Synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel FMS inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.11.037

Colony stimulating factor-1 receptor (CSF-1R or FMS) and it ligand, CSF-1, signaling regulates the differentiation and function of tumor-associated macrophages (TAMs) that play an important role in tumor progression. Derivatives of thieno[3,2-d]pyrimidine were synthesized and evaluated as kinase inhibitors of FMS. The most representative compound 21 showed strong activity (IC₅₀ = 2 nM) against FMS kinase and served as candidate for proof of concept. Anti-tumor activity alone and/or in combination with paclitaxel was examined via a tumor cell growth inhibition assay and via an in vitro tumor invasion assay using human breast adenocarcinoma cells.

Full text of DOI:10.1016/j.bmcl.2018.11.037

Accepted Manuscript
Synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel FMS
inhibitors
Yu-Yon Kim, Jaeyul Choi, Kyungjin Choi, Changhee Park, Young Hoon Kim,
Kwee Hyun Suh, Young Jin Ham, Sun Young Jang, Kyu-Hang Lee, Kwang
Woo Hwang
PII:
S0960-894X(18)30904-1
https://doi.org/10.1016/j.bmcl.2018.11.037
BMCL 26148
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 September 2018
16 November 2018
18 November 2018
Please cite this article as: Kim, Y-Y., Choi, J., Choi, K., Park, C., Hoon Kim, Y., Suh, K.H., Ham, Y.J., Jang, S.Y.,
Lee, K-H., Hwang, K.W., Synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel FMS inhibitors,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.037
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Short communication
Title
Synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel FMS inhibitors
Yu-Yon Kima,b, Jaeyul Choib, Kyungjin Choib, Changhee Parkb, Young Hoon Kimb, Kwee
Hyun Suhb, Young Jin Hamb, Sun Young Jangb, Kyu-Hang Leeb, Kwang Woo Hwang a,1
aHost Defense Modulation Lab, Collage of Pharmacy, Chung-Ang University, 84 Heukseok-
Ro, Dongjak-Gu, Seoul, 06974, Republic of Korea
bHanmi Research Center, Hanmi Pharm. Co. Ltd., 550 Dongtangiheung-Ro, Hwaseong-Si,
Gyeonggi-Do, 18469, Republic of Korea
E-mail addresses:
YYK, yykim@hanmi.co.kr; JYC, alchemistj@hanmi.co.kr; KJC, booim@hanmi.co.kr; CHP,
cchange@hanmi.co.kr; YHK, yhkeem@hanmi.co.kr; KHS, khsuh@hanmi.co.kr; YJH,
yjham@hanmi.co.kr; SYJ, jsy74@hanmi.co.kr; KHL, kyuhang@hanmi.co.kr; KWH,
khwang@cau.ac.kr
1Corresponding authors
Kwang Woo Hwang Ph.D., Host Defense Modulation Lab, College of Pharmacy, Chung-Ang
University, 84 Heukseok-Ro, Dongjak-Gu, Seoul 06974, Republic of Korea.
khwang@cau.ac.kr

Abstract
Colony stimulating factor-1 receptor (CSF-1R or FMS) and it ligand, CSF-1, signaling
regulates the differentiation and function of tumor-associated macrophages (TAMs) that play
an important role in tumor progression. Derivatives of thieno[3,2-d]pyrimidine were
synthesized and evaluated as kinase inhibitors of FMS. The most representative compound 21
showed strong activity (IC50 = 2 nM) against FMS kinase and served as candidate for proof of
concept. Anti-tumor activity alone and/or in combination with paclitaxel was examined via a
tumor cell growth inhibition assay and via an in vitro tumor invasion assay using human
breast adenocarcinoma cells.
Keywords
FMS; CSF-1R; Colony stimulating factor-1; Tumor associated macrophages (TAMs); Anti-
tumor activity; Human breast adenocarcinoma cells

