Discovery of Tetrahydroisoquinoline-Containing CXCR4 Antagonists with Improved in Vitro ADMET Properties

Article abstract of DOI:10.1021/acs.jmedchem.7b01420

CXCR4 is a seven-transmembrane receptor expressed by hematopoietic stem cells and progeny, as well as by ≥48 different cancers types. CXCL12, the only chemokine ligand of CXCR4, is secreted within the tumor microenvironment, providing sanctuary for CXCR4⁺ tumor cells from immune surveillance and chemotherapeutic elimination by (1) stimulating prosurvival signaling and (2) recruiting CXCR4⁺ immunosuppressive leukocytes. Additionally, distant CXCL12-rich niches attract and support CXCR4⁺ metastatic growths. Accordingly, CXCR4 antagonists can potentially obstruct CXCR4-mediated prosurvival signaling, recondition the CXCR4⁺ leukocyte infiltrate from immunosuppressive to immunoreactive, and inhibit CXCR4⁺ cancer cell metastasis. Current small molecule CXCR4 antagonists suffer from poor oral bioavailability and off-target liabilities. Herein, we report a series of novel tetrahydroisoquinoline-containing CXCR4 antagonists designed to improve intestinal absorption and off-target profiles. Structure-activity relationships regarding CXCR4 potency, intestinal permeability, metabolic stability, and cytochrome P450 inhibition are presented.

Full text of DOI:10.1021/acs.jmedchem.7b01420

Subscriber access provided by NAGOYA UNIV
Article
Discovery of Tetrahydroisoquinoline-Containing CXCR4
Antagonists with Improved in vitro ADMET Properties
Eric J Miller, Edgars Jecs, Valarie M Truax, Brooke M Katzman, Yesim A Tahirovic, Robert J. Wilson,
Katie M Kuo, Michelle B Kim, Huy H Nguyen, Manohar T Saindane, Huanyu Zhao, Tao Wang,
Chi S Sum, Mary Ellen Cvijic, Gretchen M Schroeder, Lawrence J Wilson, and Dennis C. Liotta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01420 • Publication Date (Web): 19 Jan 2018
Downloaded from http://pubs.acs.org on January 19, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery of TetrahydroisoquinolineꢀContaining
CXCR4 Antagonists with Improved in vitro
ADMET Properties
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
Eric J. Miller; Edgars Jecs; Valarie M. Truax; Brooke M. Katzman; Yesim A. Tahirovic;
1
1
1
1
1
Robert J. Wilson; Katie M. Kuo; Michelle B. Kim; Huy H. Nguyen; Manohar T. Saindane;
1
2
2
2
2
Huanyu Zhao; Tao Wang; Chi S. Sum; Mary E. Cvijic; Gretchen M. Schroeder; Lawrence J.
1
1*
Wilson; Dennis C. Liotta.
1
Department of Chemistry, Emory University, 1515 Dickey Dr., Atlanta, GA 30322, USA
2
BristolꢀMyers Squibb Research & Development, Princeton, NJ 08543, USA
KEYWORDS
CXCR4; CXCL12; Chemokine; Hematopoietic stem cells; Cancer; Immunomodulation;
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
CXCR4 is a 7ꢀtransmembrane receptor expressed by hematopoietic stem cells and progeny, as
well as by ≥ 48 different cancers types. CXCL12, the only chemokine ligand of CXCR4, is
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
secreted within the tumor microenvironment, providing sanctuary for CXCR4 tumor cells from
immune surveillance and chemotherapeutic elimination by 1) stimulating proꢀsurvival signaling
+
and 2) recruiting CXCR4 immunosuppressive leukocytes. Additionally, distant CXCL12ꢀrich
+
niches attract and support CXCR4 metastatic growths. Accordingly, CXCR4 antagonists can
+
potentially obstruct CXCR4ꢀmediated proꢀsurvival signaling, recondition the CXCR4 leukocyte
+
infiltrate from immunosuppressive to immunoreactive, and inhibit CXCR4 cancer cell
metastasis. Current small molecule CXCR4 antagonists suffer from poor oral bioavailability and
offꢀtarget liabilities. Herein, we report a series of novel tetrahydroisoquinolineꢀcontaining
CXCR4 antagonists designed to improve intestinal absorption and offꢀtarget profiles. Structure
activity relationships regarding CXCR4 potency, intestinal permeability, metabolic stability, and
cytochrome P450 inhibition are presented.
ACS Paragon Plus Environment
2

