Novel hydroxamic acids incorporating 1-((1H-1,2,3-Triazol-4-yl)methyl)-3-hydroxyimino-indolin-2-ones: synthesis, biological evaluation, and SAR analysis

Article abstract of DOI:10.1007/s12039-018-1472-x

Abstract: A series of seventeen novel hydroxamic acids incorporating 1-((1H-1,2,3-triazol-4-yl)methyl)-3-hydroxyimino-indolin-2-ones was designed and synthesized. Biological evaluation showed that these hydroxamic acids potently inhibited a class-I isoform of HDACs (HDAC2) with IC ₅₀ values in low micromolar range. Several compounds also exhibited good cytotoxicity. Two compounds, 5e and 5f, emerged as the most potent HDAC2 inhibitors with cytotoxicity up to 8-fold more potent than SAHA in three human cancer cell lines, including SW620 (colon cancer), PC3 (prostate cancer) and AsPC-1 (pancreatic cancer). A molecular modeling approach has been carried out which revealed some structure-activity relationships. Further investigation on absorption, distribution, metabolism, excretion and toxicity (ADMET) suggested that compounds 5e and 5f, while showing potent HDAC2 inhibitory bioactivity, hold desirable characteristics for anticancer compounds. GRAPHICAL ABSTRACT: Three series of novel hydroxamic acids incorporating 1-((1H-1,2,3-triazol-4-yl)methyl)-3-hydroxyimino-indolin-2-ones (4a-c, 5a-g, 6a-g) were synthesized. Biological evaluation showed that these hydroxamic acids potently inhibited HDAC2 with IC ₅₀ values in low micromolar range.

Full text of DOI:10.1007/s12039-018-1472-x

J. Chem. Sci. (2018) 130:63
© Indian Academy of Sciences
https://doi.org/10.1007/s12039-018-1472-x
REGULAR ARTICLE
Novel hydroxamic acids incorporating
1-((1H-1,2,3-Triazol-4-yl)methyl)-3-hydroxyimino-indolin-2-ones:
synthesis, biological evaluation, and SAR analysis
DO THI MAI DUNGa, PHAM-THE HAIa, DUONG TIEN ANHa, LE-THI-THU HUONGb,
NGUYEN THI KIM YENc, BYUNG WOO HANc, EUN JAE PARKd, YEO JIN CHOId,
JONG SOON KANGe, VAN-THI-MY HUEa,∗, SANG-BAE HANd,∗ and NGUYEN-HAI NAMa,∗
aHanoi University of Pharmacy, 13-15 Le Thanh Tong, HoanKiem, Hanoi, Vietnam
bSchool of Medicine and Pharmacy, Vietnam National University, 144 XuanThuy, Hanoi, Vietnam
cResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742,
Korea
dCollege of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 28160, Korea
eBio-EvaluationCenter, KoreaResearchInstituteofBioscienceandBiotechnology, Cheongju, Chungbuk, Korea
E-mail: shan@chungbuk.ac.kr; namnh@hup.edu.vn
MS received 5 March 2018; revised 22 April 2018; accepted 29 April 2018
Abstract. A series of seventeen novel hydroxamic acids incorporating 1-((1H-1,2,3-triazol-4-yl)methyl)-
3-hydroxyimino-indolin-2-ones was designed and synthesized. Biological evaluation showed that these
hydroxamic acids potently inhibited a class-I isoform of HDACs (HDAC2) with IC50 values in low micromolar
range. Several compounds also exhibited good cytotoxicity. Two compounds, 5e and 5f, emerged as the most
potent HDAC2 inhibitors with cytotoxicity up to 8-fold more potent than SAHA in three human cancer cell lines,
including SW620 (colon cancer), PC3 (prostate cancer) and AsPC-1 (pancreatic cancer). A molecular modeling
approach has been carried out which revealed some structure-activity relationships. Further investigation on
absorption, distribution, metabolism, excretion and toxicity (ADMET) suggested that compounds 5e and 5f,
while showing potent HDAC2 inhibitory bioactivity, hold desirable characteristics for anticancer compounds.
Keywords. Histonedeacetylase (HDAC) inhibitors; hydroxamic acids; 2-oxoindoline; triazole.
1. Introduction
into four classes based on the relative sequence
similarity.2–4 Among these four classes, class I of
HDACs, which has four members (HDAC1, 2, 3 and
HDAC8) and class II, which has six members (HDAC4,
5, 6, 7, 9 and HDAC10) have been comprehensively
investigated and demonstrated to be deeply involved
in a number of cell-related processes.3–5 Specifically,
HDAC 1, 2, 3 and 8 have been shown to promote
cellular proliferation, meanwhile HDAC 1-4, 5 and
8 prevent cellular apoptosis and differentiation. Other
HDAC isoforms, for example, HDAC 4, 6, 7 and 10,
have been demonstrated to promote angiogenesis and
cell migration, two processes very important for cancer
cell metastasis.4,5 Down-regulation of these HDAC iso-
forms has been shown to result in a number of events
related to differentiation, apoptosis and cell cycle arrest
in different types of tumor cells.6 Moreover, selective
Target-based drug design and discovery has become a
main stream in today’s new drug discovery and develop-
ment. In the field of cancer research, thanks to the recent
advances in molecular pathology, hundreds of proteins
have been validated as potential molecular targets for
anticancer drug design. These include protein kinases,
farnesyltransferases, telomerases, and histone deacety-
lases, among many others.1
Histone deacetylases (HDAC) are a group of enzymes
catalyzing removal of the acetyl groups from lysine
residues in the tails of histone proteins.2,3 Hitherto,
18 different isoforms of HDACs have been identi-
fied in eukaryotes and these isoforms are categorized
*
For correspondence
Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12039-018-1472-x) contains
supplementary material, which is available to authorized users.

