Novel N-hydroxybenzamides incorporating 2-oxoindoline with unexpected potent histone deacetylase inhibitory effects and antitumor cytotoxicity

Accepted Manuscript Novel N-Hydroxybenzamides Incorporating 2-Oxoindoline with Unexpected Potent Histone Deacetylase Inhibitory Effects and Antitumor Cytotoxicity Tran Thi Lan Huong, Do Thi Mai Dung, Nguyen Van Huan, Le Van Cuong, Pham The Hai, Le Thi Thu Huong, Jisung Kim, Yong Guk Kim, Sang-Bae Han, Nguyen-Hai Nam PII: DOI: Reference:

S0045-2068(16)30296-6 http://dx.doi.org/10.1016/j.bioorg.2017.02.002 YBIOO 2012

To appear in:

Bioorganic Chemistry

Received Date: Accepted Date:

5 December 2016 6 February 2017

Please cite this article as: T.T. Lan Huong, D.T. Mai Dung, N. Van Huan, L. Van Cuong, P. The Hai, L.T. Thu Huong, J. Kim, Y. Guk Kim, S-B. Han, N-H. Nam, Novel N-Hydroxybenzamides Incorporating 2-Oxoindoline with Unexpected Potent Histone Deacetylase Inhibitory Effects and Antitumor Cytotoxicity, Bioorganic Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bioorg.2017.02.002

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Novel N-Hydroxybenzamides Incorporating 2-Oxoindoline with Unexpected Potent Histone Deacetylase Inhibitory Effects and Antitumor Cytotoxicity Tran Thi Lan Huong,a Do Thi Mai Dung,a Nguyen Van Huan,a Le Van Cuong,a Pham The Hai,a Le Thi Thu Huong,b Jisung Kim, c Yong Guk Kim, c Sang-Bae Han, *c Nguyen-Hai Nam, *a a

Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam School of Medicine and Pharmacy, Hanoi National University, Hanoi, Vietnam c College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 28160, Korea b

Abstract:

In our search for novel small molecules targeting histone deacetylases, we have designed and synthesized two series of novel N-hydroxybenzamides incorporating 2-oxoindolines (4a-g, 6a-g). Biological evaluation showed that these benzamides potently inhibited HDAC2 with IC50 values in sub-micromolar range. In three human cancer cell lines the synthesized compounds were up to 4-fold more cytotoxic than SAHA. Docking experiments indicated that the compounds tightly bound to HDAC2 at the active binding site with binding affinities much higher than that of SAHA. Our present results demonstrate that these novel and simple N-hydroxybenzamides are potential for further development as anticancer agents and further investigation of similarly simple N-hydroxybenzamides should be warranted to obtain more potent HDAC inhibitors.

Keywords: Histone deacetylase (HDAC) inhibitors; N-hydroxybenzamides; hydroxamic acids; 2-oxoindoline; triazole; Click chemistry. *Corresponding authors: Tel.: [email protected] (S.B. Han).

+84-4-39330531;

Fax:

+84-4-39332332;

Emails:

[email protected]

(N.H.

Nam);

1. Introduction Histone deacetylases (HDAC) constitute a group of enzymes which catalyze removal of the acetyl groups from lysine residues in the tails of histone proteins [1-3]. Currently, 18 different isoforms of HDACs have been identified in eukaryotes [4]. Based on their relative sequence similarity, these isoforms are categorized into four classes. Class I of HDACs has four members which include HDAC1, 2, 3 and HDAC8. Class II of HDACs comprises of six members including HDAC4, 5, 6, 7, 9 and HDAC10. The isoforms in these two classes have been comprehensively investigated [3]. HDACs of class III, which are also known as Sirtuins, has seven members (Sirt1-7). The sirtuins are NAD+-dependent enzymes. Last, class IV of HDACs has only one member (HDAC11). This isoform is known to have properties of both class II and class I HDAC2 [3]. Different HDAc isoforms, especially of classes I and II, have been demonstrated to be involved in a number of cell-related processes. For examples, HDAC 1, 2, 3 and 8 promote cellular proliferation, meanwhile HDAC 1-4, 5 and 8 prevent cellular apoptosis and differentiation. Several other isoforms, including HDAC 4, 6, 7 and 10 have been shown to promote angiogenesis and cell migration, two processes important for cancer cell metastasis [4,5]. Supression of respective HDAC isoforms has been shown to result in a number of sequential events related to differentiation, apoptosis and cell cycle arrest in many types of tumor cells [4,5]. Furthermore, the selective effects of HDAC inhibition on the growth of tumor cells have been clearly demonstrated not only in vitro but also in a number of in vivo preclinical models and clinical settings [6,7]. Inhibition of HDACs has, therefore, become a very interesting approach in cancer treatment nowadays [8]. As a result, numerous HDAC inhibitors have been reported by medicinal chemists recently in the past years. These inhibitors range from short-chain fatty acids (like butyrate, phenylbutyrate or valproic acid) to diverse types of hydroxamic acids, or benzamides [9-15]. Among these, vorinostat (Zolinza®) (also known as SAHA or suberoylanilide hydroxamic acid) (Figure 1) was the first HDAC inhibitor approved by the U.S. FDA in October 2006 for the treatment of cutaneous T cell lymphoma (CTCL). In 2009, the second HDAC inhibitor, romidepsin (trade name Istodax®) was also approved by the U.S. FDA for the same indication. More recently, panobinostat (LBH-589, trade name Farydak®) was licensed by the US FDA in Feb 2015 for the treatment of multiple myeloma [16]. Also in 2015 chidamide (Epidaza®) was approved by the Chinese FDA for relapsed or refractory peripheral T cell lymphoma

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[17]. In addition, numerous other HDAC inhibitors such as PXD-01 (Belinostat), MS-27-527 (Entinostat) (Figure 1) are currently under clinical trials at different phases (Figure 1). In our research program to develop novel hydroxamic acids as potential inhibitors of HDACs and anticancer agents, we have designed, synthesized and evaluated several series of heterocyclic analogues of SAHA, which incorporated benzothiazole, 5-aryl-1,3,4-thiadiazole or 2-oxoindoline systems, with very potent HDAC inhibitory activity as well as cytotoxicity (Figure 2) [18-21]. Some representative compounds from these series demonstrated very potent antitumor activity when evaluated in PC-3 prostate cancer cells xenografted mice model [19]. Inspired by these results, we expanded our design to the new series of N-hydroxybenzamide type of compounds. The current paper reports the results we obtained from the synthesis, biological evaluation and representative docking study on these N-hydroxybenzamides. O CH3

O

H N O SAHA (vorinostat)

O

OH

N H

O

CH3 H3C

NH

O

O S

S CH3 O

NHOH

H N

O H3C

Romidepsin

O

HN

HN

LBH-589 (panobinostat) N H

CH3 O H N

O

NH2

H N

N

S

NHOH

Chidamide

O

O

O PXD-101 (belinostat)

N

N H

O NH2

H N

F

MS-27-275 (entinostat) O

Figure 1. Structures of some HDAC inhibitors O

O

O

N

N

R

N H

n (n = 1, 2, 4)

S

R = H, 6-CH3, 6-OCH3, 6-OC2H5, 6-SO2CH3, 6-NO2, 6-Cl, 6-CF3, 6-NO2

NHOH

O

N

N NHOH 4 H S R = H, 2-Cl, 3-Cl, 4-Cl, 4-F, 4-Br, 2-NO2, 4-NO2, 2,6-Cl2, 4-CH3, 4-OCH3, 4-N(CH3)2, 3,4-CH2OCH22,3,4-(OCH3)3, 3,4,5-(OCH3)3

