Discovery of novel N-aryl piperazine CXCR4 antagonists

Article abstract of DOI:10.1016/j.bmcl.2015.04.036

A novel series of CXCR4 antagonists with substituted piperazines as benzimidazole replacements is described. These compounds showed micromolar to nanomolar potency in CXCR4-mediated functional and HIV assays, namely inhibition of X4 HIV-1_IIIB virus in MAGI-CCR5/CXCR4 cells and inhibition of SDF-1 induced calcium release in Chem-1 cells. Preliminary SAR investigations led to the identification of a series of N-aryl piperazines as the most potent compounds. Results show SAR that indicates type and position of the aromatic ring, as well as type of linker and stereochemistry are significant for activity. Profiling of several lead compounds showed that one (49b) reduced susceptibility towards CYP450 and hERG, and the best overall profile when considering both SDF-1 and HIV potencies (6-20 nM).

Full text of DOI:10.1016/j.bmcl.2015.04.036

Accepted Manuscript
Discovery of Novel N-Aryl Piperazine CXCR4 Antagonists
Huanyu Zhao, Anthony R. Prosser, Dennis C. Liotta, Lawrence J. Wilson
PII:
S0960-894X(15)00356-X
http://dx.doi.org/10.1016/j.bmcl.2015.04.036
BMCL 22619
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 January 2015
6 April 2015
10 April 2015
Please cite this article as: Zhao, H., Prosser, A.R., Liotta, D.C., Wilson, L.J., Discovery of Novel N-Aryl Piperazine
CXCR4 Antagonists, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2015.04.036
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Discovery of Novel N-Aryl Piperazine
CXCR4 Antagonists
Leave this area blank for abstract info.
Huanyu Zhao, Anthony R. Prosser, Dennis C. Liotta and Lawrence J. Wilson*
N
N
MAGI HIV-1IIIB IC50 = 20 nM
SDF-1 Ca2+ Flux CEM-1 IC50 = 6 nM
CYP450 [3A4, 2D6, 2C19] < 20% @ 1mM
hERG ~ 50% @ 10 mM
N
N
N
N
NH2
NH2
*
HN
HN
Ar
O
8
49b

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery of Novel N-Aryl Piperazine CXCR4 Antagonists
Huanyu Zhaoa, Anthony R. Prosser,b Dennis C. Liottaa,b and Lawrence J. Wilsonb*
aEmory Institute for Drug Development, 954 Gatewood Road NE, Atlanta, GA 30329
bDepartment of Chemistry, Emory University, 1521 Dickey Drive, Atlanta, GA 30322
ARTICLE INFO
ABSTRACT
A
novel series of CXCR4 antagonists with substituted piperazines as benzimidazole
Article history:
Received
Revised
Accepted
Available online
replacements is described. These compounds showed micromolar to nanomolar potency in
CXCR4-mediated functional and HIV assays, namely inhibition of X4 HIV-1IIIB virus in MAGI-
CCR5/CXCR4 cells and inhibition of SDF-1 induced calcium release in Chem-1 cells.
Preliminary SAR investigations led to the identification of a series of N-aryl piperazines as the
most potent compounds. Results show SAR that indicates type and position of the aromatic ring,
as well as type of linker and stereochemistry are significant for activity. Profiling of several lead
compounds showed that one (43b) reduced susceptibility towards CYP450 and hERG, and the
best overall profile when considering both SDF-1 and HIV potencies (6-20 nM).
Keywords:
