at P2 and an n-butyl group at P20, a
substitution pattern that was found to give optimal
activity in our assay. Froma chemical perspective, our
initial goals were to prepare compounds bearing reactive
functionality in the meta-position of the benzenesulf-
onamide, thereby allowing for late-stage derivatization
at this position of the molecule.
fold more active than 6d, and the meta-nitro analog 7e,
showed a 20-fold improvement in activity over 6f. The
most active compound in this series was the ketoxime
analog 7a, with an EC50 of 0.024 lM in absence of
human serum. In the presence of human serum, activity
was attenuated some 17-fold (0.40 lM), which was in
line with serumeffects for other compounds in the series
(typically 10–15-fold). The amino derivative 7c, which
was very active in the absence of serum(0.037 lM),
displayed a very large serumeffect (>60-fold), indicating
this compound is highly bound to serum proteins. The
meta-cyano derivative is not reported in this series due
to difficulties encountered in synthesis of this analog
using our synthetic method. Overall, the improvement in
activity with the series of compounds in Table 2 (relative
to compounds in Table 1) may be attributed primarily to
the modification of P2/P20 groups rather than the sub-
stituents at the meta-position of the benzenesulfon-
amide.
Table 2 shows a series of meta-substituted benz-
enesulfonyl analogs prepared with optimized substituents
at P2 and P20.11 The antiviral activities of these com-
pounds was determined both in the absence of human
serum(0% HS) and in the presence of 50% human serum
(50% HS) in order to estimate the effect of protein
binding on in vivo potency. In general, these compounds
showed a considerable improvement in antiviral activity
over the 4-hydroxybenzyl series (compare the 0% HS
activities for compounds in Table 2 with those in Table
1). For example, the meta-methoxy analog 7b was 15-
In summary, we have synthesized a series of novel P10
benzenesulfonyl azacyclic urea HIV protease inhibitors
using a parallel synthesis approach. Attempts to increase
antiviral potency by making modifications on the phenyl
ring of the benzenesulfonyl group resulted in very lim-
ited success in this series (none of the substituted ana-
logs was more than 2-fold more active than the
unsubstituted analog 1c). Although the compounds
presented in Tables 1 and 2 represent a fairly diverse set
of substituted benzenesulfonamides, both series of
compounds show a lack of dynamic range in activity. X-
ray crystallographic study of compound 6b bound in the
enzyme active site revealed that the meta-cyano group of
this inhibitor was projecting into a novel binding pocket
in the S10 site. This structural data was used as the basis
to design new analogs for synthesis that were aimed at
exploring this novel binding interaction. Initial analogs
in this series (see Table 2) did not result in improvements
in activity, suggesting that displacing water fromthis
site is insufficient for potency gains. Further derivati-
zation with substituents that replace water molecules in
the S10 subsite with appropriate hydrogen bond coor-
dination and/or stack more effectively against the resi-
dues that define the pocket may result in better
inhibitors.
Table 2. SAR for meta-substituted benzenesulfonyl analogs designed
to optimize potency
HO
N
O
H2N
N
N
N
O
S
R
O
HO
Compd
R
EC50 (lM)a
(0%HS)b
EC50 (lM)a
(50%HS)c
7a
7b
7c
7d
7e
7f
C(@NOH)CH3
OCH3
NH2
0.024
0.033
0.037
0.04
0.401
0.563
2.38
CH(OH)CH3
NO2
OH
0.852
0.375
2.56
0.041
0.156
0.157
0.162
0.221
7g
7h
7i
COCH3
Br
1.09
2.49
CH@CH2
1.86
a For assay procedures, see Ref. 4.
b Assay performed in the absence of human serum.
c Assay performed in the presence of 50% human serum.

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P. P. Huang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4075–4078
ꢀ
ꢀ
with unit cell dimensions a ¼ 57:326 A, b ¼ 86:001 A,
Acknowledgements
ꢀ
c ¼ 46:077 A. Calculation of initial electron density maps
and refinement were done using CNX14;15 and refined to a
We thank Kent Stewart for carrying out some of the
preliminary computational modeling experiments in this
work. X-ray crystallography data were collected at
beamline 17-ID in the facilities of the Industrial Mac-
romolecular Crystallography Association Collaborative
Access Team(IMCA-CAT) at the Advanced Photon
Source. These facilities are supported by the companies
of the Industrial Macromolecular Crystallography
Association through a contract with Illinois Institute of
Technology (IIT), executed through IIT’s Center for
Synchrotron Radiation Research and Instrumentation.
Use of the Advanced Photon Source was supported by
the US Department of Energy, Basic Energy Sciences,
Office of Science, under Contract No W-31-109-Eng-38.
ꢀ
final Rwork ¼ 25:35 and Rfree ¼ 27:69–2:2 A resolution.
Coordinates for the crystal structure of HIV protease
complexed with 6b have been deposited in the RCSB
protein data bank, PDB ID 1T7K.
8. (a) Suguna, K.; Bott, R. B.; Padlan, E. A.; Subramanian,
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11. Compounds in Table 2 were prepared via an analogous
procedure to that described below for 7a:
References and notes
O
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O
NC
HN
O
NH
N
b
O
a
N
O
N
N
O
O
O
SEM
SEM
3
HO
N
O
NC
O
H2N
N
N
N
N
N
N
O
c
O
S
NOH
S
Br
O
O
O
OH
SEM
7a
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Reagents and conditions: (a) (i) butyl iodide, potassium
t-butoxide, THF, 45 °C, (ii) 3-cyanobenzyl bromide, NaH,
DMF; (b) (i) 10% Pd/C, H2, MeOH, (ii) 3-bromo-
benzenesulfonyl chloride, NEt3, DMAP, ClCH2CH2Cl,
40–50 °C, (iii) polyamine resin, rt, 1 h; (c) (i) tributyl-
(1-ethoxyvinyl)tin, Pd(Ph3P)4, toluene, 100 °C, (ii) 4 N
HCl, MeOH, 1 h, (iii) NH2OHÆHCl, NEt3, EtOH, 50 °C.
12. Stoll, V.; Qin, W.; Stewart, K. D.; Jakob, C.; Park, C.;
Walter, K.; Simmer, R. L.; Helfrich, R.; Bussiere, D.; Kao,
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Chem. 2002, 10, 2803.
13. Otwinowski, K. Z.; Minor, W. In Methods in Enzymology,
Macromolecular Crystallography, Part A; Carter, C. W.,
Jr.; Sweet, R. M., Eds.; Academic: New York, 1997; Vol.
276, p 307.
14. Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano,
J.-S.; Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.;
Rice, L. M.; Simonson, T.; Warren, G. L. Acta Cryst.
Sect. D: Biol. Cryst. 1998, D54, 905.
7. HIV protease was purified and crystallized in the presence
of compound 6b according to the procedures described by
Stoll et al.12 Data were collected at the Argonne National
Laboratory synchrotron on the IMCA ID17 beamline
using a Mar 165 CCD detector. Data were processed using
HKL2000.13 The co-crystals of HIV protease and com-
pound 6b belong to the orthorhombic space group P21212
15. Badger, J.; Berard, D.; Kumar, R. A.; Szalma, S.; Yip, P.;
Griesinger, C.; Junker, J. CNX software manual, Molec-
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(www.accelrys.com).