Discovery of tetrahydroisoquinoline-based CXCR4 antagonists

Article abstract of DOI:10.1021/ml400183q

A de novo hit-to-lead effort involving the redesign of benzimidazole- containing antagonists of the CXCR4 receptor resulted in the discovery of a novel series of 1,2,3,4-tetrahydroisoquinoline (TIQ) analogues. In general, this series of compounds show good potencies (3-650 nM) in assays involving CXCR4 function, including both inhibition of attachment of X4 HIV-1_IIIB virus in MAGI-CCR5/CXCR4 cells and inhibition of calcium release in Chem-1 cells. Series profiling permitted the identification of TIQ-(R)-stereoisomer 15 as a potent and selective CXCR4 antagonist lead candidate with a promising in vitro profile. The drug-like properties of 15 were determined in ADME in vitro studies, revealing low metabolic liability potential. Further in vivo evaluations included pharmacokinetic experiments in rats and mice, where 15 was shown to have oral bioavailability (F = 63%) and resulted in the mobilization of white blood cells (WBCs) in a dose-dependent manner.

Full text of DOI:10.1021/ml400183q

Letter
pubs.acs.org/acsmedchemlett
Discovery of Tetrahydroisoquinoline-Based CXCR4 Antagonists
†
‡
†
†
†
Valarie M. Truax, Huanyu Zhao, Brooke M. Katzman, Anthony R. Prosser, Ana A. Alcaraz,
‡
‡
‡
‡
Manohar T. Saindane, Randy B. Howard, Deborah Culver, Richard F. Arrendale,
Prahbakar R. Gruddanti, Taylor J. Evers, Michael G. Natchus, James P. Snyder, Dennis C. Liotta,
and Lawrence J. Wilson*
‡
‡
‡
†
†,‡
,
†
†
Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
Emory Institute for Drug Development, 954 Gatewood Road NE, Atlanta, Georgia 30329, United States
‡
*
S Supporting Information
ABSTRACT: A de novo hit-to-lead effort involving the redesign of
benzimidazole-containing antagonists of the CXCR4 receptor resulted
in the discovery of a novel series of 1,2,3,4-tetrahydroisoquinoline
(
TIQ) analogues. In general, this series of compounds show good
potencies (3−650 nM) in assays involving CXCR4 function, including
both inhibition of attachment of X4 HIV-1 virus in MAGI-CCR5/
IIIB
CXCR4 cells and inhibition of calcium release in Chem-1 cells. Series
profiling permitted the identification of TIQ-(R)-stereoisomer 15 as a
potent and selective CXCR4 antagonist lead candidate with a
promising in vitro profile. The drug-like properties of 15 were
determined in ADME in vitro studies, revealing low metabolic liability
potential. Further in vivo evaluations included pharmacokinetic
experiments in rats and mice, where 15 was shown to have oral
bioavailability (F = 63%) and resulted in the mobilization of white blood cells (WBCs) in a dose-dependent manner.
KEYWORDS: Tetrahydroisoquinolines, CXCR4 receptor, CXCR4 antagonists, HIV, WBC mobilization
he chemokine receptor CXCR4 belongs to class A of G-
protein coupled receptors (GPCRs) and is broadly
Chart 1. Selected Small-Molecule CXCR4 Antagonists
T
expressed on most hematopoietic cell types including
neutrophils, monocytes and macrophages, immature and
mature T and B-lymphocytes, hematopoietic and endothelial
progenitor stem cells, vascular endothelial cells, neurons,
1
neuronal stems cells, microglia, and astrocytes. The natural
ligand for CXCR4 is the chemokine CXCL12 (SDF-1, stromal
cell-derived factor-1), a key regulator of hematopoietic stem cell
2
,3
(
HSC) homing and retention in the bone marrow.
Importantly, the CXCR4/CXC12 axis synchronizes many
essential physiological roles, such as homeostatic regulation of
leukocyte trafficking, hematopoiesis, and embryonic develop-
9
multiple myeloma. This unexpected finding resulted in
unprecedented therapeutic use and prompted a paradigm
shift in CXCR4 research, significantly expanding the breadth
and scope from both biological and medicinal chemistry
standpoints. More recent CXCR4 discoveries include the
involvement of the CXCR4/CXCL12 axis in the progression of
4
−6
ment.
