Discovery of IACS-9439, a Potent, Exquisitely Selective, and Orally Bioavailable Inhibitor of CSF1R

Article abstract of DOI:10.1021/acs.jmedchem.0c00936

Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.

Full text of DOI:10.1021/acs.jmedchem.0c00936

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Article
Discovery of IACS-9439, a potent, exquisitely
selective and orally bioavailable inhibitor of CSF1R
Barbara Czako, Joseph R Marszalek, Jason P Burke, Pijus K Mandal, Paul G. Leonard, Jason B Cross, Faika
Mseeh, Yongying Jiang, Edward Q Chang, Erika Suzuki, Kovacs J Jeffrey, Ningping Feng, Sonal Gera, Angela
L Harris, Zhen Liu, Robert A Mullinax, Jihai Pang, Connor A Parker, Nakia D Spencer, Simon S Yu, Qi Wu,
Martin M Tremblay, Keith Mikule, Keith Wilcoxen, Timothy P Heffernan, Giulio Draetta, and Philip Jones
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00936 • Publication Date (Web): 29 Jul 2020
Downloaded from pubs.acs.org on August 2, 2020
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Tremblay, Martin; Tesaro Inc
Mikule, Keith; Tesaro Inc
Wilcoxen, Keith; Tesaro Inc
Heffernan, Timothy; University of Texas MD Anderson Cancer Center,
TRACTION
Draetta, Giulio; University of Texas MD Anderson Cancer Center,
Department of Genomic Medicine
Jones, Philip; University of Texas MD Anderson Cancer Center, Institute
of Applied Cancer Science
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Discovery of IACS-9439, a potent, exquisitely
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selective and orally bioavailable inhibitor of CSF1R
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Barbara Czako* , Joseph. R. Marszalek , Jason P. Burke , Pijus Mandal , Paul G. Leonard ,
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Jason B. Cross , Faika Mseeh , Yongying Jiang , Edward Q. Chang , Erika Suzuki , Jeffrey J.
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Kovacs , Ningping Feng , Sonal Gera , Angela L. Harris , Zhen Liu , Robert A. Mullinax , Jihai
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Pang , Connor A. Parker , Nakia D. Spencer , Simon S. Yu , Qi Wu , Martin R. Tremblay , Keith
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Mikule , Keith Wilcoxen , Timothy P. Heffernan , Giulio F. Draetta and Philip Jones
1IACS (Institute of Applied Cancer Science), University of Texas, MD Anderson Cancer
Center, 1881 East Road, Houston, TX, 77054 USA
2TRACTION (Translational Research to AdvanCe Therapeutics and Innovation in Oncology)
University of Texas, MD Anderson Cancer Center, 1881 East Road, Houston, TX, 77054 USA
3Tesaro Inc., 1000 Winter Street, Waltham, MA, 02451 USA
4Department of Genomic Medicine, The University of Texas MD Anderson Cancer
Center, Houston, Texas, 77030, USA.
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Keywords: CSF1, CSF1R, CSF1R inhibitor, colony-stimulating factor 1, immune-oncology,
macrophage, tumor associated macrophage, TAM
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ABSTRACT
Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma
across multiple human malignancies and are believed to be beneficial to tumor growth.
Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially
the pro-tumor, immune-suppressive M2 TAMs. Additionally, high expression of CSF1R
on tumor cells has been associated with poor survival in certain cancers, suggesting
tumor dependency and therefore a potential therapeutic target. The CSF1–CSF1R
signaling pathway modulates the production, differentiation and function of TAMs;
however, the discovery of selective CSF1R inhibitors devoid of Type III kinase activity
has proven to be challenging. We discovered a potent, highly selective and orally
bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose dependent
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reduction in macrophages, promoted macrophage polarization toward the M1 phenotype,
and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.
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INTRODUCTION
Strategies to enhance anti-tumor immunity by reactivating the adaptive and innate
immune compartments through checkpoint inhibitors against programmed cell death 1
(PD1), PD1 ligand (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) have shown
favorable clinical responses, yet, only a fraction of patients show durable responses.
