Structure-based design, synthesis and biological evaluation of new N-carboxyphenylpyrrole derivatives as HIV fusion inhibitors targeting gp41

Article abstract of DOI:10.1016/j.bmcl.2009.10.139

A new series of N-carboxyphenylpyrrole ligands were designed using GeometryFit based on an X-ray crystal structure of gp41. The synthesized ligands showed significant inhibitory activities against HIV gp41 6-helix bundle formation, HIV-1 mediated cell-cel

Full text of DOI:10.1016/j.bmcl.2009.10.139

Bioorganic & Medicinal Chemistry Letters 20 (2010) 189–192
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based design, synthesis and biological evaluation of new
N-carboxyphenylpyrrole derivatives as HIV fusion inhibitors targeting gp41
b,
c,
Yong Wang a, Hong Lu b, Qiang Zhu a, , Shibo Jiang , Yun Liao
*
*
*
a State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Biomedicine and Health, The Chinese Academy of Sciences, Guangzhou 510663, China
b Lindsley F. Kimball Research Institute, New York Blood Center, NY 10065, USA
c GeometryLifeSci, LLC, 3500 Barrett Dr, # 8M, Kendall Park, NJ 08824, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of N-carboxyphenylpyrrole ligands were designed using GeometryFit based on an X-ray
crystal structure of gp41. The synthesized ligands showed significant inhibitory activities against HIV
gp41 6-helix bundle formation, HIV-1 mediated cell–cell fusion and HIV-1 replication.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 12 October 2009
Revised 29 October 2009
Accepted 30 October 2009
Available online 5 November 2009
Keywords:
HIV
Gp41
Inhibitor
AIDS causes very serious public health problem and economic
burden. Globally, an estimated 33 million people are living with
HIV, with nearly 7500 new infections each day.1 So far, 33 anti-
HIV drugs (including five fixed-dose combinations) have been
approved by FDA with 30 of them belonging to reverse transcrip-
tase inhibitors (RTI) and protease inhibitors (PI).2 Combination
therapies have shown significant synergistic effects.3 However,
there is still no cure or effective vaccine for AIDS, and current
anti-HIV drugs are facing increasing prevalence of viral resistance
and side effects.4,5 Therefore, it is in urgent need to develop novel
anti-HIV drugs addressing new targets other than RTI and PI.
HIV infects cells through endocytosis and envelope glycopro-
tein- and dynamin-dependent fusion.6 Gp41, a transmembrane
subunit of HIV-1 envelope glycoprotein, plays a crucial role in
HIV fusion and entry.7 Gp41 exists as a trimer and the ectodomain
of each monomer contains an N-terminal peptide (N-peptide) and
a C-terminal peptide (C-peptide). HIV fusion involves the insertion
of the gp41 N-peptides into host cell membrane and the subse-
quent binding of the C-peptides (anchored to viral membrane) to
N-peptides, which forms a 6-helical bundle bringing the viral
membrane and host cell membrane to proximity for fusion. Gp41
is a proven drug target—the peptides derived from the gp41 C-pep-
tides were found as potent HIV fusion inhibitors able to block the
N-peptides from binding to the C-peptides.8–13 One of the peptides,
T-20 (Fuzeon/Enfuvirtide, a 36-amino acid synthetic peptide)14–16
was approved by FDA in 2003 for treating patients with multi-drug
resistant HIV. However, T-20 is not orally available and has high
production cost.17 Therefore, developing orally available nonpep-
tide small-molecule fusion inhibitors targeting gp41 is highly
desirable.
So far, no small-molecule anti-HIV drug targeting gp41 has been
successfully developed, and gp41 has long been questioned for its
druggability for (1) It is difficult for small molecules to block the
very strong protein–protein interactions between gp41 C-peptides
(red, Fig. 1) and N-peptides (blue, Fig. 1); (2) gp41 N-peptide bun-
dle is highly hydrophobic and lack of deep pockets to allow strong
binding with small molecules, which makes it an even more diffi-
cult drug target.
Despite the formidable challenges, a few small-molecule gp41
inhibitors with modest activities at micromolar concentrations
were discovered during the past 10 years mostly via high-through-
put screening and diverse compound library synthesis.18–25 A12
* Corresponding authors.
E-mail addresses: zhu_qiang@gibh.ac.cn (Q. Zhu),
(S. Jiang), leonard_liao@geometrylifesci.com (Y. Liao).
SJiang@NYBloodCenter.org
Figure 1. gp41 C- and N-peptide bundle (left); gp41 N-peptide bundle (right).
