Bioorganic & Medicinal Chemistry Letters 24 (2014) 983–988
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Synthesis and evaluation of 4-(2-pyrimidinylamino) benzamides inhibitors of hedgehog signaling pathway Minhang Xin a,b,⇑, Jun Wen b, Feng Tang b, Chongxing Tu b, Wei Huang b, Han Shen b, Xinge Zhao b, Lingfei Cheng b, Mengyu Wang b, Liandi Zhang b a b
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 Jiangsu Simcere Pharmaceutical Co. Ltd., Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No 699-18, Xuan Wu District, Nanjing 210042, P.R. China
a r t i c l e
i n f o
Article history: Received 18 September 2013 Revised 10 November 2013 Accepted 12 December 2013 Available online 17 December 2013
a b s t r a c t A novel series of hedgehog signaling pathway inhibitors has been designed based on the 4-(2-pyrimidinylamino) benzamides scaffold. The synthesis and SAR of these compounds are described. Optimization leads to the identification of compound 3c, a potent and orally available agent with improved physicochemical and pharmacokinetic properties. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Hedgehog signaling pathway Inhibitors SAR Pharmacokinetic properties
The Hedgehog (Hh) signaling pathway is receiving increasing attention because of its very role in malignant transformation of various cancers.1 Genetic mutations associated with this signaling pathway such as Ptch-mutation, Smo-mutation, and Sufumutation result in tumorigenesis in several cancers such as basal cell carcinoma (BCC), medulloblastoma, and rhabdomyosarcoma. Additionally, strong evidences link overexpression of Hh ligands to tumor aggravation of various other cancers such as hepatocellular, pancreatic, gastric, colorectal, esophageal, lung, ovarian, prostate, melanoma, glioblastoma, leukemia, lymphoma.2 Thus, inhibition of Hh signaling pathway is thought to have considerable potential for therapy of many cancer types. To date, many classes of Hh pathway inhibitors have been described.3 Vismodegib has been recently approved by FDA for metastatic BCC treatment.4 Other molecules including sonidegib (NVP-LDE225, Phase III), LY-2940680 (Phase II), BMS-833923 (XL-139, Phase II), PF-04449913(Phase I), TAK-441 (Phase I), NVP-LEQ506 (Phase I) have been already studied in clinic for many tumors treatment (Fig. 1).5 Besides, there are still some novel small molecules investigated in preclinical stage or drug discovery stage.6 In aspiration of interest in developing Hh signaling pathway inhibitors for treatment of many tumors, we recently detailed ⇑ Corresponding author. address: 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. Tel.: +86 25 85560000 3192. E-mail address: [email protected] (M. Xin). 0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.050
the discovery of a novel series of 4-(2-pyrimidinylamino) benzamides as potent Hh signaling pathway inhibitors, exemplified by 1 (Fig. 2), which inhibited the Hh signaling pathway in Gli-luciferase reporter assay with IC50 = 1.3 nM, more potent than control positive drug vismodegib. Initial SAR studies in this series showed that structural modification on A-ring of 1 seriously affected the inhibitory activity and the optimal substituents was conferred to 4-trifluoromethylphenyl, phenyl, 4-pyridinyl or others. Following SAR studies focused on D-ring modifications, especially at the position of phenyl group, demonstrating that methyl substitution at C-2 of the phenyl or several basic side chains functionalized at C-5 was favorable for high inhibitory activity.7 As a continuation of our study toward identification of potent Hh signaling pathway inhibitors, a further SAR study was conducted to explore various basic amine chains linked to D-ring, looking to identify potent inhibitors with improved physicochemical properties which was of importance for successfully developing drug candidate. Furthermore, considering that 1 exhibited unsatisfactory pharmacokinetic (PK) profile, efforts to improve the PK properties of this scaffold was also made by modification on both A-ring and D-ring. Herein, in this report we disclose the synthesis and SAR analysis of these analogs of Hh signaling pathway inhibitors, and also we report the PK evaluation in vivo. On the basis of the previous SAR study, at the outset, our strategy focused on bearing various basic substitutions at C-5 of D-ring. A number of analogs incorporating basic amine side chains at C-5 of D-ring were summarized in Table 1. The Hh signaling pathway inhibitory activity of compounds 2a–2zj were evaluated by a
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Cl
O
N
H N
Cl
N H
O
O CF 3
S O
vismodegib (GDC-0449)
N
N
O
N NH
F
N H
Exelixis /BMS Ph II
O
N
O
O
N
NH
N H BMS-833923 (XL-139)
Novartis Ph III
N N
HN O
N
N
N
OH
O
N N
CF3
O CF 3
H N
N H
N O
N N
N
sonidegib (NVP-LDE-225;LDE-225)
Curis/Genentech(Roche) launched
N N
O N
O
N N
OH
CN
LY-2940680
PF-04449913
Lilly Ph II
Pfizer Ph I
NVP-LEQ506
TAK-441 Millennium /Takeda
Ph I
Novartis Ph I
Figure 1. Chemical structures of clinical Hh signaling pathway inhibitors.
O
CF3
A
3 O
B N
N
C
2 N 1 H
D
4 5
6
N H
Figure 2. Structure of compound 1.
luciferase reporter in NIH3T3 cell carrying a stably transfected Glireporter construct (Gli-luciferase reporter cell lines).7,8 Table 1 outlined the in vitro IC50 for this series of various basic substitutions at C-5 of D-ring of 4-(2-pyrimidinylamino) benzamides9. Notably, all the analogs bearing basic amine on the molecules (2a–2zj) showed good Hh signaling inhibitory activity with IC50 varied from 0.23 to 35.35 nM. Of all the amines explored, some cyclic aliphatic amines, such as morpholine (2a), dimethyl morpholine (2b), piperidine (2c), thiomorpholine (2d), pyrrolidine (2e), were more effective than the lead compound 1 and 7–20 fold higher activity than GDC-0449. However the similar cyclic amine piperazine (2f) showed significantly decreased activity, perhaps due to relatively strong basicity of second basic center of NH. Other piperazine derivatives such as N-methyl piperazine (2g), N-ethyl piperazine (2h), N-phenyl piperazine (2i), N-cyclopropanecarbonyl piperazine (2j) and piperazin-3-one (2k) exhibited improved potency compared to 2f because of relatively suitable basicity. A survey of substituted piperidines was investigated and led to some more favorable piperidine derivatives including 4-hydroxy piperidine (2l), piperidin-4-one (2m), tetramethyl piperidine (2n), and tetramethyl piperidin-4-one (2o). However, 4-piperidyl piperidine (2p) displayed lower activity, while the activity of piperidine fusing aromatic ring such as tetrahydroquinoline (2q) and tetrahydrothienopyridine (2r) almost retained. These results indicated that adding extra basicity was not favorable to the activity. The same case was accounted by pyrrolidine derivatives 2s whose activity was weaker than 2e. Additionally, two bicyclic amine derivatives, 2t and 2u, were also investigated, and it was found 2t was 6-fold more potent than 2u. After screening a variety of cyclic amine substituents, some acyclic amines were explored. It appeared a suitable length of acyclic amines was important for maintaining activity. For examples, 2v–2z displayed subnanomolar inhibition. However, compounds 2za and 2zb, displayed much lower potency, with IC50 = 35.3 and 10.9 nM, respectively, mainly due to the larger size of amine chains. In fact, the extra basicity of these two compounds also strongly effected the decreased activity additively.
Steric hindrance could also affect the activity, for instance compound 2zc bearing a bulky dicyclohexylamine group, which showed moderate activity. Besides, secondary amines like ethylamine (2zd), hydroxyethylamine (2ze), tert-butylamine (2zf) or cyclopentylamine (2zg) were tolerated, displaying nearly comparable activity to lead compound 1, except 2zh that showing weak potency of Hh inhibitory activity, which was considered due to its length and extra basicity. In addition, interestingly, the inhibitory activity of the basic amine substituents containing aromatic groups such as furylmethamine (2zi) and phenylamine (2zj) were retained, and these results were consistent with the above reported ones 2q and 2r. In a word, this SAR study showed 30 compounds displayed better inhibitory activities than positive control GDC-0449, of which 20 exhibited superior activities to lead compound 1. To evaluate whether these compounds had an improved druggability compared to 1, of our most potent inhibitors, two compounds 2a and 2b were further investigated by profiling iv and po pharmacokinetics in SD rat.10 The results were illustrated in Table 3. After intravenous injection with 1 mg/kg in SD rats, the area-under-curve (AUC) of 2a reached 1.3 h lg/mL, larger than that of 1 (0.92 h lg/mL). Such more plasma exposure is due part to the much smaller volume of distribution (7.4 L/kg) and lower systemic clearance (12.0 mL/min/kg). Furthermore, when orally administered at 5 mg/kg dose, 2a showed higher oral exposure and lower systemic clearance. Consequently, compound 2a showed superior oral bioavailability (F = 46%) to 1 (36%). However, unfortunately, 2b bearing N-methylpiperizine group showed poor PK characteristics both by iv and po administration. Despite 2a and 2b contained much hydrophilic group morpholine and Nmethylpiperizine in structures respectively, both molecules had already possessed higher lipophilicity (2a, A log P = 7.07 and 2b, Aog P = 7.34) compared to vismodegib (Aog P = 4.265). It appeared attractive to attempt a structural modification to decrease the A log P value so as to further improve drug-like properties. Therefore, Three compounds (3a–3c) were synthesized (Table 2), beginning to replace of trifluoromethoxyphenyl of A-ring with phenyl, 4-pyridinyl and 1-methylpyrazolyl because these groups were previously reported to afford good to moderate inhibitory activity.7 Of the three compounds, both compounds 3a and 3b showed nearly equipotent inhibition with 2a in Hh signaling pathway and possessed a lower A log P values (3a, A log P = 4.95 and 3b, Aog P = 4.069), but did not improve the plasma exposure in an intravenous administration (Tables 2 and 3). Fortunately, at this point, 3c with 1-methylpyrazolyl, showing equipotent potency with 1 and a much lower A log P (3c, A log P = 3.6), was found to
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M. Xin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 983–988 Table 1 In vitro inhibition of Hh signaling pathway for compounds modification at C-5 of Dring of 4-(2-pyrimidinylamino) benzamides
Table 2 The IC50 and the AlogP values for compounds modification on A and D region of 4-(2pyrimidinylamino) benzamides9
OCF 3
R1
O N
O N N
Compds
N H
R
Compds
Gli-luc reporter IC50 (nM)
2a
N
2b
N
2c
N
2d
N
2e
N
R
N H
N
2a-2zj
O
O
S
Compds
R
Gli-luc reporter IC50 (nM)
0.53
2t
N
0.73
2u
N
1.0
2v
N
0.34
2w
0.43
2x
1.6
10.5
N
0.93 0.89
N
3a 3b
N
NH
N
N
10.5
2y
N
0.45
2g
OH
2z
0.47 OH
2h
N
N
N
0.77
2za
N
2.0
2zb
N
N
N H
2j
N
2zc
2.0
2zd
NH
O
2k
N
2.7
N
NH
0.72
2l
N
OH
0.44
2ze
2m
N
O
0.28
2zf
0.76
2zg
0.29
2zh
N H
19.5
2zi
H N
2.4
2zj
1.5
1
1.3
3.9
GDC
7.2
2n
N
2o 2p
2q
N
O
N
N
N
NH
OH
HN
HN
1.6
N O
33.6 2.5
1.1
S
2r
N
N
2s
0.68
4.95
N
N
0.61
4.069 3.6
3d
N N
N
N
10.0
3.87
3e
N N
N
9.2
4.373
3f
N N
N
5.6
4.829
N N
N
8.4
4.355
3h
N N
N
21.2
3.345
3i
N N
N
17.5
4.785
N N
N
31.9
2.833
18.1
4.308
1.3 7.2
7.795 4.265
O
OH
OH OH
1 GDC-0449
N N
HN
a A Log P values were calculated by Discovery studio 3.0 using Calculate Molecular Properties Protocol.