However, from a historical perspective, it was the
discovery elucidating CXCR4 as a coreceptor used along with
CD4 by T cell-tropic (X4) HIV-1 strains for cellular entry that
7
,8
catalyzed the observed cornucopia of research. This seminal
discovery was concurrent with elucidation of the mechanism of
the anti-HIV-1 bicyclam AMD3100, which a priori became the
first small molecule CXCR4 antagonist. Although AMD3100
2
3 different types of cancer, where the interaction between
CXCR4 and CXCL12 promotes metastasis, angiogenesis, and
tumor growth via direct and indirect mechanisms.
The unique biological properties of the CXCR4/CXCL12
axis and further research with AMD3100 (1) prompted a surge
in activity around small-molecule CXCR4 antagonists with
10−14
(1, Chart 1, Plerixafor) entered the clinic as an antiviral
treatment of X4 HIV-1 strains, during clinical trials it was
shown to mobilize various hematopoietic cells, including
CD34+ cells. Through further clinical investigations the
compound was subsequently approved by the FDA for use in
mobilizing hematopoietic stem cells (HSCs) for autologous
transplants in patients with non-Hodgkin’s lymphoma and
Received: May 14, 2013
Accepted: September 5, 2013
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ml400183q | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
1
,14,15
several entering clinical trials (Chart 1).
Systematic efforts
Table 1. MAGI Assay Results for Compounds 13−18
to replace the cyclam rings of AMD3100 with moieties that
retain basic character, while also reducing the molecular weight
and overall charge, resulted in the disclosure of a series of
compounds exemplified by AMD11070 (2). Noteworthy are
three structural modifications shown to be effective as cyclam
pharmacophore replacements: (i) a chiral tetrahydroquinoline
1
6
(
THQ); (ii) a basic heterocycle; and (iii) a butyl amine chain.
As AMD11070 (2) was the first oral CXCR4 antagonist to
enter the clinic and advance to phase II clinical trials, it paved
the way for other efforts, which produced preclinical leads, such
1
4,17−19
as the hybrid-piperazine (3) and isothiourea series (4).
Herein, we describe our efforts leading to discovery of a series
of novel and potent CXCR4 antagonists that contain a 1,2,3,4-
tetrahydroisoquinoline (TIQ) ring.
In light of the development of AMD11070 as an oral HIV
treatment, our initial focus was on replacement of the
benzimidazole, as we hypothesized this subunit to be the likely
source of CYP450 isozyme (3A4, 2D6) inhibition activities
2
0,21
a
observed with this compound.
We settled on using
All assays were performed in duplicate.
isomeric fused phenyl piperidines because they are sub-
structures found in many drug-like compounds and GPCR
ligands, as well as synthetically accessible starting from amino
acids or suitable derivatives via the Pictet−Spengler reac-
with Boc protected amino butyraldehydes 10 or 11 furnished
advanced intermediates 12a−f. This was followed by Boc
deprotection to furnish the final compounds 13−18. The
stereochemistry of these compounds was determined and
confirmed by a combination of proof by synthesis, assignment
of spectroscopic and chromatographic properties and an X-ray
crystal structure of Boc derivative 29 (Scheme 2, Supporting
Information).
2
2,23
tion.
The (S)-tetrahydroquinoline headpiece previously
disclosed for 2 was retained due to observed benefits for
16
CXCR4 potency. Our first goal was to synthesize a group of
compounds in which the isomeric heterocycle and additional
stereogenic center are variable (13−18, Scheme 1 and Table 1).
a
a
Scheme 1
Scheme 2
a
a
Reagents: (i) BH −Me S, THF (2.0 M); (ii) Dess Martin
3
2
Reagents: (i) 19, NaBH(OAc) , 1,2-DCE; (ii) TFA, DCM; (iii)
3
periodinane, DCM; (iii) (COCI) , DMSO, Et N, CH CI ; (iv) 8,
NaBH(OAc) , 1,2-DCE; (v) 10 or 11, NaBH(OAc) , 1,2-DCE; (vi)
DCM, TFA.
1
2
3
2
2
H NNH , MeOH; (iv) R CHO, NaBH(OAc) , 1,2-DCE; (v) N-
2
2
3
3
3
(CICH CO)−morpholine, K CO , DMF; (vi) (CH O) , NaBH-
2
2
3
2
n
(OAc) , DCM; (vii) acetone, NaBH(OAc) , DCM; (viii) AcCI,
3
3
Et N, DCM; (ix) KNCO, 1 N HCI; (x) Me NCOCI, NEt , DCM.