While tumor-intrinsic resistance mechanisms may exist, clinical and preclinical evidences
highlight the abundance of tumor-associated macrophages (TAMs) as critical regulatory
immune cells that promote tumor progression. Macrophages exist primarily in two main
polarization states with the alternatively activated M2 TAM responsible for promoting
tumor progression by secreting anti-inflammatory cytokines such as IL-10 and TGF,
whereas the classically activated M1 promote immune-mediated tumor killing through the
production of pro-inflammatory cytokines such as IL-1, IL-6 and tumor necrosis factor 
(TNF-).1-3 To overcome the immunosuppressive and pro-tumoral functions of TAMs,
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therapeutic strategies have focused on TAM depletion in the tumor microenvironment as
well as TAM reprogramming to favor anti-tumoral functions (polarization from M2 to M1).4,
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Colony-stimulating factor 1 receptor (also known as CSF1R, or macrophage colony-
stimulating factor receptor, M-CSFR) is a membrane-associated tyrosine kinase. It acts
as the receptor for colony-stimulating factor 1 (CSF1), a cytokine which controls the
production, differentiation and function of macrophages. Ligand binding activates CSF1R
through a process of oligomerization within the membrane, transphosphorylation of the
intracellular domain, and subsequent signaling.6
In several preclinical studies using murine breast cancer and glioblastoma models, it
was demonstrated that CSF1–CSF1R signaling blockade slowed primary tumor growth,
reduced metastatic potential and improved the long-term survival of tumor-bearing mice.7,
8,
Treatmentwith BLZ945, a potent CSF1R inhibitor, attenuated tumor growth, correlated
with decreased TAM presence, and enriched CD8+ T cells in tumor stroma in a murine
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K14-HPV-16 transgenic mouse model of cervical carcinogenesis. These findings have
been translated into a first-in-human Phase I/II study of BLZ945 alone or in combination
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with PDR001, a monoclonal antibody against PD-1 in advanced solid tumors
(
NCT02829723).10,11 Pexidartinib (PLX3397), an unselective CSF1R inhibitor that also
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inhibits cKIT, FLT3, PDGFR and PDGFR with low nanomolar activity, recently received
FDA approval.12 Pexidarinib demonstrated reduction of TAMs, efficient target
engagement, and clinical efficacy as a monotherapy, with a 38% overall response rate in
pigmented villonodular synovitis (PVNS), an orphan disease characterized by
overexpression of CSF1R.12-16 Additionally, emactuzumab (RG7155), a neutralizing
antibody against CSF1R not only reduced macrophage infiltration in mouse models but
also demonstrated similar therapeutic effects against diffuse-type giant cell tumors in
17
patients. Two other neutralizing antibodies against CSF1R, AMG 820 (NCT01444404),
andIMC-CS4 (NCT01346358) are also currently in Phase I clinical trials for the treatment
of advanced solid tumors.18, 19 While there are several clinical opportunities for single
agent activity by suppressing CSF1R biology in dependent tumor cells, full clinical benefit
is likely to come from combining CSF1R inhibition in macrophages with immune
modulating agents targeting T-cell biology, such as anti-CTLA4, anti-PD-1 and anti-PD-
L1 therapeutics. These combinations have the potential to promote tumor suppression
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through modulation of multiple arms of the immune system. These combinations
currently are being explored in numerous clinical trials (i.e. NCT02777710, NCT04301778
and NCT03927105).20-22
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Several small molecule CSF1R inhibitors have been described in the literature,
however, only a few were reported to possess high levels of selectivity (Figure 1).7-16, 23-
31
BLZ945 was shown to inhibit CSF1R enzymatic activity with an IC = 1 nM, and was
50
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reported to have a >3200-fold selectivity for CSF1R over other related kinases. GW2580
inhibited CSF1R enzymatic activity with an IC50 = 30 nM, showing 22-fold and 75-fold
selectivity towards TrkB and C, respectively, and over 150-fold selectivity against other
kinases.29,32 GW2580 displayed an unfavorable in vivo PK profile with low exposures and
rapid clearance, and has not been further developed.29 A structurally related, highly
selective inhibitor with an azetidine scaffold, JTE-952 was also reported in the literature.
In a panel consisting of 51 kinases, JTE-952 was reported to have a 20-fold selectivity for
CSF1R over TrkA, however, selectivity for TrkB and TrkC was not reported.30, 31 Analysis
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of the crystal structure of PLX3397 bound to CSF1R (PDB 4R7H) and protein-ligand
binding models of other CSF1R inhibitors (using PDB 3LCO) suggested that these
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inhibitors bind in the ATP binding pocket in the inactive, DFG-out conformation of the
kinase.