1.1 0.90
HN
O
1.3
3k 1.3
7.07 7.34
N
10.9
O N
0.53 0.45
O
3j
2i
A Log Pa
N
35.3
N
Gli-luc reporter IC50 (nM)
N N
0.23
N
R
3c
3g 2f
N
3a-3k
N H
2a 2b
0.59
N
R1
R
N H
N
significantly improve the oral exposure (AUC = 3.8 h lg/mL) by po 5 mg/kg dosage, moreover whose oral bioavailability reached almost 64%. In addition to the significant insight of low A log P value gained with the 1-methylpyrazolyl replacement, an additional structure modification at position 5 of D-ring was pursued by incorporating substituents, such as methylpiperizine, pyrrolidine, piperidine, dimethylmorpholine, etc., which were identified to have optimized activity above, then compounds 3d–3k were
prepared (Table 2). Unfortunately, although 3d–3k also demonstrated low A log P values, only displayed less potency (5.6– 31.9 nM) compared to 1, which stalled their progression of further PK studies. Therefore, based on the optimal studies of combination of activity and PK properties, compounds 3c was considered to be the ideal lead candidate, which exhibited superior potency, excellent oral exposure and improved oral bioavailability in comparison to 1. And now 3c were being evaluated in vivo in Hh pathway dependent medulloblastoma allograft and other Hh signaling operative solid tumor models. The results will be reported later. The 4-(2-pyrimidinylamino) benzamides were readily prepared according to Scheme 1. Briefly, commercially available 2,4-dichloro-5-methylpyrimidine were treated with several aryl boronic acid or boronic ester using palladium-catalyzed suzuki coupling reaction to provide 4-aryl-substituted pyrimidines 5a–5d. The intermediates 5a–5d were reacted with methyl 4-aminobenzoate under palladium catalysis to afford the buckwald coupling products 6a–6d, which were further hydrolyzed to produce the free acids 7a–7d. The condensation of 7a–7d with 3-amino-4-methylbenzyl alcohol, followed by chlorination and next acylation with various amines, afforded the designed inhibitors 2a–2zj and 3a–3k (Scheme 1).7 In summary, we have utilized 4-(2-pyrimidinylamino) benzamide core to produce novel Hh signaling pathway inhibitors. Starting from the lead compound 1, we were able to explore structural modification on A-ring and D-ring to identify potent inhibitors
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Table 3 In vivo SD rat pharmacokinetic properties for represented compounds compds GDC-0449 1 2a 2b 3a 3b 3c a b
Dose (mg/kg) 1 1 5 1 5 1 5 1 1 1 5
a
(iv) (iv)a (po)a (iv)a (po)b (iv)a (po)b (iv)a (iv)a (iv)a (po)b
Cmax (lg/mL)
AUC (hlg/mL)
Vss (L/kg)
Cl (mL/min/kg)
MRT(0–t) (h)
T1/2 (h)
1.2 0.76 0.34 1.2 0.28 0.30 0.094 0.84 0.58 2.1 1.5
2.5 0.92 1.7 1.3 2.4 0.58 1.3 0.71 0.27 1.2 3.8
0.95 14.9 27.7 7.4 16.3 20.0 42.9 2.1 5.7 0.90 2.9
6.8 16.7 46.6 12.0 32.8 24.9 51.9 23.3 61.5 14.3 23.9
2.2 4.0 5.1 4.1 6.6 7.4 9.7 1.2 0.90 0.81 2.4
1.6 10.3 7.0 7.2 5.8 9.2 9.5 1.0 1.1 0.8 1.4
F (%)
36.1 46.3 44.4
63.6
compound was formulated using 5%DMA + 5% Tween-80. Compound was formulated using 0.5%HPMC + 0.3% Tween-80, pH = 2.8.
R1
Cl a
N N
N
Cl
4
Cl
N
O N N H 8a-8d
OCH 3
c N
d
OH N H 7a-7d R1
O
O
f
e N H
N OH
O N
N H 6a-6d
R1 N H
R1
O N
5a-5d
R1
N
R1 b
N
N
N H
N Cl
N
N H
R
N H 2a-2zj;
9a-9d
3a-3k
Scheme 1. Reagents and conditions: (a) Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 6–12 h, 4-trifluoromethoxyphenylboronic acid for 5a, 73%; phenylboronic acid for 5b, 89%; pyridine-4-boronic acid for 5c, 78%; 1-methyl-1H-pyrazole-4-boronic acid pinacol ester for 5d, 43%; (b) Pd(OAc)2, BINAP, Cs2CO3, dioxane, 170 °C, microwave for 3 h, methyl 4-aminobenzoate, 43–94%; (c) NaOH, MeOH/H2O, reflux overnight, 85–100%; (d) 3-amino-4-methylbenzyl alcohol, HATU, DMF, DIPEA, 85 °C overnight, 38–94%; (e) SOCl2, CH2Cl2, rt, 2 h, 64–89%; (f) RH, DMF, K2CO3, rt, overnight; from 9a to 2a–2zj; from 9b to 3a; from 9c to 3b; from 9d to 3c–3k; 43–89%.
with improved physicochemical properties. From these compounds, some structure–activity relationships were concluded and the compound 3c was identified as a potent and orally vailable Hh signaling inhibitor with favorable pharmacokinetic properties. Nowadays, the in vivo biological evaluation and the safety investigation of 3c is being conducted both in Hh pathway dependent medulloblastoma allograft and Hh signaling operative solid tumor models, and the progress 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). The authors thank Dr. Haopeng Sun of China Pharmaceutical University for the A log P calculation. References and notes 1. (a) Li, Y.; Maitah, M. Y.; Ahmad, A.; Kong, D.; Bao, B.; Sarkar, F. H. Expert Opin. Ther. Targets 2012, 16(1), 49; (b) Zeng, X.; Goetz, J. A.; Suber, L. M.; Scott, W. J., Jr.; Schreiner, C. M.; Robbins, D. J. Nature 2001, 411, 716; (c) 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; (d) 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; (e) Onishi, H.; Katano, M. Cancer Sci. 2011, 102, 1756. 2. (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; (d) Merchant, A. A.; Matsui, W. Clin. Cancer Res. 2010, 16, 3130. 3. (a) Mahindroo, N.; Punchihewa, C.; Fujii, N. J. Med. Chem. 2009, 52, 3829; (a) Hadden, M. K. Expert Opin. Ther. Pat. 2013, 23, 345. 4. (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.;
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M. Xin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 983–988 8. 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. 9. 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 2a: MS (ESI) m/z: [M+H]+ = 578.3. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.4 Hz, ArH), 7.53 (d, 2H, J = 8.0 Hz, ArH), 7.28 (s, 1H, ArH), 7.19 (d, 1H, J = 7.6 Hz, ArH), 7.09 (d, 1H, J = 7.6 Hz, ArH), 3.57 (s, 4H, morpholine-H), 3.43 (s, 2H, PhCH2N), 2.36 (s, 4H, morpholine-H), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 95.9%; 2b: MS (ESI) m/z: [M+H]+ = 606.3. 1H NMR (400 M, DMSO-d6) d 10.01 (s, 1H, CONH), 9.78 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.95 (s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.58 (s, 1H, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.42 (d, 1H, J = 7.6 Hz, ArH), 7.36 (d, 1H, J = 8.0 Hz, ArH), 4.27 (s, 2H, PhCH2N), 3.98 (m, 2H, CHOCH), 3.26 (d, 2H, J = 7.6 Hz, CH2N), 2.68 (m, 2H, CH2N), 2.27 (s, 3H, ArCH3), 2.26 (s, 3H, ArCH3), 1.12 (s, 3H, CH3), 1.11 (s, 3H, CH3) ppm, HPLC: 95.7%; 2c: MS (ESI) m/z: [M+H]+ = 576.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 8.8 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.25 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.07 (d, 1H, J = 7.6 Hz, ArH), 3.38 (s, 2H, PhCH2N), 2.32 (m, 4H, (CH2)2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 1.48 (m, 4H, (CH2)2), 1.39 (m, 2H, CH2) ppm, HPLC: 99.5%; 2d: MS (ESI) m/z: [M+H]+ = 594.2. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 7.2 Hz, ArH), 7.54 (d, 2H, J = 6.8 Hz, ArH), 7.27 (s, 1H, ArH), 7.20 (d, 1H, J = 6.4 Hz, ArH), 7.07 (d, 1H, J = 6.4 Hz, ArH), 3.46 (s, 2H, PhCH2N), 2.60 (m, 8H, (CH2)4), 2.25 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 99.6%; 2e: MS (ESI) m/z: [M+H]+ = 562.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.63 (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), 7.29 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.09 (d, 1H, J = 7.2 Hz, ArH), 3.55 (s, 2H, PhCH2N), 2.44 (m, 4H, pyrolidine-H), 2.26 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 1.69 (m, 4H, pyrolidine-H) ppm, HPLC: 98.8%; 2f: MS (ESI) m/z: [M+H]+ = 577.3. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.93(s, 4H, ArH), 7.88 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.27 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.08 (d, 1H, J = 7.2 Hz, ArH), 3.45 (s, 2H, PhCH2N), 2.77 (m, 4H, N(CH2)2), 2.35 (m, 4H, N(CH2)2), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 98.7%; 2g: MS (ESI) m/z: [M+H]+ = 591.3. 1H NMR (400 M, DMSO-d6) d 9.96 (s, 1H, CONH), 9.62 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.4 Hz, ArH), 7.53 (d, 2H, J = 8.4 Hz, ArH), 7.26 (s, 1H, ArH), 7.18 (d, 1H, J = 7.6 Hz, ArH), 7.05 (d, 1H, J = 7.6 Hz, ArH), 3.41 (s, 2H, PhCH2N), 2.33 (m, 8H, piperazine-H), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 2.14 (s, 3H, NCH3) ppm, HPLC:99.2%; 2h: MS (ESI) m/z: [M+H]+ = 605.