R

R'ON O

X

X O

O

O

R

R

N

4

N

NHOH

NHOH 4 X = O, S R = H, 5-F, 5-Cl, 5- Br, 5-NO2, 5-CH3, 7-Cl

R' = H, CH3 R = H, 5-F, 5-Cl, 5- Br, 5-NO2, 5-CH3, 7-Cl

Figure 2. Structures of some benzothiazole-, 5-substitutedphenyl-1,3,4-thiadiazole-, and 2-oxoindoline-based hydroxamic acids

2. Results and Discussion 2.1. Chemistry The synthesis of N-hydroxybenzamides 4a-g was illustrated in scheme 1. In the first step, isatin or its 5- or 7substituted derivatives were reacted in a molar equivalence with propargyl bromide in dimethyl formamide under alkaline conditions (K2CO3) to furnish the propargyl intermediates 2a-g as brown solids in almost quantitative yields. Next, Click reaction between the intermediates 2a-g and methyl 4-azidomethylbenzoate catalyzed by CuI -2-

in acetonitrile solvent gave the esters 3a-g in good yields. Finally, the N-hydroxybenzamides 4a-g were obtained by a nucleophilic acyl substitution between 3a-g and hydroxylamine hydrochloride under alkaline conditions in a mixture of tetrahydrofurane and methanol as a solvent system. This reaction was generally smooth. The target compounds 4a-g were obtained in moderate overall yields. The N-hydroxybenzamides 6a-h were synthesized using a two-step pathway, as described in scheme 2. In the first step, isatin or its 5- or 7-substituted derivatives were also reacted in a molar equivalence with methyl 4bromomethylbenzoate in dimethyl formamide under alkaline conditions (K2CO3) with a catalytic amount of KI to give the ester intermediates 5a-h as brown oils in almost quantitative yields. The ester intermediates 5a-h were further subjected to a reaction with hydroxylamine hydrochloride under alkaline conditions in a mixture of tetrahydrofurane and methanol, similar to that applied for the synthesis of 4a-g, to obtain the target hydroxamic acids 6a-h in moderate to good yields. The structures of the final products were determined straightforward by using spectroscopic methods, including IR, MS, 1H NMR and 13C NMR. It was found that hydroxylamine reacted at both the ester and the 3-oxo groups at position 3 on the indoline moiety of the esters 3a-g and 5a-h, resulting in 3-hydroxyimino-2-oxoindoline Nhydroxybenzamides 4a-g and 6a-h. This was evidenced from the mass spectral data as well as NMR spectroscopid data. In the 13C NMR spectra of compounds 4a-g and 6a-h, it was not observed the peaks assignable for the 3-oxo carbon on the indoline ring. If the 3-oxo group on the indoline ring was still existed, the corresponding 3-oxo carbon should appear around δ 184.00-188.00 ppm in the 13C NMR spectra [21], meanwhile in the compounds 4 and 6, only peaks in a range of 162.00-168.00 ppm, which were attributable for amide and oxime moieties, were observed. In the 1H NMR spectra of compounds 4a-g and 6a-h, the downfield shifts of H-4’ (δ 8.04-7.75 ppm) were clearly observed. The downfield shift of H-4 indicated the presence of the hydroxyimino group at position 3 on the indoline ring [21]. In addition, the 1H NMR spectra of compounds 4a-g and 6a-h measured in DMSO-d6 generally showed three broad singlets around δ 13.82-13.50, 11.18-11.15 and 9.02-9.00 ppm, which were attributable to -OH (of the 3-hydroxyimino moiety on the indoline ring) and –NH, -OH (of the hydroxamic acid moiety), respectively. All these evidences confirmed the presence of the 3-hydroxyimino group on the indoline moiety. The concurrent reaction of hydroxylamine at the 3-oxo groups on the indoline ring and the ester group has been observed under the same conditions previously [21]. The condensation reaction of hydroxylamine with the 3-oxo groups on the indoline ring proceeded so readily even when the molar equivalence of hydroxylamine to compounds 3a-g or 5a-h was used. It was found that when hydroxylamine.HCl was used at 1:1 molar equivalent to the corresponding ester intermediate, a mixture of many products together with the unreacted starting materials (the ester) were observed, making it difficult to isolate the target products 4a-g and 6a-h. O

O

O

O Propargyl bromid

R

NH

N

K2CO3, DMF

R

N

CuI, AcCN

2

1

O

OCH3 N3

R

OH 2

3'

R

NaOH, MeOH/THF

5'

2'

O

4'

N

1'

N N 8 N 9

10 6'

3 4 7

1

OCH3

N N N

3

O

N H2N.OH.HCl

O

O

O

NHOH 6

5

4

7'

Scheme 1. Synthesis of novel N-hydroxybenzamides incorporating 2-oxoindoline moiety via a triazole linkage OH N O O O O 2' O O OCH3 3' OCH3 4' H2N.OH.HCl Br R R R N1' N 5' NH NaOH, MeOH/THF O K2CO3,KI, DMF 7

1

6'

5

Scheme 2. Synthesis of novel N-hydroxybenzamides incorporating 2-oxoindoline moiety

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7'

6

NHOH

2

3

1

O

4

6 5

2.2. Bioactivity The compounds synthesized were evaluated for cytotoxicty against four human cancer cell lines, including SW620 (colon cancer), PC3 (prostate cancer), AsPC-1 (pancreatic cancer), and NCI-H23 (lung adenocarcinoma). In parallel, the compounds were also evaluated for histone deacetylase inhibition using HDAC2. The results are presented in table 1. It was found that, with the exception of compound 6a, all other compounds exhibited comparable or even superior inhibitory effects against HDAC2 when compared to SAHA. Also, it was observed that introduction of either electron-withdrawing or electron-donating substituents at positions 5 or 7 all enhanced the HDAC2 inhibition in a similar manner. Substitution at position 5 seemed to be more favorable for cytotoxicity than substititution at position 7 (e.g. compound 6c vs. compound 6g; compound 4c vs. Compound 4g). From the results shown in table 1, it could be seen that the cytotoxicity of the compounds, especially compounds in series 6a-g, was relatively well correlated with the HDAC2 inhibition. Compound 6d, one of the most active HDAC2 inhibitors in the series, showed the best cytotoxic profile against all four cancer cell lines tested with the IC 50 values ranging from 1.15 to 1.61 M, which were two to three-time lower compared to that of SAHA. Other compounds in the series including 6b, 6c, and 6e also displayed attractive cytotoxicity. Within series 4a-g, the HDAC2 inhibition seemed not completely correlated with cytotoxicity. For example, compounds 4d and 4e with the best HDAC2 inhibition (IC50 values/HDAC2 of 0.91 and 0.80 M, but IC50 values/cancer cell lines of 4.18 to 16.12 M); meanwhile compound 4f with IC50 value/HDAC2 of 1.92 M, but IC50 values/cancer cell lines of 1.14 to 2.62 M. This could be explained by the aqueous solubility of the compounds, as manifested by logP values. Compound 4f has the lowest logP value (of 2.44), thus, has the highest aqueous solubility. In contrast, compounds 4d and 4e have relatively higher logP values (of 2.91 and 3.25), thus, possess the lower aqueous solubility. Furthermore, the effects of 3-hydroxyimino-2-oxoindoline moiety on other possible targets such as protein kinases or tubulins [22], might also come into play and it is remained to be investigated. In designing these compounds, we initially envisioned that compounds 4a-g would be more potent than compounds 6a-g, because the length of the linker between the 2-oxoindoline and hydroxamic acid moiety in compounds 4a-g should be more similar to the linker between the phenyl and hydroxamic acid moiety in SAHA. In compounds 6a-g, the hydroxamic acid group was connected to the 2-oxoindoline system through only a benzyl linker. Surprisingly however, the HDAC2 inhibitory effects of compounds 6a-g turned out to be more potent than that of compounds 4a-g, excepting only compound 6a. Similarly, compounds 6a-g in general were also more potent than compounds 4a-g in term of cytotoxicity. These results should serve as valuable suggestions for further design of similar very simple structures, yet potential as HDAC inhibitors and antitumor agents. Table 1. Inhibition of HDAC2 activity and cytotoxicity of the compounds synthesized against several cancer cell lines OH O R