Page 3 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Chemokines are small, soluble cytokines that bind to and signal through sevenꢀtransmembrane
1
chemokine receptors. These interactions facilitate the directional migration of chemokine
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
receptorꢀexpressing leukocytes along concentration gradients of chemokine ligands, and they
also promote the migratory aptitude and proliferation of these cells via proꢀchemotactic and proꢀ
survival gene transcription. Although most chemokine receptors interact with numerous
chemokine ligands, several others bind only to one, such as the CXC type 4 receptor (CXCR4),
which interacts solely with the CXC type 12 ligand (CXCL12, also known as stromal cellꢀ
2
derived factor 1, or SDFꢀ1). Extracellular CXCL12 binding to CXCR4 causes a conformational
change that translates to the intracellular portions of the receptor, which associate with
3
heterotrimeric G proteins. This causes dissociation of the Gα subunit from the Gβγ heterodimer.
CXCR4 predominantly signals through G proteins that contain Gαi/o subunits, which are
4
ubiquitously expressed, or Gα15 subunits, which are primarily expressed by cells of
hematopoietic origin. Once released from Gβγ, Gα proteins inhibit adenylyl cyclase (AC) and
i/o
stimulate phosphoinositide 3ꢀkinase (PI3K). Alternatively, Gα proteins activate phospholipase
1
5
+
2
C (PLC), leading to the release of intracellular Ca ions, which is commonly measured to
quantify CXCR4 activity in vitro. Through these signaling cascades, binding of CXCL12 to
CXCR4 facilitates many cellular activities comprising chemotaxis, hematopoiesis, transcription,
5
survival, and proliferation.
6
CXCR4 is the only functional chemokine receptor expressed on hematopoietic stem cells
(HSCs). As the CXCR4/CXCL12 axis is essential for HSC colonization of fetal bone marrow
7
during development, CXCL12 or CXCR4 knockout mice are embryonic lethal, exhibiting
8
defects in hematopoiesis, vascularization, and vital organ development. In adults, high levels of
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CXCL12 are secreted by stromal cells residing in the bone marrow, liver, lungs, and lymph
nodes, as well as by pathogenic, damaged, and hypoxic tissue. In contrast, CXCR4 is
predominantly expressed by HSCs and their lineages, including T and B lymphocytes, natural
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
killer cells, macrophages, and neutrophils, as well as by injured or hypoxic tissue. These
+
expression profiles result in chemoattraction of CXCR4 leukocytes and progenitors to
CXCL12ꢀrich niches where they become sequestered as a result of integrinꢀmediated adhesion to
+
stromal cell layers. CXCL12 also encourages CXCR4 leukocyte motility by stimulating
cytoskeletal rearrangements, Fꢀactin bundle organization, and leading edge formation CXCL12
further activates integrins and upregulates matrix metalloproteinases (MMPs), thereby
facilitating attachment to and penetration through proximal circulatory vessel endothelia.
Although CXCR4 has received much attention from the drug discovery and development
1
0
communities as the coꢀreceptor for Tꢀtropic HIV entry, the CXCR4/CXCL12 axis is also
5
integrally involved in the progression of many types of cancer. CXCR4 overexpression relative
1
1
to normal tissue has been detected in ≥ 48 different types of solid and hematological cancers.
Although oncogenic mutations provide the host immune system with various tumorꢀspecific
+
antigens, intratumoral CXCL12 instigates CXCR4 cancer cell adhesion to and migration
beneath tumorꢀassociated stroma in an integrinꢀdependent manner. CXCL12 also induces
CXCR4ꢀmediated transcription of growth factors, angiogenic factors, and immunosuppressive
cytokines. These events promote tumor progression, evasion of immune surveillance, and
9
resistance to chemotherapy. Privileged CXCL12ꢀrich stromal niches within the tumor
+
microenvironment additionally provide a proꢀsurvival sanctuary for quiescent CXCR4 cancer
cell subpopulations that persist after radiation therapy and/or chemotherapy, increasing the
1
2
13
14
likelihood of relapse. Accordingly, high intratumoral levels of CXCR4 and CXCL12 are
ACS Paragon Plus Environment
4

Page 5 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
independent indicators of poor clinical prognosis for patients suffering from various types of
cancer.
Tumor cells additionally evade immune surveillance and chemotherapeutic elimination by
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hijacking CXCR4/CXCL12ꢀmediated leukocyte trafficking to chemotactically recruit CXCR4
leukocytes and progenitors, many of which are immunosuppressive rather than
1
5
immunoreactive. . Although various leukocyte subsets gain access to the tumor
microenvironment, many of these cells are immunosuppressive rather than immunoreactive. For
example, intratumoral CXCL12 both repels cluster of differentiation 8 (CD8)ꢀexpressing effector
T lymphocytes and recruits immunosuppressive forkhead box P3 (FoxP3)ꢀexpressing regulatory
1
6
T lymphocytes, which facilitate both immune evasion and chemotherapeutic resistance.
+
+
Accordingly, low ratios of CD8 cytotoxic T lymphocytes to FoxP3 regulatory T lymphocytes
correlate with poor clinical prognosis, while high ratios are associated with increased survival.
+
CXCL12 also recruits CXCR4 proꢀangiogenic cells, which support tumor vascularization,
1
7
growth, and invasion.
+
CXCR4 cancer cells further hijack the normal physiological functions of the
CXCR4/CXCL12 axis to gain entry to distant CXCL12ꢀrich tissues, where they find
1
8
environments that cultivate the formation of new metastases. Stromal cell secretion of CXCL12
+
recruits CXCR4 tumor cells to, for example, the bone marrow microenvironment, which
provides a proꢀsurvival sanctuary for these cancer cells to evade immune recognition and
+
chemotherapeutic elimination. This migration of CXCR4 cancer cells from the primary tumor to
+
CXCL12ꢀrich metastatic sites occurs via a mechanism equal but opposite to normal CXCR4
1
9
+
leukocyte trafficking. Once the primary tumor is sufficiently vascularized, CXCR4 cancer
cells can enter circulation, chemotactically migrate towards distant stromal origins of CXCL12
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
secretion (typically the bone marrow, liver, lungs, and lymph nodes), and penetrate deep into the
target organ where they engraft into proꢀsurvival stromal compartments. Ultimately, stromal cell
+
secretion of CXCL12 is essential for 1) proꢀsurvival signaling within the primary CXCR4 tumor
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
microenvironment, 2) recruitment of CXCR4 immunosuppressive leukocytes and proꢀ
+
angiogenic cells to intratumoral stromal niches, and 3) CXCR4 cancer cell metastasis to distant
CXCL12ꢀrich tissues.
Figure 1. Potential CXCR4 mechanisms of action against cancer progression.
In principle therefore, small molecule CXCR4 antagonists can inhibit cancer progression via
1
2
several mechanisms of action (Figure 1). CXCR4 blockade can disrupt tumorꢀstromal
interactions that confer resistance to the host immune system and to antiꢀcancer agents by
2
0
obstructing autocrine and paracrine survival signaling mechanisms. CXCR4 antagonists can
ACS Paragon Plus Environment
6

