Novel N-substituted benzimidazole CXCR4 antagonists as potential anti-HIV agents

Article abstract of DOI:10.1016/j.bmcl.2010.02.053

The lead optimization of a series of N-substituted benzimidazole CXCR4 antagonists is described. Side chain modifications and stereochemical optimization led to substantial improvements in potency and protein shift to afford compounds with low nanomolar a

Full text of DOI:10.1016/j.bmcl.2010.02.053

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2125–2128
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel N-substituted benzimidazole CXCR4 antagonists as potential
anti-HIV agents
a
a
d
John F. Miller a, , Elizabeth M. Turner , Kristjan S. Gudmundsson , Stephen Jenkinson ,
*
Andrew Spaltenstein a, Michael Thomson b, Pat Wheelan c
a Department of Medicinal Chemistry, Infectious Diseases Center for Excellence in Drug Discovery, GlaxoSmithKline Research and Development, Five Moore Drive,
Research Triangle Park, NC 27709-3398, USA
b Department of Virology, Infectious Diseases Center for Excellence in Drug Discovery, GlaxoSmithKline Research and Development, Five Moore Drive, Research Triangle Park,
NC 27709-3398, USA
c Department of DMPK, Infectious Diseases Center for Excellence in Drug Discovery, GlaxoSmithKline Research and Development, Five Moore Drive, Research Triangle Park,
NC 27709-3398, USA
d Department of Biochemical and Analytical Pharmacology, Infectious Diseases Center for Excellence in Drug Discovery, GlaxoSmithKline Research and Development,
Five Moore Drive, Research Triangle Park, NC 27709-3398, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The lead optimization of a series of N-substituted benzimidazole CXCR4 antagonists is described. Side
chain modifications and stereochemical optimization led to substantial improvements in potency and
protein shift to afford compounds with low nanomolar anti-HIV activity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 26 January 2010
Revised 9 February 2010
Accepted 10 February 2010
Available online 14 February 2010
Keywords:
CXCR4
Benzimidazole
Tetrahydroquinoline
HIV
Antiviral
In spite of nearly three decades of intensive research toward the
treatment and eradication of HIV/AIDS, the disease remains a glo-
bal health crisis.1 The advent of highly active antiretroviral therapy
(HAART) in the mid 1990s, which employs various combinations of
reverse transcriptase and protease inhibitors, led to dramatic
improvements in clinical outcomes.2 More recently we have seen
the approval of the first examples of two newer classes of small
molecule antiretrovirals including the CCR5 antagonist Maraviroc3
and the integrase inhibitor Raltegravir,4 both in 2007. While the
diversity of drugs now available to the clinician has dramatically
improved HIV treatment, significant challenges remain. Of particu-
lar concern are long term treatment side effects5 and the emer-
gence of drug resistant viral strains.6
One area that has received a great deal of attention in recent
years is the concept of blocking viral entry by targeting the chemo-
kine receptors CCR5 and CXCR4 which function as co-receptors,
along with CD4, to facilitate fusion of the viral membrane with
the host cell.7 CXCR4 is a G-protein coupled seven-transmembrane
receptor utilized by T-tropic HIV strains to gain entry into T-cells.8
The appearance of CXCR4 utilizing strains of HIV is associated with
a decrease in the number of T-cells and accelerated disease
progression.9 In vitro studies have shown that the addition of the
natural CXCR4 ligand, SDF-1, or small molecule antagonists (e.g.,
AMD3100, AMD070) can block HIV infection.10 In addition,
AMD310011 and AMD07012 showed antiviral efficacy in recent
human clinical studies. Therefore the CXCR4 receptor represents
a promising HIV therapeutic target.
In a recent report our research group described SAR studies
based on AMD070 leading to the N-substituted benzimidazole 1
with potent anti-HIV activity.13 These studies clearly showed that
the 3-carbon tether provided the optimal distance between the
benzimidazole ring nitrogen and the distal amino group. This re-
sult prompted us to ask if further improvements in activity could
be achieved by conformationally constraining the basic amino
group by incorporating the 3-carbon chain into various ring sys-
tems (general structure A, Fig. 1). This report describes the synthe-
sis and SAR of a series of novel CXCR4 antagonists containing
various nitrogen heterocycles attached to the N-1 position of the
benzimidazole.
The first set of compounds that we prepared contained four, five
and six-membered rings attached to the benzimidazole nitrogen
* Corresponding author. Tel.: +1 919 483 2750; fax: +1 919 315 6923.