N

OH

O

N N N N

N R

4

Cpd code 4a 4b 4c 4d 4e 4f 4g 6a

LogP1

R

Molecular weight

-H 5-F 5-Cl 5-Br 5-CH3 5-OCH3 7-Cl -H

392.38 410.11 426.82 471.27 406.40 422.40 426.82 311.29

2.36 2.56 3.01 3.25 2.91 2.44 3.01 2.69

O

NHOH N

NHOH O

6

HDAC2 inhibition (IC50,2 M) 1.170.09 1.360.08 1.290.10 0.910.11 0.800.04 1.920.20 1.940.25 >30 -4-

Cytotoxicity (IC50,2 M)/Cell lines3 SW620

PC3

>30 >30 3.760.25 5.910.30 23.342.01 20.842.04 8.730.57 7.980.50 16.291.81 9.071.45 2.060.21 2.620.23 >30 >30 13.270.45 8.550.71

AsPC-1

NCI-H23

>30 3.150.24 24.282.22 4.180.61 11.421.57 1.390.17 >30 13.520.51

>30 4.210.17 22.452.11 5.460.70 12.141.72 1.140.11 >30 10.020.06

6b 6c 6d 6e 6f 6g 6h

5-F 5-Cl 5-Br 5-CH3 5-OCH3 7-Cl 5-NO2 SAHA4

329.28 345.73 390.19 325.32 341.32 345.73 365.28 264.32

2.89 3.33 3.58 3.23 2.77 3.33 2.90 1.44

0.940.03 2.730.09 1.820.09 2.410.12 2.130.09 1.160.06 2.080.09 1.530.06 0.950.12 1.360.12 0.540.03 1.330.10 1.280.08 1.150.03 1.610.05 0.580.03 1.870.46 2.460.77 1.600.49 1.320.58 0.500.06 4.480.35 6.180.12 2.900.12 2.050.12 0.440.06 3.270.35 3.410.67 2.860.26 2.950.11 0.700.05 6.050.16 10.780.02 6.240.14 6.300.14 1.060.07 3.200.0.23 3.700.38 3.750.42 3.630.34 1 2 Calculated by ChemDraw 9.0 software; The concentration (M) of compounds that produces a 50% reduction in enzyme activity or cell growth, the numbers represent the averaged results from triplicate experiments; 3Cell lines: SW620, colon cancer; PC3, prostate cancer; AsPC-1, pancreatic cancer; NCI-H23, lung adenocarcinoma cancer; 4SAHA, suberoylanilide acid, a positive control.

2.3. Docking studies It has been demonstrated that histone-H3 and histone-H4 deacetylation is principally regulated by HDAC2 and HDAC3 [23], so we decided to perform docking studies with HDAC2 in order to gain some preliminary insights into the interaction between these compounds and HDAC binding site. Previously, Lauffer and co-workers [24] have reported the crystal structure of HDAC2 in complex with SAHA (PDB ID: 4LXZ), we therefore used this crystal structure as a docking template. First, the suitability of ligand preparation procedures and docking protocol was checked by re-docking SAHA into the binding site of HDAC2 enzyme, based on the evaluation of RMSD values and interactions between this compound and the enzyme. As can be seen from Figure 3, the difference between co-crystallized and docked SAHA compounds is insignificant, displaying quite low value of RMSD (0.672 for coordinates of atoms involved in hydrogen bonding and 1.127 for coordinates of all heavy atoms). The conservation of hydrogen bond interactions with the residues Asp104, His145, His146, Asp181, and Tyr308 can be observed. In addition, the similarly close distances (< 2.5 Å) between hydroxamate groups and zinc ion, which play an important role in the zinc-binding motif of HDAC inhibitors, are conserved. Considering these results, our protocol is suitable for use in further docking experiments.

Figure 3. Superposition of SAHA X-ray structure (red) and re-docked structure (yellow) and potential interactions with HDAC2’s binding site. The distances from the zinc ion (dark grey ball) to carbonyl oxygen are 2.33 and 2.39 Å, and to hydroxyl oxygen are 1.96 and 2.33 Å for original and re-docked SAHA, respectively.

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The next step involved in docking two series of synthesized compounds into HDAC2 protein. At first sight, the docking results displayed similar interaction profiles between new compounds and reference ligand (Figure 4). The docking scores of SAHA and new ligands are given in Table 2. As can be seen in the figure there are some common amino acid residues that form the hydrogen bond network with most of N-hydroxybenzamide structures in this study, such as, His145, His146, Asp181 and Tyr308. Importantly, in all cases the hydroxamate group adopts a similar orientation as SAHA in the active site of HDAC2 X-ray structure. Average distance between oxygen atoms of carbonyl and hydroxyl groups and the zinc ion is about 2.5 Å, which is favorable to approximate to metal to chelate it and inactivate the enzyme. A different situation from SAHA is observed in hydrophobic interactions. With an aromatic ring attached to hydroxamate group, all compounds 4a-g and 6a-h preferred the same position that maximizes the van der Waals interaction in the binding pocket with residues constituted the narrow cavity of the tunnel like Phe155 and Phe210. In addition, the two aromatic rings of the capping group highly interact with various hydrophobic amino acids at the rim of active site, such as Pro34 and Leu276. In general, all compounds displayed similar to higher interactions in comparison with SAHA.

Figure 4. Superposition of the compounds 4a-g, 6a-h and reference compound SAHA docked to HDAC2’s binding site. Compounds are represented as a stick model

Table 2. Docking results of synthesized compounds 4a-g and 6a-h into human HDAC2 Distance to Binding Cpd. London Cpd. London Binding Zn2+ (Å)** Affinity *** code dG code dG Affinity dG dG*** =O -OH -78.507 -7.942 2.43 2.55 -74.243 -6.992 4a 6a -76.355 -7.448 2.36 2.72 -74.956 -7.163 4b 6b -76.511 -7.634 2.38 2.70 -75.776 -7.106 4c 6c -83.535 -7.986 2.49 2.29 -76.580 -7.174 4d 6d -80.424 -7.960 2.53 2.38 -74.939 -7.208 4e 6e -76.575 -6.439 2.38 2.58 -76.588 -7.122 4f 6f -71.347 -6.425 2.34 2.97 -77.643 -7.960 4g 6g * -73.647 -7.068 2.39 2.33 -73.899 -7.646 SAHA 6h *

Distance to Zn2+ (Å) =O -OH 2.56 2.33 2.58 2.32 2.55 2.34 2.63 2.28 2.59 2.31 2.38 2.36 2.55 2.35 2.43 2.55

SAHA: suberoylanilidehydroxamic acid; **distances from oxygen atoms (=O and –OH) of hydroxamate group to zinc ion; docking score (kcal/mol) calculated from the London (with refinement) and affinity scoring function from MOE software.