Page 7 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
also combat cancer progression through inhibition of CXCR4 immunosuppressive leukocyte
1
6
migration, thereby reconditioning the tumor microenvironment in an immunomodulatory
manner that induces a higher susceptibility to immune elimination. Furthermore, inhibition of
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CXCR4 proꢀvasculogenic and proꢀangiogenic progenitor trafficking from bone marrow stromal
niches to the tumor microenvironment can prevent tumor cell access to systemic and lymphatic
2
1
vasculature. Moreover, CXCR4 antagonists have the potential to inhibit cancer metastasis to
distant CXCL12ꢀrich tissues, blocking one of the predominant factors that contributes to poor
22
+
patient prognosis across many cancer types. Finally, since metastatic CXCR4 tumor cells
commonly find sanctuary in bone marrow compartments where they evade chemotherapeutic
elimination, CXCR4 antagonists can disrupt local tumorꢀstromal interactions, mobilizing tumor
23
cells into circulation where they are accessible to cytotoxic drugs. Each of these mechanisms of
2
4
action has been highlighted in various preclinical models of cancer by AMD3100 (Mozobil,
25
26
Plerixafor, Figure 2), a potent and selective CXCR4 antagonist that is FDAꢀapproved for
autologous HSC mobilization and transplantation for nonꢀHodgkin’s lymphoma and multiple
myeloma. Some particularly notable preclinical antiꢀtumor effects of AMD3100 include delayed
development of malignant ascites and an increased overall survival window as a single agent in
1
6
immunocompetent murine models of ovarian cancer. These results were attributed to reduced
+
+
vessel density, increased apoptosis and necrosis, and 6ꢀfold higher CD8 :FoxP3 T lymphocyte
ratios in the tumor microenvironment, enhancing susceptibility to the host immune system.
Another example involved an immunocompetent model of hepatocellular carcinoma (HCC) and
combination therapy with an antibody against immune checkpoint programmed cell death
2
7
protein 1 (PDꢀ1). HCC standard of care sorafenib causes rapid clinical resistance, which begins
with the induction of hypoxia. Hypoxia, in turn, upregulates CXCR4, CXCL12, and immune
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
checkpoint programmed death ligand 1 (PDꢀL1), all of which promote immunosuppression and
chemotherapeutic evasion. AMD3100 in combination with sorafenib or with sorafenib and antiꢀ
PDꢀ1 decreased both tumor volume and the number of metastatic growths relative to sorafenib
alone or sorafenib and antiꢀPDꢀ1 combination. In particular, the triple combination paradigm
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
substantially improved both the CD8 cytotoxic T lymphocyte infiltrate and intratumoral
apoptotic activity, emphasizing the clinical potential of combination with immune checkpoint
inhibitors.
Figure 2. Nonꢀpeptide small molecule CXCR4 antagonists.
Despite these preclinical triumphs, the utility of AMD3100 in clinical oncology as an antiꢀ
tumor agent is somewhat limited due to doseꢀlimiting cardiotoxicity and lack of oral
2
4
bioavailability. A second generation CXCR4 antagonist AMD11070 (Figure 2) with no
2
8
cyclams was developed as an orally bioavailable antiꢀHIV treatment, but potent inhibition of
2
9
cytochrome P450 (CYP450) isoforms 2D6 and 3A4 and signs of hepatotoxicity in longꢀterm
3
0
animal studies ultimately prevented clinical continuation. Various AMD11070 analogs have
ACS Paragon Plus Environment
8

Page 9 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
31
since been described with particular focus on tetrahydroquinoline modifications, side chain
3
2
33
alterations, and benzimidazole substitutions, culminating in the discovery of advanced leads
3
4
such as GSK812397. Alternative scaffolds of small molecule CXCR4 antagonists include
3
5
36
37
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
isothioureas, the most characteristic of which is IT1t, cyclic peptides, and other chemotypes.
In contrast, our research group focused on modifying the benzimidazole motif of AMD11070,
3
8
39
replacing it with Nꢀsubstituted piperazines or with tetrahydroisoquinolines. These efforts led
to the discovery of TIQꢀ15, a highly potent (IC = 6.25 nM) and selective (< 10% inhibition of
5
0
all other chemokine receptors at 10 µM) small molecule CXCR4 antagonist that exhibits robust
metabolic stability in human liver microsomes (t1/2 = 1.0 h), strong plasma stability in rats (t1/2
=
6.0 h), and rapid mobilization of leukocytes that correlated with plasma drug concentrations in
mice. Despite this impressive activity profile, TIQꢀ15 potently inhibits CYP450 subtype 2D6
(
IC = 0.320 nM), limiting its utility in combination treatment paradigms, which are commonly
50
employed in clinical oncology. TIQꢀ15 is also rapidly metabolized in mouse liver microsomes
only 17% remaining after 10 min), hindering the evaluation of antiꢀtumor efficacy after oral
(
dosing regimens in preclinical mouse models of cancer. Furthermore, despite moderate oral
bioavailability in rats, systemic exposure of TIQꢀ15 after oral administration undoubtedly
remains limited by the presence of multiple basic amines. Herein, we describe the design,
synthesis, and pharmacological evaluation of TIQꢀ15 analogs with enhanced CYP450
2
D6:CXCR4 IC ratios, improved passive membrane permeability, and elevated mouse liver
50
microsomal stability.
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RESULTS
Figure 3. Design principles of TIQꢀ15 analogs.
ACS Paragon Plus Environment
10