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.26 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.07 (d, 1H, J = 7.6 Hz, ArH), 3.41 (s, 2H, PhCH2N), 2.36 (m, 10H, CH2N(CH2)4)N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 0.96 (t, 3H, J = 7.2 Hz, CH3) ppm, HPLC: 97.7%; 2i: MS (ESI) m/z: [M+H]+ = 653.3. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.31 (s, 1H, ArH), 7.21 (m, 3H, ArH), 7.12 (t, 1H, ArH), 6.92 (d, 2H, J = 8.4 Hz, ArH), 6.76 (t, 1H, ArH), 3.50 (s, 2H, PhCH2N), 3.12 (s, 4H, N(CH2)2), 2.50 (s, 4H, N(CH2)2), 2.26 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3)ppm, HPLC: 98.6%; 2j: MS (ESI) m/z: [M+H]+ = 645.3. 1H NMR (400 M, DMSO-d6) d 9.98 (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 = 7.6 Hz, ArH), 7.30 (s, 1H, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 7.2 Hz, ArH), 3.66 (s, 2H, PhCH2N), 3.47 (s, 4H, CH2NCH2), 2.40 (m, 4H, CH2NCH2), 2.26 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 1.94 (m, 1H, cyclopropyl-H), 1.48 (m, 2H, CH2), 0.70 (m, 4H, cyclopropyl-H) ppm, HPLC: 96.6%; 2k: MS (ESI) m/z: [M+H]+ = 591.3. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.93(s, 4H, ArH), 7.88 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.31 (s, 1H, ArH), 7.23 (d, 1H, J = 8.0 Hz, ArH), 7.10 (d, 1H, J = 7.6 Hz, ArH), 3.51 (s, 2H, PhCH2N), 3.17 (s, 2H, NCH2), 2.80 (m, 2H, NCH2), 2.51 (s, 2H, NCH2), 2.26 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3) ppm, HPLC: 95.1%;2l: MS (ESI) m/z: [M+H]+ = 592.3. 1 H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.25 (s, 1H, ArH), 7.19 (d, 1H, J = 8.0 Hz, ArH), 7.06 (d, 1H, J = 7.6 Hz, ArH), 4.52 (m, 1H, CH), 3.39 (s, 2H, PhCH2N), 2.67 (t, 2H, piperidine-H), 2.26 (s, 3H, ArCH3), 2.19 (s, 3H, ArCH3), 2.07 (t, 2H, piperidine-H), 1.70 (m, 2H, CH2), 1.38 (m, 2H, CH2) ppm, HPLC: 95.9%; 2m: MS (ESI) m/z: [M+H]+ = 590.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.53 (s, 1H, ArH), 7.93 (s, 4H, ArH), 7.88 (d, 2H, J = 8.0 Hz, ArH), 7.56 (d, 2H, J = 8.0 Hz, ArH), 7.35 (s, 1H, ArH), 7.25 (d, 1H, J = 7.6 Hz, ArH), 7.13 (d, 1H, J = 7.2 Hz, ArH), 3.59 (s, 2H, PhCH2N), 2.70 (t, 4H, piperidone-H), 2.36 (t, 4H, piperidone-H), 2.27 (s, 3H, ArCH3), 2.25 (s, 3H, ArCH3) ppm, HPLC: 95.3%; 2n: MS (ESI) m/z: [M+H]+ = 632.3. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.58 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.37 (s, 1H, ArH), 7.20 (d, 1H, J = 7.2 Hz, ArH), 7.13 (d, 1H, J = 7.6 Hz, ArH), 3.74 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.16 (s, 3H, ArCH3), 1.56 (s, 2H, CH2), 1.46 (s, 4H, (CH2)2), 0.97 (s, 12H, 4*CH3) ppm, HPLC: 97.0%; 2o: MS (ESI) m/z: [M+H]+ = 646.3. 1H NMR (400 M, DMSO-d6) d 9.94 (s, 1H, CONH), 9.58 (s, 1H, NH), 8.51 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.54 (d, 2H, J = 6.8 Hz, ArH), 7.28 (s, 1H, ArH), 7.18 (d, 1H, J = 6.4 Hz, ArH), 7.07 (d, 1H, J = 6.8 Hz, ArH), 3.86 (s, 2H, PhCH2N), 2.39 (s, 4H, CH2COCH2), 2.25 (s, 3H, ArCH3), 2.18 (s, 3H, ArCH3), 1.96 (s, 12H, (CH3)4) ppm, HPLC: 95.1%; 2p: MS (ESI) m/z: [M+H]+ = 659.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.63
987
(s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 7.6 Hz, ArH), 7.29 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.11 (d, 1H, J = 6.8 Hz, ArH), 3.52 (s, 2H, PhCH2N), 3.21 (m, 1H, CHN), 2.90 (m, 2H, CH2N), 2.67 (m, 2H, NCH2), 2.52 (m, 4H, N(CH2)2), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 1.95 (m, 4H, (CH2)2), 1.56 (m, 4H, (CH2)2), 1.36 (m, 2H, CH2) ppm, HPLC: 98.1%; 2q: MS (ESI) m/z: 1/2[M+H]+ = 312.7. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.91(m, 6H, ArH), 7.54 (m, 2H, ArH), 7.24 (d, 2H, J = 14 Hz, ArH), 7.04 (s, 1H, ArH), 6.87 (m, 2H, ArH), 6.45 (m, 2H, ArH), 4.44 (s, 2H, PhCH2N), 2.72 (m, 2H, CH2N), 2.26 (s, 3H, ArCH3), 2.18 (s, 3H, ArCH3), 1.92 (m, 2H, PhCH2), 1.23 (m, 2H, CH2) ppm, HPLC: 98.3%; 2r: MS (ESI) m/z: [M+H]+ = 630.2. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.35 (s, 1H, ArH), 7.25 (m, 2H, ArH), 7.13 (d, 1H, J = 7.6 Hz, ArH), 6.76 (d, 1H, J = 5.2 Hz, ArH), 3.64 (s, 2H, PhCH2N), 3.44 (s, 2H, NCH2), 2.80 (m, 2H, NCH2), 2.76 (m, 2H, ArCH2), 2.26 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3) ppm, HPLC: 99.5%; 2s: MS (ESI) m/z: [M+H]+ = 645.3. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.26 (s, 1H, ArH), 7.19 (d, 1H, J = 8.0 Hz, ArH), 7.07 (d, 1H, J = 7.6 Hz, ArH), 4.12 (d, 1H, J = 13.2 Hz, CH), 3.31 (s, 2H, PhCH2N), 3.24 (m, 2H, CH2), 2.80 (m, 2H, CH2), 2.60 (m, 2H, CH), 2.26 (s, 3H, ArCH3), 2.19 (s, 3H, ArCH3), 2.10 (m, 1H, CH2), 1.91 (m, 1H, CH2), 1.67 (m, 4H, CH2CH2), 1.62 (m, 4H, CH2CH2) ppm, HPLC: 97.1%; 2t: MS (ESI) m/z: [M+H]+ = 602.3. 1H NMR (400 M, DMSO-d6) d 10.02 (s, 1H, CONH), 9.70 (s, 1H, NH), 8.54 (s, 1H, ArH), 7.95(s, 4H, ArH), 7.89 (d, 2H, J = 8.8 Hz, ArH), 7.57 (d, 3H, J = 8.4 Hz, ArH), 7.38 (d, 1H, J = 7.2 Hz, ArH), 7.27 (d, 1H, J = 7.2 Hz, ArH), 3.57 (s, 2H, PhCH2N), 2.71 (m, 4H, CH2NCH2), 2.30 (s, 3H, ArCH3), 2.28 (s, 3H, ArCH3) 1.71 (m, 4H, 2*CH2), 1.54 (m, 2H, CHCH), 1.44 (m, 2H, CH2) ppm, HPLC: 97.3%; 2u: MS (ESI) m/z: [M+H]+ = 617.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.54 (s, 1H, ArH), 7.94(s, 4H, ArH), 7.89 (d, 2H, J = 8.0 Hz, ArH), 7.57 (d, 2H, J = 8.0 Hz, ArH), 7.28 (s, 1H, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 7.2 Hz, ArH), 3.56 (s, 2H, PhCH2N), 2.84 (m, 2H, NCH2), 2.64 (m, 2H, NCH2), 2.22 (m, 13H, NCH3+ArCH3+CH2), 1.88 (m, 1H, CH2), 1.48 (m, 1H, CH2) ppm, HPLC: 95.3%; 2v: MS (ESI) m/z: [M+H]+ = 564.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.28 (s, 1H, ArH), 7.21 (d, 1H, J = 8.0 Hz, ArH), 7.08 (d, 1H, J = 7.6 Hz, ArH), 3.43 (s, 2H, PhCH2N), 2.30 (s, 2H, CH2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 2.12 (s, 3H, NCH3), 1.48 (m, 2H, CH2), 0.85 (t, 3H, J = 7.6 Hz, CH3) ppm, HPLC: 97.3%; 2w: MS (ESI) m/z: [M+H]+ = 564.3. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.94(s, 4H, ArH), 7.88 (d, 2H, J = 8.8 Hz, ArH), 7.56 (d, 2H, J = 8.4 Hz, ArH), 7.32 (s, 1H, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.12 (d, 1H, J = 7.2 Hz, ArH), 3.53 (s, 2H, PhCH2N), 2.91 (s, 1H, CHN), 2.26 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 2.06 (s, 3H, NCH3), 1.05 (d, 6H, (CH3)2) ppm, HPLC: 96.0%; 2x: MS (ESI) m/z: [M+H]+ = 536.3. 1HNMR (400 M, DMSO-d6) d 9.96 (s, 1H, CONH), 9.62 (s, 1H, NH), 8.51 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.4 Hz, ArH), 7.52 (d, 2H, J = 8.4 Hz, ArH), 7.29 (s, 1H, ArH), 7.19 (d, 1H, J = 8.0 Hz, ArH), 7.08 (d, 1H, J = 7.6 Hz, ArH), 3.39 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 2.17 (s, 6H, N(CH3)2) ppm, HPLC: 99.6%; 2y: MS (ESI) m/z: [M+H]+ = 564.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.93 (s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.30 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 7.2 Hz, ArH), 3.50 (s, 2H, PhCH2N), 2.51 (m, 4H, CH2NCH2), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 0.99 (s, 6H, (CH3)2) ppm, HPLC: 97.6%; 2z: MS (ESI) m/z: [M+H]+ = 596.3. 1H NMR (400 M, DMSO-d6) d 9.95 (s, 1H, CONH), 9.62 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.29 (s, 1H, ArH), 7.19 (d, 1H, J = 7.6 Hz, ArH), 7.12 (d, 1H, J = 7.6 Hz, ArH), 4.32 (t, 1H, J = 4.8 Hz, OH), 3.61 (s, 2H, PhCH2N), 3.46 (m, 4H, 2*CH2O), 2.56 (t, 4H, J = 6.0 Hz, 2*CH2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 96.3%; 2za:MS (ESI) m/z: [M+H]+ = 607.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(m, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.32 (s, 1H, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.09 (d, 1H, J = 7.6 Hz, ArH), 3.38 (s, 2H, PhCH2N), 2.82 (t, 2H, J = 7.6 Hz, CH2), 2.56 (s, 6H, 2*CH3), 2.41 (t, 2H, J = 7.2 Hz, CH2), 2.26 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 2.17 (s, 3H, CH3), 1.78 (m, 2H, CH2) ppm, HPLC: 99.0%; 2zb: MS (ESI) m/z: [M+H]+ = 635.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.88 (d, 2H, J = 8.8 Hz, ArH), 7.56 (d, 2H, J = 8.4 Hz, ArH), 7.28 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 7.6 Hz, ArH), 3.69 (s, 2H, PhCH2N), 2.93 (m, 2H, 2*CHN), 2.50 (m, 2H, 2*CH2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 0.94 (m, 12H, 4*CH3) ppm, HPLC:95.0%; 2zc: MS (ESI) m/z: [M+H]+ = 672.4. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.60 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.88 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 7.6 Hz, ArH), 7.29 (s, 1H, ArH), 7.14 (m, 2H, ArH), 3.67 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.18 (s, 3H, ArCH3), 1.79 (m, 2H, 2*NCH), 1.69 (m, 8H, 4*(CH2)), 1.23 (m, 8H, 4*(CH2)), 1.23 (m, 4H, 2*(CH2)) ppm, HPLC:94.54%; 2zd: MS (ESI) m/z: [M+H]+ = 536.2. 1H NMR (400 M, DMSOd6) d 10.01 (s, 1H, CONH), 9.72 (s, 1H, NH), 8.53 (s, 1H, ArH), 7.94 (s, 4H, ArH), 7.88 (d, 2H, J = 8.4 Hz, ArH), 7.56 (m, 3H, ArH), 7.35 (d, 1H, J = 8.0 Hz, ArH), 7.29 (d, 1H, J = 8.0 Hz, ArH), 4.11 (s, 2H, PhCH2N), 2.97 (m, 2H, CH2N), 2.26 (s, 6H, 2*ArCH3), 1.21 (t, 3H, CH3) ppm, HPLC: 98.8%; 2ze: MS (ESI) m/z: [M+H]+ = 552.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 7.2 Hz, ArH), 7.55 (s, 2H, ArH), 7.29 (s, 1H, ArH), 7.18 (s, 1H, ArH), 7.11 (s, 1H, ArH), 4.47 (s, 1H, OH), 3.68 (s, 2H, CH2O), 3.46 (s, 2H, PhCH2N), 2.57 (s, 2H, CH2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 95.6%; 2zf: MS (ESI) m/z: [M+H]+ = 564.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH),
