Introduction of fluorine to phenyl group of 4-(2-pyrimidinylamino)benzamides leading to a series of potent hedgehog signaling pathway inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.06.030

In present study, a novel series of fluorine containing 4-(2-pyrimidinylamino)benzamide analogues were designed and synthesized. The hedgehog (Hh) signaling inhibitory activities for these compounds were evaluated by a luciferase reporter method. The preliminary SAR was discussed and many compounds showed potent Hh signaling inhibitory activities. Compound 15h displayed the most potent inhibitory activity, with an IC₅₀ of 0.050?nM. This paper finds the introduction of fluorine to the 4-(2-pyrimidinylamino)benzamide scaffold can lead to a novel series of potent Hh signaling pathway inhibitors.

Full text of DOI:10.1016/j.bmcl.2017.06.030

Accepted Manuscript
Introduction of Fluorine to Phenyl Group of 4-(2-Pyrimidinylamino)benza-
mides Leading to a Series of Potent Hedgehog Signaling Pathway Inhibitors
Minhang Xin, Liandi Zhang, Jun Wen, Han Shen, Xinge Zhao, Qiu Jin, Feng
Tang
PII:
S0960-894X(17)30633-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.030
BMCL 25064
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 March 2017
27 May 2017
10 June 2017
Please cite this article as: Xin, M., Zhang, L., Wen, J., Shen, H., Zhao, X., Jin, Q., Tang, F., Introduction of Fluorine
to Phenyl Group of 4-(2-Pyrimidinylamino)benzamides Leading to a Series of Potent Hedgehog Signaling Pathway
Inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.030
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Introduction of Fluorine to Phenyl Group of 4-(2-Pyrimidinylamino)benzamides
Leading to a Series of Potent Hedgehog Signaling Pathway Inhibitors
b
b
Minhang Xin a, b,*, Liandi Zhang b, Jun Wen b, Han Shen , Xinge Zhao , Qiu Jin b, Feng
Tang b
a Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an
Jiaotong University, No 76, Yanta West Road, Xi’an 710061, P.R. China
b
Jiangsu Simcere Pharmaceutical Co. Ltd., No 699-18, Xuan Wu District, Nanjing
210042, P.R. China
Footnotes
* Corresponding author: E-mail: xmhcpu@163.com
1

Abstract: In present study,
a
novel series of fluorine containing
4-(2-pyrimidinylamino)benzamide analogues were designed and synthesized. The
hedgehog (Hh) signaling inhibitory activities for these compounds were evaluated by a
luciferase reporter method. The preliminary SAR was discussed and many compounds
showed potent Hh signaling inhibitory activities. Compound 15h displayed the most
potent inhibitory activity, with an IC50 of 0.050 nM. This paper finds the introduction of
fluorine to the 4-(2-pyrimidinylamino)benzamide scaffold can lead to a novel series of
potent Hh signaling pathway inhibitors.
Keywords: hedgehog signaling pathway inhibitors; 4-(2-pyrimidinylamino)benzamide
scaffold; fluorine; most potent.
2

Hedgehog (Hh) signaling pathway recently receives much attention due to its critical
role in the tumor growth and development. It is identified that aberrant activation of Hh
signaling pathway has been implicated in many human tumors, such as basal cell
carcinoma (BCC), medulloblastoma (MB), rhabdomysarcoma (RMS), lung cancer,
pancreatic cancer, hepatocellular cancer, gastric cancer, esophageal cancer, colorectal
1
cancer, ovarian cancer, prostate cancer, lymphoma, leukemia . Thus, Hh signaling
pathway is considered as a promising target for treating human tumors.
2
During those years, many Hh signaling pathway inhibitors have been reported .
3
Cyclopamine (1) was early reported as Hh signaling pathway inhibitors . Based on the
natural structure of cyclopamine, many cyclopamine derivatives were synthesized such as
saridegib (IPI-926, 2). However, due to disappointed therapeutic results, saridegib were
failed in clinical trials 4. In addition to cyclopamine derivatives, many chemical synthetic
small molecules were also developed as Hh signaling pathway inhibitors 5. Among these,
6
7
vismodegib (GDC-0449, 3) and sonidegib (LDE-225, 4) have now been approved by
FDA for treatment of locally advanced or metabolic BCC. Taladegib (LY-2940680, 5) 8,
9
10
11
glasdegib (PF-04449913, 6) , itraconazole (7) and BMS-833923 (XL-139, 8) are
investigated in phase II clinical studies now (Figure 1). Although the great progress for
Hh inhibitors were achieved, the evidence showed that drug resistance against Hh
12
inhibitors has emerged . Therefore, there is still an emergent need to develop novel
chemtype Hh signaling pathway inhibitors.
3