E-mail address: john.6.miller@gsk.com (J.F. Miller).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.053

2126
J. F. Miller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2125–2128
NH2
N
O
OtBu
H
H2N
NBoc
N
N
N
N
N
N
N
NBoc
c,d,e
a,b
N
N
N
N
R
N
NH2
16
(CH2)x
15
NH2
14
y
1
A
Figure 1.
N
N
N
N
N
f,g
h
N
through a methylene linker (Scheme 1). The syntheses were based
on alkylation of the previously reported racemic intermediate 313
with chlorides 2a–c which were themselves obtained by PS-PPh3
mediated chlorination of the corresponding alcohols. Potassium
carbonate promoted alkylation of 3 with 2a–c followed by acidic
deprotection gave compounds 4, 5 and 6. The secondary amines
were then methylated by reductive alkylation with formaldehyde
to afford methyl derivatives 7, 8 and 9. The second side chain motif
that we were interested in contained a piperidine ring attached to
the benzimidazole nitrogen through an ethylene linker (com-
pounds 12 and 13). The synthesis was directly analogous to that
described above and is also illustrated in Scheme 1.
N
N
N R
N
NBoc
O
17
19: R=H
18
i
20: R=Me
Scheme 2. Reagents and conditions: (a) 2-nitrochlorobenzene, K2CO3, DMF, 120 °C
(33%); (b) H2 (40 psi), 10% Pd(C), MeOH (97%); (c) CBz-glycine, BOP-chloride,
(iPr)2EtN, MeCN (86%); (d) glacial AcOH, 60 °C (87%); (e) H2 (40 psi), 10% Pd(C),
MeOH (92%); (f) compound 16, NaBH(OAc)3, AcOH, 1,2-dichloroethane (76%); (g)
37% aqueous formaldehyde, NaBH(OAc)3, 1,2-dichloroethane (78%); (h) TFA, CH2Cl2
(74%); (i) 37% aqueous formaldehyde, NaBH(OAc)3, 1,2-dichloroethane (88%).
Finally, we wanted to explore the effect of incorporating the dis-
tal nitrogen into a piperidine ring attached directly to the benz-
imidazole nitrogen (Scheme 2). This route begins with the
commercially available Boc-protected 4-aminopiperidine 14. This
compound was reacted with 2-nitrochlorobenzene under basic
conditions at elevated temperature. The resulting nitroaniline
derivative was subjected to catalytic hydrogenation to afford dia-
mine 15. Coupling of 15 with CBz-glycine followed by thermal
cyclization in AcOH and then hydrogenolysis of the CBz group gave
primary amine 16. Compound 16 was coupled with ketone 1714 by
reductive amination. This was followed by reductive methylation
of the central nitrogen to afford 18. The Boc group was cleaved
by treatment with TFA to give secondary amine 19 which was then
converted to the methyl derivative 20 by reductive methylation
with formaldehyde.
Table 1 shows antiviral and cytotoxicity data for 10 cyclic side
chain analogs along with the open chain parent compound 1 for
comparison. In general, cyclization of the amine side chain leads
to a relatively modest effect on antiviral activity ranging from two-
fold less to twofold more active than 1. A comparison of the meth-
ylene tethered N–H derivatives 4, 5 and 6 shows that the five-
membered ring is preferred providing a twofold increase in activity
relative to 1. Interestingly, methylation leads to an increase in
activity for the four and six-membered ring compounds 6 and 4
and a twofold loss in potency for the five-membered ring analog
5 suggesting that hydrophobicity alone is not a significant determi-
nant in the SAR. The ethylene linked piperidine analog 12 showed a
modest loss in activity relative to 1 with methylation conferring a
twofold potency increase. The equipotent N-piperidinyl analogs 19
and 20 were the least active in the entire set, probably due to the
higher degree of conformational rigidity associated with the side
chain. The six-membered ring N-methyl analog 7 emerged as the
most promising compound from this exercise with a 19 nM antivi-
ral activity and a 160-fold cytotoxicity window.