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M. Xin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 983–988 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.32 (s, 1H, ArH), 7.19 (d, 1H, J = 7.6 Hz, ArH), 7.13 (d, 1H, J = 8.0 Hz, ArH), 3.67 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.19 (s, 3H, ArCH3), 1.11 (s, 9H, (CH3)3) ppm, HPLC: 98.5%; 2zg: MS (ESI) m/z: [M+H]+ = 576.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.88 (d, 2H, J = 8.4 Hz, ArH), 7.56 (d, 2H, J = 8.4 Hz, ArH), 7.29 (s, 1H, ArH), 7.19 (d, 1H, J = 8.0 Hz, ArH), 7.12 (d, 1H, J = 7.6 Hz, ArH), 3.66 (s, 2H, PhCH2N), 3.00 (m, 1H, CHN), 2.26 (s, 3H, ArCH3), 2.19 (s, 3H, ArCH3), 1.73 (m, 2H, CH2), 1.64 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.34 (m, 2H, CH2) ppm, HPLC: 95.0%;2zh: MS (ESI) m/z: [M+H]+ = 619.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.31 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.11 (d, 1H, J = 7.2 Hz, ArH), 3.72 (s, 2H, PhCH2N), 2.63 (t, 2H, CH2N), 2.40 (t, 2H, NCH2), 2.32 (m, 4H, N(CH2)2), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 1.47 (m, 4H, (CH2)2), 1.36 (m, 2H, CH2) ppm, HPLC: 95.4%: 2zi: MS (ESI) m/z: [M+H]+ = 588.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.53 (s, 1H, ArH), 7.93(s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.65 (s, 1H, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.41 (s, 1H, ArH), 7.26 (d, 1H, J = 7.6 Hz, ArH), 7.18 (d, 1H, J = 8.0 Hz, ArH), 6.45 (s, 1H, furan-H), 6.42 (s, 1H, furan-H), 3.89 (s, 2H, CH2N), 3.85 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.23 (s, 3H, ArCH3) ppm, HPLC: 97.6%; 2zj: MS (ESI) m/z: [M+H]+ = 584.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.91(s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.33 (s, 1H, ArH), 7.21 (d, 1H, J = 8.0 Hz, ArH), 7.15 (d, 1H, J = 7.6 Hz, ArH), 7.02 (m, 2H, ArH), 6.56 (m, 2H, ArH), 6.51 (m, 1H, ArH), 6.24 (brs, 1H, ArNH), 4.24 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.18 (s, 3H, ArCH3) ppm, HPLC: 98.8%; 3a: MS (ESI) m/z: [M+H]+ = 494.3. 1H NMR (400 M, DMSO-d6) d 9.94 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.50 (s, 1H, ArH), 7.92 (m, 4H, ArH), 7.72 (d, 2H, J = 7.6 Hz, ArH), 7.55 (m, 3H, ArH), 7.28 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.09 (d, 1H, J = 7.6 Hz, ArH), 3.57 (m, 4H, morpholine-H), 3.43 (s, 2H, PhCH2N), 2.35 (m, 4H, morpholine-H), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 96.8%; 3b: MS (ESI) m/z: [M+H]+ = 508.3. 1H NMR (400 M, DMSO-d6) d 10.06 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.78 (d, 2H, J = 5.2 Hz, ArH), 8.57 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.70 (d, 2H, J = 5.2 Hz, ArH), 7.28 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.08 (d, 1H, J = 7.6 Hz, ArH), 3.44 (s, 2H, PhCH2N), 2.42 (m, 8H, N(CH2)2(CH2)2N), 2.26 (s, 3H, ArCH3), 2.23 (s, 3H, ArCH3), 2.20 (s, 3H, NCH3) ppm, HPLC: 97.5%; 3c: MS (ESI) m/z: [M+H]+= 413.2. 1H NMR (400 M, DMSO-d6) d 9.73 (s, 1H, CONH), 9.55 (s, 1H, NH), 8.39 (s, 1H, ArH), 8.36 (s, 1H, ArH), 8.11 (s, 1H, ArH), 7.97 (m, 4H, ArH), 7.11 (s, 3H, ArH), 3.96 (s, 3H, ArCH3), 2.33 (s, 3H, ArCH3), 2.18 (s, 6H, 2*ArCH3) ppm, HPLC: 98.4%; 3d: MS (ESI) m/z: [M+H]+ = 511.3. 1H NMR (400 M, DMSO-d6) d 9.77 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.13 (s, 1H, ArH), 7.96 (s, 4H, ArH), 7.28 (s, 1H, ArH), 7.22 (d, 1H, ArH), 7.09 (d, 1H, ArH), 3.97 (s, 3H, ArCH3), 3.43 (s, 2H, PhCH2N), 2.52 (s, 3H, NCH3), 2.35 (m, 8H, piperazine-H), 2.22 (s, 3H, ArCH3), 2.17 (s, 3H, ArCH3) ppm, HPLC: 99.4%; 3e: MS (ESI) m/z: [M+H]+ = 483.3. 1H NMR (400 M, DMSOd6) d 9.77 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.13 (s, 1H, ArH),
7.97 (s, 4H, ArH), 7.31 (s, 1H, ArH), 7.21 (d, 1H, ArH), 7.10 (d, 1H, ArH), 3.97 (s, 3H, ArCH3), 3.57 (s, 2H, PhCH2N), 2.46 (m, 4H, pyrolidine-H), 2.35 (s, 3H, ArCH3), 2.23 (s, 3H, ArCH3), 1.70 (m, 4H, pyrolidine-H) ppm, HPLC: 98.8%; 3f: MS (ESI) m/z: [M+H]+ = 496.3. 1H NMR (400 M, DMSO-d6) d 9.76 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.39 (d, 2H, ArH), 8.12 (s, 1H, ArH), 7.97 (s, 4H, ArH), 7.28 (s, 1H, ArH), 7.21 (d, 1H, ArH), 7.09 (d, 1H, ArH), 3.96 (s, 3H, ArCH3), 3.34 (s, 2H, PhCH2N), 2.50 (m, 4H, piperidine-H), 2.34 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 1.50 (m, 4H, piperidine-H), 1.40 (m, 2H, piperidine-H) ppm, HPLC: 98.4%; 3g: MS (ESI) m/z: [M+H]+ = 526.3. 1H NMR (400 M, DMSO-d6) d 9.77 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.13 (s, 1H, ArH), 7.97 (s, 4H, ArH), 7.29 (s, 1H, ArH), 7.23 (d, 1H, ArH), 7.10 (d, 1H, ArH), 3.98 (s, 3H, ArCH3), 3.58 (m, 2H, CHOCH), 3.42 (s, 2H, PhCH2N), 2.71 (m, 2H, morpholine-H), 2.35 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 1.65 (m, 2H, morpholine-H), 1.04 (d, 6H, 2*CH3) ppm, HPLC: 98.4%; 3h: MS (ESI) m/z: [M+H]+ = 512.3. 1H NMR (400 M, DMSO-d6) d 9.79 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.41 (d, 2H, ArH), 8.13 (s, 1H, ArH), 7.98 (s, 4H, ArH), 7.27 (s, 1H, ArH), 7.22 (d, 1H, ArH), 7.08 (d, 1H, ArH), 4.56 (s, 1H, OH), 3.97 (s, 3H, ArCH3), 3.41 (m, 1H, CHO), 3.36(s, 2H, PhCH2N), 2.68 (m, 2H, piperidineH), 2.35 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 2.02 (m, 2H, piperidine-H), 1.71 (m, 2H, piperidine-H), 1.39 (m, 2H, piperidine-H) ppm, HPLC: 98.6%; 3i: LC-MS (ESI) m/z: [M+H]+ = 484.3. 1H NMR (400 M, DMSO-d6) d 9.80 (s, 1H, CONH), 9.73 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.12 (s, 1H, ArH), 7.97 (s, 4H, ArH), 7.57 (s, 1H, ArH), 7.34 (d, 2H, ArH), 3.96 (s, 3H, ArCH3), 3.35 (s, 2H, PhCH2N), 2.65 (m, 2H, CH2N), 2.34(s, 3H, ArCH3), 2.28 (s, 3H, CH3N), 1.28 (m, 2H, CH2), 0.89 (m, 3H, CH3) ppm, HPLC: 99.2%; 3j: MS (ESI) m/z: [M+H]+ = 516.3. 1H NMR (400 M, DMSO-d6) d 9.77 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.12 (s, 1H, ArH), 7.98 (m, 4H, ArH), 7.29 (s, 1H, ArH), 7.20 (d, 1H, ArH), 7.13 (d, 1H, ArH), 4.37 (t, 2H, 2*OH), 3.96 (s, 3H, ArCH3), 3.61 (s, 2H, PhCH2N), 3.46 (m, 4H, 2*CH2O), 3.17 (m, 4H, 2*CH2N), 2.34(s, 3H, ArCH3), 2.21 (s, 3H, ArCH3) ppm, HPLC: 98.0%; 3k: MS (ESI) m/z: [M+H]+ = 512.3. 1H NMR (400 M, DMSO-d6) d 9.77 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.12 (s, 1H, ArH), 7.95 (s, 4H, ArH), 7.33 (s, 1H, ArH), 7.17 (d, 1H, ArH), 7.13 (d, 1H, ArH), 3.96 (s, 3H, ArCH3), 3.66 (s, 2H, PhCH2N), 2.30 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 1.11 (s, 9H, 3*CH3) ppm, HPLC: 96.9%. 10. Pharmacokinetic Studies in SD rat. Compounds were administered to SD rats at a dose of 1 mg/kg (in 5% DMA+ 5% Tween-80) for iv or 5 mg/kg (0.5%HPMC+0.3% Tween-80, pH=2.8) for po administration, respectively. After oral administration, blood samples collected for PK analyses (n = 3) were centrifuged at 4000 rpm for 5 min at 4 °C. Plasma samples were analyzed after protein precipitation with acetonitrile acidified with1% formic acid. LC/ MS/MS analysis of test compounds was performed under optimized conditions to obtain the best sensitivity and selectivity of the analyte in selected reaction monitoring mode (SRM) containing an internal standard. Plasma concentration-time data were measured by a noncompartmental approach using the software WinNonlin Enterprise, version 5.2 (Pharsight Co.,Mountain View, CA).