***

the

On the other hand, the binding scores calculated by London for compounds 4a-g can be arranged in ascending order of IC50 values and the best scores were assigned to compounds 4d (-83.5 kcal/mol) and 4e (-80.4 kcal/mol). The same results were observed for affinity scoring. Figure 5 shows hydrogen-bonding interactions of these two compounds. Both compounds 4d and 4e involve in numerous hydrogen bond interactions (Glu208, His145, -6-

His146, Tyr308 and Gly154 with 4d, andArg275, His145, His146, Tyr308 with 4e). Interestingly, compounds 4f and 4g displayed lower affinity energy (-6.4 kcal/mol) than SAHA (-7.1 kcal/mol), which coincided with their experimental results. In contrast, compounds 6a-h showed insignificant difference even though MOE London or affinity dG scoring method was used. This can be explained by observing the perfect superposition of compounds 6a-e (Figure 4). Only compound 6g with the highest London score displayed very different binding affinity. This compound formed hydrogen bonds with numerous amino acids, including His145, His146, Asp181, His183 and Tyr308, which can be considered very similar to SAHA. Images of the key interactions between compounds 6f, 6g and the enzyme are given in Figure 6. Generally, the docking scores calculated herein provide modest meaning and should be interpreted with caution as these values only reflected the structure-activity relationships in limited chemical space. It is interesting to analyze the distances from hydroxyl oxygen of hydroxamate group to zinc ion of compounds 4a-g and 6a-h. With the exception of compounds 4d and 4e, all compounds from 4a-g series display significant longer –OH-Zn distance than compounds 6a-h, raising a hypothesis to explain the higher inhibitory potency of compounds 6a-h in comparison with 4a-g. Accordingly, N-hydroxybenzamide moieties with shorter linker chain and highly hydrophobic capping group might lose the flexibility in the rim cavity of the pocket but penetrate better to the tunnel of the active site, therefore facilitated the chelation mode of hydroxamate with the zinc ion in HDAC enzyme, a crucial property related to the inhibitory activity of newly designed compounds.

Figure 5. Simulated docking poses of compounds 4d and 4e to binding pocket of HDAC2 shown as contact surface of hydrogen bonding. The zinc ion is represent as dark grey sphere, residues in the active site are shown in stick representation, hydrogen and π-π bonds are indicated as black and magenta dashed lines, respectively.

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Figure 6. Simulated docking poses of compounds 6f and 6g to binding pocket of HDAC2 shown as contact surface of hydrogen bonding. The zinc ion is represent as dark grey sphere, residues in the active site are shown in stick representation, hydrogen and π-π bonds are indicated as black and magenta dashed lines, respectively

3. Conclusions In conclusion, we have reported two series of 3-hydroxyimino-2-oxoindoline-based N-hydroxybenzamides with strong HDAC2 inhibitory effects and noted cytotoxicity against several human cancer cell lines, including SW620 (human colon cancer), PC-3 (prostate cancer), AsPC-1 (pancreas cancer), and NCI-H23 (lung adenocarcinoma). The results we obtained from this study further confirm that the 2-oxoindoline could be a good cap group for HDAC inhibitors and appropriate substituents introduced on the 2-oxoindoline moiety could result in strong HDAC inhibition effects. This study also demonstrated the simple compounds 6a-g, in which the Nhydroxybenzamide moiety linked directly to 2-oxoindoline system at position 1, turned out to be very potent both in term of HDAC inhibition and cytotoxity. These results suggest that further exploration of similarly simple structures should be warranted to find novel HDAC inhibitors as anticancer agents.

4. Experimental Part 4.1. Chemistry All reagents and solvents were purchased from Aldrich or Fluka Chemical Corp. (Milwaukee, WI, USA) or Merck unless noted otherwise. Solvents were used directly as purchased unless otherwise indicated. Thin layer chromatography which was performed using Whatman® 250 m Silica Gel GF Uniplates and visualized under UV light at 254 and 365 nm, were used to check the progress of reactions and preliminary evaluation of compounds’ homogeneity. In all cases, the compounds achieved purity of 97% or above, as estimated by HPLC method. Melting points were measured using a Gallenkamp Melting Point Apparatus (LabMerchant, London, United Kingdom) and are uncorrected. Purification of compounds were carried out using crystallization methods and/or open flash silica gel column chromatography employing Merck silica gel 60 (240 to 400 mesh) as stationary phase. Nuclear magnetic resonance spectra ( 1H NMR) were recorded on a Bruker 500 MHz spectrometer with DMSO-d6 as solvent unless otherwise indicated. Tetramethylsilane was used as an internal standard. Chemical shifts are reported in parts per million (ppm), downfield from tetramethylsilane. Mass spectra with different ionization modes including electron ionization (EI), Electrospray ionization (ESI), were recorded using PE Biosystems API2000 (Perkin Elmer, Palo Alto, CA, USA) and Mariner® (Azco Biotech, Inc. Oceanside, CA, USA) mass spectrometers, respectively. -8-

The synthesis of two series of novel series of N-hydroxybenzamides incorporating 2-oxoindoline moiety (4a-g and 6a-h) was carried out as illustrated in schemes 1 and 2. Details are described as follows: 4.1.1. General procedures for the synthesis of compounds 4a-g To a solution of 1a-g (1 mmol) in DMF (3 mL) were added K2CO3 (165.5 mg, 1.2 mmol). The resulting mixture was stirred at 80 °C for 1 hour, then KI (8.3 mg, 0.05 mmol) was added. After stirring for further 15 minutes, 0.15 ml of a solution of propargyl bromide 80% in toluene was dropped slowly into the mixture. The reaction mixture was stirred at 60 °C for 3 hours. Upon completion, the resultant mixture was cooled, poured into ice-cold water and acidified to pH~4. The orange solids formed were filtered and dried to give 2a-g, which were used for the next step without further purification. A solution of 2a-g and methyl 4-azidomethylbenzoate (1 mmol) in acetonitrile (2 mL) was stirred at room temperature for 10 minutes, then CuI (19.1 mg, 0.1 mmol) were added. The mixture was stirred at 50 °C until the reaction completed (12-24 hours). The resultant mixture was evaporated under reduced pressure to give the residues, which were re-dissolved in 50 ml of DCM. The mixture was filtered and the DCM layer was evaporated under reduced pressure to give 3a-g. The compounds 3a-g were dissolved in a mixture of methanol and tetrahydrofuran (2/1, 5 ml). Then, hydroxylamine.HCl (685 mg, 10 mmol) was added, followed by dropwise addition of a solution of NaOH (400 mg in 1 mL of water). The mixture was stirred at room temperature until the reaction completed. The resulting mixture was poured into ice-cold water, neutralised to pH ~ 7 and acidified by a solution of HCl 5% to induce precipitation. The precipitate was filtered, dried and re-crystalised from methanol to give 4a-g. 4.1.1.1. N-Hydroxy-4-((4-((3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1yl)methyl)benzamide (4a)

Yellow solid; Yield: 67.3%. mp: 176.5-178.0 oC. Rf = 0.34 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm1 ): 3381 (NH), 3170 (OH), 3036 (C-H, aren), 2954, 2853 (CH, CH2), 1694, 1639 (C=O), 1604 (C=C), 1460 (C-N). MS (ESI) m/z 391.12 [M - H]-. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.40 (1H, s, NOH); 11.19 (1H, s, NH); 9.05 (1H, s, OH); 8.19 (1H, s, H-9); 7.99 (1H, d, J = 7.5 Hz, H-4’); 7.71 (2H, d, J = 7.75 Hz, H2, H-6); 7.39 (1H, t, J = 7.5 Hz, H-6’); 7.31 (2H, d, J = 7.75 Hz, H-3, H-5); 7.11 (1H, d, J = 7.5 Hz, H-7’); 7.07 (1H, t, J = 7.5 Hz, H-5’); 5.60 (2H, s, H-7a, H-7b); 4.99 (2H, s, H-10a, H-10b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.55, 162.75, 143.40, 142.56, 142.27, 138.87, 132.55, 131.89, 127.77, 127.27, 126.85, 123.71, 122.73, 115.30, 109.60, 52.37, 34.64. Anal. Calcd. for C19H16N6O4 (392.12): C, 58.16; H, 4.11; N, 21.42. Found: C, 58.21; H, 4.17; N, 21.35. 4.1.1.2. 4-((4-((5-Fluoro-3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)-Nhydroxybenzamide (4b)