3
2
3
The group of compounds 13−18 can be synthesized according
to the sequence shown in Scheme 1 from readily available Boc-
protected amino acid based tetrahydroquinoline (THQ, 5a)
and tetrahydroisoquinoline (TIQ, 5b−d) building blocks.
Borane dimethyl sulfide reduction of chiral or racemic Boc
protected amino carboxylic acids 5a−d provided the corre-
sponding alcohols (6a−d). Oxidation to the aldehydes (7a−d),
followed by reductive amination with 8 afforded the racemic or
chiral enriched secondary amines 9a−d. Reductive amination
Initial evaluation of these compounds against CXCR4
mediated effects was performed in the viral attachment assay
with HIV-1IIIB (Table1) in CCR5/CXCR4-expressing HeLa-
CD4-LTR-β-gal (MAGI) cells measuring each compound’s
ability to block potential viral entry as well as cellular
toxicological properties. The stereochemistry, inclusion and
location of fusion of the aromatic ring all affected potency.
Unlike previous quinoline and isoquinoline heterocyclic
24
B
dx.doi.org/10.1021/ml400183q | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
replacements, the action of the tetrahydroquinoline and
tetrahydroisoquinoline moieties indicated that both the place-
ment of the phenyl ring and the need for the nitrogen atom
Scheme 2 from previously generated intermediate 9b.
Reductive amination with 19 provided the orthogonally
protected advanced intermediate 20. To access compounds
25
1
basicity are important. Surprisingly, in the case of the
tetrahydroquinolines 13 and 14, the compounds showed no
effect against viral entry in the MAGI assay up to 30 μM
indicating the importance of the basicity of the nitrogen
heterocycle. In comparison, tetrahydroisoquinolines 15−18
showed a range of antiviral potencies with therapeutic
selectivity and two important features to note. First, the site
of the phenyl ring fusion to the piperidine ring had dramatic
effects on potency, with the distal phenyl ring (R)-isomer 15
being 50−100-fold more potent than the isomeric compounds
with modification at the TIQ nitrogen (22−28, R ), Boc
removal gave advanced intermediate 21 followed by either a
reductive amination or N-alkylation. Hydrazine removal of the
phthalamide protecting group provided the final compounds.
Alternatively, to access compounds with modification at the
2
3
butyl amine nitrogen (30−35, R and R ), phthalamide removal
on 20 to give 29, followed by reductive amination or N-
acylation and subsequent Boc removal provided the final
compounds.
The data used to elucidate the structure−activity relation-
ships (SAR) of these series was generated by a combination of
two assays, namely, blockade of HIV-1IIIB attachment via the
CXCR4 receptor in MAGI cells, as well as the inhibition of
17 and 18 with the phenyl adjacent to the extending carbon.
The second important activity relationship was evident in the
dramatic effect on potency that the stereochemistry on the
tetrahydroisoquinoline demonstrated. The respective isomers
with the (R) configuration at the TIQ ring stereocenter (15 and
2+
CXCL12 induced calcium (Ca ) flux/release in Chem-1 cells
(Table 2). The compounds revealed a range of potencies and
divergent SAR. All compounds were put in both agonist and
antagonist modes in the calcium flux assay, and none showed
any agonist activity, while showing complete blockade of SDF-1
induced calcium flux at various potencies. Substitution at the
tetrahydroisoquinoline nitrogen with H-bond acceptors or
aliphatic moieties all resulted in a slight loss of potency (22-28,
2−10-fold) in the MAGI attachment assay when compared to
15 (Table 2). As a trend, bulkier heterocycles (compounds 22−
26) decreased potency more than the smaller groups (27−28).
17) are the most potent analogues, and in the case of 15, 100-
fold or more potent than the compounds bearing the
alternative stereochemistry (16 and 18). The stereochemical
preference in ligand design also led to the development of the
16
(S)-tetrahydroquinoline (8) ring in CXCR4 antagonists.
Given these results, compound 15 was chosen for further
medicinal chemistry and biological studies.
We first focused on making conservative independent
modifications to (R)-TIQ compound 15 (Scheme 2). Our
efforts involved modifications on the TIQ ring and butyl amine
side chain nitrogen atoms. Generally, these compounds (22−
2
+
A similar trend was exhibited for CXCL12 induced Ca flux;
however, the effect was much more dramatic. While there was
generally a 2−10-fold decrease in anti-HIV potency compared
to 15, there was a 10−100-fold decrease in the compounds’
abilities to block CXCL12 induced Ca2+ flux. For example,
some agents with bulky heterocycles (23−26) were ∼100-fold
less active than 15 in the calcium flux assay versus ∼10-fold less
active in the MAGI, while with the 2-pyridyl group (22) and
the methyl and isopropyl alkyl groups (27, 28), less of a
difference was observed. With regard to CXCR4 antagonist
activity, this trend is supportive of the importance of an H bond
donor at the TIQ nitrogen position and the need to retain
basicity.