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H
N
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O
O
S
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NH
NH
Cl
H
N
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CF3
N
Pexidartinib
PLX3397)
BLZ945
(
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NH2
N
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O
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N
H2N
OMe
O
O
(S)
OH
OH
GW2580
TJE953
Figure 1. Representative examples of small molecule CSF1R inhibitors.
RESULTS AND DISCUSSION
The objective of our program was to discover a highly selective small molecule CSF1R
inhibitor with excellent physicochemical and pharmacokinetic properties that potentially
could be used in combination with other immuno-oncology therapies. Herein, we describe
our efforts to identify a potent, orally bioavailable tool compound that we transformed into
a highly selective and efficacious lead candidate.
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At the outset of our program, we performed a scaffold hopping exercise using
benzothiazole as a template, with the search directed towards finding novel combinations
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of hinge binding and linker groups (Figure 2A); BREED , as implemented in MOE , was
used for this purpose. A set of 1,945 pre-aligned kinase-ligand complexes from the PDB
were compared with the benzothiazole in a molecular model of BLZ945 bound to CSF1R,
resulting in novel compounds that maintained both the position of the benzothiazole core
and the interactions with the kinase hinge region. Examination of these results suggested
that the kinase hinge residue, Cys666, could be engaged efficiently using two-atom
linkers placed between the benzothiazole core and suitable hinge-binding moieties
(Figure 2B).
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Figure 2. A) Schematic showing of scaffold hopping template and kinase hinge target
interactions B) Molecular model of 4-pyridyl hinge binding motif with -C-O- linker engaging
the backbone NH of Cys666 (PDB 3LCO).
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To test this hypothesis, a focused SAR study utilizing a variety of two-atom linkers was
performed. This study revealed that the -C-O- linker was preferred when combined with
a 4-pyridyl hinge binding motif resulting in compound 3, which exhibited an IC of 303
50
nM in a CSF1R FRET-based displacement binding assay (Table 1). There was less
preference among the two-atom linkers when combined with the 3-pyridyl hinge-binding
moiety; none of these compounds matched the enzymatic activity of compound 3.
Table 1. Enzymatic activity of compounds containing a two-atom linker between the
hinge-binding pyridine and the benzothiazole core.
Ar
B
S
N
A
NH
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Ar
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A-B
-C-O-
CSF1R IC (nM)
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CSF1R IC (nM)
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-
NH-C-
CSF1R IC (nM)
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27,712
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In an effort to improve potency, an amine substituent was incorporated onto the pyridine
of compound 3, which was predicted to form a second hinge interaction with the backbone
carbonyl of Cys666. Indeed, this modification led to a six-fold improvement in enzymatic
potency as demonstrated by compound 8. Incorporating a chloro group at the three
position of the pyridine led to similar improvement in potency as shown by compound 9.
Combining these two substituents had an additive effect with a 75-fold improvement in
potency relative to compound 3, with compound 10 exhibiting an IC50 of 4 nM in the
CSF1R FRET-based displacement binding assay. Compound 10 also inhibited mCSF1-
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mediated proliferation of MNFS-60 myelogenous leukemia cells with an IC of 228 nM,
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while it did not show significant activity against mCSF1-independent NS0 cell proliferation
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5
6
7
8
9
0
(Table 2).
Our molecular models predicted that the cyclohexyl ring extended into the deep pocket
of the DFG-out conformation of CSF1R, a region that provided additional opportunities to
improve potency and selectivity, given the modest differences in sequence among type
III kinases in this area (ie. Met637 is a Leu in cKIT, G795 is a Cys in other type III kinases).
To engage the side chain of Glu633 in the deep pocket region, a hydroxyl substituent was
incorporated on the cyclohexyl ring. To maximize the probability of engaging this
interaction, we examined all four stereoisomers of the 2-amino cyclohexan-1-ol group.
However, incorporating the hydroxyl group did not make a significant contribution to
enzymatic or cellular potency.
To evaluate whether incorporation of the hydroxyl group on the cyclohexane ring offered
any selectivity advantage, and whether the orientation of the hydroxyl group affected
selectivity, we evaluated these inhibitors in a binding assay against a representative set
of type III kinases that are related to CSF1R, including FLT3, cKIT, PDGFR and
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9
PDGFR. Overall, compound 12 exhibited a favorable selectivity profile. The cellular
activity of compound 12 was measured by monitoring inhibition of CSF1R
autophosphorylation after CSF1 stimulation of THP-1 cells, a human monocytic cell line.