Protein source: 1AIK (PDB code).8
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.139

190
Y. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 189–192
OH
COOH
N
A12
Figure 2. A12 (red) docked into gp41 N-peptide bundle (blue). Protein source:
2R5D (PDB code).28
(Fig. 2) is one of the most promising compounds discovered by Xie
and Jiang’s labs recently.26 Notably, A12 is not only able to inhibit
gp41 6-helical bundle formation (EC50 37.36 lM) but also druglike.
HOOC
Until Aug 2009, over 130 X-ray/NMR 3D structures related to
gp41 have been released from the Protein Data Bank (PDB),8,27–29
which provide invaluable information to understand gp41’s func-
tion at the atomic level as well as lay a solid foundation for struc-
ture-based design of small-molecule gp41 inhibitors.
N
GLS_12
Figure 4. Calculated most favorable binding mode of GLS_12.
However, structure-based de novo design of small-molecule
gp41 inhibitors remains extremely challenging largely due to the
lack of effective de novo drug design methodologies.30 Hamilton’s
pioneer work31 in de novo design of gp41 surface antagonists led to
hydrophobic pocket (yellow) composed of Leu29, Leu32, Thr33,
Val34 and Ile37, which happens to nicely complement the shape
of the hydrophobic pyrrole ring of A12 to form very favorable
hydrophobic interactions. These three key contact motifs may ac-
count for the binding of A12 to gp41, leading to the inhibition of
gp41 6-helix bundle formation.
a novel small-molecule gp41 inhibitor (EC50 ꢀ15
further optimization is difficult partly because its design was based
on general -helix mimicry of gp41 rather than a specific binding
pocket on gp41. Most recently, Liu et al. successfully designed a
novel small-molecule gp41 inhibitor (IC50 31 M, inhibition of
lM), however, its
a
In addition to the three key contact motifs, we then explored
additional potential contact motifs within the binding pocket in
order to find new ligands with improved binding affinities. The
most favorable binding mode of A12 suggests that its phenolic
group do not contribute significantly to its overall binding affinity
but do have plenty of room to extend into additional contact motifs
within the binding pocket, such as Arg43 (orange, Fig 3) and the
binding pocket composed of Trp35, Gln39, Leu40 (white, Fig. 3).
Using GeometryFit, we designed a new ligand GLS-12 (Fig. 4)
based on the A12 structure, in which the phenolic group of A12
was replaced by a phenyl group to not only fit into the shape of
the pocket composed of Trp35, Gln39, Leu40 (white, Fig. 4) and
Gln41 (green, Fig. 4) but also generate favorable hydrophobic inter-
actions with Trp35, Gln39, Leu40 (white, Fig. 4). The subsequent
docking study confirmed that, in the most favorable binding mode
of GLS-12 (Fig. 4), the new ligand well maintains the interactions
l
gp41 helical bundle formation) using their fragment-based de novo
ligand design technology, unfortunately, this compound failed to
show activity in a HIV mediated cell–cell fusion inhibitory assay
in the real life.32
We believe that A12 (Fig. 1) could serve as a valuable starting
point for further drug design targeting gp41 by modifying this
structure using GeometryFit, a proprietary knowledge-oriented,
computer-aided and structure-based de novo drug design method-
ology developed by GeometryLifeSci.33
Based on our docking study (gp41 N-peptide source:28 PDB ID:
2r5d) using AutoDock 4.0,34 we identified a binding mode of A12
(Fig. 3, A12: pink sticks) quite different from that proposed previ-
ously by Xie and Jiang.26 In our proposed binding mode, the calcu-
lated binding free energy of
D
G is À6.5 kcal/mol and the three key
contact motifs identified within the gp41 binding pocket are (1)
the flexible and positively-charged Lys38 (cyan sticks), which could
form a strong salt bridge with the negatively charged carboxylic
group of A12; (2) the hydrophilic Gln41 (green), which could form
two H-bonds with the same carboxylic group of A12; (3) the highly
HOOC
GLS_22
N
HOOC
Figure 3. Calculated most favorable binding mode of A12.
Figure 5. Calculated most favorable binding mode of GLS_22.