Macrophages are characterized as classical M1- and alternative M2-subtypes depending on
how they influence the immune response.1 M1 macrophages influence the inflammatory
response and anti-tumor immunity, whereas M2 macrophages are involved in
immunosuppressive and tumor promoting activities. Tumor-associated macrophages (TAMs),
which are classified as M2-polarized macrophages, are recruited by tumor cells and infiltrate
into the stromal environment of malignant tumors. TAMs in the tumor microenvironment are
known to play a crucial role in tumor progression, including tumor initiation and
development, matrix remodeling and metastasis, and immune suppression.2-3
Colony stimulating factor-1 (known as CSF-1 or macrophage-colony stimulating factor, M-
CSF) is a growth factor for monocyte and macrophage-derived cells. CSF-1 and its receptor,
colony stimulating factor-1 receptor (CSF-1R or M-CSFR, also known as FMS) have been
reported as having important roles in regulating the production, migration, differentiation,
survival, and function of macrophages and their precursors.4 Therefore, over-activation of
CSF‐1/CSF-1R signaling in macrophages has been implicated in many disease states
including osteoclast proliferation in bone osteolysis, and a number of inflammatory disorders
as well as the growth/metastasis of cancer.5-14 Especially, a number of CSF1/CSF1R-targeting
agents such as Pexidartinib and ARRY-382 are undergoing clinical trials in several cancer
including breast cancer, prostate cancer, and melanoma.15
A previous report showed that the transfer of the CSF1 gene into tumor cells induced TAM
infiltration into syngeneic BALB/c mice.16 High expressions of CSF-1 and CSF-1R in tumor
and stromal cells and a high TAM density are known to be correlated with poor prognoses in
many cancer types.17 CSF-1 promotes metastates via the regulation of TAMs in the tumor
microenvironment.18 In human metastatic breast cancer, elevated CSF-1 levels were
correlated with marked CSF-1R-positive macrophage infiltration into tumors and were
associated with poor outcomes.19 Another study also showed that macrophage-deficient/CSF-
1-deficient mice rarely developed pulmonary metastases, despite the rapid growth of primary
mammary tumors.20
Furthermore, some chemotherapies stimulate tumor cells to release CSF-1, which then
recruits TAMs expressing CSF-1R. These TAMs in the microenvironment stimulate tumor
progression by enhancing tumor resistance to chemotherapy. In combination therapy with
paclitaxel and CSF-1R antagonists on primary and metastatic tumors of mammary tumor-
bearing mice, inhibition of TAM infiltration via CSF-1R antagonists increased the anti-tumor
efficacy of cytotoxic agents.21 For these reasons, modulation of the CSF-1 signaling pathway
in the microenvironment has been of great interest in chemotherapy research.
In this paper, we describe the identification of the potent FMS inhibitor compound 21 and
evaluate its anti-tumor activities against breast cancer. By computational screening
compounds from the internal small molecule compound bank of Hanmi Pharmaceutical, we
identified 1H-indazol-4-yl)thieno[3,2-d]pyrimidine derivative 7 (Fig. 1) as a highly potent
(IC50 = 13 nM) FMS inhibitor.

Figure 1. Hit compound 7
To improve its enzyme activity and cellular activities on NFS-60 cells that express
endogenous FMS,22-23 structure-activity relationship (SAR) studies probed substituent effects
at the N-1 position (R1) and C-3 position of the indazole moiety (R2). Initially, analogues
were synthesized with substitutions at R1 (Table 1). The replacement of the phenyl moiety
with methoxy phenyl moiety (8) led to an almost two-fold loss of enzyme and cellular
activity. In contrast, the replacement of the phenyl moiety with a pyridine moiety (9-11)
showed good enzyme activity (IC50 < 10 nM). To further improve enzyme and cellular
activity, we added an adopted substituent to the pyridine moiety. Halogen substituents (12-13)
in the pyridine moiety had similar enzyme activities compared to regular pyridine moieties
(9-11), and cellular activity did not dramatically improve. In contrast, alkyl substituents (14-
15) on the pyridine moiety also showed improved cellular activity. In particular, a methyl
substituent (14) on the pyridine moiety showed good cellular activity (GI50 = 27.5 nM) in an
NFS-60 cell line whose growth depends on FMS.22-23 Next, we tried to attach an alicyclic,
heterocyclic, or alkynyl substituent (16-18) to the indazole moiety. Although all the resultant
compounds showed good enzyme activities, they did not show sufficiently strong cellular
activity.
Table 1. FMS enzyme and cellular activity of compounds 7-18
FMS IC50
(nM)
NFS-60 GI50
(nM)
94
Compound
R1
GW-2580
7
-
3
13
37
9
1005
2390
2502
77
8
9
6
10
11
12
7
135
13
233