HO
NBoc
n
a
4: n=2
5: n=1
6: n=0
N
N
N
N
b,c
N
N
N
Cl
+
NBoc
n
2a: n=2
2b: n=1
2c: n=0
NH
NH
n
3
N
d
N
N
N
NMe
7: n=2
8: n=1
9: n=0
n
N
N
OH
e,f
g,h
Cl
N
N
H
N
O
10
11
OtBu
N
N
R
Having determined that the optimal side chain structure was
the six-membered ring associated with compound 7, we next
turned our attention to stereochemical considerations. Compound
7 contains two stereocenters and was initially prepared as a mix-
ture of four stereoisomers. We sought to deconvolute any potential
stereochemical SAR by individually preparing all four discreet ster-
eoisomers. The synthesis of the S,S stereoisomer is illustrated in
Scheme 3. Reaction of the commercial, enantiopure, Boc-protected
diamine 21 with 2-nitrofluorobenzene followed by catalytic reduc-
tion afforded diamine 22. Cyclization by treatment of 22 with
12: R=H
i
13: R=Me
Scheme 1. Reagents and conditions: (a) PS-PPh3, CCl4, CH2Cl2 (93% for 2a); (b)
K2CO3, KI, DMF, 90–120 °C (79% for n = 2); (c) 4 N HCl in dioxane, MeOH, (94% for 4);
(d) 37% aqueous formaldehyde, NaBH(OAc)3, 1,2-dichloroethane (80% for 7); (e)
(Boc)2O, CH2Cl2 (91%); (f) PS-PPh3, CCl4, CH2Cl2 (90%); (g) compound 3, K2CO3, DMF,
90 °C (50%); (h) 4 N HCl in dioxane, MeOH, (87%); (i) 37% aqueous formaldehyde,
NaBH(OAc)3, 1,2-dichloroethane (83%).

J. F. Miller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2125–2128
2127
Table 1
Anti-HIV IC50
containing cyclic amine sidechains
Cl
N
s
standard deviation (n) and CC50s for N-substituted benzimidazoles
NH2
HN
NBoc
c
N
NBoc
NBoc
H2N
a,b
N
N
21
22
23
N
N
R
N
N
N
N
N
N
d,e
f
a
b
Compound
R
IC50 (nM)
CC50 (nM)
N
N
N
NH
NMe
N
1
NH2
44 3 (2)
51 2 (2)
9600
24
H
CH3
H
N
4
5
6
15,000
25
26
Scheme 3. Reagents and conditions: (a) 2-nitrofluorobenzene, K2CO3, MeCN, reflux
(98%); (b) H2 (50 psi), 10% Pd(C), MeOH (99%); (c) (MeO)3CCH2Cl, PTSA, CH2Cl2
(85%); (d) compound 23, (iPr)2EtN, KI, MeCN, 65 °C (86%); (e) TFA, CH2Cl2 (97%); (f)
37% aqueous formaldehyde, NaBH(OAc)3, 1,2-dichloroethane (94%).
H
N
24 1 (2)
84 7 (2)
7700
15,000
N
H
CH3
N
7
8
9
19 2 (2)
57 2 (2)
66 5 (2)
3000
15,000
14,000
Table 2
Anti-HIV IC50
substituted benzimidazoles
s
standard deviation (n) and CC50s for stereochemically defined N-
CH3
N
A
N
N
N
CH3
N
B
CH3
N
N
12
13
64 4 (2)
33 1 (2)
8300
4900
N
H
a
b
Compound
A config
B config
IC50 (nM)
CC50 (nM)
N
7
26
27
28
29
R/S
S
S
R
R
R/S
S
R
S
R
19 2 (2)
6.0 1 (2)
21 1 (2)
210 (1)
3000
3600
3500
3600
2800
CH3
19
20
99 6 (2)
95 8 (2)
13,000
16,000
N
N
H
500 (1)
CH3
a,b
See footnotes a and b in Table 1.
a
HOS cells expressing hCXCR4/hCCR5/hCD4/pHIV-LTR-luciferase, HIV-1, CXCR4
strain (IIIB). Compounds were tested for their ability to block infection of the HOS
cell line. IC50 is the concentration at which 50% efficacy in the antiviral assay was
observed.15
CC50 is the concentration at which 50% cytotoxicity is observed in the HOS cell
line.
While compound 26 showed an impressive 6 nM antiviral activ-
ity, it suffered from a 10-fold reduction in activity when assayed in
the presence of serum proteins (human serum albumin and -acid
b
a
glycoprotein). Therefore, our final SAR exercise was focused on
reducing the protein shift by replacing the side chain methyl group
with several alternate substituents. This was easily accomplished
via reductive alkylations of secondary amine 25 with aldehydes
and ketones (Scheme 4).
chlorotrimethylorthoacetate under acid catalysis gave benzimid-
azole derivative 23. Alkylation of the previously reported, enantio-
pure, tetrahydroquinoline 2413 with 23 under basic conditions
followed by TFA mediated Boc deprotection afforded secondary
amine 25. Reductive alkylation with formaldehyde gave methyl
derivative 26 as the S,S stereoisomer. The remaining three stereo-
isomers were prepared in a directly analogous manner using the
appropriate enantiopure starting materials.
