Discovery of novel small molecule orally bioavailable C-X-C chemokine receptor 4 antagonists that are potent inhibitors of T-tropic (X4) HIV-1 replication

Article abstract of DOI:10.1021/jm100073m

The redesign of azamacrocyclic CXCR4 chemokine receptor antagonists resulted in the discovery of novel, small molecule, orally bioavailable compounds that retained T-tropic (CXCR4 using, X4) anti-HIV-1 activity. A structure-activity relationship (SAR) was determined on the basis of the inhibition of replication of X4 HIV-1 NL4.3 in MT-4 cells. As a result of lead optimization, we identified (S)-N′-((1H-benzo[d]imidazol-2-yl)methyl)- N′-(5,6,7,8-tetrahydroquinolin-8-yl)butane-1,4-diamine (AMD070) 2 as a potent and selective antagonist of CXCR4 with an IC₅₀ value of 13 nM in a CXCR4 ¹²⁵I-SDF inhibition binding assay. Compound 2 inhibited the replication of T-tropic HIV-1 (NL4.3 strain) in MT-4 cells and PBMCs with an IC₅₀ of 2 and 26 nM, respectively, while remaining noncytotoxic to cells at concentrations exceeding 23 μM. The pharmacokinetics of 2 was evaluated in rat and dog, and good oral bioavailability was observed in both species. This compound represents the first small molecule orally bioavailable CXCR4 antagonist that was developed for the treatment of HIV-1 infection.

Full text of DOI:10.1021/jm100073m

3376 J. Med. Chem. 2010, 53, 3376–3388
DOI: 10.1021/jm100073m
Discovery of Novel Small Molecule Orally Bioavailable C-X-C Chemokine Receptor 4 Antagonists
That Are Potent Inhibitors of T-Tropic (X4) HIV-1 Replication
Renato T. Skerlj,*,† Gary J. Bridger,‡ Al Kaller,‡ Ernest J. McEachern,‡ Jason B. Crawford,‡ Yuanxi Zhou,‡ Bem Atsma,‡
Jonathon Langille,‡ Susan Nan,‡ Duane Veale,‡ Trevor Wilson,‡ Curtis Harwig,‡ Sigrid Hatse,§ Katrien Princen,§
Erik De Clercq,§ and Dominique Schols§
†Genzyme Corp., 153 Second Avenue, Waltham, Massachusetts 02451, ‡AnorMED, Inc., 200-20353 64th Avenue, Langley, British Columbia,
V2Y 1N5, Canada, and Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
§
Received January 18, 2010
The redesign of azamacrocyclic CXCR4 chemokine receptor antagonists resulted in the discovery of
novel, small molecule, orally bioavailable compounds that retained T-tropic (CXCR4 using, X4) anti-
HIV-1 activity. A structure-activity relationship (SAR) was determined on the basis of the inhibition of
replication of X4 HIV-1 NL4.3 in MT-4 cells. As a result of lead optimization, we identified (S)-N0-((1H-
benzo[d]imidazol-2-yl)methyl)-N0-(5,6,7,8-tetrahydroquinolin-8-yl)butane-1,4-diamine (AMD070) 2
as a potent and selective antagonist of CXCR4 with an IC50 value of 13 nM in a CXCR4 125I-SDF
inhibition binding assay. Compound 2 inhibited the replication of T-tropic HIV-1 (NL4.3 strain) in
MT-4 cells and PBMCs with an IC50 of 2 and 26 nM, respectively, while remaining noncytotoxic to cells
at concentrations exceeding 23 μM. The pharmacokinetics of 2 was evaluated in rat and dog, and good
oral bioavailability was observed in both species. This compound represents the first small molecule
orally bioavailable CXCR4 antagonist that was developed for the treatment of HIV-1 infection.
Introduction
a ligand-induced conformational change that is unable to
support interaction with viral env gp120.5 Given that the most
commonly transmitted HIV-1 strains (M-tropic, CCR5 using
orR5) utilize the CCR5coreceptor, most of the research effort
has focused on identifying a CCR5 antagonist,7-9 culminat-
ing in the U.S. approval of maraviroc in 2007.10,11 Maraviroc
was indicated for combination antiretroviral treatment of
adults infected with exclusively CCR5-tropic HIV-1 whose
viral loads remain detectable despite existing antiretroviral
treatmentorwho have multipledrug resistant virus. However,
the emergence of T-tropic, CXCR4-using, or X4 HIV-1
variants in a minority of HIV-1 infected persons has been
observed following treatment with maraviroc.12 This devel-
opment strongly suggests that a combination of CCR5 and
CXCR4 antagonistsfortreatment of dual/mixedtropicHIV-1
infection will be required for complete viral suppression.
We have been actively involved in the development of
CXCR4 antagonists beginning with the prototype compound
AMD3100 (1, Figure 1), which was originally developed as
an anti-HIV agent13-15 and evaluated clinically in HIV-1
infected patients.16-18 Interestingly, in these initial clinical trials
leukocytosis was observed which upon further investigation
showed that administration of 1 resulted in consistent increases
in the number of peripheral blood CD34þ stem cells.19 This led
to the clinical development of 1 in hematopoietic stem cell
mobilization and the subsequent U.S. approval of plerixafor
(1) in 2008 for use in combination with granulocyte colony-
stimulating factor to mobilize hematopoietic stem cells to the
peripheral blood for collection and subsequent autologous
transplantation in patients with non-Hodgkin’s lymphoma
(NHL) and multiple myeloma (MM).20-22 In parallel we also
discovered and developed a small-molecule, orally bioavailable
The standard of care in treating patients infected with
HIV-1a is to begin a HAART regimen following a significant
decline in CD4þ T-cells, resulting in suppression of virus
replication to low or undetectable circulating HIV-1 RNA
levels.1 Initiation of HAART regimens immediately following
infection may have additional benefits such as slowing the
decline to AIDS and reducing the transmission of HIV.2
Typical HAART regimens consist of a combination of three
or more HIV-1 protease and reverse transcriptase inhibitors.
However, poor compliance due to a high pill burden and
significant long-term toxicities have resulted in the emergence
of drug resistant virus in many patients and hence the need for
new treatment options.
Entry blockade has been validated as a viable target since
the approval of enfuvirtide, an injectable anti-HIV drug that
prevents HIV entry by blocking env gp41-mediated fusion.3
Since the discovery of the chemokine receptors CXCR4 and
CCR5 as HIV coreceptors,4 a clearer understanding of the
mechanistic details of virus cell entry has emerged and a
tremendous amount of effort has been expended on discover-
ing novel antagonists of these receptors as potential anti-HIV
agents.5,6 CXCR4 and CCR5 belong to the seven-transmem-
brane G-protein-coupled receptor (GPCR) superfamily that
uponthe allosteric binding of a ligand to thereceptor results in
*To whom correspondence should be addressed. Phone: 781-434-
3684. Fax: 781-672-5823. E-mail: renato.skerlj@genzyme.com.
a Abbreviations: HIV, human immunodeficiency virus; HAART,
highly active antiretroviral therapy; AIDS, acquired immunodeficiency
syndrome; CXCR4, C-X-C chemokine receptor 4; CCR5, C-C-R
chemokine receptor 5.
r
pubs.acs.org/jmc
Published on Web 03/19/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3377
Figure 1. Structures of AMD3100 and AMD070.
Figure 2. Redesign evolution leading to nonmacrocyclic com-
pounds.
CXCR4 antagonist (AMD070) (2)23-25 (Figure 1) that was
shown to be a potent inhibitor of T-tropic (X4) HIV-1 replica-
tion.26 In a dose range finding study in healthy volunteers it was
demonstrated that at doses ranging from 50 to 400 mg 2 is safe,
well tolerated, and orally bioavailable.24 Administration of 1
and 2 to HIV-1 infected patients, whose virus was confirmed
to use CXCR4 for viral entry, suppressed the replication of
X4 and dual/mixed strains of viruses.17,24 The following dis-
cussion will focus on our medicinal chemistry efforts leading to
the discovery of 2.
a moderate anti-HIV-1 activity with an EC50 of 40 nM against
the HIV-1 (IIIB) strain. In addition, we had shown that
substitution of one of the nitrogen atoms with a pyridine ring
where the side chain was now linked to one of the proximal
nitrogen atoms as exemplified by compound B (Figure 2)
resulted in an anti-HIV-1 activity comparable to that of
compound A, EC50 of 63 nM vs 40 nM.27 Hence, compound
B represented the ideal starting point in our redesign efforts.
In the following discussion we will explain the rationale that
resulted in the discovery of nonmacrocyclic analogues repre-
sented by structure C. These initial compounds had anti-HIV-
1 activity equivalent to that of macrocycle B, thus supporting
our initial redesign criteria.
