Function-oriented development of CXCR4 antagonists as selective human immunodeficiency virus (HIV)-1 entry inhibitors

Article abstract of DOI:10.1021/jm501772w

Motivated by the pivotal role of CXCR4 as an HIV entry co-receptor, we herein report a de novo hit-to-lead effort on the identification of subnanomolar purine-based CXCR4 antagonists against HIV-1 infection. Compound 24, with an EC₅₀ of 0.5 nM against HIV-1 entry into host cells and an IC₅₀ of 16.4 nM for inhibition of radioligand stromal-derived factor-1α (SDF-1α) binding to CXCR4, was also found to be highly selective against closely related chemokine receptors. We rationalized that compound 24 complementarily interacted with the critical CXCR4 residues that are essential for binding to HIV-1 gp120 V3 loop and subsequent viral entry. Compound 24 showed a 130-fold increase in anti-HIV activity compared to that of the marketed CXCR4 antagonist, AMD3100 (Plerixafor), whereas both compounds exhibited similar potency in mobilization of CXCR4⁺/CD34⁺ stem cells at a high dose. Our study offers insight into the design of anti-HIV therapeutics devoid of major interference with SDF-1α function.

Full text of DOI:10.1021/jm501772w

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Article
Function-oriented development of CXCR4 antagonists as
selective Human Immunodeficiency Virus (HIV)-1 entry inhibitors
Chien-Huang Wu, Chuan- Jen Wang, Chun-Ping Chang, Yung-Chi Cheng, Jen-Shin
Song, Jiing-Jyh Jan, Ming-Chen Chou, Yi-Yu Ke, Jing Ma, Ying-Chieh Wong, Tsung-
Chih Hsieh, Yun-Chen Tien, Elizabeth A. Gullen, Chen-Fu Lo, Chia-Yi Cheng, Yu-Wei Liu,
Amit A. Sadani, Chia-Hua Tsai, Hsing-Pang Hsieh, Lun Kelvin Tsou, and Kak-Shan Shia
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501772w • Publication Date (Web): 13 Jan 2015
Downloaded from http://pubs.acs.org on January 15, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Tsou, Lun; National Health Research Institutes, Institute of Biotechnology
and Pharmaceutical Research
Shia, Kak-Shan; National Health Research Institutes, Institute of
Biotechnology and Pharmaceutical Research
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Function-oriented development of CXCR4 antagonists as selective
Human Immunodeficiency Virus (HIV)-1 entry inhibitors
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†a
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ChienꢀHuang Wu, ChuanꢀJen Wang, ChunꢀPing Chang, YungꢀChi Cheng, JenꢀShin Song, Jiingꢀ
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Jyh Jan, MingꢀChen Chou, YiꢀYu Ke, Jing Ma, YingꢀChieh Wong, TsungꢀChih Hsieh, YunꢀChen
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Tien, Elizabeth A. Gullen, ChenꢀFu Lo, ChiaꢀYi Cheng
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YuꢀWei Liu, Amit A. Sadani, ChiaꢀHua
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Tsai, HsinꢀPang Hsieh, Lun K. Tsou,* and KakꢀShan Shia*
a
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli
County 35053, Taiwan, R.O.C.
b
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
c
Liver Diseases Center, Department of Infectious Diseases, Second Xiangya Hospital, Central South
University, Hunan Province, China.
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ABSTRACT
Motivated by the pivotal role of CXCR4 as an HIV entry coreceptor, we herein report a de
novo hitꢀtoꢀlead effort on the identification of subnanomolar purineꢀbased CXCR4 antagonists against
HIVꢀ1 infection. Compound 24, with an EC50 of 0.5 nM against HIVꢀ1 entry into host cells and an
IC of 16.4 nM for inhibition of radioligand stromalꢀderived factorꢀ1α (SDFꢀ1α) binding to CXCR4,
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was also found to be highly selective against closely related chemokine receptors. We rationalized
that compound 24 complementarily interacted with the critical CXCR4 residues that are essential for
binding to HIVꢀ1 gp120 V3 loop and subsequent viral entry. Compared to the marketed CXCR4
antagonist AMD3100 (Plerixafor), compound 24 showed a 130ꢀfold increase in antiꢀHIV activities,
while both compounds only exhibited similar potency in mobilization of CXCR4+/CD34+ stem cells
at a high dose. Our study offers insights for antiꢀHIV therapeutic design to be devoid of major
interferences with SDFꢀ1α function.
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INTRODUCTION
The battle against HIV/AIDS remains a formidable task and is of paramount importance to
develop efficacious therapies with minimal resistance. With significant progress in elucidating
molecular insights into HIV pathogenesis and druggable targets, the Highly Active Antiretroviral
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Therapy (HAART) arsenal has evolved beyond the traditional drugs that target three main viral
1, 2
enzymes: reverse transcriptase, integrase and protease.
In particular, the HIV entry mechanism
involving multiple conformational changes has provided potential targets and propelled a plethora of
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ꢀ8
therapeutic developments for the disruption of HIV viral attachment, coreceptor binding and fusion.
HIVꢀ1 infection is initiated by the association of viral glycoprotein 120 (gp120) with CD4 cell
receptor, which in turn triggers a conformational change in gp120, allowing exposure of the third
variable loop (V3 loop) of gp120 to bind to the chemokine receptors including T cellꢀtropic CXCR4
or macrophageꢀtropic CCR5. The subsequent conformational change in gp41 leads to a fusion of the
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viral envelope and host cell membrane. Indeed, enfuvirtide, a peptidomimetic of gp41, was approved
10, 11
by FDA in 2003 to block HIVꢀ1 viral fusion.
A decade after uncovering the critical roles of
chemokine receptors CXCR4 and CCR5 in mediating HIV entry, the firstꢀinꢀclass CCR5 chemokine
receptor antagonist maraviroc was approved in 2007 to provide additional antiꢀHIV treatment
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arsenal. The clinical observation of the predominant CXCR4ꢀutilizing strains in HIVꢀ1 infected
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patients after maraviroc administration suggests a mixedꢀtropic viral population and thus a necessity
in the development of CXCR4 antagonists for complete viral suppression.
The induced chemotactic signaling mediated by the chemokine SDFꢀ1α (also known as CꢀXꢀC
motif chemokine 12α, CXCL12α) and its receptor CXCR4 is of significant biological importance
14ꢀ16
involved in tumor metastasis, angiogenesis, tumor progression and survival.
Interestingly, this
pathway is also exploited by pathogens to alter the signaling patterns of the hosts in various disease
17, 18
progressions.
The imperative need for selective CXCR4 antagonists is not limited to therapeutic
applications in blocking HIVꢀ1 entry, but also in modulating SDFꢀ1α:CXCR4 axis’ involvement in
14ꢀ16
tumor metastasis, angiogenesis, tumor progression and survival.
Notably, in about 50% of the late
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stage HIVꢀ1 infected patients, HIVꢀ1 used either CXCR4 alone or in combination with CCR5 to
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facilitate viral entry into host cells and accelerate disease progression. The emergence of CXCR4ꢀ
utilizing strains also coincides with the onset of immune deficiency accompanying by a marked drop
20ꢀ22
of CD4+ Tꢀcell counts, thereby facilitating HIVꢀ1 replication.
It was shown that SDFꢀ1α, at low
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concentration, could effectively disrupt the association of HIVꢀ1 with CXCR4 and therefore reduce
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the infection.
The disruption comes from the steric hindrance caused by the binding of SDFꢀ1α to
CXCR4. Furthermore, SDF-1α-mediated down regulation of cellꢀsurface CXCR4 by inducing its
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endocytosis could lead to inhibition of HIV infection. CXCR4 has been validated as a viable target
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in that the selective CXCR4 antagonists, such as AMD3100 (Plerixafor) , or oralꢀbioavailable
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AMD0070 , could significantly reduce viral load in Tꢀtropic (X4) HIVꢀinfected patients. However,
AMD3100 failed as an HIV drug in phase II clinical trials for lacking oral availability and cardiac
disturbance.
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The recently reported coꢀcrystal structures of CXCR4 with its antagonists , IT1t, a small
molecule, and CVX15, a cyclic peptide, have provided key design features for new compounds. Both
SDFꢀ1α and the V3 loop of HIVꢀ1 gp120 complement a substantial portion of the CXCR4 acidic
extracellular domain, forming multiple saltꢀbridge contacts with predominantly the aspartate and
1
8, 29, 30
glutamate residues.
In turn, a myriad of highly basic CXCR4 antagonists were developed to
31, 32
exploit these chargeꢀcharge interactions as they play pivotal roles in binding to CXCR4 receptor.