Piperazine
G-protein coupled receptor
CXC chemokine receptor 4
CXCR4 antagonists
HIV
2009 Elsevier Ltd. All rights reserved.
The CXC chemokine receptor 4 (CXCR4) is an alpha G-
protein coupled chemokine receptor that is widely expressed on
most hematopoietic cells, predominantly leukocytes including
neutrophils, monocytes, and macrophages.[1] As the specific
ligand for CXCR4, CXCL12 (SDF-1, stromal cell-derived factor-
1) plays a vital role in hematopoietic stem cell (HSC) homing to
the bone marrow and HSC quiescence,[2-4] making the
CXCR4/CXCL12 axis key to many essential physiological
activities such as homeostatic regulation of leukocyte trafficking,
hematopoiesis and embryonic development.[5,6] It has also been
shown that CXCR4 assists the entry of HIV into CD4+ T cells as
a co-receptor with CD4. [7,8] Not surprisingly, the discovery of the
involvement of CXCR4 in HIV infection triggered tremendous
research efforts in the development of CXCR4 antagonists,
primarily as potential HIV entry inhibitors.[9] The compound
AMD 3100 (1, Perlixafor™, Chart 1), a CXCR4 antagonist, first
entered clinical trials as an anti-T-tropic HIV agent, but was later
approved by FDA in a different therapeutic setting as supportive
treatment for autologous stem cell transplantation due to its
efficacy in mobilizing HSC from bone marrow to the peripheral
blood stream.[10,11] This serendipity dramatically broadened the
breadth and scope of CXCR4 research, leading to the discovery
of various CXCR4 antagonists both peptide-based[12] and small
molecules[13]. Recently, the CXCR4/CXCL12 axis has been
linked to more than 23 types of cancer including ovarian,
prostate, oesophageal, melanoma, neuroblastoma and renal cell
carcinoma, where the receptor/ligand interaction is a key factor in
cancer metastasis, angiogenesis, and tumor growth via direct and
indirect mechanisms.[14-19] Furthermore, the involvement of
CXCR4/CXCL12 in various, yet essential signaling pathways
and physiological activities has made it an attractive target for the
development of novel treatments in the areas of HSC
mobilization during autologous transplantation, T-tropic HIV
infection, metastatic tumors, tissue regeneration, inflammation
and WHIM syndrome.[20]
NH HN
N
N
N
H
NH
N
N
N
N
N
N
NH2
S
N
OH
HN
N
N
N
S
N
HN
N
3 (GSK812397)
2 (AMD11070)
NH HN
4 (IT1t)
1 (AMD3100)
N
N
N
N
N
NH2
NH2
R1
N
X
*
H
H
HN
N
X
N
N
R2
8 (PIP)
N
5 (X=CH; WZ811)
6 (X=N; MSX-122)
7 (TIQ-15)
Chart 1. Selected small molecule CXCR4 antagonists.
Efforts in the search for CXCR4 antagonists has resulted in
several small molecules being discovered (Chart 1, 2-7), with one
of these (2, AMD11070) entering human clinical trials.[1,19,21-23]
Further work revealed other analogs of note including a 11070
congener (GSK812397, 3), IT1t (4), and AMD3100 homologues
WZ811 (5) and MSX-122 (6).[24-27] Recently, we reported a
series of TIQ analogs represented by TIQ-15 (7), where the
*Corresponding Author Contact Information: e-mail – ljwilso@emory.edu; telephone – 404-727-6689.

benzimidazole portion was replaced by a tertahydro-isoquinoline
ring.[28] Considering the complexity of the many roles of CXCR4,
further research is still greatly needed for the discovery of
CXCR4 antagonists that have increased affinity with the ability
to target CXCR4 differentially, while showing validated drug-
like properties including bioavailability, pharmacokinetics and
pharmacodynamics. Inspired by previous work in our group and
others,[24, 26-28] we herein describe a series of novel CXCR4 small
molecule antagonists 8 consisting of chiral N-aryl piperazines as
replacements for the benzimidazole ring. As a complementary
study to our previous research on CXCR4 antagonists, which
contain a fused N-heterocycle and aromatic ring system (7) and
those done on AMD11070 (2), current efforts involved the
incorporation of a piperazine moiety linked with an aromatic ring
with various linkers as the replacement of the 1,2,3,4-
tetrahydroisoquinoline[28] and benzimidazole ring systems[22]. The
goals were to improve the ADMET properties of these molecules
as piperazines have shown privileged pharmacological merits in
drug development, particularly within GPCR ligands.[29,30]
available racemic N-Boc piperazine carboxylic acid 9.
