Bioorganic & Medicinal Chemistry Letters 23 (2013) 6777–6783
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
The discovery of novel N-(2-pyrimidinylamino) benzamide derivatives as potent hedgehog signaling pathway inhibitors Minhang Xin ⇑, Jun Wen, Feng Tang, Chongxing Tu, Han Shen, Xinge Zhao Jiangsu Simcere Pharmaceutical Co. Ltd, Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No. 699-18, Xuan Wu District, Nanjing 210042, PR China
a r t i c l e
i n f o
Article history: Received 6 August 2013 Revised 23 September 2013 Accepted 9 October 2013 Available online 17 October 2013 Keywords: Hedgehog signaling Hedgehog signaling pathway inhibitors Synthesize Lead compound
a b s t r a c t Hedgehog signaling pathway inhibitors are emerging as new therapeutic intervention against cancer. A novel series of N-(2-pyrimidinylamino) benzamide derivatives as hedgehog signaling pathway inhibitors were designed and synthesized. Most compounds presented significant inhibitory effect on hedgehog signaling pathway, among which 21 compounds exhibited more potent than vismodegib. Furthermore, compound 6a showed moderate pharmacokinetic properties in vivo, representing a promising lead compound for further exploration. Ó 2013 Elsevier Ltd. All rights reserved.
The Hedgehog (Hh) pathway is a very important signaling pathway which is critical to cell proliferation and control of survival signals in embryonic development and tissue patterning, and critical to maintenance of stem cells and acquisition of epithelialto-mesenchymal transition (EMT) in adults.1 The process of Hh signaling activation has been widely described. Signaling activation on Hh pathway is initiated by ligands-induced stimulation of membrane associated receptor patched (Ptch). The binding of extracellular Hh family proteins, such as Sonic Hh (Shh), Desert Hh (Dhh) and Indian Hh (Ihh) to Ptch, relieves the suppressed GPCR-like receptor smoothened (Smo), which moves into the primary cilia to promote the dissociation of the complex of Suppressor of fused (Sufu) and Glioma-associated oncogene homologue (Gli), and activates Gli. The activated Gli then triggers downstream signals and contributes to cellular proliferation, differentiation and survival.2 Despite Hh pathway is silent in most adult tissues, abnormal activation of it contributes to uncontrolled cell growth and leads to malignant transformation, which has been commonly observed in a number of cancer types.3 Hence, Hh signaling pathway inhibition has been regarded as an important therapeutic strategy for cancer treatment. To date, two mechanisms for Hh signaling aberrantly activated in cancers have been elucidated. One is mutation-driven manner through mutation of members of the pathway such as Ptch-mutation, Smo-mutation, and Sufu-mutation, leading to tumorigenesis
⇑ Corresponding author. Tel./fax: +86 25 85560000 3192. E-mail address: [email protected] (M. Xin). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.022
involving basal cell carcinoma (BCC), medulloblastoma, and rhabdomyosarcoma. The other is Hh ligand-driven manner in which the overexpression of Hh ligand induced by autocrine or paracrine stimulation results in tumor aggravation including colorectal, esophageal, gastric, hepatocellular, pancreatic, prostate, lung, ovarian, glioblastoma, melanoma, lymphoma, leukemia, and so on.4 Besides, there are several evidences suggested that Hh signaling activation may influence cancer stem cells which is responsible for cancer recurrence.5 These research results demonstrated that Hh signaling pathway inhibitor not only can provide significant clinical benefit for BCC treatment, but also has good promising potential combating a number of other cancers for a single or combinational therapy. During these years, many novel types of Hh pathway inhibitors are being studied.6 Vismodegib was the first approved Hh pathway inhibitor by FDA in 2012 for use in metastatic BCC treatment.7 Moreover, several other synthetic small molecule inhibitors are advancing into clinical trials, including Novartis’s NVP-LDE225 (sonidegib, Phase III), Lilly’s LY-2940680 (Phase II), BMS’s BMS833923 (XL-139, Phase II), Infinity’s saridegib (IPI-926, discontinued in 2012), Pfizer’s PF-04449913(Phase I), Takeda’s TAK-441 (Phase I) and Novartis’s NVP-LEQ506 backup for sonidegib (Phase I) (Fig. 1).8 Nowadays, these represented drugs are being investigated on other haematological malignancies and solid tumors, such as BCC, medulloblastoma, leukemia, pancreatic, gastric, breast, and small-cell lung cancers in Phase I, Phase II and Phase III,8 which raises researchers’ more confidence about the therapeutic potential of targeting Hh. Since there are already a lot of Hh inhibitors structures published, we aimed to discover new Hh signaling pathway inhibitors with improved inhibition in order to amplify
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Cl
O
N
H N
Cl
N H
O S O
vismodegib (GDC-0449)
N
NH
N
O
O LY-2940680
BMS-833923 (XL-139)
Lilly Ph II
Exelixis /BMS Ph II
H
O HN
N
N OH
N
N N
N
CN
O
S
O
H
N H
NVP-LEQ506 Novartis Ph I
TAK-441 Millennium /Takeda Ph I
O H
H
OH
CF3 PF-04449913 Pfizer Ph I
HN
N
O
O
N N
CF3
O
N
NH
N
F
N H
Novartis Ph III
O N H
N
O
N
H N
N H
N
sonidegib (NVP-LDE-225;LDE-225)
Curis/Genentech (Roche) launched
N
N
O
O CF3
N N
O N
H
IPI-926 Infinity (dicontinued)
Figure 1. Chemical structures of FDA-approved and clinical Hh signaling inhibitors.
O
O
CF3
CF3
CF3 from ALLO-2 fragment
N N
O
H N N
N H ALLO-2
O
N H CF3
O
N N O
O
1b
N
N
N N
1a
from LDE-225 fragment
N
H N O
H N N
N N
N H CF3
N
from BMS-833923 similar fragment
LDE-225
O N H
N N
6a
N H
Figure 2. A design for N-(2-pyrimidinylamino) benzamide scaffold.
antitumor profiles in the future. Herein we describe the successful identification of a novel series of N-(2-pyrimidinylamino) benzamide derivatives. On the basis of deeply understanding the structure–activity relationship (SAR), fragment-based design and screening is always highly attractive design tool in traditional medicinal chemistry exploration.9 Recently, a compound containing pyrimidinylamino unit named ALLO-2 was reported to show good Hh signaling inhibition with distinct binding mode which could inhibit the D473H mutant Smo.10 The pyrimidinylamino group was considered as a privileged structure commonly occurring in drugs such as imatinib, nilotinib and so on. Consequently, we attempted to synthesize some compounds containing 4-(trifluoromethoxy)phenyl pyrimidinylamine moiety and change the structural fragment such as indazole, pyridinylmorpholine, phenylbenzamide to bear molecular diversity and complexity for scaffold-detection (Fig. 2). The target scaffold N-(2-pyrimidinylamino) benzamide possessed outstanding advantage and the compound 6a demonstrated leading inhibitory activity with an IC50 of 1.3 nM in a Gli-luciferase reporter assay,11 better than GDC-0449 (7.2 nM) and LDE-225 (5.5 nM) (Table 1). Encouraged by this excellent potency, a systematic study of SAR exploration was carried out for understanding the structural modification window for future optimization. Initially, compound 6a was divided into four regions: 4-trifluromethoxylphenyl (A), pyrimidinyl (B), 4-aminobenzamide (C) and 2, 6-dimethylphenyl groups (D) (Fig. 3). At the outset, our focus was on exploring modifications to A moiety, including the substitutions on the aromatic residue and alkyl instead. It was important to detail the contribution of the diverse modification of A moiety influ-
Table 1 Identification of pioneer Hh signaling inhibitors Compounds
Structures
Gli-luciferase reporter IC50 (nM)
OCF3
1a N
125
H N
N
N N H
OCF3
O
1b
N
N N
N H OCF3
N N GDC-0449 LDE-225
N
O
6a
>500
1.3
N H N H 7.2 5.5
encing the activity. Besides, it was worth investigating the fragment B pyrimidinyl core. An attractive option was to test the
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OCH 3
OCF3 F O CN
N N
OCF3 F
N
N CF 3
O
N
A moitey A N
D moiety O
N N
N
N B
O
N
C
N
N
D
N
N
N H N
N H
Bn
C moiety
B moiety
N
N
N
N
N
N
N
F
HN
F3C
O
N N
N
N H
N N
N
N
N
NH
N N
N
O
N
Figure 3. Designed structural modification of N-(2-pyrimidinylamino) benzamide derivatives.