To identify efficacious agents against Tꢀtropic (X4) HIVꢀ1 infections, herein, from the discovery of
quinazolineꢀbased polyamine CXCR4 antagonists as HIVꢀ1 entry inhibitors, we will describe the
design, synthesis and structureꢀactivity relationship (SAR) to culminate in a novel series of HIVꢀ1
selective, CXCR4 specific, purineꢀbased antagonists with a broad therapeutic window. Our study
provides tantalizing insights into developing antagonists that selectively target key CXCR4 residues
that govern the HIVꢀ1 entry process.
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Chemistry
Side chains A-H in Figure 1 and test compounds 1ꢀ8 in Table 1 were prepared according to a
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general synthetic route shown in previous literature. Test Compounds 9-11 were prepared according
to a general synthetic method shown in Scheme 1 using compound Ia and its corresponding 2,4ꢀ
diamino quinazoline 9, respectively, as a typical example. The commercially available 2ꢀaminoꢀ5ꢀ
methoxy benzoic acid (Ia) was coupled with urea to provide 6ꢀmethoxyꢀquinazolineꢀ2,4ꢀdiol (IIa) in
85% yield. Treatment of IIa with phosphorus oxychloride in the presence of 2ꢀethylꢀpyridine as a base
gave 2,4ꢀdichloroꢀ6ꢀmethoxyꢀquinazoline (IIIa), which without purification was first coupled with 4ꢀ
aminoꢀ1ꢀBocꢀpiperidine in a chemoselective manner to give intermediate IVa in 59% yield over two
steps, followed by a second coupling with protected side chain D under microwave irradiation to
afford Va in 63% yield. Subsequent acidic deprotection afforded the desired compound 9 in 92%
yield. Compounds 12-14 and 15-17 were synthesized respectively from IVa-IVc by coupling with
side chains E and F, and deprotection with HCl/ether following a similar synthetic procedure for Va
and 9.
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Test compounds 18 and 21-27 were prepared according to a general synthetic method shown in
Scheme 2 using compound 18 as a typical example. The commercially available 2,6ꢀdichloropurine
was protected with 3,4ꢀdihydroꢀ2Hꢀpyran to provide 18-I in quantitative yield, which in turn was
coupled with side chain D in a chemoselective manner to give intermediate 18-II in 62% yield,
o
followed by a second coupling with piperazine under microwave irradiation (15 min, 100 C) or
o
heating at 100 C in 1ꢀpentanol for 15 hours to afford 18-III in 64% yield. After acidic deprotection,
compound 18 was obtained in 94% yield. Compound 22 and 25-27 were synthesized respectively
from 18-I by coupling with different side chains followed by a second coupling with piperazine and
deprotection with HCl/ether in a procedure similar to that for 18. The synthesis of compound 19 was
shown in scheme 3.
RESULTS AND DISCUSSION
Quinazoline-based antagonists.
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Our group has previously reported a series of potent quinazolineꢀbased CXCR4 antagonists on
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their abilities to mobilize stem cells. In the present study, we evaluated the potential of these
quinazolineꢀbased CXCR4 antagonists (Table 1) in the blockade of HIVꢀ1 entry by performing
luciferase activity assays using TZMꢀbl cells (see Materials and Methods). The binding affinities of
compounds 1ꢀ5 toward the CXCR4 receptor increased significantly by 10ꢀ to 20ꢀfold through terminal
ringꢀsize expansion from threeꢀ to sevenꢀmembered ring, which also showed a relatively good
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correlation with their antiꢀHIVꢀ1 activities. Despite the nitrogen atoms at the R position of
compounds 4’s and 6’s terminal sixꢀmembered rings displaying distinct binding modes towards
CXCR4, they both harnessed similar CXCR4 binding affinities and antiꢀHIVꢀ1 activities. Compounds
7
with a terminal piperazine ring and 8 with an exposed tertiary amine all showed dramatic decreases
in CXCR4 binding and antiꢀHIV activities, suggesting a hydrophobic pocket existing in CXCR4 to
facilitate the terminal binding of the antagonists.
We further addressed the influence of the quinazoline core in CXCR4 binding by synthesizing
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a series of compounds, 9-17. We installed electronꢀdonating methoxy group added either at the R or
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R , or both R and R positions, in combination with modifications at the R position by terminal
cyclohexyl, cycloheptyl, or piperidine ring, respectively (Table 1). Compared to compound 4, the
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addition of a methoxy group either at the R or R position led to a 4~5ꢀfold moderate increase in
CXCR4 binding affinity for both compounds 9 and 10 with terminal cyclohexyl ring and compounds
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2 and 13 with cycloheptyl ring at the R position. The terminal ring could situate in a hydrophobic
cleft of CXCR4, and the mounting of a methoxy group to the quinazoline core might induce an
allosteric conformational change of the compound to facilitate its interaction with CXCR4. Moreover,
the increased electron density by the introduced methoxy group might play a role in the enhanced
1
2
binding of the compound to CXCR4. However, double modifications at both the R and R positions
with methoxy groups did not further increase, but only maintained (compound 11), or even decreased
(
compounds 14 and 17) the binding affinity of the compounds to CXCR4, suggesting that
hydrophobic interactions could be involved for the binding. Moreover, the introduction of two
methoxy groups could affect the binding orientations of the compounds by shifting the larger
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cycloheptyl ring (compound 14) out of position or extending the piperidine tertiary amine to an
unfavourable binding mode (compound 17). Interestingly, such effect was tolerated for compound 11
with the cyclohexyl ring, since the CXCR4 binding affinity was maintained.
Despite the strong CXCR4 bindings by compounds 9, 10, 12 and 13 (IC50 ~ 10 nM), only
compound 10 with the terminal cyclohexyl ring showed a significant 8ꢀfold increase in the blockade
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of HIVꢀ1 infection (EC = 8.6 nM). The addition of a methoxy group at the R position presumably
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allowed the cyclohexyl ring to adopt a favourable binding orientation, resulting in better interactions
with key residue(s) on CXCR4 for HIVꢀ1 entry. From the SAR studies, we identified that compound
10, a quinazolineꢀbased CXCR4 antagonist, had a higher CXCR4 binding affinity and potent antiꢀHIV
activity when compared to the documented CXCR4 antagonists AMD3100 and IT1t (Table 1).
However, with the exception of compound 4, the quinazolineꢀbased antagonists had moderate
cytotoxicities to CEM and TZMꢀbl cells, and were deemed an appropriate replacement of the core and
other functional groups. Taken together, we based the next design on an incorporation of a different
core scaffold while maintained the terminal cyclohexyl ring on the projected arm.
Purine-based antagonists
In an effort to increase the therapeutic index of the CXCR4 antagonists and maintain the
extended twoꢀarm projections of the quinazolineꢀbased structures, we employed purine as a new core
and synthesized a series of potent CXCR4 antagonists, 18ꢀ27 (Table 2). Gratifyingly, there was a
significant increase of the therapeutic index as many of the purineꢀbased antagonists exhibited no
toxicities at concentrations up to 50 ꢁM in CEM and TZMꢀbl cells. With the purine scaffold, we first
tested the effects of the side chain modifications. Compound 18A was first synthesized to carry similar
two side chains from compound 4, however, its CXCR4 binding affinity was dramatically decreased
to 230 nM (Figure 2). This is likely due to the loss of hydrophobic interactions of the purine scaffold
to the CXCR4 cleft as compared to the quinazoline core. Surprisingly, the CXCR4 binding affinity of
compound 18 was restored to 18.1 nM by swapping the orientations of the two side chains, and its
potency of antiꢀHIVꢀ1 activity was improved to 2.0 nM (Table 2). The binding orientation of
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compound 18 may form strong interactions with residues in the extracellular domain of CXCR4 that
are important for binding to the HIVꢀ1 gp120 V3 loop.
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The methyl group incorporation either at the R position of the purine imidazole ring in
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compound 19 or at both the R and piperazine ringꢀlinked R positions in compound 20 resulted in a
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~5ꢀfold decrease in CXCR4 binding affinity. The potency of HIVꢀ1 inhibition was generally
maintained in the low nanomolar range with a 2ꢀfold decrease in antiꢀHIV activity as compared to that
of compound 18. However, these methyl group modifications also led to unfavourable cytotoxicities.
The maintaining of the antiꢀHIV activities for compounds 18-20 implied the importance of the
orientation and binding modes of the two side chains. The elongation of the lower side chain by 4ꢀ
methylpiperidine in compound 21 led to a 2~3ꢀfold decrease in CXCR4 binding and yet a 40ꢀfold
decrease in antiꢀHIV activity (Table 2), suggesting a small binding pocket in the lower side chain that
allows for limited substitutions.