Y. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 189–192
191
R
OH
OH
N
OH
OH
COOCH3
COOH
COOCH3
COOH
ii, 76%
ii, 74%
i, 94%
COOH
N
NH2
2
NH2
1
GLS_12, R=H, 61%
N
A12
GLS_18, R=CH3, 55%
GLS_21, R=COOH, 63%
3
COOH
iii, 86%
vi
R
CHO
CHO
R
OTf
COOCH3
(HO)2B
v, 86%
iv
5d
iv
(HO)2B
5a, R=H
5b, R=CH3
5c, R=COOH
COOCH3
COOCH3
COOCH3
N
N
N
7
N
6a, R=H, 94%
6d, 88%
vii, 95%
6b, R=CH3, 95%
6c, R=COOH, 67%
4
vi, 66%
COOEt
COOH
COOEt
COOH
viii, 80%
vi, 61%
COOCH3
COOH
COOCH3
COOH
N
N
N
N
GLS_22
8
9
GLS_23
Scheme 1. Reagents and conditions: (i) H2SO4, MeOH, reflux; (ii) 2,5-hexanedione, p-TsOH, Toluene, reflux; (iii) Tf2O, Et3N, DCM, 0 °C; (iv) PdCl2(PPh3)2, K2CO3, THF/H2O
(2:1), reflux; (v) malonic acid, piperidine, pyridine, reflux; (vi) (1) NaOH, EtOH, (2) HCl; (vii) ethyl (triphenylphosphoranyliden)acetate, DCM; (viii) Pd/C, H2, THF/MeOH (4:1).
with the previous three key contact motifs while docking nicely
into the new binding pocket with its phenyl group. The calculated
GLS-22 (Fig. 5), the new ligand’s carboxylic group could form a salt
bridge with Arg43 and at the same time well maintain the interac-
tions with the previous four key contact motifs. As a result, the cal-
binding free energy of
D
G was increased to À7.0 kcal/mol.
To further increase the binding affinity, we decided to recruit
the positively charged Arg43 (orange, Fig. 4) as an additional con-
tact motif within the binding pocket. Using GeometryFit, we de-
signed another new ligand GLS-22 (Fig. 5) based on the structure
of GLS-12, in which a negatively charged carboxylic acid handle
was added at the para position of the phenyl ring in order to form
a strong electrostatic interaction with Arg43. The subsequent dock-
ing study confirmed that, in the most favorable binding mode of
culated binding free energy of
D
G was increased to À8.9 kcal/mol.
In this preliminary study, total of five new ligands were de-
signed, synthesized and screened for their inhibitory activities
against (1) HIV gp41 six-helix bundle (6-HB) formation, (2) HIV-
1-mediated cell-cell fusion (CF) and (3) HIV-1 replication (p24
and CPE), as previously described,21,26 using A12 as a control. The
synthesis is illustrated in Scheme 1 and the bio-assay results are
summarized in Table 1.
Table 1
Anti-HIV bioassay results versus calculated binding
D
G
GLS_12 R = H
GLS_18 R = Me
N
R
GLS_21 R = COOH
GLS_22 R = CH=CHCOOH
GLS_23 R = CH2CH2COOH
O
HO
Ligand35
D
G (calcd) (kcal/mol)
IC50
(
lM)
CC50
(l
M)
SIb
a
p24
CPE
CF
6-HB
CC50/IC50 (p24)
A12
À6.5
À7.0
À6.9
À8.7
À8.9
À8.6
28.19 3.79
17.76 2.74
18.19 2.04
25.63 1.92
4.91 0.69
8.30 1.34
55.45 16.59
37.92 7.55
79.54 8.16
33.93 6.66
7.71 1.64
43.24 2.28
59.17 2.00
51.61 3.38
26.01 1.31
3.60 0.27
8.30 0.11
29.39 5.47
28.96 2.27
21.26 2.88
20.50 1.11
20.73 2.11
21.75 0.75
333.84 22.88
289.80 13.18
280.04 38.11
355.23 24.47
255.28 3.73
227.27 22.86
11.84
16.31
15.39
13.85
51.99
27.38
GLS-12
GLS-18
GLS-21
GLS-22
GLS-23
13.08 3.19
a
CC50: 50% cytotoxicity concentration.
SI: selectivity index.
b

192
Y. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 189–192
8. Chan, D. C.; Fass, D.; Berger, J. M.; Kim, P. S. Cell 1997, 89, 263.
All 5 designed new ligands showed better activities than A12 in
9. Chan, D. C.; Kim, P. S. Cell 1998, 93, 681.
all assays except GLS_18 in the CPE assay and GLS_12 & 18 in the
CF assay. The assay results are in accordance with our predictions
using GeometryFit in most cases. The designed new ligands
(GLS_21,22,23) targeting two more contact motifs within the bind-
ing pocket than those of A12 all showed better activities across all
four assays, whereas the ligands (GLS_12,18) targeting only one
more hydrophobic contact motif than those of A12 mostly showed
comparable activities, which indicates that the second additional
contact motif, Arg43, may contribute more significantly to the
increased binding affinity. The best ligand, GLS_22, showed ꢀ6–
7-folds better inhibitory activity than A12 in the HIV replication
assays, ꢀ12-folds in the HIV cell–cell fusion assay and ꢀ1.4-folds
in the gp41 six-helix bundle formation assay. All five new ligands
10. Jiang, S.; Zhao, Q.; Debnath, A. K. Curr. Pharm. Des. 2002, 8, 563.
11. Jiang, S.; Lin, K.; Strick, N.; Neurath, A. R. Nature 1993, 365, 113.
12. Wild, C. T.; Shugars, D. C.; Greenwell, T. K.; McDanal, C. B.; Matthews, T. J. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 9770.