Page 11 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
To surmount the indicated shortcomings of TIQꢀ15, a series of conformationally restricted
TIQꢀ15 derivatives with decreased basicity and/or increased lipophilicity were designed (Figure
3). While improving basicity and lipophilicity parameters was expected to increase intestinal
permeability, rigidifying the butylamine side chain was anticipated to maximize CXCR4
specificity and to limit CYP450 2D6 inhibition and mouse metabolism. These design principles
led to the conception of TIQꢀ15 analogs bearing benzylic amine (1), allylic amine (2), or gemꢀ
dimethylbutylamine (3ꢀ5) side chains, as well as carbocyclic or heterocyclic amine
functionalization. Synthesis of these compounds was envisioned to be conducted in a modular
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
9
fashion, quite similar to the previously described preparation of TIQꢀ15 analogs, which utilized
butylamine 6 as a common synthetic intermediate (Scheme 1).
a
Scheme 1. Arylmethyl functionalization of the butylamine side chain
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Reagents and Conditions: a) C H NCHO or PhCHO, Na(OAc) BH, DCM, rt, 24 h, 26ꢀ68%
5
5
3
yield; b) TFA, DCM, rt, 24 h, 67ꢀ88% yield; c) C H NCHO, Na(OAc) BH, DCM, rt, 5 h; d)
BOC O, DMAP, DIPEA, DCM, rt, 6 h, 78ꢀ79% yield over 2 steps; e) TFA, DCM, rt, 24 h, 14ꢀ
4% yield; f) PhCHO, Ti(OiPr) , NaBH , DCM, MeOH, rt, 4 h, 44% yield; g) TFA, DCM, rt, 24
h, 20% yield.
5
5
3
2
2
4 4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Sodium triacetoxyborohydride (STAB)ꢀmediated reductive aminations involving amine 6 and
benzaldehyde or 2ꢀ, 3ꢀ, or 4ꢀpyridinecarboxaldehydes were anticipated to deliver the monoꢀ
substituted analogs after BOC group removal. In contrast, the corresponding bis(pyridylmethyl)
(
7aꢀc) and dibenzyl (7d) analogs predominated over the desired monoꢀsubstituted derivatives,
even when only 0.95 eq of aldehyde were added, indicating that the monoꢀsubstituted products
retained comparable reactivity to the unsubstituted starting material. Although the disubstituted
compounds were undesired byproducts, they were still considered useful probes to investigate
the CXCR4 binding pocket that interacts with the butylamine side chain of TIQꢀ15. While
STABꢀmediated reductive amination of butylamine 6 and pyridinecarboxaldehydes favored
disubstituted over monoꢀsubstituted products, column chromatography provided ample quantities
of pure disubstituted products for biological evaluation, as well as smaller quantities of difficult
to separate mixtures (~2:1) of monoꢀ to bis(pyridylmethyl) congeners. This permitted selective
masking of the only remaining nucleophilic amines via DMAPꢀcatalyzed BOC protection, which
enabled chromatographic purification of the desired monoꢀpyridylmethyl BOCꢀprotected amines.
BOC protecting groups were subsequently removed using TFA, which cleanly provided the
40
desired monoꢀpyridylmethyl TIQꢀ15 analogs 8aꢀc . While this BOC protection strategy
delivered the desired compounds in the face of unexpected overreactivity during STABꢀmediated
reductive amination, this was clearly a problem that needed to be addressed for future analogs. A
particularly attractive option was to first preform the imine intermediate and subsequently add a
4
1
reducing agent. Accordingly, butylamine 6 was stirred with benzaldehyde and titanium
isopropoxide to ensure complete imine formation, followed by addition of sodium borohydride
ACS Paragon Plus Environment
12

Page 13 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
to rapidly reduce both the imine and any remaining benzaldehyde to prevent overreactivity. This
protocol facilitated isolation of the desired monoꢀbenzyl product, which was then subjected to
BOC deprotection conditions, generating the desired monoꢀbenzyl TIQꢀ15 analog 9. This
titanium isopropoxide/sodium borohydride procedure was additionally applied toward the
syntheses of carbocyclic and heterocyclic TIQꢀ15 derivatives (Scheme 2). Butylamine 6
underwent titanium isopropoxide/sodium borohydrideꢀmediated reductive amination with
aldehydes 10aꢀf, producing the corresponding monoꢀsubstituted products without detection of
any undesired disubstituted byproducts. These intermediates were then treated with TFA to
remove the BOC protecting groups, delivering the desired monoꢀsubstituted TIQꢀ15 analogs
11aꢀf. Finally, reductive amination between butylamine 6 and ketones 12aꢀg did not require the
titanium isopropoxide/sodium borohydride protocol, but was rather afforded using STAB with
acetic acid as an additive, generating the intermediate monoꢀsubstituted products. Typical BOC
deprotection was carried out to yield the resulting monoꢀsubstituted TIQꢀ15 analogs 13aꢀg,
thereby furnishing this series of substitutedꢀbutylamine derivatives of TIQꢀ15.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 2. Saturated cyclic functionalization of the butylamine side chain
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: a) 10, Ti(OiPr) , NaBH , DCM, MeOH, rt, 4 h, 27ꢀ62% yield; b)
4
4
TFA, DCM, rt, 24 h, 42ꢀ98% yield; c) 12, Na(OAc) BH, AcOH, DCM, rt, 24 h, 40ꢀ55% yield; d)
TFA, DCM, rt, 24 h, 19ꢀ98% yield.
3
a
Scheme 3. Synthesis of benzylic amine side chain analogs
ACS Paragon Plus Environment
14

Page 15 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: a) 2ꢀCyanobenzaldehyde, Na(OAc) BH, DCM, rt, 4 h, 64% yield;
3
b) NiCl ꢀ(H O) , NaBH , BOC O, NH(CH CH NH ) , MeOH, 0˚C to rt, 96 h, 26% yield; c)
2
2
6
4
2
2
2
2 2
TFA, DCM, rt, 24 h, 62% yield; d) BH ꢀMe S, DCM, rt, 24 h, 49ꢀ74% yield; e) DessꢀMartin
3
2
periodinane, DCM, rt, 2.5 h; f) 14, Na(OAc) BH, AcOH, DCM, rt, 24 h, 51ꢀ55% yield over 2
3
steps; g) TFA, DCM, rt, 24 h, 49ꢀ65% yield.
3
9
Secondary amine 14 was used as the common starting material for the syntheses of benzylic
and allylic amine side chain analogs. First, secondary amine 14 underwent STABꢀmediated
reductive amination with 2ꢀcyanobenzaldehyde to produce the corresponding nitrile 15 in 64%
yield (Scheme 3a). The nitrile was subsequently reduced using nickel(II) chloride hexahydrate
4
2
and sodium borohydride, and the forming amine was trapped in situ with BOC O. The resulting
2
intermediate was deprotected using TFA to deliver the desired orthoꢀbenzylic amine side chain
analog 16 in 62% yield. Alternatively, the metaꢀ and paraꢀbenzylic amine side chain derivatives
1
9a and 19b were prepared using a different approach (Scheme 3b). Commercially available
43
carboxylic acids 17a and 17b were reduced to the corresponding alcohols 18a and 18b using
44
boraneꢀdimethylsulfide complex, followed by DessꢀMartin oxidation of the resulting alcohols
to the analogous aldehydes. With the metaꢀ and paraꢀsubstituted benzaldehyde derivatives in
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hand, each of them was subjected to STABꢀmediated reductive amination with secondary amine
1
4 using an acetic acid additive to yield the desired bisꢀBOCꢀprotected intermediates. BOC
group removal using TFA generated metaꢀ and paraꢀbenzylic amine congeners 19a and 19b,
respectively. To prepare the cisꢀallylic and transꢀallylic amine side chain analogs of TIQꢀ15,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
32
45
secondary amine 14 was reacted with cisꢀallylic chloride 20 or transꢀallylic bromide 22 in
3
2
SN2 fashion under Finkelsteinꢀlike conditions to generate the corresponding tertiary amines
Scheme 4). The BOC protecting groups of the resulting cisꢀallylic amine were removed using
(
TFA to deliver derivative 21 in 59% yield. Alternatively, the amines of the transꢀallylic
intermediate were differentially protected as phthalimide and BOC. Accordingly, the phthalimide
group was removed using aqueous hydrazine, generating allylic amine 23, followed by BOC
group deprotection using TFA to afford transꢀallylic amine analog 24.
a
Scheme 4. Synthesis of allylic amine side chain analogs
ACS Paragon Plus Environment
16

