European Journal of Medicinal Chemistry 86 (2014) 639e652

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Design, synthesis, and biological evaluation of 1, 3-disubstitutedpyrazole derivatives as new class I and IIb histone deacetylase inhibitors Yiwu Yao a, 1, Chenzhong Liao b, 1, Zheng Li c, 1, Zhen Wang a, Qiao Sun a, Chunping Liu a, Yang Yang b, Zhengchao Tu a, *, Sheng Jiang a, * a b c

Laboratory of Medicinal Chemistry, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China School of Medical Engineering, Hefei University of Technology, Hefei, Anhui 230009, China The Methodist Hospital Research Institute, Houston, TX 77030, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 June 2014 Received in revised form 6 September 2014 Accepted 7 September 2014 Available online 8 September 2014

A novel series of HDAC inhibitors demonstrating class I and IIb subtype selectivity have been identified using a scaffold-hopping strategy. Several designed compounds showed better selectivity for class I and IIb over class IIa HDAC isoforms comparing to the FDA approved HDAC targeting drug SAHA. A representative lead compound 22 bearing a biphenyl moiety demonstrated promising class I and IIb HDAC isoforms selectivity and in vitro anticancer activities against several cancer cell lines. This work could serve as a fundamental platform for further exploration of selective HDAC inhibitors using designed molecular scaffold. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: HDAC Isoforms selectivity Structureeactivity relationship Scaffold-hopping

1. Introduction Histone deacetylases (HDACs) are a family of enzymes that remove the acetyl groups of acetylated histones or other nonhistone substrates leading to transcriptional repression, and play important roles in the upstream control of chromosome remodeling, gene transcription, cell proliferation and apoptosis [1,2]. HDACs have been indicated as one of the major players in tumorigenesis and HDACs function inhibition has been proved to be an effective strategy in cancer therapy [3]. Thus far, 18 HDACs have been identified in human and divided into five groups: class I (HDAC1, HDAC2, HDAC3, HDAC8), class IIa (HDAC4, HDAC5, HDAC7, HDAC9), class IIb (HDAC6, HDAC10), class III (SIRT 1e7), and class IV (HDAC11) [4,5]. Classes I, II, and IV enzymes are Zn2þ-dependent metallohydrolases, whereas class III enzymes are NADþ-dependent Sir2-like deacetylases [5]. Class I HDACs primarily localize to the nucleus and are expressed ubiquitously, and generally involved in cell proliferation and differentiation [6]. Of note, class I and IIb

* Corresponding authors. E-mail addresses: [email protected] (Z. Tu), [email protected], [email protected] (S. Jiang). 1 Yiwu Yao, Chenzhong Liao and Zheng Li contributed equally to this publication. http://dx.doi.org/10.1016/j.ejmech.2014.09.024 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

HDAC isoforms are over expressed in most solid and hematological tumors, highly correlating with a worse prognosis [7e11], but not resting endothelial cells and normal organs. Therefore, selective targeting class I and IIb HDACs by directly inhibiting their functions has become a major focus in cancer chemotherapy [12e14]. The suberoylanilide hydroxamic acid (SAHA, vorinostat) is a HDAC inhibitor which was approved for cutaneous T cell lymphoma treatment (CTCL) in Oct 2006. To date, about 11 HDAC inhibitors (HDACi) are in various stages of clinical development for therapy of multiple cancer types [2,15]. Most clinical trial HDACi is pan-HDAC inhibitors, which seem to lack selectivity when assessed in biochemical or cellular assays [15,16]. On the other hand, pan-HDAC inhibitors showed promising anti-tumor effects in clinical trials, but side effects such as diarrhea, electrolyte changes, and cardiac arrhythmias resulting from their off-target activity might limit their further clinical application [17,18]. Therefore, developing isoform and class-selective HDACis to reduce undesirable side effects from off-target activity is highly in demand [19]. Although many HDACs inhibitors have been developed, only a few class I HDACs inhibitors were reported [20e31]. We have recently reported the synthesis and the biological activity of triazole 1, which selectively binds HDAC 1 with low-nanomolar affinity [32]. Similar with many reported HDACis, compound 1 was

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also composed by three parts: zinc binding group (ZBG), linker and capping group (also called surface recognition group, Fig. 1). In this study, we envisioned that replacement of the triazole ring with different heterocycles may increase the selectivity for HDAC isoforms. In addition, the effect of different linker length and surface recognition group were also investigated to explore the detailed structure activity relationship (SAR). Herein, we describe the design, synthesis and biological characterization of a series of disubstituted-pyrazole derivatives as novel selective HDAC I and HDAC IIb inhibitors using compound 1 as the molecular scaffold.

2. Chemistry We initially designed compounds 2e6 as new HDAC1 inhibitors, in which the triazole ring of compound 1 was replaced with pyrrole, imidazole, and pyrazole by using a scaffold-hopping strategy (Fig. 1). Compounds 2e6 were synthesized by using the procedure described in Scheme 1. The 3-(bromomethyl)biphenyl 38 was reacted with aldehyde 39e42, respectively, to afford the building blocks 43e47 in 80e98% yields. The extension of the hydrocarbon chain was achieved by Wittig reaction of 43e47 to give 48e52 in 43e74% yields, which were further hydrogenated over 10% PdeC, hydrolysis of ester and afforded acids 53e57 in 81e98% yields, respectively. Subsequently, 53e57 were treated with oxalyl chloride or isobutyl chloroformate to give the corresponding acyl chloride or mixed anhydride, which were reacted with hydroxylamine hydrochloride to furnish the final products 2e6 in 73e82% yields, respectively. The structure of 6 was confirmed by X-ray crystallographic analysis, as shown in Fig. 2. The crystal data were deposited at the Cambridge Crystallographic Data Centre. The deposited number is CCDC 957445. In order to explore the effect of the rigidness of the surface recognition group for the enzymatic inhibitory activities and selectivity, we designed and synthesized compounds 7 and 8 (Scheme 2). Intermediates 61 and 62 were prepared from commercially available 1-bromo-2-(bromomethyl) benzene 58 in a two-step procedure, respectively, involving base mediated coupling with aldehydes 39 and 40 followed by subsequent extension of the hydrocarbon chain. The intermediates 61 and 62 were further hydrogenated over 10% PdeC, Pd catalyzed cyclization and hydrolysis of ester to afford acids 63 and 64 in 30e40% yields,

Surface recognition group O Ph

N H

N

3. Results and discussion First, compounds 1e6 were tested using the isolated enzymes against HDAC1 and HDAC7 using SAHA as the positive control. The results are summarized in Table 1 and showed interesting HDACs isoform selectivity. Compound 1 displayed good selectivity for HDAC1 (IC50 ¼ 0.112 mM) over HDAC7 (not active up to 10 mM). By replacing the triazole ring with pyrrole and imidazole respectively, compounds 2 and 3 showed 1.7 and 3.3 times less potent than the

Zinc binding group (ZBG)

OH

OH N N H N N Linker 1 HDAC1: IC50 = 0.112 μM HDAC7: not active

N N 4

N H

Ph

N 3

Ph NH OH

OH

O

N N

O

N H

N

O

Ph

5

O

O

2

Ph

respectively. Subsequently, 63 and 64 were transformed to the final products 7 and 8 in 82% yields, respectively. To study the SAR of compound 5, a series of analogs 9e32 were designed and prepared via multi-step synthesis starting from the bromo-substituted benzyl derivatives 65aes or iodobenzene 65t (Scheme 3). The coupling reaction of 65aet with pyrazole aldehydes gave the pyrazole derivatives 68aet, respectively, which were then transformed by Wittig reaction into the derivatives 69aet in yields of 34e87%. Hydrogenation of double bond in the presence of over 10% PdeC provided compounds 70aet in high yields. Compound 70e or 70n were coupled with morpholine, aniline and indole to give 71uex. Hydrolysis of the ethyl esters and final coupling with hydroxylamine hydrochloride furnished the pyrazole derivatives 9e32 in 75e86% yields. In order to investigate the optimal length of the alkyl chain, we designed and synthesized compounds 33e36, whose fatty chains have 5e8 carbon atoms respectively. The synthetic route was similar to that of compound 22 as described in Scheme 4. The 4(bromomethyl)biphenyl 65k was coupled with 1H-pyrazole-3carbaldehyde 41 following extension of the hydrocarbon chain by Wittig reaction. Hydrogenation of double bond followed by hydrolysis of the methyl or ethyl esters and final coupling with hydroxylamine hydrochloride furnished the hydroxamic acid derivatives 33e36. Compound 37 was prepared as illustrated in Scheme 5. Condensation of 1H-pyrazole-3-carboxylic acid 81 with methyl 3aminopropanoate in the presence of HATU gave compound 82, which was further connected with benzyl bromide in the presence of K2CO3 to furnish intermediate 83 in 68% yield. Hydrolysis of the methyl esters and subsequent coupling with hydroxylamine hydrochloride gave the 1H-pyrazole-3-carboxamide derivative 37.

N N 6

Fig. 1. Design of new class I and IIb HDACis using a scaffold-hopping strategy.

OH NH

OH

Y. Yao et al. / European Journal of Medicinal Chemistry 86 (2014) 639e652

641

Scheme 1. General procedure for the synthesis of compounds 2e6. Reagents and conditions: (a) TBAB, NaOH, DCM, 0  C to room temp, 39, 98%; (b) K2CO3, CH3CN, room temp, 40e42, 80e98%; (c) [Ph3PþCH2CH2CH2CO2Et]Br, NaHMDS, THF, 78  C to room temp, 43e74%; (d) (i) H2, 10% PdeC, MeOH, room temp; (ii) LiOH, MeOH, H2O, room temp, 81e98% in two steps; (e) (i) 54e57, (COCl)2, DMF, DCM, reflux; (ii) NH2OH$HCl, DIPEA, DCM, 0  C, 73e80% in two steps; (f) (i) 53, ClCO2i-Bu, Et3N, THF, 0  C; (ii) NH2OH$HCl, KOH, MeOH, room temp, 82% in two steps.

Fig. 2. The crystal structure of 6 confirmed by X-ray crystallographic analysis.

original lead compound 1 (2, IC50 ¼ 0.191 mM; 3, IC50 ¼ 0.367 mM) against HDAC1. Both of them have no effect against HDAC7. When replacing the triazole ring with pyrazole ring to achieve compounds 4e6, it was observed that compound 4 with alkyl chain in the 4th position of the pyrazole ring showed a decreased binding of HDAC1 in comparison with compound 1 (2.4-fold). While compound 5, with alkyl chain in the 3rd position, inhibited the HDAC1 with an IC50 value of 0.064 mM, which was about 1.8 times more potent than compound 1. Compound 6 showed the best HDAC1 potency but lower selectivity between HDAC1 (IC50 ¼ 0.035 mM) and HDAC7 (IC50 ¼ 7.94 mM).