12
23
243
28
13
14
10
30
34
15
16
254
19
26
62
17
18
>10000
The synthetic route of R1-changed derivatives is shown in Scheme 1. Compounds 7-18 were
prepared from commercially available 4-nitro-1H-indazole (1). Bromination of 1 was
performed using bromine gas, and gave an excellent yield (94%). The 1-substituted indazole
of 2 was easily derived from an SN2 reaction using an alkyl-halide. Reduction of 3 was
performed using Pd/C, and the amide coupling of 4 and 5 using commercially available
HATU. Each step was isolated via column chromatography.
Scheme 1. Preparation of compounds 7-18. Reagents and conditions: (a) Br2, NaOAc,
AcOH/CHCl3(1:1), 20°C; (b) R1CH2Cl, K2CO3, DMF, rt; (c) H2, Pd/C, THF/MeOH(1:1), rt;
(d) HATU, DIPEA, DMF, rt.
Compound 14 was the most potent compound of the R1 changed-derivatives, based on
cellular activity. This compound also showed good oral bioavailability (37%)
(Supplementary data).
Next, we fixed R1 as 2-methylpyridine and replaced R2 with the C-3 position of an indazole
moiety (compound 14: Br). We then tried halogen (23-24) and alkyl (25-27) substituents at
R2 (Table 2). Compound 23 with a halogen moiety showed more potent cellular activity (3.4
fold) than compound 14, and compound 25 with an alkyl moiety had the most potent enzyme
activity (IC50 = 2 nM). Compound 25 showed more stable liver metabolism than compound
23 in microsomal stability test using human hepatocytes. Remaining value of parent molecule
at 60 minutes of compound 25 and 23 were 87% and 66%, respectively.

Table 2. FMS enzyme and cellular activity of compounds 23-27
FMS IC50
NFS-60 GI50
Compound
R2
(nM)
20
9
2
9
(nM)
8
30
80
>10000
Cl
I
Me
Et
23
24
25
26
27
Vinyl
12
4458
The synthetic route of R2-changed derivatives is shown in Scheme 2. Compounds 25-27 were
prepared from commercially available 4-nitro-1H-indazole (1). After same two steps with
scheme 1, the N-substituted indazole of 19 was produced using the Suzuki reaction in a good
yield (85%). Reduction of 20 was performed using Pd/C, and the amide coupling of 21 and 5
using commercially available HATU. Each step was isolated via column chromatography.
Scheme 2. Preparation of compounds 25-27. Reagents and conditions: (a) R2B(OH)2, K3PO4,
Pd(OAc)2, PhMe/H2O, 100°C; (b) H2, Pd/C, THF/MeOH(1:1), rt; (c) HATU, DIPEA, DMF,
rt.
Having successfully achieved good enzyme and/or cellular activities, the drug metabolism
and pharmacokinetic profiles for the more potent compounds (10 and 25) were evaluated. To
understand the potential drug-drug interaction liabilities of these compounds, a CYP
(Cytochromes P450) reversible inhibition assay using human liver microsomes was
conducted. In this test, compound 25 showed weak inhibition of five major CYP enzymes,
whereas compound 10 inhibited four CYP enzymes either strongly or moderately (IC50 range
from < 0.25 to 2.02 µM) except CYP2D6 (Table 3). These results indicate that compound 25
has a lower risk of drug-drug interactions than compound 10. Moreover, compound 25
showed both good oral bioavailability (F = 36%) in mice (Table 4) and also an IC50 value >
50 µM for the binding capacity to the hERG (Human ether-a-go-go-related gene) membrane.
Based on these results, compound 25 was chosen for further study.