OCF3
OCF3
OCF3 Cl N N 2
a 73%
Cl
ArNH 2
N
B(OH)2 N
Cl
c
3a
a (for 3a-3n) b (for 3o-3p) R1
R1
N
Cl
3a-3p
Ar N H 1a (43%, Ar=1H-indazol-5-yl ) 1b (70%, Ar=6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl) 1c (62%, ArNH 2=N-(4-aminophenyl)-2-methylbenzamide) R1
O
R1
O
e
d
N
N N
N
N
OCH3
N N H 4a-4p
N
N H 5a-5p
OH
f
O N H
N N
N H 6a-6p
Scheme 1. Reagents and conditions: (a) 4-trifluoromethoxyphenylboronic acid for 3a, 3-trifluoromethoxyphenylboronic acid for 3b, 2-trifluoromethoxyphenylboronic acid for 3c, 3-fluoro-4-trifluoromethoxyphenylboronic acid for 3d, 4-cyanophenylboronic acid for 3e, 4-morpholinophenyl boronic acid for 3f, 4-fluorophenylboronic acid for 3g, 4-methylphenylboronic acid for 3h, phenylboronic acid for 3i, pyridine-4-boronic acid for 3j, pyridine-3-boronic acid for 3k, 1-methyl-1H-pyrazole-4-boronic acid pinacol ester for 3l, 3-furanboronic acid for 3m, Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 6–12 h; cyclopropylboronic acid for 3n, Pd(dppf)Cl2, TEA, DMF, H2O, 100 °C for 6 h; 43–97%; (b) CH3MgCl for 3o, BnMgCl for 3p, Fe(acac)3, dry THF, 0 °C to rt,12 h; 77%, 58%; (c) 1H-indazol-5-amine for 1a, 6-((2S,6R)-2,6-dimethylmorpholino) pyridine-3-amine for 1b, N(4-aminophenyl)-2-methylbenzamide for 1c, n-BuOH, p-toluenesulfonic acid, 200 °C microwave for 2 h; 43%, 70%; (d) methyl 4-aminobenzoate, Pd(OAc)2, BINAP, Cs2CO3, dioxane, 170 °C, microwave for 3 h; 38–89%; (e) NaOH, MeOH/H2O, reflux overnight; 85–100%; (f) 2,6-dimethylaniline, HATU, DIPEA, DMF, 85 °C overnight; 40–95%.
substituent group and to acquire the role of nitrogen of pyrimidinyl. Subsequently, some replacements of moiety C and D were made to identify the applicability of these functionality changes (Fig. 3). As summarized in Figure 3, up to 39 compounds (1a–1c, 6a–6p, 15a–15j, 18a, 19a–19g, 20a and 20o) were synthesized.12 The general synthetic methods to build the designed compounds were illustrated in Schemes 1–3. The preparation compounds 1a–1c involved two transformations, Suzuki coupling and primary arylamine substitution (Scheme 1). A library compounds modification on A region were synthesized began with Suzuki coupling (3a–3n) under Pd catalyst and Kumada coupling (3o–3p) under Fe(acac)3, followed by Buchwald coupling (4a–4p) and hydrolysis to generate free acid (5a–5p), subsequently acylation with anilines to afford target compounds 6a–6p (Scheme 1). The similar method used to build the pyrimidinyl derivatizing was based on commercially or readily available starting materials dichloropyrimidines and halogeno pyridines. One key building block 11a used in this study was prepared according to literature
method13 (Scheme 2A). The synthesis of target compounds 15a–15i employed the similar procedures as described above for the preparation of 6a–6p (Scheme 2B). While 15j was prepared using modified steps shown in Scheme 2C, mainly involving reactions of substitution on position 2 and followed Suzuki coupling on position 4, and subsequently the same operation as formerly employed to yield compound 15j (Scheme 2C). The synthesis of compounds of structural alteration on C and D moiety was described on Scheme 3. The synthesis of 18a originated with former intermediate 4a, which was converted to 18a after three steps of methylation, hydrolysis and condensation in turn. Compounds 19a–19g were installed by condensation reactions with free acid 5a and readily available anilines. Compounds 22a and 22o were synthesized using N-acylation, chlorination and nucleophilic substitution as depicted in Scheme 3. The Hh signaling pathway inhibitory activity of synthetic compounds were assessed using a luciferase reporter in NIH3T3 cell carrying a stably transfected Gli-reporter construct (Gli-luciferase reporter cell lines). Table 2 outlined the in vitro IC50 for a series of A-moiety structural modification of N-(2-pyrimidinylamino) benz-
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M. Xin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6777–6783 O
O
(A)
O
a
O
O
N H 9
23%
O
90%
7 (B)
O c
NH
b
8
S
N H 10
70%
N
O
N
90%
Cl
11a OCF3
OCF3
OCF3
OCF3
Cl d
NH
Cl(Br) R2
Y
OCF 3 R
Cl R2 11a-11i
f
R2
Cl
R3
X
R
e
B(OH) 2 12
Y
3
X
R2
h OCH 3
Y X
12a-12i
O
O
O 3
OH
Y
R3
N H 13a-13i
X
R2
i
14a-14i
OCF3
N H
Y
R3
N H
X
N H 15a-15i
OCF3
OCF3
(C) Cl
Cl g
F3C
N N
Cl
O
O
e
F3C
N
49%
N
11j
F3C
O
N
h O
N
N H
12j
N
13j
O
i OH
N
58%
62%
N H
O F3C
F 3C
N H
N
29%
N H
N
N H 15j
14j
Scheme 2. Reagents and conditions: (a) ethyl formate, THF, LDA, 78 °C to rt, 2 h, 90%; (b) thiocarbamide, MeOH, reflux, overnight, 23%; (c) chloroacetic acid, EtOH/H2O, reflux, 7 h, 70%; (d) POCl3, DMF, 100 °C, 2 h, 90%; (e) 2,4-dichloro-5-cyclopropylpyrimidine for 12a, 2,4-dichloro-5,6-dimethylpyrimidine for 12b, 2,4-dichloropyrimidine for 12c, 2,4-dichloro-6-methylpyrimidine for 12d, 2,4-dichloro-5-fluoro pyrimidine for 12e, 2,4-dichloro-6-propylpyrimidine for 12f, 2,4-dichloro-5-ethylpyrimidine for 12g, 4bromo-2-chloropyridine for 12h, 2-bromo-6-chloropyridine for 12i, Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 6–12 h; 40–89%; (f) methyl 4-aminobenzoate, Pd(OAc)2, BINAP, Cs2CO3, dioxane, 170 °C, microwave for 3 h; 62–88%; (g) methyl 4-aminobenzoate, DIPEA, t-BuOH, rt, overnight; 49%; (h) NaOH, MeOH/H2O, reflux overnight; 58–95%; (i) 2,6dimethylaniline, HATU, DIPEA, DMF, 85 °C overnight; 26–73%.