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 4-(2-pyrimidinylamino) benzamides inhibitors of hedgehog signaling pathway Minhang Xin a,b,⇑, Jun Wen b, Feng Tang b, Chongxing Tu b, Wei Huang b, Han Shen b, Xinge Zhao b, Lingfei Cheng b, Mengyu Wang b, Liandi Zhang b a b
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 Jiangsu Simcere Pharmaceutical Co. Ltd., Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No 699-18, Xuan Wu District, Nanjing 210042, P.R. China
a r t i c l e
i n f o
Article history: Received 18 September 2013 Revised 10 November 2013 Accepted 12 December 2013 Available online 17 December 2013
a b s t r a c t A novel series of hedgehog signaling pathway inhibitors has been designed based on the 4-(2-pyrimidinylamino) benzamides scaffold. The synthesis and SAR of these compounds are described. Optimization leads to the identification of compound 3c, a potent and orally available agent with improved physicochemical and pharmacokinetic properties. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Hedgehog signaling pathway Inhibitors SAR Pharmacokinetic properties
The Hedgehog (Hh) signaling pathway is receiving increasing attention because of its very role in malignant transformation of various cancers.1 Genetic mutations associated with this signaling pathway such as Ptch-mutation, Smo-mutation, and Sufumutation result in tumorigenesis in several cancers such as basal cell carcinoma (BCC), medulloblastoma, and rhabdomyosarcoma. Additionally, strong evidences link overexpression of Hh ligands to tumor aggravation of various other cancers such as hepatocellular, pancreatic, gastric, colorectal, esophageal, lung, ovarian, prostate, melanoma, glioblastoma, leukemia, lymphoma.2 Thus, inhibition of Hh signaling pathway is thought to have considerable potential for therapy of many cancer types. To date, many classes of Hh pathway inhibitors have been described.3 Vismodegib has been recently approved by FDA for metastatic BCC treatment.4 Other molecules including sonidegib (NVP-LDE225, Phase III), LY-2940680 (Phase II), BMS-833923 (XL-139, Phase II), PF-04449913(Phase I), TAK-441 (Phase I), NVP-LEQ506 (Phase I) have been already studied in clinic for many tumors treatment (Fig. 1).5 Besides, there are still some novel small molecules investigated in preclinical stage or drug discovery stage.6 In aspiration of interest in developing Hh signaling pathway inhibitors for treatment of many tumors, we recently detailed ⇑ Corresponding author. address: 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. Tel.: +86 25 85560000 3192. E-mail address: [email protected] (M. Xin). 0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.050
the discovery of a novel series of 4-(2-pyrimidinylamino) benzamides as potent Hh signaling pathway inhibitors, exemplified by 1 (Fig. 2), which inhibited the Hh signaling pathway in Gli-luciferase reporter assay with IC50 = 1.3 nM, more potent than control positive drug vismodegib. Initial SAR studies in this series showed that structural modification on A-ring of 1 seriously affected the inhibitory activity and the optimal substituents was conferred to 4-trifluoromethylphenyl, phenyl, 4-pyridinyl or others. Following SAR studies focused on D-ring modifications, especially at the position of phenyl group, demonstrating that methyl substitution at C-2 of the phenyl or several basic side chains functionalized at C-5 was favorable for high inhibitory activity.7 As a continuation of our study toward identification of potent Hh signaling pathway inhibitors, a further SAR study was conducted to explore various basic amine chains linked to D-ring, looking to identify potent inhibitors with improved physicochemical properties which was of importance for successfully developing drug candidate. Furthermore, considering that 1 exhibited unsatisfactory pharmacokinetic (PK) profile, efforts to improve the PK properties of this scaffold was also made by modification on both A-ring and D-ring. Herein, in this report we disclose the synthesis and SAR analysis of these analogs of Hh signaling pathway inhibitors, and also we report the PK evaluation in vivo. On the basis of the previous SAR study, at the outset, our strategy focused on bearing various basic substitutions at C-5 of D-ring. A number of analogs incorporating basic amine side chains at C-5 of D-ring were summarized in Table 1. The Hh signaling pathway inhibitory activity of compounds 2a–2zj were evaluated by a
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Cl
O
N
H N
Cl
N H
O
O CF 3
S O
vismodegib (GDC-0449)
N
N
O
N NH
F
N H
Exelixis /BMS Ph II
O
N
O
O
N
NH
N H BMS-833923 (XL-139)
Novartis Ph III
N N
HN O
N
N
N
OH
O
N N
CF3
O CF 3
H N
N H
N O
N N
N
sonidegib (NVP-LDE-225;LDE-225)
Curis/Genentech(Roche) launched
N N
O N
O
N N
OH
CN
LY-2940680
PF-04449913
Lilly Ph II
Pfizer Ph I
NVP-LEQ506
TAK-441 Millennium /Takeda
Ph I
Novartis Ph I
Figure 1. Chemical structures of clinical Hh signaling pathway inhibitors.
O
CF3
A
3 O
B N
N
C
2 N 1 H
D
4 5
6
N H
Figure 2. Structure of compound 1.
luciferase reporter in NIH3T3 cell carrying a stably transfected Glireporter construct (Gli-luciferase reporter cell lines).7,8 Table 1 outlined the in vitro IC50 for this series of various basic substitutions at C-5 of D-ring of 4-(2-pyrimidinylamino) benzamides9. Notably, all the analogs bearing basic amine on the molecules (2a–2zj) showed good Hh signaling inhibitory activity with IC50 varied from 0.23 to 35.35 nM. Of all the amines explored, some cyclic aliphatic amines, such as morpholine (2a), dimethyl morpholine (2b), piperidine (2c), thiomorpholine (2d), pyrrolidine (2e), were more effective than the lead compound 1 and 7–20 fold higher activity than GDC-0449. However the similar cyclic amine piperazine (2f) showed significantly decreased activity, perhaps due to relatively strong basicity of second basic center of NH. Other piperazine derivatives such as N-methyl piperazine (2g), N-ethyl piperazine (2h), N-phenyl piperazine (2i), N-cyclopropanecarbonyl piperazine (2j) and piperazin-3-one (2k) exhibited improved potency compared to 2f because of relatively suitable basicity. A survey of substituted piperidines was investigated and led to some more favorable piperidine derivatives including 4-hydroxy piperidine (2l), piperidin-4-one (2m), tetramethyl piperidine (2n), and tetramethyl piperidin-4-one (2o). However, 4-piperidyl piperidine (2p) displayed lower activity, while the activity of piperidine fusing aromatic ring such as tetrahydroquinoline (2q) and tetrahydrothienopyridine (2r) almost retained. These results indicated that adding extra basicity was not favorable to the activity. The same case was accounted by pyrrolidine derivatives 2s whose activity was weaker than 2e. Additionally, two bicyclic amine derivatives, 2t and 2u, were also investigated, and it was found 2t was 6-fold more potent than 2u. After screening a variety of cyclic amine substituents, some acyclic amines were explored. It appeared a suitable length of acyclic amines was important for maintaining activity. For examples, 2v–2z displayed subnanomolar inhibition. However, compounds 2za and 2zb, displayed much lower potency, with IC50 = 35.3 and 10.9 nM, respectively, mainly due to the larger size of amine chains. In fact, the extra basicity of these two compounds also strongly effected the decreased activity additively.
Steric hindrance could also affect the activity, for instance compound 2zc bearing a bulky dicyclohexylamine group, which showed moderate activity. Besides, secondary amines like ethylamine (2zd), hydroxyethylamine (2ze), tert-butylamine (2zf) or cyclopentylamine (2zg) were tolerated, displaying nearly comparable activity to lead compound 1, except 2zh that showing weak potency of Hh inhibitory activity, which was considered due to its length and extra basicity. In addition, interestingly, the inhibitory activity of the basic amine substituents containing aromatic groups such as furylmethamine (2zi) and phenylamine (2zj) were retained, and these results were consistent with the above reported ones 2q and 2r. In a word, this SAR study showed 30 compounds displayed better inhibitory activities than positive control GDC-0449, of which 20 exhibited superior activities to lead compound 1. To evaluate whether these compounds had an improved druggability compared to 1, of our most potent inhibitors, two compounds 2a and 2b were further investigated by profiling iv and po pharmacokinetics in SD rat.10 The results were illustrated in Table 3. After intravenous injection with 1 mg/kg in SD rats, the area-under-curve (AUC) of 2a reached 1.3 h lg/mL, larger than that of 1 (0.92 h lg/mL). Such more plasma exposure is due part to the much smaller volume of distribution (7.4 L/kg) and lower systemic clearance (12.0 mL/min/kg). Furthermore, when orally administered at 5 mg/kg dose, 2a showed higher oral exposure and lower systemic clearance. Consequently, compound 2a showed superior oral bioavailability (F = 46%) to 1 (36%). However, unfortunately, 2b bearing N-methylpiperizine group showed poor PK characteristics both by iv and po administration. Despite 2a and 2b contained much hydrophilic group morpholine and Nmethylpiperizine in structures respectively, both molecules had already possessed higher lipophilicity (2a, A log P = 7.07 and 2b, Aog P = 7.34) compared to vismodegib (Aog P = 4.265). It appeared attractive to attempt a structural modification to decrease the A log P value so as to further improve drug-like properties. Therefore, Three compounds (3a–3c) were synthesized (Table 2), beginning to replace of trifluoromethoxyphenyl of A-ring with phenyl, 4-pyridinyl and 1-methylpyrazolyl because these groups were previously reported to afford good to moderate inhibitory activity.7 Of the three compounds, both compounds 3a and 3b showed nearly equipotent inhibition with 2a in Hh signaling pathway and possessed a lower A log P values (3a, A log P = 4.95 and 3b, Aog P = 4.069), but did not improve the plasma exposure in an intravenous administration (Tables 2 and 3). Fortunately, at this point, 3c with 1-methylpyrazolyl, showing equipotent potency with 1 and a much lower A log P (3c, A log P = 3.6), was found to
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M. Xin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 983–988 Table 1 In vitro inhibition of Hh signaling pathway for compounds modification at C-5 of Dring of 4-(2-pyrimidinylamino) benzamides
Table 2 The IC50 and the AlogP values for compounds modification on A and D region of 4-(2pyrimidinylamino) benzamides9
OCF 3
R1
O N
O N N
Compds
N H
R
Compds
Gli-luc reporter IC50 (nM)
2a
N
2b
N
2c
N
2d
N
2e
N
R
N H
N
2a-2zj
O
O
S
Compds
R
Gli-luc reporter IC50 (nM)
0.53
2t
N
0.73
2u
N
1.0
2v
N
0.34
2w
0.43
2x
1.6
10.5
N
0.93 0.89
N
3a 3b
N
NH
N
N
10.5
2y
N
0.45
2g
OH
2z
0.47 OH
2h
N
N
N
0.77
2za
N
2.0
2zb
N
N
N H
2j
N
2zc
2.0
2zd
NH
O
2k
N
2.7
N
NH
0.72
2l
N
OH
0.44
2ze
2m
N
O
0.28
2zf
0.76
2zg
0.29
2zh
N H
19.5
2zi
H N
2.4
2zj
1.5
1
1.3
3.9
GDC
7.2
2n
N
2o 2p
2q
N
O
N
N
N
NH
OH
HN
HN
1.6
N O
33.6 2.5
1.1
S
2r
N
N
2s
0.68
4.95
N
N
0.61
4.069 3.6
3d
N N
N
N
10.0
3.87
3e
N N
N
9.2
4.373
3f
N N
N
5.6
4.829
N N
N
8.4
4.355
3h
N N
N
21.2
3.345
3i
N N
N
17.5
4.785
N N
N
31.9
2.833
18.1
4.308
1.3 7.2
7.795 4.265
O
OH
OH OH
1 GDC-0449
N N
HN
a A Log P values were calculated by Discovery studio 3.0 using Calculate Molecular Properties Protocol.