We next turned our attention to the appropriate placement for the peripheral functionalities in
the upper side chain of the purineꢀbased antagonists. Compound 22 with a pyridine substitute of the
phenyl ring moiety showed a 2ꢀfold decrease in both CXCR4 binding and antiꢀHIV activities as
compared to compound 18, suggesting that the N atom in the pyridine ring might not significantly
change the binding role of the original phenyl ring moiety (Table 2). To further access the possible
binding motifs and to increase the antiꢀHIV activity, we investigated the effects of varying chain
length by installing extra methylene groups at positions n and m of the upper functional chain (Table
2
). With an introduced methylene group at position n, compound 23 showed an around 2ꢀfold decrease
in both CXCR4 binding and antiꢀHIV activities, suggesting that the orientation of the πꢀπ stacking
interactions arising from neighbouring aromatic rings might be important in CXCR4 binding.
However, with the introduced methylene group at position m, compound 24 showed a 4ꢀfold
enhancement of antiꢀHIV activity (EC50 = 0.5 nM), while its CXCR4 binding affinity was not changed
as compared to that of compound 18 (Table 2). The results suggested that the degree of rotation and
flexibility of terminal chain from the m position allow particular interactions with residues on the
CXCR4 receptor that can closely control HIVꢀ1 entry. Encouraged by the above findings, we
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envisioned that compound 25 with metaꢀsubstitution would harness similar projection of the
peripheral upper side chain to that of compound 24. Indeed, compound 25 exhibited a comparable
antiꢀHIV activity to that of compound 24, while there was a 4ꢀfold increase in CXCR4 binding
affinity. The less correlation between the CXCR4 binding affinity and antiꢀHIV activity for
compounds 18, 24 and 25, as an example, suggested that additional mechanisms are involved in
determining the antiꢀHIV activity. As compared to compound 25, compounds 26 and 27, with the
same projection of the peripheral upper side chain, exhibited a 4~10ꢀfold decrease in both CXCR4
binding and antiꢀHIV activities, suggesting that the presence of a tertiary amine in the pyridine moiety
would either hinder the πꢀπ stacking interactions between neighbouring aromatic rings of the
compound or interfere with the hydrophobic cleft of CXCR4. Interestingly, compounds 26 bearing a
pyridine substitution and a metaꢀsubstitution of the peripheral upper side chain, showed similar
CXCR4 binding and antiꢀHIV activities as compound 22. The above findings implied that the
projection of the metaꢀsubstituted upper side chain from a phenyl ring in compound 25 produced a
favourable binding orientation with CXCR4.
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In comparison to the initially quinazolineꢀbased compound 4, our hitꢀtoꢀlead generation effort
resulted in the identification of compound 24 with improved CXCR4 binding affinity and HIVꢀ1 entry
inhibition by 2.2ꢀfold and 134ꢀfold (Table 1 vs Table 2), respectively. Furthermore, we greatly
reduced the cytotoxicities of the quinazolineꢀbased CXCR4 antagonists by the substitution with a
purine core. Intriguingly, compound 18A with a purine core swapping of the quinazoline core in
compound 4 resulted in a significant loss of CXCR4 binding. However, compound 18 with a
switching of the two peripheral appendages in compound 18A readily restored the CXCR4 binding
activity. Moreover, such employment increased the antiꢀHIVꢀ1 entry activity by 34.2ꢀfold (Figure 2 vs
Table 2). Taken together, from the SAR studies of the CXCR4 antagonists, we have shown: 1) the
importance of a terminal cyclohexyl ring group; 2) the cytotoxic effects of the quinazoline core
resolved by a purine core replacement; 3) the orientation of the side chains with projected peripheral
functionalities involved in CXCR4 binding and antiꢀHIV activities and 4) the effects of hydrophobic
interactions between the antagonists and CXCR4. Furthermore, the SAR studies could lead to the
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exploration of other potential binding sites on the CXCR4 receptor for HIVꢀ1 entry by introducing
flexibility and optimization of the upper side chain of the purineꢀbased antagonists.
Time of addition assays
To validate that these CXCR4 antagonists are HIVꢀ1 entry inhibitors through interactions with
CXCR4, we performed time of addition assays over one HIV replication cycle by monitoring the
effects of compound 24 in TZMꢀbl cells. Compound 24, at various concentrations ranging from 0 to
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20 nM, was added three hours before, simultaneously with, and three hours after HIVꢀ1 infection, and
showed a doseꢀdependent inhibition of viral replication (Figure 3). When added three hours before or
simultaneously with HIVꢀ1 infection, compound 24 maintained its potent antiꢀHIV activities at EC50
concentrations consistent with the observation in Table 2. In contrast, the antiꢀHIV activity by
compound 24 was significantly reduced, when added three hours after HIVꢀ1 infection. The preꢀ and
coꢀtreatments with compound 24 in HIVꢀ1 infection provided more protection than the postꢀtreatment,
confirming that the CXCR4 antagonist indeed protected against viral entry in the early phase of HIVꢀ1
infection.
Molecular dynamics simulation study
We then carried out molecular dynamics (MD) simulations to investigate the interacting
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9
CXCR4 (PDB code 3OE0) residues to gain insights into the possible binding mode of compound 24
(Figure 4 and Figure S1). Intriguingly, compound 24 docked towards the Nꢀterminal domain of
CXCR4 and formed hydrogen bonding with the CXCR4 residues Asp193, His281, and Glu288. As
shown in Figure 4A, from purine core to lower piperazine ring of compound 24, of which two
hydrogen bonds formed with CXCR4: the N3 of the purine core interacting with the side chain of
His281 and the terminal piperazine nitrogen with the side chain of Glu288. In particular, Glu288 of
CXCR4 has been shown to be one of the most important residues for HIVꢀ1 coreceptor activity. A
significant loss (>50%) of this activity was observed by the substitution of Glu288 with either alanine
34, 35
or even aspartic acid that preserves the physicochemical properties.
Moreover, the nitrogens from
the projected upper side chain of compound 24 formed two hydrogen bonds with Asp193 side chain.
From a simulation study, Asp193 is found to form a highly interacting salt bridge with V3 loop residue
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Lys10. Moreover, alanine substitution of Asp193 resulted in a dramatic reduction of the HIVꢀ1
36
coreceptor activity of CXCR4. While alanine substitutions at Asp187 and Phe189 of the CXCR4
residues also experimentally impaired >60% of the HIVꢀ1 coreceptor activity, site directed
mutagenesis studies demonstrated the crucial roles of Arg188, Tyr190, and Pro191 in HIVꢀ1
3
5, 37ꢀ40
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binding.
Interestingly, the terminal cyclohexyl ring and the upper side chain of compound 24
picked up several significant hydrophobic interactions with the CXCR4 residues Phe189, Tyr190 and
Pro191. The phenyl ring moiety linked to the purine core in compound 24 was involved in
hydrophobic interactions with Tyr190, Gln200 and Leu266. The alanine substitution of Gln200 was
39
found to significantly affect HIVꢀ1 coreceptor binding. Moreover, Gln200 is calculated to form a
30
strong nonpolar interaction with the aromatic ring of the V3 loop residue Trp20. Taken together, the
MD results provided great insight into the molecular interactions between compound 24 and CXCR4
receptor. We reasoned that the potent antiꢀHIV activity of compound 24 could come from its
interactions with the critical CXCR4 residues in the Nꢀterminal domain that are exploited by HIVꢀ1
V3 loop for viral entry. To provide further insight into the potent HIV entry inhibition by these
purineꢀbased antagonists, compound 25 was subjected to MD study (Figure S2). Gratifyingly,
compound 25, similar to that of compound 24, docked to the Nꢀterminal extracellular loop II domain
and interacted with majority of the abovementioned CXCR4 residues. MD simulations showed that
compound 25 hydrogen bonded to several CXCR4 residues that were reported to be critical for HIV
entry. By modifying the orientation of the upper side chain in compound 24 from para to meta in
compound 25, compound 25’s upper side chain gained additional strong hydrogen bond interactions
35, 37, 39
with Asp187, Glu277 and hydrophobic interaction with Arg188.
The terminal piperazine
nitrogens of both compounds 24 and 25 were anchored towards the interior of CXCR4 by interacting
with the side chain of Glu288, an important residue for HIV coreceptor activity. In all, by targeting a
distinct domain of CXCR4 that could be pivotal for HIVꢀ1 entry, we greatly improved the antiꢀHIV
activities of compounds 24 and 25 to the subnanomolar range, a 130ꢀfold enhancement in potency
relative to that of AMD3100. Furthermore, we envisioned that a lower dose of compound 24 could be
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employed for treatment of chronic HIVꢀ1 infection to minimize adverse effects of normal
physiological functions of SDFꢀ1α:CXCR4 axis.