Figure 1. Representative Hh signaling pathway inhibitors.
We recently reported a series of novel potent Hh signaling pathway inhibitors
13
composed of 4-(2-pyrimidinylamino)benzamides, such as compound 9 . In an effort to
develop more novel chemical structures, many fused hetero-aromatic rings systems, such
as pyrrolo[2,1-f][1,2,4]triazines 14, dihydro-5H-pyrano[2,3-d]pyrimidines 15, purines,
pyrrolo[2,3-d]pyrimidines,
pyrrolo[3,2-d]pyrimidines,
thieno[2,3-d]pyrimidines,
16
thieno[3,2-d]pyrimidines, and furo[3,2-d]pyrimidines were explored. Although some
derivatives displayed high inhibitory activity 17, the drug-like properties were unsatisfied
18. Considering the big molecular planarity and high aromaticity of these fused rings, our
drug design interest was again turned back to the 4-(2-pyrimidinylamino)benzamide
scaffold, which featured with decreased planarity and aromaticity 19, 20
.
The introduction of fluorine element into the bioactive small molecules is a very
useful drug design strategy. Many drugs contain the fluorinated substructures such as
4

gefitinib, sunitinib, lapatinib, afatinib 21. In fact, the C-F bond has specific bioactivity for
its special size and electronegativity. Fluorine containing compounds generally
demonstrate better metabolic stability and membrane permeability than the corresponding
fluorine-free ones. Considering these knowledge and in connect to our drug discovery
22
program of new Hh signaling pathway inhibitors , we intend to introduce fluorine
element to the 4-(2-pyrimidinylamino)benzamide backbone. Herein, we describe the a
series of novel Hh signaling pathway inhibitors which was fluorine containing
4-(2-pyrimidinylamino)benzamide derivatives (Figure 2).
Figure 2. The design of a series of fluorine containing 4-(2-pyrimidinylamino)benzamides
The
synthetic
routes
for
the
novel
fluorine
containing
4-(2-pyrimidinylamino)benzamides were outlined in Scheme 1. In the presence of
triethylamine (or sodium carbonate), the commercially available 2,4-dichloropyrimidine
10 was reacted with corresponding boronic reagents using Pd catalyst to give Suzuki
coupling product 11a and 11c-g, while intermediate 11b was prepared by Stille coupling
reaction under microwave irradiation. Subsequently, Buchwald-Hartwig coupling of 11
with 4-amino-3-fluorobenzoic acid under basic condition afforded fluorine containing 12.
Condensation 12 with corresponding 3-aminobenzyl alcohol generated 13. Chlorination
of 13 with thionyl chloride provided 14, which was then reacted with corresponding
secondary amines to yield target compounds 15a-15l. 15m-15n were prepared by
intermediate 13a. Condensation of 12a with 5-hydroxy-2-methylaniline gave 16,
following by O-alkylation to afford compound 15m-15n (Scheme 1).
5