Yellow solid; Yield: 70%. mp: 190.0-191.5 oC. Rf = 0.40 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm-1): 3419 (NH), 3197 (OH), 3045 (C-H, aren), 2921, 2852 (CH, CH2), 1695, 1642 (C=O), 1619 (C=C), 1473 (CN). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.77 (1H, s, NOH); 11.20 (1H, s, NH); 9.04 (1H, s, OH); 8.19 (1H, s, H-9); 7.75 (1H, dd, J = 8.25 Hz, J’ = 2.75 Hz, H-4’); 7.71 (2H, d, J = 8.25 Hz, H-2, H-6); 7.32 (2H, d, J = 8.25 Hz, H-3, H-5); 7.28 (1H, td, J = 9.25 Hz, J’ = 2.75 Hz, H-6’); 7.13 (1H, dd, J = 8.5 Hz, J = 4.00 Hz, H-7’); 5.60 (2H, s, H-7a, H-7b); 4.99 (2H, s, H-10a, H-10b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.79, 162.63, 158.96, 157.07, 143.13, 142.13, 138.88, 132.57, 127.80, 127.30, 123.75, 118.12, 115.80, 113.86, 110.69, 52.40, 34.79. HR-MS (ESI) m/z calculated for C19H15FN6O4, [M - H]- 409.1066. Found, 409.1068. 4.1.1.3. 4-((4-((5-Chloro-3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)-Nhydroxybenzamide (4c)

Yellow solid; Yield: 68%. mp: 198.0-199.0 oC. Rf = 0.43 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm-1): 3429 (NH), 3204 (OH), 3040 (C-H, aren), 2856 (CH, CH2), 1696, 1642 (C=O), 1610 (C=C), 1466 (C-N). MS (ESI) m/z 425.08 [M - H]-. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.82 (1H, s, NOH); 11.19 (1H, s, -9-

NH); 9.03 (1H, s, OH); 8.19 (1H, s, H-9); 7.95 (1H, d, J = 1.5 Hz, H-4’); 7.71 (2H, d, J = 7.5 Hz, H-2, H-6); 7.48 (1H, dd, J = 8.5 Hz, J = 1.5 Hz, H-6’); 7.32 (2H, d, J = 7.5 Hz, H-3, H-5); 7.15 (1H, d, J = 8.5 Hz, H-7’); 5.60 (2H, s, H-7a, H-7b); 4.99 (2H, s, H-10a, H-10b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.79, 162.42, 142.68, 142.04, 141.34, 138.84, 132.58, 131.36, 127.80, 127.30, 126.58, 126.13, 123.72, 116.43, 111.23, 52.40, 34.81. Anal. Calcd. for C19H15ClN6O4 (426.08): C, 53.47; H, 3.54; N, 19.69. Found: C, 53.51; H, 3.52; N, 19.65. 4.1.1.4. 4-((4-((5-Bromo-3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)-Nhydroxybenzamide (4d)

Yellow solid; Yield: 65%. mp: 203.5-204.5 oC. Rf = 0.45 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm-1): 3425 (NH), 3231 (OH), 2921, 2854 (CH, CH2), 1719, 1637 (C=O), 1607 (C=C), 1464 (C-N). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.82 (1H, s, NOH); 11.18 (1H, s, NH); 9.03 (1H, s, OH); 8.19 (1H, s, H-9); 8.08 (1H, d, J = 2.0 Hz, H-4’); 7.71 (2H, d, J = 8.0 Hz, H-2, H-6); 7.61 (1H, dd, J = 8.5 Hz, J = 2.0 Hz, H-6’); 7.32 (2H, d, J = 8.0 Hz, H-3, H-5); 7.10 (1H, d, J = 8.5 Hz, H-7’); 5.59 (2H, s, H-7a, H-7b); 4.99 (2H, s, H-10a, H-10b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.79, 162.31, 142.56, 142.03, 141.74, 138.85, 134.20, 132.58, 128.84, 127.82, 127.31, 123.73, 116.88, 114.24, 111.75, 52.41, 34.80. HR-MS (ESI) m/z calculated for C19H15BrN6O4, [M - H]- 469.0265. Found, 469.0246. 4.1.1.5. N-Hydroxy-4-((4-((3-(hydroxyimino)-5-methyl-2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1yl)methyl)benzamide (4e)

Yellow solid; Yield: 59%. mp: 204.5-206.0 oC. Rf = 0.30 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm-1): 3405 (NH), 3192 (OH), 3035 (C-H, aren), 2858 (CH, CH2), 1693, 1643 (C=O), 1619 (C=C), 1476 (C-N). 1 H-NMR (500 MHz, DMSO-d6, ppm): δ 9.04 (1H, s, OH); 8.16 (1H, s, H-9); 7.83 (1H, s, H-4’); 7.70 (2H, d, J = 8.0 Hz, H-2, H-6); 7.31 (2H, d, J = 8.0 Hz, H-3, H-5); 7.20 (1H, d, J = 7.75 Hz, H-6’); 6.99 (1H, d, J = 7.75 Hz, H-7’); 5.59 (2H, s, H-7a, H-7b); 4.96 (2H, s, H-10a, H-10b); 2.27 (3H, s, CH3). 13C NMR (125 MHz, DMSOd6, ppm): δ 162.79, 143.55, 142.34, 140.33, 138.88, 132.57, 132.09, 131.79, 127.80, 127.38, 127.28, 123.66, 115.36, 109.38, 52.37, 34.66, 20.50. HR-MS (ESI) m/z calculated for C20H18N6O4, [M + Na]+ 429.1280, [M + H]+ 407.1462. Found, 429.1299, 407.1480. 4.1.1.6. N-Hydroxy-4-((4-((3-(hydroxyimino)-5-methoxy-2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1yl)methyl)benzamide (4f)

Yellow solid; Yield: 52%. mp: 172.5-174.0 oC. Rf = 0.41 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm-1): 3393 (NH), 3193 (OH), 3037 (C-H, aren), 2920, 2850 (CH, CH2), 1707, 1662 (C=O), 1598 (C=C), 1438 (CN). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.17 (1H, s, H-9); 7.71 (2H, d, J = 8.25 Hz, H-2, H-6); 7.59 (1H, d, J = 2.0 Hz, H-4’); 7.31 (2H, d, J = 8.25 Hz, H-3, H-5); 7.03-6.96 (2H, m, H-6’, H-7’); 5.59 (2H, s, H-7a, H7b); 4.95 (2H, s, H-10a, H-10b); 3.73 (3H, s, OCH3). 13C NMR (125 MHz, DMSO-d6, ppm): δ 162.68, 155.22, 143.64, 142.37, 138.86, 136.12, 132.50, 129.29, 127.79, 127.27, 123.67, 116.75, 115.90, 113.01, 110.19, 52.64, 52.36, 34.68. HR-MS (ESI) m/z calculated for C20H18N6O5, [M - H]- 421.1266. Found, 421.1258. 4.1.1.7. 4-((4-((7-Chloro-3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)-Nhydroxybenzamide (4g)