OCF3
OCF3 O
N N
O
a O
N H
N OCF3
4a
O
N
N OCF3
N N
N
5a, 5o
N
N
N H N 18a
R4
N H 19a-19g
e
1
N N
R1
O
R
OH N H
O N
23%
17
N H
N
N H
O
OH
O
d OH
5a R1
c
O N
95%
N 16
N
b O
N
50%
OCF3
OCF3
N H N H
O g
f N H
N OH
20a, 20o
N
N H 21a, 21o
R1
O N
Cl N H 22a, 22o
N H
N
Bn
N
a (R 1=4-trifluoromethoxyphenyl) o (R 1=methyl)
Scheme 3. Reagents and conditions: (a) MeI, DMF, K2CO3, 60 °C, 12 h, 50%; (b) NaOH, MeOH/H2O, reflux overnight; 95%; (c) HATU, DIPEA, DMF, 85 °C overnight; 23%; (d) otoluidine for 19a, 2-methyl-5-(1-methyl-1H-pyrazol-4-yl)aniline for 19b, 4-((4-methylpiperazin-1-yl)methyl)aniline for 19c, 2-((4-methylpiperazin-1-yl)methyl)aniline for 19d, 3-((4-methylpiperazin-1-yl)methyl) aniline for 19e, isopropyl amine for 19f, cyclohexylamine for 19g, HATU, DIPEA, DMF, 85 °C overnight; 39–78%; (e) 3-amino-4methylbenzyl alcohol, HATU, DMF, DIPEA, 85 °C overnight; 20a, 94%; 20o, 60%; (f) SOCl2, CH2Cl2, rt, 2 h; 21a, 64%; 21o, 85%; (g) MeBnNH, DMF, K2CO3, rt, overnight; 22a, 78%; 22o, 43%.
amides. Substituents on phenyl of the region A played an essential role in regulating Hh signaling inhibitory activity. SARs suggested the para-OCF3 and meta-OCF3 substituent showed comparably good inhibition, while ortho-OCF3 led to a significant loss of activity (6a, 6b vs 6c). This clear SAR trend appeared introducing one electronwithdrawing group adjacent to 4-position or 3-position of A-ring was a preference. However both para-OCF3 and meta-F disubstituents had weak inhibitory activity (6a, 6b vs 6d), indicating that adding another electrophilic group could be harmful to activity. A survey of some substituents at para-position was investigated and led to a few favorable electron-withdrawing substituents including –CN (6e), –F (6g). Although compound 6i was lack of electrophilic group at para-position, it showed tolerable. Compared with these, morpholino (6f) produced moderate activity likely due to the steric
hindrance at para-position, and methyl (6h) showed no activity almost because of methyl serving as electron-donating group. Replacement of phenyl with 4-pyridinyl (6j) appeared tolerant, but an attempt of 3-pyridinyl (6k) was unsuccessful, which appeared the position of the nitrogen in the pyridine was critical for the inhibitory activity. Other heteroaryl such as pyrazolyl and furyl demonstrated less potency. Replacement of phenyl with alkyl such as cyclopropyl, methyl and benzyl was undesirable (6n, 6o, 6p) because of no A-ring aromaticity directly linked to B-ring. In this study, 6 compounds showed better inhibitory activity than positive control GDC-0449, and the essential SAR offered some clear substituents at the A-ring for future optimization. Table 3 summarized IC50 of compounds with substituents on the central pyrimidinyl ring (region B). Integral SARs suggested that a
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R1
Table 4 In vitro inhibition of Hh signaling pathway for compounds modification on C and D region of N-(2-pyrimidinylamino) benzamides
OCF3
O N H
N N
O
N H 6a-6p
N
R1
Compds 6a
N H
N
Gli-luc reporter IC50 (nM)
Compds
R4
N H
R4
Gli-luc reporter IC50 (nM)
1.3
F3CO
6b
1.2
6a
1.3
F3CO
OCF3
>500
6c OCF3
6d
O
18a N
F3CO
158.5
N
F
6e
NC
6f
O
6g
F
6h
H3C
N
OCF
2.8
3
20.2
N
1c
6i
1.3
O
N H
>500 19a
1.0
0.54
N
19b 6k
N N
10.6
O
6m
N
19c
118.3 N
15.7
6n
1.2
N N
>500
N
6l
42.1
H N
N
2.2 N
6j
2.7 N H
32.8
H3 C
6o
151.5
2.3
19d N
N
34.3
6p GDC-0449
7.2
353.6
19e N
Table 3 In vitro inhibition of Hh signaling pathway for compounds modification on B region of N-(2-pyrimidinylamino) benzamides
N
19f
73.1
19g
2.2
22a
N
OCF3
1.1 Bn
O O R2 R3
Y X
N
N H N
GDC-0449
Compds
R2
R3
X
Y
6a
Me
H
N
N
1.3
H
N
N
1.3
Me H Me H Pr H H H H
N N N N N N N C N
N N N N N N C N N
1.6 1.2 1.4 1.6 0.8 5.3 1.6 23.4 0.53 7.2
15a 15b 15c 15d 15e 15f 15g 15h 15i 15j GDC-0449
Me H H F H Et H H CF3
N
22o
N H
N
Bn
100.6
N H
7.2
Gli-luc reporter IC50 (nM)
small hydrophobic group, such as –H, –F, –Me, –CF3, –Et, –Pr, cyclopropyl (15a, 15b, 15c, 15d, 15e, 15f, 15g, 15j), was favored at the 5- or 6-positions of pyrimidinyl, all of which showed comparable activity to 6a. It appeared to be a presence of hydrophobic cavity for suitable size, which even allowed the propyl group incorporated in. Interestingly, it is noticeable that compound 15h showed equipotent activity to 15c, while compound 15i led to a significant loss of activity. This appreciable drop demonstrated the 1position of the nitrogen in pyrimidinyl ring played a more important role than 3-position of it for modulating Hh signaling inhibi-
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Table 5 Pharmacokinetic properties for represented compound 6a in SD rats
a
Dosea (mg/kg)
Cmax (ng/mL)
AUC (h ng/mL)
Vss (mL/kg)
Cl (mL/h/kg)
MRT(0–t) (h)
T1/2 (h)
1 (iv) 5 (po)
764 342
915 1705
14,898 27,738
1004 2797
4.0 5.1
10.3 7.0
F (%)
36
Compound 6a was formulated using 5% DMA + 5% Tween-80.
tory activity (15c vs 15h vs 15i). In this series, 9 compounds demonstrated improved Hh signaling inhibition compared to GDC0449. The SAR studies showed that hydrophobic and suitable length substituents incorporated in 5- or 6-positions exhibited considerable potential for structural modification. Table 4 depicted IC50 of N-(2-pyrimidinylamino) benzamides derivatizing on region C and D. N-Methyl analogs 18a showed potency in vitro activity nearly equal to 6a. We speculated that the free hydrogen of NH appeared to play almost no role in its potency. Based on the intrinsic observation it was evident that benzamide functionality of C region was favorable to phenyl aminoacyl of C (1c vs 6a, 19a). Exploration of D-moiety demonstrated that 3-position of phenyl bearing some N-functionality such as pyrazole, piperizine, benzylamine was tolerable (19b, 19d, 22a vs 19c, 19e), which suggested that some hydrophilic basic side chains incorporated in this position were worth to synthesized for further water-soluble optimization. Replacement of phenyl with isopropyl resulted less potency (19f), however, interestingly the activity of substituent cyclohexyl retained. Compared 22a with 22o, it was again obvious that methyl instead of A-moiety phenyl made the activity largely diminished. In this SAR exploration, 6 compounds revealed enhanced potency compared to GDC0449. The SAR exploration demonstrated hydrophilic basic side chains incorporated in D-moiety was tolerable for improved activity. Having established the initial SAR of N-(2-pyrimidinylamino) benzamide derivatives, it was found that many compounds showed enhancing inhibitory activity compared to GDC-0449. Among these compounds, 6a was chosen to study the pharmacokinetic properties in vivo. As illustrated in Table 5, 6a displayed moderate pharmacokinetic properties, with an acceptable bioavailability (F = 36%) and half-life (T1/2 = 7 h), but a large volume of distribution (Vss = 27738 mL/kg) and low oral exposure (1705 h ng/mL) (Table 5). In an attempt to optimize the pharmacokinetic properties is next due course and the work will be described in the accompanying paper. In conclusion, a novel class of N-(2-pyrimidinylamino) benzamide derivatives as Hh signaling pathway inhibitors was identified. Thorough SAR exploration identified the key functionalities necessary for potency, enabling 21 compounds with significant inhibitory activity compared to GDC-0449 to be identified. Compound 6a with good potency, distinctive structure, moderate pharmacokinetic properties, was deserved as one promising lead compound. In the light of our encouraging results, and exploiting the information arising from the SARs, the further design of highly efficient and improved pharmacokinetic N-(2-pyrimidinylamino) benzamide corebased Hh signaling pathway inhibitory agents is currently progress and will be reported in due course. Acknowledgments This work was supported by the National Major Science and Technology Project of China (Innovation and Development of New Drugs, No. 2011ZX09401-008).