Page 17 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: a) 20, KI, DIPEA, MeCN, 50˚C, 72 h, 69% yield; b) TFA, DCM, rt,
2
4 h, 59% yield; c) 22, KI, DIPEA, 50˚C, 24 h, 51% yield; d) NH ꢀNH (aq), MeOH, rt, 24 h,
2 2
7
6% yield; e) TFA, DCM, rt, 24 h, 25% yield.
To prepare the 2,2ꢀdimethylbutyl side chain analog, lactone 25 preliminarily underwent
4
6
trimethylaluminumꢀmediated ring opening with 1ꢀoctanethiol (Scheme 5a), followed by
4
7
Mitsunobu reaction of the resulting alcohol with nucleophilic phthalimide to yield desired
48
intermediate 26. Fukuyama reduction of the thioester to the corresponding aldehyde proceeded
cleanly, and subsequent STABꢀmediated reductive amination with amine 27 generated
anticipated secondary amine 28 in high yield. Ensuing reductive amination with aldehyde 29
using titanium isopropoxide and STAB furnished the differentially protected scaffold of the 2,2ꢀ
dimethylbutyl side chain analog. Phthalimide deprotection with aqueous hydrazine, followed by
TFAꢀmediated BOC group removal, yielded derivative 30. To prepare the 3,3ꢀdimethylbutyl side
chain analog, the least stericallyꢀencumbered alcohol of 2,2ꢀdimethylbutaneꢀ1,4ꢀdiol was
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
49
selectively silyl protected in 67% isolated yield (Scheme 5b). The remaining alcohol was
4
7
subjected to Mitsunobu reaction using phthalimide as a nucleophile, which was followed by
silyl group removal using aqueous hydrochloric acid to generate desired intermediate 32. Sulfur
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
trioxideꢀpyridine complex was subsequently employed in a ParikhꢀDoering oxidation to deliver
the corresponding aldehyde, which was then subjected to reductive amination involving
secondary amine 14 and STAB, followed by aqueous hydrazineꢀmediated phthalimide
deprotection to yield the desired primary amine 33. Finally, analog 34 was afforded via removal
of the BOC protecting group with TFA. Reduction of nitropentanoate 35 via Raney Nickelꢀ
catalyzed hydrogenolysis generated the corresponding amine in situ, which spontaneously
5
1
cyclized onto the ester to deliver the related pyrrolidinone (Scheme 5c). BOC protection of this
γꢀlactam furnished desired intermediate 36. DIBALHꢀmediated amide reduction to
corresponding hemiaminal proceeded cleanly in 71% isolated yield. Subsequent STABꢀmediated
reductive amination with secondary amine 14 delivered the resulting bisꢀBOC protected scaffold,
which was finally deprotected with TFA to yield the desired 4,4ꢀdimethylbutylamine 37.
a
Scheme 5. Synthesis of gemꢀdimethylbutylamine side chain analogs
ACS Paragon Plus Environment
18

Page 19 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: a) HSC H , Me Al, DCM, 0˚C to rt, 12 h, 66% yield; b) PPh ,
8
17
3
3
DIAD, phthalimide, THF, rt, 2.5 h, 93% yield; c) PdCl , Et SiH, Et N, DCM, rt, 20 min, 86%
2
3
3
yield; d) 27, Na(OAc) BH, DCE, rt, 1 h, 99% yield; e) 29, Ti(OiPr) , Na(OAc) BH, DCM, rt, 12
3
4
3
h, 56% yield; f) NH ꢀNH (aq), MeOH, rt, 12 h; g) TFA, DCM, rt, 24 h, 85% over 2 steps; h)
2
2
TBSCl, imidazole, DMF, 0˚C to rt, 1 h, 67% yield; i) PPh , DIAD, phthalimide, THF, rt, 36 h; j)
3
HCl (aq), THF, rt, 2 h, 84% yield over 2 steps; k) SO ꢀC H N, Et N, DCM, DMSO, 0˚C, 3 h,
3
5
5
3
77% yield; l) 14, Na(OAc) BH, AcOH, DCE, rt, 12 h, 81% yield; m) NH ꢀNH (aq), MeOH, rt,
3
2
2
24 h; n) TFA, DCM, rt, 24 h, 91% yield over 2 steps; o) Raney Ni, H , EtOH, rt, 18 h, 88%
2
yield; p) BOC O, DMAP, Et N, DCM, 18 h, 40˚C, 82% yield; q) DIBALH, Et O, ꢀ78˚C to rt, 6
2
3
2
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
h, 71% yield; r) 14, Na(OAc) BH, AcOH, DCM, rt, 60 h, 30% yield; s) TFA, DCM, rt, 24 h,
3
3
6% yield.
Table 1. Activity profiles of monoꢀ and bis(arylmethyl)ꢀsubstituted butylamines
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CXCR4 mAChR
Ca Flux Ca Flux
a
CYP450 Metabolic Stability
+
2
+2
PAMPA (nm/s)
b
b
1
2
IC (µM)
(% Remaining)
ID
R
R
50
a
b
IC (nM) IC (µM) pH = 7.4 pH = 5.5 2D6 3A4 Human
Mouse
17.0
5
0
50
b
b
TIQ-15
H
H
6.25 ± 2.05
> 30.0
0
6.00
0.320 > 20.0
77.0
0.548
0.432
0.521
4.84
7
7
a
b
2ꢀPyridylmethyl 2ꢀPyridylmethyl 664 ± 123
3ꢀPyridylmethyl 3ꢀPyridylmethyl 471 ± 190
12.9
~11.5
11.4
294 ± 128 48.0 ± 7.00 1.16
435 ± 14.0 91.0 ± 23.0 0.348
6.01
1.29
0.374
1.40
7c
4ꢀPyridylmethyl 4ꢀPyridylmethyl 832 ± 522
774 ± 303 180 ± 7.00 0.120 0.622
1.55
b
7
8
8
d
a
b
Benzyl
Benzyl
> 16,700
> 33.3
> 30.0
~19.9
> 33.3
~3.43
ND
39.0 ± 51.0 0.494
2.24
4.50
8.45
4.50
6.15
0.298
8.60
b
b
H
H
H
H
2ꢀPyridylmethyl 89.5 ± 24.8
109
0
58.0
0
0.350
0.217
26.0
3ꢀPyridylmethyl 15.3 ± 5.00
21.4
10.2
b
8c
9
4ꢀPyridylmethyl
Benzyl
22.7
0
6.50 ± 5.00 0.145
0.141
1.92
6.44
20.1 ± 10.5
ND
0
71.7
31.2
a
b
n=2; n=1; ND = not determined; Reported error represents standard deviation.
Each of these final compounds was evaluated in vitro for CXCR4 inhibition using a
+
2
52
fluorometric Ca flux assay, for intestinal permeability via parallel artificial membrane
5
3
permeability assay (PAMPA), for CYP450 isoform inhibition using a fluorescenceꢀbased
54
assay, and for metabolic stability via human and mouse liver microsome assays using liquid
5
5
chromatography tandemꢀmass spectrometry (LCꢀMS/MS) quantification.
Muscarinic
acetylcholine receptors (mAChR) were considered surrogates for offꢀtarget activity due to
previously observed CXCR4 antagonist promiscuity at these receptors (unpublished data), and
+2
inhibition was measured using fluorometric Ca flux assays. Table 1 includes the results
obtained for the monoꢀ and bis(arylmethyl) substituted analogs. Amongst this initial series of
TIQꢀ15 derivatives, monoꢀ3ꢀpyridylmethyl (8b, IC = 15.3 ± 5.00 nM), monoꢀ4ꢀpyridylmethyl
50
ACS Paragon Plus Environment
20