Yellow solid; Yield: 67%. mp: 197-198.56 oC. Rf = 0.42 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm-1): 3223 (OH), 3171 (C-H, aren), 2959, 2858 (CH, CH2), 1704, 1635 (C=O), 1603 (C=C), 1469 (C-N). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.20 (1H, s, NH); 9.06 (1H, s, OH); 8.15 (1H, s, H-9); 8.07 (1H, d, J = 7.5 Hz, H-4’); 7.71 (2H, d, J = 7.5 Hz, H-2, H-6); 7.39 (1H, d, J = 8.5 Hz, H-6’); 7.29 (2H, d, J = 7.5 Hz, H-3, H5); 7.10 (1H, t, J = 7.5 Hz, H-5’); 5.60 (2H, s, H-7a, H-7b); 5.32 (2H, s, H-10a, H-10b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.68, 143.80, 142.17, 139.01, 138.35, 133.66, 132.50, 127.61, 127.26, 125.77, 124.23, 122.98, 118.34, 114.75, 52.38, 37.05. HR-MS (ESI) m/z calculated for C19H15ClN6O4, [M - H]- 425.0771. Found, 425.0758. -10-

4.1.2. General procedures for the synthesis of 6a-g To a solution of 1a-g (147 mg, 1 mmol) in DMF (3 mL) were added K 2CO3 (165.5 mg, 1.2 mmol). After stirring for 45 minutes, KI (8.3 mg, 0.05 mmol) and a solution of methyl 4-bromomethylbenzoate (229 mg, 1 mmol) in DMF (1 mL) were added. The mixture was stirred at 80 °C for 1-3 hours. Upon completion, the resultant mixture was cooled, poured into ice-cold water (20 mL) and acidified to pH~4 to induce precipitation. The precipitates were filtered and dried to give orange-yellowish solids 5a-g which were used for the next step without further purification. The orange-yellowish solids 5a-g obtained above were dissolved in methanol/tetrahydrofurane (2/1, 10 ml) and the mixture was cooled to 0 °C. Then, hydroxylamine.HCl (685 mg, 10 mmol) was added, followed by dropwise addition of a solution of NaOH (400 mg in 1 mL of water). The resultant mixture was stirred at room temperature for 2 hours. Upon completion, the reaction mixture was poured into ice-cold water, neutralised to pH~7 to yield yellow solids. The precipitates was filtered, dried and recrystalised from methanol to give the target compounds 6a-g. 4.1.2.1. N-Hydroxy-4-((3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)benzamide (6a) Yellow solid; Yield: 80 %, mp: 167.0-168.5 oC; Rf = 0.55 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3205 (OH), 3045 (C-H, aren), 2854 (CH, CH2), 1721 (C=O), 1606 (C=C), 1435 (C-N). ESI-MS (m/z): 310 [M-H]-. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.54 (1H, s, N-OH), 11.17 (1H, s, NH), 8.01 (1H, d, J = 7.5 Hz, H-4’), 7.71 (2H, d, J = 8.0 Hz, H-2, H-6), 7.39 (2H, d, J = 8.0 Hz, H-3, H-5), 7.36 (1H, td, J = 8.0 Hz, J’ = 1.0 Hz, H-6’), 7.08 (1H, t, J = 7.5 Hz, H-5’), 6.97 (1H, d, J = 8.0 Hz, H-7’), 4.99 (2H, s, H-7a, H-7b); 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.92, 163.23, 143.39, 142.61, 139.37, 132.03, 131.97, 127.30, 127.13, 126.97, 122.87, 115.35, 109.50, 42.28. Anal. Calcd. for C16H13N3O4 (311.09): C, 61.73; H, 4.21; N, 13.50. Found: C, 61.75; H, 4.24; N, 13.47. 4.1.2.2. 4-((5-Fluoro-3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)-N-hydroxybenzamide (6b) Yellow solid; Yield: 89 %, mp: 166.5-167.5 oC; Rf = 0.57 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3232 (OH), 3068 (C-H, aren), 2899 (CH, CH2), 1708, 1652 (C=O), 1474 (C-N). ESI-MS (m/z): 328 [M-H]-. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.82 (1H, s, N-OH), 11.17 (1H, s, NH), 9.01 (1H, s, OH), 7.90 (1H, d, JH-4’ - F = 6.0 Hz, H-4’), 7.78 – 7.70 (2H, m, H-2, H-6), 7.43 – 7.40 (2H, m, H-3, H-5), 7.24 (1H, s, H-6’), 6.97 (1H, s, H-7’), 4.99 (2H, s, H-7a, H-7b); 13C NMR (125 MHz, DMSO-d6, ppm): δ 166.98, 163.93, 163.13, 159.02, 157.13, 143.12, 141.05, 139.19, 138.93, 132.08, 130.02, 129.75, 127.33, 127.27, 127.15, 118.28, 118.09, 115.94, 115.86, 114.13, 113.92, 110.57, 110.51, 42.42. Anal. Calcd. for C16H12FN3O4 (329.08): C, 58.36; H, 3.67; N, 12.76. Found: C, 58.38; H, 3.68; N, 12.74. 4.1.2.3. 4-((5-Chloro-3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)-N-hydroxybenzamide (6c) Yellow solid; Yield: 82%, mp: 172.0-173.5 oC; Rf = 0.48 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3408 (NH), 3269 (OH), 2866 (CH, CH2), 1712, 1666 (C=O), 1610 (C=C), 1466 (C-N). ESI-MS (m/z): 346 [M+H]+. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.19 (1H, s, NH), 9.04 (1H, s, OH), 7.98 (1H, s, H-4’), 7.70 (2H, d, J = 8.0 Hz, H-2, H6), 7.43 (1H, d, J = 8.5 Hz, H-6’), 7.38 (2H, d, J = 8.0 Hz, H-3, H-5), 6.98 (1H, d, J = 8.5 Hz, H-7’), 4.99 (2H, s, H-7a, H-7b); 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.04, 142.76, 141.41, 139.13, 132.11, 131.48, 129.69, 127.40, 127.20, 126.80, 126.31, 116.61, 111.15, 42.50. Anal. Calcd. for C16H12ClN3O4 (345.05): C, 55.58; H, 3.50; N, 12.15. Found: C, 55.56; H, 3.52; N, 12.19. 4.1.2.4. 4-((5-Bromo-3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)-N-hydroxybenzamide (6d) Yellow solid; Yield: 75%, mp: 178.0-179.0 oC; Rf = 0.51 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3265 (NH), 3215 (OH), 3054 (C-H, aren), 2855 (CH, CH2), 1711, 1664 (C=O), 1606 (C=C), 1463 (C-N). ESI-MS (m/z): 388 [MH]-. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.88 (1H, s, NOH), 11.18 (1H, s, NH), 8.10 (1H, d, J = 2.0 Hz, H4’), 7.70 (2H, d, J = 8.0 Hz, H-2, H-6), 7.55 (1H, dd, J = 8.25 Hz, J’ = 2.0 Hz, H-6’), 7.38 (2H, d, J = 8.0 Hz, H-3, H-5), 6.94 (1H, d, J = 8.25 Hz, H-7’), 4.98 (2H, s, H-7a, H-7b); 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.80, 162.81, 142.55, 141.76, 139.00, 134.23, 132.07, 128.97, 127.31, 127.10, 116.96, 114.35, 111.56, 42.41. Anal. Calcd. for C16H12BrN3O4 (389.00): C, 49.25; H, 3.10; N, 10.77. Found: C, 49.27; H, 3.17; N, 10.72. -11-