28 and 30−35, Table 2) can be synthesized as shown in
Table 2. Assay Results for Compounds 15 and 22−35
Regarding the SAR of the carbamide and N-alkyl butyl amine
moieties (30−35), the simple N-alkylated compounds 30 and
3
1 retained both potent HIV activity and CXCL12 induced
2+
Ca flux, although slightly less potent in the MAGI assay (2−
5
-fold). To our surprise, the 2-pyridyl and carbamide based
derivatives (32−34) were 40−80-fold less potent than 15 in the
MAGI assay but remained equipotent in blocking CXCL12
induced Ca2+ flux. These results potentially signify that basicity
and steric environment on the nitrogen of the butyl amine side
chain may be important for anti-HIV activity but not for normal
CXCL12 mediated receptor signaling since nearly all
2+
substitutions (30−34) resulted in potent Ca flux inhibition,
with the only exception being the dimethyl urea (35). Although
the complete nature of this difference is not well understood,
taken together with the TIQ nitrogen substitution effects, it is
both significant and novel from the perspective of antagonist
design.
We next undertook a computational docking study in order
to better understand this preliminary SAR and develop a
working hypothesis on how our compounds interact with the
CXCR4 receptor. Using the recently reported CXCR4 receptor
crystal structure, we developed a computational model of 15
a
b
All compunds had TC50 values >10 μM. All assays were run in
duplicte.
26
bound to CXCR4. Because of the importance of the
C
dx.doi.org/10.1021/ml400183q | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
interaction of IT1t with Asp97 and Glu288 in the crystal
structure, we hypothesized that 15 would interact with the
same residues. Thus, we flexibly docked compound 15 into the
IT1t subsite of CXCR4 (PDB ID 3ODU). In our best pose,
the positively charged primary butyl amine of 15 interacts with
the Glu288 carboxylate via a salt bridge and forms a hydrogen
bond with the Tyr116 side chain phenol oxygen at the bottom
of the pocket, while the protonated TIQ ring nitrogen forms a
salt bridge with the Asp97 carboxylate (Figure 1). The THQ
consistent with maintaining the TIQ nitrogen-Asp97 inter-
action and altering the butyl amine-Glu288 interaction.
As lead compound 15 was identified as the most potent in
both assays, we decided to examine other in vitro CXCR4
based properties of the compound (Table 3). As a confirmation
of antiviral properties, screening in peripheral blood mono-
nuclear cells (PBMCs), resulted in IC50 and IC90 values of 35
27
and 240 nM, respectively, for blockage of infection of HIV1IIIB
.
Similar to the TC result observed in the MAGI assay, at these
50
concentrations cytotoxicity was not observed in PBMCs,
resulting in a therapeutic index of >1000 (TC50 = 47 μM).
Concerning anti-HIV activity, the potencies of 15, AMD3100
(
1), and 11070 (2) are comparable and equal to both the
literature and in-house assays (30−40 nM for all three
compounds). Further validation of CXCR4 antagonist behavior
was provided by a competitive binding assay and other
functional assays. Competitive binding studies with radiolabeled
125
I-SDF-1 showed displacement of the chemokine with an IC50
of 112 nM for 15. In the forskolin/CXCL12 stimulated cAMP
production assay, a measure of Gα signaling, 15 inhibited
i
cAMP production, with an IC value of 19 nM. Compound 15
50
also inhibited β-arrestin recruitment with an IC50 value of 15
nM. Finally, to measure chemokine family and type selectivity,
compound 15 was screened in a panel of chemokine receptor
assays in both CXCR and CCR types with no observed cross-
activity up to 10 μM indicating high selectivity toward CXCR4.