In order to assess the compound’s selectivity against PDGFR, we measured agonist
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
(PDGF-DD) induced PDGFR autophosphorylation in a HEK293 cell line expressing
human recombinant PDGFR PDGFR cells). BLZ945 was also tested in this
assay as a competitor with reported selectivity against PDGFR. Our results confirmed
that both BLZ945 and 12 exhibited minimal selectivity against PDGFR in a cellular
setting, with 12 inhibiting CSF1 stimulated CSF1R autophosphorylation in THP-1 cells
with an IC50 of 183 nM, while inhibiting PDGFR autophosphorylation in
HEK293/PDGFR cells with an IC of 446 nM. BLZ945 displayed an IC of 155 nM in
50
50
the cellular phospho-CSF1R (THP) assay and IC of 579 nM in the phospho-PDGFRβ
50
(HEK293/ PDGFRβ) assay.
Table 2. SAR studies in the hinge region and deep pocket area of the 6-(pyridin-4-
ylmethoxy)benzo[d]thiazole scaffold lead to significant improvement in potency.
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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
N
O
S
X
Z
Y
N
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
Compound
8
9
10
11
12
13
14
X
Y
-NH2
-H
-NH2
-NH2
-NH2
-NH2
-NH2
-H
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
NH
NH
NH
NH OH
NH OH
NH OH
NH OH
Z
CSF1R IC50
52
30
4
2
1.4
0.6
1.1
1.2
2.4
1.9
[
nM]
CSF1R Kd
nM] (fold)
ND
ND
ND
2.5
1.1
[
7
9
44
(40)
65
180
(300)
74
170
(141)
400
(333)
28
1000
(526)
320
(168)
94
cKIT Kd [nM]
fold)
ND
ND
ND
ND
(
(
31)
70
(68)
1
FLT3 Kd [nM]
fold)
ND
ND
ND
(
(59)
1.3
(86)
12
4
6
PDGFR Kd
nM] (fold)
[
(
18)
.8
1.5)
(1.2)
0.84
(0.76)
(20)
19
(23)
1.2
(49)
5.6
3
PDGFR Kd
[nM] (fold)
(
(32)
(1)
(3)
Phospho-
CSF1R (THP-
ND
ND
ND
ND
183
ND
ND
1
) IC [nM]
5
0
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Page 15 of 122
Journal of Medicinal Chemistry
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8
9
Phospho-
PDGFRβ
446
ND
ND
ND
ND
50
ND
ND
(
HEK293) IC50
(2.4)
[nM] (Fold X)
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
MNSF60 IC50
2269
1204
228
133
98
189
ND
[
nM]
NS0 IC [nM] 18,809 14,989 41,666 27,500 47,945
ND
50
To assess broad kinase selectivity, compound 12 was evaluated against a panel of 468
kinases (Figure 3). It exhibited an acceptable selectivity profile, with significant activity
against only a handful of related type III kinases at 1 uM including c-KIT, FLT3, PDGFR
and PDGFR (Table S1).
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Journal of Medicinal Chemistry
Page 16 of 122
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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
Figure 3. Selectivity profile of compound 12 against a panel of 468 kinases at 1 M test
concentration. The larger the red circle, the greater the inhibition. Six kinases with percent
of control < 10: CSF1R, cKIT, FLT3, AURB, PDGFR and PDGFR. See also Table S1,
Supporting information.
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
Compound 12 displayed low to moderate microsomal and hepatic clearance in in vitro
microsomal and hepatocyte preparations and high plasma protein binding. It exhibited a
suitable in vivo PK profile across species, with low clearance, reasonable half-life and
good oral bioavailability (Table 3).
Table 3. Microsomal and hepatic stablity, plasma protein binding and in vivo PK profile of
compound 12.