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.10. 022. References and notes 1. (a) Zeng, X.; Goetz, J. A.; Suber, L. M.; Scott, W. J., Jr; Schreiner, C. M.; Robbins, D. J. Nature 2001, 411, 716; (b) Li, Y.; Maitah, M. Y.; Ahmad, A.; Kong, D.; Bao, B.; Sarkar, F. H. Exp. Opin. Ther. Targets 2012, 16, 49. 2. (a) Stone, D. M.; Hynes, M.; Armanini, M.; Swanson, T. A.; Gu, Q.; Johnson, R. L.; Scott, M. P.; Pennica, D.; Goddard, A.; Phillips, H.; Noll, M.; Hooper, J. E.; Sauvage, F.; Rosenthal, A. Nature 1996, 384, 129; (b) Tian, H.; Callahan, C. A.; DuPree, K. J.; Darbonne, W. C.; Ahn, C. P.; Scales, S. J.; Sauvage, F. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 4254. 3. Onishi, H.; Katano, M. Cancer Sci. 2011, 102, 1756. 4. (a) Rubin, L. L.; de Sauvage, F. J. Nat. Rev. Drug Disc. 2006, 5, 1026; (b) Ng, J. M.; Curran, T. Nat. Rev. Cancer 2011, 11, 493; (c) Metcalfe, C.; de Sauvage, F. J. Cancer Res. 2011, 71, 5057. 5. Merchant, A. A.; Matsui, W. Clin. Cancer Res. 2010, 16, 3130. 6. (a) Guerlet, G.; Spangenberg, T.; Mann, A.; Faure, H.; Ruat, M. Bioorg. Med. Chem. Lett. 2011, 21, 3608; (b) Malancona, S.; Altamura, S.; Filocamo, G.; Kinzel, O.; Hernando, J. I.; Rowley, M.; Scarpelli, R.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett. 2011, 21, 4422; (c) Kinzel, O.; Alfieri, A.; Altamura, S.; Brunetti, M.; Bufali, S.; Colaceci, F.; Ferrigno, F.; Filocamo, G.; Fonsi, M.; Gallinari, P.; Malancona, S.; Hernando, J. I.; Monteagudo, E.; Orsale, M. V.; Palumbi, M. C.; Pucci, V.; Rowley, M.; Sasso, R.; Scarpelli, R.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett. 2011, 21, 4429; (d) Brown, M. L.; Aaron, W.; Austin, R. J.; Chong, A.; Huang, T.; Jiang, B.; Kaizerman, J. A.; Lee, G.; Lucas, B. S.; McMinn, D. L.; Orf, J.; Rong, M.; Toteva, M. M.; Xu, G.; Ye, Q.; Zhong, W.; Degraffenreid, M. R.; Wickramasinghe, D.; Powers, J. P.; Hungate, R.; Johnson, M. G. Bioorg. Med. Chem. Lett. 2011, 21, 5206; (e) Ontoria, J. M.; Bufi, L. L.; Torrisi, C.; Bresciani, A.; Giomini, C.; Rowley, M.; Serafini, S.; Bin, H.; Hao, W.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett. 2011, 21, 5274; (f) Muraglia, E.; Ontoria, J. M.; Branca, D.; Dessole, G.; Bresciani, A.; Fonsi, M.; Giuliano, C.; Llauger Bufi, L.; Monteagudo, E.; Palumbi, M. C.; Torrisi, C.; Rowley, M.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett. 2011, 21, 5283; (g) Wang, J.; Mook, R. A., Jr.; Lu, J.; Gooden, D. M.; Ribeiro, A.; Guo, A.; Barak, L. S.; Lyerly, H. K.; Chen, W. Bioorg. Med. Chem. 2012, 20, 6751; (h) Peukert, S.; Jain, R. K.; Geisser, A.; Sun, Y.; Zhang, R.; Bourret, A.; Carlson, A.; Dasilva, J.; Ramamurthy, A.; Kelleher, J. F. Bioorg. Med. Chem. Lett. 2009, 19, 328; (i) 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, 106; (j) Peukert, S.; He, F.; Dai, M.; Zhang, R.; Sun, Y.; Miller-Mislin, K.; McEwan, M.; Lagu, B.; Wang, K.; Yusuff, N.; Bourret, A.; Ramamurthy, A.; Vattay, A.; Wang, A.; Guo, R.; Yuan, J.; Green, J.; Williams, J.; Buonamici, S.; Kelleher, J. F.; Dorsch, M. ChemMedChem 2013, 8, 1261. 7. Dlugosz, A.; Agrawal, S.; Kirkpatrick, P. Nat. Rev. Drug Disc. 2012, 11, 437. 8. https://www.thomson-pharma.com/. 9. (a) Hajduk, P. J.; Greer, J. Nat. Rev. Drug Disc. 2007, 6, 211; (b) Möcklinghoff, S.; van Otterlo, W. A.; Rose, R.; Fuchs, S.; Zimmermann, T. J.; Dominguez, Seoane. M.; Waldmann, H.; Ottmann, C.; Brunsveld, L. J. Med. Chem. 2011, 54, 2005. 10. Tao, H.; Jin, Q.; Koo, D. I.; Liao, X.; Englund, N. P.; Wang, Y.; Ramamurthy, A.; Schultz, P. G.; Dorsch, M.; Kelleher, J.; Wu, X. Chem. Biol. 2011, 18, 432. 11. (a) 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, 5576; (b) Ohashi, T.; Oguro, Y.; Tanaka, T.; Shiokawa, Z.; Shibata, S.; Sato, Y.; Yamakawa, H.; Hattori, H.; Yamamoto, Y.; Kondo, S.; Miyamoto, M.; Tojo, H.; Baba, A.; Sasaki, S. Bioorg. Med. Chem. 2012, 20, 5496. 12. Purity of all final compounds was assessed by HPLC and determined to be >95%. The structures were confirmed by 1H NMR and LC/MS. Analytic data of represented compounds 6a: MS (ESI) m/z: [M+H]+ = 493.1. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.57 (s, 1H, NH), 8.53 (s, 1H, ArH), 7.95 (m, 4H, ArH), 7.89 (d, 2H, J = 8.8 Hz, ArH), 7.57 (d, 2H, J = 8.8 Hz, ArH), 7.12 (s, 3H, ArH),
M. Xin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6777–6783 2.27 (s, 3H, ArCH3), 2.18 (s, 6H, 2ArCH3) ppm, HPLC: 97.6%; 15a: MS (ESI) m/z: [M+H]+ = 519.2. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.54 (s, 1H, NH), 8.39 (s, 1H, ArH), 7.99 (d, 2H, ArH), 7.96 (m, 4H, ArH), 7.56 (d, 2H, ArH), 7.11 (s, 3H, ArH), 2.17 (s, 6H, 2⁄CH3), 1.90 (m, 1H, cyclopropyl-CH), 0.88 (m, 2H, cyclopropyl-CH2), 0.71 (m, 2H, cyclopropyl-CH2) ppm, HPLC: 99.0%; 18a: MS (ESI) m/z: [M+H]+ = 507.2. 1H NMR (400 M, DMSO-d6) d 9.71 (s, 1H, CONH), 8.39 (s, 1H, ArH), 8.01 (d, 2H, ArH), 7.81 (d, 2H, ArH), 7.57 (d, 2H, ArH), 7.50 (d,
6783
2H, ArH), 7.12 (s, 3H, ArH), 3.57 (s, 3H, NCH3), 2.23 (s, 3H, ArCH3), 2.19 (s, 6H, 2ArCH3) ppm, HPLC: 98.0%; 19a: MS (ESI) m/z: [M+H]+ = 479.2. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.93 (s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.35 (d, 1H, J = 7.6 Hz, ArH), 7.27 (d, 1H, J = 7.2 Hz, ArH), 7.21 (m, 1H, ArH), 7.17 (m, 1H, ArH), 2.26 (s, 3H, ArCH3), 2.23 (s, 3H, ArCH3) ppm, HPLC: 96.2%. 13. Fenick, D. J.; Falvey, D. E. J. Org. Chem. 1994, 59, 479.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
The discovery of novel N-(2-pyrimidinylamino) benzamide derivatives as potent hedgehog signaling pathway inhibitors Minhang Xin ⇑, Jun Wen, Feng Tang, Chongxing Tu, Han Shen, Xinge Zhao Jiangsu Simcere Pharmaceutical Co. Ltd, Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No. 699-18, Xuan Wu District, Nanjing 210042, PR China
a r t i c l e
i n f o
Article history: Received 6 August 2013 Revised 23 September 2013 Accepted 9 October 2013 Available online 17 October 2013 Keywords: Hedgehog signaling Hedgehog signaling pathway inhibitors Synthesize Lead compound
a b s t r a c t Hedgehog signaling pathway inhibitors are emerging as new therapeutic intervention against cancer. A novel series of N-(2-pyrimidinylamino) benzamide derivatives as hedgehog signaling pathway inhibitors were designed and synthesized. Most compounds presented significant inhibitory effect on hedgehog signaling pathway, among which 21 compounds exhibited more potent than vismodegib. Furthermore, compound 6a showed moderate pharmacokinetic properties in vivo, representing a promising lead compound for further exploration. Ó 2013 Elsevier Ltd. All rights reserved.