63 Page 2 of 13
J. Chem. Sci. (2018) 130:63
Figure 1. Structures of some HDAC inhibitors.
suppression of the growth of tumor cells caused by analogues of SAHA, which incorporated benzothiazole
HDAC inhibition has been clearly demonstrated not or 5-aryl-1,3,4-thiadiazole systems (Figure 2). These
only in vitro but also in a number of in vivo preclini- compounds exhibited very potent HDAC inhibitory
cal models and clinical settings.6 Design of compounds activity as well as cytotoxicity.17–20 Several represen-
to inhibit appropriate HDACs has, therefore, become tative compounds from these series also demonstrated
a very interesting approach in cancer drug design and very potent antitumor activity in PC-3 prostate cancer
development nowadays.7 As a result, a number of cells xenografted mice model.18 Encouraged by these
HDAC inhibitors have been reported in the past decades. results, we expanded our design to the new series of
These inhibitors are diverse, from short-chain fatty 17 hydroxamic acids. In these hydroxamic acids the
acids (like butyrate, phenylbutyrate or valproic acid) to 2-oxoindoline system is employed as a cap group and
hydroxamic acids, or benzamides.8–14 To date, at least 4 linkers of different lengths incorporating a triazole moi-
HDAC inhibitors have been approved for use in clinical ety are probed. The current paper reports the results we
settings. The first HDAC inhibitor approved by the U.S. obtained from the synthesis, biological evaluation and
FDA in October 2006 for the treatment of cutaneous computational study on these novel hydroxamic acids.
T cell lymphoma (CTCL) was vorinostat (trade name,
Zolinza®) (also known as SAHA or suberoylanilidehy-
droxamic acid) (Figure 1). In 2009, the second HDAC
inhibitor, romidepsin (trade name, Istodax®) was also
2. Experimental
approved by the U.S. FDA for the same indication. In
Feb 2015, panobinostat (LBH-589, trade name Fary-
dak®) was licensed by the US FDA for the treatment
of multiple myeloma.15 Also in 2015 chidamide (Epi-
daza®) was approved by the Chinese FDA for relapsed
or refractory peripheral T cell lymphoma.16 In addition,
a number of other HDAC inhibitors such as PXD-01
(belinostat), MS-27-527 (entinostat) (Figure 1) are cur-
rently under different phases of clinical trials for several
types of cancer (Figure 1).
2.1 Chemistry
2.1a Chemicals and instruments: Thin layer chro-
matography (TLC) was performed using Whatman® 250 µm
Silica Gel GF Uniplates and visualized under UV light at 254
and 365 nm. TLC was used to check the progress of reactions
and preliminary evaluation of compounds’ homogeneity.
Melting points were measured using a Gallenkamp Melting
Point Apparatus (LabMerchant, London, United Kingdom)
and are uncorrected. Purification of compounds was carried
out using crystallization methods and/or open silica gel col-
umn flash chromatography employing Merck silica gel 60
(240 to 400 mesh) as stationary phase. Nuclear magnetic reso-
nance spectra (1H NMR) were recorded on a Bruker 500 MHz
spectrometer with DMSO-d6 as solvent unless otherwise
In continuation of our research program to develop
novel hydroxamic acids as potential inhibitors of
HDACs and anticancer agents, we have designed, syn-
thesized and evaluated several series of heterocyclic