1.1 0.90
HN
O
1.3
3k 1.3
7.07 7.34
N
10.9
O N
0.53 0.45
O
3j
2i
A Log Pa
N
35.3
N
Gli-luc reporter IC50 (nM)
N N
0.23
N
R
3c
3g 2f
N
3a-3k
N H
2a 2b
0.59
N
R1
R
N H
N
significantly improve the oral exposure (AUC = 3.8 h lg/mL) by po 5 mg/kg dosage, moreover whose oral bioavailability reached almost 64%. In addition to the significant insight of low A log P value gained with the 1-methylpyrazolyl replacement, an additional structure modification at position 5 of D-ring was pursued by incorporating substituents, such as methylpiperizine, pyrrolidine, piperidine, dimethylmorpholine, etc., which were identified to have optimized activity above, then compounds 3d–3k were
prepared (Table 2). Unfortunately, although 3d–3k also demonstrated low A log P values, only displayed less potency (5.6– 31.9 nM) compared to 1, which stalled their progression of further PK studies. Therefore, based on the optimal studies of combination of activity and PK properties, compounds 3c was considered to be the ideal lead candidate, which exhibited superior potency, excellent oral exposure and improved oral bioavailability in comparison to 1. And now 3c were being evaluated in vivo in Hh pathway dependent medulloblastoma allograft and other Hh signaling operative solid tumor models. The results will be reported later. The 4-(2-pyrimidinylamino) benzamides were readily prepared according to Scheme 1. Briefly, commercially available 2,4-dichloro-5-methylpyrimidine were treated with several aryl boronic acid or boronic ester using palladium-catalyzed suzuki coupling reaction to provide 4-aryl-substituted pyrimidines 5a–5d. The intermediates 5a–5d were reacted with methyl 4-aminobenzoate under palladium catalysis to afford the buckwald coupling products 6a–6d, which were further hydrolyzed to produce the free acids 7a–7d. The condensation of 7a–7d with 3-amino-4-methylbenzyl alcohol, followed by chlorination and next acylation with various amines, afforded the designed inhibitors 2a–2zj and 3a–3k (Scheme 1).7 In summary, we have utilized 4-(2-pyrimidinylamino) benzamide core to produce novel Hh signaling pathway inhibitors. Starting from the lead compound 1, we were able to explore structural modification on A-ring and D-ring to identify potent inhibitors
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Table 3 In vivo SD rat pharmacokinetic properties for represented compounds compds GDC-0449 1 2a 2b 3a 3b 3c a b
Dose (mg/kg) 1 1 5 1 5 1 5 1 1 1 5
a
(iv) (iv)a (po)a (iv)a (po)b (iv)a (po)b (iv)a (iv)a (iv)a (po)b
Cmax (lg/mL)
AUC (hlg/mL)
Vss (L/kg)
Cl (mL/min/kg)
MRT(0–t) (h)
T1/2 (h)
1.2 0.76 0.34 1.2 0.28 0.30 0.094 0.84 0.58 2.1 1.5
2.5 0.92 1.7 1.3 2.4 0.58 1.3 0.71 0.27 1.2 3.8
0.95 14.9 27.7 7.4 16.3 20.0 42.9 2.1 5.7 0.90 2.9
6.8 16.7 46.6 12.0 32.8 24.9 51.9 23.3 61.5 14.3 23.9
2.2 4.0 5.1 4.1 6.6 7.4 9.7 1.2 0.90 0.81 2.4
1.6 10.3 7.0 7.2 5.8 9.2 9.5 1.0 1.1 0.8 1.4
F (%)
36.1 46.3 44.4
63.6
compound was formulated using 5%DMA + 5% Tween-80. Compound was formulated using 0.5%HPMC + 0.3% Tween-80, pH = 2.8.
R1
Cl a
N N
N
Cl
4
Cl
N
O N N H 8a-8d
OCH 3
c N
d
OH N H 7a-7d R1
O
O
f
e N H
N OH
O N
N H 6a-6d
R1 N H
R1
O N
5a-5d
R1
N
R1 b
N
N
N H
N Cl
N
N H
R
N H 2a-2zj;
9a-9d
3a-3k
Scheme 1. Reagents and conditions: (a) Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 6–12 h, 4-trifluoromethoxyphenylboronic acid for 5a, 73%; phenylboronic acid for 5b, 89%; pyridine-4-boronic acid for 5c, 78%; 1-methyl-1H-pyrazole-4-boronic acid pinacol ester for 5d, 43%; (b) Pd(OAc)2, BINAP, Cs2CO3, dioxane, 170 °C, microwave for 3 h, methyl 4-aminobenzoate, 43–94%; (c) NaOH, MeOH/H2O, reflux overnight, 85–100%; (d) 3-amino-4-methylbenzyl alcohol, HATU, DMF, DIPEA, 85 °C overnight, 38–94%; (e) SOCl2, CH2Cl2, rt, 2 h, 64–89%; (f) RH, DMF, K2CO3, rt, overnight; from 9a to 2a–2zj; from 9b to 3a; from 9c to 3b; from 9d to 3c–3k; 43–89%.
with improved physicochemical properties. From these compounds, some structure–activity relationships were concluded and the compound 3c was identified as a potent and orally vailable Hh signaling inhibitor with favorable pharmacokinetic properties. Nowadays, the in vivo biological evaluation and the safety investigation of 3c is being conducted both in Hh pathway dependent medulloblastoma allograft and Hh signaling operative solid tumor models, and the progress 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). The authors thank Dr. Haopeng Sun of China Pharmaceutical University for the A log P calculation. References and notes 1. (a) Li, Y.; Maitah, M. Y.; Ahmad, A.; Kong, D.; Bao, B.; Sarkar, F. H. Expert Opin. Ther. Targets 2012, 16(1), 49; (b) Zeng, X.; Goetz, J. A.; Suber, L. M.; Scott, W. J., Jr.; Schreiner, C. M.; Robbins, D. J. Nature 2001, 411, 716; (c) 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; (d) 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; (e) Onishi, H.; Katano, M. Cancer Sci. 2011, 102, 1756. 2. (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; (d) Merchant, A. A.; Matsui, W. Clin. Cancer Res. 2010, 16, 3130. 3. (a) Mahindroo, N.; Punchihewa, C.; Fujii, N. J. Med. Chem. 2009, 52, 3829; (a) Hadden, M. K. Expert Opin. Ther. Pat. 2013, 23, 345. 4. (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) http://www.fda.gov/. 5. (a) Pan, W.; 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.; Fangxian, S.; 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, 130; (b) Rocconi, R. P.; Samant, L.; WO 2013/043255, 2013.; (b) Ohashi, T.; Oguro, Y.; Tanaka, T.; Shiokawa, Z.; Tanaka, Y.; Shibata, S.; Sato, Y.; Yamakawa, H.; Hattori, H.; Yamamoto, Y.; Kondo, S.; Miyamoto, M.; Nishihara, M.; Ishimura, Y.; Tojo, H.; Baba, A.; Sasaki, S. Bioorg. Med. Chem. 2012, 20, 5507; (c) Munchhof, M. J.; Li, Q.; Shavnya, A.; Borzillo, G. V.; Boyden, T. L.; Jones, C. S.; LaGreca, S. D.; MartinezAlsina, L.; Patel, N.; Pelletier, K.; Reiter, L. A.; Robbins, M. D.; Tkalcevic, G. T. ACS Med. Chem. Lett. 2012, 3, 106; (d) 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. 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) Schaefer, G. I.; Perez, J. R.; Duvall, J. R.; Stanton, B. Z.; Shamji, A. F.; Schreiber, S. L. J. Am. Chem. Soc. 2013, 135, 9578. 7. Xin, M.; Wen, J.; Tang, F.; Tu, C.; Shen, H.; Zhao, X. Bioorg. Med. Chem. Lett. 2013, 23, 6777.
M. Xin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 983–988 8. 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. 9. 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 2a: MS (ESI) m/z: [M+H]+ = 578.3. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.4 Hz, ArH), 7.53 (d, 2H, J = 8.0 Hz, ArH), 7.28 (s, 1H, ArH), 7.19 (d, 1H, J = 7.6 Hz, ArH), 7.09 (d, 1H, J = 7.6 Hz, ArH), 3.57 (s, 4H, morpholine-H), 3.43 (s, 2H, PhCH2N), 2.36 (s, 4H, morpholine-H), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 95.9%; 2b: MS (ESI) m/z: [M+H]+ = 606.3. 1H NMR (400 M, DMSO-d6) d 10.01 (s, 1H, CONH), 9.78 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.95 (s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.58 (s, 1H, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.42 (d, 1H, J = 7.6 Hz, ArH), 7.36 (d, 1H, J = 8.0 Hz, ArH), 4.27 (s, 2H, PhCH2N), 3.98 (m, 2H, CHOCH), 3.26 (d, 2H, J = 7.6 Hz, CH2N), 2.68 (m, 2H, CH2N), 2.27 (s, 3H, ArCH3), 2.26 (s, 3H, ArCH3), 1.12 (s, 3H, CH3), 1.11 (s, 3H, CH3) ppm, HPLC: 95.7%; 2c: MS (ESI) m/z: [M+H]+ = 576.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 8.8 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.25 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.07 (d, 1H, J = 7.6 Hz, ArH), 3.38 (s, 2H, PhCH2N), 2.32 (m, 4H, (CH2)2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 1.48 (m, 4H, (CH2)2), 1.39 (m, 2H, CH2) ppm, HPLC: 99.5%; 2d: MS (ESI) m/z: [M+H]+ = 594.2. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 7.2 Hz, ArH), 7.54 (d, 2H, J = 6.8 Hz, ArH), 7.27 (s, 1H, ArH), 7.20 (d, 1H, J = 6.4 Hz, ArH), 7.07 (d, 1H, J = 6.4 Hz, ArH), 3.46 (s, 2H, PhCH2N), 2.60 (m, 8H, (CH2)4), 2.25 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 99.6%; 2e: MS (ESI) m/z: [M+H]+ = 562.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.63 (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), 7.29 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.09 (d, 1H, J = 7.2 Hz, ArH), 3.55 (s, 2H, PhCH2N), 2.44 (m, 4H, pyrolidine-H), 2.26 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 1.69 (m, 4H, pyrolidine-H) ppm, HPLC: 98.8%; 2f: MS (ESI) m/z: [M+H]+ = 577.3. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.93(s, 4H, ArH), 7.88 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.27 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.08 (d, 1H, J = 7.2 Hz, ArH), 3.45 (s, 2H, PhCH2N), 2.77 (m, 4H, N(CH2)2), 2.35 (m, 4H, N(CH2)2), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 98.7%; 2g: MS (ESI) m/z: [M+H]+ = 591.3. 