Redesign Criteria
Theimportance of maintaininga basic aminemoietyaspart
of the redesign criteria is highlighted by several studies that
have shown that binding of 1 and related analogues is highly
dependent upon the amino acids aspartic acid (Asp) 171 and
Asp 262, located in the transmembrane regions TM-IV and
TM-VI.30-34 In these studies it was concluded, on the basis of
receptor mutagenesis, that 1 acts on the CXCR4 receptor by
binding to the aspartic acid moieties D171 in TM-IV and
D262 in TM-VI and that these residues are essential for the
ability of 1 to block the binding of the chemokine ligand
stromal-cell-derived factor (SDF) 1R as well as the binding of
the CXCR4-specific antibody clone 12G5. More importantly
it was also found that in U87 cells stably transfected with CD4
and the mutant CXCR4 receptors CXCR4[D171N] and
CXCR4[D262N] the ability of 1 to inhibit HIV infection
was diminished confirming that 1 binds in a region of the
receptor that is critical for X4 HIV-1 coreceptor function.31
Recently we reported the structure-activity relationship of a
series of azamacrocyclic CXCR4 chemokine receptor antago-
nists and identified the pharmacophore necessary for potent
anti-HIV activity.27 Our original pharmacophore hypothesis
was that (1) the minimum macrocyclic requirements for
potent activity are the protonated secondary amine groups
in a 14-membered ring and (2) the activity is improved by
H-bond acceptors which presumably lock the ring in a favorable
conformation for antiviral activity.27 In terms of identifying a
small molecule antagonist of CXCR4, these compounds repre-
sented a substantial advance over the original bicyclams,28,29
since a 2-aminomethylpyridine group was identified as a repla-
cement for one of the macrocyclic rings (Figure 2). Although
the prototype compound AMD3465 exhibited potent inhibi-
tion against X4 HIV-1 replication,27 the oral bioavailability
was still poor, since the overall charge at physiological pH was
þ2 to þ3. As a consequence, a research effort was initiated to
find a nonmacrocyclic analogue with an overall reduced
charge at physiological pH that retained potent anti-HIV
activity and was orally bioavailable. The SAR that we had
earlier established was important in our redesign efforts
because we had previously shown that one of the nitrogen
atoms could be replaced by carbon or a phenyl group as
exemplified by compound A (Figure 2) while still maintaining
Chemistry
The five- to seven-membered bicyclic amines 3-5 were
synthesized by catalytic hydrogenation of the corresponding
acetamido substituted quinolines followed by acetamide
hydrolysis, as previously reported for the synthesis of 8-amino-
5,6,7,8-tetrahydroquinoline35 4. The bicyclic amines 3-5 under-
went reductive amination with N-(4-formylbenzyl)-2-nitro-
N-(pyridinyl-2-ylmethyl)benzenesulfonamide 6, which was
synthesized by oxidation of the corresponding alcohol,27
followed by nosyl group deprotection to afford the corres-
ponding secondary amines 10-12 (Scheme 1). The functio-
nalization of the intermediate scaffold 8 was performed by
N-alkylation with the appropriate alkyl halide or mesylate
using either K2CO3 or DIPEA in hot DMF or alternatively
using K2CO3 in CH3CN. Deprotection of the nosyl protecting
group was accomplished using thiophenol, and the resultant
amine 15 was isolated as the HBr salt (Scheme 1). Alterna-
tively the bicyclic amines 3 and 4 could be elaborated by first
undergoing reductive amination with pyridine-2-carboxalde-
hyde (imine formation followed by hydrogenation using 10%
Pd/C in MeOH) to afford the corresponding secondary
amine. N-Alkylation with N-[1-methylene-4-chloromethyl-
enephenylene]-N-(2-nitrobenzenesulfonyl)-2-(aminomethyl)-
pyridine27 orthesamemoietywith the corresponding diethyl-
phosphoryl protecting group27 followed by subsequent
deprotection and salting gave the corresponding HBr salts
14 and 13, respectively (Scheme 1).
The functionalization of the scaffolds 8 and 17 was per-
formed by one of the two following methods (Schemes 1
and 2). In the first method, the scaffold was N-alkylated with
the appropriate alkyl halide or mesylate and base in hot DMF
or CH3CN. Alternatively, the amine was alkylated by reduc-
tive amination with the appropriate aldehyde using either
NaBH3CN or NaBH(OAc)3. For intermediates where R =
nosyl, the protecting group was removed with thiophenol
as described above and the resultant amine isolated as its
HBr salt. The Boc-protected derivatives were simply treated

3378 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Skerlj et al.
Scheme 1a
a Reagents: (a) NaBH3CN, MeOH; (b) PhSH, K2CO3, CH3CN; (c) HBr, AcOH; (d) pyridine-2-carboxaldehyde, MeOH, Pd/C, H2; (e) N-[1-
methylene-4-chloromethylenephenylene]-N-(diethylphosphoryl)-2-(aminomethyl)pyridine, K2CO3, CH3CN; (f) N-[1-methylene-4-chloromethylene-
phenylene]-N-(2-nitrobenzenesulfonyl)-2-(aminomethyl)pyridine, K2CO3, CH3CN; (g) R0X, DIPEA or K2CO3, DMF; (h) R0X, K2CO3, CH3CN.
Scheme 2a
a Reagents: (a) 4, NaBH(OAc)3, MeOH; (b) R0CHO, NaBH3CN, MeOH; (c) HBr, AcOH.
Scheme 3a
with HBr/AcOH to afford the salts directly. For instance, in
the cases of compounds 15c and 15e a tert-butoxycarbonyl
protecting group was utilized (Scheme 2). Reductive amina-
tion of the Boc-protected aldehyde 16 with 8-amino-5,6,7,8-
tetrahydroquinoline 4 using NaBH(OAc)3 in MeOH gave the
Boc-protected scaffold 17. Subsequent reductive amination
a Reagents: (a) Dess-Martin periodinane, CH2Cl2; (b) 8, NaBH-
(OAc)3, MeOH; (c) PhSH, K2CO3, CH3CN; (d) HBr, AcOH.
with 2-imidazolecarboxaldehyde and N-(tert-butyloxy-
carbonyl)-N-benzylaminoacetaldehyde followed by concomi-
tant deprotection and salting afforded the HBr salts 15c and
15e, respectively (Scheme 2).
Similarly, compound 15f was synthesized by oxidation of
tert-butyl 2-oxoethyl(pyridine-2-ylmethylcarbamate 18 to
reverse amides 30 and 31 were prepared by first coupling
the corresponding aldehyde 19 with Dess-Martin periodi-
nane. Reductive amination with the secondary amine 8
using NaBH(OAc)3 in MeOH followed by sequential nosyl
group and Boc deprotection afforded the HBr salt 15f
afford the amides 30 and 31 (Scheme 6).
(Scheme 3).
The chain-extended benzimidazole analogues 22 and 23
amines 24 and 25, which were then coupled with 1H-benzi-
midazole-2-carbaldehyde by reductive amination to afford
the freebases 26 and 27, respectively (Scheme 5). Similarly the
4-chloromethylbenzoyl chloride with the appropriate amino-
pyridine and then N-alkylating the resultant benzyl chlorides
followed by concomitant Boc deprotection and salting to
The benzylic amines 32 and 33 were synthesized by reduc-
tive amination of 8-amino-5,6,7,8-tetrahydroquinoline 4 with
were prepared as described in Scheme 4. For example, con-
the regioisomeric cyanobenzaldehydes using NaBH(OAc)3
densation of phenylenediamine with 4-chlorobutyric acid in
refluxing HCl afforded the alcohol (1H-benzimidazol-2-yl)-
propan-1-ol, which was then protected with Boc2O and
oxidized to the aldehyde 21. Standard reductive amination
of 17 followed by salting gave the propyl linker 23. On the
other hand, condensation of phenylenediamine with 4-chlor-
opropionic acid in refluxing HCl afforded the chloride 2-(2-
chloroethyl)-1H-benzimidazole 20 which was protected with
Boc2O and used for N-alkylation with 17 followed by salting
to give the ethyl linker 22 (Scheme 4).
The amide analogues 26 and 27 were prepared in four steps
as outlined in Scheme 5. The coupling of 2-picolinic acid with
the appropriate aminobenzyl alcohol using EDC/HOBt to
afford the amide followed by mesylation and N-alkylation
with 8-amino-5,6,7,8-tetrahydroquinoline 4 gave the desired
in CH2Cl2 followed by N-alkylation with N-Boc-2-chloro-
methylbenzimidazole36 and subsequent reduction of the cya-
no group to the primary amine using H2 in the presence of
Raney Ni (Scheme 7).
Compounds 36-42, 46, and 47 were synthesized from the
key secondary amine intermediate 35 which was obtained
by N-alkylation of 4 with N-tert-butoxycarbonyl-2-chloro-
methylbenzimidazole.36,37 The desired side chain could be
installed by either N-alkylation or reductive amination of
the secondary amine followed by functional group manipula-
tion to afford the desired target compounds (Scheme 8). For
example, the (Z)-alkene 37 was readily prepared by N-alkyla-
tion of 35 with (Z)-tert-butyl 4-chlorobut-2-enylcarbamate
and subsequent deprotection with TFA to afford the corres-
ponding freebase 37. Similarly the (E)-alkene 38 was prepared

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3379
Scheme 4a
a Reagents: (a) ClCH2(CH2)nCO2H, 4 N HCl, reflux; (b) Boc2O, DlPEA, DMF; (c) Dess-Martin periodinane CH2Cl2; (d) 17, DlPEA, Nal, CH3CN,
80 °C; (e) 17, NaBH(OAc)3, AcOH, CH2Cl2; (f) HBr, AcOH.
Scheme 5a
a Reagents: (a) 2-PyCO2H, EDC, HOBt, NMM, DMF; (b) MsCl, Et3N, CH2Cl2; (c) 4, DlPEA, CH3CN, 80 °C; (d) 1H-benzimidazole-2-
carbaldehyde, NaBH(OAc)3, THF, 60 °C.