Page 21 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
8c, IC = 22.7 nM), and monoꢀbenzyl (9, IC = 20.1 ± 10.5 nM) analogs demonstrated the
5
0
50
most potent inhibition of CXCR4, while bis(pyridylmethyl) analogs were much less potent (471
± 190 nM ≤ IC ≤ 832 ± 522 nM), and dibenzyl congener 7d was completely inactive. None of
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
these compounds strongly inhibited mAChR activity (IC > 10 µM) except for the monoꢀbenzyl
5
0
analog, which still proved relatively inactive (9, IC ≈ 3.43 µM). Although the monoꢀsubstituted
50
derivatives poorly permeated artificial intestinal membranes, disubstituted amines were
substantially more permeable, likely due to increased lipophilicity and decreased amine basicity.
None of the monoꢀarylmethyl derivatizations of the TIQꢀ15 butylamine improved the CYP450
profile, each potently inhibiting the 2D6 isoform with IC values in the nanomolar range and
5
0
picking up micromolar activity against the 3A4 isoform. Perhaps not surprisingly, the
disubstituted analogs not only retained potent 2D6 and modest 3A4 inhibition, but also
introduced antagonist activity at several other CYP450 isoforms (unpublished data) and suffered
from extremely rapid metabolism by both human and mouse liver microsomes. Although monoꢀ
benzyl congener 9 demonstrated reasonable metabolic stability for both species relative to TIQꢀ
1
5, the other monoꢀsubstituted analogs were metabolized much more rapidly. Despite suboptimal
PAMPA, CYP450, and metabolic properties, monoꢀpyridylmethyl functionalization produced
the most interesting and attractive activity profiles relative to other compounds within this series,
marking them for further investigation with alternative side chains.
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Activity profiles of saturated carbocyclic and heterocyclic substituted butylamines
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CXCR4 mAChR
Ca Flux Ca Flux
a
CYP450 Metabolic Stability
+
2
+2
PAMPA (nm/s)
b
b
IC (µM)
(% Remaining)
ID
X
n
50
a
b
IC (nM) IC (µM) pH = 7.4 pH = 5.5 2D6 3A4 Human
Mouse
17.0
41.8
37.7
48.5
37.9
2.82
39.3
43.8
34.8
55.8
38.0
23.2
17.6
50.0
5
0
50
b
b
TIQ-15 NA
1a
CH
NA 6.25 ± 2.05
> 30.0
0
6.00
0.320 > 20.0
77.0
50.0
49.2
57.6
84.6
49.4
74.0
46.1
57.2
53.0
100
1
2
1
1
1
1
1
1
0
0
0
0
0
0
0
158 ± 22.8
168 ± 83.1
52.9 ± 45.0
> 16.7
> 16.7
> 33.3
> 16.7
> 16.7
> 16.7
> 33.3
~28.2
> 33.3
> 33.3
> 33.3
> 33.3
~19.9
9.00 ± 13.0 21.0 ± 7.00 0.354 2.78
11b
11c
CF
2
ND
0
ND
0.538 19.2
O
6.00 ± 8.00 2.72 > 20.0
b
1
1d
1e
NH
S
36.5
ND
ND
ND
ND
0
ND
0
2.53 > 20.0
0.629 > 20.0
1.17 > 20.0
1
69.6 ± 2.78
17.0 ± 13.4
75.2 ± 53.9
3.71 ± 0.620
29.4 ± 16.5
63.4 ± 48.7
54.7 ± 1.93
45.5 ± 28.8
1
1f
SO
2
13a
CH
2
14.5 ± 11.0 0.524 11.7
1
1
1
3b
3c
3d
CF
2
32.0 ± 9.00 17.0 ± 9.00 0.189 11.3
O
0
0
0.657 > 20.0
NH
S
ND
19.0 ± 26.0 2.61 > 20.0
13e
3f
3g C(CH
2.00 ± 3.00 46.0 ± 17.0 0.434 > 20.0
39.9
11.7
30.1
1
SO
2
0
0
0
0.853 > 20.0
0.090 12.9
b
1
3
)
2
77.6
3.00 ± 4.00
a
b
n=2; n=1; ND = not determined; Reported error represents standard deviation.
Activity profiles of final compounds containing saturated carbocyclic and heterocyclic
substituted butylamines are outlined in Table 2. All TIQꢀ15 analogs in this second series
demonstrated poor passive permeability, as well as virtual inactivity at mAChR. In contrast, each
of these congeners potently inhibited CXCR4 activity (IC50 < 100 nM), except for
cyclohexylmethyl (11a, IC = 158 ± 22.8 nM) and difluorocyclohexylmethyl (11b, IC = 168 ±
50
50
8
3.1 nM) derivatives. Notably, difluorocyclohexyl analog 13b and sulfonyl analog 11f were the
only compounds with comparable CXCR4 potency (IC = 3.71 ± 0.620 nM and 17.0 ± 13.4 nM,
5
0
respectively) to TIQꢀ15 (IC50 = 6.25 ± 2.05 nM). All other derivatives amongst this series
exhibited relatively similar CXCR4 antagonist activity (29.4 ± 16.5 nM ≤ IC50 ≤ 77.6 nM).
ACS Paragon Plus Environment
22