The Hedgehog (Hh) pathway is a very important signaling pathway which is critical to cell proliferation and control of survival signals in embryonic development and tissue patterning, and critical to maintenance of stem cells and acquisition of epithelialto-mesenchymal transition (EMT) in adults.1 The process of Hh signaling activation has been widely described. Signaling activation on Hh pathway is initiated by ligands-induced stimulation of membrane associated receptor patched (Ptch). The binding of extracellular Hh family proteins, such as Sonic Hh (Shh), Desert Hh (Dhh) and Indian Hh (Ihh) to Ptch, relieves the suppressed GPCR-like receptor smoothened (Smo), which moves into the primary cilia to promote the dissociation of the complex of Suppressor of fused (Sufu) and Glioma-associated oncogene homologue (Gli), and activates Gli. The activated Gli then triggers downstream signals and contributes to cellular proliferation, differentiation and survival.2 Despite Hh pathway is silent in most adult tissues, abnormal activation of it contributes to uncontrolled cell growth and leads to malignant transformation, which has been commonly observed in a number of cancer types.3 Hence, Hh signaling pathway inhibition has been regarded as an important therapeutic strategy for cancer treatment. To date, two mechanisms for Hh signaling aberrantly activated in cancers have been elucidated. One is mutation-driven manner through mutation of members of the pathway such as Ptch-mutation, Smo-mutation, and Sufu-mutation, leading to tumorigenesis
⇑ Corresponding author. Tel./fax: +86 25 85560000 3192. E-mail address: [email protected] (M. Xin). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.022
involving basal cell carcinoma (BCC), medulloblastoma, and rhabdomyosarcoma. The other is Hh ligand-driven manner in which the overexpression of Hh ligand induced by autocrine or paracrine stimulation results in tumor aggravation including colorectal, esophageal, gastric, hepatocellular, pancreatic, prostate, lung, ovarian, glioblastoma, melanoma, lymphoma, leukemia, and so on.4 Besides, there are several evidences suggested that Hh signaling activation may influence cancer stem cells which is responsible for cancer recurrence.5 These research results demonstrated that Hh signaling pathway inhibitor not only can provide significant clinical benefit for BCC treatment, but also has good promising potential combating a number of other cancers for a single or combinational therapy. During these years, many novel types of Hh pathway inhibitors are being studied.6 Vismodegib was the first approved Hh pathway inhibitor by FDA in 2012 for use in metastatic BCC treatment.7 Moreover, several other synthetic small molecule inhibitors are advancing into clinical trials, including Novartis’s NVP-LDE225 (sonidegib, Phase III), Lilly’s LY-2940680 (Phase II), BMS’s BMS833923 (XL-139, Phase II), Infinity’s saridegib (IPI-926, discontinued in 2012), Pfizer’s PF-04449913(Phase I), Takeda’s TAK-441 (Phase I) and Novartis’s NVP-LEQ506 backup for sonidegib (Phase I) (Fig. 1).8 Nowadays, these represented drugs are being investigated on other haematological malignancies and solid tumors, such as BCC, medulloblastoma, leukemia, pancreatic, gastric, breast, and small-cell lung cancers in Phase I, Phase II and Phase III,8 which raises researchers’ more confidence about the therapeutic potential of targeting Hh. Since there are already a lot of Hh inhibitors structures published, we aimed to discover new Hh signaling pathway inhibitors with improved inhibition in order to amplify
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Cl
O
N
H N
Cl
N H
O S O
vismodegib (GDC-0449)
N
NH
N
O
O LY-2940680
BMS-833923 (XL-139)
Lilly Ph II
Exelixis /BMS Ph II
H
O HN
N
N OH
N
N N
N
CN
O
S
O
H
N H
NVP-LEQ506 Novartis Ph I
TAK-441 Millennium /Takeda Ph I
O H
H
OH
CF3 PF-04449913 Pfizer Ph I
HN
N
O
O
N N
CF3
O
N
NH
N
F
N H
Novartis Ph III
O N H
N
O
N
H N
N H
N
sonidegib (NVP-LDE-225;LDE-225)
Curis/Genentech (Roche) launched
N
N
O
O CF3
N N
O N
H
IPI-926 Infinity (dicontinued)
Figure 1. Chemical structures of FDA-approved and clinical Hh signaling inhibitors.
O
O
CF3
CF3
CF3 from ALLO-2 fragment
N N
O
H N N
N H ALLO-2
O
N H CF3
O
N N O
O
1b
N
N
N N
1a
from LDE-225 fragment
N
H N O
H N N
N N
N H CF3
N
from BMS-833923 similar fragment
LDE-225
O N H
N N
6a
N H
Figure 2. A design for N-(2-pyrimidinylamino) benzamide scaffold.
antitumor profiles in the future. Herein we describe the successful identification of a novel series of N-(2-pyrimidinylamino) benzamide derivatives. On the basis of deeply understanding the structure–activity relationship (SAR), fragment-based design and screening is always highly attractive design tool in traditional medicinal chemistry exploration.9 Recently, a compound containing pyrimidinylamino unit named ALLO-2 was reported to show good Hh signaling inhibition with distinct binding mode which could inhibit the D473H mutant Smo.10 The pyrimidinylamino group was considered as a privileged structure commonly occurring in drugs such as imatinib, nilotinib and so on. Consequently, we attempted to synthesize some compounds containing 4-(trifluoromethoxy)phenyl pyrimidinylamine moiety and change the structural fragment such as indazole, pyridinylmorpholine, phenylbenzamide to bear molecular diversity and complexity for scaffold-detection (Fig. 2). The target scaffold N-(2-pyrimidinylamino) benzamide possessed outstanding advantage and the compound 6a demonstrated leading inhibitory activity with an IC50 of 1.3 nM in a Gli-luciferase reporter assay,11 better than GDC-0449 (7.2 nM) and LDE-225 (5.5 nM) (Table 1). Encouraged by this excellent potency, a systematic study of SAR exploration was carried out for understanding the structural modification window for future optimization. Initially, compound 6a was divided into four regions: 4-trifluromethoxylphenyl (A), pyrimidinyl (B), 4-aminobenzamide (C) and 2, 6-dimethylphenyl groups (D) (Fig. 3). At the outset, our focus was on exploring modifications to A moiety, including the substitutions on the aromatic residue and alkyl instead. It was important to detail the contribution of the diverse modification of A moiety influ-
Table 1 Identification of pioneer Hh signaling inhibitors Compounds
Structures
Gli-luciferase reporter IC50 (nM)
OCF3
1a N
125
H N
N
N N H
OCF3
O
1b
N
N N
N H OCF3
N N GDC-0449 LDE-225
N
O
6a
>500
1.3
N H N H 7.2 5.5
encing the activity. Besides, it was worth investigating the fragment B pyrimidinyl core. An attractive option was to test the
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OCH 3
OCF3 F O CN
N N
OCF3 F
N
N CF 3
O
N
A moitey A N
D moiety O
N N
N
N B
O
N
C
N
N
D
N
N
N H N
N H
Bn
C moiety
B moiety
N
N
N
N
N
N
N
F
HN
F3C
O
N N
N
N H
N N
N
N
N
NH
N N
N
O
N
Figure 3. Designed structural modification of N-(2-pyrimidinylamino) benzamide derivatives.