A
number of divergent routes could be utilized with this starting
material to access different derivatives. For example: route one
involves simple conversion to the methyl ester (10); route two
consisted of conversion to the alcohol followed by TBS
protection of the alcohol to give 11; and route three consists of
simultaneous di-benzylation of both the carboxylic acid and
piperazine nitrogen moieties to give compound 12. The other
intermediates (10,11) were then utilized to make various N-
piperazine building blocks (13-17). The methyl ester 10 was
converted to N-phenyl sulfonamide, phenyl (via Buchwald-
Hartwig) and carboxybenzyl (CBZ) derivatives 15-17 followed
by reduction to the corresponding alcohols (21-23) and oxidation
to give piperazine aldehydes 27-29. The silyl ether 11 was
acylated to give benzoyl and furanoyl compounds 13 and 14,
followed by deprotection (19, 20) and oxidation to give
piperazine aldehydes 25 and 26. Finally, N-benzyl derivative 12
could be converted to the aldehyde 24 by either reduction to the
alcohol (18) and oxidation or directly by di-isobutyl aluminum
hydride reduction. The first of two consecutive reductive
aminations with the piperazine aldehydes 24-29 with chiral S-
THQ 30 gave intermediates 31-36 that were now
diastereomerically differentiated (syn and anti). In the case of the
N-benzyl derivative 31, these diastereomers were readily
separable by chromatography. An X-ray structure determination
on suitable crystals of the lower Rf isomer of 31 revealed this to
Our synthetic efforts focused on providing a versatile route
that would allow substitution at both piperazine nitrogen atoms,
while allowing access to both stereoisomers on the piperazine
ring. Although the anti-diastereomer/R-stereoisomer is preferred
in the TIQ series (7), we were not certain how this would affect
activity in this series. The general synthesis of the piperazines
(8) is shown in Scheme 1. Syntheses commenced from readily
be
the
syn-isomer
(31b,
Supplementary
Material).
Scheme 1.a
H
O
N
OH
R
R
*
*
*
ix or x
*
vii or viii
iv or v or vi
Boc
Boc
Boc
Boc
N
N
N
N
N
NH
N
Ar
Ar
Ar
9 (R=CO2H)
10 (R=CO2Me)
11 (R=CH2OTBS)
12 (R=CO2Bn, Ar=CH2Ph)
18 (Ar=CH2Ph)
19 (Ar=COPh)
20 (Ar=CO-(2)-furyl)
21 (Ar=SO2Ph)
22 (Ar=Ph)
24 (Ar=CH2Ph)
25 (Ar=COPh)
26 (Ar=CO-(2)-furyl)
27 (Ar=SO2Ph)
28 (Ar=Ph)
i
13 (R=CH2OTBS, Ar=COPh)
14 (R=CH2OTBS, Ar=CO-(2)-furyl)
15 (R=CO2Me, Ar=SO2Ph)
16 (R=CO2Me, Ar=Ph)
ii, iii
17 (R=CO2Me, Ar=Cbz)
23 (Ar=Cbz)
29 (Ar=Cbz)
OH
Boc
N
N
N
N
NH2
N
xvi
37
30
NH
N
N
NHBoc
NH2
xi
xii
*
Boc
*
Boc
*
N
N
HN
N
N
N
Ar
Ar
Ar
38 (Ar=CH2Ph)
39 (Ar=COPh)
48 (Ar=CH2Ph)
49 (Ar=COPh)
31 (Ar=CH2Ph)
32 (Ar=COPh)
40 (Ar=CO-(2)-furyl)
41 (Ar=SO2Ph)
42 (Ar=Ph)
50 (Ar=CO-(2)-furyl)
51 (Ar=SO2Ph)
52 (Ar=Ph)
33 (Ar=CO-(2)-furyl)
34 (Ar=SO2Ph)
35 (Ar=Ph)
43 (Ar=Cbz)
53 (Ar=Cbz)
36 (Ar=Cbz)
44a (Ar=SO2-2-naphthyl)
45a (Ar=SO2-2-thiophenyl)
46a (Ar=SO2-(4Cl)Ph)
47a (Ar=CONHPh)
54a (Ar=SO2-2-naphthyl)
55a (Ar=SO2-2-thiophenyl)
56a (Ar=SO2-(4Cl)Ph)
57a (Ar=CONHPh)
xiii,
xiv,
xv
a Reagents. i. MeOH, EDCI, THF, DCM; ii. BH3-SMe2, THF; iii. TBSCl, NEt3, DCM; iv. BnBr, K2CO3, DMF;
v. PhCOCl or PhSO2Cl or 2-Furyl-COCl or CbzCl, NEt3, DCM; vi. PhBr, Pd2dba3, DavePhos, Cs2CO3,
1,4-dioxane; vii. NaBH4, CaCl2, THF, EtOH; viii. Bu4NF, THF; ix. Dess-Martin, NaHCO3, DCM; x. (COCl)2,
DMSO, NEt3, DCM; xi. 30, NaBH(OAc)3, DCM; xii. 37, NaBH(OAc)3, DCM, AcOH; xiii. H2, Pd(C), MeOH;
xiv. Cl-SO2-2-naphthyl or Cl-SO2-2-thiophenyl or Cl-SO2-(4Cl)Ph, NEt3, DCM; xv. PhNCO, DCM; xvi. TFA, DCM.