Page 23 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Attractively, none of these congeners inhibited CYP450 1A4, 2C19, 2C8, or 2C9 isoforms
(
unpublished data), nor did any of them substantially inhibit the 3A4 isoform (IC values > 10
50
µM), except for cyclohexylmethyl analog 11a (IC50 = 2.78 µM). Although each of the
compounds retained antagonist activity against the 2D6 isoform, piperidinyl (13d),
piperidinylmethyl (11d), and tetrahydropyranylmethyl (11c) modifications in particular limited
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
this activity (2.53 µM ≤ IC ≤ 2.72 µM). Furthermore, none of these compounds demonstrated
50
metabolic liabilities on the order of the arylmethyl analogs. Rather, metabolic stability was
considerably improved across the board, perhaps with the exception of sulfonyl analog 13f,
which was metabolized relatively extensively. While piperidinyl and piperidinylmethyl
derivatives 13d and 11d were particularly resistant to human microsomal metabolism,
tetrahydropyranyl (13c), tetrahydropyranylmethyl (11c), and dimethylcyclohexyl (13g)
congeners were the least susceptible to mouse microsomal metabolism relative to the rest of the
compounds in this series. Although piperidinyl and piperidinylmethyl functionalization of the
butylamine resulted in reasonably attractive activity profiles, each of these modifications
introduces an additional basic amine, which would likely make robust oral bioavailability
extremely difficult to achieve. Accordingly, these compounds represent important data points,
but were not marked for further investigation. In contrast, difluorocyclohexyl (13b, most potent
against CXCR4), dimethylcyclohexyl (13g, good mouse metabolic stability), tetrahydropyranyl
(13c, good mouse metabolic stability), and tetrahydropyranylmethyl (11c, good mouse metabolic
stability and most optimal 2D6 activity) modifications warranted further exploration with
alternative side chains due to relatively attractive full activity profiles with particular emphasis
on the indicated properties.
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Activity profiles of TIQꢀ15 amine side chain analogs
CXCR4 mAChR
Ca Flux Ca Flux
a
CYP450 Metabolic Stability
PAMPA (nm/s)
b
b
Amine
Side Chain
+2
+2
IC (µM) (% Remaining)
ID
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
IC (nM) IC (µM) pH = 7.4 pH = 5.5 2D6 3A4 Human
Mouse
17.0
1.29
89.2
94.2
4.36
10.7
5.22
51.2
3.32
50
50
b
b
TIQ-15
Butyl
6.25 ± 2.05
> 30.0
0
6.00
214 ± 1.00 184 ± 41.5 0.318 15.2
ND ND 0.032 4.63
301 ± 22.5 337 ± 61.0 0.048 7.44
0.320 > 20.0
77.0
50.5
97.3
100
1
6
orthoꢀBenzylic
metaꢀBenzylic
paraꢀBenzylic
cisꢀAllylic
50.8 ± 37.1
292 ± 82.9
115 ± 85.9
1.20
~25.7
> 33.3
1
1
9a
9b
b
a
21
9.26
0.605 ± 0.287 569 ± 129 39.0 ± 5.00 0.333 12.7
100
2
3
3
4
0
4
transꢀAllylic
129 ± 109
> 16.7
> 33.3
> 16.7
> 16.7
829 ± 100 264 ± 27.0 0.510 9.29
255 ± 106 268 ± 95.0 0.197 10.8
100
2,2ꢀDimethylbutyl > 33,300
3,3ꢀDimethylbutyl 69.0 ± 54.4
76.1
88.7
73.5
405 ± 144
0
0
0.498 > 20.0
37 4,4ꢀDimethylbutyl 24.6 ± 8.46
20.0 ± 28.0 0.345 > 20.0
a
b
n=2; n=1; ND = not determined; Reported error represents standard deviation.
Activity profiles of the TIQꢀ15 side chain analogs are presented in Table 3. This series of
compounds retained moderately potent CXCR4 antagonist activity (50.8 ± 37.1 nM ≤ IC ≤ 129
5
0
±
109 nM), with the exceptions of 2,2ꢀdimethylbutyl analog 30 (IC > 33.3 µM) and the metaꢀ
50
benzylic amine 19a (IC = 292 ± 82.9 nM), which were particularly weak inhibitors, as well as
5
0
cisꢀallylic amine 21 and 4,4ꢀdimethylbutyl derivative 37, which were particularly potent (IC =
5
0
9
.26 nM and 24.6 ± 8.46 nM, respectively). All of these congeners, except for cisꢀallylic amine
21 (IC = 0.605 ± 0.287 nM) and orthoꢀbenzylic amine 16 (IC = 1.20 µM), were virtually
5
0
50
inactive at mAChR (IC50 ≥ 16.7 µM). Notably, intestinal permeability was dramatically
improved across the board, with the interesting exception of 4,4ꢀdimethylbutyl congener 37,
which marked only a marginal improvement over TIQꢀ15 passive membrane diffusion. Although
potent inhibition of the CYP450 2D6 isoform was demonstrated by all of the compounds within
this series, only analogs containing benzylic amine side chains inhibited other CYP450 isoforms
(unpublished data). Inhibition of the 3A4 isoform was variable across the series, but only metaꢀ
benzylic amine 19a (IC = 4.63 µM) exhibited < 5 µM antagonist activity. Furthermore, each of
5
0
ACS Paragon Plus Environment
24