Br

Br Br

(a) or (b)

N

X

X

(e) or (f)

N 63: X=N; 64: X=CH

OH O

Br

CHO

59: X=N; 60: X=CH

58

Structure-based docking methods are the effective ways to explore the binding modes of ligands [33]. To understand the binding modes of compounds 5 and 6 with HDAC1, these two compounds were docked into the active site of a homology model of HDAC1 complexed with TSA [24]. Nevertheless, when we were writing this manuscript, the X-ray crystal structure of HDAC1 in complex with the dimeric ELM2-SANT domain of MTA1 from the NuRD complex was published (PDB code: 4bkx) [34]. In the binding site of this complex, however, there is no inhibitor being accommodated there except an acetate anion which chelates the zinc cation. To employ this structure information for our study, we compared and superimposed this structure with the human HDAC2 structure complexed with SAHA (PDB code: 4lxz) [35] and then incorporated this compound into the active site of HDAC1. This model was minimized and done a 10 ns molecular dynamic simulation by employing the program of Amber 12. The final modeled HDAC1-SAHA was used for the docking study. Before docking, these two compounds were treated as negatively charged since DFT studies indicated that the hydroxamic acid would be deprotonated when chelating the zinc ion, which then was pentacoordinated at the active site of HDAC1 [36,37]. Aliphatic hydroxzmic acid had a pKa value of 9.4; and the acidity of hydroxamic acids could increase ~3.3 pKa units upon complexation to zinc in the active site of TACE. In the cases of HDAC1 and HDAC7, the protonation states of the hydroxamic acids were not only regulated by the binding to the positive charged zinc ion, but also by the coordinations of the His and Tyr residues in the active sites. Such a scene can also been observed when the metal ion is

N

(c)

OEt X

O

(d)

61: X=N; 62: X=CH X N 7: X=N; 8: X=CH

NHOH O

Scheme 2. Synthesis of compounds 7 and 8. Reagents and conditions: (a) TBAB, NaOH, DCM, 39, 0  C to room temp, 96%; (b) K2CO3, CH3CN, 40, room temp, 82%; (c) [Ph3PþCH2CH2CH2CO2Et]Br, NaHMDS, THF, 78  C to room temp, 85e89%; (d) (i) H2, 10% PdeC, MeOH, room temp; (ii) Pd(OAc)2, PPh3, K2CO3, DMF, 120  C; (iii) LiOH, MeOH, H2O, room temp, 30e40% in three steps; (e) (i) 63, (COCl)2, DMF, DCM, reflux; (ii) NH2OH$HCl, DIPEA, DCM, 0  C, 82% in two steps; (f) (i) 64, ClCO2i-Bu, Et3N, THF, 0  C; (ii) NH2OH$HCl, KOH, MeOH, room temp, 82% in two steps.

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(a) or (b)

R1CH2Br or PhI

or (c)

65a-t (e) or (f)

R1

N n N

CHO

N n N

R1

R2 68a-t

O R1

(d)

nN N R2 70a-t R

1 =4

O

69a-t O

(i)

R1

OEt (g)

or (h ) -B ro r3 R1 -B r-P h-

OEt

R2

nN N R2

O nN N R2

OH 72a-x

(i) OEt

71u: R1=4-Morpholinophenyl-,R 2=H, n=1, 3-sub; 71v: R1=4-(1H-indole-1-yl)-Ph-, R2=H, n=1, 3-sub; 71w: R1=4-PhNH-Ph-, R2=H, n=1, 3-sub; 71x: R1=3-PhNH-Ph-, R2=H, n=1, 3-sub;

(j) or (k)

O R1

n N N R2 9-32

NHOH

68a, 69a, 70a: R1=Ph-, R2=H, n=1, 3-sub; 68b, 69b, 70b: R1=Ph-, R2=H, n=0, 3-sub; 68c, 69c, 70c: R1=t-Bu-Ph-, R2=H, n=1, 3-sub; 68d, 69d, 70d: R1=4-Me-Ph-, R2=H, n=1, 3-sub; 68e, 69e, 70e: R1=4-Br-Ph-, R2=H, n=1, 3-sub; 68f, 69f, 70f: R1=4-NO2-Ph-, R2=H, n=1, 3-sub; 68g, 69g, 70g: R1=4-CF3-Ph-, R2=H, n=1, 3-sub; 68h, 69h, 70h: R1=4-F-Ph-, R2=H, n=1, 3-sub; 68i, 69i, 70i: R1=4-BnO-Ph-, R2=H, n=1, 3-sub; 68j, 69j, 70j: R1=4-PhO-Ph-, R2=H, n=1, 3-sub; 68k, 69k, 70k: R1=4-Biph-, R2=H, n=1, 3-sub; 68l, 69l, 70l: R1=3-PhO-Ph-, R2=H, n=1, 3-sub; 68m,69m, 70m: R1=3-MeO-Ph-, R2=H, n=1, 3-sub; 68n, 69n, 70n: R1=3-Br-Ph-, R2=H, n=1, 3-sub; 68o, 69o, 70o: R1=3-Cl-4-F-Ph-, R2=H, n=1, 3-sub; 68p, 69p, 70p: R1=3,4-methylenedioxyl-Ph-, R2=H, n=1, 3-sub; 68q, 69q, 70q: R1=2-Nap-,R2=H, n=1, 3-sub; 68r, 69r, 70r: R1=2,4-di-F-Ph-, R2=H, n=1, 3-sub; 68s, 69s, 70s: R1=Ph-, R2=4-Me, n=1, 3-sub; 68t, 69t, 70s: R1=Ph-, R2=3-Ph, n=1, 4-sub;

HN N 41

CHO

HN N

Ph

66 CHO

O

HN N 67

OEt

72a, 9: R1=Ph-, R2=H, n=1, 3-sub-; 72b, 10: R1=Ph-, R2=H, n=0, 3-sub-; 72c, 11: R1=t-Bu-Ph-, R2=H, n=1, 3-sub; 72d, 12: R1=4-Me-Ph-, R2=H, n=1, 3-sub; 72e, 13: R1=4-Br-Ph-, R2=H, n=1, 3-sub; 72f, 14: R1=4-NO2-Ph-, R2=H, n=1, 3-sub; 72g, 15: R1=4-CF3-Ph-, R2=H, n=1, 3-sub; 72h, 17: R1=4-F-Ph-, R2=H, n=1, 3-sub; 72i, 18: R1==4-BnO-Ph-, R2=H, n=1, 3-sub; 72j, 19: R1=4-PhO-Ph-, R2=H, n=1, 3-sub; 72k, 22: R1=4-Biph-, R2=H, n=1, 3-sub; 72l, 24:R1=3-PhO-Ph-, R2=H, n=1, 3-sub; 72m, 25: R1=3-MeO-Ph-, R2=H, n=1, 3-sub; 72n, 26: R1=3-Br-Ph-, R2=H, n=1, 3-sub; 72o, 27: R1=3-Cl-4-F-Ph-, R2=H, n=1, 3-sub; 72p, 28: R1=3,4-methylenedioxyl-Ph-, R2=H, n=1, 3-sub; 72q, 29: R1=2-Nap-,R2=H, n=1, 3-sub; 72r, 30: R1=2,4-di-F-Ph-, R2=H, n=1, 3-sub; 72s, 31: R1=Ph-, R2=4-Me, n=1, 3-sub; 72t, 32: R1=Ph-, R2=3-Ph, n=1, 4-sub; 72u, 16: R1=4-Morpholinophenyl-,R2=H, n=1, 3-sub; 72v, 20: R1=4-(1H-indole-1-yl)-Ph-, R2=H, n=1, 3-sub; 72w, 21: R1=4-PhNH-Ph-, R2=H, n=1, 3-sub; 72x, 23: R1=3-PhNH-Ph-, R2=H, n=1, 3-sub;

Scheme 3. Synthesis of compounds 9e32. Reagents and conditions: (a) CuI, Cs2CO3, PhI, 41, 8-hydroxyquinonine-N-oxide, DMSO, 90  C, 46%; (b) K2CO3, CH3CN, 41 or 66, room temp, 25e80%; (c) (i) 67, K2CO3, CH3CN, room temp, 69%; (ii) LiAlH4, THF, 0  C to room temp, 86%; (iii) PCC, 4A molecular sieve, DCM, room temp, 45%. (d) [Ph3PþCH2CH2CH2CO2Et] Br, NaHMDS, THF, 78  C to room temp, 34e87%; (e) H2, 10% PdeC, MeOH, room temp; (f) NaOAc, TsNHNH2, DME/H2O, reflux; (g) 70e/70n, Pd(OAc)2, BINAP, Cs2CO3, morpholine/ aniline, PhMe, 120  C; (h) 70e, CuI, K3PO4, indole, N, N0 -dimethylethane-1, 2-diamine, PhMe, 110  C; (i) LiOH, MeOH, H2O, room temp, 47e99% in two or three steps; (j) (i) (COCl)2, DMF, DCM, reflux; (ii) NH2OH$HCl, DIPEA, DCM, 0  C, 68e87% in two steps; (k) (i)TBTU, Et3N, DMF, room temp; (ii) NH2OH$HCl, KOH, MeOH, room temp, 75e86% in two steps.

magnesium [38]. When doing the docking, the other three residues were treated as following: Asp 176 and Asp 264 were deprotonated, and His 178 was protonated. As shown in Fig. 3A, the docking result indicated that compounds 5 and 6 had very similar binding modes

to HDAC1. Both of the two oxygen atoms of the hydroxamic acid chelated the catalytic zinc ion and had hydrogen bond interactions with His 141 and Tyr 303 respectively; the fatty chain and the pyrazole ring, as the linker, fitted in the hydrophobic tube

Scheme 4. Synthesis of compounds 33e36. Reagents and conditions: (a) K2CO3, CH3CN, room temp, 43%; (b) [Ph3PþCH2CH2(CH2)nCO2R]Br, NaHMDS, THF, 78  C to room temp, 37e64%; (c) (i) H2, 10% PdeC, MeOH, room temp; (ii) LiOH, MeOH, H2O, room temp, 57e84% in two steps; (d) (i) (COCl)2, DMF, DCM, reflux; (ii) NH2OH$HCl, DIPEA, DCM, 0  C, 68e88% in two steps.