This stereochemistry observation for 31b was consistent with
compounds in the TIQ series (7) where the upper and lower Rf
stereoisomers corresponded to anti and syn diastereomers
respectively. Most of the other isomers (32-36) were separable at
the next step upon subsequently attaching the Boc protected butyl
amine side chain (37). Attachment of this side chain was
accomplished by reacting the secondary amines 31-36 with
derivative 37 to give the di-Boc protected compounds 38-43. As
mentioned before, the N-benzyl isomers (31) were separated
prior to this step and then carried through individually. The N-
acyl and sulfonyl compounds (39-41, and 43) were separated into
their anti and syn diastereomers at this stage. The phenyl
derivative 42 was inseparable and carried forward as a mixture.
All these intermediates were N-Boc deprotected by treatment
with trifluoro acetic acid to give the corresponding final
compounds (48-53a/b, Table 1) as free amines. The phenyl
derivative 52 was inseparable at either stage and tested as a
diastereomeric mixture.
Alternatively, the two derivatives, one with no piperazine
substituents (68) and the other with a N-benzoyl group
transposed to the top piperazine nitrogen (69) were synthesized
from another commercially available N-Boc protected piperazine
carboxylic acid (58, Scheme 2). Compound 58 was first
protected by Cbz-Cl (59), followed by reduction (60) and Parikh-
Doering oxidation to furnish aldehyde 61. Similar to Scheme 1,
intermediate 61 was sequentially reacted with tetrahydro-
quinoline 30 and Boc/Cbz-protected amino butyraldehydes 37/63
to give differentially protected derivatives 64 and 65. These were
followed by hydrogenolysis removal of Cbz on the top piperazine
nitrogen to give 66, followed by reaction with benzoyl chloride
for compound 67. TFA-facilitated Boc deprotection of 67
provided the final product 69. Alternatively, the bis-Cbz
derivative 65 was Boc-deprotected and then hydrogenated to give
68. In these cases, the stereoisomers were separated at
compounds 65 and 67. Since the stereochemistry of intermediate
31b was determined and confirmed by X-ray crystal structure
(Scheme 1, Supplementary material) the stereochemical
assignments of all other compounds were deduced from this
observation and the lower Rf compound was assigned as the syn
daistereomer (Table 1, URf (a) and LRf (b)). The labeling of the
compounds by the R,S-nomenclature system is based on priority
rankings of the substituents around the chiral center on the
piperazine ring, which changed from compound to compound.
This assignment along with the Rf properties is listed along with
the compound structure in Table 1.
A modular modification on Scheme 1 utilized a late stage
intermediate, the upper Rf isomer of the N-Boc/N-Cbz
differentially protected piperazine compound 43a, to make
several variations on the bottom nitrogen (54a-57a, Scheme 1).
This was accomplished by first deprotecting the Cbz group by
hydrogenation followed by treatment with the appropriate
electrophile (ArSO2Cl or PhNCO) to give the di-Boc compounds
(44a-47a). Subsequent Boc deprotection with trifluoroacetic acid
gave the corresponding final compounds.