OCF3
OCF3
OCF3 Cl N N 2
a 73%
Cl
ArNH 2
N
B(OH)2 N
Cl
c
3a
a (for 3a-3n) b (for 3o-3p) R1
R1
N
Cl
3a-3p
Ar N H 1a (43%, Ar=1H-indazol-5-yl ) 1b (70%, Ar=6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl) 1c (62%, ArNH 2=N-(4-aminophenyl)-2-methylbenzamide) R1
O
R1
O
e
d
N
N N
N
N
OCH3
N N H 4a-4p
N
N H 5a-5p
OH
f
O N H
N N
N H 6a-6p
Scheme 1. Reagents and conditions: (a) 4-trifluoromethoxyphenylboronic acid for 3a, 3-trifluoromethoxyphenylboronic acid for 3b, 2-trifluoromethoxyphenylboronic acid for 3c, 3-fluoro-4-trifluoromethoxyphenylboronic acid for 3d, 4-cyanophenylboronic acid for 3e, 4-morpholinophenyl boronic acid for 3f, 4-fluorophenylboronic acid for 3g, 4-methylphenylboronic acid for 3h, phenylboronic acid for 3i, pyridine-4-boronic acid for 3j, pyridine-3-boronic acid for 3k, 1-methyl-1H-pyrazole-4-boronic acid pinacol ester for 3l, 3-furanboronic acid for 3m, Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 6–12 h; cyclopropylboronic acid for 3n, Pd(dppf)Cl2, TEA, DMF, H2O, 100 °C for 6 h; 43–97%; (b) CH3MgCl for 3o, BnMgCl for 3p, Fe(acac)3, dry THF, 0 °C to rt,12 h; 77%, 58%; (c) 1H-indazol-5-amine for 1a, 6-((2S,6R)-2,6-dimethylmorpholino) pyridine-3-amine for 1b, N(4-aminophenyl)-2-methylbenzamide for 1c, n-BuOH, p-toluenesulfonic acid, 200 °C microwave for 2 h; 43%, 70%; (d) methyl 4-aminobenzoate, Pd(OAc)2, BINAP, Cs2CO3, dioxane, 170 °C, microwave for 3 h; 38–89%; (e) NaOH, MeOH/H2O, reflux overnight; 85–100%; (f) 2,6-dimethylaniline, HATU, DIPEA, DMF, 85 °C overnight; 40–95%.
substituent group and to acquire the role of nitrogen of pyrimidinyl. Subsequently, some replacements of moiety C and D were made to identify the applicability of these functionality changes (Fig. 3). As summarized in Figure 3, up to 39 compounds (1a–1c, 6a–6p, 15a–15j, 18a, 19a–19g, 20a and 20o) were synthesized.12 The general synthetic methods to build the designed compounds were illustrated in Schemes 1–3. The preparation compounds 1a–1c involved two transformations, Suzuki coupling and primary arylamine substitution (Scheme 1). A library compounds modification on A region were synthesized began with Suzuki coupling (3a–3n) under Pd catalyst and Kumada coupling (3o–3p) under Fe(acac)3, followed by Buchwald coupling (4a–4p) and hydrolysis to generate free acid (5a–5p), subsequently acylation with anilines to afford target compounds 6a–6p (Scheme 1). The similar method used to build the pyrimidinyl derivatizing was based on commercially or readily available starting materials dichloropyrimidines and halogeno pyridines. One key building block 11a used in this study was prepared according to literature
method13 (Scheme 2A). The synthesis of target compounds 15a–15i employed the similar procedures as described above for the preparation of 6a–6p (Scheme 2B). While 15j was prepared using modified steps shown in Scheme 2C, mainly involving reactions of substitution on position 2 and followed Suzuki coupling on position 4, and subsequently the same operation as formerly employed to yield compound 15j (Scheme 2C). The synthesis of compounds of structural alteration on C and D moiety was described on Scheme 3. The synthesis of 18a originated with former intermediate 4a, which was converted to 18a after three steps of methylation, hydrolysis and condensation in turn. Compounds 19a–19g were installed by condensation reactions with free acid 5a and readily available anilines. Compounds 22a and 22o were synthesized using N-acylation, chlorination and nucleophilic substitution as depicted in Scheme 3. The Hh signaling pathway inhibitory activity of synthetic compounds were assessed using a luciferase reporter in NIH3T3 cell carrying a stably transfected Gli-reporter construct (Gli-luciferase reporter cell lines). Table 2 outlined the in vitro IC50 for a series of A-moiety structural modification of N-(2-pyrimidinylamino) benz-
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M. Xin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6777–6783 O
O
(A)
O
a
O
O
N H 9
23%
O
90%
7 (B)
O c
NH
b
8
S
N H 10
70%
N
O
N
90%
Cl
11a OCF3
OCF3
OCF3
OCF3
Cl d
NH
Cl(Br) R2
Y
OCF 3 R
Cl R2 11a-11i
f
R2
Cl
R3
X
R
e
B(OH) 2 12
Y
3
X
R2
h OCH 3
Y X
12a-12i
O
O
O 3
OH
Y
R3
N H 13a-13i
X
R2
i
14a-14i
OCF3
N H
Y
R3
N H
X
N H 15a-15i
OCF3
OCF3
(C) Cl
Cl g
F3C
N N
Cl
O
O
e
F3C
N
49%
N
11j
F3C
O
N
h O
N
N H
12j
N
13j
O
i OH
N
58%
62%
N H
O F3C
F 3C
N H
N
29%
N H
N
N H 15j
14j
Scheme 2. Reagents and conditions: (a) ethyl formate, THF, LDA, 78 °C to rt, 2 h, 90%; (b) thiocarbamide, MeOH, reflux, overnight, 23%; (c) chloroacetic acid, EtOH/H2O, reflux, 7 h, 70%; (d) POCl3, DMF, 100 °C, 2 h, 90%; (e) 2,4-dichloro-5-cyclopropylpyrimidine for 12a, 2,4-dichloro-5,6-dimethylpyrimidine for 12b, 2,4-dichloropyrimidine for 12c, 2,4-dichloro-6-methylpyrimidine for 12d, 2,4-dichloro-5-fluoro pyrimidine for 12e, 2,4-dichloro-6-propylpyrimidine for 12f, 2,4-dichloro-5-ethylpyrimidine for 12g, 4bromo-2-chloropyridine for 12h, 2-bromo-6-chloropyridine for 12i, Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 6–12 h; 40–89%; (f) methyl 4-aminobenzoate, Pd(OAc)2, BINAP, Cs2CO3, dioxane, 170 °C, microwave for 3 h; 62–88%; (g) methyl 4-aminobenzoate, DIPEA, t-BuOH, rt, overnight; 49%; (h) NaOH, MeOH/H2O, reflux overnight; 58–95%; (i) 2,6dimethylaniline, HATU, DIPEA, DMF, 85 °C overnight; 26–73%.