Scheme 1. Reagents and conditions: (a) Pd(PPh3)2Cl2, NEt3 or 2M aq Na2CO3, dioxane, reflux, 14h,
34-77 %; 4-trifluoromethoxyphenylboronic acid for 11a and 11d; 2-(tributylstannyl)pyridine,
Pd(PPh3)4, toluene, N2, microwave 150 oC, 20 min, for 11b; phenylboronic acid for 11c;
4-Pyridinylboronic acid for 11e; 3-Pyridinylboronic acid for 11f; 1-methyl-1H-pyrazole-4-boronic
acid pinacol ester for 11g; (b) 4-amino-3-fluorobenzoic acid, Pd(OAc)2, BINAP, Cs2CO3, dioxane,
reflux, 16 h, 20-88 %; (c) 3-Amino-4-methylbenzyl alcohol, HATU, DMF, DIPEA, 80 oC
15h,13-92 %; (e) secondary amine, SOCl2, CH2Cl2, rt, 5h, 36-96 %; (f) 5-Hydroxy-2-methylaniline,
HATU, DMF, DIPEA, 80 oC 15h, 81 %; (g) R4Cl, DMF, K2CO3, 80 oC, 5h, 30-97 %.
Thus, the fluorine containing 4-(2-pyrimidinylamino)benzamide analogues 15a-n were
illustrated in Table 1. All targeted compounds were test for their Hh signaling pathway
inhibition by a luciferase reporter in NIH3T3 cell carrying a stably transfected
Gli-reporter construct (Gli-luciferase reporter cell lines) and vismodegib was used as the
positive control. The IC50 data in Table 1 showed all fluorine containing analogues of
4-(2-pyrimidinylamino)benzamides exhibited good Hh signaling pathway inhibitory
6

activity with IC50 values range from 0.050 nM to 48.85 nM. Based on our previously
SAR understanding, the hydrophilic substituent of R4 at D-ring was initiatively
investigated in the fluorine containing 4-(2-pyrimidinylamino)benzamides. It was found
that all the aliphatic tertiary amine side chain including dimethylamino (15a, 0.78 nM),
diethylamino (15b, 0.90 nM), pyrrolidinyl (15c, 2.03 nM), piperidyl (15d, 0.94 nM),
N-methyl piperazinyl (15e, 0.93 nM) and morpholinyl (15f, 0.50 nM) afforded more
potent than vismodegib (7.2 nM). Especially, the morpholinyl (15f) displayed the
strongest potency in the subclass, 14-fold more than vismodegib. Subsequently, the
alkyl-bridged morpholinyl derivatives with ethoxy and propoxy were also explored. It
showed that morpholinylethoxy (15m, 0.71 nM) and morpholinylpropoxy (15n, 1.02 nM)
also showed very potent inhibition. This results seem that the variable spacer length
between D-ring and hydrophilic substituent of R4 were tolerable. The change of R2 group
was attempted to reduce the molecule weight and the liposolubility. The 2-pyridyl group
(15g, 1.15 nM) showed decreased potency compared to 15f, while the phenyl group (15h)
showed an IC50 value of 0.05 nM, which was 10-fold more potent than 15f and
approximately 150-fold than vismodegib, was the most potent inhibitory activity in the
class. Additionally, to determine combination influence of multiple variable groups at R1,
R2, R3 and R4 on the inhibitory activity, several compounds (15i, 15j, 15k and 15l) were
synthesized. However, all of four compounds displayed decreased inhibitory activity
compared to the above compounds. Among these, compound 15j (42.69 nM) and 15l
(48.85 nM) gave the weakest inhibitory activity, which was less potent than vismodegib,
while compound 15i (5.67 nM) and 15k (7.20 nM) afforded comparable activity to
vismodegib. On the whole, based the above discussion, the introduction of fluorine to
3-position of phenyl group of 4-(2-pyrimidinylamino)benzamides can lead to potent Hh
signaling pathway inhibition. Noticeably, the phenyl group in R2 position and morpholine
group in R4 give rise to the most potent inhibitory activity (IC50 = 0.05 nM) in this
fluorine containing 4-(2-pyrimidinylamino)benzamide series. This study suggests that the
introduction of fluorine element is a very used drug-design strategy in the discovery of
novel 4-(2-pyrimidinylamino)benzamide-based Hh signaling pathway inhibitors.
Table 1. Hh signaling pathway inhibition of 4-(2-pyrimidinylamino)benzamide derivatives
7