In an effort to assess the drug potential of compound 15,
several ADME based tests were performed (Table 3). In terms
of physical parameters, the aqueous solubility of the free base
was investigated by nephelometry and found to be >100 μg/
mL at physiological pH (7.4). The metabolic stability of
compound 15 was assessed in mouse, rat, and human liver
microsomes, where it exhibited species-dependent stability
behavior. In both rodents, lower half-lives (20−30 min) and
higher clearances were observed. In human liver microsomes,
the result was a longer half-life (t1/2 > 60 min) and lower
clearance rate. Also, the human CYP450 inhibition for isozymes
2C19, 2D6, and 3A4 by compound 15 was found to be minimal
(<10%, Table 3). These results are significant, as AMD11070
(2) has been shown to inhibit both 3A4 and 2D6 isozymes at
levels with low therapeutic difference and to alter drug levels in
humans with the known CYP inhibitors midazolam (CYP3A4)
Figure 1. Docking pose of compound 15 in the IT1t/3ODU crystal
structure showing hydrogen bonding and key amino acids.
moiety is situated in a hydrophobic pi-stacking pocket
comprising mainly the indole of Trp94 and the imidazole of
His113. The key role of the TIQ nitrogen in salt bridge
formation is consistent with the SAR of the compounds in
Table 1, where activity drops off significantly for the nonbasic
tetrahydroquinolines 13 and 14. Furthermore, the SAR from
the alteration of substitution on both nitrogen atoms of 15
(
Table 2) provides some support for this model. Since Glu288
is a key residue for HIV interaction while Asp97 is more
important for SDF-1 function, the modifications of the TIQ
nitrogen (22−28), which reduce calcium flux potency more
than HIV activity, are consistent with altering the Asp97-TIQ
interaction while maintaining the integrity of the Glu288-butyl
2
6,28
amine interaction.
The modifications of the butyl amine
side chain that alter the basicity (32−34), which reduce HIV
blocking effects while maintaining calcium flux potency, are
21
and dextromethorphan (CYP2D6). On the basis of the
Table 3. Data Summary of CXCR4 Biological and ADME Assays on Compound 15
CXCR4 biology or ADME assay
result(s)
inhibition of infection of HIV-1IIIB virus in PBMCs
cytotoxicity in PBMCs/therapeutic Index
IC50 = 0.035 μM/IC90 = 0.24 μM
TC50 = 47 μM/TI = 1334
IC50 = 0.12 μM
[125
I]-SDF-1α displacement Chem-1 cells
effect on SDF-1 blockade of cAMP
EC50 = 0.019 μM
IC50 = 0.015 μM
blockade of SDF-1 induced β-arrestin-2
chemokine family selectivity panel: CCR1,2b,3,4,5,6,7,8,9/CXCR1,2,3,5,6
<10% @ 10 μM
aqueous solubility (pH 7.4)
>274 μM/>100 μg/mL
CLint (mL/min/mg)/t1/2
0.064/22 min
liver microsomal digestion by species:
Mouse
Rat
0.035/39 min
Human
<0.02/>60 min
human CYP450 [3A4, 2D6, 2C19] % inhibition
rat PK (i.v.-2 mg/kg): t1/2, AUC (0−∞)
rat PK (p.o.-10 mg/kg): t1/2, AUC (0−∞)
F
6%, 8%, 0% @ 1 μM
1.2 h, 108 h·ng/mL
6 h, 328 h·ng/mL
63%
D
dx.doi.org/10.1021/ml400183q | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
excellent potency and encouraging in vitro metabolism data, 15
was profiled in rat pharmacokinetic (PK) studies dosed both
intravenously and by oral gavage. The area under the curve
values (328 h·ng/mL for 10 mg/kg p.o. and 108 h·ng/mL for 2
mg/kg i.v.) were calculated for the experiments. Normalizing
for dose levels, this translates into a good oral bioavailability
value (F = 63%).
motif may also provide compounds with unique biological
selectivity and initial modifications to the TIQ nitrogen and
butyl amine side chain provide tantalizing insights for the
potential design of T-tropic HIV selective antagonists that do
not interfere with SDF-1 based receptor signaling, as well as
more potent antagonists of the CXCR4/CXCL12 axis. On the
basis of the encouraging in vivo and in vitro properties of
compound 15, it has become the target for further lead
Clinical trial observations with AMD3100 (1) led to its
development as a mobilization agent of hematopoietic
progenitor (CD34+) cells. Therefore, we became interested
in measuring the mobilization effects of compound 15. We
chose white blood cell (WBC) mobilization studies in mice as a
proxy for CD34+ mobilization since both phase I human
studies and preclinical mouse studies with AMD3100 showed
that the WBCs mobilized into the peripheral blood exhibited
30
optimization studies and future communications.
ASSOCIATED CONTENT
Supporting Information
■
*
S
Experimental and characterization data for all new compounds
and all biological data, computational docking studies, and the
X-ray of compound 29 is provided. This material is available
free of charge via the Internet at http://pubs.acs.org.