J. Chem. Sci. (2018) 130:63
Page 3 of 13 63
Figure 2. Structures of some benzothiazole- and 5-substituted phenyl-1,3,4-thiadiazole-based hydroxamic acids.
indicated. Tetramethylsilane was used as an internal standard. NaOH (400mg in 1mL of water). The mixtures were stirred at
Chemical shifts are reported in parts per million (ppm), down- room temperature until the reaction completed. The resulting
field from tetramethylsilane. The elemental (C, H, N) analyses reaction mixtures were poured into ice-cold water, neutralised
were performed on a Perkin Elmer model 2400 elemental ana- to pH ∼ 7 and acidified by dropwise addition of a solution
lyzer. IR spectra were recorded on a Perkin Elmer FTIR model of HCl 5% to induce precipitation. The precipitates were fil-
RX1spectrometer(KBr disc, 4000−400 cm−1). Mass spectra tered, dried and re-crystallized in methanol to give the target
with different ionization modes including electron ionization compounds 4-6.
(EI), Electrospray ionization (ESI), were recorded using PE
Biosystems API2000 (Perkin Elmer, Palo Alto, CA, USA)
andMariner®(AzcoBiotech, Inc.Oceanside, CA, USA)mass
spectrometers, respectively. All the reagents (of synthetic
grade with purity of 97% minimum) and solvents (of technical
grade) were purchased from Aldrich, Fluka Chemical Corp.
(Milwaukee, WI, USA) or Merck unless noted otherwise.
Solvents were used directly as purchased unless otherwise
indicated.
2.1.2a N-Hydroxy-3-(4-((3-(hydroxyimino)-2-oxoindo-
lin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propanamide
(4a): Yellow solid; Yield: 64%. M.p.: 185.5–186.0 C. R f
◦
= 0.40 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm−1):
3423 (NH), 3287, 3197 (OH), 3064 (C-H, aren), 2895 (CH,
CH2), 1723, 1672 (C=O), 1609, 1549 (C=C), 1462 (C-N). 1
H-NMR (500 MHz, DMSO-d6 , ppm): δ 10.47 (1H, s, NH);
8.82 (1H, s, OH); 8.02 (1H, s, H-5’); 7.99 (1H, d, J = 7.0 Hz,
H-4”); 7.39 (1H, t, J = 8.0 Hz, H-6”); 7.11 (1H, t, J = 8.5
Hz, H-5”); 7.07 (1H, d, J = 7.5 Hz, H-7”); 4.96 (2H, s, H-6’a,
H-6’b); 4.51 (2H, t, J = 7.0 Hz, H-3a, H-3b); 2.57 (2H, t, J =
7.0 Hz, H-2a, H-2b). 13C NMR (125 MHz, DMSO-d6 , ppm):
δ 165.82, 162.75, 143.39, 142.54, 141.69, 131.88, 126.77,
123.51, 122.69, 115.27, 109.62, 45.58, 34.57, 32.49. HR-MS
(ESI) m/z calculated for C14H14N6O4[M+Na]+ 353.0969.
Found 353.1001.
2.1b General procedures for the synthesis of com-
pounds 4–6: The synthesis of the series of 17 N-
hydroxyalkanamides incorporating 1-((1H-1,2,3-triazol-4-
yl)methyl)-3-hydroxyiminoindolin-2-one moities (4-6) was
carried out as illustrated in Scheme 1. Details are as follows:
To the respective solution of isatins 1a–g (1 mmol) in DMF
(3mL) were added K2CO3 (165.5mg, 1.2 mmol). The result-
ing mixtures were stirred at 80 ◦C for 1 h, then KI (8.3mg,
0.05 mmol) was added. After stirring for further 15 min,
0.15mL of a solution of propargyl bromide 80% in toluene
was added dropwise slowly into the mixtures. The reaction
mixtures were again stirred at 60 ◦C for 3 h. Upon completion
of the reaction, the resulting mixtures were cooled, poured
into ice-cold water and acidified to pH∼4. The orange solids
formed were filtered and dried to give the propargylatedis-
atins 2, which were used for the next step without further
purification.
A respective solution of compounds 2 and methyl azi-
doesters (1 mmol) in acetonitrile (2mL) was stirred at room
temperature for 10 min, then CuI (19.1mg, 0.1 mmol) was
added. The mixture was stirred at 50 ◦C until the reaction
completed (12–24 h). The corresponding resulting mixtures
were evaporated under reduced pressure to give the residues,
which were re-dissolved in 50mL of DCM. The mixtures
were filtered and the DCM layers were evaporated under
2.1.2b 3-(4-((5-Fluoro-3-(hydroxyimino)-2-oxoindolin-
1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxypropan-
amide (4b): Yellow solid; Yield: 63%. M.p.: 187.5–188.5
◦C. R f = 0.42 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr,
cm−1): 3293 (OH), 3069 (C-H, aren), 2921, 2856 (CH, CH2),
1719 (C=O), 1616 (C=C), 1480, 1439 (C-N). 1 H-NMR (500
MHz, DMSO-d6 , ppm): δ 10.38 (1H, s, NH); 8.05 (1H, s, H-
5’); 7.74 (1H, dd, J = 8.0 Hz, Jꢀ = 2.0 Hz, H-4”); 7.28 (1H, td,
J = 8.5 Hz, Jꢀ = 2.0 Hz, H-6”); 7.13 (1H, dd, J = 8.5 Hz, Jꢀ
= 4.0 Hz, H-7”); 4.97 (2H, s, H-6’a, H-6’b); 4.53–4.48 (2H,
m, H-3a, H-3b); 2.86 (1H, t, J = 6.75 Hz, H-2a) 2.57 (2H, t,
J = 6.75 Hz, H-2b). 13C NMR (125 MHz, DMSO-d6 , ppm):
δ 165.85, 162.64, 157.06, 143.07, 141.55, 138.90, 123.62,
118.15, 115.77, 113.78, 110.77, 45.65, 34.72, 32.55. HR-MS
(ESI) m/z calculated for C14H13FN6O4[M-H]− 347.0909.
Found 347.0897.
reduced pressure to give the intermediate esters 3, which 2.1.2c 3-(4-((5-Chloro-3-(hydroxyimino)-2-oxoindolin-
were dissolved in a mixture of methanol and tetrahydrofuran 1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxypropan-
(2/1, 5mL). Then, hydroxylamine.HCl (685mg, 10 mmol) amide (4c): Yellow solid; Yield: 63%. M.p.: 187.0–189.0
was added, followed by dropwise addition of a solution of ◦C. R f = 0.41 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr,