The SAR data associated with the four individual stereoisomers
and the isomer mixture 7 is illustrated in Table 2. The absolute
configuration at the tetrahydroquinoline attachment point (posi-
tion A) has the largest effect on potency showing a 20–40-fold
activity preference for the S configuration. This is consistent with
our previous observations of stereochemical SAR with the tetrahy-
droquinoline ring system.13,16 The effect of the absolute configura-
tion of the side chain (position B) is more subtle but shows a
consistent 2–4-fold preference for the S configuration. The S,S iso-
mer 26 showed a greater than sevenfold increase in activity over
the original racemic, open chain parent compound 1.
Antiviral and protein shift data for our final set of analogs is
shown in Table 3. Replacement of the side chain methyl group with
a hydrogen atom (compound 25) leads to a nearly complete loss of
protein shift with retention of antiviral activity. Substitution with
an isopropyl group (compound 30) reduced the protein shift down
to twofold and provided a threefold activity increase. The larger
branched alkyl analogs 31 and 32 gave protein shifts in the 5–6-
fold range accompanied by modest reductions in potency. Interest-
ingly, the polar side chains associated with compounds 33 and 34
provided minimal improvements in protein shift relative to 26.
From this study, compound 30 was identified as the most promis-
ing analog showing a very impressive 2 nM antiviral activity and a
twofold protein shift.
In order to further assess its viability as a potential drug candi-
date, compound 30 was subjected to pharmacokinetic analysis in

2128
J. F. Miller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2125–2128
and N-substituent optimization, it was possible to substantially
improve the antiviral activity (22-fold relative to 1). From these
studies, compound 30 emerged as a promising candidate showing
a 2 nM antiviral activity, a 1000-fold cytotoxicity window, and an
attractive twofold protein shift. In addition, compound 30 showed
acceptable pharmacokinetic properties in both rats and dogs. The
results described herein led us to pursue several additional struc-
ture–activity directions in the tetrahydroquinoline series. These
studies will be discussed in future reports.
N
N
N
N
a for 30
b for 31,32,33
c,d for 34
N
N
N
N
R
NH
N
25
30 - 34
Scheme 4. Reagents and conditions: (a) acetone, NaBH(OAc)3, 1,2-dichloroethane
(80%); (b) RCHO, NaBH(OAc)3, 1,2-dichloroethane (89% for 31); (c) TBSOCH2CHO,
NaBH(OAc)3, 1,2-dichloroethane; (d) TBAF, THF (56% for two steps).
Acknowledgements
We thank Richard Hazen, Wendell Lawrence, Mark Edelstein
and David McCoy for providing HIV-1 antiviral data and protein
shift data.
Table 3
Anti-HIV IC50
s
standard deviation (n), CC50
s
and protein shifts for distal N-
substituent optimization
References and notes
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HIV/AIDS (UNAIDS): Geneva, 2008.
N
N
2. (a) Detels, R.; Tarwater, R.; Phair, J. P.; Margolick, J.; Riddler, S. A.; Munoz, A.
Aids 2001, 15, 347; (b) Palella, F. J.; Delaney, K. M.; Moorman, A. C.; Loveless, M.
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N
R
N
3. Vandekerckhove, L.; Verhofstede, C.; Vogelaers, D. J. Antimicrob. Chemother.
2009, 63, 1087.
4. Chirch, L. M.; Morrison, S. A.; Steigbigel, R. T. Exp. Opin. Pharmacother. 2009, 10,
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Pirmohamed, M. J. Antimicrob. Chemother. 2008, 62, 648; (b) Johnson, M. O.;
Neilands, T. B. Aids Behav. 2007, 11, 575.
a
b
Compound
R
IC50 (nM)
CC50 (nM)
Fold IC50 protein
shiftc
26
25
Me
H
6.0 1 (2)
8.0 2 (2)
3600
3500
10
1.2
6. Carlo-Federico, P.; Moyle, G.; Tsoukas, C.; Ratanasuwan, W.; Gatell, J.;
Schechter, M. J. Med. Virol. 2008, 80, 565.