Scheme 6a
a Reagents: (a) 2-Py(CH2)nNH2, EtN, THF, 0 °C; (b) Ns-4, K2CO3, CH3CN, 80 °C; (c) PhSH, K2CO3, CH3CN; (d) N-Boc-2-chloromethylbenzi-
midazole, K2CO3, CH3CN, 80 °C; (e) HBr, AcOH.
Scheme 7a
aldehyde which underwent reductive amination with 8-amino-
5,6,7,8-tetrahydroquinoline 4in the presence of NaBH(OAc)3.
N-Alkylation of 35a with 5-bromovaleronitrile followed by
Raney Ni hydrogenation, SEM deprotection with 4 N HCl at
50 °C for 6 h, and salting afforded the HBr salt 43. In contrast
compound 44 was prepared by reductive amination of 35a
with tert-butyl (6-oxohexyl)carbamate followed by concomi-
tant deprotection of the Boc and SEM groups using 4 N HCl at
50 °C for 3 h to afford the freebase which was converted to the
HBr salt 44 using standard conditions.
a Reagents:(a) cyanobenzaldehyde, NaBH(OAc)3, CH2Cl2;(b) N-Boc-
The unprotected intermediate 35b, prepared by the reaction
2-chloromethylbenzimidazole, DIPEA, KI, CH3CN, 60 °C; (c) H2, Raney
of 2-(aminomethyl)benzimidazole with 6,7-dihydro-5H-qui-
Ni, MeOH, NH3; (d) benzaldehyde, NaBH4, MeOH; (e) HBr, AcOH.
nolin-8-one39 in dry MeOH to form the imine followed by
NaBH4 reduction, was used to prepare compounds 45 and 48.
by N-alkylation of 35 with (E)-N-(4-bromo-2-butenyl)phtha-
Compound 45 was prepared by a series of reactions starting
limide followed by phthalimide deprotection with hydrazine
with the reductive amination of 35b with 4-(tert-butyldi-
to afford the freebase 38. Compound 42 was prepared by
methylsilyloxy)butanal, TBDMS deprotection using HF-pyri-
N-alkylation of 35 with 4-bromovaleronitrile followed by
dine, N-Boc protection, Swern oxidation of the alcohol to the
Raney Ni catalyzed hydrogenation to afford the primary amine
aldehyde, reductive amination with N,N-dimethylamine, and
which was converted to the HBr salt 42. On the other hand the
finally Boc deprotection andsalting using HBrin aceticacid to
aliphatic amines 40 and 41 were prepared by reductive amina-
afford the HBr salt 45. In addition, 35b also underwent EDC
tion with N-(tert-butoxycarbonyl)-2-aminoacetaldehyde and
coupling with 4-(tert-butoxycarbonylamino)butanoic acid to
tert-butyl 3-oxopropylcarbamate, respectively, followed by
afford the corresponding amide which after standard depro-
deprotection and salting to afford the HBr salts.
tection and salting afforded the HBr salt 48.
The SEM38 protected intermediate 35a was used to prepare
compounds 43 and 44. Compound 35a was prepared by
Results and Discussion
N-protection of 2-hydroxymethylbenzimidazole with SEM-
Cl in the presence of DIPEA followed by oxidation with
MnO2 in CH2Cl2 to afford the corresponding SEM protected
The strategy that we employed in redesigning compound B
was to fix the side chain as the 2-aminomethylpyridine group

3380 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Skerlj et al.
Scheme 8a
Table 1. Anti-HIV-1 Activity of Small Molecule Antagonists of
CXCR4
a Reagents: (a) RX, DIPEA, KI, CH3CN, 60 °C;(b) TFA;(c) H2NNH2
3
H2O, EtOH, reflux; (d) H2, Raney Ni, MeOH, NH3; (e) RCHO, NaBH-
(OAc)3, CH2Cl2; (f) 4 N HCl; (g) HBr, AcOH; (h) HF-pyridine, THF;
(i) (Boc)2O, DIPEA, THF; (j) (COCl)2, DMSO, CH2Cl2; (k) NMe2,
NaBH(OAc)3, CH2Cl2; (l) HOBT, EDC, NMM, DMF.
and disconnect the pyridine C-C bond, thus opening up the
macrocyclic ring (Figure 2). However, we also recognized that
in order to maintain some degree of rigidity, modification of
the pyridine ring into a synthetically accessible bicyclic ring
system such as the amino substituted cycloalkylpyridines
would be necessary. These ring systems, e.g., 8-aminotetra-
hydroquinoline, satisfied our pharmacophore criteria (H-bond
acceptor capability and rigidity) and as a consequence were
deemed an appropriate replacement for the macrocylic ring. A
logical first step was to investigate the effect of varying the
cycloalkylpyridines ring size from 5 to 7. Although the amino
group is attached to a stereocenter, we made no attempt to resolve
the stereocenter at this early stage of the research program.
The primary data used to drive the SAR were the ability of
these compounds to inhibit replication of HIV-1 NL4.3 in
MT-4 cells, a strain of HIV that uses exclusively CXCR4 for
fusion and viral entry into target cells. The ability of these
compounds to inhibit replication of HIV-1 NL4.3 in MT-4
cells showed that the six- and seven-membered rings (11 and
12, Table 1) had an IC50 of 2.7 and 2.5 μM whereas the five-
membered ring 10 was less potent with an IC50 of 14 μM. A
close examination of molecular models revealed that the “bite
angle” of the primary amine increases with decreasing ring
size, suggesting that a bite angle that exceeds the six-mem-
bered ring is detrimental to anti-HIV-1 activity. To assess
whether this compound class would have good absorption,
results from the Caco-2 cell permeability assay for 11 pre-
dicted a high absorption potential in human and did not show
efflux transport potential (ratio Papp (B-A/A-B) < 3.0),
suggesting that 11 is a not a P-glycoprotein substrate. On the
basis of these encouraging preliminary results and ease of
synthesis, we prepared a series of simple analogues of 11 to
further test our pharmacophore hypothesis (Table 1).
We were gratified to find that the simple addition of a
methylpyridine moiety to the secondary amine to afford 14
increased the antiviral potency by an order of magnitude to an
IC50 of 0.27 μM (Table 1). The importance of the H-bond
acceptor was confirmed by comparing the antiviral activity of
the phenyl derivative 15b (IC50 > 4.3) versus the pyridine
derivative 14. In addition the length of the linker was im-
portant; the two-carbon linker 15a was 10-fold less active than
the one-carbon linker 14, suggesting that the optimal spacer
between the tertiary nitrogen atom and adjoining N was two
carbons. Substitution of the pyridine moiety with an alter-
native H-bond acceptor, an imidazole moiety 15c, resulted in
comparable activity: IC50 of 0.20 μM vs 0.27 μM. However,
incorporation of an H-bond donor such as the basic amino
moiety 15d rendered the compound inactive (IC50 > 25 μM).
Interestingly some activity was restored upon substitution of
the amine with a lipophilic benzylic moiety as in 15e, resulting
in an IC50 of 0.92 μM which could be attributed to a
combination of increased lipophilicity and decreased pKa
of the secondary benzylic amine (1 log reduction). A further
15-foldenhancement in antiviral activitywas accomplishedby
replacement of the phenyl group with an H-bond acceptor
moiety such as pyridine 15f which had an IC50 of 0.06 μM. A
similar antiviral activity (0.06 μM) was obtained with 15g
containing a more rigid and lipophilic benzimidazole moiety.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3381
Table 2. Anti-HIV-1 Activity of Benzimidazole Analogues of 15g
HIV-1 (NL4.3/MT-4)
IC50 (μM)
MT-4 cells
CC50 (μM)
compd
n
X
Y
15g
15i
15j
15k
22
1
1
1
1
2
3
N
NH
NH
O
0.06
1.6
9.1
4.6
1.1
1.2
23
8.0
Figure 3. Structure of compound 15g.
CH
N
204
22.3
23.1
101
that the ideal moiety on the top left-hand side of the molecule
was an unsubstituted tetrahydroquinoline containing a ter-
tiary amine at C8.
N
S
N
NH
NH
23
N
Substitution of the benzimidazole moiety of 15g was
also investigated to determine the impact of electronic and/
or steric parameters. However, a clear structure-activity
relationship was not evident, and since no enhancement in
antiviral activity was observed, we determined that the opti-
mum moiety for the southern quadrant was the unsubstituted
benzimidazole. In addition the effect of heteroatom replace-
ment on the benzimidazole moiety was investigated to further
understand the impact of basicity and charge on anti-HIV-1
activity and to test our pharmacophore hypothesis (Table 2).
It appears that the activity is directly related to the basicity of
the heteroaromatic moiety. As the basicity increases through
the series O to S to N (15j, 15k, 15g) the anti-HIV-1 activity
(IC50) in MT-4 cells also increases from 9.1 to 4.6 to 0.06 μM,
respectively, supporting our original pharmacophore hypo-
thesis of the necessity of a basic moiety linked to the tertiary
nitrogen. Linker length was also investigated (Table 2), and it
was found that the optimum linker length was one carbon,
since the addition of a two-carbon linker 22 or three-carbon
linker 23 reduced the anti-HIV-1 activity by approximately
20-fold, supporting the earlier hypothesis (Table 1) that a two-
carbon spacer between the tertiary N and adjoining N atoms
was the pharmacophore necessary for potency.