Page 25 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the compounds amongst this series showed robust human metabolic stability, the most
susceptible of which was orthoꢀbenzylic amine 16 (50.5% remaining after 10 min). In stark
contrast, mouse microsomal stability was particularly poor (< 10% remaining after 10 min) for
the cisꢀallylic amine (21), orthoꢀbenzylic amine (16), 4,4ꢀdimethylbutyl (37), and 2,2ꢀ
dimethylbutyl (30) side chain derivatives. Although transꢀallylic amine 24 was only modestly
more stable in mouse liver microsomes than these compounds, 3,3ꢀdimethylbutyl congener 34,
as well as the metaꢀ and paraꢀbenzylic amines 19a and 19b were quite stable in comparison.
Ultimately, the paraꢀbenzylic amine (19b), transꢀallylic amine (24), and 3,3ꢀdimethylbutylamine
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(34) side chains elicited more attractive full activity profiles than cisꢀallylic amine 21, as well as
the other gemꢀdimethylbutyl and benzylic amine side chain analogs, marking these three
particular side chains to be incorporated in subsequent series of TIQꢀ15 analogs.
a
Scheme 6. Preparation of the paraꢀbenzylic amine side chain scaffold
a
Reagents and Conditions: a) 39, Et N, THF, rt, 24 h; b) DessꢀMartin periodinane, DCM, rt, 3
3
h; c) 14, Na(OAc) BH, DCM, rt, 24 h, 65% yield over 3 steps; d) NH ꢀNH (aq), MeOH, rt, 24 h,
3
2
2
5
5% yield.
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 7. Functionalization of the paraꢀbenzylic amine side chain
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: a) 10c, Ti(OiPr) , NaBH , DCM, MeOH, rt, 4 h, 37% yield; b) TFA,
DCM, rt, 24 h, 88% yield; c) 12b, 12c, or 12g, Na(OAc) BH, AcOH, DCM, rt, 24 h, 59ꢀ84%
yield; d) TFA, DCM, rt, 24 h, 71ꢀ87% yield; e) C H NCHO, Ti(OiPr) , NaBH , DCM, MeOH,
4
4
3
5
5
4
4
rt, 4 h, 61ꢀ65% yield; f) TFA, DCM, rt, 24 h, 70ꢀ92% yield.
At this point, three arylmethyl substitutions (2ꢀ, 3ꢀ, and 4ꢀmonoꢀpyridylmethyl), four saturated
carbocyclic and heterocyclic modifications (difluorocyclohexyl, dimethylcyclohexyl,
tetrahydropyranyl, and tetrahydropyranylmethyl), and three side chains (paraꢀbenzylic amine,
transꢀallylic amine, and 3,3ꢀdimethylbutylamine) were selected as the subject of second
generation investigation. Accordingly, amine 38 and phthalimide 39 underwent phthalimide
5
6
44
transfer reaction (Scheme 6), which was followed by DessꢀMartin oxidation of the remaining
benzylic alcohol to corresponding aldehyde 40. Subsequent reductive amination with secondary
ACS Paragon Plus Environment
26

Page 27 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
amine 14 and STAB furnished the differentially protected scaffold of the paraꢀbenzylic amine
side chain analog of TIQꢀ15. Subsequent phthalimide protecting group removal using aqueous
hydrazine delivered corresponding primary amine 41, which was primed for selective
functionalization of the benzylic amine. Amine 41 and aldehyde 10c were then subjected to
reductive amination using titanium isopropoxide and sodium borohydride to yield the
corresponding monoꢀadduct, which was subsequently deprotected with TFA to deliver the
desired tetrahydropyranylmethyl benzylic amine 42 (Scheme 7). Alternatively, amine 41
underwent STABꢀmediated reductive amination with difluorocyclohexanone 12b,
tetrahydropyranone 12c, and dimethylcyclohexanone 12g, followed by TFAꢀmediated BOC
deprotection to furnish the corresponding substituted benzylic amines 43b, 43c, and 43g.
Benzylic amine 41 and 2ꢀ, 3ꢀ, and 4ꢀpyridinecarboxaldehydes were also subjected to titanium
isopropoxide and sodium borohydride reductive amination conditions to generate solely the
corresponding monoꢀadducts, which were then deprotected using TFA to afford the desired
bis(arylmethyl) amines 44aꢀc. In parallel, second generation substituted transꢀallylic amines
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Scheme 8) and substituted 3,3ꢀdimethylbutylamines (Scheme 9) were prepared in similar
fashion. All final compounds were pharmacologically evaluated according to the previously
described assay paradigm, and activity profiles of these second generation TIQꢀ15 analogs are
presented in Table 4.
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 8. Functionalization of the transꢀallylic amine side chain
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: a) 10c, Ti(OiPr) , NaBH , DCM, MeOH, rt, 4 h, 40% yield; b) TFA,
DCM, rt, 24 h, 75% yield; c) 12b, 12c, or 12g, Na(OAc) BH, AcOH, DCM, rt, 24 h, 51ꢀ63%
yield; d) TFA, DCM, rt, 24 h, 19ꢀ98% yield; e) C H NCHO, Ti(OiPr) , NaBH , DCM, MeOH,
4
4
3
5
5
4
4
rt, 4 h, 54ꢀ63% yield; f) TFA, DCM, rt, 24 h, 70ꢀ92% yield.
ACS Paragon Plus Environment
28

Page 29 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Scheme 9. Functionalization of the 3,3ꢀdimethylbutylamine side chain
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: a) 10c, Ti(OiPr) , NaBH , DCM, MeOH, rt, 4 h, 73% yield; b) TFA,
DCM, rt, 24 h, 74% yield; c) 12b, 12c, or 12g, Na(OAc) BH, AcOH, DCM, rt, 24 h, 54ꢀ60%
4
4
3
yield; d) TFA, DCM, rt, 24 h, 96ꢀ98% yield.
ACS Paragon Plus Environment
29