We wanted to determine the ability of these compounds to
block CXCR4 signaling as well as HIV entry. The activities of
Scheme 2.a
these compounds against CXCR4 were evaluated by
a
CO2H
N
R
combination of two assays: (1) the viral attachment assay with
HIV-1IIIB in CCR5/CXCR4-expressing HeLa-CD4-LTR-β-gal
(MAGI) cells measuring the compound’s ability to block
potential viral entry;[31,32] (2) inhibition of CXCL12 induced
calcium (Ca2+) flux/release in Chem-1 cells showing the
compound’s potential to initiate the receptor’s G-protein
function.[33] The results of these two assays for the piperazine
compounds is shown in Table 1 and indicate stereochemistry, as
well as inclusion and location of the aromatic substitution on the
piperazine ring are of central importance to potency in both
assays. Some of the compounds with substituents at the lower
nitrogen atom (48-57a) showed moderate to potent activities in
blocking viral entry with IC50 values below 100 nM in one or
both assays, showing that the aromatic substituent contributed to
a 10-100 fold increase in activity. This is demonstrated by
comparing these compounds to the ones with an N-H at this
position (63a/b). The compound with no linkage to the phenyl
ring (52) gave no significant contribution to activity, but
introduction of a linker with and without heteroatoms, such as
oxygen and sulfur, to the aromatic group did slightly improve the
activity especially when the linker length was one atom (48, 49,
51 compared to 52). Furthermore, the linkage between the
piperazine ring and nature of the aromatic group played a
significant role as hetero cycles, multiple rings (50, 54a, 55a),
carbamate and urea linkages (53, 57a) resulted in a drop off in
activity of 10-fold or more. The only variation that seemed
tolerable in addition to benzyl, bezamide and phenyl sulfonamide
linkages-groups (48, 49, 51 a/b) was para substitution on the
aromatic ring (p-chlorophenyl, 56a), albeit with no increase in
activity though. When the group was placed on the top
piperazine nitrogen (64) all activity is eliminated confirming our
choice in focusing on the bottom nitrogen of the piperazine ring.
Marginal differences were observed when comparing
stereoisomers but this difference in the most potent compounds
(49a/b and 51a/b) was only 2-3 fold. This is significant when
*
Cbz
R
*
i-iii
N
N
N
Boc
Boc
58 (R=H)
59 (R=COPh)
60 (R=CH2OH)
61 (R=CHO)
iv
OH
N
R
37 (R=Boc)
63 (R=Cbz)
N
N
N
NH
NHR
v
Cbz
*
Cbz
*
N
N
N
N
Boc
Boc
64 (R=Boc)
65 (R=Cbz)
62
viii, vi
vi, vii
N
N
N
N
viii
NH2
NHBoc
R
R
*
*
N
N
NH
N
Boc
68 (R=H)
69 (R=COPh)
66 (R=H)
67 (R=COPh)
a Reagents: (i) CbzCl, NEt3, 1,4-Dioxane; (ii) BH3-SMe2, THF,
0oC; (iii) SO3-pyridine, NEt3, DCM, DMSO; (iv) 30, NaBH(OAc)3,
1,2-DCE; (v) 37 or 63, NaBH(OAc)3, AcOH, DCM; (vi) H2, Pd(C),
EtOH; (vii) BzCOCl, NEt3, DCM; (viii) TFA, DCM.

compared to the TIQ scaffold (7) where a 100-fold difference
was observed between R and S stereoisomers.
This smaller difference in calcium flux potency also applies to
linker atoms and stereochemistry supporting the general trend.
This trend is not observed in the TIQ scaffold where ring position
and stereochemistry contribute to a 100-fold or more in potency
differences. Furthermore, compound 48b shows a unique
property when compared to the rest showing a preference of
blocking HIV versus SDF-1 effects (HIV selectivity = 0.19/0.03
= 6 fold) compared to 49b (HIV selectivity = 0.006/0.02 = 0.3
fold). This effect is further supported by the observation that
compound 48b has a less potent ability to displace 125I-SDF-1
than TIQ-15 (IC50 = 2,920 nM for 48b versus 112 nM for 7). This
discrepancy in activity might become the gateway to find more
selective CXCR4 based HIV entry blockers.
Generally speaking, most compounds behaved slightly better
as CXCR4 ligands in blocking G-protein-mediated calcium flux.
All compounds showed CXCR4 antagonist behavior with
varying potencies (Table 1, 1 uM down to 2 nM). In contrast to
the MAGI-HIV results, almost all aromatic groups increased
potency, and most had IC50 values below 100 nM in the calcium
flux assay. An example of this difference is seen in comparing
sulfonamide groups on the upper Rf isomer (51a, 54a, 55a, 56a)
where the calcium flux potency range is about 30-fold (2-71 nM),
while the HIV assay range is about 100-fold (50-4,600 nM).
Table 1. Structures and biological activities of piperazine derivatives from Schemes 1 and 2.