13. Lu, M.; Blacklow, S. C.; Kim, P. S. Nat. Struct. Biol. 1995, 2, 1075.
14. Kilby, J. M.; Eron, J. J. N. Eng. J. Med. 2003, 348, 2228.
15. Lalezari, J. P.; Henry, K.; O’Hearn, M.; Montaner, J. S.; Piliero, P. J.; Trottier, B.;
Walmsley, S.; Cohen, C.; Kuritzkes, D. R.; Eron, J. J., Jr.; Chung, J.; DeMasi, R.;
Donatacci, L.; Drobnes, C.; Delehanty, J.; Salgo, M. N. Eng. J. Med. 2003, 348,
2175.
16. Weissenhorn, W.; Dessen, A.; Harrison, S. C.; Skehel, J. J.; Wiley, D. C. Nature
1997, 387, 426.
17. Liu, S.; Wu, S.; Jiang, S. Curr. Pharm. Des. 2007, 13, 143.
18. Kazmierski, W. M.; Kenakin, T. P.; Gudmundsson, K. S. Chem. Biol. Drug Des.
2006, 67, 13.
19. Debnath, A. K.; Radigan, L.; Jiang, S. J. Med. Chem. 1999, 42, 3203.
20. Zhao, Q.; Ernst, J. T.; Hamilton, A. D.; Debnath, A. K.; Jiang, S. AIDS Res. Hum.
Retroviruses 2002, 18, 989.
had low cytotoxicity (CC50: 227–355 lM), and the most potent
ligand, GLS_22, had the highest Selectivity Index (SI) (CC50/IC50
(p24): 51.99).
21. Jiang, S.; Lu, H.; Liu, S.; Zhao, Q.; He, Y.; Debnath, A. K. Antimicrob. Agents
Chemother. 2004, 48, 4349.
22. Jin, B. S.; Lee, W. K.; Ahn, K.; Lee, M. K.; Yu, Y. G. J. Biomol. Screen. 2005, 10, 13.
23. Xu, Y.; Lu, H.; Kennedy, J. P.; Yan, X.; McAllister, L. A.; Yamamoto, N.; Moss, J. A.;
Boldt, G. E.; Jiang, S.; Janda, K. D. J. Comb. Chem. 2006, 8, 531.
24. Liu, S.; Lu, H.; Zhao, Q.; He, Y.; Niu, J.; Debnath, A. K.; Wu, S.; Jiang, T. S. Biochim.
Biophys. Acta 2005, 1723, 270.
25. Jiang, S.; Debnath, A. K.; Lu, H. U.S. Patent 0287319 A1, 2006.
26. Liu, K.; Lu, H.; Hou, L.; Qi, Z.; Teixeira, C.; Barbault, F.; Fan, B.-T.; Liu, S.; Jiang, S.;
Xie, L. J. Med. Chem. 2008, 51(24), 7843.
27. (a) Tan, K.; Liu, J.; Wang, J.; Shen, S.; Lu, M. Proc. Natl. Acad. Sci. U.S.A. 1997,
94(23), 12303; (b) Shu, W.; Ji, H.; Lu, M. J. Biol. Chem. 2000, 275(3), 1839; (c)
Shu, W.; Liu, J.; Ji, H.; Radigen, L.; Jiang, S.; Lu, M. Biochemistry 2000, 39(7),
1634; (d) Wang, S.; York, J.; Shu, W.; Stoller, M. O.; Nunberg, J. H.; Lu, M.
Biochemistry 2002, 41(23), 7283.
Summary: In this preliminary study, we identified a new bind-
ing mode of A12 involving three key contact motifs within the
binding pocket, based on which we explored two additional poten-
tial contact motifs within the binding pocket and designed five
new derivatives of A12 using GeometryFit. All five new ligands
showed improved anti-HIV activities in almost all assays, which
suggests that our design model and methodology are reliable, pav-
ing the way for our de novo design of novel small-molecule HIV
inhibitors targeting gp41 in the future.