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643

Scheme 5. Synthesis of compound 37. Reagents and conditions: (a) NH2CH2CH2CO2Me$HCl, DIPEA, HATU, DMF, 0  C to room temp, 64%; (b) benzyl bromide, K2CO3, CH3CN, room temp, 68%; (c) LiOH, MeOH, H2O, room temp, 92%; (d) (i) TBTU, Et3N, DMF, room temp; (ii) NH2OH$HCl, KOH, MeOH, room temp, 85% in two steps.

comprised of Phe 205, Phe 150, and Leu 271; the biphenyl group had hydrophobic interactions with His 28, Pro 29 and Leu 271 which were in the rim of the binding pocket. Besides those interactions, there maybe were two unusual heterocyclic CH$$$OeC hydrogen bonds [39] existed between the pyrazole ring of the ligands and the residue of Asp 99. Therefore, compound 5 represented a new class I HDAC inhibitor with a new chemical scaffold and further structural optimization was conducted to improve its HDCA1 inhibitory activity. We then further investigated why most of our compounds selectively inhibit HDAC1 but not HDAC7. SAHA did have activity against HDAC7, but with a much worse IC50 value than HDAC1 (38.9 mM versus 0.131 mM). A crystal structure of SAHA-cdHDAC7 complex was available (PDB code: 3c0z) [40], however, the capping group of SAHA could not be identified because of the poor electron density. Yet, TSA-cdHDAC7 complex (PDB code: 3c10) showed how TSA binds to HDAC7. Unlike the way that SAHA interacted with HDAC1, TSA formed monodentate complex with the zinc cation, and TSA did not have hydrophobic interaction with His 541 (but SAHA had such an interaction with the corresponding His 28 of HDAC1). His 541 of HDAC7 had a different orientation from the His 28 of HDAC1 (Fig. 3B). In the crystal structure of HDAC7, it could be observed that this residue chelated another zinc ion (the other three chelants are Cys 533, Cys 535 and Cys 618 of the protein of HDAC7). Because of the chelating interaction, the position of His 541 of HDAC7 was almost fixed (but His 28 of HDAC1 is freer). So although the binding pockets of HDAC1 and HDAC7 were very similar based on the residues forming them, we assumed the opposite orientations of His 28 of HDAC1 and His 541 of HDAC7 could lead to different selectivity profiles of HDACis against HDAC1 and HDAC7. For compound 5, it had stronger hydrophobic interactions against His 28 of HDAC1 than SAHA based on the plausible binding mode, however, it would have van der Walls confliction with His 541 of HDAC7 if compound 5 interacts with HDAC7 in the same way with HDAC1 (Fig. 3B). That possibly could be the reason why compound 5 lost inhibition against HDAC7. We then hypothesized that, if the rigid biphenyl group of compound 5 was modified to much flexible groups, the inhibition against HDAC7 could be regained. Our hypothesis was proved by compound 19 with more flexible 4-phenoxybenzyl group (no selectivity between HDAC1 and HDAC7), which showed that the flexibility of the capping group was an important factor for the selectivity to HDAC7. We continued to explore the effect of the rigidness of the surface recognition group on the enzymatic inhibitory activities and selectivity by designing compounds 7 and 8, both of which contained a fused and inflexible ring in the capping region. Unfortunately, enzymatic and cellular assays revealed that both of them are less potent than compound 5 (7, IC50 ¼ 1.155 mM; 8, IC50 ¼ 1.758 mM, see Table 2) against HDAC1, which also indicated the importance of the flexibility of the capping region.

Based on the binding mode of compound 5 to HDAC1, we assumed if knocking out a phenyl ring from the biphenyl ring of 5 could attenuate the affinity since both of these two rings interact with the protein via hydrophobic interaction. When the biphenyl was substituted with benzyl or phenyl, the resulting compounds 9 and 10 showed almost 10 times less potent than compound 5. The position of substitution in phenyl ring had great impact on the effectiveness of the set of compounds against HDAC1 and HDAC7. It was observed that different functional groups at the para-position of phenyl ring showed different selectivity between HDAC1 and HDAC7. When a variety of functional groups such as tert-butyl, methyl, bromo and nitro groups (11e14) were introduced at paraposition of compound 9, respectively, their selectivities between HDAC1 and HDAC7 were changed slightly in comparison with compound 9. Conversely, compounds 15e17 having trifluoromethyl, fluoro, and morpholine groups were introduced at para-position showed poor selectivity between HDAC1 and HDAC7. Interestingly, the aromatic functional groups at the paraposition of phenyl ring (18e22) could significantly increase inhibition activities, which suggested that para-position might be a feasible position for further optimization. Compound 18 with benzyloxy group at para-position displayed an IC50 value of 0.067 mM against HDAC1, which was about 4.7 times more potent than compound 9. However, compound 19 having a phenoxy group at the para-position showed great loss of selectivity between HDAC1 and HDAC7. Compound 21, bearing a phenylamino at para-position, displayed an IC50 value of 0.035 mM, which was about 9 times more potent than compound 9. Compound 22, with phenyl group substituted at para-position, was the most potent in this series (IC50 ¼ 0.033 mM) and superior to its meta counterpart 5. It was observed that meta-position substitution in the phenyl ring of compound 9 decreased the selectivity between HDAC1 and HDAC7 remarkably (23e29). For instance, when a methoxy group was introduced at meta-position of compound 9, the resulting compound 25 exhibited the reverse selectivity between HDAC1 and HDAC7 (HDAC1: IC50 ¼ 0.342 mM; HDAC7: IC50 ¼ 0.311 mM). Molecular modeling indicated that the meta-position substituted methoxyl group could fit a small hydrophobic cavity comprised of Pro 809 and Leu 810 of HDAC7 (Fig. 4B). Such a seemingly additive minor hydrophobic interaction leaded to significant improvement of the inhibition to HDAC7. In the binding mode of 25 to HDAC7, the hydroxamate group formed a monodentate complex with the zinc ion, which was consistent with the reported result [40]. However, compound 25 formed bidentate chelating complexes with HDAC1, as indicated by our modeling results. These different chelating modes could be one of the reasons why these inhibitors had weaker affinities against HDAC7 than HDAC1. Yet, some HDAC7 inhibitors could form a hydrogen bond with a crystallized water molecule and had indirect interaction with the protein of HDAC7 (Fig. 4C). If increasing the hydrophobicity of the surface recognition group of compound 5, the overall trend was that the

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Table 1 In vitro HDAC1 and HDAC7 enzyme inhibitory activities of compounds 1e6.a,b Compd

SAHA 1 2 3 4 5 6

IC50 (mM) HDAC1

HDAC7

0.131 0.112 0.191 0.367 0.266 0.064 0.035

38.9 NA NA NA NA NA 7.94

a SAHA was used as the positive control. Values are means of three experiments, standard error of the IC50 was generally less than 10%. b NA, not active up to the highest concentration tested (10 mM).

Fig. 3. (A) Plausible binding modes of compounds 5 (green), 6 (magenta) and 22 (yellow) to HDAC1. The zinc ion is displayed as a cyan ball. (B) The orientations of His 28 of HDAC1 and the corresponding His 541 of HDAC7 are opposite. SAHA, TSA and the docking pose of compound 5 against HDAC1 are shown as cyan, yellow and gray respectively. Three cysteine residues chelating another zinc ion also are shown as yellow. This figure was made after superimposing the proteins of HDAC1 and HDAC7. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the side chain, showed 290 times less potent against HDAC1 than compound 5. Docking results indicated that the fatty linear chain of 5 was sandwiched by two phenylalanine residues, Phe 679 and Phe 738, and had hydrophobic interaction with the protein. So the attenuating of the affinity of 37 to HDAC1 was not a surprise, since the amide was a polar group, which could not fit the hydrophobic channels well into the binding pockets of not only HDAC1, but also HDAC7. However, slightly extending the alkyl chain could put the amide group into the rim of the binding pocket. In such a situation, the affinity would not be attenuated, and SAHA was an example. To determine the HDACs isoform selectivity, five representative compounds 5, 10, 20, 21, and 22 were selected for a HDAC profiling against the full panel of 11 HDAC isoforms to investigate the selectivity of the new serial of HDACis using SAHA as the positive control (Table 3). The isoform assays showed that all 5 compounds displayed good selectivity on class I HDAC isoforms (HDAC1, 2, 3 and 8) and class IIb HDAC isoforms (HDAC6 and 10) over class IIA HDACs (HDAC4, 5, 7 and 9), and class IV HDAC11. Compound 22 displayed modest preference for class I and IIb HDAC isoforms but exhibited no obvious inhibition against class IIa HDACs up to 10 mM. To further validate the function of the compounds for HDAC isoforms in cells, we examined the effects of the representative compounds 5 and 22 on the acetylation levels of histone H3 and atubulin in the HCT116 cell lines using western blotting. The presence of acetylated histone H3 indicated inhibition of the class I HDACs, whereas the presence of acetylated tubulin was an indicator of HDAC6 inhibition. As expected, both compounds 5 and 22 caused a dose-dependent increase in acetylated H3 and acetylated atubulin in a dose-dependent manner (Fig. 5). The result further suggested that both of compounds 5 and 22 were class I and class IIb selective inhibitors. We further investigated the antiproliferative activities of these derivatives using 8 human tumor cell lines, Hela (human cervical cancer cell line), MCF7 (breast cancer cell line), BGC823 (human stomach cancer cell line), A549 (human lung cancer cell line), HT1080 (human sarcoma cell line), K562 (human immortalized myelogenous leukemia cell line), U937 (human histiocytic leukemia cell line) and Molt-4 (human T cell leukemia cell line), with SAHA as the positive control. As showed in Table 4, analogs 5, 6, 9, 12, 13, 18e29 and 32 showed very good GI50 values in the nanomolar range against human T cell leukemia molt-4 cancer cell line. It was remarkable that compounds 5, 21 and 22, especially in view of its higher class I and IIb activities, had much better anticancer activities than that of SAHA against several cancer cell lines. In contrast, compounds 33e37, the poor inhibitors of HDAC1, show much weaker activity against these cell lines as we expected. Thus, taken together, the results of the enzyme and cell-based assays verified the high correlation between the in vitro HDAC1 inhibitory activity and cellular cytotoxicity of HDACis. 4. Conclusion

inhibition against HDAC1 will increase. For example, compound 29, in which the surface recognition group was a naphthyl group, regained the affinity with an IC50 value of 0.043 mM. In addition, compounds 31 and 32, having other substituent group in the pyrazole, displayed lower selectivity between HDAC1 and HDAC7 in comparison with compound 9. The impact of the length separating the pyrazole ring and the hydroxamate moiety of compound 22 was also investigated, and the results indicated that exceeding six methylene spacers caused dramatic decrease of the selectivity between HDAC1 and HDAC7 (compounds 33e36). Compound 37, an analog with amide bond in

Previously, we reported a series of triazole-based hydroxamic acid derivatives as HDAC1 inhibitors [32]. In the present study, a series of disubstituted-pyrazole derivatives were designed, synthesized, and evaluated as novel selective HDACirs using a scaffoldhopping strategy. SAR was thoroughly studied by changing the length of the linker and the flexibility of the surface recognition group. We identified a representative lead 22 with a biphenyl group, which selectively inhibited class I and IIb isoforms (HDAC1: IC50 ¼ 0.033 mM; HDAC2: IC50 ¼ 0.226 mM; HDAC3: IC50 ¼ 0.030 mM; HDAC6: IC50 ¼ 0.034 mM; HDAC10: IC50 ¼ 0.029 mM). Compound 22 showed promising in vitro

Y. Yao et al. / European Journal of Medicinal Chemistry 86 (2014) 639e652

645

Table 2 In vitro HDAC1 and HDAC7 enzyme inhibitory activities of compounds 7e37.a,b Table 2 (continued )

IC50 (mM)

Compd

SAHA N N

Ph

H N O

5

OH

HDAC1

HDAC7

0.131

38.9

0.064

NA

IC50 (mM)

Compd

N

PhHN

N NHOH

N O

7

1.16

N

PhO

NHOH

N O

8

NHOH

0.32

N

NA

O

9

0.719

NA

0.342

0.311

0.068

7.68

0.116

0.770

NHOH

0.195

0.36

NHOH

0.043

4.40

0.539

1.46

0.646

0.530

0.202

0.487

NHOH

0.227

5.60

NHOH

0.233

4.49

NHOH

2.66

12.68

NHOH

2.49

7.98

9.58

NA

O

NA MeO

N

0.96

NHOH

N

NHOH

N

O

25

N

0.145

O

24

1.76

HDAC7

NHOH

N 23

NA

HDAC1

N

Br

NHOH

N

O

26

F

0.313

NA

N

Cl

NHOH

N

O

27 N

NHOH

N

1.31

NA

O

11

O N

O

N

O

28 N

NHOH

N

0.323

NA

N

O

12

N

O

29 Br N

NHOH

N

0.218

NA

F N

O

13

F

0.242

NHOH

N

O

30

NHOH

NA N

O

N 31

0.197

2.92

N N

NHOH O

Ph 32

O N N

NHOH

N

0.376

0.854

O

16

Ph

O N

F N

NHOH

N

0.293

0.355 Ph

O N

BnO N

NHOH

N

0.067

Ph

O N

NHOH

N

0.086

O

N N 20

NHOH

0.075

21

N

N 22

6

O

N N

N 36

NA

PhHN N

5

Ph N

N

N 35

0.628

O

19

4

NA

PhO N

N 34

O

18

3

33

O

17

N

NHOH

0.035

NA

H N O 37

O

NHO H

SAHA was used as the positive control. Values are means of three experiments, standard error of the IC50 was generally less than 10%. b NA, not active up to the highest concentration tested (10 mM).