SDF-1
Ca2+ Flux
CEM-1
IC50
MAGI
HIV1IIIB
IC50
Compound
Number
(µM)a,b
(µM)a
a (URf)
b (LRf)
R1
R2
(*)-R/S
68a
68b
H
H
H
H
R
S
0.31
1.1
0.255
1.2
52a/b
H
Ph
Mix. (1:1)
0.72
0.13
48a
48b
H
H
CH2Ph
CH2Ph
R
S
0.15
0.03
0.23
0.19
49a
49b
H
H
C(O)Ph
C(O)Ph
S
R
0.06
0.02
0.002
0.006
69a
69b
C(O)Ph
C(O)Ph
H
H
R
S
>100
>100
-
-
50a/b
50b
H
H
Mix. (2:1)
R
0.21
0.15
-
0.035
53a
53b
H
H
CO2CH2Ph
CO2CH2Ph
S
R
0.34
1.35
0.018
-
51a
51b
H
H
SO2Ph
SO2Ph
S
R
0.05
0.02
0.002
0.023
54a
H
S
4.6
0.071
55a
56a
57a
H
H
H
S
S
S
3.44
0.05
0.35
0.039
0.002
21
C(O)NHPh
aAll assays were performed in duplicate. bThe cytotoxcities (TC50’s) for all compounds were greater than 10 M.
To better understand the SAR of our efforts we engaged in a
molecular modeling study using the existing CXCR4 X-ray
crystal structures.34 As we have previously published studies
using both the small molecule and peptide crystal structures with
AMD11070, we utilized these findings to focus our efforts here.35
We selected the most potent diastereomeric pair with the

benzamide (49a and 49b) for docking studies with the
CXCR4:CVX15 grid, which we hypothesize will adopt “peptide-
mimetic” binding poses. Docking these isomers and optimizing
the resulting grids resulted in unique and different docking poses
(Figure 1). In the case of the upper Rf (49a) diastereomer, the
molecule sits in the receptor and makes hydrogen bond
interactions with three residues and is depicted in Figure 1A as
follows: (i) the butyl amine side chain forms a salt bridge with
aspartic acid residue 97 (Asp97); (ii) the top piperizne N-H forms
a salt bridge with glutamic acid residue 288 (Glu288); and (iii)
the benzoyl carbonyl forms a H-bond interaction with the side
chain of arginine 188 (Arg188). The lower Rf diastereomer
(49b) forms similar interactions but with a few differences and is
depicted in Figure 1B as follows: (i) the butyl amine side chain
forms a salt bridge with aspartic acid 187 (asp187); and (ii) the
piperazine N-H forms a salt bridge with glutamic acid residue
288 (Glu288). To compare and contrast the two seemingly
significant and different poses for these isomers, an overlay of
49a and 49b was generated and is shown in Figure 1C. From this
overlay and the previous pictures (Figure 1A and 1B), it can be
seen that the two molecules occupy similar positions in the
for the butyl amine and THQ substituents. These two poses are
consistent with findings of the crystal structure determinations
with the CVX15 peptide and IT1t with CXCR4, which include
the following residues: (i) for IT1t – Asp97 and 187, Glu288; and
(ii) for CVX15 – Asp187, Arg188. Given these parameters it
seems both isomers 49a and 49b are hybrid binders in that they
interact with residues identified in both ligand-crystal structure
complexes.
Our final efforts in this series involved accessing the drug-
like characteristics of our best leads to identify and compare to
other series of compounds. We selected the two compounds 49b
and 51b since they had the best selection of potency in both
assays and contained different aryl groups on the piperazine ring
(benzamide versus sulfonamide). We decided upon a small but
significant subset of ADMET assays, consisting of CYP450
inhibition, and hERG channel binding. The assays consisted of
measuring the effect of these compounds and the corresponding
results are shown in Table 2 along with previous reported values
for compounds 2 and 7. The findings are supportive of the
piperazine addition as a better drug-like fragment than the
benzimidazole on AMD11070 (2) in regards to the CYP450
activities. First, when comparing the CYP450 inhibition for the
three isozymes (3A4, 2D6, and 2C19), both 49b and 51b show
marked improvement compared to 2, with the benzamide 49b
having the lowest response and similar to TIQ-15 (7).28,36 The
sulfonamide (51b) had slightly higher inhibition of 2D6 in
comparison but both had low responses against 3A4 and 2C19.