Gli-luc reporter
IC50 (nM)
compds
R1
R2
R3
R4
CH3
CH3
0.78
15a
CH3
CH3
CH3
CH3
CH3
CH3
0.90
15b
15c
15d
2.03
0.94
CH3
CH3
0.93
15e
CH3
CH3
CH3
CH3
CH3
CH3
0.50
1.15
15f
15g
15h
0.050
F
F
CH3
H
5.67
15i
15j
42.69
H
H
H
7.20
48.85
0.71
15k
15l
H
CH3
CH3
CH3
15m
CH3
-
1.02
7.2
15n
-
-
vismodegib
8

After establishing structure-activity relationship, many compounds displayed
outstandingly potent Hh inhibitory activities. In particular, seven compounds (15a, 15b,
15d, 15e, 15f, 15h, and 15m) showed IC50 values < 1 nM, which was
approximately >8-fold more effective than the launched drug vismodegib. Considering
compound 15e having a N-methyl piperazinyl group in its chemical structure which
might be easily salified under acidic condition, therefore, compound 15e was picked out
as a representative in this series to evaluate its pharmacokinetic properties in SD rats by
23
dosed iv (1 mg/kg) and po (5 mg/kg) administration in different formulated condition .
As shown in Table 2, compound 15e showed a much long half-life (t1/2 = 14.89 h) and
large volume of distribution (Vz = 71687.7 mL/kg) which might attribute to the fluorine
effect. Otherwise, compound 15e displayed ordinary plasma exposure (AUC(0-t)
=
956.17 hr×ng/mL) and high clearance (Cl = 3342.38 mL/hr/kg) suggesting further
structural optimization should concentrate on the pharmacokinetic properties.
Table 2. Pharmacokinetic properties for compound 15e in SD rats
Dose
(mg/kg)
1 (iv)a
T1/2
(h)
Cmax
AUC(0-t)
Vz
Cl
MRT(0-t)
(h)
F
(%)
compds
(ng/mL) (hr×ng/mL) (mL/kg) (mL/hr/kg)
13.52 224.97
383.25
37425.8
1933.41
7.93
5 (po)a
5 (po)b
15e
14.89
11.71
62.42
57.11
956.17
882.40
71687.7
69464.7
3342.38
4121.86
10.71
10.27
-
-
a Compound was formulated using 5 % DMA + 5 % Tween-80, b Compound was formulated using 0.5%
HPMC+0.3% Tween-80, pH = 2.8.
In conclusion, a new series of fluorine containing 4-(2-pyrimidinylamino)benzamide
analogues were designed and synthesized. The Hh signaling inhibitory activities for all
compounds were evaluated by a luciferase reporter method. The preliminary SAR was
discussed and most of compounds displayed more potent Hh signaling inhibitory
activities than positive control vismodegib. Compound 15h showed the most potent
activity, with an IC50 value of 0.050 nM. Our findings show the introduction of fluorine
element to 4-(2-pyrimidinylamino)benzamide can lead to potent Hh signaling pathway
inhibition. This study provides a new thought in designing novel potent anticancer agents
especially in the Hh signaling pathway inhibitors.
9