Binding specificity and functional activity tests
Since compounds 24 and 25 were identified with strong binding affinities for CXCR4 and
potent antiꢀHIV entry activities in subnanomolar range, further studies were conducted to address their
binding specificities and functional activities. As shown in Table 3, compounds 24 and 25 exhibited
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>609ꢀfold and >2,380ꢀfold selectivities for CXCR4 binding, respectively, versus a panel of closely
related chemokine receptors including CXCR2, CCR2, CCR4 and CCR5 whose binding affinities
were >10,000 nM (as IC50). Furthermore, compounds 24 and 25 showed specific inhibitions for
CXCR4. Recent reports had suggested that CXCR7 is the most closelyꢀrelated chemokine receptor to
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CXCR4. Using the established SDFꢀ1αꢀdependent βꢀarrestin assay of CXCR7, compound 24 did
not antagonize SDFꢀ1α binding to CXCR7 and the triggering of βꢀarrestin recruitment even at fortyꢀ
fold higher SDFꢀ1α concentration, providing a >1,960ꢀfold functional selectivity over CXCR4ꢀ
dependent HIV entry inhibition by compound 24 (Figure S3). These results, together with several
31
reported CXCR4 antagonists, strongly suggested that the introduction of nitrogenꢀcontaining
appendages with a purine core or other skeletons might be pivotal in designing potent and specific
CXCR4 antagonists. The nitrogenꢀcontaining fragments could mimic the Lys/Argꢀrich nature of the
4
3
SDFꢀ1α, the natural ligand involved in the binding and activation of CXCR4. Moreover, since TZMꢀ
bl cells express both CXCR4 and CCR5 coreceptors, the lack of binding of compounds 24 and 25 to
CCR5 from the abovementioned results provided strong support that the observed potent antiꢀHIV
entry effects (EC ~0.5 nM) were correlated with their binding to CXCR4 expressed on the TZMꢀbl
50
cell surface.
Despite the significant therapeutic potential of SDFꢀ1α in blocking CXCR4 in the HIV field,
growing evidences have accumulated that SDFꢀ1α:CXCR4 axis is involved in tumor progression,
1
4, 15
angiogenesis and metastasis.
are migrated to metastasis target tissues that release SDFꢀ1α.
newly developed CXCR4 antagonists overlapped with the SDFꢀ1α binding sites on the CXCR4
Recent findings have validated that cancer cells expressing CXCR4
14, 15
A question concerning whether our
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receptor was raised. We addressed this issue by investigating the inhibitory activity of compound 24
on SDFꢀ1αꢀinduced cell migration. The chemotaxis inhibition assay was performed using CCRFꢀCEM
cells that express endogenous human CXCR4. As shown in Figure 5A, while there was no apparent
toxicities to CEM cells (CC50 > 50ꢁM), compound 24 exerted an IC50 of 3.1 nM to block SDFꢀ1αꢀ
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induced CEM migration. This was fairly comparable to the IC50 (16.4 nM) for the inhibition of
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radioligand
IꢀSDFꢀ1α binding by compound 24 to hCXCR4ꢀtransfected membrane (Table 2).
AMD3100 was measured at 24.6 nM in this assay system. Thus, compound 24 was 8ꢀfold more potent
in CEM chemotaxis inhibition, and 13ꢀfold more potent in CXCR4 binding than AMD3100 (Table 1
1
25
vs Table 2). Interestingly, the IC50 (213.1 nM) for 50% inhibition of
IꢀSDFꢀ1α binding by
AMD3100 to hCXCR4 (Table 1) was not comparable to its IC value for chemotaxis inhibition. This
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0
observation suggested that compound 24 might partially overlap with the SDFꢀ1α binding sites on the
CXCR4 receptor and that AMD3100 might access different binding sites.
Several observations on AMD3100’s disruption of the homing of stem and progenitor cells had
led to its serendipitous development as a mobilization agent of stem cells (CD34+). We were
interested to find out if the significant increase of CXCR4 binding affinity by compound 24 would
greatly enhance its mobilization abilities of stem cells. Using C57BL/6 mice for stem cell
3
3
mobilization assay as previously described, to our surprise, compound 24 was only found to
mobilize CXCR4+/CD34+, the stem cells of interest, as efficiently as AMD3100 in the linear range
(Figure 5B). CXCR4+/CD34+ cells were isolated in almost equal number from collected peripheral
blood 2h after the indicated dosing (0.1 milligram per kilogram (mpk), 1 mpk and 5 mpk). Although
compound 24 showed a 13ꢀfold and 130ꢀfold increase in CXCR4 binding affinity and antiꢀHIVꢀ1
activity, respectively, it did not translate into an increased mobilization of stem cells. An explanation
for this could be that compound 24 targeted residues of the distinct CXCR4 domains crucial for viral
entry mediated by HIVꢀ1 V3 loop since studies have shown that binding and signalling domains in
3
0, 35, 37
CXCR4 are possibly distinct and separate.
This observation strongly suggested the decoupling
of two CXCR4ꢀmediated biological processes and the functional studies of compound 24
demonstrated the possibility of designing potent and selective CXCR4 antagonists.
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CONCLUSION
In summary, we successfully designed and synthesized of a novel series of selective CXCR4
antagonists with a purine core scaffold that potently inhibited HIVꢀ1 infection (18-20 and 22-27).
Compound 24, the most active inhibitor, displayed a strong binding affinity for CXCR4 (IC50 = 16.4 ±
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3
.5 nM) and a potent antiꢀHIVꢀ1 activity as viral entry blocker (EC50 = 0.51 ± 0.02 nM), with no
cytotoxicities up to 50 ꢁM. Although more detailed structural studies are required to dissect the
interactions between compound 24 and CXCR4, our MD simulations showed a high degree of
molecular complementarity of compound 24 with CXCR4 residues essential for HIVꢀ1 entry mediated
by HIVꢀ1 V3 loop. Compound 24, when added prior to or simultaneously as in a time course
experiment, showed potent inhibitions, confirming it as an HIVꢀ1 entry inhibitor. Compared to
AMD3100, the subnanomolar and functionꢀoriented increase in HIV entry inhibition (>130ꢀfold) of
compound 24 should afford a significant dosage decrease in antiꢀHIV treatment. Since a much higher
dose (5mpk) of compound 24 is needed to mobilize stem cells, the lower dosage of compound 24 may
improve the therapeutic window of inhibiting HIV entry while reducing the disruption to stem cells
homing in the bone marrow. Furthermore, these results strongly suggests that structural insights into
the conformational states of CXCR4 towards compound 24 could aid future therapeutic design to
minimize interference of normal physiological functions of SDFꢀ1α:CXCR4 axis.
EXPERIMENTAL SECTION
General. Unless otherwise stated, all materials used were commercially obtained and used as
supplied. Reactions requiring anhydrous conditions were performed in flameꢀdried glassware and
cooled under an argon or nitrogen atmosphere. Unless otherwise stated, reactions were carried out
under argon or nitrogen and monitored by analytical thin layer chromatography performed on glassꢀ
backed plates (5 × 10 cm) precoated with silica gel 60 F254 as supplied by Merck (Merck & Co., Inc.,
Whitehouse Station in Readington Township, NJ). Visualization of the resulting chromatograms was
performed by looking under an ultraviolet lamp (λ = 254 nm) followed by dipping in an ethanol
solution of vanillin (5% w/v) containing sulfuric acid (3% v/v) or phosphomolybdic acid (2.5% w/v)
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and charring with a heat gun. Solvents for reactions were dried and distilled under an argon or
nitrogen atmosphere prior to use as follows: THF, diethyl ether (ether), and DMF from a dark blue
solution of sodium benzophenone ketyl; toluene, dichromethane, and pyridine from calcium hydride.
Flash chromatography was used routinely for purification and separation of product mixtures using
silica gel 60 of 230−400 mesh size as supplied by Merck. Eluent systems are given in volume/volume
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concentrations. Melting points were determined using a KRUSS KIP1N melting point meter. H and
1
3
C NMR spectra were recorded on a Varian Mercuryꢀ300 (300 MHz) and a Varian Mercuryꢀ400 (400
MHz). Chloroformꢀd or dimethyl sulfoxideꢀd6 was used as the solvent and TMS (δ 0.00 ppm) as an
internal standard. Chemical shift values are reported in ppm relative to the TMS in delta (δ) units.
Multiplicities are recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd
(doublet of doublets), dt (doublet of triplets), and m (multiplet). Coupling constants (J) are expressed
in hertz. Electrospray mass spectra (ESMS) were recorded as m/z values using an Agilent 1100 MSD
mass spectrometer. All test compounds displayed more than 95% purity as determined by Agilent
1100 series HPLC system using a C18 column (Thermo Golden, 4.6 mm × 250 mm). The gradient
system for HPLC separation was composed of MeOH (mobile phase A) and H O solution containing
2
0.1% trifluoroꢀacetic acid (mobile phase B). The starting flow rate was 0.5 mL/min and the injection
volume was 10 ꢁL. During first 2 min the percentage of phase A was 10%. At 6 min, the percentage of
phase A was increased to 50%. At 16 min, the percentage of phase A was increased to 90% over 9 min.
o
The system was operated at 25 C. Peaks were detected at 254 nm. IUPAC nomenclature of
compounds was determined with ACD/Name Pro software.