29
almost identical kinetics and egress to CD34+ cells. The
experiments for 15 were performed by giving the compound to
Harlan mice subcutaneously at each of three different dose
levels (5, 10, and 20 mg/kg) and taking blood samples at
multiple time points followed by measurement of both WBC
and drug levels. The WBC response of 15 was found to be dose
dependent over the dose range used (Figure 2, solid lines).
AUTHOR INFORMATION
Corresponding Author
(L.J.W.) E-mail: ljwilso@emory.edu. Phone: 404-727-6689.
■
*
Author Contributions
The manuscript was written through contributions of all
authors and all have given approval to the final version.
Funding
We acknowledge the use of shared instrumentation provided by
grants from the NIH and the NSF.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to Open Eye Scientific Software for generous
provision of software enabling several molecular design tasks.
■
ABBREVIATIONS
Figure 2. Plots of WBC counts (solid lines) and drug-plasma levels
■
(
dotted lines) versus time for three subcutaneous dose levels of
GPCR, G-protein coupled receptor; CXCR4, CXC chemokine
receptor 4; TIQ, tetrahydroisoquinoline; THQ, tetrahydroqui-
noline; Boc, t-butyloxycarbonyl; CXCL12, CXC chemokine
ligand 12; SDF-1, stromal cell-derived factor 1; HIVIIIB, Human
Immunodeficiency Virus strain IIIB; PBMC, peripheral blood
mononuclear cells; WBC, white blood cells; HSC, hema-
topoietic stem cells; Phth, phthalimide; MAGI, multinuclear
activation of a galactosidase indicator; ADME, absorption−
distribution−metabolism−excretion; CYP450, cyto-chrome
P450; cAMP, cyclic adenosine monophosphate; AUC, area
under the curve; PK, pharmacokinetic; i.v., intravenous; p.o.,
oral gavage; DCE, 1,2-dichloroethane; DCM, dichloromethane;
TFA, trifluoroacetic acid
compound 15 in mice.
Two hours after injection, total circulating WBC counts
increased and reached a maximum for each dose. This
corresponds to increases of 1.6-fold at 5 mg/kg, 2.3-fold at
1
0 mg/kg, and 2.7-fold at 20 mg/kg. Maximal WBC
mobilization occurred at the one to two hour time range,
similar to the slight delay observed in mobilization studies with
2
9
AMD3100. Furthermore, this data shows that the WBC
response seems dose-related, with the 10 and 20 mg/kg doses
giving similar responses that appear to be near maximal. When
measuring drug levels (Figure 2, dotted lines), maximal
amounts were seen at the one-hour time point for all three
doses, while WBC mobilization occurred at the one to two-
hour time range. The maximum plasma concentrations of 15
were as follows: 1542, 1021, and 725 ng/mL for the 20, 10, and
REFERENCES
■
(
1) Peled, A.; Wald, O.; Burger, J. Development of novel CXCR4-
based therapeutics. Expert Opin. Invest. Drugs 2012, 21, 341−353.
(2) Lapidot, T.; Petit, I. Current understanding of stem cell
mobilization: the roles of chemokines, proteolytic enzymes, adhesion
molecules, cytokines, and stromal cells. Exp. Hematol. 2002, 30, 973−
81.
3) Tzeng, Y.; Li, H.; Kang, Y. L.; Chen, W. C.; Chen, W. C.; Lai, D.
5
mg/kg doses, respectively (Supporting Information). The
lowest concentrations measured were at the 6 to 8 h time
points (87−318 ng/mL range), where little to no WBC
mobilization was observed.
In summary, a novel series of highly potent and selective
CXCR4 antagonists based on a chiral tetrahydroisoquinoline
9
(
M. Loss of CXCL12/SDF-1 in adult mice decreases the quiescent state
of hematopoietic stem/progenitor cells and alters the pattern on
hematopoietic regeneration after myelosuppresion. Blood 2011, 117,
(
(R)-TIQ) scaffold (15, 22−28, and 30−35) has been
identified through a hit-to-lead effort focused on benzimidazole
replacements. This novel series makes use of a GPCR
chemotype with a chiral linkage that may exploit unique and
efficient contacts with amino acid residues in the receptor. The
4
(
29−439.