OCF3
OCF3 O
N N
O
a O
N H
N OCF3
4a
O
N
N OCF3
N N
N
5a, 5o
N
N
N H N 18a
R4
N H 19a-19g
e
1
N N
R1
O
R
OH N H
O N
23%
17
N H
N
N H
O
OH
O
d OH
5a R1
c
O N
95%
N 16
N
b O
N
50%
OCF3
OCF3
N H N H
O g
f N H
N OH
20a, 20o
N
N H 21a, 21o
R1
O N
Cl N H 22a, 22o
N H
N
Bn
N
a (R 1=4-trifluoromethoxyphenyl) o (R 1=methyl)
Scheme 3. Reagents and conditions: (a) MeI, DMF, K2CO3, 60 °C, 12 h, 50%; (b) NaOH, MeOH/H2O, reflux overnight; 95%; (c) HATU, DIPEA, DMF, 85 °C overnight; 23%; (d) otoluidine for 19a, 2-methyl-5-(1-methyl-1H-pyrazol-4-yl)aniline for 19b, 4-((4-methylpiperazin-1-yl)methyl)aniline for 19c, 2-((4-methylpiperazin-1-yl)methyl)aniline for 19d, 3-((4-methylpiperazin-1-yl)methyl) aniline for 19e, isopropyl amine for 19f, cyclohexylamine for 19g, HATU, DIPEA, DMF, 85 °C overnight; 39–78%; (e) 3-amino-4methylbenzyl alcohol, HATU, DMF, DIPEA, 85 °C overnight; 20a, 94%; 20o, 60%; (f) SOCl2, CH2Cl2, rt, 2 h; 21a, 64%; 21o, 85%; (g) MeBnNH, DMF, K2CO3, rt, overnight; 22a, 78%; 22o, 43%.
amides. Substituents on phenyl of the region A played an essential role in regulating Hh signaling inhibitory activity. SARs suggested the para-OCF3 and meta-OCF3 substituent showed comparably good inhibition, while ortho-OCF3 led to a significant loss of activity (6a, 6b vs 6c). This clear SAR trend appeared introducing one electronwithdrawing group adjacent to 4-position or 3-position of A-ring was a preference. However both para-OCF3 and meta-F disubstituents had weak inhibitory activity (6a, 6b vs 6d), indicating that adding another electrophilic group could be harmful to activity. A survey of some substituents at para-position was investigated and led to a few favorable electron-withdrawing substituents including –CN (6e), –F (6g). Although compound 6i was lack of electrophilic group at para-position, it showed tolerable. Compared with these, morpholino (6f) produced moderate activity likely due to the steric
hindrance at para-position, and methyl (6h) showed no activity almost because of methyl serving as electron-donating group. Replacement of phenyl with 4-pyridinyl (6j) appeared tolerant, but an attempt of 3-pyridinyl (6k) was unsuccessful, which appeared the position of the nitrogen in the pyridine was critical for the inhibitory activity. Other heteroaryl such as pyrazolyl and furyl demonstrated less potency. Replacement of phenyl with alkyl such as cyclopropyl, methyl and benzyl was undesirable (6n, 6o, 6p) because of no A-ring aromaticity directly linked to B-ring. In this study, 6 compounds showed better inhibitory activity than positive control GDC-0449, and the essential SAR offered some clear substituents at the A-ring for future optimization. Table 3 summarized IC50 of compounds with substituents on the central pyrimidinyl ring (region B). Integral SARs suggested that a
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M. Xin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6777–6783 Table 2 In vitro inhibition of Hh signaling pathway for compounds modification on A region of N-(2-pyrimidinylamino) benzamides
R1
Table 4 In vitro inhibition of Hh signaling pathway for compounds modification on C and D region of N-(2-pyrimidinylamino) benzamides
OCF3
O N H
N N
O
N H 6a-6p
N
R1
Compds 6a
N H
N
Gli-luc reporter IC50 (nM)
Compds
R4
N H
R4
Gli-luc reporter IC50 (nM)
1.3
F3CO
6b
1.2
6a
1.3
F3CO
OCF3
>500
6c OCF3
6d
O
18a N
F3CO
158.5
N
F
6e
NC
6f
O
6g
F
6h
H3C
N
OCF
2.8
3
20.2
N
1c
6i
1.3
O
N H
>500 19a
1.0
0.54
N
19b 6k
N N
10.6
O
6m
N
19c
118.3 N
15.7
6n
1.2
N N
>500
N
6l
42.1
H N
N
2.2 N
6j
2.7 N H
32.8
H3 C
6o
151.5
2.3
19d N
N
34.3
6p GDC-0449
7.2
353.6
19e N
Table 3 In vitro inhibition of Hh signaling pathway for compounds modification on B region of N-(2-pyrimidinylamino) benzamides
N
19f
73.1
19g
2.2
22a
N
OCF3
1.1 Bn
O O R2 R3
Y X
N
N H N
GDC-0449
Compds
R2
R3
X
Y
6a
Me
H
N
N
1.3
H
N
N
1.3
Me H Me H Pr H H H H
N N N N N N N C N
N N N N N N C N N
1.6 1.2 1.4 1.6 0.8 5.3 1.6 23.4 0.53 7.2
15a 15b 15c 15d 15e 15f 15g 15h 15i 15j GDC-0449
Me H H F H Et H H CF3
N
22o
N H
N
Bn
100.6
N H
7.2
Gli-luc reporter IC50 (nM)
small hydrophobic group, such as –H, –F, –Me, –CF3, –Et, –Pr, cyclopropyl (15a, 15b, 15c, 15d, 15e, 15f, 15g, 15j), was favored at the 5- or 6-positions of pyrimidinyl, all of which showed comparable activity to 6a. It appeared to be a presence of hydrophobic cavity for suitable size, which even allowed the propyl group incorporated in. Interestingly, it is noticeable that compound 15h showed equipotent activity to 15c, while compound 15i led to a significant loss of activity. This appreciable drop demonstrated the 1position of the nitrogen in pyrimidinyl ring played a more important role than 3-position of it for modulating Hh signaling inhibi-
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M. Xin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6777–6783
Table 5 Pharmacokinetic properties for represented compound 6a in SD rats
a
Dosea (mg/kg)
Cmax (ng/mL)
AUC (h ng/mL)
Vss (mL/kg)
Cl (mL/h/kg)
MRT(0–t) (h)
T1/2 (h)
1 (iv) 5 (po)
764 342
915 1705
14,898 27,738
1004 2797
4.0 5.1
10.3 7.0
F (%)
36
Compound 6a was formulated using 5% DMA + 5% Tween-80.
tory activity (15c vs 15h vs 15i). In this series, 9 compounds demonstrated improved Hh signaling inhibition compared to GDC0449. The SAR studies showed that hydrophobic and suitable length substituents incorporated in 5- or 6-positions exhibited considerable potential for structural modification. Table 4 depicted IC50 of N-(2-pyrimidinylamino) benzamides derivatizing on region C and D. N-Methyl analogs 18a showed potency in vitro activity nearly equal to 6a. We speculated that the free hydrogen of NH appeared to play almost no role in its potency. Based on the intrinsic observation it was evident that benzamide functionality of C region was favorable to phenyl aminoacyl of C (1c vs 6a, 19a). Exploration of D-moiety demonstrated that 3-position of phenyl bearing some N-functionality such as pyrazole, piperizine, benzylamine was tolerable (19b, 19d, 22a vs 19c, 19e), which suggested that some hydrophilic basic side chains incorporated in this position were worth to synthesized for further water-soluble optimization. Replacement of phenyl with isopropyl resulted less potency (19f), however, interestingly the activity of substituent cyclohexyl retained. Compared 22a with 22o, it was again obvious that methyl instead of A-moiety phenyl made the activity largely diminished. In this SAR exploration, 6 compounds revealed enhanced potency compared to GDC0449. The SAR exploration demonstrated hydrophilic basic side chains incorporated in D-moiety was tolerable for improved activity. Having established the initial SAR of N-(2-pyrimidinylamino) benzamide derivatives, it was found that many compounds showed enhancing inhibitory activity compared to GDC-0449. Among these compounds, 6a was chosen to study the pharmacokinetic properties in vivo. As illustrated in Table 5, 6a displayed moderate pharmacokinetic properties, with an acceptable bioavailability (F = 36%) and half-life (T1/2 = 7 h), but a large volume of distribution (Vss = 27738 mL/kg) and low oral exposure (1705 h ng/mL) (Table 5). In an attempt to optimize the pharmacokinetic properties is next due course and the work will be described in the accompanying paper. In conclusion, a novel class of N-(2-pyrimidinylamino) benzamide derivatives as Hh signaling pathway inhibitors was identified. Thorough SAR exploration identified the key functionalities necessary for potency, enabling 21 compounds with significant inhibitory activity compared to GDC-0449 to be identified. Compound 6a with good potency, distinctive structure, moderate pharmacokinetic properties, was deserved as one promising lead compound. In the light of our encouraging results, and exploiting the information arising from the SARs, the further design of highly efficient and improved pharmacokinetic N-(2-pyrimidinylamino) benzamide corebased Hh signaling pathway inhibitory agents is currently progress and will be reported in due course. Acknowledgments This work was supported by the National Major Science and Technology Project of China (Innovation and Development of New Drugs, No. 2011ZX09401-008).