Acknowledgments
This work was partly supported by Shaanxi Province Postdoctoral Science
Foundation and New Teachers Supporting Program of Xi’an Jiaotong University.
Supplementary data
Supplementary data associated with this article can be found, in the online version.
References and notes
1. Ruch, J.M.; Kim, E.J. Drugs. 2013, 73(7), 613.
2. Xin, M. Expert Opin. Ther. Pat. 2015, 25(5), 549.
3. Tremblay, M.R.; Nevalainen, M.; Nair, S.J.; Porter, J.R.; Castro, A.C.; Behnke,
M.L.; Yu, L.C.; Hagel, M.; White, K.; Faia, K.; Grenier, L.; Campbell, M.J.; Cushing,
J.; Woodward, C.N.;Hoyt, J.; Foley, M.A.; Read, M.A.; Sydor, J.R.; Tong,
J.K.; Palombella, V.J.; McGovern, K.; Adams, J. J. Med. Chem. 2008, 51(21), 6646.
4. Tremblay, M.R.; Lescarbeau, A.; Grogan, M.J.; Tan, E.; Lin, G.; Austad, B.C.; Yu,
L.C.; Behnke, M.L.; Nair, S.J.; Hagel, M.; White, K.; Conley, J.; Manna,
J.D.; Alvarez-Diez, T.M.; Hoyt, J.; Woodward, C.N.; Sydor, J.R.; Pink,
M.; MacDougall,
J.; Campbell,
M.J.; Cushing,
J.; Ferguson,
J.; Curtis,
M.S.; McGovern, K.; Read, M.A.; Palombella, V.J.; Adams, J.; Castro, A.C. J. Med.
Chem. 2009, 52(14), 4400.
5. Banerjee, U.; Hadden, M.K. Expert Opin. Drug Discov. 2014, 9(7), 751.
6. Robarge, K.D.; Brunton, S.A.; Castanedo, G.M.; Cui, Y.; Dina, M.S.; Goldsmith, R.;
Gould, S.E.; Guichert, O.; Gunzner, J.L.; Halladay, J.; Jia, W.; Khojasteh, C.; Koehler,
M.F.; Kotkow, K.; La, H.; Lalonde, R.L.; Lau, K.; Lee, L.; Marshall, D.; Marsters, J.C.
Jr.; Murray, L.J.; Qian, C.; Rubin, L.L.; Salphati, L.; Stanley, M.S.; Stibbard, J.H.;
Sutherlin, D.P.; Ubhayaker, S.; Wang, S.; Wong, S.; Xie, M. Bioorg. Med. Chem. Lett.
2009, 19(19), 5576.
7. Pan, S.; Wu, X.; Jiang, J.; Gao, W.; Wan, Y.; Cheng, D.; Han, D.; Liu, J.; Englund,
N.P.; Wang. Y.; Peukert, S.; Miller-Moslin, K.; Yaun, J.; Guo, R.; Matsumoto, M.;
Vattay, A.; Jiang, Y.; Tsao, J.; Sun, F.; Pferdekamper, A.C.; Dodd, S.; Tuntland, T.;
Wieslawa, M.; Kelleher, J.F.; Yao, Y.; Warmuth, M.; Williams, J.; Dorsch, M. ACS
Med. Chem. Lett. 2010, 1(3), 130.
8. Wang, C.; Wu, H.; Katritch, V.; Han, G.W.; Huang, X.P.; Liu, W.; Siu, F.Y.; Roth,
B.L.; Cherezov, V.; Stevens, R.C. Nature 2013, 497(7449), 338.
9. Munchhof, M.J.; Li, Q.; Shavnya, A.; Borzillo, G.V.; Boyden, T.L.; Jones,
C.S.; LaGreca,
S.D.; Martinez-Alsina,
L.; Patel,
N.; Pelletier,
K.; Reiter,
L.A.; Robbins, M.D.;Tkalcevic, G.T. ACS Med. Chem. Lett. 2012, 3(2), 106.
10. Kim, J.; Tang, J.Y.; Gong, R.; Kim, J.; Lee, J.J.; Clemons, K.V.; Chong, C.R.; Chang,
K.S.; Fereshteh, M.; Gardner, D.; Reya, T.; Liu, J.O.; Epstein, E.H.; Stevens, D.A.;
Beachy, P.A. Cancer Cell 2010, 17(4), 388.
11. Rocconi, R.P.; Samant, L. WO943255, 2013.
10