Interestingly linking the benzimidazole side chain directly
through the N atom as in 15h rendered the compound inactive
(IC50 of 18.8 uM).
With the discovery of several potential lead compounds,
these were further evaluated in a Caco-2 cell permeability
assay to predict for absorption and P-glycoprotein substrate
specificity. Although the antiviral activity of 15f was good, the
compound was predicted to have medium absorption and be a
substrate for P-glycoprotein (ratio Papp (B-A/A-B) > 3.0).
In a pharmacokinetic study todetermineoral bioavailability it
was observed that 15f was acutely toxic when given by the iv
route with an MTD of 1 mg/kg, suggesting that toxicity is
Cmax related. On the other hand compound 15g in the Caco-2-
cell permeability assay was predicted to have high absorption
and not be a substrate for P-glycoprotein (ratio Papp (B-A/
A-B) < 3.0). The high absorption was confirmed by the in
vivo evaluation of the (S)-enantiomer of 15g in rat and dog,
demonstrating an excellent oral bioavailability of 47% and
55%, respectively (Table 8). As a result of the good PK data
and anti-HIV-1 activity, this compound was selected as a lead
for further optimization.
Our strategy to optimize the antiviral activity of 15g was to
modify the tetrahydroquinoline, benzimidazole, and side
chain quadrants of the molecule as depicted in Figure 3.
Modification of the tetrahydroquinoline moiety was investi-
gated by synthesizing several analogues according to pre-
viously published methodology to determine the following:
the optimum point of attachment of the amino moiety,35 the
effect of saturation,40 the effect of replacing pyridine with
other heterocycles (bicyclic amines containing fused pyridine,
pyrazine, furan, and tetrahydropyran heterocycles), and hetero-
atom incorporation.41 The compounds were ranked, relative
to 11, according to the ability to inhibit replication of HIV-1
NL4.3 in MT-4 cells. However, none of the compounds
synthesized exhibited an antiviral activity superior to 11.
These results are consistent with our original pharmacophore
hypothesis, the requirement for an H-bond acceptor and a
protonated amine that are proximal to each other as demon-
strated by the 8-aminotetrahydroquinoline moiety. In addi-
tion the effect of substitution of the tetrahydroquinoline
moiety was also investigated. It was found that substitution
had an adverse effect on antiviral activity. The effect was
independent of the electronic nature of the substituents,
suggesting that steric factors had a detrimental impact on
antiviral activity. We concluded, on the basis of these results,
The final quadrant explored was the nature of the side chain
with the intent to determine the effect of varying the basicity
and length as shown by structures A and B (Table 3). For
instance, was a basic amino moiety a requirement for anti-
HIV-1 activity? To address this question, an amide was
introduced adjacent to the phenyl ring (structure B, com-
pound 31), resulting in a 25-fold loss in anti-HIV-1 activity
(IC50 of 1.44 μM). Similarly, the incorporation of an amide
adjacent to the pyridine group (structure A, compound 27)
resulted in an 18-fold loss in anti-HIV-1 activity (IC50 of 0.88
μM). On the other hand, truncation of the side chain amides
26 and 30 resulted in comparable activity to the full length side
chains 27 and 31 (approximately 1.3-fold difference), indicat-
ing that the length of the side chain was not critical for anti-
HIV-1 activity. On the basis of these results, it is apparent that
a basic amino moiety is required for potent antiviral activity
because the introduction of an amide, a neutral H-bond
donor, renders the compounds considerably less active.
More importantly, the necessity of the pyridine nitro-
gen was probed by substitution with a phenyl group, 34,
resulting in a 1.6-fold reduction in anti-HIV-1 activity, IC50
of 0.095 μM vs 0.059 μM, suggesting that the pyridine
nitrogen was not crucial for anti-HIV-1 activity (Table 3)
and the structure could be truncated while still retaining

3382 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Table 3. Anti-HIV-1 Activity of Side Chain Modification of 15g
Skerlj et al.
Table 4. Anti-HIV-1 Activity of Side Chain Modification
HIV-1 (NL4.3/MT-4) MT-4 cells
IC50 (μM)
compd structure
n
X
Y
CC50 (μM)
26
27
15g
34
30
31
A
A
A
A
B
0
1
1
1
0
1
CO
CO
CH2
CH2
CO
CO
N
N
N
C
0.66
0.88
0.059
0.095
0.98
1.44
37.5
36.8
23.0
26.4
34.3
126.8
N
N
B
potency. As a result, the truncated benzylic primary amine 32
was synthesized and found to have comparable anti-HIV-1
activity to 34 with an IC50 of 0.167 μM (1.7-fold difference),
supporting the notion that an unsubstituted basic amine is
sufficient for anti-HIV-1 activity (Table 4). Interestingly,
when the meta 33 and ortho 36 substituted benzylic amines
were evaluated, 36 was foundtobeapproximately 3-foldmore
active then 15g, IC50 of 0.021 μM vs 0.059 μM. This result led
to the conclusion that the phenyl group may not be necessary
for anti-HIV-1 activity, and as a consequence, a series of
closely related aliphatic analogues were prepared. We were
pleased to find that the direct analogue of the ortho benzylic
amine 36, the (Z)-alkene 37, had an anti-HIV-1 activity of
0.015 μM vs 0.021 μM whereas the anti-HIV-1 activity of the
(E)-isomer 38 was within 2-fold of that for 37. Evaluation of
the comparable saturated compound containing a butylamine
chain 42 resulted in an anti-HIV-1 activity of 0.0048 μM, a
3-fold increase compared to 37, which was the most potent
compound identified within this series. The chain length was
investigated to determine the optimal length required for anti-
HIV-1 activity. The ethylamine analogue 40 was 275-fold less
potent, whereasthe propylanalogue41was16-foldlesspotent
then the butylamine 42. Similarly the pentyl and hexyl analo-
gues 43 and 44 were 15-fold and 20-fold less potent than 42,
indicating that the optimum chain length was the butyl
analogue as originally hypothesized. Substitution of the pri-
mary amine as the N,N-dimethyl 45 or N-methyl 46 analogues
led to a 10-fold and 2-fold loss in potency. The importance of
the primary amine was further corroborated by substitution
with the nitrile 47 which resulted in a 155-fold reduction in
potency. In addition the importance of the tertiary amine was
substantiated by the amide 48 which was 550-fold less active
then 42, supporting our initial pharmacophore hypothesis.
All the compounds synthesized at this stage of the research
program were racemic mixtures. As a consequence, we set out
to separate the enantiomers in order to determine which
enantiomer provided more potent antiviral activity. We were
able to separate the two enantiomers of 15g using preparative
chiral HPLC to obtain (S)-15g and (R)-15g. It was gratifying
to find that there was a dramatic difference in anti-HIV-1
activity in MT-4 cells (Table 5); the (R)-enantiomer had an
IC50 of 1.08 μM (18-fold higher than the racemate), whereas
the (S)-enantiomer had an IC50 of 0.03 μM (2-fold more active
than the racemate). We were able to assign the absolute
configuration of (S)-15g by analogy to the key intermediate,
(S)-8-aminotetrahydroquinoline, where wehadunequivocally
established the absolute configuration based on a single X-ray
crystal structure of a rhenium complex and reported on the
synthesis of (S)-8-aminotetrahydroquinoline using a lipase
based enzymatic resolution of the racemate.41 This methodo-
logy was used to synthesize the single enantiomers of 15g and
42. As expected, a comparable difference in anti-HIV-1
activity was observed for the two enantiomers of 42; the (R)-
enantiomer was 30-fold less potent (0.144 μM), whereas the
(S)-enantiomer was 2-fold more potent (0.002 μM).