1H NMR (400 M, DMSO-d6) d 9.96 (s, 1H, CONH), 9.62 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.4 Hz, ArH), 7.53 (d, 2H, J = 8.4 Hz, ArH), 7.26 (s, 1H, ArH), 7.18 (d, 1H, J = 7.6 Hz, ArH), 7.05 (d, 1H, J = 7.6 Hz, ArH), 3.41 (s, 2H, PhCH2N), 2.33 (m, 8H, piperazine-H), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 2.14 (s, 3H, NCH3) ppm, HPLC:99.2%; 2h: MS (ESI) m/z: [M+H]+ = 605.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.26 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.07 (d, 1H, J = 7.6 Hz, ArH), 3.41 (s, 2H, PhCH2N), 2.36 (m, 10H, CH2N(CH2)4)N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 0.96 (t, 3H, J = 7.2 Hz, CH3) ppm, HPLC: 97.7%; 2i: MS (ESI) m/z: [M+H]+ = 653.3. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.31 (s, 1H, ArH), 7.21 (m, 3H, ArH), 7.12 (t, 1H, ArH), 6.92 (d, 2H, J = 8.4 Hz, ArH), 6.76 (t, 1H, ArH), 3.50 (s, 2H, PhCH2N), 3.12 (s, 4H, N(CH2)2), 2.50 (s, 4H, N(CH2)2), 2.26 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3)ppm, HPLC: 98.6%; 2j: MS (ESI) m/z: [M+H]+ = 645.3. 1H NMR (400 M, DMSO-d6) d 9.98 (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 = 7.6 Hz, ArH), 7.30 (s, 1H, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 7.2 Hz, ArH), 3.66 (s, 2H, PhCH2N), 3.47 (s, 4H, CH2NCH2), 2.40 (m, 4H, CH2NCH2), 2.26 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 1.94 (m, 1H, cyclopropyl-H), 1.48 (m, 2H, CH2), 0.70 (m, 4H, cyclopropyl-H) ppm, HPLC: 96.6%; 2k: MS (ESI) m/z: [M+H]+ = 591.3. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.93(s, 4H, ArH), 7.88 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.31 (s, 1H, ArH), 7.23 (d, 1H, J = 8.0 Hz, ArH), 7.10 (d, 1H, J = 7.6 Hz, ArH), 3.51 (s, 2H, PhCH2N), 3.17 (s, 2H, NCH2), 2.80 (m, 2H, NCH2), 2.51 (s, 2H, NCH2), 2.26 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3) ppm, HPLC: 95.1%;2l: MS (ESI) m/z: [M+H]+ = 592.3. 1 H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.25 (s, 1H, ArH), 7.19 (d, 1H, J = 8.0 Hz, ArH), 7.06 (d, 1H, J = 7.6 Hz, ArH), 4.52 (m, 1H, CH), 3.39 (s, 2H, PhCH2N), 2.67 (t, 2H, piperidine-H), 2.26 (s, 3H, ArCH3), 2.19 (s, 3H, ArCH3), 2.07 (t, 2H, piperidine-H), 1.70 (m, 2H, CH2), 1.38 (m, 2H, CH2) ppm, HPLC: 95.9%; 2m: MS (ESI) m/z: [M+H]+ = 590.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.53 (s, 1H, ArH), 7.93 (s, 4H, ArH), 7.88 (d, 2H, J = 8.0 Hz, ArH), 7.56 (d, 2H, J = 8.0 Hz, ArH), 7.35 (s, 1H, ArH), 7.25 (d, 1H, J = 7.6 Hz, ArH), 7.13 (d, 1H, J = 7.2 Hz, ArH), 3.59 (s, 2H, PhCH2N), 2.70 (t, 4H, piperidone-H), 2.36 (t, 4H, piperidone-H), 2.27 (s, 3H, ArCH3), 2.25 (s, 3H, ArCH3) ppm, HPLC: 95.3%; 2n: MS (ESI) m/z: [M+H]+ = 632.3. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.58 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.37 (s, 1H, ArH), 7.20 (d, 1H, J = 7.2 Hz, ArH), 7.13 (d, 1H, J = 7.6 Hz, ArH), 3.74 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.16 (s, 3H, ArCH3), 1.56 (s, 2H, CH2), 1.46 (s, 4H, (CH2)2), 0.97 (s, 12H, 4*CH3) ppm, HPLC: 97.0%; 2o: MS (ESI) m/z: [M+H]+ = 646.3. 1H NMR (400 M, DMSO-d6) d 9.94 (s, 1H, CONH), 9.58 (s, 1H, NH), 8.51 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.54 (d, 2H, J = 6.8 Hz, ArH), 7.28 (s, 1H, ArH), 7.18 (d, 1H, J = 6.4 Hz, ArH), 7.07 (d, 1H, J = 6.8 Hz, ArH), 3.86 (s, 2H, PhCH2N), 2.39 (s, 4H, CH2COCH2), 2.25 (s, 3H, ArCH3), 2.18 (s, 3H, ArCH3), 1.96 (s, 12H, (CH3)4) ppm, HPLC: 95.1%; 2p: MS (ESI) m/z: [M+H]+ = 659.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.63
987
(s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 7.6 Hz, ArH), 7.29 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.11 (d, 1H, J = 6.8 Hz, ArH), 3.52 (s, 2H, PhCH2N), 3.21 (m, 1H, CHN), 2.90 (m, 2H, CH2N), 2.67 (m, 2H, NCH2), 2.52 (m, 4H, N(CH2)2), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 1.95 (m, 4H, (CH2)2), 1.56 (m, 4H, (CH2)2), 1.36 (m, 2H, CH2) ppm, HPLC: 98.1%; 2q: MS (ESI) m/z: 1/2[M+H]+ = 312.7. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.91(m, 6H, ArH), 7.54 (m, 2H, ArH), 7.24 (d, 2H, J = 14 Hz, ArH), 7.04 (s, 1H, ArH), 6.87 (m, 2H, ArH), 6.45 (m, 2H, ArH), 4.44 (s, 2H, PhCH2N), 2.72 (m, 2H, CH2N), 2.26 (s, 3H, ArCH3), 2.18 (s, 3H, ArCH3), 1.92 (m, 2H, PhCH2), 1.23 (m, 2H, CH2) ppm, HPLC: 98.3%; 2r: MS (ESI) m/z: [M+H]+ = 630.2. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.35 (s, 1H, ArH), 7.25 (m, 2H, ArH), 7.13 (d, 1H, J = 7.6 Hz, ArH), 6.76 (d, 1H, J = 5.2 Hz, ArH), 3.64 (s, 2H, PhCH2N), 3.44 (s, 2H, NCH2), 2.80 (m, 2H, NCH2), 2.76 (m, 2H, ArCH2), 2.26 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3) ppm, HPLC: 99.5%; 2s: MS (ESI) m/z: [M+H]+ = 645.3. 1H NMR (400 M, DMSO-d6) d 9.97 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.26 (s, 1H, ArH), 7.19 (d, 1H, J = 8.0 Hz, ArH), 7.07 (d, 1H, J = 7.6 Hz, ArH), 4.12 (d, 1H, J = 13.2 Hz, CH), 3.31 (s, 2H, PhCH2N), 3.24 (m, 2H, CH2), 2.80 (m, 2H, CH2), 2.60 (m, 2H, CH), 2.26 (s, 3H, ArCH3), 2.19 (s, 3H, ArCH3), 2.10 (m, 1H, CH2), 1.91 (m, 1H, CH2), 1.67 (m, 4H, CH2CH2), 1.62 (m, 4H, CH2CH2) ppm, HPLC: 97.1%; 2t: MS (ESI) m/z: [M+H]+ = 602.3. 1H NMR (400 M, DMSO-d6) d 10.02 (s, 1H, CONH), 9.70 (s, 1H, NH), 8.54 (s, 1H, ArH), 7.95(s, 4H, ArH), 7.89 (d, 2H, J = 8.8 Hz, ArH), 7.57 (d, 3H, J = 8.4 Hz, ArH), 7.38 (d, 1H, J = 7.2 Hz, ArH), 7.27 (d, 1H, J = 7.2 Hz, ArH), 3.57 (s, 2H, PhCH2N), 2.71 (m, 4H, CH2NCH2), 2.30 (s, 3H, ArCH3), 2.28 (s, 3H, ArCH3) 1.71 (m, 4H, 2*CH2), 1.54 (m, 2H, CHCH), 1.44 (m, 2H, CH2) ppm, HPLC: 97.3%; 2u: MS (ESI) m/z: [M+H]+ = 617.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.54 (s, 1H, ArH), 7.94(s, 4H, ArH), 7.89 (d, 2H, J = 8.0 Hz, ArH), 7.57 (d, 2H, J = 8.0 Hz, ArH), 7.28 (s, 1H, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 7.2 Hz, ArH), 3.56 (s, 2H, PhCH2N), 2.84 (m, 2H, NCH2), 2.64 (m, 2H, NCH2), 2.22 (m, 13H, NCH3+ArCH3+CH2), 1.88 (m, 1H, CH2), 1.48 (m, 1H, CH2) ppm, HPLC: 95.3%; 2v: MS (ESI) m/z: [M+H]+ = 564.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.28 (s, 1H, ArH), 7.21 (d, 1H, J = 8.0 Hz, ArH), 7.08 (d, 1H, J = 7.6 Hz, ArH), 3.43 (s, 2H, PhCH2N), 2.30 (s, 2H, CH2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 2.12 (s, 3H, NCH3), 1.48 (m, 2H, CH2), 0.85 (t, 3H, J = 7.6 Hz, CH3) ppm, HPLC: 97.3%; 2w: MS (ESI) m/z: [M+H]+ = 564.3. 1H NMR (400 M, DMSO-d6) d 10.00 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.94(s, 4H, ArH), 7.88 (d, 2H, J = 8.8 Hz, ArH), 7.56 (d, 2H, J = 8.4 Hz, ArH), 7.32 (s, 1H, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.12 (d, 1H, J = 7.2 Hz, ArH), 3.53 (s, 2H, PhCH2N), 2.91 (s, 1H, CHN), 2.26 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 2.06 (s, 3H, NCH3), 1.05 (d, 6H, (CH3)2) ppm, HPLC: 96.0%; 2x: MS (ESI) m/z: [M+H]+ = 536.3. 1HNMR (400 M, DMSO-d6) d 9.96 (s, 1H, CONH), 9.62 (s, 1H, NH), 8.51 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.85 (d, 2H, J = 8.4 Hz, ArH), 7.52 (d, 2H, J = 8.4 Hz, ArH), 7.29 (s, 1H, ArH), 7.19 (d, 1H, J = 8.0 Hz, ArH), 7.08 (d, 1H, J = 7.6 Hz, ArH), 3.39 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 2.17 (s, 6H, N(CH3)2) ppm, HPLC: 99.6%; 2y: MS (ESI) m/z: [M+H]+ = 564.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.93 (s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.30 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 7.2 Hz, ArH), 3.50 (s, 2H, PhCH2N), 2.51 (m, 4H, CH2NCH2), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 0.99 (s, 6H, (CH3)2) ppm, HPLC: 97.6%; 2z: MS (ESI) m/z: [M+H]+ = 596.3. 1H NMR (400 M, DMSO-d6) d 9.95 (s, 1H, CONH), 9.62 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.29 (s, 1H, ArH), 7.19 (d, 1H, J = 7.6 Hz, ArH), 7.12 (d, 1H, J = 7.6 Hz, ArH), 4.32 (t, 1H, J = 4.8 Hz, OH), 3.61 (s, 2H, PhCH2N), 3.46 (m, 4H, 2*CH2O), 2.56 (t, 4H, J = 6.0 Hz, 2*CH2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 96.3%; 2za:MS (ESI) m/z: [M+H]+ = 607.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(m, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.32 (s, 1H, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.09 (d, 1H, J = 7.6 Hz, ArH), 3.38 (s, 2H, PhCH2N), 2.82 (t, 2H, J = 7.6 Hz, CH2), 2.56 (s, 6H, 2*CH3), 2.41 (t, 2H, J = 7.2 Hz, CH2), 2.26 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 2.17 (s, 3H, CH3), 1.78 (m, 2H, CH2) ppm, HPLC: 99.0%; 2zb: MS (ESI) m/z: [M+H]+ = 635.