2
4
N -{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-6-methoxy-N -piperidin-4-yl-
quinazoline-2,4-diamine hydrochloride salt (9). To a magnetically stirred solution of Va (0.53 g,
0
.64 mmole) in CH
2
Cl
2
(10 mL) was added 1N HCl/diethylether (20 mL, 20 mmole) dropwise at 25
o
o
C under an atmosphere of argon. The resulting mixture was stirred at 25 C for 15 h and concentrated
1
by removing the solvent to afford of compound 9 (0.38 g, 92%); H NMR (300 MHz, D O) δ
2
7.56−7.50 (m, 4H), 7.15−6.93 (m, 3H), 4.71 (s, 2H), 4.26 (m, 2H), 4.24 (m, 1H), 3.73 (s, 3H), 3.51 (m,
13
2H), 3.20−3.05 (m, 6H), 2.13−1.70 (m, 10H), 1.56 (m, 1H), 1.33−1.11 (m, 6H); C NMR (75 MHz,
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9
D O) δ 158.69, 155.42, 151.48, 140.27, 132.41, 130.21, 129.52, 127.50, 124.55, 117.57, 108.96,
2
103.77, 57.33, 56.12, 50.61, 46.92, 44.02, 43.79, 43.18, 41.13, 28.72, 27.19, 24.32, 23.77, 22.75;
ESMS m/z: 532.3 (M+1); HPLC purity = 95.35 %, tR = 14.35 min.
2
4
N -{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-7-methoxy-N -piperidin-4-yl-
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quinazoline-2,4-diamine hydrochloride salt (10). Compound 10 was synthesized from Vb (0.56 g,
0.67 mmol) following a similar synthetic procedure for 9 and obtained as a white solid (0.36 g, 83%);
1
H NMR (300 MHz, D
O) δ 7.65 (d, J = 9.0 Hz, 1H), 7.55−7.48 (m, 4H), 6.50 (dd, J = 9.0, 2.1 Hz,
2
1H), 6.38 (d, J = 2.1 Hz, 1H), 4.67 (s, 2H), 4.26 (s, 2H), 4.21 (m, 1H), 3.74 (s, 3H), 3.53 (m, 2H),
1
3
3.17−3.01 (m, 6H), 2.13−1.71 (m, 10H), 1.56 (m, 1H), 1.33−1.13 (m, 6H); C NMR (75 MHz, D
2
O)
δ 164.03, 158.85, 152.18, 140.29, 140.11, 130.20, 129.53, 127.53, 125.11, 113.62, 102.28, 97.72,
57.34, 55.96, 50.60, 46.78, 44.05, 43.75, 43.15, 41.15, 28.62, 27.33, 24.34, 23.77, 22.74; ESMS m/z:
532.3 (M+1); HPLC purity = 96.25 %, tR = 14.26 min.
2
4
N -{4-[(3-Cyclohexylamino-propylamino)-methyl]-benzyl}-6,7-dimethoxy-N -piperidin-4-yl-
quinazoline-2,4-diamine hydrochloride salt (11). Compound 11 was synthesized from Vc (0.53 g,
0.61 mmol) following a similar synthetic procedure for 9 and obtained as a white solid (0.37 g, 90%);
1
H NMR (300 MHz, D O) δ 7.57−7.48 (m, 4H), 7.26 (s, 1H), 6.65 (s, 1H), 4.75 (s, 2H), 4.26 (s, 2H),
2
4
.23 (m, 1H), 3.83 (s, 6H), 3.48 (m, 2H), 3.19−3.00 (m, 6H), 2.13−1.96 (m, 6H), 1.93−1.71 (m, 4H),
1
3
1.65 (m, 1H), 1.33−1.13 (m, 6H); C NMR (75 MHz, D
O) δ 158.55, 154.62, 151.75, 146.11, 140.12,
34.66, 130.15, 129.39, 127.30, 103.61, 101.54, 97.33, 57.38, 56.40, 56.22, 50.67, 46.89, 44.03, 43.92,
3.19, 41.18, 28.69, 27.34, 24.33, 23.78, 22.78; ESMS m/z: 562.3 (M+1); HPLC purity = 95.27 %, tR
2
1
4
=
14.23 min.
2
4
N -{4-[(3-Cycloheptylamino-propylamino)-methyl]-benzyl}-6-methoxy-N -piperidin-4-yl-
quinazoline-2,4-diamine hydrochloride salt (12). Compound 12 was synthesized from IVa by
coupling with side chain E and deprotection with HCl/diethylether following a similar synthetic
1
procedure for 9 and obtained as a white solid (0.37 g, 56% over 2 steps); H NMR (300 MHz, D O) δ
2
7.56−7.50 (m, 4H), 7.14 (d, J = 2.1 Hz, 1H), 7.06 (dd, J = 9.3, 2.1 Hz, 1H), 6.94 (d, J = 9.3 Hz, 1H),,
4.71 (s, 2H), 4.26 (m, 2H), 4.24 (m, 1H), 3.72 (s, 3H), 3.53 (m, 2H), 3.20−2.96 (m, 6H), 2.13−1.80 (m,
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H), 1.68−1.31 (m, 10H); C NMR(75 MHz, D O) δ 158.75, 155.47, 151.61, 140.30, 132.50, 130.23,
2
29.35, 127.58, 124.61, 117.65, 109.08, 103.81, 59.59, 56.15, 50.59, 46.93, 44.02, 43.70, 43.19, 41.58,
0.20, 27.22, 27.12, 23.12, 22.77; ESMS m/z: 546.4 (M+1); HPLC purity = 95.36 %, tR = 15.00 min.
2
4
N -{4-[(3-Cycloheptylamino-propylamino)-methyl]-benzyl}-7-methoxy-N -piperidin-4-yl-
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quinazoline-2,4-diamine hydrochloride salt (13). Compound 13 was synthesized from IVb by
coupling with side chain E and deprotection with HCl/diethylether following a similar synthetic
1
procedure for 9 and obtained as a white solid (0.36 g, 57% over 2 steps); H NMR (300 MHz, D
2
O) δ
7
.89 (d, J = 9.0 Hz, 1H), 7.55−7.49 (m, 4H), 6.94 (dd, J = 9.0, 2.1 Hz, 1H), 6.82 (d, J = 2.1 Hz, 1H),
4.78 (s, 2H), 4.35 (m, 1H), 4.26 (s, 2H), 3.91 (s, 3H), 3.47 (m, 2H), 3.17−2.99 (m, 6H), 2.13−1.77 (m,
1
3
7
1
3
H), 1.90−1.39 (m, 12H); C NMR (75 MHz, D O) δ 164.04, 158.87, 152.19, 140.34, 140.12, 130.23,
2
29.36, 127.63, 125.16, 113.63, 102.32, 97.73, 59.59, 55.97, 50.67, 46.78, 44.05, 43.65, 43.16, 41.58,
0.20, 27.36, 27.11, 23.13, 22.77; ESMS m/z: 546.4 (M+1); HPLC purity = 96.98 %, tR = 15.07 min.
2
4
N -{4-[(3-Cycloheptylamino-propylamino)-methyl]-benzyl}-6,7-methoxy-N -piperidin-4-yl-
quinazoline-2,4-diamine hydrochloride salt (14). Compound 14 was synthesized from IVc by
coupling with side chain E and deprotection with HCl/diethylether following a similar synthetic
1
procedure for 9 and obtained as a white solid (0.36 g, 52% over 2 steps); H NMR (400 MHz, D O) δ
2
7
.54−7.49 (m, 4H), 7.22 (s, 1H), 6.58 (s, 1H), 4.73 (s, 2H), 4.25 (s, 2H), 4.24 (m, 1H), 3.80 (s, 6H),
3.48 (m, 2H), 3.23−3.00 (m, 6H), 2.14−1.96 (m, 7H), 1.93−1.77 (m, 2H), 1.71−1.59 (m, 2H),
13
1
1
4
2
.55−1.37 (m, 8H); C NMR (75 MHz, D O) δ 160.99, 157.14, 154.21, 148.56, 142.75, 137.13,
32.75, 131.95, 129.92, 105.68, 103.95, 99.80, 62.20, 59.02, 58.82, 53.20, 49.47, 46.61, 46.46, 45.79,
4.22, 32.81, 29.94, 29.70, 25.70, 25.39; ESMS m/z: 576.4 (M+1); HPLC purity = 95.59 %, tR =
14.80 min.