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.10. 022. References and notes 1. (a) Zeng, X.; Goetz, J. A.; Suber, L. M.; Scott, W. J., Jr; Schreiner, C. M.; Robbins, D. J. Nature 2001, 411, 716; (b) Li, Y.; Maitah, M. Y.; Ahmad, A.; Kong, D.; Bao, B.; Sarkar, F. H. Exp. Opin. Ther. Targets 2012, 16, 49. 2. (a) Stone, D. M.; Hynes, M.; Armanini, M.; Swanson, T. A.; Gu, Q.; Johnson, R. L.; Scott, M. P.; Pennica, D.; Goddard, A.; Phillips, H.; Noll, M.; Hooper, J. E.; Sauvage, F.; Rosenthal, A. Nature 1996, 384, 129; (b) Tian, H.; Callahan, C. A.; DuPree, K. J.; Darbonne, W. C.; Ahn, C. P.; Scales, S. J.; Sauvage, F. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 4254. 3. Onishi, H.; Katano, M. Cancer Sci. 2011, 102, 1756. 4. (a) Rubin, L. L.; de Sauvage, F. J. Nat. Rev. Drug Disc. 2006, 5, 1026; (b) Ng, J. M.; Curran, T. Nat. Rev. Cancer 2011, 11, 493; (c) Metcalfe, C.; de Sauvage, F. J. Cancer Res. 2011, 71, 5057. 5. Merchant, A. A.; Matsui, W. Clin. Cancer Res. 2010, 16, 3130. 6. (a) Guerlet, G.; Spangenberg, T.; Mann, A.; Faure, H.; Ruat, M. Bioorg. Med. Chem. Lett. 2011, 21, 3608; (b) Malancona, S.; Altamura, S.; Filocamo, G.; Kinzel, O.; Hernando, J. I.; Rowley, M.; Scarpelli, R.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett. 2011, 21, 4422; (c) Kinzel, O.; Alfieri, A.; Altamura, S.; Brunetti, M.; Bufali, S.; Colaceci, F.; Ferrigno, F.; Filocamo, G.; Fonsi, M.; Gallinari, P.; Malancona, S.; Hernando, J. I.; Monteagudo, E.; Orsale, M. V.; Palumbi, M. C.; Pucci, V.; Rowley, M.; Sasso, R.; Scarpelli, R.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett. 2011, 21, 4429; (d) Brown, M. L.; Aaron, W.; Austin, R. J.; Chong, A.; Huang, T.; Jiang, B.; Kaizerman, J. A.; Lee, G.; Lucas, B. S.; McMinn, D. L.; Orf, J.; Rong, M.; Toteva, M. M.; Xu, G.; Ye, Q.; Zhong, W.; Degraffenreid, M. R.; Wickramasinghe, D.; Powers, J. P.; Hungate, R.; Johnson, M. G. Bioorg. Med. Chem. Lett. 2011, 21, 5206; (e) Ontoria, J. M.; Bufi, L. L.; Torrisi, C.; Bresciani, A.; Giomini, C.; Rowley, M.; Serafini, S.; Bin, H.; Hao, W.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett. 2011, 21, 5274; (f) Muraglia, E.; Ontoria, J. M.; Branca, D.; Dessole, G.; Bresciani, A.; Fonsi, M.; Giuliano, C.; Llauger Bufi, L.; Monteagudo, E.; Palumbi, M. C.; Torrisi, C.; Rowley, M.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett. 2011, 21, 5283; (g) Wang, J.; Mook, R. A., Jr.; Lu, J.; Gooden, D. M.; Ribeiro, A.; Guo, A.; Barak, L. S.; Lyerly, H. K.; Chen, W. Bioorg. Med. Chem. 2012, 20, 6751; (h) Peukert, S.; Jain, R. K.; Geisser, A.; Sun, Y.; Zhang, R.; Bourret, A.; Carlson, A.; Dasilva, J.; Ramamurthy, A.; Kelleher, J. F. Bioorg. Med. Chem. Lett. 2009, 19, 328; (i) 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, 106; (j) Peukert, S.; He, F.; Dai, M.; Zhang, R.; Sun, Y.; Miller-Mislin, K.; McEwan, M.; Lagu, B.; Wang, K.; Yusuff, N.; Bourret, A.; Ramamurthy, A.; Vattay, A.; Wang, A.; Guo, R.; Yuan, J.; Green, J.; Williams, J.; Buonamici, S.; Kelleher, J. F.; Dorsch, M. ChemMedChem 2013, 8, 1261. 7. Dlugosz, A.; Agrawal, S.; Kirkpatrick, P. Nat. Rev. Drug Disc. 2012, 11, 437. 8. https://www.thomson-pharma.com/. 9. (a) Hajduk, P. J.; Greer, J. Nat. Rev. Drug Disc. 2007, 6, 211; (b) Möcklinghoff, S.; van Otterlo, W. A.; Rose, R.; Fuchs, S.; Zimmermann, T. J.; Dominguez, Seoane. M.; Waldmann, H.; Ottmann, C.; Brunsveld, L. J. Med. Chem. 2011, 54, 2005. 10. Tao, H.; Jin, Q.; Koo, D. I.; Liao, X.; Englund, N. P.; Wang, Y.; Ramamurthy, A.; Schultz, P. G.; Dorsch, M.; Kelleher, J.; Wu, X. Chem. Biol. 2011, 18, 432. 11. (a) 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, 5576; (b) Ohashi, T.; Oguro, Y.; Tanaka, T.; Shiokawa, Z.; Shibata, S.; Sato, Y.; Yamakawa, H.; Hattori, H.; Yamamoto, Y.; Kondo, S.; Miyamoto, M.; Tojo, H.; Baba, A.; Sasaki, S. Bioorg. Med. Chem. 2012, 20, 5496. 12. Purity of all final compounds was assessed by HPLC and determined to be >95%. The structures were confirmed by 1H NMR and LC/MS. Analytic data of represented compounds 6a: MS (ESI) m/z: [M+H]+ = 493.1. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.57 (s, 1H, NH), 8.53 (s, 1H, ArH), 7.95 (m, 4H, ArH), 7.89 (d, 2H, J = 8.8 Hz, ArH), 7.57 (d, 2H, J = 8.8 Hz, ArH), 7.12 (s, 3H, ArH),
M. Xin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6777–6783 2.27 (s, 3H, ArCH3), 2.18 (s, 6H, 2ArCH3) ppm, HPLC: 97.6%; 15a: MS (ESI) m/z: [M+H]+ = 519.2. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.54 (s, 1H, NH), 8.39 (s, 1H, ArH), 7.99 (d, 2H, ArH), 7.96 (m, 4H, ArH), 7.56 (d, 2H, ArH), 7.11 (s, 3H, ArH), 2.17 (s, 6H, 2⁄CH3), 1.90 (m, 1H, cyclopropyl-CH), 0.88 (m, 2H, cyclopropyl-CH2), 0.71 (m, 2H, cyclopropyl-CH2) ppm, HPLC: 99.0%; 18a: MS (ESI) m/z: [M+H]+ = 507.2. 1H NMR (400 M, DMSO-d6) d 9.71 (s, 1H, CONH), 8.39 (s, 1H, ArH), 8.01 (d, 2H, ArH), 7.81 (d, 2H, ArH), 7.57 (d, 2H, ArH), 7.50 (d,
6783
2H, ArH), 7.12 (s, 3H, ArH), 3.57 (s, 3H, NCH3), 2.23 (s, 3H, ArCH3), 2.19 (s, 6H, 2ArCH3) ppm, HPLC: 98.0%; 19a: MS (ESI) m/z: [M+H]+ = 479.2. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.93 (s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.35 (d, 1H, J = 7.6 Hz, ArH), 7.27 (d, 1H, J = 7.2 Hz, ArH), 7.21 (m, 1H, ArH), 7.17 (m, 1H, ArH), 2.26 (s, 3H, ArCH3), 2.23 (s, 3H, ArCH3) ppm, HPLC: 96.2%. 13. Fenick, D. J.; Falvey, D. E. J. Org. Chem. 1994, 59, 479.