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.88 (d, 2H, J = 8.8 Hz, ArH), 7.56 (d, 2H, J = 8.4 Hz, ArH), 7.28 (s, 1H, ArH), 7.20 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 7.6 Hz, ArH), 3.69 (s, 2H, PhCH2N), 2.93 (m, 2H, 2*CHN), 2.50 (m, 2H, 2*CH2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 0.94 (m, 12H, 4*CH3) ppm, HPLC:95.0%; 2zc: MS (ESI) m/z: [M+H]+ = 672.4. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.60 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.88 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 7.6 Hz, ArH), 7.29 (s, 1H, ArH), 7.14 (m, 2H, ArH), 3.67 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.18 (s, 3H, ArCH3), 1.79 (m, 2H, 2*NCH), 1.69 (m, 8H, 4*(CH2)), 1.23 (m, 8H, 4*(CH2)), 1.23 (m, 4H, 2*(CH2)) ppm, HPLC:94.54%; 2zd: MS (ESI) m/z: [M+H]+ = 536.2. 1H NMR (400 M, DMSOd6) d 10.01 (s, 1H, CONH), 9.72 (s, 1H, NH), 8.53 (s, 1H, ArH), 7.94 (s, 4H, ArH), 7.88 (d, 2H, J = 8.4 Hz, ArH), 7.56 (m, 3H, ArH), 7.35 (d, 1H, J = 8.0 Hz, ArH), 7.29 (d, 1H, J = 8.0 Hz, ArH), 4.11 (s, 2H, PhCH2N), 2.97 (m, 2H, CH2N), 2.26 (s, 6H, 2*ArCH3), 1.21 (t, 3H, CH3) ppm, HPLC: 98.8%; 2ze: MS (ESI) m/z: [M+H]+ = 552.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 7.2 Hz, ArH), 7.55 (s, 2H, ArH), 7.29 (s, 1H, ArH), 7.18 (s, 1H, ArH), 7.11 (s, 1H, ArH), 4.47 (s, 1H, OH), 3.68 (s, 2H, CH2O), 3.46 (s, 2H, PhCH2N), 2.57 (s, 2H, CH2N), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 95.6%; 2zf: MS (ESI) m/z: [M+H]+ = 564.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH),
988
M. Xin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 983–988 7.92(s, 4H, ArH), 7.87 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.32 (s, 1H, ArH), 7.19 (d, 1H, J = 7.6 Hz, ArH), 7.13 (d, 1H, J = 8.0 Hz, ArH), 3.67 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.19 (s, 3H, ArCH3), 1.11 (s, 9H, (CH3)3) ppm, HPLC: 98.5%; 2zg: MS (ESI) m/z: [M+H]+ = 576.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.88 (d, 2H, J = 8.4 Hz, ArH), 7.56 (d, 2H, J = 8.4 Hz, ArH), 7.29 (s, 1H, ArH), 7.19 (d, 1H, J = 8.0 Hz, ArH), 7.12 (d, 1H, J = 7.6 Hz, ArH), 3.66 (s, 2H, PhCH2N), 3.00 (m, 1H, CHN), 2.26 (s, 3H, ArCH3), 2.19 (s, 3H, ArCH3), 1.73 (m, 2H, CH2), 1.64 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.34 (m, 2H, CH2) ppm, HPLC: 95.0%;2zh: MS (ESI) m/z: [M+H]+ = 619.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.64 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.31 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.11 (d, 1H, J = 7.2 Hz, ArH), 3.72 (s, 2H, PhCH2N), 2.63 (t, 2H, CH2N), 2.40 (t, 2H, NCH2), 2.32 (m, 4H, N(CH2)2), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 1.47 (m, 4H, (CH2)2), 1.36 (m, 2H, CH2) ppm, HPLC: 95.4%: 2zi: MS (ESI) m/z: [M+H]+ = 588.3. 1H NMR (400 M, DMSO-d6) d 9.99 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.53 (s, 1H, ArH), 7.93(s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.65 (s, 1H, ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.41 (s, 1H, ArH), 7.26 (d, 1H, J = 7.6 Hz, ArH), 7.18 (d, 1H, J = 8.0 Hz, ArH), 6.45 (s, 1H, furan-H), 6.42 (s, 1H, furan-H), 3.89 (s, 2H, CH2N), 3.85 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.23 (s, 3H, ArCH3) ppm, HPLC: 97.6%; 2zj: MS (ESI) m/z: [M+H]+ = 584.3. 1H NMR (400 M, DMSO-d6) d 9.98 (s, 1H, CONH), 9.65 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.91(s, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.33 (s, 1H, ArH), 7.21 (d, 1H, J = 8.0 Hz, ArH), 7.15 (d, 1H, J = 7.6 Hz, ArH), 7.02 (m, 2H, ArH), 6.56 (m, 2H, ArH), 6.51 (m, 1H, ArH), 6.24 (brs, 1H, ArNH), 4.24 (s, 2H, PhCH2N), 2.26 (s, 3H, ArCH3), 2.18 (s, 3H, ArCH3) ppm, HPLC: 98.8%; 3a: MS (ESI) m/z: [M+H]+ = 494.3. 1H NMR (400 M, DMSO-d6) d 9.94 (s, 1H, CONH), 9.63 (s, 1H, NH), 8.50 (s, 1H, ArH), 7.92 (m, 4H, ArH), 7.72 (d, 2H, J = 7.6 Hz, ArH), 7.55 (m, 3H, ArH), 7.28 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.09 (d, 1H, J = 7.6 Hz, ArH), 3.57 (m, 4H, morpholine-H), 3.43 (s, 2H, PhCH2N), 2.35 (m, 4H, morpholine-H), 2.26 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3) ppm, HPLC: 96.8%; 3b: MS (ESI) m/z: [M+H]+ = 508.3. 1H NMR (400 M, DMSO-d6) d 10.06 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.78 (d, 2H, J = 5.2 Hz, ArH), 8.57 (s, 1H, ArH), 7.92(s, 4H, ArH), 7.70 (d, 2H, J = 5.2 Hz, ArH), 7.28 (s, 1H, ArH), 7.21 (d, 1H, J = 7.6 Hz, ArH), 7.08 (d, 1H, J = 7.6 Hz, ArH), 3.44 (s, 2H, PhCH2N), 2.42 (m, 8H, N(CH2)2(CH2)2N), 2.26 (s, 3H, ArCH3), 2.23 (s, 3H, ArCH3), 2.20 (s, 3H, NCH3) ppm, HPLC: 97.5%; 3c: MS (ESI) m/z: [M+H]+= 413.2. 1H NMR (400 M, DMSO-d6) d 9.73 (s, 1H, CONH), 9.55 (s, 1H, NH), 8.39 (s, 1H, ArH), 8.36 (s, 1H, ArH), 8.11 (s, 1H, ArH), 7.97 (m, 4H, ArH), 7.11 (s, 3H, ArH), 3.96 (s, 3H, ArCH3), 2.33 (s, 3H, ArCH3), 2.18 (s, 6H, 2*ArCH3) ppm, HPLC: 98.4%; 3d: MS (ESI) m/z: [M+H]+ = 511.3. 1H NMR (400 M, DMSO-d6) d 9.77 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.13 (s, 1H, ArH), 7.96 (s, 4H, ArH), 7.28 (s, 1H, ArH), 7.22 (d, 1H, ArH), 7.09 (d, 1H, ArH), 3.97 (s, 3H, ArCH3), 3.43 (s, 2H, PhCH2N), 2.52 (s, 3H, NCH3), 2.35 (m, 8H, piperazine-H), 2.22 (s, 3H, ArCH3), 2.17 (s, 3H, ArCH3) ppm, HPLC: 99.4%; 3e: MS (ESI) m/z: [M+H]+ = 483.3. 1H NMR (400 M, DMSOd6) d 9.77 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.13 (s, 1H, ArH),
7.97 (s, 4H, ArH), 7.31 (s, 1H, ArH), 7.21 (d, 1H, ArH), 7.10 (d, 1H, ArH), 3.97 (s, 3H, ArCH3), 3.57 (s, 2H, PhCH2N), 2.46 (m, 4H, pyrolidine-H), 2.35 (s, 3H, ArCH3), 2.23 (s, 3H, ArCH3), 1.70 (m, 4H, pyrolidine-H) ppm, HPLC: 98.8%; 3f: MS (ESI) m/z: [M+H]+ = 496.3. 1H NMR (400 M, DMSO-d6) d 9.76 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.39 (d, 2H, ArH), 8.12 (s, 1H, ArH), 7.97 (s, 4H, ArH), 7.28 (s, 1H, ArH), 7.21 (d, 1H, ArH), 7.09 (d, 1H, ArH), 3.96 (s, 3H, ArCH3), 3.34 (s, 2H, PhCH2N), 2.50 (m, 4H, piperidine-H), 2.34 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 1.50 (m, 4H, piperidine-H), 1.40 (m, 2H, piperidine-H) ppm, HPLC: 98.4%; 3g: MS (ESI) m/z: [M+H]+ = 526.3. 1H NMR (400 M, DMSO-d6) d 9.77 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.13 (s, 1H, ArH), 7.97 (s, 4H, ArH), 7.29 (s, 1H, ArH), 7.23 (d, 1H, ArH), 7.10 (d, 1H, ArH), 3.98 (s, 3H, ArCH3), 3.58 (m, 2H, CHOCH), 3.42 (s, 2H, PhCH2N), 2.71 (m, 2H, morpholine-H), 2.35 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 1.65 (m, 2H, morpholine-H), 1.04 (d, 6H, 2*CH3) ppm, HPLC: 98.4%; 3h: MS (ESI) m/z: [M+H]+ = 512.3. 1H NMR (400 M, DMSO-d6) d 9.79 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.41 (d, 2H, ArH), 8.13 (s, 1H, ArH), 7.98 (s, 4H, ArH), 7.27 (s, 1H, ArH), 7.22 (d, 1H, ArH), 7.08 (d, 1H, ArH), 4.56 (s, 1H, OH), 3.97 (s, 3H, ArCH3), 3.41 (m, 1H, CHO), 3.36(s, 2H, PhCH2N), 2.68 (m, 2H, piperidineH), 2.35 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 2.02 (m, 2H, piperidine-H), 1.71 (m, 2H, piperidine-H), 1.39 (m, 2H, piperidine-H) ppm, HPLC: 98.6%; 3i: LC-MS (ESI) m/z: [M+H]+ = 484.3. 1H NMR (400 M, DMSO-d6) d 9.80 (s, 1H, CONH), 9.73 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.12 (s, 1H, ArH), 7.97 (s, 4H, ArH), 7.57 (s, 1H, ArH), 7.34 (d, 2H, ArH), 3.96 (s, 3H, ArCH3), 3.35 (s, 2H, PhCH2N), 2.65 (m, 2H, CH2N), 2.34(s, 3H, ArCH3), 2.28 (s, 3H, CH3N), 1.28 (m, 2H, CH2), 0.89 (m, 3H, CH3) ppm, HPLC: 99.2%; 3j: MS (ESI) m/z: [M+H]+ = 516.3. 1H NMR (400 M, DMSO-d6) d 9.77 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.12 (s, 1H, ArH), 7.98 (m, 4H, ArH), 7.29 (s, 1H, ArH), 7.20 (d, 1H, ArH), 7.13 (d, 1H, ArH), 4.37 (t, 2H, 2*OH), 3.96 (s, 3H, ArCH3), 3.61 (s, 2H, PhCH2N), 3.46 (m, 4H, 2*CH2O), 3.17 (m, 4H, 2*CH2N), 2.34(s, 3H, ArCH3), 2.21 (s, 3H, ArCH3) ppm, HPLC: 98.0%; 3k: MS (ESI) m/z: [M+H]+ = 512.3. 1H NMR (400 M, DMSO-d6) d 9.77 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.40 (d, 2H, ArH), 8.12 (s, 1H, ArH), 7.95 (s, 4H, ArH), 7.33 (s, 1H, ArH), 7.17 (d, 1H, ArH), 7.13 (d, 1H, ArH), 3.96 (s, 3H, ArCH3), 3.66 (s, 2H, PhCH2N), 2.30 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 1.11 (s, 9H, 3*CH3) ppm, HPLC: 96.9%. 10. Pharmacokinetic Studies in SD rat. Compounds were administered to SD rats at a dose of 1 mg/kg (in 5% DMA+ 5% Tween-80) for iv or 5 mg/kg (0.5%HPMC+0.3% Tween-80, pH=2.8) for po administration, respectively. After oral administration, blood samples collected for PK analyses (n = 3) were centrifuged at 4000 rpm for 5 min at 4 °C. Plasma samples were analyzed after protein precipitation with acetonitrile acidified with1% formic acid. LC/ MS/MS analysis of test compounds was performed under optimized conditions to obtain the best sensitivity and selectivity of the analyte in selected reaction monitoring mode (SRM) containing an internal standard. Plasma concentration-time data were measured by a noncompartmental approach using the software WinNonlin Enterprise, version 5.2 (Pharsight Co.,Mountain View, CA).