In vitro ADME properties of
compound 12
In vivo pharmacokinetic data of compound 12
Specie
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Page 17 of 122
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s
Plasma Hepato Microso Dose
protein cytes mes IV/PO
binding CLint,s CLint,sc [mg/kg
CL
T1/2 [h]
[
mL/mi
n/kg]
Vss
Oral F
[%]
[L/kg]
(IV)
(%
caled
aled
]
(IV)
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
bound) [mL/min [mL/min
Mouse
Rat
Dog
Monke
Human
>99
>99
>99
>99
>99
11
30
55
68
0.3/10
0.3/3
0.3/3
0.3/1
23
3.2
12
1.8
0.75
1.3
1.6
3
4.8
7.0
>90
67
34
72
155
58
10
293
95
25
1.2
0.6
Given its in vitro and in vivo profiles, compound 12 was used to probe CSF1R biology,
with BLZ945 serving as a comparitor in these experiements. An immunohistochemistry
(
IHC) assay was used to measure the well-established macrophage marker F4/80 in
PANC02 tumors from mice that had been treated with daily oral doses of 200 mg/kg
compound 12 or BLZ945 for 7 days (n=4 mice/group). Treatment with each compound
resulted in similar extent of reduction of F4/80 positive cells within the tumors (Figures
4
A-D), consistent with macrophage depletion. As an orthogonal measure of compound
activity, CSF1R protein levels were measured by ELISA in tumors from mice similarly
treated, and were found to be depleted to a similar extent by both compounds (Figure 4E)
(n=4 mice/group). Furthermore, as an additional measure of anti-macrophage activity,
mRNA transcripts of well known macrophage expressed genes were measured, and
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Journal of Medicinal Chemistry
Page 18 of 122
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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
similar to the IHC and ELISA results, 200 mg/kg of compound 12 or BLZ945 (n=3
mice/group) reduced the expression of the panel by 62-93% (Figure 4F). Similar
depletion of macrophages was observed for MC38 and EMT6 tumors treated with
compound 12 or BLZ945 (data not shown). Taken together, treatment of PANC02 tumors
with compound 12 reduced tumor macrophages to a similar extent as BLZ945.
Treatment of mice bearing MC38 allograft tumors with 200 mg/kg compoud 12 (n=10
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
mice/group) resulted in 62% reduction in tumor growth (Figure 4G), consistent with the notion
that inhibition of CSF1R has therapeutic potential, possibly through macrophage depletion.
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Page 19 of 122
Journal of Medicinal Chemistry
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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
Figure 4. CSF1R inhibitors depletes tumor macrophages and reduces tumor growth. IHC
for F4/80 for PANC02 tumors in mice that were treated daily for 7 days with (A) vehicle,
(B) 200 mg/kg BLZ945 or (C) 200 mg/kg compound 12 (n=4 mice/group). (D) Pathologist
scored quantification of F4/80 IHC signal in PANC02 tumors from mice treated daily for 7
days with vehicle, 200 mg/kg BLZ945 or 200 mg/kg compound 12 (n=4 mice/group). (E)
Relative CSF1R protein expression in PANC02 tumors from mice treated daily for 7 days
with vehicle, 200 mg/kg BLZ945, or 200 mg/kg compound 12 as measured using ELISA
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Journal of Medicinal Chemistry
Page 20 of 122
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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
(
n = 4 mice/group). (F) Relative mRNA expression of known macrophage genes in
PANC02 tumors from mice treated daily for 7 days with vehicle, 200 mg/kg BLZ945, or
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
2
00 mg/kg compound 12 (n=3 mice/group) as measured using a nanostring code set. (G)
Growth of MC38 tumors implanted subcutaneously in mice and treated daily with vehicle
or 200 mg/kg compound 12 (n=10 mice/group).
While the anti-tumor impact of compound 12 was encouraging, we were not satisfied
with the overall potency, physicochemical properties and the level of selectivity that this
inhibitor exhibited toward related type III RTKs, especially toward PDGFR. Therefore,
we continued our quest to identify inhibitors with significantly improved potency,
properties and selectivity. In order to improve the selectivity profile of the benzothiazole
scaffold, we used a combination of X-ray structures from the PDB and the patent
literature. Sequence and structural comparison of the CSF1R active site with c-KIT, FLT3,
PDGFR and PDGFR revealed that CSF1R is unique among these kinases; the
CSF1R Gly795 residue aligns with a cysteine in the other related kinases (Figures 5A
and 5B). Substitution at the 4-position of the benzothiazole core would fill the space
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Page 21 of 122
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9
adjacent to Gly795 in CSF1R, while this substitution pattern would not be tolerated in c-
KIT, FLT3, PDGFR or PDGF as it would lead to a steric clash with the cysteine residue
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
(Figure 5C). Furthermore, overlaying models of compound 12 and GW2580, (Figure 5D),
an inhibitor that showed high selectivity toward CSF1R over other related kinases, but not
TrkA,
led
to
the
same
conclusion.