-Methoxy-N -piperidin-4-yl-N -{4-[(3-piperidin-1-yl-propylamino)-methyl]-benzyl}-
4
2
6
quinazoline-2,4-diamine hydrochloride salt (15). Compound 15 was synthesized from IVa by
coupling with side chain F and deprotection with HCl/diethylether following a similar synthetic
1
procedure for 9 and obtained as a white solid (0.33 g, 51% over 2 steps); H NMR (300 MHz, D O) δ
2
7.57−7.52 (m, 4H), 7.31 (s, 1H), 7.23−7.21 (m, 2H), 4.75 (s, 2H), 4.29 (m, 1H), 4.27 (s, 2H), 3.82 (s,
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H), 3.56−3.47 (m, 4H), 3.20−2.92 (m, 8H), 2.20−2.10 (m, 2H), 2.05−1.65 (m, 9H), 1.47 (m, 1H); C
NMR (75 MHz, D O) δ 158.70, 155.45, 151.50, 140.24, 132.40, 130.20, 129.48, 127.40, 124.56,
17.54, 108.99, 103.80, 61.61, 56.18, 53.29, 50.74, 46.96, 44.05, 44.00, 43.22, 27.21, 22.71, 20.97,
20.69; ESMS m/z: 518.3 (M+1); HPLC purity = 96.36 %, tR = 13.88 min.
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-Methoxy-N -piperidin-4-yl-N -{4-[(3-piperidin-1-yl-propylamino)-methyl]-benzyl}-
quinazoline-2,4-diamine hydrochloride salt (16). Compound 16 was synthesized from IVb by
coupling with side chain F and deprotection with HCl/diethylether following a similar synthetic
1
procedure for 9 and obtained as a white solid (0.31 g, 49% over 2 steps); H NMR (300 MHz, D
2
O) δ
7.66 (d, J = 9.0 Hz, 1H), 7.57−7.51 (m, 4H), 6.17 (dd, J = 9.0, 2.1 Hz, 1H), 6.37 (d, J = 2.1 Hz, 1H),
4
2
1
.66 (s, 2H), 4.27 (s, 2H), 4.21 (m, 1H), 3.70 (s, 3H), 3.56−3.51 (m, 4H), 3.23−2.91 (m, 8H),
13
.27−2.20 (m, 2H), 2.05−1.65 (m, 9H), 1.49 (m, 1H); C NMR (75 MHz, D O) δ 164.06, 158.81,
2
52.19, 140.24, 140.06, 130.18, 129.46, 127.82, 125.14, 113.66, 102.25, 97.73, 61.59, 55.97, 53.33,
53.29, 50.72, 44.06, 44.00, 43.18, 27.33, 22.72, 20.97, 20.69; ESMS m/z: 518.3 (M+1); HPLC purity
=
99.05 %, tR = 12.73 min.
4
2
6
,7-Dimethoxy-N -piperidin-4-yl-N -{4-[(3-piperidin-1-yl-propylamino)-methyl]-benzyl}-
quinazoline-2,4-diamine hydrochloride salt (17). Compound 17 was synthesized from IVc by
coupling with side chain F and deprotection with HCl/diethylether following a similar synthetic
1
procedure for 9 and obtained as a white solid (0.32 g, 45% over 2 steps); H NMR (400 MHz, D
2
O) δ
7
3
1
.54−7.44 (m, 4H), 7.29 (s, 1H), 6.68 (s, 1H), 4.76 (s, 2H), 4.35 (m, 1H), 4.27 (s, 2H), 3.86 (s, 6H),
.57−3.49 (m, 4H), 3.25−3.16 (m, 4H), 3.10−2.91 (m, 4H), 2.27−2.20 (m, 2H), 2.05−1.65 (m, 9H),
13
.51 (m, 1H); C NMR (75 MHz, D O) δ 158.49, 154.61, 151.71, 146.01, 140.06, 134.60, 130.15,
2
129.43, 127.25, 103.13, 101.45, 97.27, 57.35, 56.43, 56.23, 53.30, 50.72, 46.92, 44.08, 44.02, 43.22,
27.35, 22.72, 20.95, 20.68; ESMS m/z: 548.3 (M+1); HPLC purity = 95.99 %, tR = 13.84 min.
N-Cyclohexyl-N'-{4-[(2-piperazin-1-yl-9H-purin-6-ylamino)-methyl]-benzyl}-propane-1,3-
diamine hydrochloride salt (18). A solution of 1N HCl/diethyl ether (4.8 mL) was added to the
solution of 18-III (240 mg, 0.31 mmole) in CH Cl (9.6 mL). The reaction mixture was stirred for 15
2
2
1
hours and concentrated by removing the solvent to afford of compound 18 (174 mg, y: 94% ); H
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NMR (400 MHz, D O) δ 8.40 (s, 1H), 7.52−7.44 (m, 4H), 4.82 (s, 2H), 4.26 (s, 2H), 4.01 (m, 4H),
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.29 (m, 4H), 3.21−3.08 (m, 4H), 2.18−2.02 (m, 4H), 1.82 (m, 2H), 1.63 (m, 1H), 1.40−1.12 (m, 6H);
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C NMR (75 MHz, D O) δ 154.39, 151.63, 147.61, 139.56, 139.26, 130.13, 129.51, 127.95, 105.36,
57.39, 50.78, 44.06, 43.99, 42.56, 41.73, 41.20, 28.73, 24.37, 23.79, 22.77; ESMS m/z: 478.3 (M+1);
HPLC purity = 96.8 %, tR = 13.07 min.
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N-Cyclohexyl-N'-{4-[(9-methyl-2-piperazin-1-yl-9H-purin-6-ylamino)-methyl]-benzyl}-
propane-1,3-diamine hydrochloride salt (19). A solution of 1N HCl/diethyl ether (3 mL) was added
to the solution of 19-III (156 mg, 0.23 mmole) in CH
2
Cl
2
(6 mL). The reaction mixture was stirred at
o
1
25 C for 15h and concentrated by removing the solvent to afford 19 (123 mg, y: 91%), H NMR (400
MHz, D O) δ 8.75 (s, 1H), 7.55−7.45 (m, 4H), 4.82 (s, 2H), 4.27 (s, 2H), 4.04 (m, 4H), 3.85 (s, 3H),
2
13
3
.26−3.11 (m, 8H), 2.18−2.04 (m, 4H), 1.84 (m, 2H), 1.66 (m, 1H), 1.40−1.12 (m, 6H); C NMR (75
2
MHz, D O) δ 159.02, 151.60, 149.92, 140.24, 137.51, 130.12, 129.40, 127.95, 104.67, 57.42, 50.83,
44.00, 43.91, 42.83, 41.38, 41.24, 30.78, 28.78, 24.40, 23.84, 22.80; ESMS m/z: 492.3 (M+1); HPLC
purity = 97.1 %, tR = 13.34 min.
N-Cyclohexyl-N'-(4-{[9-methyl-2-(4-methyl-piperazin-1-yl)-9H-purin-6-ylamino]-methyl}-
benzyl)-propane-1,3-diamine hydrochloride salt (20). Compound 20 was synthesized from 19-II
following a similar synthetic procedure for 19 with two steps and obtained as a white solid (143 mg,
1
65% over 2 steps); H NMR (400 MHz, D
2
O) 8.75 (s, 1H), 7.53 (d, J = 7.2 Hz, 2H), 7.48 (d, J = 7.2
Hz, 2H), 4.82 (s, 2H), 4.27 (s, 2H), 3.85 (s, 3H), 3.54 (m, 2H), 3.33 (m, 2H), 3.22−3.08 (m, 6H), 3.01
13
(
m, 2H), 2.92 (s, 3H), 2.18−2.04 (m, 4H), 1.85 (m, 2H), 1.66 (m, 1H), 1.40−1.12 (m, 6H); C NMR
(
75 MHz, D O) δ 159.49, 152.14, 150.30, 140.74, 137.95, 130.58, 129.85, 128.38, 105.21, 57.88,
2
53.25, 51.27, 44.45, 44.32, 43.38, 42.17, 41.67, 31.22, 29.22, 24.86, 24.28, 23.24; ESMS m/z: 506.3
(M+1); HPLC purity = 97.0 %, tR = 13.39 min.