28. Welch, B. D.; VanDemark, A. P.; Heroux, A.; Hill, C. P.; Kay, M. S. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 16828.
Acknowledgement
29. http://www.rcsb.org/pdb.
30. Mauser, H.; Guba, W. Curr. Opin. Drug Disc. Dev. 2008, 11(3), 365.
31. Ernst, J. T.; Kutzki, O.; Debnath, A. K.; Jiang, S.; Lu, H.; Hamilton, A. D. Angew.
Chem., Int. Ed. 2002, 41(2), 278.
32. Liu, B.; Joseph, R. W.; Dorsey, B. D.; Schiksnis, R. A.; Katrina, N.; Bukhtiyarova,
M.; Springman, E. B. Bioorg. Med. Chem. Lett. 2009, 19, 5693.
33. http://geometrylifesci.com.
This work was supported by GeometryLifeSci, LLC, an NIH grant
(AI46221) and grant funding from National Science Foundation of
China (20942001) and Start-up Foundation for New Investigators
from Guangzhou Institute of Biomedicine and Health (GIBH).
34. Morris, G.; Goodsell, D.; Halliday, R.; Huey, R.; Hart, W.; Belew, R.; Olson, A. J.
Comput. Chem. 1998, 19, 1639.
References and notes
35. Spectral data of the ligands synthesized: A12: 1H NMR (CDCl3, 400 MHz) d 10.46
(br, 1H), 7.80 (s 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 5.91 (s, 2H),
2.04 (s, 6H). MS: [M+1]+. 232 (ES+APCI). GLS_12: 1H NMR (CDCl3, 400 MHz) d
7.80 (s 1H), 7.46–7.38 (m, 7H), 5.93 (s, 2H), 2.17(s, 6H). MS: [M+1]+. 292
(ES+APCI). GLS_18: 1H NMR (CDCl3, 400 MHz) d 7.78 (s 1H), 7.46 (d, J = 8.0 Hz,
1H), 7.39 (d, J = 8.0 Hz, 1H), 7.30–7.22 (m, 4H), 5.93 (s, 2H), 2.41 (s, 3H), 2.09(s,
6H). MS: [M+1]+. 306 (ES+APCI). GLS_21: 1H NMR (DMSO-d6, 400 MHz) d 7.9 (d,
J = 8.0 Hz, 2H), 7.60–7.51 (m, 5H), 5.85 (s, 2H), 2.03 (s, 6H). MS: [M+1]+. 336
(ES+APCI). GLS_22: 1H NMR (DMSO-d6, 400 MHz) d 7.75 (d, J = 8.0 Hz, 2H), 7.64
(d, J = 16.0 Hz, 1H), 7.56–7.51 (m, 3H), 7.45 (d, J = 8.4 Hz, 2H), 6.58 (d,
J = 16.0 Hz, 1H), 5.84 (s, 2H), 2.02 (s, 6H). MS: [M+1]+. 362 (ES+APCI).
GLS_23: 1H NMR (DMSO-d6, 400 MHz) d 7.52–7.47 (m, 3H), 7.40–7.28 (m,
4H), 5.84 (s, 2H), 2.88 (t, 2H), 2.59 (t, 2H), 2.09(s, 6H). MS: [M+1]+. 364
(ES+APCI).
1. World Health Statistics; WHO, 2009, p 11.
2. http://www.hivandhepatitis.com/hiv_and_aids/hiv_treat.html.
3. De Clercq, E. J. Clin. Virol. 2004, 30, 115.
4. Richman, D. D.; Morton, S. C.; Wrin, T.; Hellmann, N.; Berry, S.; Shapiro, M. F.;
Bozzette, S. A. AIDS 2004, 18, 1393.
5. (a) Carr, A.; Cooper, D. A. Lancet 2000, 356, 1423;
strengthens warning on HIV drug, Wed Aug 26, 2009 5:32 pm EDT. http://
www.reuters.com/article/marketsNews/idINN2628355520090826?rpc=44.
6. (a) Miyauchi, K.; Kim, Y.; Latinovic, O.; Morozov, V.; Melikyan, G. B. Cell 2009,
137(3), 433. and references cited therein; (b) Fackler, O. T.; Peterlin, B. M. Curr.
Biol. 2000, 10, 1005.
b Johnson & Johnson
7. Moore, J. P.; Jameson, B. A.; Weiss, R. A.; Sattentau, Q. J. In Viral Fusion
Mechanisms; Bentz, J., Ed.; CRC Press: Boca Raton, FL, 1993; pp 233–289.