The effects of compounds 15g and 42 and the enantiomers
regarding the specific interaction with CXCR4 were studied in
more detail. The effects on SDF-1 induced Ca2þ flux in
CD4þCXCR4þ T cells (CEM-CCRF) cells were found to
correlate with the anti-HIV-1 activity in CD4þ CXCR4þ
T
cells (Table 5). Compounds 15g and (S)-15g both inhibited
Ca2þ flux with a comparable IC50 of 0.28 and 0.38 μM,
respectively, whereas the (R)-enantiomer was about 10-fold
less active with an IC50 of 2.47 μM. Similarly, compounds 42
and 2 both inhibited Ca2þ flux with a comparable IC50 of
0.017 and 0.012 μM, respectively, whereas the (R)-enantiomer

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3383
Table 5. Correlation of Anti-HIV-1 Activity and CXCR4 Specific Interactions of 2, 15g, and 42
IC50 (μM)
12G5 mAb inhibition
HIV-1 (NL4.3/
MT-4)
Ca-Flux
CEM-CCRF
compd
stereochemistry
125I-SDF-1 binding
15g
R/S
S
0.06
0.03
0.28
0.38
0.25
0.12
29.2
na
na
na
(S)-15g
(R)-15g
42
R
1.08
2.47
na
R/S
S
0.005
0.002
0.144
0.017
0.012
0.108
0.022
0.013
0.206
2
(R)-42
na
na
R
Table 6. Chemokine Receptor Selectivity Profiles of 2 and 15g
IC50 (μM)
compd
CCR1/MIP-1R
CCR2b/MCP-1
CCR4/TARC
CCR5/MIP-1β
CXCR1/interleukin-8
CXCR2/interleukin-8
(S)-15g
2
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Table 7. Anti-HIV-1 Activity of 2 and 15g against Other HIV-1 Strains, in PBMCs and in the Presence of 10% Human Serum
10% human serum
IC50 (μM)
HIV-1 NL4.3
HIV-1 IIIb
PBMC NL4.3
compd
IC50 (μM)
IC90 (μM)
IC50 (μM)
IC90 (μM)
IC50 (μM)
IC90 (μM)
HIV-1 NL4.3
HIV-1 IIIb
15g
2
0.051
0.001
0.137
0.003
0.060
na
0.108
na
0.277
0.009
0.559
0.026
0.069
0.016
0.081
na
was about 5-fold less active with an IC50 of 0.108 μM,
demonstrating a good correlation with the anti-HIV-1 acti-
vity. To provide further evidence that these compounds were
acting through antagonism of the CXCR4 receptor, the ability
to inhibit binding of a CXCR4-specific mAb (12G5) to CXCR4
on SUP-T1 cells and competitive binding with 125I-SDF-1 in
CD4þCXCR4þ T cells (CEM-CCRF) cells was also investi-
gated (Table 5). A good correlation between inhibition of
HIV-1 replication and inhibition of 12G5 binding to CXCR4
and competitive binding with 125I-SDF-1 was observed. For
example, compounds 15g and (S)-15g exhibited IC50 values of
0.25 and 0.12 μM for 12G5 mAb binding to CXCR4 and IC50
values of 0.06 and 0.03 μM for anti-HIV-1 activity. Similarly,
compounds 42 and 2 exhibited exhibited IC50 values of 0.022
and 0.013 μM for 125I-SDF-1 binding and IC50 values of 0.005
and 0.002 μM for anti-HIV-1 activity.
The selectivity of 15g and 2 against a series of other closely
related G-protein-coupled chemokine receptors (GPCRs)
(CCR1, CCR2b, CCR4, CCR5, CXCR1, and CXCR2) was
evaluated in radiolabeled chemokine-receptor binding assays,
and the IC50 binding affinities were found to be consistently
>10 μM, indicating that the compounds are selective for
CXCR4 (Table 6).
NL4.3 was 0.051 μM vs 0.060 μM in HIV-1 IIIb. The presence
of 10% human serum had little effect on the activity of 15g
against either virus strain. However, in the presence of 10%
human serum, 2was 16-fold less potent (0.016 μM vs 0.001 μM)
against the NL4.3 strain. The IC50 and IC90 were also
evaluated for X4 HIV-1 infection in PBMCs. The IC50 values
for 15g were 5-fold higher (0.277 μM vs 0.051 μM) and the
IC90 values were 4-fold higher (0.559 μM vs 0.137 μM) in
PBMCs compared toMT-4cells. Similarly, the IC50 valuesfor
2 were 9-fold higher (0.009 μM vs 0.001 μM) and 8.7-fold
higher (0.003 μM vs 0.026 μM) in PBMCs compared to MT-4
cells. This difference is comparable to that seen with other
compounds when comparing inhibition of infection in the
MT-4 CD4þ T cell tumor line and in PBMCs.
Because of ease of handling, the hydrochloride salts of 15g
and 2 were synthesized and evaluated in rat and dog pharma-
cokinetics (Table 8). The oral bioavailability of 15g was good
in both rat (F = 48%) and dog (F = 55%), resulting in the
selection of this compound for lead optimization as discussed
previously. Compound 2 showed promising oral bioavailabi-
lity in ratanddog. The rateof clearance was speciesdependent
with compound 2 having lower clearance in dog compared to
rat (1.3 vs 3.7 (mL/min)/kg), and this was reflected in a longer
half-life (9.9 h) and excellent oral bioavailability (80%). On
the basis of the pharmacokinetic data and ADME data, 2 was
selected for safety studies in rat and dog.
The antiviral activity of 15g and 2 was confirmed against
another X4 strain of HIV-1, HIV-1 IIIb in MT-4 cells. The
repeat studies were conducted in duplicate and both IC50 and
IC90 calculated (Table 7). The activity of 15g was comparable
with both strains of HIV-1. For example, the IC50 in HIV-1
In conclusion we successfully redesigned a class of azama-
crocyclic compounds that are antagonists of the chemokine

3384 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Table 8. Pharmacokinetics of 2 and 15g in Rat and Doga
Skerlj et al.
solution was stirred at room temperature (or 50 °C) for 1-24 h.
The solvent was removed under reduced pressure, and CH2Cl2
(10 mL/mmol amine) and water (2 mL/mmol amine) were added
to the residue. The phases were separated, and the aqueous
phase was extracted with CH2Cl2 (3 ꢀ 5 mL). The combined
organic phases were dried (Na2SO4) and concentrated. Purifica-
tion of the crude material by chromatography provided the free
base. An alternative workup is as follows: The reaction mixture
was filtered and concentrated to provide a yellow oil which was
purified by chromatography on basic alumina (eluant CH2Cl2,
then 20:1 CH2Cl2/CH3OH) to provide the free base as a color-
less oil.
Cmax
AUC0-inf
Cl
V
T1/2
F
compd (μg/mL) (mg h/mL) ((mL/min)/kg) (L/kg) (h) (%)
3
PK in Rat
(S)-15g
2
4.5
1.2
8.7
5.8
2.2
3.7
5.5
14.3
2.9
3.5
48
22
PK in Dog
(S)-15g
2
0.8
1.7
4.4
3.4
4.4
1.3
17.5
19.1
5.7
9.9
55
80
a Clearance (Cl), volume of distribution (Vdss) and half life (T1/2
)
General Procedure C: Salt Formation Using Saturated HBr (g)
in Acetic Acid. To a solution of the free base in glacial acetic acid
(or dioxane) (2 mL) was added a saturated solution of HBr (g) in
acetic acid (or dioxane) (2 mL). A large volume of ether (25 mL)
was then added to precipitate a solid, which was allowed to settle
to the bottom of the flask, and the supernatant solution was
decanted. The solid was washed by decantation with ether (3 ꢀ
25 mL), and the remaining traces of solvent were removed under
vacuum. For additional purification (where necessary), the solid
can be dissolved in methanol and reprecipitated with a large
volume of ether. Washing the solid with ether by decantation,
followed by drying of the solid in vacuo (0.1 Torr) gave the
desired compound.
General Procedure D: N-Alkylation of Amines with Mesylates
or Alkyl Halides. To a solution of the amine (1-1.4 equiv), N,N,-
diisopropylethylamine (or K2CO3) (1.5-2 equiv), and KI
(0.05-0.16 equiv) in CH3CN (∼0.1-0.2 M) was added the
mesylate or alkyl halide (such as 1-N-tert-butoxycarbonyl-2-
chloromethylbenzimidazole) (1-1.4 equiv). The mixture was
stirred at 50-70 °C for 3-25 h, as monitored by analytical thin
layer chromatography. The reaction mixture was cooled, di-
luted with CH2Cl2 (10 mL/mmol amine), and poured into either
saturated aqueous NaHCO3 or brine (10 mL/mmol alcohol).
The phases were separated, and the aqueous phase was extracted
with CH2Cl2 (3 ꢀ 10 mL/mmol amine). The combined organic
phases were dried (Na2SO4 or MgSO4) and concentrated under
reduced pressure. The crude material was purified by chroma-
tography to afford the desired N-alkylated product.
General Procedure E: Direct Reductive Amination with NaBH-
(OAc)3. To a stirred solution of the amine (1 equiv) in CH2Cl2
(∼0.2 M), at room temperature, were added the carbonyl
compound (∼1-2 equiv), glacial acetic acid (0-2 equiv), and
NaBH(OAc)3 (∼1.5-3 equiv). The resultant solution was stir-
red at room temperature. The reaction mixture was poured
into either saturated aqueous NaHCO3 or 1.0 M aqueous NaOH
(10 mL/mmol amine). The phases were separated, and the
aqueous phase was extracted with CH2Cl2 (3 ꢀ 10 mL /mmol
amine). The combined organic phases were dried (Na2SO4) and
concentrated. The crude material was purified by chromato-
graphy on silica gel.
General Procedure F: EDC Coupling. To a stirred solution of
the amine (1 equiv), acid (1.1 equiv), 1-hydroxybenzotriazole
(1.1 equiv), 4-methylmorpholine (1.5 equiv) in anhydrous DMF
(∼0.3 M), at room temperature under nitrogen atmosphere,
was added N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
(EDC) (1.1 equiv). The resultant solution was stirred at room
temperature. DMF was removed under vacuum. The mixture
was diluted with CH2Cl2 (100 mL/mmol), washed with NaH-
CO3, dried (Na2SO4), concentrated, and purified by chromato-
graphy on silica gel.
calculated following a 10 mg/kg iv dose in rat and 5 mg/kg iv dose in dog
for 15g and 5 mg/kg iv dose in rat and 2.5 mg/kg iv dose in dog for 2. Oral
bioavailability (F) calculated following solution doses of 40 mg/kg in rat
and 25mg/kg in dog for 15g and 100 mg/kginratand 6 mg/kg indog for 2.
receptor CXCR4 and potent inhibitors of (T-tropic, CXCR4
using, or X4) HIV-1 replication. The azamacrocyclic com-
pounds are not orally bioavailable because at physiological
pH they contain a charge of þ2 to þ3. Through our redesign
efforts we synthesized a novel series of small molecule orally
bioavailable antagonists of the chemokine receptor CXCR4,
based on inhibition of SDF-1 induced chemotaxis and cal-
cium flux and 12G5 binding data, that retained potent
inhibition of X4 HIV-1 replication. Recently others have also
reported on the discovery of oral CXCR4 agents.42 In addi-
tion these compounds were determined to be selective for
CXCR4 and displayed favorable pharmacokinetic properties.