O

NHOH O

a

N

0.033

NA

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Y. Yao et al. / European Journal of Medicinal Chemistry 86 (2014) 639e652

5. Experimental procedure 5.1. Chemistry Reagents and solvents were used as purchased without further purification. The progress of all reactions was monitored by TLC using ethyl acetate/n-hexane as solvent system, and spots were visualized by irradiation with ultraviolet light (254 nm). Flash chromatography was performed using silica gel (300e400 mesh). 1 H NMR and 13C NMR spectra were recorded on Bruker Avance ARX-400 (or Bruker Avance ARX-500). The low or high resolution of ESIMS was recorded on an Agilent 1200 HPLC-MSD mass spectrometer or Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer, respectively. Anhydrous solvents were obtained as follows: THF by distillation from sodium and benzophenone; dichloromethane, toluene and N, N-dimethylformamide from CaH2. All other solvents were reagent grade. All moisture sensitive reactions were carried out in flame dried flask under argon atmosphere. 5.1.1. 5-(1-([1,10 -biphenyl]-3-ylmethyl)-1H-pyrrol-2-yl)-Nhydroxypentanamide (2) To a cooled (0  C) solution of 53 (120 mg, 0.36 mmol) in dry THF (3 mL) was added tert-butyl carbonochloridate (70 ul, 0.54 mmol) and triethylamine (75 ul, 0.54 mmol). After stirring for 10 min, the solid was filtered off and the filtrate was added to a freshly prepared solution of hydroxylamine, obtained by reaction between hydroxylamine hydrochloride (75 mg, 1.08 mmol) and KOH (61 mg, 1.08 mmol) in methanol (0.6 ml). After being stirred at room temperature for 2 h, the mixture was evaporated under reduced pressure and the residue was purified by silica gel chromatography to afford 2 (103 mg, 82%) as a white solid. 1H NMR (400 MHz, CD3OD): d 7.52e7.50 (m, 2H), 7.47 (d, J ¼ 7.6 Hz, 1H), 7.41e7.33 (m, 3H), 7.32e7.28 (m, 1H), 7.19 (s, 1H), 6.95 (d, J ¼ 7.6 Hz, 1H), 6.68 (dd, J ¼ 2.0, 2.4 Hz, 1H), 6.04 (t, J ¼ 3.2 Hz, 1H), 5.90 (s, 1H), 5.14 (s, 2H), 2.48 (t, J ¼ 7.2 Hz, 2H), 2.02 (t, J ¼ 7.0 Hz, 2H), 1.62e1.53 (m, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 168.9, 140.4, 140.0, 139.8, 132.3, 129.1, 128.9, 127.5, 126.6, 125.4, 124.7, 120.9, 106.7, 105.7, 49.2, 32.0, 28.0, 25.2, 24.8 ppm. HRMS (ESI): calcd for C22H25N2O2 [MþH]þ 349.1838; found 349.1839.

Fig. 4. (A) Plausible binding mode of compound 9 to HDAC7. (B) The methoxyl group of compound 25 can fit a small hydrophobic cavity comprised of Pro 809 and Leu 810, leading to significant improvement of inhibition. (C) Comparison of the docking pose of compound 25 (green) and the binding pose of TSA (cyan, PDB ID: 3C10). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

anticancer activities against several cancer cell lines. Biological results demonstrated that compounds with strong inhibition of class I and IIb HDAC isoforms also exhibited high levels of potency in the cell-based assay. In contrast, the weaker inhibitors exhibited reduced potency against cancer cells. Enzyme inhibitory study correlated well with the cell-based result. We believe that more selective HDAC isozyme inhibitors are more likely to provide better effective chemotherapy with less side effects compared to paninhibitors, and also are useful as tools for probing the biology of the enzymes. Further therapeutic study with compound 22 on metastatic tumors in animal models is currently underway in our laboratory and will be reported in due course.

5.1.2. 5-(1-([1,10 -biphenyl]-3-ylmethyl)-1H-imidazol-2-yl)-Nhydroxypentanamide (3) To a stirred solution of 5-(1-([1,10 -biphenyl]-3-ylmethyl)-1Himidazol-2-yl) pentanoic acid 54 (200 mg, 0.60 mmol) in dry CH2Cl2 (6 mL) was added oxalyl dichloride (0.26 ml, 2.99 mmol) and 1 drop of DMF at 0  C. The mixture was stirred at reflux for 2 h and then evaporated and the residue was dried in vacuo and dissolved in CH2Cl2 (8 ml). In another vessel, to a suspension of hydroxylamine hydrochloride (166 mg, 2.39 mmol) in DCM (8 ml) was added DIPEA (0.79 mL, 4.78 mmol), and the resulting mixture was stirred at r.t. for 15 min. The contents of both vessels were combined. After stirring at 0  C for 2 h, the solvent was removed and the residue was diluted with water, extracted with ethyl acetate (3  30 mL). The combined organic phases were washed successively with 2 M HCl, water and brine, dried with Na2SO4, filtered, concentrated and purified by silica gel chromatography to afford 3 as a white solid (159 mg, 76%). 1H NMR (400 MHz, CD3OD): d 7.63 (d, J ¼ 7.8 Hz, 1H), 7.61e7.57 (m, 2H), 7.53 (s, 1H), 7.49 (t, J ¼ 7.8 Hz, 1H), 7.46e7.40 (m, 3H), 7.37e7.33 (m, 2H), 7.24e7.19 (m, 1H), 5.43 (s, 2H), 2.95 (t, J ¼ 7.2 Hz, 2H), 2.08 (t, J ¼ 6.8 Hz, 2H), 1.68e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 172.3, 149.1, 143.6, 141.6, 136.8, 130.9, 130.0, 128.8, 128.4, 128.0, 127.5, 127.3, 123.3, 121.6, 51.7, 32.9, 27.4, 25.8 ppm. HRMS (ESI): calcd for C21H24N3O2 [MþH]þ 350.1790, found 350.1792. HPLC analysis: 97.3% in purity.

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Table 3 Inhibition activities (IC50) of compounds 5, 10, 20, 21, 22 and SAHA against HDAC isoforms 1e11.a,b Compd

IC50 (mM) Class I

5 10 20 21 22 SAHA a b

Class IIa

Class IIb

Class IV

HDAC1

HDAC2

HDAC3

HDAC8

HDAC4

HDAC5

HDAC7

HDAC9

HDAC6

HDAC10

HDAC11

0.064 0.314 0.075 0.035 0.033 0.131

0.397 2.747 0.558 0.204 0.226 0.483

0.068 0.367 0.246 0.013 0.030 0.182

2.984 2.515 4.918 1.024 5.238 4.89

81.5 NA 86.4 56.3 83.1 NA

NA 9.85 NA 3.54 NA 2.48

NA NA NA NA NA 38.9

48.9 55.8 NA 18.3 47.2 22.8

5.218 0.064 0.158 0.065 0.034 0.035

0.094 0.774 0.136 0.025 0.029 0.686

2.342 NA 0.897 NA 1.725 5.84

SAHA was used as the positive control. Values are means of three experiments, standard error of the IC50 was generally less than 10%. NA, not active up to the highest concentration tested.

5.1.3. 5-(1-([1,10 -biphenyl]-3-ylmethyl)-1H-pyrazol-4-yl)-Nhydroxypentanamide (4) The procedure was the same as described above for the synthesis of 3. Compound 4 was obtained as a white solid (153 mg, 73%). 1H NMR (400 MHz, CD3OD): d 7.84 (s, 1H), 7.69 (s, 1H), 7.59e7.56 (m, 3H), 7.50 (s, 1H), 7.43 (q, J ¼ 7.6 Hz, 3H), 7.33 (t, J ¼ 7.2 Hz, 1H), 7.21 (d, J ¼ 7.6 Hz, 1H), 5.47 (s, 2H), 2.54 (t, J ¼ 6.8 Hz, 2H), 2.41 (t, J ¼ 6.8 Hz, 2H), 1.63e1.53 (m, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 169.0, 140.3, 139.9, 138.6, 138.4, 129.1, 128.9, 128.2, 127.5, 126.6, 126.5, 125.9, 125.8, 120.8, 54.6, 32.0, 30.0, 24.7, 23.3 ppm. HRMS (ESI): calcd for C21H24N3O2 [MþH]þ 350.1790; found 350.1792. HPLC analysis: 97.1% in purity. 5.1.4. 5-(1-([1,10 -biphenyl]-3-ylmethyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (5) The procedure was the same as described above for the synthesis of 3. Compound 5 was obtained as a white solid (157 mg, 75% for two steps). 1H NMR (400 MHz, CD3OD): d 7.60 (d, J ¼ 2.0 Hz, 1H), 7.56e7.51 (m, 3H), 7.45e7.36 (m, 4H), 7.34e7.28 (m, 1H), 7.14 (d, J ¼ 7.6 Hz, 1H), 6.14 (d, J ¼ 2.0 Hz, 1H), 5.32 (s, 2H), 2.62 (t, J ¼ 7.0 Hz, 2H), 2.09 (t, J ¼ 6.8 Hz, 2H), 1.64 (t, J ¼ 3.6 Hz, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 169.0, 152.0, 140.3, 139.9, 138.7, 130.8, 129.1, 128.9, 127.5, 126.6, 126.4, 125.8, 104.0, 54.4, 32.0, 28.7, 27.4, 24.8 ppm. HRMS (ESI): calculated for C21H23N3O2 [MþH]þ 350.1790, found 350.1791. HPLC analysis: 96.2% in purity. 5.1.5. 5-(1-([1,10 -biphenyl]-3-ylmethyl)-1H-pyrazol-5-yl)-Nhydroxypentanamide (6) The procedure was the same as described above for the synthesis of 3. Compound 6 was obtained as a white solid (85 mg, 80%). 1 H NMR (400 MHz, CD3OD): d 7.56e7.49 (m, 3H), 7.46 (d, J ¼ 1.9 Hz, 1H), 7.39 (q, J ¼ 7.9 Hz, 3H), 7.35e7.25 (m, 2H), 7.02 (d, J ¼ 7.6 Hz, 1H), 6.19 (d, J ¼ 1.6 Hz, 1H), 5.41 (s, 2H), 2.63 (t, J ¼ 6.8 Hz, 2H), 2.04

Fig. 5. Western blot detection of acetylated histone H3 and a-tubulin levels in HCT116 cells after 8 h treatment with selected compounds 5, 22 and controls. Histone H3 (bottom) serves as a control for protein loading.