Figure 5. A) Model of compound 12 bound to CSF1R (PDB 3LCO). B) Sequence
alignment of class III receptor tyrosine kinases highlighting a key difference in the xDFG
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Journal of Medicinal Chemistry
Page 22 of 122
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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
motif. C) Overlay of cKIT Cys809 (orange; PDB 4HVS) with model of compound 12 bound
to CSF1R shows a small pocket that is present in CSF1R that is filled by a cysteine side
chain in other class III RTKs. D) Overlay of GW2580 (pink) with model of compound 12
shows a methoxy group filling this pocket.
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
To test this hypothesis, we synthesized the 4-methoxy substituted derivative of 12,
compound 15. Indeed, this modification maintained high affinity towards CSF1R in
binding and cellular activity assays, and led to a dramatic improvement in selectivity
against related kinases cKIT, FLT3, PDGFR and PDGFR. The same high level of
selectivity was observed in the cellular phospho-PDGFR target engagement assay
(Table 4).
To understand whether introducing other substituents into the 4-position would lead to
the same level of selectivity increase, we performed a focused SAR study. We found that
introduction of a trifluoromethoxy substituent into the 4-position (16) leads to decreased
cellular potency. Incorporation of a fluoro- (17) or chloro (18) substituent was tolerated
for potency, however, only lower levels of selectivity was achieved compared to that of
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Page 23 of 122
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9
the methoxy-substitued analog 15. Further structural analysis revealed that a hydrophobic
pocket formed by the sidechains of Thr663 and Met637 could potentially accommodate
a substituent in the 7-position of the benzothiazole core and that filling this pocket could
lead to a potency improvement. To test this hypothesis, the 7-chloro- (19) and 7-fluoro
benzothiazole (20) analogs were prepared. These inhibitors revealed that while a
substituent in the 7-position was indeed tolerated for potency, this modification was
detrimental to selectivity.
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
Table 4. SAR study of substituted benzothiazole analogs leads to dramatic improvement
of selectivity.
NH2
Cl
N
X
Y
O
S
N
NH OH
Compound #
15
-H
16
17
-H
18
-H
-F
19
-Cl
-H
20
-F
-H
X
Y
-H
-OMe
-OCF3
-Cl
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Journal of Medicinal Chemistry
Page 24 of 122
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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
CSF1R IC50
nM]
[
4
17
1
1
1.2
3.4
1.1
1.3
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
CSF1R
Kd [nM]
2.5
8.1
0.6
ND
(Fold X)
FLT3
30,000
5
90
1300
(520)
5.4
23
Kd [nM]
ND
ND
ND
(
72)
(1.58)
(17)
(
>1875)
(Fold X)
cKit
3
0,000
>30,000
(>3703)
2100
(840)
530
(98)
790
Kd [nM]
Fold X)
(>1875)
(607)
(
PDGFR
3
0,000
>30,000
(>3703)
370
12
32
Kd [nM]
(>1875)
(148)
(3.5)
(24)
(Fold X)
PDGFR
Kd [nM]
(Fold X)
30,000
>30,000
(>3703)
230
(92)
4.1
6.7
ND
(
>1875)
(1.2)
(5.1)
MNSF60 IC50
[nM]
3
45
930
288
125
104
194
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2
3
4
5
6
7
8
9
Phospho-
CSF1R (THP-
2
43
ND
ND
191
127
176
ND
ND
1)
[
nM]
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
Phospho-
PDGFRβ
(HEK293)
>50,000
4114
(20)
1339
(10)
250
(
>200)
(1.4)
[
nM] (Fold X)
cLogP
3.5
5.2
ND
ND
4.32
>99 / >99
78/32
3.8
4.3
ND
ND
3.8
ND
ND
PPB [%
>99 /
98.7
>99 /
98.6
bound], (r/h)
CLint,scaled
[
mL/min/kg]
r/h)
99/38
91/19
(
Introducing a substituent into the 4-position led to the desired selectivity improvement,
however, these inhibitors possessed suboptimal in vitro DMPK properties, particularly
unfavorable metabolic turnover in rat and human microsomes (Table 4).