N-{4-[(2-[4,4']Bipiperidinyl-1-yl-9H-purin-6-ylamino)-methyl]-benzyl}-N'-cyclohexyl-
propane-1,3-diamine hydrochloride salt (21). Compound 21 was synthesized from 18-I following a
similar synthetic procedure for 18 with three steps and obtained as a white solid (186 mg, 36% over 3
1
steps); H NMR (400 MHz, D
2
O) δ 8.06 (s, 1H), 7.52−7.44 (m, 4H), 4.82 (s, 2H), 4.34 (m, 2H), 4.25
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(
s, 2H), 3.46 (m, 2H), 3.22−2.90 (m, 8H), 2.18−1.76 (m, 10H), 1.65 (m, 1H), 1.61−1.10 (m, 12H); C
NMR (75 MHz, D O) δ 152.01, 151.05, 146.37, 140.88, 139.51, 130.17, 129.54, 128.18, 105.98,
7.39, 50.77, 46.66, 45.70, 44.17, 43.96, 41.19, 39.19, 37.59, 28.75, 27.99, 25.58, 24.39, 23.81, 22.77;
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ESMS m/z: 560.4 (M+1); HPLC purity = 99.3 %, tR = 13.69 min.
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N-Cyclohexyl-N'-{6-[(2-piperazin-1-yl-9H-purin-6-ylamino)-methyl]-pyridin-3-ylmethyl}-
propane-1,3-diamine hydrochloride salt (22). Compound 22 was synthesized from 18-I following a
similar synthetic procedure for 18 with three steps and obtained as a white solid (150 mg, 29% over 3
1
steps); H NMR (400 MHz, D
2
O) δ 8.91 (s, 1H), 8.72−7.64 (m, 2H), 8.17 (d, J = 8.4 Hz, 1H), 5.27 (s,
2H), 4.57 (s, 2H), 3.93 (m, 4H), 3.39−3.12 (m, 8H), 2.24−2.06 (m, 4H), 1.86 (m, 2H), 1.68 (m, 1H),
1
3
1
1
2
.42−1.18 (m, 6H); C NMR (75 MHz, D O) δ 156.44, 155.40, 151.71, 149.16, 147.43, 143.02,
2
38.67, 129.21, 125.82, 105.08, 57.50, 47.19, 44.95, 42.64, 42.48, 41.38, 41.19, 28.80, 24.42, 23.84,
2.86; ESMS m/z: 479.3 (M+1); HPLC purity =98.6 %, tR = 13.69 min.
N-Cyclohexyl-N'-{4-[2-(2-piperazin-1-yl-9H-purin-6-ylamino)-ethyl]-benzyl}-propane-1,3-
diamine hydrochloride salt (23). Compound 23 was synthesized from 18-I following a similar
synthetic procedure for 18 with three steps and obtained as a white solid (197 mg, 38% over 3 steps);
1
H NMR (400 MHz, D O) δ 8.28 (s, 1H), 7.41−7.38 (m, 4H), 4.23 (s, 2H), 4.06 (m, 4H), 3.93 (s, 2H),
2
3
.40 (m, 4H), 3.21−3.03 (m, 6H), 2.16−2.04 (m, 4H), 1.83 (m, 2H), 1.64 (m, 1H), 1.40−1.12 (m, 6H);
1
3
C NMR (75 MHz, D
2
O) δ 154.29, 152.14, 147.64, 140.88, 139.31, 129.86, 129.80, 128.44, 105.93,
57.38, 50.79, 43.83, 43.55, 41.67, 41.43, 41.16, 34.32, 28.73, 24.37, 23.79, 22.75; ESMS m/z: 492.3
(M+1); HPLC purity = 95.4 %, tR = 13.31 min.
N-Cyclohexyl-N'-(2-{4-[(2-piperazin-1-yl-9H-purin-6-ylamino)-methyl]-phenyl}-ethyl)-
propane-1,3-diamine hydrochloride salt (24). Compound 24 was synthesized from 18-I following a
similar synthetic procedure for 18 with three steps and obtained as a white solid (195 mg, 38% over 3
1
steps); H NMR (400 MHz, D
2
O) δ 8.39 (s, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H),
4.72 (s, 2H), 4.03 (m, 4H), 3.39−3.24 (m, 6H), 3.23−3.08 (m, 4H), 3.02 (m, 2H), 2.18−2.06 (m, 4H),
13
1
.83 (m, 2H), 1.64 (m, 1H), 1.41−1.12 (m, 6H); C NMR (75 MHz, D O) δ 154.21, 151.36, 147.53,
2
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39.24, 136.81, 135.45, 129.05, 127.91, 105.19, 57.43, 48.57, 44.49, 44.13, 42.58, 41.75, 41.23, 31.25,
8.76, 24.40, 23.82, 22.77; ESMS m/z: 492.3 (M+1); HPLC purity = 99.6 %, tR = 13.34 min.
N-Cyclohexyl-N'-{3-[(2-piperazin-1-yl-9H-purin-6-ylamino)-methyl]-benzyl}-propane-1,3-
2
diamine hydrochloride salt (25). Compound 25 was synthesized from 18-I following a similar
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synthetic procedure for 18 with three steps and obtained as a white solid (178 mg, 34% over 3 steps);
1
H NMR (400 MHz, D O) δ 8.34 (s, 1H), 7.52−7.36 (m, 4H), 4.82 (s, 2H), 4.21 (s, 2H), 3.99 (m, 4H),
2
3
.27 (m, 4H), 3.21−3.09 (m, 4H), 2.18−2.02 (m, 4H), 1.81 (m, 2H), 1.63 (m, 1H), 1.40−1.12 (m, 6H);
1
3
2
C NMR (75 MHz, D O) δ 154.40, 151.56, 147.74, 139.30, 139.01, 130.92, 129.54, 128.81, 128.55,
128.40, 105.37, 57.44, 51.04, 44.20, 44.11, 42.60, 41.77, 41.25, 28.78, 24.42, 23.84, 22.81; ESMS
m/z: 478.3 (M+1); HPLC purity = 95.3 %, tR = 13.30 min.
N-Cyclohexyl-N'-{6-[(2-piperazin-1-yl-9H-purin-6-ylamino)-methyl]-pyridin-2-ylmethyl}-
propane-1,3-diamine hydrochloride salt (26). Compound 26 was synthesized from 18-I following a
similar synthetic procedure for 18 with three steps and obtained as a white solid (152 mg, 30% over 3
1
steps); H NMR (400 MHz, D
2
O) δ 8.49 (s, 1H), 8.04 (t, J = 7.6 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H),
7
2
1
.58 (d, J = 7.6 Hz, 1H), 4.98 (s, 2H), 4.47 (s, 2H), 3.92 (m, 4H), 3.24−3.10 (m, 6H), 3.08 (m, 2H),
13
.18−2.02 (m, 4H), 1.77 (m, 2H), 1.60 (m, 1H), 1.36−1.08 (m, 6H); C NMR (75 MHz, D O) δ
2
56.81, 155.07, 151.68, 148.50, 148.15, 141.16, 139.04, 123.50, 123.16, 105.30, 57.44, 49.90, 44.89,
44.63, 42.57, 41.61, 41.19, 28.75, 24.37, 23.81, 22.77; ESMS m/z: 479.3 (M+1); HPLC purity = 98.5
, tR = 13.11 min.
%
N-Cyclohexyl-N'-{5-[(2-piperazin-1-yl-9H-purin-6-ylamino)-methyl]-pyridin-3-ylmethyl}-
propane-1,3-diamine hydrochloride salt (27). Compound 27 was synthesized from 18-I following a
similar synthetic procedure for 18 with three steps and obtained as a white solid (154 mg, 30% over 3
1
steps); H NMR (400 MHz, D
2
O) δ 8.96−8.93 (m, 2H), 8.79 (s, 1H), 8.56 (s, 1H), 5.14 (s, 2H), 4.57
(s, 2H), 4.02 (m, 4H), 3.38−3.26 (m, 6H), 3.10 (m, 2H), 2.23−2.06 (m, 4H), 1.84 (m, 2H), 1.67 (m,
1
3
1
1
H), 1.40−1.15 (m, 6H); C NMR (75 MHz, D O) δ 156.26, 152.33, 149.09, 148.44, 142.15, 141.80,
2
40.20, 139.39, 131.28, 105.92, 57.93, 47.77, 45.42, 43.18, 41.99, 41.62, 41.51, 29.22, 24.86, 24.28,
23.29; ESMS m/z: 479.3 (M+1); HPLC purity = 99.0 %, tR = 12.98 min.
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Establishment of Human CXCR4 Stable Cell Line and Membrane Purification. The
hCXCR4 cDNA was subcloned into pIRES2ꢀEGFP vector (Clontech Laboratories, Inc., Mountain
View, CA). Transfected HEKꢀ293 cells stably expressed hCXCR4 (HEKꢀ293 CXCR4) were selected
by EGFP and 1 mg/ml G418 sulfate. The selected clone was maintained in DMEM supplemented with
o
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0% fetal bovine serum and 0.5 mg/ml G418 sulfate with 5% CO at 37 C in the humidified
2
incubator. For membrane purification, cells were homogenized in iceꢀcold buffer A (50 mM TrisꢀHCl,
pH 7.4, 5 mM MgCl
2
, 2.5 mM EDTA, 10% sucrose) with freshly prepared 1 mM PMSF. The
o
homogenate was centrifuged at 3500 x g for 15 min at 4 C. The pellet was removed and the
o
supernatant then was centrifuged at 43000 x g for additional 30 min at 4 C. The final pellet was
o
resuspended in buffer A and stored at ꢀ80 C.