(t, J ¼ 6.6 Hz, 2H), 1.61e1.58 (m, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 168.8, 142.5, 140.3, 139.8, 138.4, 137.9, 128.9, 128.8, 127.3, 126.4, 125.7, 125.5, 125.0, 104.1, 51.7, 31.8, 27.5, 24.5, 24.2 ppm. HRMS (ESI): C21H23N3O2 calculated [MþH]þ 350.1790, found 350.1791. HPLC analysis: 96.5% in purity. 5.1.6. N-hydroxy-5-(5H-imidazo[5,1-a]isoindol-3-yl) pentanamide (7) The procedure was the same as described above for the synthesis of 3. Compound 7 was obtained as a white solid (273 mg, 82%). 1H NMR (400 MHz, DMSO-d6): d 10.08 (s, 1H), 8.71 (s, 1H), 7.85 (d, J ¼ 5.6 Hz, 1H), 7.84 (s, 1H), 7.68 (d, J ¼ 7.2 Hz, 1H), 7.56e7.48 (m, 2H), 5.43 (s, 2H), 3.05 (t, J ¼ 7.6 Hz, 2H), 2.03 (t, J ¼ 7.2 Hz, 2H), 1.83e1.75 (m, 2H), 1.59e1.52 (m, 2H) ppm. 13C NMR (125 MHz, DMSO-d6): d 168.8, 143.9, 140.8, 137.5, 128.9, 128.8, 127.5, 124.3, 121.4, 108.9, 50.5, 31.6, 24.8, 24.4 ppm. HRMS (ESI): C15H18N3O2 calculated [MþH]þ 272.1321, found 272.1322. HPLC analysis: 95.1% in purity. 5.1.7. N-hydroxy-5-(5H-pyrrolo[2,1-a]isoindol-3-yl) pentanamide (8) The procedure was the same as described above for the synthesis of 2. Compound 8 was obtained as a white solid (273 mg, 82%). 1H NMR (400 MHz, DMSO-d6): d 10.36 (s, 1H), 8.67 (s, 1H), 7.44 (d, J ¼ 7.6 Hz, 2H), 7.29 (t, J ¼ 7.4 Hz, 1H), 7.13 (t, J ¼ 7.6 Hz, 1H), 6.16 (d, J ¼ 3.2 Hz, 1H), 5.94 (d, J ¼ 3.2 Hz, 1H), 4.88 (s, 2H), 2.60 (t, J ¼ 6.6 Hz, 2H), 2.00 (t, J ¼ 6.4 Hz, 2H), 1.59 (s, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 169.0, 140.2, 135.5, 133.6, 130.2, 127.7, 124.1, 123.5, 117.7, 109.0, 97.9, 48.3, 32.0, 27.0, 25.9, 24.9 ppm. HRMS (ESI): C16H19N2O2 calculated [MþH]þ 271.1368, found 271.1369. HPLC analysis: 96.5% in purity. 5.1.8. 5-(1-benzyl-1H-pyrazol-3-yl)-N-hydroxypentanamide (9) The procedure was the same as described above for the synthesis of 3. Compound 9 was obtained as a white solid (157 mg, 79%). 1H NMR (400 MHz, CD3OD): d 7.53 (d, J ¼ 2.4 Hz, 1H), 7.33e7.24 (m, 3H), 7.16 (d, J ¼ 6.8 Hz, 2H), 6.12 (d, J ¼ 2.0 Hz, 1H), 5.25 (s, 2H), 2.61 (t, J ¼ 7.0 Hz, 2H), 2.10 (t, J ¼ 7.0 Hz, 2H), 1.66e1.62 (m, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 169.0, 151.9, 138.0, 130.7, 128.4, 127.4, 127.3, 103.9, 54.4, 32.1, 28.7, 27.4, 24.9 ppm. HRMS (ESI): C15H20N3O2 calculated [MþH]þ 274.1477, found 274.1478. HPLC analysis: 99.8% in purity. 5.1.9. N-hydroxy-5-(1-phenyl-1H-pyrazol-3-yl)pentanamide (10) The procedure was the same as described above for the synthesis of 3. Compound 10 was obtained as yellow wax (254 mg, 85%). 1H NMR (400 MHz, CD3OD): d 8.06 (d, J ¼ 2.4 Hz, 1H), 7.68e7.66 (m, 2H), 7.45 (t, J ¼ 8.0 Hz, 2H), 7.30e7.26 (m, 1H), 6.34 (d, J ¼ 2.4 Hz, 1H), 2.71 (t, J ¼ 7.0 Hz, 2H), 2.14 (t, J ¼ 6.8 Hz, 2H),

648

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Table 4 Antiproliferative activities of compounds 1e37 against eight different cancer cell lines.aed Compd

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 SAHA

GI50 (mM) Hela

MCF7

BGC823

A549

HT1080

K562

U937

Molt-4

1.54 1.77 >10 3.27 1.70 2.54 NA NA 6.70 19.3 10.1 5.73 4.48 14.7 9.33 14.4 8.59 6.89 4.94 4.46 2.20 1.23 5.23 6.94 5.26 2.88 1.79 5.87 1.28 8.67 11.3 3.20 2.65 3.25 10 3.88 NA 3.20

8.76 5.41 >10 1.34 3.16 NA NA NA 5.54 14.5 9.29 5.76 5.65 10.7 10.6 7.33 4.3 6.9 2.49 8.03 2.61 1.81 4.99 9.98 2.82 3.56 2.98 3.22 1.76 4.25 6.24 1.82 >10 >10 >10 >10 NA 2.20

1.55 1.60 >10 1.11 0.87 1.25 NA >10 5.79 9.62 10.4 6.51 4.72 11.2 8.80 14.6 9.16 4.25 3.41 4.92 1.55 0.26 6.05 11.4 5.42 3.60 1.97 6.01 1.41 9.34 11.1 2.39 11.1 19.6 >80 >80 NA 4.26

1.81 3.58 >10 3.71 2.37 ND NA >10 7.04 13.6 16.1 8.08 5.94 14.9 9.63 16.7 10.3 5.33 4.54 4.93 1.96 1.33 7.81 11.7 6.21 3.46 2.24 7.41 1.40 10.6 16.4 3.23 6.02 >10 >10 >10 NA 5.27

1.66 2.45 9.42 2.98 1.88 10.0 >10 >10 8.98 4.61 10.0 6.85 4.93 13.7 9.49 5.00 4.73 6.62 1.82 4.54 2.04 1.69 6.15 6.60 2.91 4.04 2.65 2.91 2.11 4.66 5.99 1.21 3.68 >10 >10 >10 10.2 1.79

0.58 0.59 >10 1.21 0.74 1.05 NA NA 2.84 3.65 4.27 3.03 2.14 4.19 3.97 4.66 4.20 2.24 2.11 1.92 0.71 0.46 3.45 4.24 3.06 1.70 1.04 3.66 0.76 4.36 5.32 1.25 3.68 >10 >10 10 NA 1.97

0.62 1.43 6.28 1.51 0.90 11.8 NA 10.1 1.85 4.46 2.09 1.79 1.49 3.45 1.51 2.62 2.45 1.94 1.52 1.58 0.62 0.51 1.75 1.43 1.43 0.62 0.50 1.63 0.44 2.50 3.31 0.76 2.65 3.25 10 3.88 NA 1.42

0.58 1.10 4.09 1.34 0.67 0.95 14.1 4.18 0.69 1.62 1.08 0.81 0.57 1.09 1.08 1.63 1.38 0.82 0.68 0.59 0.24 0.17 0.65 0.97 0.97 0.38 0.33 0.97 0.23 1.35 1.54 0.40 1.82 3.63 >10 3.39 NA 0.68

a

SAHA was used as the positive control. Inhibition of cell growth by the listed compounds was determined by using CCK-8 assay. c NA, not active up to the highest concentration tested. d Standard error of the GI50 was generally less than 10%. b

1.73e1.69 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 168.8, 152.2, 137.5, 126.5, 125.7, 123.4, 116.2, 103.6, 29.5, 26.1, 24.6, 22.4 ppm. HRMS (ESI): C14H18N3O2 calculated [MþH]þ 260.1321, found 260.1322. HPLC analysis: 97.2% in purity. 5.1.10. 5-(1-(4-(tert-butyl)benzyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (11) The procedure was the same as described above for the synthesis of 3. Compound 11 was obtained as a white solid (201 mg, 71%). 1H NMR (400 MHz, CD3OD): d 7.50 (d, J ¼ 2.4 Hz, 1H), 7.35 (d, J ¼ 8.4 Hz, 2H), 7.10 (d, J ¼ 8.4 Hz, 2H), 6.11 (d, J ¼ 2.4 Hz, 1H), 5.20 (s, 2H), 2.60 (t, J ¼ 6.8 Hz, 2H), 2.11e2.08 (m, 2H), 1.66e1.62 (m, 4H), 1.28 (s, 9H) ppm. 13C NMR (125 MHz, CD3OD): d 172.8, 154.3, 151.9, 135.5, 132.2, 128.2, 126.6, 105.5, 55.8, 35.3, 33.5, 31.7, 30.2, 28.5, 26.3 ppm. HRMS (ESI): C19H28N3O2 calculated [MþH]þ 330.2103, found 330.2102. HPLC analysis: 98.2% in purity. 5.1.11. N-hydroxy-5-(1-(4-methylbenzyl)-1H-pyrazol-3-yl) pentanamide (12) The procedure was the same as described above for the synthesis of 3. Compound 12 was obtained as a white solid (174 mg, 85%). 1H NMR (400 MHz, CD3OD): d 7.49 (d, J ¼ 2.4 Hz, 1H), 7.13 (d,

J ¼ 8.0 Hz, 2H), 7.06 (d, J ¼ 8.0 Hz, 2H), 6.11 (d, J ¼ 2.0 Hz, 1H), 5.19 (s, 2H), 2.60 (t, J ¼ 7.0 Hz, 2H), 2.29 (s, 3H), 2.09 (t, J ¼ 6.8 Hz, 2H), 1.65e1.62 (m, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 168.9, 151.6, 136.4, 134.6, 130.1, 128.7, 127.2, 103.6, 54.1, 31.9, 28.5, 27.2, 24.7, 20.4 ppm. HRMS (ESI): C16H22N3O2 calculated [MþH]þ 287.1634, found 287.1635. HPLC analysis: 99.1% in purity.

5.1.12. 5-(1-(4-bromobenzyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (13) The procedure was the same as described above for the synthesis of 3. Compound 13 was obtained as a white solid (165 mg, 78%). 1H NMR (400 MHz, CD3OD): d 7.56 (d, J ¼ 2.0 Hz, 1H), 7.47 (d, J ¼ 8.4 Hz, 2H), 7.08 (d, J ¼ 8.8 Hz, 2H), 6.14 (d, J ¼ 2.0 Hz, 1H), 5.23 (s, 2H), 2.61 (t, J ¼ 7.0 Hz, 2H), 2.11e2.08 (m, 2H), 1.65e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 172.8, 154.8, 138.0, 132.8, 132.5, 130.2, 122.6, 105.8, 55.3, 33.5, 30.2, 28.5, 26.3 ppm. HRMS (ESI): C15H19BrN3O2 calculated [MþH]þ 352.0582, found 352.0583. HPLC analysis: 95.8% in purity.

5.1.13. N-hydroxy-5-(1-(4-nitrobenzyl)-1H-pyrazol-3-yl) pentanamide (14) The procedure was the same as described above for the synthesis of 3. Compound 14 was obtained as a pale yellow solid (95 mg, 71%). 1H NMR (400 MHz, CD3OD): d 8.19 (d, J ¼ 8.8 Hz, 2H), 7.64 (d, J ¼ 2.0 Hz, 1H), 7.34 (d, J ¼ 8.8 Hz, 2H), 6.18 (d, J ¼ 2.4 Hz, 1H), 5.41 (s, 2H), 2.62 (t, J ¼ 6.8 Hz, 2H), 2.10 (t, J ¼ 6.8 Hz, 2H), 1.64 (t, J ¼ 3.4 Hz, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 172.8, 155.3, 148.9, 146.3, 133.0, 129.1, 124.8, 106.0, 55.2, 33.5, 30.1, 28.5, 26.3 ppm. HRMS (ESI): C15H19N4O4 calculated [MþH]þ 319.1328, found 319.1329. HPLC analysis: 96.8% in purity.

5.1.14. N-hydroxy-5-(1-(4-(trifluoromethyl)benzyl)-1H-pyrazol-3yl)pentanamide (15) The procedure was the same as described above for the synthesis of 3. Compound 15 was obtained as a white solid (250 mg, 80%). 1H NMR (400 MHz, CD3OD): d 7.62 (m, 3H), 7.31 (d, J ¼ 8.0 Hz, 2H), 6.17 (d, J ¼ 2.0 Hz, 1H), 5.36 (s, 2H), 2.62 (t, J ¼ 7.0 Hz, 2H), 2.10 (t, J ¼ 6.8 Hz, 2H), 1.67e1.63 (m, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 169.0, 152.4, 142.8, 131.1, 128.0 (q, J ¼ 31.5 Hz), 127.9, 125.3( q, J ¼ 3.7 Hz), 124.2 (q, J ¼ 272.1 Hz), 104.1, 53.8, 32.0, 28.7, 27.4, 24.8 ppm. HRMS (ESI): C16H19F3N3O2 calculated [MþH]þ 342.1351, found 342.1352. HPLC analysis: 96.6% in purity.

5.1.15. N-hydroxy-5-(1-(4-morpholinobenzyl)-1H-pyrazol-3-yl) pentanamide (16) To a stirred solution of 72u (323 mg, 0.94 mmol) in DMF (9.4 ml) were added TBTU (453 mg, 1.41 mmol) and TEA (0.26 ml, 1.88 mmol). The mixture was stirred at room temperature for 2 h and then added to a freshly prepared solution of hydroxylamine, obtained by reaction between hydroxylamine hydrochloride (131 mg, 1.88 mmol) and KOH (106 mg, 1.88 mmol) in methanol (1 ml). After stirring at room temperature overnight, the mixture was evaporated under reduced pressure and the residue was purified by silica gel chromatography to afford 16 as a colorless oil (259 mg, 77%). 1H NMR (400 MHz, CD3OD): d 7.46 (d, J ¼ 2.0 Hz, 1H), 7.11 (d, J ¼ 8.8 Hz, 2H), 6.91 (d, J ¼ 8.8 Hz, 2H), 6.09 (d, J ¼ 2.0 Hz, 1H), 5.15 (s, 2H), 3.80 (t, J ¼ 4.8 Hz, 4H), 3.10 (t, J ¼ 4.8 Hz, 4H), 2.60 (t, J ¼ 6.8 Hz, 2H), 2.09 (t, J ¼ 6.8 Hz, 2H), 1.65e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 168.8, 150.3, 148.6, 128.0, 125.7, 125.6, 113.0, 101.4, 63.9, 51.7, 46.6, 29.5, 26.2, 24.5, 22.3 ppm. HRMS (ESI): C19H27N4O3 calculated [MþH]þ 359.2005, found 359.2006. HPLC analysis: 95.6% in purity.

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5.1.16. 5-(1-(4-fluorobenzyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (17) The procedure was the same as described above for the synthesis of 3. Compound 17 was obtained as a white solid (214 mg, 76%). 1H NMR (400 MHz, CD3OD): d 7.54 (d, J ¼ 2.0 Hz, 1H), 7.22e7.18 (m, 2H), 7.06e7.02 (m, 2H), 6.12 (d, J ¼ 2.4 Hz, 1H), 5.23 (s, 2H), 2.61 (t, J ¼ 7.0 Hz, 2H), 2.11e2.08 (m, 2H), 1.65e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 168.8, 159.8 (d, J ¼ 244.8 Hz), 150.7, 130.7 (d, J ¼ 3.2 Hz), 128.3, 126.4 (d, J ¼ 8.3 Hz), 112.4 (d, J ¼ 21.9 Hz), 101.7, 51.3, 29.5, 26.2, 24.5, 22.3 ppm. HRMS (ESI): C15H19FN3O2 calculated [MþH]þ 292.1383, found 292.1382. HPLC analysis: 98.7% in purity.

5.1.21. 5-(1-([1,10 -biphenyl]-4-ylmethyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (22) The procedure was the same as described above for the synthesis of 3. Compound 22 was obtained as a white solid (200 mg, 87%). 1H NMR (400 MHz, CD3OD): d 7.59e7.56 (m, 5H), 7.41 (t, J ¼ 7.6 Hz, 2H), 7.31 (t, J ¼ 7.2 Hz, 1H), 7.25 (d, J ¼ 8.0 Hz, 2H), 6.14 (d, J ¼ 2.0 Hz, 1H), 5.30 (s, 2H), 2.63 (t, J ¼ 7.0 Hz, 2H), 2.10 (t, J ¼ 6.8 Hz, 2H), 1.65 (t, J ¼ 3.6 Hz, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 168.8, 150.6, 138.1, 137.9, 133.7, 128.4, 125.9, 124.9, 124.4, 124.3, 124.0, 101.6, 51.8, 29.5, 26.2, 24.5, 22.3 ppm. HRMS (ESI): C21H23N3O2 calculated [MþH]þ 350.1863, found 350.1864. HPLC analysis: 99.6% in purity.

5.1.17. 5-(1-(4-(benzyloxy)benzyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (18) The procedure was the same as described above for the synthesis of 3. Compound 18 was obtained as a white solid (595 mg, 76%). 1H NMR (400 MHz, CD3OD): d 7.49 (d, J ¼ 2.0 Hz, 1H), 7.41e7.26 (m, 5H), 7.12 (d, J ¼ 8.8 Hz, 2H), 6.94 (d, J ¼ 8.4 Hz, 2H), 6.10 (d, J ¼ 2.0 Hz, 1H), 5.17 (s, 2H), 5.05 (s, 2H), 2.60 (t, J ¼ 6.8 Hz, 2H), 2.09 (t, J ¼ 6.8 Hz, 2H), 1.65e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 172.8, 159.9, 154.4, 138.6, 132.1, 130.8, 129.9, 129.5, 128.9, 128.5, 116.1, 105.5, 71.0, 55.6, 33.5, 30.2, 28.5, 26.3 ppm. HRMS (ESI): C22H26N3O3 calculated [MþH]þ 380.1896, found 380.1895. HPLC analysis: 96.8% in purity.

5.1.22. N-hydroxy-5-(1-(3-(phenylamino)benzyl)-1H-pyrazol-3-yl) pentanamide (23) The procedure was the same as described above for the synthesis of 16. Compound 23 was obtained as a white foam (287 mg, 86%). 1H NMR (400 MHz, DMSO-d6): d 10.35 (s, 1H), 8.67 (s, 1H), 8.18 (s, 1H), 7.65 (d, J ¼ 2.0 Hz, 1H), 7.22e7.15 (m, 3H), 7.03 (d, J ¼ 8.0 Hz, 2H), 6.95 (dd, J ¼ 1.4, 8.2 Hz, 1H), 6.87 (s, 1H), 6.81 (t, J ¼ 7.4 Hz, 1H), 6.65 (d, J ¼ 7.6 Hz, 1H), 6.04 (d, J ¼ 2.4 Hz, 1H), 5.17 (s, 2H), 2.50 (t, J ¼ 1.4 Hz, 2H), 1.96 (t, J ¼ 6.4 Hz, 2H), 1.53 (d, J ¼ 3.2 Hz, 4H) ppm. 13 C NMR (125 MHz, DMSO-d6): d 169.1, 151.9, 143.5, 143.1, 140.0, 130.7, 129.2, 129.1, 119.8, 118.5, 116.8, 115.6, 115.3, 103.8, 54.5, 32.0, 28.7, 27.4, 24.9 ppm. HRMS (ESI): C21H25N4O2 calculated [MþH]þ 365.1899, found 365.1899. HPLC analysis: 96.0% in purity.

5.1.18. N-hydroxy-5-(1-(4-phenoxybenzyl)-1H-pyrazol-3-yl) pentanamide (19) The procedure was the same as described above for the synthesis of 3. Compound 19 was obtained as a white solid (280 mg, 82%). 1H NMR (400 MHz, CD3OD): d 7.54 (d, J ¼ 2.0 Hz, 1H), 7.35e7.31 (m, 2H), 7.18 (d, J ¼ 8.8 Hz, 2H), 7.09 (t, J ¼ 7.4 Hz, 1H), 6.97e6.92 (m, 4H), 6.12 (d, J ¼ 2.0 Hz, 1H), 5.23 (s, 2H), 2.61 (t, J ¼ 7.0 Hz, 2H), 2.10 (t, J ¼ 6.8 Hz, 2H), 1.66e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 163.3, 149.0, 145.1, 123.9, 122.7, 121.4, 120.6, 115.1, 110.5, 110.3, 96.1, 46.0, 24.0, 20.7, 19.0, 16.8 ppm. HRMS (ESI): C21H24N3O3 calculated [MþH]þ 366.1739, found 366.1738. HPLC analysis: 97.5% in purity. 5.1.19. 5-(1-(4-(1H-indol-1-yl)benzyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (20) The procedure was the same as described above for the synthesis of 16. Compound 20 was obtained as a yellow oil (266 mg, 77%). 1H NMR (400 MHz, CD3OD): d 7.63 (d, J ¼ 2.0 Hz, 1H), 7.60 (d, J ¼ 8.0 Hz, 1H), 7.51e7.49 (m, 3H), 7.40 (d, J ¼ 3.2 Hz, 1H), 7.36 (d, J ¼ 8.4 Hz, 2H), 7.18e7.14 (m, 1H), 7.11e7.07 (m, 1H), 6.63 (d, J ¼ 3.6 Hz, 1H), 6.16 (d, J ¼ 2.0 Hz, 1H), 5.35 (s, 2H), 2.64 (t, J ¼ 6.8 Hz, 2H), 2.11 (t, J ¼ 6.8 Hz, 2H), 1.66 (t, J ¼ 3.6 Hz, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 174.7, 156.6, 142.6, 139.0, 138.7, 134.4, 132.8, 131.7, 130.7, 127.2, 125.2, 123.9, 123.2, 113.1, 107.6, 106.5, 57.4, 35.4, 32.1, 30.4, 28.2 ppm. HRMS (ESI): C23H24N4O2 calculated [MþH]þ 389.1972, found 389.1974. HPLC analysis: 96.5% in purity. 5.1.20. N-hydroxy-5-(1-(4-(phenylamino)benzyl)-1H-pyrazol-3-yl) pentanamide (21) The procedure was the same as described above for the synthesis of 16. Compound 21 was obtained as a white foam (270 mg, 81%). 1H NMR (400 MHz, CD3OD): d 7.47 (d, J ¼ 2.0 Hz, 1H), 7.21e7.17 (m, 2H), 7.08e7.00 (m, 6H), 6.83 (t, J ¼ 7.4 Hz, 1H), 6.09 (d, J ¼ 2.0 Hz, 1H), 5.14 (s, 2H), 2.61 (t, J ¼ 6.8 Hz, 2H), 2.10 (t, J ¼ 6.8 Hz, 2H), 1.66e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 172.8, 154.2, 145.2, 144.8, 131.9, 130.1, 129.7, 129.4, 121.5, 118.7, 118.0, 105.4, 55.9, 33.5, 30.2, 28.5, 26.3 ppm. HRMS (ESI): C21H24N4O2 calculated [MþH]þ 365.1972, found 365.1975. HPLC analysis: 96.0% in purity.

5.1.23. N-hydroxy-5-(1-(3-phenoxybenzyl)-1H-pyrazol-3-yl) pentanamide (24) The procedure was the same as described above for the synthesis of 3. Compound 24 was obtained as a white solid (151 mg, 82%). 1H NMR (400 MHz, CD3OD): d 7.54 (d, J ¼ 2.0 Hz, 1H), 7.34e7.27 (m, 3H), 7.10 (t, J ¼ 7.4 Hz, 1H), 6.95e6.85 (m, 4H), 6.73 (s, 1H), 6.11 (d, J ¼ 2.4 Hz, 1H), 5.23 (s, 2H), 2.59 (t, J ¼ 6.8 Hz, 2H), 2.11e2.07 (m, 2H), 1.64e1.61 (m, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 170.0, 156.7, 156.2, 151.9, 140.0, 130.5, 129.8, 123.3, 122.0, 118.5, 117.1, 103.7, 53.9, 28.5, 27.2, 20.5, 13.8 ppm. HRMS (ESI): C21H24N3O3 calculated [MþH]þ 366.1739, found 366.1738. HPLC analysis: 96.0% in purity. 5.1.24. N-hydroxy-5-(1-(3-methoxybenzyl)-1H-pyrazol-3-yl) pentanamide (25) The procedure was the same as described above for the synthesis of 3. Compound 25 was obtained as a white solid (258 mg, 79%). 1H NMR (400 MHz, CD3OD): d 7.53 (d, J ¼ 2.4 Hz, 1H), 7.22 (t, J ¼ 7.8 Hz, 1H), 6.82 (dd, J ¼ 2.2, 8.2 Hz, 1H), 6.74e6.70 (m, 2H), 6.13 (d, J ¼ 2.4 Hz, 1H), 5.22 (s, 2H), 3.74 (s, 3H), 2.62 (t, J ¼ 7.0 Hz, 2H), 2.11e2.08 (m, 2H), 1.66e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 168.8, 157.5, 150.5, 136.2, 128.4, 126.7, 116.5, 110.3, 110.0, 101.6, 52.0, 51.7, 29.5, 26.2, 24.5, 22.3 ppm. HRMS (ESI): C16H22N3O3 calculated [MþH]þ 304.1583, found 304.1584. HPLC analysis: 98.1% in purity. 5.1.25. 5-(1-(3-bromobenzyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (26) The procedure was the same as described above for the synthesis of 3. Compound 26 was obtained as a white solid (220 mg, 84%). 1H NMR (400 MHz, CD3OD): d 7.59 (d, J ¼ 2.0 Hz, 1H), 7.42 (d, J ¼ 8.0 Hz, 1H), 7.30 (s, 1H), 7.24 (t, J ¼ 7.8 Hz, 1H), 7.13 (d, J ¼ 7.6 Hz, 1H), 6.15 (d, J ¼ 2.4 Hz, 1H), 5.25 (s, 2H), 2.62 (t, J ¼ 7.2 Hz, 2H), 2.10 (t, J ¼ 6.8 Hz, 2H), 1.67e1.63 (m, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 169.0, 152.3, 140.7, 130.9, 130.6, 130.2, 130.0, 126.4, 121.6, 104.0, 53.6, 32.0, 28.7, 27.4, 24.8 ppm. HRMS (ESI): C15H18BrN3O2 calculated [MþH]þ 352.0655, found 352.0654. HPLC analysis: 99.8% in purity.

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5.1.26. 5-(1-(3-chloro-4-fluorobenzyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (27) The procedure was the same as described above for the synthesis of 3. Compound 27 was obtained as a white solid (293 mg, 76%). 1H NMR (400 MHz, CD3OD): d 7.59 (d, J ¼ 2.0 Hz, 1H), 7.28 (dd, J ¼ 2.0, 6.8 Hz, 1H), 7.19 (t, J ¼ 8.8 Hz, 1H), 7.15e7.11 (m, 1H), 6.15 (d, J ¼ 2.4 Hz, 1H), 5.23 (s, 2H), 2.61 (t, J ¼ 7.0 Hz, 2H), 2.10 (t, J ¼ 7.0 Hz, 2H), 1.65e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 163.3, 149.4 (d, J ¼ 247.9 Hz), 145.5, 126.8 (d, J ¼ 4.4 Hz), 123.0, 121.2, 119.2 (d, J ¼ 7.4 Hz), 112.4 (d, J ¼ 18.2 Hz), 108.3 (d, J ¼ 21.8 Hz), 96.4, 45.2, 24.0, 20.6, 19.0, 16.8 ppm. HRMS (ESI): C15H18ClFN3O2 calculated [MþH]þ 326.0993, found 326.0995. HPLC analysis: 99.8% in purity.

5.1.27. 5-(1-(benzo[d][1,3]dioxol-5-ylmethyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (28) The procedure was the same as described above for the synthesis of 16. Compound 28 was obtained as a white solid (206 mg, 75%). 1H NMR (400 MHz, CD3OD): d 7.50 (d, J ¼ 2.4 Hz, 1H), 6.77e6.67 (m, 3H), 6.11 (d, J ¼ 2.0 Hz, 1H), 5.91 (s, 2H), 5.14 (s, 2H), 2.61 (t, J ¼ 7.0 Hz, 2H), 2.10 (t, J ¼ 6.8 Hz, 2H), 1.66e1.62 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 168.8, 150.5, 145.4, 144.8, 128.3, 128.1, 118.1, 105.2, 104.9, 101.6, 98.5, 51.9, 29.5, 26.2, 24.5, 22.3 ppm. HRMS (ESI): C15H18N3O4 calculated [MþH]þ 304.1219, found 304.1217. HPLC analysis: 96.3% in purity.

5.1.28. N-hydroxy-5-(1-(naphthalen-2-ylmethyl)-1H-pyrazol-3-yl) pentanamide (29) The procedure was the same as described above for the synthesis of 3. Compound 29 was obtained as a white solid (251 mg, 77%). 1H NMR (400 MHz, CD3OD): d 7.82e7.78 (m, 3H), 7.62 (s, 1H), 7.60 (d, J ¼ 2.4 Hz, 1H), 7.48e7.43 (m, 2H), 7.29 (dd, J ¼ 1.6, 8.4 Hz, 1H), 6.16 (d, J ¼ 2.4 Hz, 1H), 5.42 (s, 2H), 2.65e2.61 (m, 2H), 2.10 (t, J ¼ 7.0 Hz, 2H), 1.65 (q, J ¼ 3.4 Hz, 4H) ppm. 13C NMR (125 MHz, DMSO-d6): d 169.0, 152.0, 135.5, 132.8, 132.3, 130.7, 128.1, 127.7, 127.5, 126.3, 126.1, 126.0, 125.6, 104.0, 54.6, 32.0, 28.7, 27.4, 24.9 ppm. HRMS (ESI): C19H21N3O2 calculated [MþH]þ 324.1707, found 324.1709. HPLC analysis: 95.3% in purity.

5.1.29. 5-(1-(2,4-difluorobenzyl)-1H-pyrazol-3-yl)-Nhydroxypentanamide (30) The procedure was the same as described above for the synthesis of 3. Compound 30 was obtained as a white solid (231 mg, 68%). 1H NMR (400 MHz, CD3OD): d 7.55 (d, J ¼ 2.4 Hz, 1H), 7.16e7.10 (m, 1H), 7.00e6.90 (m, 2H), 6.13 (d, J ¼ 2.4 Hz, 1H), 5.28 (s, 2H), 2.60 (t, J ¼ 7.0 Hz, 2H), 2.09 (t, J ¼ 7.0 Hz, 2H), 1.65e1.61 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 168.8, 160.3 (dd, J ¼ 248.0, 12.0 Hz), 158.0 (dd, J ¼ 249.0, 12.2 Hz), 150.9, 128.5, 128.2 (dd, J ¼ 9.9, 5.3 Hz), 117.8 (dd, J ¼ 14.9, 3.9 Hz), 108.5 (dd, J ¼ 21.6, 3.9 Hz), 101.7, 100.7 (t, J ¼ 25.9 Hz), 29.5, 26.2, 24.5, 22.3 ppm. HRMS (ESI): C15H18F2N3O2 calculated [MþH]þ 310.1289, found 310.1287. HPLC analysis: 97.5% in purity.

5.1.30. 5-(1-benzyl-4-methyl-1H-pyrazol-3-yl)-Nhydroxypentanamide (31) The procedure was the same as described above for the synthesis of 3. Compound 31 was obtained as a white solid (67 mg, 76%). 1H NMR (400 MHz, CD3OD): d 7.32e7.23 (m, 4H), 7.14 (d, J ¼ 7.2 Hz, 2H), 5.18 (s, 2H), 2.57 (t, J ¼ 7.0 Hz, 2H), 2.10 (t, J ¼ 6.8 Hz, 2H), 2.00 (s, 3H), 1.63e1.59 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 168.8, 148.4, 134.9, 127.2, 125.7, 124.7, 124.3, 111.3, 51.9, 29.5, 25.8, 22.7, 22.4, 4.4 ppm. HRMS (ESI): C16H22N3O2 calculated [MþH]þ 288.1634, found 288.1636. HPLC analysis: 99.3% in purity.

5.1.31. 5-(1-benzyl-3-phenyl-1H-pyrazol-4-yl)-Nhydroxypentanamide (32) The procedure was the same as described above for the synthesis of 3. Compound 32 was obtained as a white solid (190 mg, 76%). 1H NMR (400 MHz, CD3OD): d 7.55e7.53 (m, 3H), 7.42e7.24 (m, 8H), 5.31 (s, 2H), 2.61 (t, J ¼ 7.2 Hz, 2H), 2.05 (t, J ¼ 7.0 Hz, 2H), 1.64e1.54 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): d 168.8, 147.4, 134.5, 131.1, 127.4, 125.8, 125.5, 125.1, 124.9, 124.7, 124.5, 116.7, 52.4, 29.5, 27.0, 22.4, 21.0 ppm. HRMS (ESI): C21H24N3O2 calculated [MþH]þ 349.1790, found 349.1791. HPLC analysis: 97.6% in purity. 5.1.32. 6-(1-([1,10 -biphenyl]-4-ylmethyl)-1H-pyrazol-3-yl)-Nhydroxyhexanamide (33) The procedure was the same as described above for the synthesis of 3. Compound 33 was obtained as a white solid (238 mg, 88%). 1H NMR (400 MHz, CD3OD): d 7.59e7.57 (m, 5H), 7.41 (t, J ¼ 7.8 Hz, 2H), 7.31 (t, J ¼ 7.2 Hz, 1H), 7.25 (d, J ¼ 8.0 Hz, 2H), 6.14 (d, J ¼ 2.0 Hz, 1H), 5.30 (s, 2H), 2.61 (t, J ¼ 7.6 Hz, 2H), 2.07 (t, J ¼ 7.4 Hz, 2H), 1.69e1.59 (m, 4H), 1.40e1.33 (m, 2H) ppm. 13C NMR (125 MHz, CD3OD): d 172.9, 154.9, 142.1, 141.9, 137.7, 132.4, 129.9, 128.9, 128.4, 128.3, 128.0, 105.6, 55.8, 33.7, 30.5, 29.7, 28.8, 26.5 ppm. HRMS (ESI): C22H26N3O2 calculated [MþH]þ 364.1947, found 364.1949. HPLC analysis: 97.8% in purity. 5.1.33. 7-(1-([1,10 -biphenyl]-4-ylmethyl)-1H-pyrazol-3-yl)-Nhydroxyheptanamide (34) The procedure was the same as described above for the synthesis of 3. Compound 34 was obtained as a white solid (191 mg, 75%). 1H NMR (400 MHz, CD3OD): d 7.57 (d, J ¼ 6.0 Hz, 5H), 7.41 (t, J ¼ 7.6 Hz, 2H), 7.31 (t, J ¼ 7.4 Hz, 1H), 7.25 (d, J ¼ 8.0 Hz, 2H), 6.13 (d, J ¼ 1.6 Hz, 1H), 5.30 (s, 2H), 2.60 (t, J ¼ 7.4 Hz, 2H), 2.05 (t, J ¼ 7.4 Hz, 2H), 1.64e1.57 (m, 4H), 1.35 (s, 4H). 13C NMR (125 MHz, CD3OD): d 173.0, 155.0, 142.1, 141.9, 137.7, 132.4, 129.9, 128.9, 128.4, 128.3, 127.9, 105.6, 55.8, 33.7, 30.7, 29.9, 29.8, 28.8, 26.7 ppm. HRMS (ESI): C23H28N3O2 calculated [MþH]þ 378.2103, found 378.2105. HPLC analysis: 96.6% in purity. 5.1.34. 8-(1-([1,10 -biphenyl]-4-ylmethyl)-1H-pyrazol-3-yl)-Nhydroxyoctanamide (35) The procedure was the same as described above for the synthesis of 3. Compound 35 was obtained as a white solid (343 mg, 68%). 1H NMR (400 MHz, CD3OD): d 7.59e7.56 (m, 5H), 7.41 (t, J ¼ 7.6 Hz, 2H), 7.31 (t, J ¼ 7.4 Hz, 1H), 7.25 (d, J ¼ 8.0 Hz, 2H), 6.13 (d, J ¼ 2.4 Hz, 1H), 5.30 (s, 2H), 2.60 (t, J ¼ 7.6 Hz, 2H), 2.05 (t, J ¼ 7.4 Hz, 2H), 1.65e1.57 (m, 4H), 1.33 (s, 6H). 13C NMR (125 MHz, DMSO-d6): d 169.7, 152.6, 140.0, 139.6, 137.3, 131.0, 129.2, 128.3, 127.7, 127.0, 126.8, 104.3, 54.3, 32.5, 29.3, 28.8, 28.7, 27.8, 25.3 ppm. HRMS (ESI): C24H30N3O2 calculated [MþH]þ 392.2260, found 392.2262. HPLC analysis: 97.1% in purity. 5.1.35. 9-(1-([1,10 -biphenyl]-4-ylmethyl)-1H-pyrazol-3-yl)-Nhydroxynonanamide (36) The procedure was the same as described above for the synthesis of 3. Compound 36 was obtained as a white solid (191 mg, 69%). 1H NMR (400 MHz, CD3OD): d 7.58e7.56 (m, 5H), 7.41 (t, J ¼ 7.6 Hz, 2H), 7.31 (t, J ¼ 7.2 Hz, 1H), 7.25 (d, J ¼ 8.0 Hz, 2H), 6.13 (d, J ¼ 2.0 Hz, 1H), 5.30 (s, 2H), 2.60 (t, J ¼ 7.6 Hz, 2H), 2.05 (t, J ¼ 7.4 Hz, 2H), 1.65e1.58 (m, 4H), 1.31 (s, 8H) ppm. 13C NMR (125 MHz, DMSOd6): d 169.3, 152.3, 139.8, 139.4, 137.2, 130.8, 129.0, 128.1, 127.5, 126.8, 126.7, 104.0, 54.2, 32.3, 29.2, 28.8, 28.7, 28.6, 27.7, 25.1 ppm. HRMS (ESI): C25H32N3O2 calculated [MþH]þ 406.2416, found 406.2418. HPLC analysis: 98.9% in purity.

Y. Yao et al. / European Journal of Medicinal Chemistry 86 (2014) 639e652

5.1.36. 1-Benzyl-N-(3-(hydroxyamino)-3-oxopropyl)-1H-pyrazole3-carboxamide (37) The procedure was the same as described above for the synthesis of 16. Compound 37 was obtained as a white solid (323 mg, 85%). 1H NMR (400 MHz, DMSO-d6): d 10.43 (s, 1H), 8.74 (s, 1H), 8.02 (s, 1H), 7.91 (d, J ¼ 2.4 Hz, 1H), 7.37e7.22 (m, 5H), 6.65 (d, J ¼ 2.0 Hz, 1H), 5.39 (s, 2H), 3.40 (t, J ¼ 6.4 Hz, 2H), 2.22 (t, J ¼ 7.0 Hz, 2H) ppm. 13 C NMR (125 MHz, DMSO-d6): d 168.0, 161.7, 147.1, 137.5, 132.7, 129.1, 128.3, 127.9, 106.6, 55.6, 35.7, 32.8 ppm. HRMS (ESI): C14H17N4O3 calculated [MþH]þ 289.1222, found 289.1225. HPLC analysis: 98.0% in purity. 5.2. Biological assays 5.2.1. Reagents Trichostatin A was purchased from Sigma. Vorinostat was synthesized according to known procedures [19]. All other reagents were supplied by Sigma (Germany) unless otherwise stated. 5.2.2. In vitro HDAC inhibition assay The Purified recombinant Human HDACs 1 to 11 and their corresponding substrates were purchased from BPS Bioscience (BPS Bioscience Inc., USA). The assays were carried out in 384-well format using the BPS fluorescent-based HDAC activity assay according to the manufacturer's protocol. In brief, 10 ml of the HDAC reaction mixture was composed of HDAC assay buffer, 100 mg BSA, serial diluted test compounds, appropriate concentration of HDACs, and 20 mM fluorogenic substrate, the mixture was incubated at 37  C for 60 min, and then stopped by addition of developer containing trypsin and TSA. After 20 min incubation, the fluorescence was detected at the excitation wavelength of 360 nm and the emission wavelength of 460 nm using EnVision Multi label Reader (PerkinElmer Inc., USA). The analytical software, GraphPad Prism 5.0 (GraphPad Software, Inc., USA) was used to generate IC50 value via non-linear regression analysis.

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After incubated, the PVDF membranes were washed four times with PBST buffer, then incubated with goat anti-rabbit or antimouse IgG-horseradish peroxidase conjugates (1:2000) for 1 h at room temperature and washed 4 times. The immunoblots were visualized by enhanced chemiluminescence. 5.3. Docking studies The deprotonated forms of the compounds described in this article were docked into the active sites of the HDAC1 (PDB code: 4bkx) and the crystal structure of human HDAC7 (PDB code: 3C10) €dinger suite. [40], by employing the program of Glide 5.9 of Schro Since there is no ligand in the active site of HDAC1, we incorporated SAHA by superimposing the crystal structure of HDAC1 with a crystal structure of HDAC2 bound with SAHA. The formed complex was done a 10 ns molecular dynamic simulation by employing the program of Amber 12. In all cases, all ions except for the catalytic zinc ion were deleted. For the human HDAC7 crystal structure, three water molecules, which are in the tube-like active site and have direct or indirect interactions with the bound ligand trichostatin A, were kept in the docking study Before docking the designed HDACis into the active sites, trichostatin A was docked back to both of the receptors to choose suitable scoring function and other docking parameters and to evaluate the docking accuracy. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 21172220 and 21472191), the National Basic Research Program of China (2009CB940900), the Strategic Leading Science & Technology Program of the Chinese Academy of Sciences and the “Fundamental Research Funds for the Central Universities” (Chunhua project 2013HGCH0015). Appendix A. Supplementary data

5.2.3. Cell culture and cell viability assay Cell lines, Hela, MCF7, BGC823, A549, HT1080, K562, U937 and Molt-4 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator with 5% CO2 in air at 37  C. Cell viability assay was determined by the CCK8 (DOJINDO LABORATORIES, Japan) assay. Cells were seeded at a density of 400e800 cells/well in 384 well plates and treated with various concentration of compounds or solvent control. After 72 h incubation, CCk8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (Perkin Elmer, USA). Dose response curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., USA). 5.2.4. Western bolt analysis Human colon cancer HCT116 cells were cultivated in McCoy5A culture medium with antibiotics and 10% fetal bovine serum. Cells were seeded at a destiny of 6  104/well in six well plates and incubated overnight to allow attachment. The HCT116 cells were treated with various concentration of compounds 5, 22 and TSA for 24 h at 37  C. After treatment, the cells were washed, harvested and lysed. The 10 mg total proteins for each sample were loaded on 15% SDS-polyacrylamide gel, then transferred onto PVDF membranes. The transferred membranes were blocked for 1 h with 5% skimmed milk, and then incubated overnight at 4  C with Acetyl-Histone H3 Antibody (Cell Signaling) (1:1000), Anti-Acetylated Tubulin Antibody (Sigma) (1:1000), or Histone H3 (Cell Signaling) (1:2000).

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