Table 3. CYP enzyme inhibition assay for compounds 10 and 25
IC50 (µM)
Compound
CYP1A2
< 0.25
CYP2C9
1.38
CYP2C19
2.02
CYP2D6
> 20.00
> 20.00
CYP3A4
0.31
10
25
> 20.00
> 20.00
> 20.00
> 20.00
CYP enzyme inhibition assay used the HLM with LC/MS/MS analysis method.
Table 4. Pharmacokinetic parameters for compound 25
t1/2
(hr)
Cmax
(ng/ml)
Tmax
(h)
AUC0-7h Vd
(ng·hr/ml) (L/kg)
Cl
(L/hr/kg)
BA
(%)
Compound Route
IV
1.6
2.6
2546.5
900
NA
2.0
3367.2
4037.8
2.0
7.5
0.9
2.0
NA
36
25a
PO
Male CD-1 mice treated with 3 mg/kg IV and 10 mg/kg PO.
a 5 % DMSO in 30 % PEG400 solution for IV and 0.5 % HPMC in 0.05 % Tween80 solution for PO
To determine the biochemical selectivity for compound 25, twenty eight kinases were
tested.24 As summarized in Table 5, compound 25 was highly selective against FMS and
inhibited FMS with an IC50 of 2 nM. It also inhibited the receptor tyrosine kinases c-RAF and
DDR2 (IC50 < 100 nM for both) and FGFR1, KDR, MEKL, and Tie2 (IC50 100 ~ 600 nM).
However, the other twenty one kinases were inhibited only at much higher concentrations of
25.
Table 5. Kinase selectivity of compound 25
Kinase
IC50 (nM)
FMS
2
DDR2
c-RAF
FGFR1
64
80
145

KDR
MELK
Tie2
527
258
208
IC50 values were measured at the respective ATP Km for each kinase.
The inhibition potential of compound 25 on the migration of tumor cells by TAMs was
evaluated via an invasion assay using MDA-MB-231 cells (human breast adenocarcinoma
cells) and RAW264.7 cells (mouse macrophage cells). Compound 25 inhibited the migration
of MDA-MB-231 cells in a dose-dependent manner; the inhibitory effect after treatment with
1 µM was 67%. (Fig. 2)
100
50
0
nM
1000
0
100
500
Figure 2. Tumor invasion assay in MDA-MB-231/Luc cells
The anti-tumor effects of compound 25 against breast cancer in combination with cytotoxic
agents was evaluated using an in vitro tumor cell growth inhibition assay. As shown in Figure
3, paclitaxel dose- dependently induced FMS expression in MDA-MB-231 cells, and
compound 25 effectively reduced paclitaxel-induced FMS expression.

Figure 3. (A) FMS expression levels after treatment with paclitaxel in MDA-MB-231 cells
was increased. (B) Compound 25 dose-dependently inhibited overexpressed FMS by
paclitaxel treatment in MDA-MB-231 cells. GAPDH, Glyceraldehyde 3-phosphate
dehydrogenase
In a combination study with paclitaxel and compound 25 in MDA-MB-231 cells, treatment
with compound 25 (5 µM) alone showed a reduction in cell growth by 20%, whereas
treatment with paclitaxel in combination with compound 25 showed a synergistic growth
inhibition effect (CI < 0.1) compared with treatment with paclitaxel alone. (Fig. 4)
100
80
60
Compound 21 5uM
Paclitaxel
Paclitaxel + Compound 21 5uM
40
20
0
CI < 0.1 at all conc. of paxlitaxel
-4.0
-3.5
-3.0
-2.5
-2.0
Log conc. of paclitaxel (uM)
Figure 4. In vitro combination study of compound 25 and/or paclitaxel on cell growth
inhibition in MDA-MB-231 cells. The synergistic effect was determined by calculating the
combination index (CI) using the Calcusyn program (Cambridge.UK). The CI value was
considered statistically significant when it was less than 1.0.
In summary, we identified and optimized a novel and potent class of FMS kinase inhibitors
based on a thieno[3,2-d]pyrimidine core. One of these, compound 25, was employed in an in
vitro efficacy study to demonstrate its potential benefit in the treatment of cancer. The
inhibition of FMS signaling by compound 25 was efficacious in reducing the migration of
cancer cells and showed a synergistic anti-tumor activity against breast cancer in combination
with a cytotoxic agent.

Declarations of interest: none.
Funding: not applicable
A. Supplementary data
Supplementary data associated with this article can be found in the online version.
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Graphic Abstract