Radioligand Binding Assay. An amount of 2ꢀ4 ꢀg of purified membrane with CXCR4 was
125
incubated with 0.16 nM IꢀSDFꢀ1α and compounds of interest in the incubation buffer (50 mM
HEPESꢀNaOH, pH 7.4, 100 mM NaCl, 5 mM MgCl , 1 mM CaCl , 0.5% BSA) The nonspecific
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binding was defined in the presence of 50 ꢁM AMD3100 (plerixafor). The reaction mixtures were
o
incubated for 1.5 h at 30 C and then transferred to a 96ꢀwell GF/B filter plate (Millipore Corp.,
Billerica, MA). The reaction mixtures were terminated by manifold filtration and washed with iceꢀ
cold wash buffer (50 mM HEPESꢀNaOH, pH 7.4, 100 mM NaCl) for four times. The radioactivity
bound to the filter was measured by Topcount (PerkinElmer Inc., Waltham, MA). IC50 values were
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determined by the concentrations of compounds required to inhibit 50% of the specific binding of Iꢀ
SDFꢀ1α and calculated by nonlinear regression (GraphPad software, San Diego, CA).
Luciferase Activity Assay. Using TZMꢀbl cells, the luciferase activity assays were performed
for studying the efficacy of the CXCR4 antagonists on HIVꢀ1 infectivity. TZMꢀbl is a HeLaꢀderived
cell line expressing high levels of the introduced CD4, CCR5 and CXCR4 receptors, and contains
HIVꢀ1 long terminal repeatꢀdriven β−galactosidase and luciferase reporter cassettes that are activated
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by HIVꢀ1 Tat expression. For the luciferase reporter experiments, 5×10 cells/well were plated in 96ꢀ
well plates and cultured at 37°C with 5% CO in the humidified incubator. After one or two overnight
incubation and usually 30 min prior to HIV infection, TZMꢀbl cells were treated with CXCR4
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antagonists in serial dilution and then infected by HIVꢀ1IIIB at a multiplicity of infection (M.O.I.) of
1. The HIVꢀ1 IIIB strain was used for infection. FARCyte machine (Amersham Pharmacia) was used
for data reading and at least three independent experiments were carried out to get the standard
deviation.
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Cytotoxicity Assay. CEM, a human Tꢀlymphoblast leukemia, and TZMꢀbl cells were used for
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cell cytotoxicity assays. 1×10 cells/well were plated in 24ꢀwell plates. After overnight incubation,
cells were treated with CXCR4 antagonists for 72 hours. The cells were then fixed and stained with
0.5% methylene blue in 50% ethanol for 2 hours at room temperature, followed by washing with tap
water to remove excess color. Plates were dried, then resuspended in 1% sarkosyl and incubated for 3
hrs at room temperature. Methylene blue was oxidized by living cells to a colorless product, while
dead cells remained blue colored. Cell growth was quantitated based on the amount of methylene blue
adsorbed into cells measured by a spectrophotometer (Molecular Devices) at 595 nm. All experiments
were performed in triplicate wells and repeated at least three times to get the standard deviation.
Docking analysis of compounds with CXCR4. The protein structures of CXCR4 (Protein Data
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Bank identifier [PDB ID] no. 3OE0) was applied for this study. All the calculations were performed
using Discovery Studio 2.1 (DS 2.1) (Accelrys, Inc., San Diego, CA). The docking analysis was
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conducted using the DS/LigandFit program with the CHARMm force field. The number of docking
poses was set as 100 with default parameters. The decision of the best pose was according the binding
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information from the Wu et al.
Molecular dynamics simulations. The molecular dynamics (MD) simulations were carried out
46, 47
using GROMACS v4.5.4 to refine the docked structures.
The topology of docked ligand was
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generated by PRODRG serve. The force field for the whole system was GROMOS 43a1. The
proteinꢀligand complex was restrained in a box of cubic shape whose edges were placed at 1 nm from
the complex and SPC/E water model was performed. The system was electrically neutralized by
ꢀ
adding 11 Cl ion. A twoꢀstep energy minimization was performed using steepest descent and
−1
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conjugate gradient algorithms to converge the system upto 10 kJ mol nm . After a short energy
minimization step, the system was subjected to NVT (300K) and NPT (1 bar) equilibration with 100
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ps running, and LINCS algorithm was used to constrain the hydrogen bond lengths. The time step
was kept at 2 fs for the simulation. The cutꢀoff distance of 10 Å was used for all short range nonꢀ
bonded interactions and 12 Å Fourier grid spacing in PME method for long range electrostatics.
Finally the restraints of complex structure were removed and performed to a 20 ns MD calculation
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(
Supporting Information 1).
Chemotaxis Assay. CCRFꢀCEM (Tꢀcell acute lymphoblastic leukemia) cells were suspended in
RPMI 1640 containing 10% FBS and then preincubated with indicated concentrations of compounds
o
for 10 min at 37 C. The assay was performed in Millicell Hanging Cell Culture Inserts (pore size 5
ꢁm; 24ꢀwell plate; Millipore, Bedford, MA, USA). Compounds containing 10 nM SDFꢀ1 were plated
in the lower chambers of inserts, and cells with compounds were plated in the upper chamber of
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inserts at a density of 2.5 × 10 cells/well. After 2.5 h incubation at 37 C, cells in both chambers of
inserts were measured by flow cytometer (Guava Technologies, Hayward, CA, USA).
Flow Cytometry Analysis for Stem/progenitor cell counting. C57BL/6 male mice were treated
with potential CXCR4 antagonist individually by subcutaneous injection, and then blood samples
containing mobilized stem/progenitor cells were collected 2 hours later. After labeled with specific
antibodies, including APCꢀconjugated antiꢀCXCR4 (clone 2B11; eBioscience), FITCꢀconjugated antiꢀ
CD34 (clone RAM34; eBioscience), PEꢀconjugated antiꢀCD133 (clone 13A4; eBioscience) and antiꢀ
KDR (clone Avsa12a1; eBioscience), antiꢀcꢀKit (clone 2B8; eBioscience), antiꢀScaꢀ1 (clone D7;
eBioscience), antiꢀlinage (Mouse Hematopoietic Lineage Biotin Panel, eBioscience) and Streptavidin
PEꢀCy7 (eBioscience), cells were washed, characterized and quantified by flow cytometer (Guava
Technologies, Hayward, CA, USA). Each data point included at least 60,000 events for analysis of
mobilized cells.
Use of Animal Subjects. All experimental protocols were approved and performed in
accordance with the guidelines defined by the Institutional Animal Care and Use Committee (IACUC)
of National Health Research Institutes (NHRI), Taiwan, R.O.C.
ASSOCIATED CONTENT
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Supporting Information
MD of compound 25:CXCR4 (PDB code 3OE0) complex and the RMSD of the protein backbone of
αC atom of amino acids during the MD simulations; the NMR spectra of the key compounds, 18, 24,
and 25 and the synthesis of compounds IIa-IIc, IVa-IVc, Va-Vc, 18-I, 18-II, 18-III, 19-I, 19-II and
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19-III; procedures of CXCR2, CCR2, CCR4, CCR5 radioligand binding assays and CXCR7 betaꢀ
arrestin assay are described in supporting information. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*
Telephone: +886ꢀ37ꢀ246ꢀ166 ext. 35709. Fax: + 886ꢀ37ꢀ586ꢀ456. Eꢀmail: ksshia@nhri.org.tw
* Telephone: +886ꢀ37ꢀ246ꢀ166 ext. 35769. Fax: + 886ꢀ37ꢀ586ꢀ456. Eꢀmail: kelvintsou@nhri.org.tw
Author Contributions
†
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to the National Health Research Institutes and Ministry of Science and Technology of
the Republic of China (MOST 101ꢀ2325ꢀBꢀ400ꢀ016) and Public Health Service grant AIꢀ38204 from
the National Institutes of Health for financial support. YungꢀChi Cheng is a fellow of National
Foundation for Cancer Research (USA) and thanks NIH (5R01A1038204ꢀ20) for financial support.
ABBREVIATIONS USED
HAART, Highly Active Antiretroviral Therapy; gp120, glycoprotein 120; SDFꢀ1α, stromalꢀderived
factorꢀ1α; SAR, structure−activity relationships; MD, molecular dynamics; mpk, milligram per
kilogram; V3 loop, third variable loop.
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