Furthermore, when comparing these three series (benzimidazole,
TIQ, and piperazine) series, one can conclude that the
benzimidazole moiety results in substantial CYP450 inhibition
supporting our efforts to alter this section of the molecule to
improve ADMET properties. When tested against the hERG
channel, both compounds showed beneficial effects in
comparison to both AMD11070 (2) and TIQ-15 (7) in an
absolute sense. In these cases, both compounds show less than
50% at 1 M. Whereas, compound 49b has the best value (55%
at 10 M), the results must be compared to on-target potencies to
provide a fair comparison. In the case of 49b, the anti-viral
potency is slightly higher than 7 (PBMC-HIV-1IIIB IC50 = 88 nM
versus 24 nM for 7) by about 3-fold while the hERG activity is
about 10-fold different (55% at 10 M versus 50% at 1 M).
Therefore, the benzamide 49b may hold a slight advantage in the
therapeutic index in comparison to compound 7 with more potent
anti-viral activity. A pharmacokinetic (PK) study of 49b may
provide further evidence of drug-like potential.
(A)
(B)
Table 2. CYP450 and hERG Parameters for 49b, 51b, 2, and 7.
hERG (%)b
10mM
CYP450 (% @ 1mM)a
Compound
2C19
2
2
20
0
2D6
19
41
100
3A4
0
0
35
6
1mM
18
37
-
49b
51b
2
55
79
-
7
8
50
93
aIsolated human enzymes and fluorometric substrates. bDisplacement of 3H-dofetilide in HEK293
cells.
Figure 1. Docking poses of diasteromeric piperazines 49a and
49b in the CVX15:CXCR4 crystal structure showing critical
amino acids and key hydrogen bonds between protein and ligand
(green dotted lines). (A) Compound 49a. (B) Compound 49b.
In conclusion, we have presented the discovery of a novel
series of N-aryl piperazine based CXCR4 antagonists (8). These
compounds showed potent effects against blocking the functional
responses of SDF-1 and blockade of HIV entry in assays
measuring these effects. Preliminary SAR provided an optimal
linker between the piperazine nitrogen and aromatic group as
being a carbonyl or sulfonamide. Furthermore, our initial efforts
in aryl SAR show some tolerance but also a limitation on the size
and type of group. Finally, assessment of drug-like properties
show two potential lead compounds (49b and 51b) where a
reduction in CYP activity is observed as well as reduction in
receptor with regards to the piperazine ring, as both involve
interaction of the piperazine N-H with the glutamic acid residue
(Glu288). However, there are notable differences. One is that
the carbonyl of the benzoyl group on 49a picks up a van der
waals interaction with the side chain of Arg188, while 49b does
not. The other main difference is that the butyl amine side chains
interact with different aspartic acid residues (Asp97 for 49a vs.
Asp 187 for 49b) showing a fundamentally different positioning

20. Tamamura, H.; Tsutsumi, H.; Nomura, W.; Tanaka, T.; Fujii, N. A
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hERG potential. Our results also show that to fully ascertain the
ability to deliver these agents orally, more experiments are
needed. The optimization of these compounds as both anti-HIV
entry agents and CXCR4 antagonists will be reported in due
course.
22. Skerlj. RT.; Bridger, GJ.; Kaller, A.; McEachem, EJ.; Crawford,
JB.; Zhou, Y.; Atsma, B.; Langille, J.; Nan, S.; Veale, D.; Wilson,
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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 Chemotherapy 2007, 51(7), 2351-
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24. 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
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25. 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
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26. Zhan, W.; Liang, Z.; Zhu, A.; Kurtkaya, S.; Shim, H.; Snyder,
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Antagonists. J. Med. Chem. 2007, 50, 5655-5664.
27. Liang, Z.; Zhan, W.; Zhu, A.; Yoon, Y.; Lin, S.; Sasaki, M.;
Klapproth, J.-M.A.; Yang, H.; Grossniklaus, H.E.; Xu, J.; Rojas,
M.; Voll, R.J.; Goodman, M.M.; Arrendale, R.F.; Liu, J.; Yun,
C.C.; Snyder, J.P.; Liotta, D.C.; Shim, H. Development of a
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28. Truax. V.M.; Zhao, H.; Katzman, B.M.; Prosser, A.R.; Alcaraz,
A.A.; Saindane, M.T.; Howard, R.B.; Culver, D.; Arrendale, R.F.;
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Acknowledgments. ARP would like to thank the National
Science Foundation for funding.
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Supplementary Material. Procedures and characterization data
for the compounds in Table 1, Schemes 1 and 2, as well as the x-
ray crystal structure of 31b are included.