4) Tachibana, K.; Hirota, S.; Iizasa, H.; Yoshia, H.; Kawabata, K.;
Kataoka, Y.; Kitamura, Y.; Matsushima, K.; Yoshida, N.; Nishikawa, S.;
Kishimoto, T.; Nagasawa, T. The chemokine receptor CXCR4 is
E
dx.doi.org/10.1021/ml400183q | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
essential for vascularization of the gastrointestinal tract. Nature 1998,
(23) Thomas, J. B.; Atkinson, R. N.; Rothman, R. B.; Fix, S. E.;
Mascarella, S. W.; Vinson, N. A.; Xu, H.; Dersch, C. M.; Lu, Y.-F.;
Cantrell, B. E.; Zimmerman, D. M.; Carroll, F. I. Identification of the
first trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine derivative
to possess highly potent and selective opioid κ receptor antagonism
activity. J. Med. Chem. 2001, 44, 2687−2690.
3
(
93, 591−594.
5) Zou, Y.; Kottman, A. H.; Kuroda, M.; Taniuchi, I.; Littman, D. R.
The function of the chemokine receptor CXCR4 in hematopoiesis and
in cellular development. Nature 1998, 393, 595−599.
(
6) Nagasawa, T.; Tachibana, K.; Kawabata, K. A CXC chemokine
SDF-1/PBSF: a ligand for a HIV co-receptor, CXCR4. Adv. Immunol.
999, 71, 211−228.
7) De Clercq, E. The bicyclam AMD3100 story. Nat. Rev. Drug
Discovery 2003, 2, 581−587.
8) Oberlin, E.; Amara, A.; Bachelerie, F.; Bessia, C.; Virelizier, J. L.;
(24) Lackman-Smith, C.; Osterling, C.; Luckenbaugh, K.;
Mankowski, M.; Snyder, B.; Lewis, G.; Paull, J.; Profy, A.; Ptak, R.
G.; Buckheit, R. W.; Watson, K. M.; Cummins, J. E.; Sanders-Beer, B.
E. Development of a comprehensive human immunodeficiency virus
type I screening algorithm for discovery and preclinical testing of
topical microbicides. Antimicrob. Agents Chemother. 2008, 52 (5),
1768−1781.
1
(
(
Arenzana-Seisdedos, F.; Schwartz, O.; Heard, J. M.; Clark-Lewis, I.;
Legler, D. F.; Loetscher, M.; Baggiollni, M.; Moser, B. The CXC
chemokine SDF-1 is the ligand for LESTR/fusin and prevents
infection by T-cell-line-adapted HIV-1. Nature 1996, 382, 833−935.
(25) Miller, J. F.; Gudmundson, K. S.; D’Aurora Richardson, L.;
Jenkinson, S.; Spaltenstein, A.; Thomson, M.; Wheelan, P. Synthesis
and SAR of novel isoquinoline CXCR4 antagonists with potent anti-
HIV activity. Bioorg. Med. Chem. Lett. 2010, 20, 3026−3030.
(26) Wong, R.; Bodart, V.; Metz, M.; Labrecque, J.; Bridger, G.;
Fricker, S. Comparison of the multiple binding modes of bicyclam,
monocyclam, and noncyclam small-molecule CXC chemokine
receptor 4 inhibitors. Mol. Pharmacol. 2008, 74, 1485−1495.
(
9) Dipersio, J. F.; Yasothan, U.; Kirkpatrick, D. Plerixafor. Nat. Rev.
Drug Discovery 2009, 8, 105−106.
10) Balkwell, F. Cancer and the chemokine network. Nat. Rev.
Cancer 2004, 4, 540−550.
11) Zlotnik, A. New Insights on the role of CXCR4 in cancer
metastasis. J. Pathol. 2008, 215, 211−213.
12) Teicher, B. A.; Fricker, S. P. CXCL12(SDF-1)/CXCR4 pathway
in cancer. Clin. Cancer Res. 2010, 16, 2927−2931.
13) Burger, J. A.; Peled, A. CXCR4 antagonists: targeting the
microenvironment in leukemia and other cancers. Leukemia 2009, 23,
3−52.
14) Wilson, L. J.; Liotta, D. C. Emergence of small-molecule
(
(
(27) Wu, B.; Chein, E. Y.; Mol, C. D.; Fenalti, G.; Liu, W.; Katritch,
(
V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F. C.; Hamel, D. J.; Kuhn,
P.; Handel, T. M.; Cherezov, V.; Stevens, R. C. Structures of the
CXCR4 chemokine GPCR with small-molecule and cyclic peptide
antagonists. Science 2010, 330, 1066−1071.