The lead compound 2 was selected for preclinical safety
evaluation and ultimately entered the clinic for assessment
as an antiviral agent in HIV infected patients.23,24
Experimental Section
Unless otherwise noted, most of the reagents and solvents were
purchased at the highest commercial quality and used without
further purification. All reactions involving air- or moisture-
sensitive reagents were performed under a nitrogen or argon
atmosphere. Silica gel chromatography was performed using
glass columns packed with silica gel 60 (40-63 μM, VWR
1
International). H and 13C NMR spectra were recorded at 300
and 75 MHz, respectively, on a Bruker Avance 300 spectrometer
with shifts referenced to the residual proton shift of the internal
deuterated solvent. Electrospray mass spectra were recorded on a
Bruker-HP Esquire-LC ion trap mass spectrometer. Microana-
lyses for C, H, N, and halogen were performed by Atlantic
Microlab, Inc. (Norcross, GA) and were within 0.4% of theo-
retical values. Purity was determined by reversed phase HPLC
and was g95% for all compounds tested.
General Procedure A: Direct Reductive Amination with
NaBH3CN. To a stirred solution of the amine (∼1-2 equiv) in
anhydrous MeOH (concentration ∼0.1 M), at room tempera-
ture, was added the carbonyl compound (∼1-2 equiv) in one
portion. Once the carbonyl compound had dissolved (∼5 min),
NaBH3CN (∼2-4 equiv) was added in one portion and the
resultant solution was stirred at room temperature. The solvent
was removed under reduced pressure and CH2Cl2 (20 mL/mmol
of amine) and brine or 1.0 M aqueous NaOH (10 mL/mmol
amine) were added to the residue. The phases were separated,
and the aqueous phase was extracted with CH2Cl2 (3 ꢀ 10 mL/
mmol amine). The combined organic phases were dried
(Na2SO4) and concentrated. The crude material was purified
by chromatography on silica gel.
General Procedure G: Removal of Boc Group with TFA. A
solution of the Boc-protected amine in CH2Cl2 (∼0.05 M) and
TFA (∼ 1-2 mL/mmol) was stirred at room temperature for
∼1-3 h. The reaction mixture was diluted with CH2Cl2 and then
concentrated in vacuo. The residue was diluted with CH2Cl2 and
1 N NaOH to pH > 11. The aqueous layer was extracted with
CH2Cl2 and the combined organic layers were dried (Na2SO4),
filtered, and concentrated in vacuo to afford the desired amine,
General Procedure B: Deprotection of the 2-Nitrobenzenesul-
fonyl Group (Nosyl). To a stirred solution of the nosyl-protected
amine (1 equiv) in anhydrous CH3CN (or DMF) (∼0.05 M), at
room temperature was added thiophenol (4-8 equiv) followed
by powdered K2CO3 (8-12 equiv). The resulting bright-yellow

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3385
which was either used without purification in the next step or
purified by chromatography on silica gel.
(d, 1H, J = 14.1 Hz), 3.85 (s, 2H), 3.90 (s, 2H), 4.05 (dd, 1H, J =
9.0, 5.7 Hz), 6.24 (s, 1H), 7.02 (t, 1H, J = 7.5 Hz), 7.10-7.15 (m,
3H), 7.24-7.31 (m, 2H), 7.36-7.51 (m, 6H), 7.61 (dt, 1H, J =
N-(2-Nitrobenzenesulfonyl)-N-(2-pyridinylmethyl)-N0-(5,6,7,8-
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine (8). By use
of general procedure A, reaction of 4 (21.2 g, 51 mmol) with 6
(7.61 g, 51 mmol) followed by purification using silica gel
chromatography (20:1 CH2Cl2/CH3OH) gave 8 (11 g, 40%) as
an orange oil. 1H NMR (CDCl3) δ 1.74-1.84 (m, 2H), 1.99-2.05
(m, 1H), 2.02-2.05 (m, 1H), 2.72-2.86 (m, 2H), 3.13 (br s, 1H),
3.79-3.94 (m, 3H), 4.57 (s, 2H), 4.60 (s, 2H), 7.07-7.11 (m, 4H),
7.20-7.24 (m, 3H), 7.37 (d, 1,H, J = 7.4 Hz), 7.53, (t, 2H, J = 8.4
Hz) 7.64 (br s, 2H), 7.94 (d, 1H, J = 7.8 Hz), 8.40 (t, 2H, J = 5.9
Hz); 13C NMR (CDCl3) δ 19.70, 28.61, 28.85, 51.43, 51.54, 52.37,
57.56, 122.26, 122.78, 122.82, 124.55, 128.91 (2), 129.12 (2),
131.39, 131.98, 132.87, 133.65, 133.98, 134.60, 136.98, 137.28,
140.87, 147.20, 148.30, 149.60, 156.34, 157.77. ES-MS m/z 544
7.5, 1.8 Hz), 8.55 (d, 1H, J = 7.2), 8.63 (d, 1H, J = 7.2 Hz); 13
C
NMR (CDCl3) δ 21.09, 23.69, 29.15, 47.41, 53.25, 53.82, 54.48,
59.15, 99.14, 110.98, 118.85, 119.65, 120.57, 121.87, 121.89,
122.35, 128.15, 128.68, 128.85, 134.44, 135.89, 136.40, 136.86,
138.75, 138.77, 139.11, 147.01, 149.25, 158.04, 159.78. ES-MS
m/z 488 (M þ H). Anal. (C32H33N5 0.6H2O) C, H, N.
3
Pyridine-2-carboxylic Acid 4-{[(1H-Benzimidazol-2-ylmethyl)-
(5,6,7,8-tetrahydroquinolin-8-yl)amino]methyl}benzylamide (27).
By use of the procedure described for compound 26, (4-amino-
methylphenyl)methanol (200 mg, 1.46 mmol) and picolinic acid
(195 mg, 1.61 mmol) gave the corresponding amido alcohol
which was converted directly to 4-(picolinamidomethyl)benzyl
methanesulfonate (214 mg, 60%, 2 steps). By use of general
procedure D, reaction of 4 (148 mg, 1.00 mmol) and 4-(picolin-
amidomethyl)benzyl methanesulfonate (214 mg, 0.7 mmol) fol-
lowed by purification on silica gel (CH2Cl2/CH3OH/NH4OH
97:1.5:1.5) gave 25 (81 mg, 33%) as a yellow oil. By use of general
procedure E, reaction of N-(4-((5,6,7,8-tetrahydroquinolin-8-
ylamino)methyl)benzyl)picolinamide (25) (40 mg, 0.11 mmol)
and 1H-benzimidazole-2-carbaldehyde (16 mg, 0.11 mmol) fol-
lowed by purification on silica gel (CH2Cl2/CH3OH/NH4OH
198:1:1) gave 27 (28 mg, 52%) as a white foam. 1H NMR
(CD3OD) δ 1.60-1.63 (m, 1H), 1.95-2.06 (m, 2H), 2.20-2.22
(m, 1H), 2.66-2.83 (m, 2H), 3.51 (d, 1H, J = 13.2 Hz), 3.58 (d,
1H, J = 13.2 Hz), 3.94 (d, 1H, J = 15.3 Hz), 4.04-4.09 (m, 3H),
4.42 (br s, 2H), 7.09-7.15 (m, 4H), 7.20 (dd, 1H, J = 7.8, 4.8 Hz),
7.20 (d, 1H, J = 8.1 Hz), 7.28 (d, 2H, J = 8,1 Hz), 7.41-7.47 (m,
2H), 7.48-7.52 (m, 2H), 7.91 (ddd, 1H, J = 7.5, 7.5, 1.8 Hz),
8.06 (d, 1H, J = 7.8 Hz), 8.55 (d, 1H, J = 4.5 Hz), 8.60 (d, 1H,
J = 4.5 Hz); 13C NMR (CD3OD) δ 22.84, 24.33, 30.62, 44.13,
51.41, 56.15, 63.13, 123.55, 124.03, 128.13, 128.61, 130.80, 137.39,
138.95, 139.14, 139.51, 139.67, 148.35, 150.17, 151.40, 156.31,
(M þ H). Anal. (C29H29N5O4S 0.1CH2Cl2) C, H, N.
3
N-(tert-Butoxycarbonyl)-N-(2-pyridinylmethyl)-N0-(5,6,7,8-
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine (17). By use
of general procedure E, reaction of 4 (4.16 g, 28.1 mmol) with 16
(9.15 g, 28.1 mmol) followed by purification on silica gel
(EtOAc) gave 17 (9.65 g, 75%) as a yellow oil. 1H NMR
(CDCl3) (mixture of rotational isomers) δ 1.41 (br s) and 1.48
(br s) (total 9H), 1.76-1.83 (m, 2H), 2.02-2.06 (m, 1H),
2.15-2.18 (m, 1H), 2.75-2.83 (m, 2H), 3.81-3.85 (m, 1H),
3.86 (d, 1H, J = 12 Hz), 3.97 (d, 1H, J = 12 Hz), 4.44 (br s, 2H),
4.53 (br s, 2H), 7.04 (dd, 1H, J = 7.8, 4.8 Hz), 7.12-7.25 (m,
4H), 7.33-7.37 (m, 3H), 7.62 (td, 1H, J = 7.5, 1.8 Hz), 8.38 (dd,
1H, J = 4.8, 1.2 Hz), 8.52 (dd, 1H, J = 5.7, 1.8 Hz); 13C NMR
(CDCl3) (mixture of rotational isomers) δ 19.37, 28.09, 28.34,
28.56, 49.83, 50.08, 51.22, 51.45, 57.21, 79.71, 79.83, 120.47,
121.49, 121.66, 121.73, 127.36, 128.12, 132.10, 136.18, 136.31,
136.52, 139.50, 146.49, 148.89, 155.62, 157.17, 157.91, 158.27.
ES-MS m/z 459 (M þ H).
N-(2-Pyridinylmethyl)-N0-(1H-benzimidazol-2-ylmethyl)-N0-
(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine Tetra-
hydrobromide Dihydrate (15g). By use of general procedure D,
reaction of 8 (425 mg, 0.78 mmol) with 2-chloromethylbenzimi-
dazole (129 mg, 0.77 mmol) followed by purification by radial
chromatography on silica gel (2 mm plate, 20:1 CH2Cl2/CH3OH
containing 1% NH4OH) gave the protected amine (169 mg, 31%)
as a yellow solid. By use of general procedure B, reaction of the
protected amine (161 mg, 0.239 mmol) followed by purification
using radial chromatography on silica gel (2 mm plate, 50:1:1
CH2Cl2/CH3OH/NH4OH) gave the free base (61 mg, 52%) as a
yellow oil. By use of general procedure C, conversion of the free
base (61 mg, 0.125 mmol) to the HBr salt gave 15g (79 mg, 74%) as
a white solid. 1H NMR (D2O) δ 1.93-1.98 (m, 1H), 2.19-2.31 (m,
2H), 2.41-2.46 (m, 1H), 3.20 (br s, 2H), 3.77-3.88 (m, 4H), 4.16
(s, 2H), 4,44 (d, 1H, J = 16.5 Hz), 4.63 (d, 1H, J = 16.5 Hz),
4.73-4.79 (m, 1H, overlaps with HOD), 7.04 (d, 2H, J = 8.1 Hz),
7.23 (d, 2H, J = 7.8 Hz), 7.37 (dd, 2H, J = 3.0, 6.3 Hz), 7.54 (dd,
2H, J = 3.0, 6.3 Hz), 7.67 (d, 1H, J = 7.8 Hz), 7.72 (dd, 1H, J =
6.3, 6.9 Hz), 7.91 (dd, 1H, J = 6.0, 7.8 Hz), 8.20 (t, 1H, J =
7.8 Hz), 8.39 (d, 1H, J = 8.1 Hz), 8.67 (d, 1H, J = 5.1 Hz), 8.75
(d, 1H, J = 5.7 Hz); 13C NMR (D2O) δ 20.46, 20.97, 27.87, 48.88,
50.22, 50.44, 56.71, 63.26, 113.92, 126.15, 126.43, 126.52, 126.65,
130.04, 130.22, 130.47, 130.92, 138.23, 139.70, 141.05, 142.99,
147.15, 147.95, 148.32, 150.80, 151.79. ES-MS m/z 489 (M þ H).
Anal. (C31H32N6 4.0HBr 2.0H2O) C, H, N, Br.
158.31, 166.94. ES-MS m/z 503 (M þ H). Anal. (C31H30N6O
3
0.8H2O 0.3CH2Cl2) C, H, N.
3
4-{[(1H-Benzo[d]imidazol-2-ylmethyl)(5,6,7,8-tetrahydro-
quinolin-8-yl)amino]methyl}-N-(pyridin-2-yl)benzamide (30). To
a solution of 2-aminopyridine (304 mg, 3.22 mmol) and Et3N
(0.8 mL, 5.70 mmol) in anhydrous THF (5 mL) at 0 °C was added
a solution of 4-(chloromethyl)benzoyl chloride (282 mg, 1.40
mmol) in THF (5 mL), and the mixture was stirred at 0 °C for 3 h.
The mixture was diluted with EtOAc (75 mL) and saturated
aqueous NH4Cl (50 mL). The organic layer was washed with
brine (1 ꢀ 50 mL), dried (Na2SO4), concentrated, and purified
on silica gel (hexanes/EtOAc, 9:1) to give 4-(chloromethyl)-
N-(pyridinyl-2-yl)benzamide (170 mg, 49%) as a white solid. By
use of general procedure D, reaction of 8 (230 mg, 0.69 mmol)
with 4-(chloromethyl)-N-(pyridinyl-2-yl)benzamide (170 mg,
0.69 mmol) followed by purification on silica gel (15:1 CH2Cl2/
EtOAc) gave the protected amine (344 mg, 92%) as a white foam.
By use of general procedure B, reaction of the protected amine
(340 mg, 0.62 mmol) followed by purification using radial
chromatography on silica gel (2 mm plate, 3:3:94 MeOH/
NH4OH/CH2Cl2) afforded 28 (210 mg, 93%) as a pale-yellow
oil. By use of general procedure D, reaction of N-(pyridin-2-yl)-
4-[(5,6,7,8-tetrahydroquinolin-8-ylamino)methyl]benzamide
(28) (155 mg, 0.43 mmol) with N-Boc-2-chloromethylbenzimid-
azole36 (115 mg, 0.43 mmol) followed by purification using radial
chromatography on silica gel (2 mm plate, 3:3:94 MeOH/
NH4OH/CH2Cl2) afforded 30 (100 mg, 47%) as a white foam.
1H NMR (CDCl3) δ 1.68-1.75 (m, 1H), 1.97-2.08 (m, 2H),
2.26-2.32 (m, 1H), 2.71-2.84 (m, 1H), 2.86-2.91 (m, 1H), 3.84
(s, 2H), 3.95 (d, 1H, J = 16.5 Hz), 4.08-4.14(m, 1H), 4.22 (d, 1H,
J = 16.0 Hz), 7.03-7.07 (m, 1H), 7.16-7.22 (m, 3H), 7.45 (d, 1H,
J = 6.9 Hz), 7.52-7.61 (m, 3H), 7.65 (d, 1H, J = 7.5 Hz), 7.73
(ddd, 1H, J = 1.8, 7.2, 8.7 Hz), 7.80 (d, 2H, J = 8.1 Hz),
8.27-8.29 (m, 1H), 8.33 (d 1H, J = 8.4 Hz), 8.44 (br s, 1H),
8.72-8.80 (m, 1H); 13C NMR (CDCl3) δ 21.72, 23.30, 29.56,
3
3
N-(2-Pyridinylmethyl)-N0-(indol-2-ylmethyl)-N0-(5,6,7,8-tetra-
hydro-8-quinolinyl)-1,4-benzenedimethanamine (15i). By use of
general procedure E, reaction of 8 (320 mg, 0.58 mmol) and
3H-indole-2-carboxaldehyde (85 mg, 0.58 mmol) gave the pro-
tected amine (300 mg, 77%) as a yellow oil. By use of general
procedure B, reaction of the protected amine (150 mg, 0.22
mmol) gave the free base 15i (55 mg, 51%) as a pale-yellow
1
solid. H NMR (CDCl3) δ 1.63-1.72 (m, 1H), 1.85-1.95 (m,
1H), 1.97-2.09 (m, 1H), 2.12-2.22 (m, 1H), 2.68-2.72 (m, 1H),
2.77-2.90 (m, 1H), 3.65 (d, 1H, J = 13.5 Hz), 3.78 (s, 2H), 3.80

3386 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Skerlj et al.
49.29, 54.16, 60.83, 111.39, 114.52, 120.17, 122.29, 122.81, 127.72,
129.28, 133.58, 135.17, 137.76, 138.77, 144.61, 147.38, 148.21,
152.00, 156.13, 157.54, 165.99. ES-MS m/z 489.2 (M þ H). Anal.
(C30H28N6O 0.6H2O 0.7CHCl3) C, H, N.
against viral cytopathicity. The 50% cytotoxic concentration
(CC50) was defined as the compound concentration required to
reduce the viability of mock-infected PBMCs or MT-4 cells by
50%. The “greater than” symbol (>) is used to indicate the
highest concentrations at which the compounds were tested and
still found to be noncytotoxic. Average IC50 and CC50 values for
several separate experiments are presented as defined above.
125I-Chemokine Competition Binding Assay. Competition
binding studies against CXCR4 were performed in human
CD4þCXCR4þ CEM-CCRF cells. CXCR4 inhibitors in a con-
centration range were incubated for 3 h at 4 °C in binding buffer
(PBS containing 5 mM MgCl2, 1 mM CaCl2, 0.25% BSA, pH
7.4) with 5 ꢀ 105 cells and 100 pM 125I-SDF-1R (Perkin-Elmer,
2200 Ci/mmol) in Millipore Durapore filter plates. Unbound
125I-SDF-1R was removed by washing with 50 mM HEPES,
0.5 M NaCl, pH 7, at 4 °C. The bound radioactivity was counted
using a 1450 MicroBeta liquid scintillation counter (Wallac).
3
3
N-[(1H-Benzo[d]imidazol-2-yl)methyl]-N-(4-(aminomethyl)-
benzyl)-5,6,7,8-tetrahydroquinolin-8-amine (32). By use of general
procedure E, reaction of 4 (24.3 g, 164 mmol) with 4-cyanoben-
zaldehyde (21.5 g, 164 mmol) followed by purification on silica
gel (5% CH3OH/CH2Cl2) afforded 4-[(5,6,7,8-tetrahydroquino-
lin-8-ylamino)methyl]benzonitrile (30.9 g, 72%) as a pale-yellow
solid. By use of general procedure D, reaction of 4-[(5,6,7,8-
tetrahydroquinolin-8-ylamino)methyl]benzonitrile (30.1 g, 114
mmol) with N-(tert-butoxycarbonyl)-2-chloromethylbenzimida-
zole (30.5 g, 114 mmol) followed by purification on silica gel (3%
CH3OH/CH2Cl2) gave the coupled product (38.0 g, 67%) as a
pale-orange foam. This material (35 g, 70.9 mmol) was subse-
quently dissolved in NH3 saturated methanol, treated with Raney
nickel, and placed under 50 psi of H2 on a Parr shaker for 16 h.
The mixture was filtered through Celite 521, concentrated, and
purified on silica gel (5% CH3OH/ CH2Cl2) to give 32 (25.1 g,
ꢀ
The data were analyzed by nonlinear regression using PRISMO
4.0 (GraphPAD Software, San Diego, CA).
Radioligand binding studies for CCR1, CCR2b, CCR4,
CCR5, CXCR1, and CXCR2 were performed at MDS Labora-
tories, Bothell, WA.
1
90%) as a yellow powder. H NMR (CDCl3) δ 1.63-1.74 (m,
1H), 1.98-2.06 (m, 2H), 2.24-2.30 (m, 1H), 2.67-2.91 (m, 2H),
3.69 (s, 2H), 3.75 (s, 2H), 3.91-4.17 (m, 3H), 7.14-7.19 (m, 5H),
7.33 (d, 2H, J = 7.5 Hz), 7.43 (d, 1H, J = 7.5 Hz), 7.54-7.58 (m,
2H), 8.69 (d, 1H, J = 4.2 Hz); 13C NMR (CDCl3) δ 21.66, 23.57,
29.53, 44.85, 49.00, 54.06, 60.93, 115.15, 115.26, 122.09(2),
122.66, 128.46(2), 129.36(2), 135.21, 137.71, 137.78, 138.75,
138.95, 147.12, 155.90, 157.35. ES-MS m/z 398 (M þ H). Anal.
(C25H27N5 0.6H2O 0.4CH2Cl2) C, H, N.
Anti-CXCR4 mAb (Clone 12G5) Binding Assay. To study the
effect of the compounds on anti-CXCR4 mAb (clone 12G5)
binding, SUP-T1 T cells were first preincubated with the com-
pounds (with 1 as a control) for 30 min on ice, washed with PBS
with 2% FCS, and incubated with PE-conjugated anti-CXCR4
mAb (R&D Systems Europe, Oxon, U.K.) for 30 min on ice.
After being washed with PBS, the cell samples were fixed with
1% paraformaldehyde in PBS and analyzed on a FACS Calibur
flow cytometery. The dose-dependent inhibitory effects of the
compounds on mAb binding were determined using the mean
fluorescence intensity values, as described previously.14
SDF-1 Calcium Flux Induced Signaling Assay. For further
characterization of the CXCR4 inhibitors, CEM-CCRF cells
were resuspended (10 ꢀ 106 cells/mL) in serum-reduced media
(RPMI 1640 containing 2% fetal calf serum) and loaded with
the calcium indicator, Fluo-4/AM (1 μM) (Molecular Probes,
Inc., Eugene, OR), for 30 min at 37 °C. The dye-loaded cells were
washed twice with Hanks balanced salt solution in 20 mM
HEPES, 0.2% BSA, 2.5 mM probenecid, pH 7.4. The cells were
resuspended in the same buffer (7 ꢀ 106 cells/mL) followed by a
20 min incubation at room temperature in the dark. The cells
were then incubated in the dark at 37 °C for 15 min with CXCR4
inhibitors. Changes in intracellular calcium concentration upon
addition of SDF-1 (15 nM), the specific ligand for CXCR4, were
monitored using the FLEXstation (Molecular Devices, Sunny-
vale, CA) at 525 nm (excitation λ = 485 nm). The fluorescence
data were analyzed using the program Softmax PRO 4.0 (Mole-
cular Devices) and IC50 values calculated using GraphPad Prism
4.0 software (San Diego, CA).
Caco-2 Assay. Caco-2 monolayers were grown to confluency
on collagen-coated microporous polycarbonate membranes in
12-well Costar Transwell plates. The permeability assay was run
in Hank’s balanced salt solution (HBSS) containing 10 mM
HEPES and 15 mM glucose at a pH of 7.0 ( 0.2. Dosing
solution concentrations were 10-100 μM in assay buffer. Cells
were dosed on the apical side (A-to-B) or basolateral side (B-to-
A) and incubated at 37 °C with 5% CO2 and 90% relative
humidity. After 2 h a 200 μL sample was taken from the receiver
chamber. Each determination was performed in duplicate. All
samples were assayed by LC/MS using electrospray ionization.
The dosing solution was diluted 100-fold and analyzed as an
analytical standard. The assay was performed by Absorption
Systems (Exton, PA).
3
3
N1-(1H-Benzo[d]imidazol-2-ylmethyl)-N1-(5,6,7,8-tetrahydro-
quinolin-8-yl)butane-1,4-diamine Trihydrobromide Dihydrate
(42). By use of general procedure D, reaction of 35 (169 mg,
0.451 mmol) and 4-bromovaleronitrile (0.10 mL, 1.01 mmol)
followed by purification on silica gel (30:1:1 CH2Cl2/CH3OH/
NH4OH) provided the tertiary amine (108 mg, 54%) as a yellow
foam. The amine (108 mg, 0.24 mmol) was dissolved in NH3
saturated MeOH (4 mL), treated with Raney nickel (100 mg),
and placed under 50 psi of H2 on a Parr shaker for 24 h.
Purification by radial chromatography on silica gel (1 mm plate,
20:1:1 CH2Cl2/CH3OH/NH4OH) provided the free base (33 mg,
39%) as a white foam. By use of general procedure C, conversion
of the free base (33 mg, 0.094 mmol) to the HBr salt gave 42
(40 mg, 68%) as a white solid. 1H NMR (D2O) δ 1.52 (br s, 4H),
1.74-1.88 (m, 1H), 1.95-2.08 (m, 1H), 2.15-2.21 (m, 1H),
2.34-2.39 (m, 1H), 2.50-2.61 (m, 1H), 2.79-2.86 (m, 3H),
2.99-3.02 (m, 2H), 4.38 (d, 1H, J = 16.8 Hz), 4.47-4.56 (m,
2H), 7.58-7.63 (m, 2H), 7.76-7.88 (m, 3H), 8.34 (d, 1H, J =
7.8 Hz), 8.62 (d, 1H, J = 5.7 Hz); 13C NMR (D2O) δ 20.42 (2 C),
25.03, 25.42, 27.64, 39.50, 48.20, 51.71, 60.64, 114.26, 125.93,
126.93, 131.05, 139.32, 140.62, 148.09, 150.31, 151.82; ES-MS
m/z 350 (M þ H). Anal. (C21H27N5 2.9HBr 2.2H2O) C, H, N.
3
3
Anti-HIV Activity Assays. The CD4þCXCR4þ lymphocytic
MT-4 cell line was obtained from the American Type Culture
Collection (Rockville, MD). PBMCs from healthy donors were
isolated by density gradient centrifugation and stimulated with
PHA at 1 μg/mL (Sigma Chemical Co., Bornem, Belgium) for 3
days at 37 °C. The activated cells (PHA-stimulated blasts) were
washed three times with PBS, and viral infections were done as
described by Schols et al.15 HIV-infected or mock-infected
PHA-stimulated blasts were cultured in the presence of 25 U/
mL of IL-2 and varying concentrations of compounds. Super-
natant was collected at day 10, and HIV-1 core antigen in the
culture supernatant was analyzed by the p24 viral Ag ELISA kit
(Perkin-Elmer, Boston, MA). Inhibition of HIV-1 replication in
MT-4 cells was performed as previously described.14 Anti-HIV-
1 activity and cytotoxicity measurements were carried out in
parallel. They were based on the viability of MT-4 cells that had
been infected with HIV-1 in the presence of various concentra-
tions of the test compounds. The IC50 was defined as the
concentration required to inhibit 50% of the virus-infected cells
Supporting Information Available: Experimental procedures
and characterization data for compounds 10-14,15a-f,15j,15k,16,
18, 20-23, 26, 31, 33-35, 35a, 35b, 36-41, and43-48. Thismaterial
is available free of charge via the Internet at http://pubs.acs.org.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3387
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