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2
3
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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
Analyzing the correlation between liver microsomal intrinsic clearance and cLogP for
nearly one hundred analogs that were synthesized in the 6-methoxy benzothiazole series
revealed that inhibitors with a cLogP > 3.5 tended to have high clearance in human liver
microsomes (Figure S1). Based on this observation we targeted the design and synthesis
of inhibitors with a cLogP of less than 3.5 to improve metabolic stability, while retaining
favorable enzymatic and cellular potency.
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
To increase polarity and modulate physicochemical properties, we devised SAR studies
in the hinge as well as deep pocket area. Decreasing the cLogP by structural
modifications in the hinge region quickly led to improved microsomal stability, while
selectivity was preserved as demonstrated by compound 21 (Table 5). Reversing the
stereochemistry of the amino- and hydroxyl- substituents on the cyclohexyl ring led to
improved potency, while maintaining selectivity and favorable in vitro DMPK properties
as shown by compound 22. Introduction of a 7-chloro substituent onto the benzothiazole
core led to further improvement in potency, as exemplified by compound 23, without a
significant deleterious effect on selectivity or physicochemical properties.
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7
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9
Since compounds 22 and 23 exhibited acceptable enzymatic and cellular potency, as
well as a promising in vitro ADME profile, we decided to evaluate these inhibitors in vivo.
Both inhibitors had a suitable in vivo PK profiles across species with low to moderate
clearance, reasonable half-life and excellent oral bioavailability in rat and mouse, and with
moderate oral bioavailability in dog (Table S2).
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
Table 5. Property optimization of benzothiazole analogs.
NH2
NH2
NH2
N
N
N
N
N
N
Cl
O
S
N
O
S
N
O
S
N
NH OH
NH OH
NH OH
OMe
OMe
22
7
OMe
Compound #
21
21
23
CSF1R IC [nM]
2
50
4.1
2.6
CSF1R Kd [nM]
FLT3 [nM]
6.7
>30,000
(>4,477)
4,800
>30,000
(7,317)
>30,000
(7,317)
>30,000
>30,000
(>11,538)
>30,000
(>11,538)
24,000
(Fold X)
cKit [nM]
(Fold X)
(750)
PDGFR [nM]
4,100
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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
(
Fold X)
(611)
(7,317)
>30,000
(7,317)
178
(9,230)
27,000
(10,384)
60
PDGFR [nM]
(Fold X)
4,900
(731)
504
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
MNSF60 IC [nM]
50
Phospho-CSF1R
[nM] (THP-1)
486
215
63
Phospho-PDGFRβ
nM] (HEK293)
41000
>50000
(>23)
34254
(543)
[
(84)
2.41
(
Fold X)
cLogP
2.41
3.02
PPB [% bound],
r/h)
9
0.4/77.1
92.2/77.6
98.3/97.2
(
CLint,scaled
24/10
20/9.5
43/33
[mL/min/kg] (r/h)
These compounds exhibited excellent overall properties, however, we were not
satisfied with the cellular potency of these inhibitors. SAR studies performed earlier on
aminopyridine derivative 24 revealed that introduction of an acetamide onto the
aminopyridine moiety of 24 resulted in a significant boost in potency (Table 6).
Table 6. Potency improvement by introducing an acetamide onto compound 24.
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O
NH2
HN
Me
N
O
S
N
N
O
S
N
NH OH
NH OH
OMe
OMe
Compound #
CSF1R IC50
24
25
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
[
nM]
76
4
MNSF60 IC50
1
455
82
[
nM]
Although compound 25 possessed excellent potency, it suffered from significant non-
NADPH-dependant microsomal metabolism; incubation with human and mouse liver
microsomes for 45 min led to 80% and 54% parent remaining, respectively, even in the
absence of NADPH (Table S4.) Metabolite identification studies in human and mouse
liver microsomes confirmed that the major metabolite is the hydrolysis product of the
amide of compound 25 (Table S5).
Based on this information we hypothesized that we would be able to further boost the
potency, while maintaining optimal properties, if we introduced a substituent onto the
amino pyrimidine moiety of inhibitor 22. However, instead of introducing a hydrolytically
unstable amide, we decided to incorporate a urea, as well as a series of heterocyclic
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