30
2.0 1 (4)
2100
2.0
7. (a) Qian, K.; Morris-Natschke, S. L.; Lee, K. Med. Res. Rev. 2009, 29, 369; (b)
Kuritzkes, D. R. Curr. Opin. HIV AIDS 2009, 4, 82; (c) Kazmierski, W. M.;
Gudmundsson, K. S.; Piscitelli, S. C. Ann. Rep. Med. Chem. 2007, 42, 301; (d)
Mosley, C. A.; Wilson, L. J.; Wiseman, J. M.; Skudlarek, J. W.; Liotta, D. C. Expert
Opin. Ther. Patents 2009, 19, 23; (e) Grande, F.; Garofalo, A.; Neamati, N. Curr.
Pharm. Des. 2008, 14, 385.
31
32
8.1 2 (2)
15 2 (2)
>2000
2300
6.2
5.1
8. Shaheen, F.; Collman, R. G. Curr. Opin. Infect. Dis. 2004, 17, 7.
9. Schuitemaker, H.; Koot, M.; Kootstra, N. A.; Dercksen, M. W.; de Goede, R. E.;
van Steenwijk, R. P.; Lange, J. M.; Schattenkerk, J. K.; Miedema, F.; Tersmette, M.
J. Virol. 1992, 66, 1354.
10. Grande, F.; Garofalo, A.; Neamati, N. Curr. Pharm. Des. 2008, 14, 385.
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S.; Jackson, J. B.; Coombs, R. W.; Glesby, M. J.; Flexner, C. W.; Bridger, G. J.;
Badel, K.; MacFarland, R. T.; Henson, G. W.; Calandra, G. J. Acquir. Immune Defic.
Syndr. 2004, 37, 1253.
N
33
34
33 3 (2)
12 3 (2)
2500
2500
8.7
9.1
OH
a,b
See footnotes a and b in Table 1.
Protein shift is the shift in concentration at which 50% efficacy in the antiviral
assay is observed in the presence of human serum albumin (45 mg/mL) and
glycoprotein (1 mg/mL).
c
a-acid
12. Moyle, G.; DeJesus, E.; Boffito, M.; Wong, R. S.; Gibney, C.; Badel, K.;
MacFarland, R.; Calandra, G.; Bridger, G.; Becker, S. Clin. Infect. Dis. 2009, 48,
798.
13. Gudmundsson, K. S.; Sebahar, P. R.; Richardson, L. D.; Miller, J. F.; Turner, E. M.;
Catalano, J. G.; Spaltenstein, A.; Lawrence, W.; Thomson, M.; Jenkinson, S.
Bioorg. Med. Chem. Lett. 2009, 19, 5048.
both rats and dogs. Following a 1 mg/kg IV dose in rats, 30 showed
a 5.8 h half life and an 8.5 mL/min/kg clearance value. Oral dosing
in rats at 3 mg/kg produced an 11% oral bioavailability. After IV
dosing at 1 mg/kg in dogs, an 11 h half life and a 9.4 mL/min/kg
clearance value was observed. Dog oral dosing at 1 mg/kg yielded
a 22% oral bioavailability. In addition, screening of compound 30
against a panel of enzymes and receptors showed little risk of
undesirable off-target activity including hERG.
14. Kelly, T. R.; Lebedev, R. L. J. Org. Chem. 2002, 67, 2197.
15. All compounds were also characterized in a cell fusion CXCR4 receptor binding
assay. The resulting SAR tracked very closely with the HOS antiviral data. For
example, compounds 1, 20 and 30 showed IC50s in the receptor binding assay
of 47 nM, 76 nM, and 4.5 nM, respectively. In addition, representative
compounds were characterized in a cell based functional assay and were
shown to be non-competitive antagonists of the CXCR4 receptor. For assay
protocols see: Gudmundsson, K.; Miller, J. F.; Turner, E. M. International Patent
WO 2006/020415, 2006.
Using the open chain analog 1 as a starting point for SAR stud-
ies, we have demonstrated that by employing the appropriate con-
formational modifications, in combination with stereochemical
16. Gudmundsson, K. S.; Boggs, S. D.; Catalano, J. G.; Svolto, A.; Spaltenstein, A.;
Thomson, M.; Wheelan, P.; Jenkinson, S. Bioorg. Med. Chem. Lett. 2009, 19, 6399.