(
4
(
(28) Brelot, A.; Heveker, N.; Montes, M.; Alizon, M. Identification of
residues of CXCR4 critical for human immunodeficiency virus
coreceptor and chemokine receptor activities. J. Biol. Chem. 2000,
CXCR4 antagonists as novel immune and hematopoietic system
regulatory agents. Drug Dev. Res. 2011, 72, 598−602.
2
(
75, 23736−23744.
(
15) Choi, W.-T.; Duggineni, S.; Xu, Y.; Huang, Z.; An, J. Drug
29) Lack, N. A.; Green, B.; Dale, D. C.; Calandra, G. B.; Lee, H.;
discovery research targeting the CXC chemokine receptor 4
CXCR4). J. Med. Chem. 2012, 55, 977−994.
16) Skerlj, R. T.; Bridger, G. J.; Kaller, A.; McEachem, E. J.;
MacFarland, R. T.; Badel, K.; Liles, W. C.; Bridger, G. A
pharmacokinetic-pharmacodynamic model for the mobilization of
CD34+ hematopoietic progenitor cells by AMD3100. Clin. Pharmacol.
Ther. 2005, 77, 427−436.
(
(
Crawford, J. B.; Zhou, Y.; Atsma, B.; Langille, J.; Nan, S.; Veale, D.;
Wilson, T.; Harwig, C.; Hatse, S.; Princen, K.; DeClercq, E.; Schols, D.
Discovery of a novel small molecule orally bioavailable C−X−C
chemokine receptor 4 antagonists that are potent inhibitors of T-tropic
(
30) For example, in an initial hERG channel evaluation, compound
3
1
5 showed 50% and 93% inhibition at 1 and 10 μM in a [ H]dofetilide
displacement assay in HEK-293 cells.
(
X4) HIV-1 replication. J. Med. Chem. 2010, 53, 3376−3388.
(
17) Stone, N. D.; Dunaway, S. B.; Flexner, C.; Tierney, C.; Calandra,
G. B.; Becker, S.; Cao, Y.-J.; Wiggins, I. P.; Conley, J.; MacFarland, R.
T.; Park, J.-G.; Lalama, C.; Snyder, S.; Kallungal, B.; Klingman, K. L.;
Hendrix, C. W. Multiple-dose escalation study of the safety,
pharmacokinetics, and biologic activity of oral AMD070, a selective
CXCR4 receptor inhibitor, in human subjects. Antimicrob. Agents
Chemother. 2007, 51 (7), 2351−2358.
(
18) Jenkinson, S.; Thomson, M.; McCoy, D.; Edelstein, M.;
Danehower, S.; Lawrence, W.; Wheelan, P.; Spaltenstein, A.;
Gudmundsson, K. Blockade of X4-tropic HIV-1 cellular entry by
GSK812397, a potent noncompetitive CXCR4 receptor antagonist.
Antimicrob. Agents Chemother. 2010, 54 (2), 817−824.
(
19) Thoma, G.; Streiff, M. B.; Kovarik, J.; Glickman, F.; Wagner, T.;
Beerli, C.; Zerwes, H.-G. Orally bioavailable isothioureas block
function of the chemokine receptor CXCR4 in vitro and in vivo. J.
Med. Chem. 2008, 51, 7915−7920.
(
20) Nyunt, M.; Becker, S.; MacFarland, R. T.; Chee, P.;
Scarborough, R.; Everts, S.; Calandra, G. B.; Hendrix, C. W.
Pharmacokinetic effect of AMD070, an oral CXCR4 antagonist, on
CYP3A4 and CYP2D6 substrates midazolam and dextromorphan in
healthy volunteers. J. Acquired Immune Defic. Syndr. 2008, 47, 559−
565.
(
21) Cao, Y. J.; Flexner, C. W.; Dunaway, S.; Park, J. G.; Klingman,
K.; Wiggins, I.; Conley, J.; Radebaugh, C.; Kashuba, A. D.;
MacFarland, R.T.; Becker, S.; Hendrix, C. W. Effect of low-dose
ritonavir on the pharmakokinetics of the CXCR4 antagonist AMD070
in healthy volunteers. Antimicrob. Agents Chemother. 2008, 52 (5),
1
(
630−1634.
22) Kettle, J. G.; Ward, R. A.; Griffen, E. Data-mining patent
literature for novel chemical reagents for use in medicinal chemistry
design. Med. Chem. Commun. 2010, 1, 331−338.
F
dx.doi.org/10.1021/ml400183q | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX