Design and Synthesis of Orally Bioavailable Aminopyrrolidinone

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Design and Synthesis of Orally Bioavailable Aminopyrrolidinone Histone Deacetylase 6 Inhibitors Xianfeng Lin, Wenming Chen, Zongxing Qiu, Lei Guo, Wei Zhu, Wentao Li, Zhanguo Wang, Weixing Zhang, Zhenshan Zhang, Yiping Rong, Meifang Zhang, Lingjie Yu, Sheng Zhong, Rong Zhao, Xihan Wu, Jason C. Wong, and Guozhi Tang* Roche Pharmaceutical Research and Early Development, Roche Innovation Center Shanghai, 720 Cailun Road, Shanghai 201203, China S Supporting Information *

ABSTRACT: Histone deacetylase 6 (HDAC6) removes the acetyl group from lysine residues in a number of non-histone substrates and plays important roles in microtubule dynamics and chaperone activities. There is growing interest in identifying HDAC6-selective inhibitors as chemical biology tools and ultimately as new therapeutic agents. Herein we report the design, synthesis, and phenotypic screening of a novel class of 3-aminopyrrolidinone-based hydroxamic acids as HDAC6 inhibitors. In particular, the α-methyl-substituted enantiomer 33 (3-S) showed significant in-cell tubulin acetylation (Tub-Ac) with an EC50 of 0.30 μM but limited impact on p21 levels at various concentrations. In enzyme inhibition assays, 33 demonstrated high selectivity for HDAC6 with an IC50 of 0.017 μM and selectivity indexes of 10 against HDAC8 and over 4000 against HDAC1−3 isoforms. Moreover, 33 has suitable drug metabolism and pharmacokinetics properties compared with other hydroxamic acid-based HDAC inhibitors, warranting further biological studies and development as a selective HDAC6 inhibitor.

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INTRODUCTION Inhibition of histone deacetylase 6 (HDAC6) has recently emerged as an attractive approach for the treatment of cancer, Alzheimer’s disease, and autoimmune disorders.1,2 HDAC6 is a cytoplasmic HDAC enzyme that uniquely features two deacetylase domains, a dynein motor binding domain to enable HDAC6 to shuttle cargo along the microtubule and a zinc finger ubiquitin-binding domain at the C-terminus.3 Functionally, HDAC6 removes the acetyl group from lysine residues in a number of non-histone substrates, including α-tubulin, Hsp90, cortactin, and peroxiredoxin, and plays important roles in microtubule dynamics and chaperone activities.4,5 In contrast to the lethal effect of HDAC1−3 genetic ablation, it has been reported that mice with HDAC6 knocked out are effectively normal.6 Indeed, HDAC6 inhibitors do not seem to be cytotoxic toward normal cells and may have fewer side effects than pan-HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) and HDAC1−3-selective inhibitors such as romidepsin.1,7 More recently, it has been demonstrated that inhibition of HDAC6 results in not only antiproliferative effects but also antidepressant and immunosuppressive effects with potential therapeutic applications.8,9 To date, a number of HDAC6-selective inhibitors have been reported.7,10 The first HDAC6-selective inhibitor, tubacin (1) (Figure 1), achieved selectivity through a bulky, relatively complex capping group, which unfortunately conferred poor © XXXX American Chemical Society

pharmacokinetic properties to the molecule overall and limited its application to enzyme- and cell-based chemical genetic studies.11 In contrast, ACY-1215 (rocilinostat, 2) has a rigid and less cumbersome capping group contributing to its HDAC6 selectivity; nevertheless, it exhibited a selectivity index of only 10 against HDAC1−3.12 Xenograft studies in nude mice indicated that 2 had synergistic antiproliferative activities in combination with bortezomib, and this molecule is currently in phase I/II clinical trials to treat patients with multiple myeloma. HDAC6 selectivity can also be generated by means of conformational restrictions at the linker and capping groups simultaneously, and recent examples include 3-oxo-2,4dihydroquinoxaline 3, tubastatin (4), HPOB (5), ACY-775 (6), BRD9575 (7), and quinazolin-4-one-linked analogue 8 (Figure 1).13−18 Among them, compounds 3−6 have parasubstituted aromatic linkers and rigid capping groups. Notably, 4 is a state-of-the-art HDAC6 inhibitor that showed excellent selectivity over HDAC1 (>3000×) and was initially disclosed for its neuroprotective effects.14 More recently, 4 and its β- and γ-carboline analogues were reported to have anti-inflammatory effects through enhancement of the immunosuppressive activity of Foxp3+ regulatory T cells.19 Studies of 5 revealed that Received: December 30, 2014

A

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Journal of Medicinal Chemistry

Figure 1. Structures of reported HDAC6 inhibitors.

Figure 2. Design of the prototype aminopyrrolidinone-linked HDAC inhibitors.

exposure. In fact, i.p. administration is frequently used and is probably the only practical option to achieve sufficient plasma exposure for many HDAC6 inhibitors and to demonstrate in vivo efficacy in animal models. We previously reported the identification and functional studies of aminotetraline 9 as an HDAC6 and HDAC8 dual inhibitor with 100-fold selectivity over other Zn2+-dependent HDACs.20 In our endeavor to develop orally available HDACisoform-selective inhibitors, we identified a novel class of aminopyrrolidinones as HDAC6 inhibitors by scaffold hopping from bicyclic-linked 9. Herein we report the rational design, synthesis, and structure−activity relationship (SAR) and drug metabolism and pharmacokinetics (DMPK) studies of this class of HDAC6 inhibitors.

HDAC6 inhibition did not induce cell death by itself but enhanced the cytotoxicity of DNA-damaging anticancer drugs.15 With better brain bioavailability than 4, compound 6 showed antidepressant effects in mouse models upon intraperitoneal (i.p.) administration.16 The “capless” 7 featuring a cyclopentene linker was also reported as an HDAC6 inhibitor and showed mediocre selectivity in enzymatic inhibition.17 In a recent report, 8 showed about 60-fold selectivity toward HDAC6 over HDAC1 as a result of a more extended linker compared with other phenyl-linker-based HDAC inhibitors.18 In vivo efficacy studies indicated that 8 was able to block Zn2+mediated β-amyloid aggregation and enhance the learningbased performance of mice without producing toxic or mitogenic effects. These results suggest that HDAC6 is a promising new target for a growing number of disease indications that may require different molecular properties and selectivity profiles and that HDAC6 inhibition could be a viable approach to mitigate toxicity associated with pan-HDAC inhibitors. However, a major hurdle in the development of hydroxamic acid-based HDAC6 inhibitors is their limited oral availability and drug

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RESULTS AND DISCUSSION HDAC6 inhibitors have to date been restricted to the use of hydroxamic acids and thiolates21 as Zn2+-binding groups. The hydroxamic acid class has long been considered a challenging functional group in drug research and development, in large B

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Journal of Medicinal Chemistry Scheme 1. Synthesis of Analogues 11−14a

Reagents and conditions: (a) K3PO4, CH3CN, rt, 1 h; (b) NaOH, rt, overnight, 65%; (c) K2CO3, DMF, 120 °C, MW, 10 min, 23%; (d) HONH2, KOH, MeOH, 60 °C, >50%; (e) NaH, MeI, DMF, rt to 60 °C, 46%; (f) t-BuOK, DMF, 100 °C, MW, 10 min, 26%.

a

Scheme 2. Synthesis of 4-Aminopyrrolidinone Analogue 15a

Reagents and conditions: (a) neat, 180 °C, 30 min, 98%; (b) SOCl2, 1,2-dichloroethane, rt to 80 °C, 1 h; (c) NH4OH, THF, rt 1 h, 85%; (d) DIB, THF, H2O, overnight, 81%; (e) (S)-MonoPhos, CuBr, Cs2CO3, DMF, 100 °C, overnight, 32%; (f) HONH2, KOH, MeOH, rt to 60 °C, 28%.

a

precursors 9 and 10, and was only weakly active against HDAC1 with an IC50 of 19.5 μM. DMPK studies indicated that 11 had good microsomal stability and solubility (human liver microsomal test (HLM) = 4.6 mL min−1 kg−1, mouse liver microsomal test (MLM) = 26 mL min−1 kg−1; see Table 3), but like many other hydroxamic acid-based inhibitors, it showed high in vivo clearance and poor oral bioavailability with F < 1%. On the basis of its attractive chemistry scaffold, low molecular weight, and selectivity profile, we designed additional analogues of 11 in an attempt to improve the HDAC6 inhibition and DMPK properties. We quickly explored the SAR and the possible role of the pNH moiety as a hydrogen-bond donor and synthesized a few close analogues of 11 according to the synthetic methods shown in Scheme 1. Briefly, α-bromopyrrolidinone 37 was obtained by coupling of aniline (35) and acyl chloride 36 and subsequent cyclization under basic conditions. Then 37 was treated with methyl 4-aminobenzoate (38) and K2CO3 in a microwave reactor to give methyl ester 39, which was converted to 11 by treatment with aqueous hydroxylamine and KOH. We also blocked the NH moiety with a methyl group and synthesized analogue 12 through N-methylation of ester 39 followed by hydroxylamination. By a similar method, bromide

part because of inactivation by enzyme-catalyzed hydrolysis and limited drug exposure. In our structural optimization of 9, it was found that tetrahydroquinoline-linked compounds showed better plasma stability and clearance profiles in mice, as exemplified by analogue 10. Tetrahydroquinoline 10 also had strong inhibitory effects on HDAC6 with an IC50 of 12 nM in biochemical assays (Figure 2). However, the enhanced enzymatic inhibition did not translate into in-cell tubulin acetylation (Tub-Ac) effects, as 10 showed an EC50 of 11.8 μM in A549 cells, likely because of its high polar surface area (PSA)22 and limited cell permeability. It was postulated the p-NH is involved in hydrogen bonding with D567 of HDAC6, and therefore, we intended to keep the aniline substructure and reshuffled the local conformational constriction of 10 as guided by molecular docking and conformational overlay. Thus, the aminopyrrolidinone-based analogue 11 was designed and synthesized with a phenyl moiety to probe the rim region of HDAC6 (see Figure 5A) and then evaluated for cellular effects on Tub-Ac. 11 exhibited moderate in-cell Tub-Ac with an EC50 of 5.9 μM, which correlated well to the enzymatic inhibition of HDAC6 (IC50 = 0.38 μM; see Table 1). In HDAC isoform profiling, 11 showed dual inhibition of HDAC6 and HDAC8, similar to its C

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Journal of Medicinal Chemistry 37 was treated with phenol 40 and t-BuOK to give phenyl ether 41, and subsequent hydroxylamination gave the ether-linked analogue 13. To confirm that the N-phenylpyrrolidinone group is important for HDAC6 binding and isoform selectivity, the “capless” analogue 14 was made by hydroxylamination of commercially available methyl 4-(methylamino)benzoate. Moreover, we synthesized 4-aminopyrrolidinone analogue 15 to probe the effect of the linker position with respect to the pyrrolidinone group (Scheme 2). Thus, 5-oxo-1-phenylpyrrolidine-3-carboxylic acid (43) was made by Michael addition of 35 and itaconic acid (42) and subsequent condensation under neat conditions. After acid 43 was converted to amide 44, a (diacetoxyiodo)benzene (DIB)mediated Hofmann degradation of 44 afforded amine 45 in 69% yield over three steps. It is noteworthy that a direct Curtius rearrangement of 43 gave a much lower yield of 45. Then amine 45 was coupled with iodide 46 by a coppercatalyzed Ullmann reaction to afford ester 47. Finally, 47 was treated with hydroxylamine and KOH to give hydroxamic acid 15. As shown in Table 1, both N-methylated analogue 12 and ether analogue 13 showed poor Tub-Ac activity with EC50

Figure 3. Effects on p21 induction by compounds 9−32. p21-RP10 represents the relative luciferase signal in the p21 reporter gene assay by individual example compared with that of 61 at 10 μM and normalized by internal GFP transfection control. Experiments were carried out in triplicate, and the data varied by 10% or less.

Table 1. Effects on Tubulin Acetylation and Enzymatic Inhibition by 9−15 IC50 (μM)

On the basis of the preliminary SAR data for the pyrrolidinone analogues, we decided to conduct additional structural modifications of 11 in order to explore important π−π or hydrophobic interactions with HDAC6 residues at the hydrophobic channel as well as the rim region. Thus, analogues 16−29 were synthesized according to the general procedure shown in Scheme 3. As described previously, α-bromopyrrolidinones 49 were prepared from amines 48 and acyl chloride 36 and then treated with ammonia to give α-aminopyrrolidinones 50. Ester intermediates 52 were prepared by copper-catalyzed Ullmann coupling of 50 with substituted phenyl halides 51. Compounds 16−28 with structural variations in the capping group and the phenyl linker were obtained by treating 52 with hydroxylamine under basic conditions. To further increase our understanding of the SAR for this scaffold, we also reduced the pyrrolidinone moiety and synthesized 3-aminopyrrolidine-based analogues such as 29 by borane−THF-mediated reduction and hydroxylamination of methyl esters 53. Analogues 16−29 were then evaluated for their effects on Tub-Ac and p21 induction in cells. Analogues 16 and 17 with N-benzyl and N-phenethyl terminal groups had 2−3 times less Tub-Ac activity than 11 (Table 2). From the structural finetuning of the N-phenyl group, it was found that p-Cl substitution improved the Tub-Ac activity and HDAC6 inhibition, with analogue 18 having an EC50 of 1.4 μM in the Tub-Ac assay and no effect on p21 induction at 3 and 10 μM. However, 18 had poor solubility in the lyophilized solubility assay (LYSA). It seems that the meta position could tolerate various substitutions, including dimethylaminoalkoxy, that contributed to the solubility, while ortho substituents such as −Cl were detrimental to Tub-Ac activity (analogues 19−21). It was also found that p-CN could be tolerated and increased the compound solubility, as analogue 25 showed an EC50 of 3.1 μM and LYSA of 28 μg/mL. However, bulkier groups such as trifluoromethyl and methylsulfonyl led to a significant drop in Tub-Ac activity (analogues 22 and 24). Analogue 26 with a naphthyl capping group showed potent Tub-Ac activity, consistent with our hypothesis that bicyclic aromatic groups

b

ID

Tub-Ac EC50 (μM)a

HDAC1

HDAC6

HDAC8

LYSA (μg/mL)c

SAHA 9 10 11 12 13 14 15

1.0 1.6 11.8 5.9 >30 >30 19.2 18.1

0.24 6.31 9.96 19.5 >100 >100 52.3 >100

0.054 0.048 0.012 0.38 48.8 5.74 1.77 2.26

0.66 0.08 0.47 0.21 8.2 4.92 0.94 6.25

>385 59 210 33 − 203 − >350

a

EC50 values for tubulin acetylation based on ELISA experiments run in duplicate (standard deviation (SD) below 15%). bIC50 values for enzymatic inhibition of HDAC enzymes. Experiments were run in duplicate (SD ± 15%). Assays were performed by Reaction Biology Corporation (Malvern, PA, USA). cLyophilized solubility assay (LYSA) mean values from duplicate runs (SD ± 10%).

values above 30 μM, and the results were in good agreement with their moderate enzymatic inhibition against HDAC6. Not surprisingly, 14 was several times less active against HDAC6 and HDAC8 than 11, and it showed weak Tub-Ac effects with an EC50 of 19.2 μM. 4-Aminopyrrolidinone analogue 15 was also less active than 11 in Tub-Ac. Overall, the results suggest that both conformational constraint and hydrogen bonding by the p-NH moiety contribute to the HDAC6 selectivity. To further confirm the selectivity profile, we tested the analogues in a p21 reporter gene assay that served as a surrogate of HDAC1−3 inhibition and compared the effects with that of the HDAC1 inhibitor N-{4-[N-(2-aminophenyl)carbamoyl]benzyl}carbamic acid 3-pyridylmethyl ester (SNDX275, 61) at 3, 10, and 30 μM in HeLa cells.23 It was obvious that 9−15 did not significantly induce p21 relative to 61 at 10 μM (Figure 3; for clarity, data at other concentrations are not shown). In contrast, the pan-HDAC inhibitor SAHA had a strong p21 induction effect that was 8.5 times that of 61 at 10 μM. D

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Journal of Medicinal Chemistry Scheme 3. Synthesis of 3-Aminopyrrolidinone Analogues 16−29a

Reagents and conditions: (a) K3PO4, CH3CN, rt, 1 h; (b) NaOH, rt, overnight; (c) NH4OH, CH3CN, 40 °C; (d) (S)-MonoPhos, CuBr, Cs2CO3, DMF, 90 °C, overnight; (e) HONH2, KOH, MeOH, 60 °C; (f) BH3, THF, reflux, overnight.

a

and p-Cl and -CN substituents can enhance molecular interactions with the rim residues of HDAC6. Different from the SAR of the capping groups that bind to the relatively open rim region, there was little room to modify the phenyl linker, as substituents as small as −F (27) and −Cl (28) resulted in a significant drop in Tub-Ac activity. It is likely that those substituents may have direct steric clashes with residues at the hydrophobic channel, or as in the case of 3-Cl, they may force the capping group to adopt an unfavorable binding pose. In general, reduction of the pyrrolidinone moiety also turned out to be harmful to both HDAC6 inhibition and metabolic stability. For example, pyrrolidine 29 had less HDAC6 inhibition and moderate microsomal clearance (MLM = 53 mL min−1 kg−1) compared with its counterpart 18. Having improved the Tub-Ac potency through capping modifications, we revisited the pyrrolidinone moiety and conducted a “methyl walk” to explore the steric effects on HDAC6 inhibition and HDAC isoform selectivity. Specifically, analogues 30−32 were synthesized from intermediates 52 (Scheme 4) on the basis of the assumption that an α-methyl substituent would have minimal impact on the overall molecular conformation. N-Boc-protected 54 was treated with lithium diisopropylamide (LDA) and then with MeI to give ester 55, which was then treated with trifluoroacetic acid (TFA) to remove the protecting group, and subsequent hydroxylamination gave 30−32. To our delight, α-methylation not only enhanced the in-cell Tub-Ac activity but also the aqueous solubility (30 vs 11 and 31 vs 18). Especially, 31 and 32 demonstrated highly potent Tub-Ac activities with EC50 values in the submicromolar range and only marginal p21 induction at 10 μM (Figure 3). Given the beneficiary impact of α-methylation, we then carried out a chiral synthesis of S enantiomer 33 and its stereoisomer 34 to investigate the effect of stereochemistry on HDAC6 inhibition (Scheme 4). Thus, a copper-catalyzed coupling between (S)-methionine 56 and methyl 4-iodobenzoate gave carboxylic acid 57, which was coupled with 4chloroaniline to afford 58. After methylation of 58, the resulting

sulfonium salt was treated with NaH to give pyrrolidinone 59, which was then converted to 33 with hydroxylamine and KOH. By means of the same procedure, 34 was made from racemic methionine 60 and chiral separation of the pyrrolidinone intermediate by supercritical fluid chromatography (SFC). In our cellular assays, 33 demonstrated 6 times more potent TubAc activity than 34 with an EC50 of 0.30 μM, and neither compound showed significant p21 induction at 3 and 10 μM. The S enantiomer 33 was also about 15 times more potent than 34 in HDAC6 enzyme inhibition assays with an IC50 of 17 nM and an attractive selectivity profile over HDAC1 and other isoforms. In fact, 33 did not affect the enzymatic activity of other Zn2+-dependent HDAC isoforms except HDAC8, with respect to which an IC50 of 0.18 μM was obtained (see the Supporting Information). Finally, the effects of 33 and 34 on HDAC6 inhibition were further confirmed by Western blot study and compared with those of analogues 11, 14, and 18 and SAHA. Thus, NCI-H929 cells were treated with compounds at 1 and 10 μM for 24 h and checked for HDAC6 and Tub-Ac levels by immunoblot (Figure 4). In agreement with their relative potency in HDAC6 inhibition and Tub-Ac assays, these compounds induced Tub-Ac in a concentration-dependent manner, with 33 having the strongest effects. To understand the SAR of the aminopyrrolidinone series, we performed computational docking in an HDAC6 homology model built from PDB entry 1ZZ124 using Glide by Schrödinger. In our docking prediction, 3-aminopyrrolidinone 11 has a good overlay with 9 and 10 in the catalytic domain of HDAC6, and the capping group of 11 fits into the same cavity at the rim region as occupied by the pyridylpyrimidinyl group of 9 (Figure 5A). In comparison with the larger capping group of 9, it is clear that small substituents such as −Cl and −CN can be tolerated at the para position of the terminal phenyl group and contribute to the ligand−HDAC6 interaction. In the binding models of 33 and 34, the 3-methyl group points to a small cavity, but the capping group of 34 takes distinct orientations from that of 33 and is not in close contact with the rim residues of HDAC6, except for F566 (Figure 5B). This E

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Journal of Medicinal Chemistry Table 2. Effects on Tubulin Acetylation and HDAC Inhibition by 16−34a

a EC50 values for tubulin acetylation based on ELISA experiments run in duplicate. Values are in μM with SD below 15%. bIC50 values for enzymatic inhibition of HDAC enzymes. Experiments were run in duplicate. Values are in μM with of SD ± 15%. Assays were performed by Reaction Biology Corporation. cLYSA mean values from duplicate runs, reported in μg/mL with SD ± 10%. dNot determined.

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Scheme 4. Synthesis of α-Methylated Analogues 30−34a

Reagents and conditions: (a) DMAP, Boc2O, CH3CN, rt, 12 h, >80%; (b) LDA, THF, −78 °C for 30 min, then MeI, −78 °C to rt, ∼20%; (c) TFA, DCM, 0 °C, 2 h, >90%; (d) HONH2, KOH, MeOH, 60 °C, 2 h, >50%; (e) methyl 4-iodobenzoate, K2CO3, CuI, DMSO, MW, 150 °C, 90%; (f) 4chloroaniline, HATU, NEt3, DMF, rt, 60%; (g) MeI, CH3CN, rt; (h) NaH, DMF, rt, 70%. a

Figure 4. Western blots of HDAC6 and Tub-Ac in NCI-H929 cells. NCI-H929 cells were treated with SAHA, aminopyrrolidinone analogues 11, 18, 33, 34, and “capless” analogue 14 for 24 h at the indicated concentrations, and their effects on Tub-Ac and HDAC6 were compared with those of the DMSO control.

Figure 5. Docking models of 3-aminopyrrolidinone analogues in complex with the HDAC6 catalytic domain. (A) Overlay of analogue 11 (purple) with aminotetralin analogue 9 (yellow) in the HDAC6 homology model, with key residues in the hydrophobic channel and rim region labeled in yellow. The p-NH moiety is probably involved in hydrogen-bonding interactions with D567. (B) Docking models of 33 (cyan) and 34 (magenta) in the catalytic pocket of HDAC6. For clarity, the HDAC6 binding site is shown as a surface representation. Oxygen, nitrogen, and chlorine atoms are shown in red, blue, and green, respectively.

poor fit probably accounts for the reduced inhibitory activity of 34 against HDAC6. With increased local conformational restriction, the α-methyl group likely contributes to isoform selectivity over other HDACs, as indicated by the HDAC panel data. The DMPK properties of selected aminopyrrolidinone-based HDAC6 inhibitors were evaluated in male ICR mice following intravenous and oral administration. Analogues 11 and 18 showed high plasma clearance (Cl) in mice despite their good microsomal stability, and none of them gave sufficient oral exposure, with oral availabilities (F) below 3% (Table 3). On the other hand, the α-methyl substituent led not only to improved physicochemical properties but also to better DMPK profiles. Despite having a higher clogP value, 33 demonstrated good human and mouse microsomal stabilities (HLM = 3.0 mL min−1 kg−1, MLM = 38 mL min−1 kg−1) as well as significantly

improved plasma clearance compared with 18. In particular, 33 had high oral bioavailability with F = 55% and area under the curve (AUC(0−∞)) of 10500 μg L−1 hr−1, which are significantly better than the oral bioavailability and exposure of 18 by 10 mg/kg (mpk) intravenous and 100 mpk oral adminstrations. It is possible that the methyl group may disrupt the crystal packing of the aminopyrrolidinone-based hydroxamic acids and thus enhance compound permeation through the intestinal G

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Journal of Medicinal Chemistry Table 3. DMPK Profiles of HDAC Inhibitors compd

HLMa (mL min−1 kg−1)

MLMa (mL min−1 kg−1)

Cl (mouse)b (mL min−1 kg−1)

SAHAc 11 18 33

0.1 4.6 8 3.0

33 26 44 38

112 108 156 87

AUC(0−∞)b (μg L−1 hr−1, i.v.) 1486 3850 1065 1900

(10 (25 (10 (10

mpk) mpk) mpk) mpk)

T1/2b (h) 0.4 0.7 1.1 2.1

AUC(0−∞)b (μg L−1 hr−1, p.o.) 619 125 278 10500

(50 mpk) (50 mpk) (100 mpk) (100 mpk)

Cmaxb (μg/L)

Fb (%)

501 34 57 626

8 1 3 55

a

Scaled intrinsic clearance of compounds in human liver microsome (HLM) and mouse liver microsome (MLM). Experiments were run in duplicate. bThe single-dose pharmacokinetics (SDPK) study in male ICR mice was carried out according to the standard procedures. Major parameters, including plasma clearance (Cl), AUC (i.v.), T1/2 (i.v.), AUC (p.o.), Cmax (p.o.) and oral bioavailability (F) are reported. cThe SDPK data for SAHA were taken from ref 25. bohydroxamic Acid (10). This compound was prepared from 3aminoquinoline as described in the Supporting Information. MS: calcd (MH+) 363.1, exp (MH+) 363.2. HRMS: calcd (MH+) 363.1564, exp (MH+) 363.1561. 1H NMR (400 MHz, CD3OD): δ 9.44 (s, 1H), 8.95 (d, J = 8.0 Hz, 1H), 8.50 (d, J = 4.8 Hz, 1H), 8.48 (d, J = 4.8 Hz, 1H), 7.92 (m, 1H), 7.42 (m, 1H), 6.59 (t, J = 5.2 Hz, 3H), 4.61 (m, 1H), 3.60 (m, 1H), 3.30 (m, 1H), 3.18 (m, 1H), 2.98 (m, 1H). 4-[(2-Oxo-1-phenylpyrrolidin-3-yl)amino]benzenecarbohydroxamic Acid (11). To a suspension of aniline (35) (465 mg, 5.0 mmol) and K3PO4 (530 mg, 2.5 mmol) in CH3CN (20 mL) was added 2,4-dibromobutyryl chloride (36) (1.32 g, 5.0 mmol) at 0 °C. The mixture was brought to rt and stirred for 1 h. Then NaOH (1 mL, 50% aqueous solution) was added, and the mixture was stirred overnight. After the mixture was filtered, the solid was washed with CH3CN (10 mL), and the combined filtrate was concentrated. The residue was purified by column chromatography to afford 37 as a white solid (0.78 g, 65%). Then a mixture of 37 (216 mg, 0.9 mmol), methyl 4-aminobenzoate (38) (151 mg, 1.0 mmol), and K2CO3 (128 mg, 1.0 mmol) in 5 mL of DMF was stirred at 120 °C for 10 min in a microwave reactor. After removal of DMF, the crude product was purified by column chromatography to afford 39 as a white solid (64 mg, 23%). Next, to a solution of 39 (50 mg, 0.16 mmol) in MeOH (2 mL) were added hydroxylamine (0.5 mL, 50% aqueous solution) and KOH (10 mg), and the mixture was heated at 60 °C for 3 h. After workup with EtOAc and water, the crude product was purified by preparative HPLC to afford 11 as a white solid (31 mg, 62%). MS: calcd (MH+) 312.1, exp (MH+) 312.1. HRMS: calcd (MH+) 312.1343, exp (MH+) 312.1344. 1H NMR (400 MHz, DMSO-d6): δ 10.82 (s, 1H), 8.71 (s, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.17 (t, J = 7.2 Hz, 2H), 6.71 (d, J = 8.8 Hz, 1H), 6.53 (d, J = 7.2 Hz, 1H), 4.53−4.51 (m, 1H), 3.85−3.82 (m, 2H), 2.64−2.58 (m, 1H), 1.94−1.88 (m, 1H). 4-[Methyl(2-oxo-1-phenylpyrrolidin-3-yl)amino]benzenecarbohydroxamic Acid (12). To a mixture of 39 (155 mg, 0.5 mmol) and MeI (160 μL, 2.57 mmol) in 2 mL of DMF was added NaH (80 mg, 60%, 0.5 mmol) at rt. After 3 h of stirring at 60 °C, the solution was partitioned between EtOAc and water. The organic phase was washed with brine, dried over anhydrous Na2SO4, and concentrated. Flash chromatography gave the ester intermediate, which was treated with hydroxylamine and KOH as described previously. 12 was obtained as a white solid after HPLC purification of the crude product. MS: calcd (MH+) 326.1, exp (MH+) 326.1. HRMS: calcd (MH+) 326.1499, exp (MH+) 326.1502. 1H NMR (400 MHz, CD3OD): δ 7.70 (d, J = 8.8 Hz, 2H), 7.43 (m, 2H), 7.24 (m, 2H), 7.11 (m, 1H), 6.95 (d, J = 9.2 Hz, 2H), 4.64 (m, 1H), 3.50 (m, 1H), 3.31 (m, 1H), 2.98 (s, 3H), 2.60 (m, 1H), 1.98 (m, 1H). 4-(2-Oxo-1-phenylpyrrolidin-3-yl)oxybenzenecarbohydroxamic Acid (13). To a solution of methyl 4-hydroxybenzoate (40) (228 mg, 1.5 mmol) in 5 mL of DMF was added tert-BuOK (168 mg, 1.5 mmol), and the mixture was stirred at rt for 30 min. Then 37 (240 mg, 1.0 mmol) was added to the solution, and the reaction mixture was stirred at 100 °C for 3 h. After removal of DMF, the crude product was purified by flash chromatography to give 41 (81 mg, 26%), which was then treated with hydroxylamine and KOH as described previously. 13 was obtained as a white solid after HPLC purification of the crude product. MS: calcd (MH+) 313.1, exp (MH+) 313.1. HRMS: calcd

membrane. Moreover, 33 showed medium plasma protein binding and had low potential to perpetrate drug−drug interactions, with IC50 values above 50 μM against five major CYP enzymes (see the Supporting Information). In conclusion, 33 is a valuable orally available probe to study the in vivo effects of HDAC6 inhibition in relevant disease models.

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CONCLUSIONS In summary, we have designed and evaluated a novel class of aminopyrrolidinone-based hydroxamic acids as HDAC6 inhibitors. Similar to the aminotetralin analogue 9 disclosed previously, the reported chemical series showed preferential inhibition of HDAC6 over the HDAC1−3 isoforms and demonstrated strong induction of Tub-Ac and minimal effects on p21 levels. Through structural modifications of the chemical scaffold, the α-methyl substituent was found to increase the HDAC6 inhibition and isoform selectivity, presumably by fitting into a previously underexplored cavity in the rim region of HDAC6 on the basis of docking studies. In particular, the S enantiomer 33 demonstrated over 4000-fold selectivity over HDAC1−3 and 10-fold selectivity over HDAC8. Compound 33 also has a suitable DMPK profile, high oral bioavailability, and low risk of CYP-related drug−drug interactions. In view of the increasing understanding of the disease biology of HDAC6, the identification of orally available and highly potent aminopyrrolidinone-based HDAC6 inhibitors warrants further in vitro and in vivo functional studies and the development of novel therapeutic agents.

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EXPERIMENTAL SECTION

Synthetic Chemistry General Comments. All of the starting materials were obtained commercially and were used without further purification. All of the reported yields are for isolated products and are not optimized. All of the intermediates were purified by silica gel chromatography using either a Biotage SP1 system or an ISCO CombiFlash chromatography instrument. All of the final compounds were purified by preparative HPLC on a reversed-phase column using a Waters XBridge OBD Phenyl (30 mm × 100 mm, 5 μm) or OBD RP18 (30 mm × 100 mm, 5 μm) column under acidic conditions (A, 0.1% formic acid in H2O; B, 0.1% formic acid in acetonitrile) or basic conditions (A, 0.01% ammonia in H2O; B, acetonitrile). For SFC chiral separation, the intermediates were separated using a chiral column (Daicel Chiralpak IC, 30 mm × 250 mm, 5 μm) on a Mettler Teledo SFC-Multigram system (solvent system of 95% CO2 and 5% IPA (0.5% TEA in IPA), backpressure of 100 bar, UV detection at 254 nm). Optical rotation was measured using a Rudolph Autopol V automatic polarimeter at a wavelength of 589 nm. LC−MS spectra were obtained using a MicroMass Platform LC (Waters Alliance 2795ZQ2000). NMR spectra were obtained using a Bruker Avance 400 MHz NMR spectrometer. All of the final compounds had purities greater than 95% based upon LC−MS and 1H NMR analyses. Synthetic Procedures for Compounds 10−13 and 15. 3-{[4(3-Pyridyl)pyrimidin-2-yl]amino}-1,2,3,4-tetrahydroquinoline-6-carH

DOI: 10.1021/jm502011f J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Journal of Medicinal Chemistry (MH+) 313.1183, exp (MH+) 313.1182. 1H NMR (CD3OD, 400 MHz): δ 8.53 (s, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 7.44 (m, 2H), 7.26 (m, 1H), 7.18 (d, J = 8.8 Hz, 2H), 5.31 (t, J = 7.96 Hz, 1H), 3.94−4.01 (m, 2H), 2.85 (m, 1H), 2.17−2.29 (m, 1H). 4-[(5-Oxo-1-phenylpyrrolidin-3-yl)amino]benzenecarbohydroxamic Acid (15). A mixture of itaconic acid (42) (2.60 g, 20 mmol) and 35 (1.86 g, 20 mmol) was heated at 180−200 °C for 0.5 h. When the mixture was brought to rt, the resulting product was recrystallized from EtOAc to afford 43 as a white solid (4.1 g, 98%). To a suspension solution of 43 (4.0 g, 19.5 mmol) in 1,2dichloroethane (30 mL) was added dropwise thionyl chloride (4 mL), and the mixture was stirred at 80 °C for 1 h. After 1,2-dichloroethane and thionyl chloride were removed in vacuo, the residue was dissolved in anhydrous THF (10 mL) and added dropwise to a solution of aqueous ammonia (28%, 20 mL) in THF (10 mL). The mixture was stirred at rt for 1 h. After removal of THF, the white solid was collected and dried over anhydrous Na2SO4 to afford 44 (3.4 g, 85%). Data for 44: MS: calcd (MH+) 205.1, exp (MH+) 205.1. 1H NMR (400 MHz, DMSO-d6): δ 8.53 (br s, 2H), 7.64 (d, J = 7.6 Hz, 2H), 7.41 (t, J = 8.1 Hz, 2H), 7.09−7.24 (m, 1H), 4.23 (dd, J1 = 11.1 Hz, J2 = 7.1 Hz, 1H), 3.95−4.11 (m, 1H), 3.84 (dd, J1 = 11.1 Hz, J2 = 2.5 Hz, 1H), 3.02 (dd, J1 = 17.7 Hz, J2 = 8.3 Hz, 1H), 2.59 (dd, J1 = 17.7 Hz, J2 = 3.0 Hz, 1H). Next, to a suspension of 44 (1.3 g, 6.4 mmol) in THF (20 mL) and water (20 mL) was added (diacetoxyiodo)benzene (DIB) (2.1 g, 6.4 mmol), and the mixture was stirred at rt overnight. After removal of THF, the remaining aqueous solution was acidified with HCl (1 N) to pH 2 and washed with EtOAc twice (15 mL). The aqueous phase was separated and concentrated to afford 45 as the HCl salt (1.1 g, 81%). Subsequently, a mixture of 45 (155 mg, 0.73 mmol), methyl 4iodobenzoate (46) (190 mg, 0.73 mmol), Cs2CO3 (714 mg, 2.2 mmol), CuBr (5.3 mg, 0.04 mmol), and (3,5-dioxa-4phosphacyclohepta[2,1-a;3,4-a′]dinaphthalen-4-yl)dimethylamine ((S)-MonoPhos) (28 mg, 0.08 mmol) in DMF (3 mL) was stirred at 100 °C overnight. The mixture was cooled to rt, diluted with EtOAc (30 mL), and washed with water (10 mL). The organic layer was separated, dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography to afford 47 as a white solid (72 mg, 32%). After treatment of 47 with hydroxylamine and KOH as described previously, the crude product was purified by preparative HPLC to afford 15 as a white solid (21 mg). MS: calcd (MH+) 312.1, exp (MH+) 312.1. HRMS: calcd (MH+) 312.1343, exp (MH+) 312.1347. 1H NMR (CD3OD, 400 MHz): δ 7.63−7.60 (m, 4H), 7.40 (t, J = 8.0 Hz, 2H), 7.20 (t, J = 7.6 Hz, 1H), 6.71 (d, J = 8.8 Hz, 2H), 4.39−4.32 (m, 2H), 3.80 (dd, J1 = 9.6 Hz, J2 = 2.8 Hz, 1H), 3.12 (dd, J1 = 17.2 Hz, J2 = 7.2 Hz, 1H), 2.58 (dd, J1 = 17.6 Hz, J2 = 3.2 Hz, 1H). General Synthetic Procedures for Compounds 16−29. To a solution of 49 (4.4 mmol) in CH3CN (20 mL) was added aqueous ammonia solution (10 mL). The mixture was stirred at 40 °C overnight. After removal of CH3CN, the remaining aqueous solution was extracted with DCM (20 mL twice). The organic layer was separated, dried over anhydrous Na2SO4, and concentrated to give 50, which was used in next step directly. A mixture of 50 (1.0 mmol), methyl 4-iodo/bromobenzoate (51) (1.0 mmol), Cs2CO3 (714 mg, 2.2 mmol), CuBr (5.3 mg, 0.04 mmol), and (S)-MonoPhos (28 mg, 0.08 mmol) in DMF (3 mL) was stirred at 100 °C overnight. Then the mixture was diluted with EtOAc (30 mL) and washed with water. The organic layer was separated, dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography to afford 52. To a solution of 52 (0.76 mmol) in MeOH (2 mL) were added hydroxylamine (1 mL, 50% aqueous solution) and KOH (10 mg), and the mixture was stirred at 60 °C for 3 h. After workup of the reaction mixture, the crude product was purified by preparative HPLC. 4-[(1-Benzyl-2-oxopyrrolidin-3-yl)amino]benzenecarbohydroxamic Acid (16). MS: calcd (MH+) 326.1, exp (MH+) 326.1. HRMS: calcd (MH+) 326.1499, exp (MH+) 326.1497. 1 H NMR (CD3OD, 400 MHz): δ 7.59 (d, J = 8.4 Hz, 2H), 7.40−7.30

(m, 5H), 6.76 (d, J = 8.4 Hz, 2H), 4.58−4.49 (m, 2H), 4.38 (t, J = 8.8 Hz, 1H), 3.37−3.33 (m, 2H), 2.61−2.57 (m, 1H), 1.90−1.84 (m, 1H). 4-{[2-Oxo-1-(2-phenylethyl)pyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (17). MS: calcd (MH+) 340.1, exp (MH+) 340.1. HRMS: calcd (MH+) 340.1656, exp (MH+) 340.1659. 1 H NMR (CD3OD, 400 MHz): δ 7.57 (d, J = 8.8 Hz, 2H), 7.34−7.22 (m, 5H), 6.70 (d, J = 8.8 Hz, 2H), 4.20 (d, J = 8.8 Hz, 1H), 3.57 (d, J = 7.6 Hz, 2H), 3.36−3.33 (m, 2H), 2.90 (d, J = 7.2 Hz, 2H), 2.56− 2.52 (m, 1H), 1.85−1.79 (m, 1H). 4-{[1-(4-Chlorophenyl)-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (18). MS: calcd (MH+) 346.1, exp (MH+) 346.1. HRMS: calcd (MH+) 346.0953, exp (MH+) 346.0951. 1 H NMR (DMSO-d6, 400 MHz): δ 10.82 (b, 1H), 8.72 (b, 1H), 7.78 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H), 6.51 (d, J = 7.6 Hz, 1H), 4.55−4.48 (m, 1H), 3.87−3.79 (m, 2H), 2.68−2.62 (m, 1H), 1.95−1.92 (m, 1H). 4-{[1-(3-Chlorophenyl)-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (19). MS: calcd (MH+) 346.1, exp (MH+) 346.1. HRMS: calcd (MH+) 346.0953, exp (MH+) 346.0952. 1 H NMR (CD3OD, 400 MHz): δ 7.91 (t, J = 2.0 Hz, 1H), 7.62−7.57 (m, 3H), 7.40 (t, J = 8.0 Hz, 1H), 7.23−7.21 (m, 1H), 6.79 (d, J = 8.8 Hz, 1H), 4.55−4.48 (m, 1H), 3.95−3.90 (m, 2H), 2.79−2.74 (m, 1H), 2.07−2.02 (m, 1H). 4-[(1-{3-[2-(Dimethylamino)ethoxy]phenyl}-2-oxopyrrolidin-3yl)amino]benzenecarbohydroxamic Acid (20). MS: calcd (MH+) 399.2, exp (MH+) 399.2. 1H NMR (CD3OD, 400 MHz): δ 7.61 (d, J = 8.0 Hz, 2H), 7.49 (s, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 2H), 4.52 (t, J = 8.4 Hz, 1H), 3.96 (t, J = 5.6 Hz, 2H), 3.94−3.90 (m, 2H), 2.98−2.95 (m, 2H), 2.77−2.73 (m, 1H), 2.23 (s, 6H), 2.07−2.02 (m, 1H). 4-{[1-(2-Chlorophenyl)-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (21). MS: calcd (MH+) 346.1, exp (MH+) 346.1. HRMS: calcd (MH+) 346.0953, exp (MH+) 346.0952. 1 H NMR (DMSO-d6, 400 MHz): δ 10.82 (b, 1H), 7.62−7.60 (m, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.48−7.39 (m, 3H), 6.73 (d, J = 8.8 Hz, 2H), 6.51 (b, 1H), 4.55−4.48 (m, 1H), 3.87−3.66 (m, 2H), 2.68−2.64 (m, 1H), 2.07−2.01 (m, 1H). 4-({2-Oxo-1-[4-(trifluoromethyl)phenyl]pyrrolidin-3-yl}amino)benzenecarbohydroxamic Acid (22). MS: calcd (MH+) 380.1, exp (MH+) 380.1. HRMS: calcd (MH+) 380.1217, exp (MH+) 380.1217. 1 H NMR (CD3OD, 400 MHz): δ 7.95 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 4.55−4.48 (m, 1H), 3.95−3.90 (m, 2H), 2.79−2.74 (m, 1H), 2.07−2.02 (m, 1H). 4-{[1-(3,4-Dichlorophenyl)-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (23). MS: calcd (MH+) 380.1, exp (MH+) 380.2. HRMS: calcd (MH+) 380.0563, exp (MH+) 380.0561. 1 H NMR (CD3OD, 400 MHz): δ 8.08 (d, J = 2.4 Hz, 2H), 7.65−7.55 (m, 2H), 6.79 (d, J = 8.8 Hz, 2H), 4.55−4.52 (m, 1H), 3.95−3.90 (m, 2H), 2.79−2.74 (m, 1H), 2.07−2.02 (m, 1H). 4-{[1-(4-Methylsulfonylphenyl)-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (24). MS: calcd (MH+) 390.1, exp (MH+) 390.2. HRMS: calcd (MH+) 390.1118, exp (MH+) 390.1113. 1 H NMR (CD3OD, 400 MHz): δ 8.74 (d, J = 9.2 Hz, 2H), 8.69 (d, J = 9.2 Hz, 2H), 8.32 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 4.68 (m, 2H), 4.04 (m, 1H), 3.46 (m, 1H), 2.79 (m, 1H). 4-{[1-(4-Cyanophenyl)-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (25). MS: calcd (MH+) 337.1, exp (MH+) 337.1. HRMS: calcd (MH+) 346.0953, exp (MH+) 346.0951. 1 H NMR (CD3OD, 400 MHz): δ 7.96 (d, J = 9.2 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 4.52 (t, J = 8.4 Hz, 1H), 3.87−4.00 (m, 2H), 2.76 (m, 1H), 2.06 (m, 1H). 4-{[1-(2-Naphthyl)-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (26). MS: calcd (MH+) 362.1, exp (MH+) 362.1. HRMS: calcd (MH+) 337.1295, exp (MH+) 337.1295. 1 H NMR (CD3OD, 400 MHz): δ 8.09 (s, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.96 (m, 3H), 7.67 (d, J = 8.4 Hz, 2H), 7.55 (m, 2H), 6.87 (d, J = 8.4 Hz, 2H), 4.52 (t, J = 8.4 Hz, 1H), 4.00−3.87 (m, 2H), 2.76 (m, 1H), 2.06 (m, 1H). 4-{[1-(4-Chlorophenyl)-2-oxopyrrolidin-3-yl]amino}-2-fluorobenzenecarbohydroxamic Acid (27). MS: calcd (MH+) 364.1, exp I

DOI: 10.1021/jm502011f J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Journal of Medicinal Chemistry (MH+) 364.1. 1H NMR (CD3OD, 400 MHz): δ 7.79−7.67 (m, 2H), 7.64−7.55 (m, 1H), 7.48−7.33 (m, 2H), 6.70−6.61 (m, 1H), 6.59− 6.49 (m, 1H), 4.59−4.46 (m, 1H), 4.00−3.85 (m, 2H), 2.82−2.69 (m, 1H), 2.13−1.98 (m, 1H). 3-Chloro-4-{[1-(4-chlorophenyl)-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (28). MS: calcd (MH+) 380.0, exp (MH+) 380.1. HRMS: calcd (MH+) 380.0563, exp (MH+) 380.0562. 1 H NMR (CD3OD, 400 MHz): δ 7.78−7.68 (m, 3H), 7.63 (dd, J1 = 8.6 Hz, J2 = 2.0 Hz, 1H), 7.49−7.36 (m, 2H), 6.95 (d, J = 8.6 Hz, 1H), 4.60 (dd, J1 = 10.6 Hz, J2 = 8.3 Hz, 1H), 4.07−3.83 (m, 2H), 2.95− 2.76 (m, 1H), 2.14 (s, 1H). 4-{[1-(4-Chlorophenyl)pyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (29). Borane−THF complex (2 mL) was added to a solution of methyl 4-{[1-(4-chlorophenyl)-2oxopyrrolidin-3-yl]amino}benzoate (52) (172 mg) in THF (20 mL), and the mixture was refluxed overnight. After removal of THF, the residue was dissolved in 5 mL of MeOH, and to this solution were added hydroxylamine (1 mL, 50% aqueous solution) and KOH (20 mg). The reaction mixture was stirred at 60 °C for 3 h. The product was purified by preparative HPLC to afford 29 as a white solid (52 mg, 31%). MS: calcd (MH+) 332.1, exp (MH+) 332.1. HRMS: calcd (MH+) 332.1160, exp (MH+) 332.1148. 1H NMR (CD3OD, 400 MHz): δ 7.60 (d, J = 9.6 Hz, 2H), 7.14 (d, J = 10 Hz, 2H), 7.05 (d, J = 9.6 Hz, 2H), 6.56 (d, J = 10 Hz, 2H), 4.26 (m, 1H), 3.66 (m, 1H), 3.48 (m, 1H), 3.78 (m, 1H), 3.18 (m, 1H), 2.39 (m, 1H), 2.06 (m, 1H). General Synthetic Procedures for Compounds 30−32. Ester 52 (1 mmol) was dissolved in CH3CN (10 mL). To the solution were added (Boc)2O (2.0 mmol) and DMAP (0.20 mmol), and the mixture was stirred at rt for 12 h. After removal of the solvent, the residual oil was purified by flash chromatography to afford 54. To a solution of 54 (0.8 mmol) in dry THF (3 mL) was added LDA (1.8 M in THF, 1.0 mmol) at −78 °C, and the mixture was stirred for 30 min. Then MeI (2 mmol) was added dropwise, and the reaction mixture was stirred overnight with the temperature raised to rt spontaneously. The reaction was quenched with saturated NH4Cl aqueous solution, and the resulting mixture was extracted with EtOAc, dried over Mg2SO4, and concentrated. After purification by column chromatography, the product 55 was dissolved in a mixed solvent of CH2Cl2 and TFA (4 mL, 3:1) and stirred at 0 °C for 2 h. After removal of the solvent, the residue was diluted with CH2Cl2, and the organic phase was washed with satd. NaHCO3, dried over Mg2SO4, and concentrated. The residue was dissolved in a mixture of MeOH (5 mL), hydroxylamine (1 mL, 50% aqueous solution), and KOH (20 mg), and the reaction mixture was stirred at 60 °C for 3 h. The product was purified by preparative HPLC to afford 30−32 as a white powder. 4-[(3-Methyl-2-oxo-1-phenylpyrrolidin-3-yl)amino]benzenecarbohydroxamic Acid (30). MS: calcd (MH+) 326.1, exp (MH+) 326.1. HRMS: calcd (MH+) 326.1499, exp (MH+) 326.1496. 1 H NMR (CD3OD, 400 MHz): δ 7.70 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.45 (t, J = 8.0 Hz, 2H), 7.25 (t, J = 7.6 Hz, 1H), 6.66 (d, J = 8.8 Hz, 2H), 4.03−3.95 (m, 2H), 2.72 (dt, J1 = 13.2 Hz, J2 = 9.6 Hz, 1H), 2.19 (qd, J1 = 6.4, J2 = 1.6 Hz, 1H), 1.57 (s, 3H). 4-{[1-(4-Chlorophenyl)-3-methyl-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (31). MS: calcd (MH+) 360.1, exp (MH+) 360.1. HRMS: calcd (MH+) 360.1109, exp (MH+) 360.1109. 1 H NMR (CD3OD, 400 MHz): δ 7.74 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 4.00− 3.96 (m, 2H), 2.72 (dt, J1 = 12.8 Hz, J2 = 9.6 Hz, 1H), 2.22−2.16 (m, 1H), 1.56 (s, 3H). 4-{[3-Methyl-2-oxo-1-(6-quinolyl)pyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (32). MS: calcd (MH+) 377.1, exp (MH+) 377.1. HRMS: calcd (MH+) 377.1608, exp (MH+) 377.1607. 1 H NMR (CD3OD, 400 MHz): δ 9.06 (dd, J1 = 4.9 Hz, J2 = 1.4 Hz, 1H), 8.95 (d, J = 8.3 Hz, 1H), 8.70 (dd, J1 = 9.2 Hz, J2 = 2.4 Hz, 1H), 8.46 (d, J = 2.3 Hz, 1H), 8.24 (d, J = 9.3 Hz, 1H), 7.94 (dd, J1 = 8.6 Hz, J2 = 5.1 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H), 4.31−4.11 (m, 2H), 2.88−2.76 (m, 1H), 2.31−2.26 (m, 1H), 1.62 (s, 3H).

Synthetic Procedures for Compounds 33 and 34. 4-{[(3S)-1(4-Chlorophenyl)-3-methyl-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (33). A mixture of methyl 4iodobenzoate (2.62 g, 10 mmol), (2S)-2-amino-2-methyl-4-methylsulfanylbutanoic acid (56) (2.44 g, 15 mmol), CuI (95 mg, 0.5 mmol), and K2CO3 (2.07 g, 15 mmol) in 10 mL of DMSO was stirred at 150 °C in a microwave reactor for 10 min. After cooling down, the DMSO solution was partitioned between EtOAc and aqueous HCl (0.1 N). The organic phase was washed with water and brine, dried, and concentrated. Flash chromatography gave 2.68 g of 57 (90%). MS: calcd (MH+) 298.1, exp (MH+) 298.1. 1H NMR (CD3OD, 400 MHz): δ 7.76 (d, J = 8.0 Hz, 2H), 6.63 (d, J = 8.0 Hz, 2H), 3.83 (s, 3H), 2.56−2.18 (m, 4H), 2.03 (s, 3H), 1.57 (s, 3H). To a solution of 57 (2.5 g, 8.4 mmol), 4-chloroaniline (1.27 g, 10 mmol), and triethylamine (3 mL) in 20 mL of DMF was added 2-(7aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (3.82 g, 10 mmol). The solution was stirred at rt for 6 h, and then water was added. The organic phase was washed with aqueous HCl (0.1 N), water, and brine, dried over anhydrous Na2SO4, and concentrated. Flash chromatography gave 2.06 g of 58 (60%). MS: calcd (MH+) 407.0, exp (MH+) 407.0. 1H NMR (CD3OD, 400 MHz): δ 7.85−7.73 (m, 2H), 7.53−7.43 (m, 2H), 7.35−7.23 (m, 2H), 6.71− 6.40 (m, 2H), 3.83 (s, 3H), 2.57 (s, 1H), 2.49−2.32 (m, 2H), 2.30− 2.16 (m, 1H), 2.01 (s, 3H), 1.58 (s, 3H). A mixture of 58 (2.03 g, 5 mmol) and MeI (1.2 mL, 20 mmol) in 20 mL of CH3CN was stirred overnight at rt and then concentrated in vacuo. The residue was then dissolved in 15 mL of anhydrous DMF, and NaH (240 mg, 60%, 6 mmol) was added to the solution. The mixture was stirred at rt for 6 h and then poured into ice water, and the aqueous phase was extracted with EtOAc. The organic phases were combined, washed with brine, dried over anhydrous Na2SO4, and concentrated. Flash chromatography afforded pyrrolidinone 59 (1.8 g, 70%). MS: calcd (MH+), 359.1 exp (MH+) 359.1. 1H NMR (CDCl3, 400 MHz): δ 7.98−7.82 (m, 2H), 7.76−7.60 (m, 2H), 7.48−7.33 (m, 2H), 6.72−6.58 (m, 2H), 3.98−3.83 (m, 5H), 2.71−2.47 (m, 2H), 1.57 (s, 3H). 59 (1.44 g, 4 mmol) was treated with hydroxylamine (3 mL, 50% aqueous solution) and KOH (50 mg) as described above, and HPLC purification afforded 33 (1.01 g, 70%). Optical rotation: [α]20 D = 99.3 (0.9 mg/mL, MeOH). MS: calcd (MH+) 360.1, exp (MH+) 360.1. HRMS: calcd (MH+) 360.1109, exp (MH+) 360.1107. 1H NMR (CD3OD, 400 MHz): δ 7.76−7.71 (m, 2H), 7.56 (d, J = 8.8 Hz, 2H), 7.46−7.39 (m, 2H), 6.65 (d, J = 8.8 Hz, 2H), 4.03−3.91 (m, 2H), 2.79−2.65 (m, 1H), 2.35−2.12 (m, 1H), 1.56 (s, 3H). 4-{[(3R)-1-(4-Chlorophenyl)-3-methyl-2-oxopyrrolidin-3-yl]amino}benzenecarbohydroxamic Acid (34). By means of the procedures for the synthesis of 33, analogue 34 was synthesized from racemic 2-amino-2-methyl-4-methylsulfanylbutanoic acid (60) and SFC chiral separation of the pyrrolidinone intermediate. Optical + rotation: [α]20 D = −101.5 (1.3 mg/mL, MeOH). MS: calcd (MH ) 360.1, exp (MH+) 360.1. HRMS: calcd (MH+) 360.1109, exp (MH+) 360.1110. 1H NMR (CD3OD, 400 MHz): δ 7.74 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 4.00−3.96 (m, 2H), 2.72 (dt, J1 = 12.8, J2 = 9.6 Hz, 1H), 2.22− 2.16 (m, 1H), 1.56 (s, 3H). Molecular Modeling. Homology modeling and molecular dynamics simulations were conducted in the Molecular Operating Environment (MOE), and geometry optimizations of compounds and molecular docking were finished by MacroModel and Schrödinger Glide, respectively. The HDAC6 homology model was constructed using PDB entry 1ZZ1 as a template, and the sequence corresponding to HDAC6b was aligned with that of HDAH in the MOE-Align module. The final model was taken as the Cartesian average of all of the intermediate models and was further refined by molecular dynamics simulations and energy minimizations, during which only the side chains of all of the residues were allowed to move. The molecular dynamics simulations were set to 5 ps of heating to 300 K, 10 ps of equilibrium at 300 K, and 5 ps of cooling to 0 K. The OPLS_2005 force field of MacroModel was used to optimize the initial geometries of the compounds with a final root-mean-square J

DOI: 10.1021/jm502011f J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry (rms) gradient of 0.01 kcal mol−1 Å−1. The default values of the optimization parameters and thresholds were kept. All of the torsion angles of the compound were released to freely rotate, and 10000 docking runs were adopted during the XP docking with the NE2 atom of His residues and Zn2+ as docking constraints. Docking poses were clustered on the basis of heavy-atom rms deviation values (0.5 Å), and the top-ranked poses of each compound were selected and viewed graphically within Schrödinger Maestro and finally displayed in PyMol. Microsomal Stability Assay. Each incubated mixture contained 0.5 mg/mL liver microsome (human or mouse), 100 mM potassium phosphate buffer (pH 7.4), 10 mM NADPH, and 1 μM test compound in a total volume of 400 μL. After prewarming at 37 °C for 10 min, the NADPH was added to initiate the reaction. The reaction was terminated after 0, 3, 6, 9, 15, or 30 min by adding 150 μL of 100 ng/mL tolbutamide (internal standard) in ice-cold methanol into 300 μL of incubation mixture. The sample was then centrifuged at 4000 rpm for 10 min at 4 °C. The supernatant was then analyzed by LC− MS/MS. LYSA Solubility Assay. A 150 μL aliquot of 10 mM DMSO stock solution of the compound was prepared and divided into two portions. For one portion, the DMSO solution was evaporated to dryness at 35 °C in a centrifugal vacuum evaporator from Genevac Technologies, and the residue was redissolved in 50 mM phosphate buffer (pH 6.5). The mixture was stirred and shaken for 1−2 h. The solution was allowed to stand overnight and then filtered before HPLC analysis. The other portion was used to prepare a calibration curve by dilution of the DMSO stock solution using the same PBS buffer mentioned above to obtain a series of solutions with concentrations in the range of 50−500 μM. Pharmacokinetic Analysis. All of the compounds were evaluated in male ICR mice (18−25 g; three mice in each group were used for blood collection at each time point). Compounds were dissolved in 0.1 M PBS containing 5% DMSO and 40% PEG400 to yield a final concentration of 2 mg/mL for intravenous administration (at 10 or 25 mpk) and in a water solution containing 0.445% microcrystalline cellulose, 0.055% carboxymethyl cellulose sodium, and 1.6% lactose to yield a suspension for oral gavage at 50 or 100 mpk. In each species, blood samples (150 μL) were collected at eight time points (p.o.) or nine time points (i.v.) into sodium heparin centrifuge tubes, and plasma samples were then isolated by centrifugation and stored at −20 °C prior to analysis. Plasma concentrations were determined by LC− MS/MS, and the data were analyzed by noncompartmental methods using WinNonlin Pro (Pharsight Corp., Mountain View, CA). All of the animal studies were approved and regularly reviewed by the Institutional Animal Care and Use Committee (IACUC) of Roche Pharma Research and Early Development (pRED) China. Biology General Comments. Brief descriptions of the biological protocols used to generate data in this study can be found below. All growth media included 100 units/mL penicillin and 100 μg/mL streptomycin, and all cell lines were grown in 5% CO2 at 37 °C. NCIH929, A549, and HeLa cells were obtained from ATCC. Mammalian protein extraction reagent and tissue protein extraction reagent were obtained from Thermo Scientific. EDTA-free protease inhibitors were obtained from Roche Diagnostics. Monoclonal anti-p21 antibody and antiacetylated histone H3 (Ac 1−20) antibodies were purchased from Calbiochem. Anti-HDAC6 was obtained from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated monoclonal anti-GAPDH, peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG were purchased from KangChen Biotech. The enhanced chemiluminescence (ECL) detection kit was obtained from Thermo Scientific. Tubulin Acetylation Cytoblot Assay. Tubulin acetylation (TubAc) was detected by the antiacetylated tubulin antibody (Sigma) and HRP-conjugated secondary antibody (KangChen Bio-tech). A549 cells were seeded into assay plates (Corning 3912) at a concentration of 1 × 105 cells/mL and incubated for 16−18 h at 37 °C in the presence of 5% CO2. Then 20 μL of diluted compound solution was transferred to the cell culture plate and incubated for 17−18 h. After medium removal and fixation by formaldehyde (3.7% paraformaldehyde in TBS), the cells in the plates were treated with 180 μL of −20 °C

MeOH and incubated for 5 min at rt. The cell lysis was incubated with 75 μL of primary antiacetylated tubulin antibody and secondary HRPconjugated antibody solution (1:750 antiacetylated tubulin, 1:750 HRP-conjugated anti-mouse IgG in antibody dilution buffer not containing sodium azide) for 4 h at 4 °C. Then 75 μL of ECL reagent was added into the wells, and the luminescence from each well was immediately quantified by the plate reader. On the basis of the luminescence readings, the EC50 values against Tub-Ac were calculated by plotting the curves with XLfit 4.0 software. p21 Reporter Gene Assay. Compounds were tested for their ability to induce p21 gene expression using a reporter gene assay involving HeLa cells transfected with a p21 promoter−luciferase construct. The p21 promoter has the Sp1/Sp3 binding site for HDAC but not the upstream p53 binding site. Briefly, the day before transfection, HeLa cells were seeded at 11000 cells/well in a 96-well culture plate and incubated at 37 °C in 5% CO2 overnight. For transfection, the medium was removed and replaced with 100 μL/well transfection medium prepared by the following procedure: 5 μL of serum-free DMEM, 0.15 μL of Fugene 6 reagent, 40 ng of p21-luc, and 10 ng of GFP were mixed and incubated at room temperature for 30 min. Then 98 μL of DMEM (with 10% FBS, 1% penicillin, and streptomycin) was added to the DNA/Fugene 6 reagent complex and mixed gently. After incubation of the cells for 24 h at 37 °C under 5% CO2, fresh medium and test compounds were added to the wells and incubated for 15 h. Cells were lysed by adding 80 μL of culture lysis reagent (Promega) to each well. An amount of 50 μL of each lysate was taken for GFP detection using an excitation wavelength of 486 nm and detection at 527 nm. Then 100 μL of luciferase assay reagent (Promega) was added to 20 μL of cell lysate for luminometer reading. The relative gene level of p21 induced by individual compound was compared to that induced by 61 at 1, 3, and 10 μM concentrations. Western Blotting. NCI-H929 cells (1 × 106) were seeded overnight and then incubated with indicated concentrations of compounds for 24 h. Cell extract was prepared by lysing cultured cells with a mammalian protein extraction reagent supplemented with EDTA-free protease inhibitor for 15 min. Supernatants were collected following centrifugation of lysed cells at 15000g for 10 min at 4 °C. To analyze the cell lysate, 30 μg of total protein per sample was resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes with immobilized proteins were probed with antibodies for HDAC6 and Tub-Ac. The reactive protein bands were visualized using an ECL detection system.

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ASSOCIATED CONTENT

* Supporting Information S

Detailed experimental procedures for the synthesis and characterization of 10, X-ray structure of intermediate XXV, selected 1H NMR spectra, experimental conditions for enzymatic inhibition, HDAC panel profiling of 33 and 34 against 11 Zn2+-dependent HDACs, CYP inhibition, and plasma protein binding and permeability tests of the aminopyrrolidinone series. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 21 38954910 extension 3350. Fax: +86 21 50790291. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Wenzhi Cheng, Qinshan Gao, and Peilan Ding for their analytical assistance and purification of final compounds. We also thank Yuxia Zhang, Yi Zhang, Hongxia K

DOI: 10.1021/jm502011f J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry

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Qiu, and Xiaoyue Wu for carrying out the physicochemical and metabolic stability studies.

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ABBREVIATIONS USED DIB, (diacetoxyiodo)benzene; HDAC, histone deacetylase; HLM, human liver microsomal test; LYSA, lyophilized solubility assay; MLM, mouse liver microsomal test; SAHA, suberoylanilide hydroxamic acid; SAR, structure−activity relationship; SFC, supercritical fluid chromatography

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