4.1.2.5. N-Hydroxy-4-((3-(hydroxyimino)-5-methyl-2-oxoindolin-1-yl)methyl)benzamide (6e) Yellow solid; Yield: 77%, mp: 159.5-160.5 oC; Rf = 0.53 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3227 (OH), 3073 (C-H, aren), 2920, 2863 (CH, CH2), 1712 (C=O), 1616 (C=C), 1479 (C-N). ESI-MS (m/z): 324 [M-H]-. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.50 (1H, s, NOH), 11.16 (1H, s, NH), 9.01 (1H, s, OH), 7.85 (1H, s, H-4’), 7.69 (2H, d, J = 8.0 Hz, H-2, H-6), 7.37 (2H, d, J = 8.0 Hz, H-3, H-5), 7.16 (1H, d, J = 7.75 Hz, H-6’), 6.85 (1H, d, J = 7.75 Hz, H-7’), 4.96 (2H, s, H-7a, H-7b), 2.26 (3H, s, CH3); 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.95, 163.25, 143.53, 140.39, 139.45, 132.16, 131.94, 129.72, 127.52, 127.29, 127.10, 115.40, 109.29, 42.29, 20.05. Anal. Calcd. for C17H15N3O4 (325.11): C, 62.76; H, 4.65; N, 12.92. Found: C, 62.73; H, 4.68; N, 12.94. 4.1.2.6. N-Hydroxy-4-((3-(hydroxyimino)-5-methoxy-2-oxoindolin-1-yl)methyl)benzamide (6f) Yellow solid; Yield: 65%, mp: 165.5-167.5 oC; Rf = 0.48 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3212 (OH), 2897 (CH, CH2), 1704, 1630 (C=O), 1437 (C-N). ESI-MS (m/z): 340 [M-H]-. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.59 (1H, s, NOH), 11.17 (1H, s, NH), 9.01 (1H, s, OH); 7.70 (2H, d, J = 8.25 Hz, H-2, H-6), 7.60 (1H, d, J = 2.5 Hz, H-4’), 7.38 (2H, d, J = 8.25 Hz, H-3, H-5), 6.94 (1H, dd, J = 8.5 Hz, J’ = 2.5 Hz, H-6’), 6.87 (1H, d, J = 8.5 Hz); 4.95 (2H, s, H-7a, H-7b); 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.98, 163.13, 155.33, 143.667, 139.47, 136.23, 132.02, 129.73, 127.31, 127.13, 116.90, 115.94, 113.29, 110.14, 55.67, 42.35. Anal. Calcd. for C17H15N3O5 (341.10): C, 59.82; H, 4.43; N, 12.31. Found: C, 59.85; H, 4.46; N, 12.28. 4.1.2.7. 4-((7-Chloro-3-(hydroxyimino)-2-oxoindolin-1-yl)methyl)-N-hydroxybenzamide (6g) Yellow solid; Yield: 73%, mp: 167.0-168.5 oC; Rf = 0.54 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3384 (NH), 3298 (OH), 3104 (C-H, aren), 2923, 2853 (CH, CH2), 1710, 1648 (C=O), 1603 (C=C), 1444 (C-N). ESI-MS (m/z): 344 [M-H]-. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 13.91 (1H, s, NOH), 11.16 (1H, s, NH), 9.00 (1H, s, OH), 8.10 (1H, d, J = 8.0 Hz, H-4’), 7.70 (2H, d, J = 8.25 Hz, H-2, H-6), 7.38 (1H, d, J = 8.0 Hz, H-6’), 7.28 (2H, d, J = 8.25 Hz, H-3, H-5), 7.12 (1H, t, J = 8.0 Hz, H-5’), 5.32 (2H, s, H-7a, H-7b); 13C NMR (125 MHz, DMSO-d6, ppm): δ 164.04, 163.92, 142.13, 140.78, 138.41, 133.80, 131.65, 127.24, 125.98, 125.88, 124.43, 118.30, 114.56, 44.02. Anal. Calcd. for C16H12ClN3O4 (345.05): C, 55.58; H, 3.50; N, 12.15. Found: C, 55.56; H, 3.52; N, 12.19. 4.1.2.8. N-hydroxy-4-((3-(hydroxyimino)-5-nitro-2-oxoindolin-1-yl)methyl)benzamide (6h) Yellow solid; Yield: 72 %, mp: 182.0-183.0 oC; Rf = 0.40 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3315 (NH), 3209 (OH), 2879 (CH, CH2), 1716, 1655 (C=O), 1614 (C=C), 1470 (C-N). ESI-MS (m/z): 355 [M-H]-. 1H-NMR (500 MHz, DMSO-d6, ppm): δ 14.22 (1H, s, NOH), 11.18 (1H, s, NH), 8.72 (1H, s, H-4’), 8.31 (1H, d, J = 7.75 Hz, H-6’), 7.72 (2H, d, J = 7.75 Hz, H-2, H-6), 7.42 (2H, d, J = 7.75 Hz, H-3, H-5), 7.20 (1H, d, J = 7.75 Hz, H-7’), 5.08 (2H, s, H-7a, H-7b); 13C NMR (125 MHz, DMSO-d6, ppm): δ 168.47, 163.47, 147.73, 142.65, 142.06, 138.56, 132.13, 128.24, 127.31, 127.09, 126.88, 121.52, 115.30, 109.74, 42.74. Anal. Calcd. for C16H12BrN3O4 (356.08): C, 53.94; H, 3.39; N, 15.73 Found: C, 53.91; H, 3.37; N, 15.73.

4.2. Cytotoxicity assay The cytotoxicity of the synthesized compounds was evaluated against three human cancer cell lines, including SW620 (colon cancer), PC3 (prostate cancer), and AsPC-1 (pancreatic cancer). The cell lines were purchased from a Cancer Cell Bank at the Korea Research Institute of Bioscience and Biotechnology (KRIBB). The media, sera and other reagents that were used for cell culture in this assay were obtained from GIBCO Co. Ltd. (Grand Island, New York, USA). The cells were culture in DMEM (Dulbecco’s Modified Eagle Medium) until confluence. The cells were then trypsinized and suspended at 3 × 104 cells/mL of cell culture medium. On day 0, each well of the 96-well plates was seeded with 180 L of cell suspension. The plates were then incubated in a 5% CO2 incubator at 37 oC for 24 h. Compounds were initially dissolved in dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations by culture medium. Then 20 L of each compounds’ samples, which were prepared as described above, were added to each well of the 96-well plates, which had been seeded with cell suspension and incubated for 24-h, at various concentrations. The plates were further incubated for 48 h. Cytotoxicity of the compounds was measured by the colorimetric method, as described previously [25] with slight modifications [26,27]. The IC50 values were calculated using a Probits method [28] and were averages of three independent -12-

determinations (SD ≤ 10%). 4.3. HDAC2 enzyme assay The HDAC2 enzyme was purchased from BPS Bioscience (San Diego, CA, USA). The HDAC enzymatic assay was performed using a Fluorogenic HDAC Assay Kit (BPS Bioscience) according to the manufacturer’s instructions. Briefly, HDAC2 enzymes were incubated with vehicle or various concentrations of the assayed samples or SAHA for 30 min at 37 oC in the presence of an HDAC fluorimetric substrate. The HDAC assay developer (which produces a fluorophore in reaction mixture) was added, and the fluorescence was measured using VICTOR3 (PerkinElmer, Waltham, MA, USA) with excitation at 360 nm and emission at 460 nm. The measured activities were subtracted by the vehicle-treated control enzyme activities and IC50 values were calculated using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

4.4. Docking studies Docking study was carried out by using MOE 2009.10 software [29]. The 3D crystal structure of HDAC2 protein in complex with SAHA [24] was retrieved from the Protein Data Bank (PDB ID: 4LXZ). After removing SAHA from the complex structure, the enzyme was prepared with the aid of ICM-pro 3.8-3. All water molecules were removed and the hydrogen atoms were aided to the protein. The active site of the en zyme includes key residues His145, His146, Asp181, His183 and Asp269 which were appropriately protonated according to the general catalytic transformations of Zinc metalloenzymes described by Pottel et al [30]. Afterwards, the binding site was energy-minimized applying AMBER99 force field based on truncated Newton method in MOE (convergence criteria of 0.01 Å root-mean-square deviation, RMSD). For ligand preparation we followed the same procedures mentioned in previous works [18-21]. As the ionization state of hydroxamic acids in complex with Zn 2+ was strongly suggested as negative hydroxamate coordination [31], herein their hydroxyl groups were deprotonated. Docking experiments were performed based on MOE Triangle Matcher placement method and the other parameters were default settings, retaining fifty poses for each ligand. London dG Score with refinement was used as main criteria for selecting the best ligand configuration with minimum binding energy [32]. In addition, the selected conformations were rescored using the affinity dG scoring function [29]. For interaction visualization, all the figures were built using the snapshots taken in BIOVIA Discovery Studio v.3.5 program [33].