12. Yauch, R.L.; Dijkgraaf, G.J.; Alicke, B.; Januario, T.; Ahn, C.P.; Holcomb, T.; Pujara,
K.; Stinson, J.; Callahan, C.A.; Tang, T.; Bazan, J.F.; Kan, Z.; Seshagiri, S.; Hann,
C.L.; Gould, S.E.; Low, J.A.; Rudin, C.M.; de Sauvage, F.J. Science 2009, 326(5952),
572.
13. Xin, M.; Wen, J.; Tang, F.; Tu, C.; Shen, H.; Zhao, X. Bioorg. Med. Chem. Lett. 2013,
23(24), 6777.
14. Xin, M.; Zhang, L.; Tang, F.; Tu, C.; Wen, J.; Zhao, X.; Liu, Z.; Cheng, L.; Shen, H.
Bioorg Med Chem 2014, 22(4), 1429.
15. Xin, M.; Zhang, L.; Shen, H.; Wen, J.; Tu, C.; Liu, Z.; Cheng, L.; Zhao, X. Med.
Chem. Res. 2014, 23(8), 3784.
16. Zhang, L.; Xin, M.; Shen, H.; Wen, J.; Tang, F.; Tu, C.; Zhao, X.; Wei, P. Bioorg. Med.
Chem. Lett. 2014, 24(15), 3486.
17. Xin, M.; Wen, J.; Tang, F.; Tu, C.; Huang, W.; Shen, H.; Zhao, X.; Cheng, L.; Wang,
M.; Zhang, L. Bioorg. Med. Chem. Lett. 2014, 24(3), 983.
18. Zhang, L.; Xin, M.; Wen, J.; Tang, F.; Tu, C.; Shen, H.; Wei, P. Chin. J. Org. Chem.
2014, 34(7), 1407.
19. Xin, M.; Zhang, L.; Jin, Q.; Tang, F.; Wen, J.; Gu, L.; Cheng, L.; Zhao, Y. Eur. J. Med.
Chem. 2016, 110,115.
20. Xin, M.; Zhang, L.; Wen, J.; Shen, H.; Liu, Z.; Zhao, X.; Wang, M.; Cheng, L.;
Huang, W.; Tang, F. Bioorg. Med. Chem. 2016, 24(5), 1079.
21. Wang, J.; Sanchez-Rosello, M.; Acena, J.L.; del Pozo, C.; Sorochinsky, A.E.; Fustero,
S.; Soloshonok, V.A.; Liu, H. Chem. Rev. 2014, 114(4), 2432.
22. Lou, Y.; Sweeney, Z.K.; Kuglstatter, A.; Davis, D.; Goldstein, D.M.; Han, X.; Hong,
J.; Kocer, B.; Kondru, R.K.; Litman, R.; McIntosh, J.; Sarma, K.; Suh, J.; Taygerly, J.;
Owens, T.D. Bioorg. Med. Chem. Lett. 2015, 25(2), 367.
23. The experimental description of in vivo pharmacokinetic evaluation was found in the
supporting information.
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Graphical Abstract
Introduction of Fluorine to Phenyl Group of 4-(2-Pyrimidinylamino)benzamides
Leading to a Series of Potent Hedgehog Signaling Pathway Inhibitors
Minhang Xin a, b, *, Liandi Zhang b, Jun Wen b, Han Shen b, Xinge Zhao b, Qiu Jin b, Feng
Tang b
a Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an
Jiaotong University, No 76, Yanta West Road, Xi’an 710061, P.R. China
b
Jiangsu Simcere Pharmaceutical Co. Ltd., No 699-18, Xuan Wu District, Nanjing
210042, P.R. China
12