Acknowledgements We acknowledge the principal financial support from the National Foundation for Science and Technology of Vietnam (NAFOSTED, Grant number 104.01-2015.08). A grant No 2008-0062275 from the Korean Government is also greatly appreciated. Declaration of interest The authors report no conflict of interest. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

P.A. Marks, R.A. Rifkind, V.M. Richon, R. Breslow, T. Miller, W.K. Kelly. Nature Rev. Cancer. 1 (2001) 194202. O. Witt, H.E. Deubzer, T. Milde, I. Oehme. Cancer Lett. 277 (2009) 8-21. A.J.M. De Ruijter, A.H.V. Gennip, H.N. Caron, S. Kemp, A.B.P.V. Kuilenburg. Biochem. J. 370 (2003) 737-739. J.E. Bolden, M.J. Peart, R.W. Johnstone. Nat. Rev. Drug Disc. 5 (2006) 769-784. M. Dokmanovic, P.A. Marks. Expert Opin. Investig. Drug. 14 (2005) 1497-1511. R.W. Johnstone. Nature Rev. Drug Disc. 1 (2002) 287-299. K.B. Glaser. Biochem. Pharmacol. 74 (2007) 659-671. A.C. West, R.W. Johnstone. J. Clin. Invest. 124 (2014) 30-39. S. Dallavalle, R. Cincinelli, R. Nannei, L. Merlini, G. Morini, S. Penco, C. Pisano, L. Vesci, M. Barbarino, V. Zuco, M. De Cesare, F. Zunino. Eur J. Med. Chem. 44 (2009) 1900-1912. T.U. Bracker, A. Sommer, I. Fichtner, H. Faus, B. Haendler, H. Hess-Stumpp. Int. J. Oncol. 35 (2009) 909-920. M.S. Finnin, J.R. Donigian, A. Cohen, V.M. Richon, R.A. Rifkind, P.A. Marks, R. Breslow, N.P. Pavietich. Nature 401 (1999) 188-193. S. Valente, A. Mai. Expert. Opin. Ther. Pat. 24 (2014) 401-415. -13-

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J. Li, G. Li, X. Xu. Curr. Med. Chem. 20 (2013) 1858-1886. K. Ververis, A. Hiong, T.C. Karagiannis, P.V. Licciardi. Biologics 7 (2013) 47-60. T. Qiu, L. Zhou, W. Zhu, T. Wang, J. Wang, Y. Shu, P. Liu. Future Oncol. 9 (2013) 255-269. FDA approves Farydak for treatment of multiple myeloma. fda.gov. Retrieved 6 October 2015. G. Malini. Nature Reviews Drug Dis. 14 (2015) 225-226. D.T.K. Oanh, H.V. Hai, V.T.M. Hue, S.H. Park, H.J. Kim, B.W. Han, H.S. Kim, J.T. Hong, S.B. Han, N.H. Nam. Bioorg. Med. Chem. Lett. 21 (2011) 7509-7512. [19] T.T. Tung, D.T. Oanh, P.T. Dung, V.T. Hue, S.H. Park, B.W. Han, Y. Kim, J.T. Hong, S.B. Han, N.H. Nam. Med. Chem. 9 (2013) 1051-1057. [20] N.H. Nam, T.L. Huong, D.T.M. Dung, D.T.K. Oanh, P.T.P. Dung, K.R. Kim, B.W. Han, Y.S. Kim, J.T. Hong, S.B. Han. J. Enzyme Inhib. Med. Chem. 29 (2014) 611-618. [21] N.H. Nam, T.L. Huong, D.T.M. Dung, D.T.K. Oanh, P.T.P. Dung, D. Quyen, K.R. Kim, B.W. Han, Y.S. Kim, J.T. Hong, S.B. Han. Eur. J. Med. Chem. 70 (2013) 477-486. [22] Vine, K. L., Matesic, L., Locke, J. M. & Skropeta, D. (2013). Recent highlights in the development of isatinbased anticancer agents. In M. Prudhomme (Eds.), Advances in Anticancer Agents in Medicinal Chemistry (pp. 254-312). Sharjah, UAE: Bentham Science Publishers. [23] H.R. Pelzel, C.L. Schlamp, R.W. Nickells. BMC Neuroscience 11 (2010) 62-69. [24] B.E. Lauffer, R. Mintzer, Fong, R. Fong, S. Mukund, C. Tam, I. Zilberleyb, B. Flicke, A. Ritscher, G.; Vallero, R. Fedorowicz, D.F. Ortwine, J. Gunzner, Z. Modrusan, L. Neumann, C.M. Koth, P.J. Lupardus,J.S.; Heise, C.E. Kaminker, P. Steiner. J. Biol. Chem. 288 (2013) 26926-26943. [25] P. Skehan, R. Storeng, D. Scudiero, A. Monk, J. MacMahon, D. Vistica, J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd. J. Natl. Cancer Inst. 82 (1990) 1107-1112. [26] G. Ye, N.H. Nam, A. Kuma, A. Saleh, D.B. Shenoy, M.M. Amiji, X. Lin, G. Sun, K. Parang. J. Med. Chem. 50 (2007) 3604-3617. [27] Y.J. You, Y. Kim, N.H. Nam, B.Z. Ahn. Bioorg. Med. Chem. Lett. 13 (2003) 2629-2632. [28] L. Wu, A.M. Smythe, S.F. Stinson, L.A. A. Mullendore, D.A. Monks, K.D. Scudiero, A.D. Koutsoukos, L.V. Rubinstein, M.R. Boyd, R.H. Shoemaker. Cancer Res. 52 (1992) 3029-3034. [29] Chemical Computing Group Inc. Molecular Operating Environment (MOE). https://www.chemcomp.com. [30] J. Pottel, E. Thierrein, J.L. Gleason, N. Moitessier. J. Chem. Inf. Model. 54 (2014) 254−265. [31] R. Wu, Z. Lu, Z. Cao, Y. Zang. J. Am. Chem. Soc. 133 (2011) 6110-6113. [32] A.D. Mackerell, M. Feig, C.L. Brooks. J. Comput. Chem. 25 (2004) 1400-1415. [33] Discovery Studio, version 3.5. Accelrys, Inc; San Diego, CA, USA: 2014. http://accelrys.com/products/collaborative-science/biovia-discovery-studio.

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Graphical abstract OH O R

N

OH

O

N N N N

4

N O

NHOH R

N

NHOH O

6

Two series of novel, simple N-hydroxybenzamides incorporating 2-oxoindolines (4a-g, 6a-g) were designed and synthesized. Biological evaluation showed that these benzamides potently inhibited HDAC2 with IC50 values in sub-micromolar range. A number of compounds also exhibited cytotoxicity comparable to that of SAHA, a positive control.

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Highlights   

Two series of novel N-hydroxybenzamides were synthesized. Simple N-hydroxybenzamides exhibited potent HDAC2 inhibition Simple N-hydroxybenzamides also exhibited good cytotoxicity.

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