63 Page 4 of 13
J. Chem. Sci. (2018) 130:63
Scheme1. Synthesisofhydroxamicacidsincorporating1-((1H-1,2,3-triazol-4-yl)methyl)-3-hydroxyimino-
indolin-2-ones (4–6).
cm−1): 3420 (NH), 3255, 3151 (OH), 3035 (C-H, aren), 2851 4.98 (2H, s, H-6’a, H-6’b); 4.32–4.29 (2H, m, H-4a, H-4b);
(CH, CH2), 1713, 1647 (C=O), 1609 (C=C), 1464 (C-N).1 1.99–1.92 (4H, m, H-3a, H-3b, H-2a, H-2b). 13C NMR (125
H-NMR (500 MHz, DMSO-d6 , ppm): δ 14.70 (1H, s, NH); MHz, DMSO-d6 , ppm): δ 168.05, 162.64, 157.06, 143.13,
10.48 (1H, s, NH); 8.83 (1H, s, OH); 8.03 (1H, s, H-5’); 141.72, 138.92, 123.43, 118.14, 115.79, 113.80, 110.75,
7.96 (1H, d, J = 1.75 Hz, H-4”); 7.48 (1H, dd, J = 8.5 Hz, 48.91, 34.79, 28.96, 25.77. MS (ESI) m/z361.11 [M-H]−.
Jꢀ = 1.75 Hz, H-6”); 7.14 (1H, d, J = 8.5 Hz, H-7”); 4.98 Anal. Calcd. For C15H15FN6O4 (362.32): C, 49.72; H, 4.17;
(2H, s, H-6’a, H-6’b); 4.52 (2H, t, J = 6.75 Hz, H-3a, H-3b); N, 23.20. Found: C, 49.75; H, 4.21; N, 23.24.
2.57 (2H, t, J = 6.75 Hz, H-2a, H-2b). 13C NMR (125 MHz,
DMSO-d6 , ppm): δ 165.83, 162.40, 142.66, 141.43, 141.31,
131.37, 126.55, 126.08, 123.59, 116.40, 111.27, 45.62, 34.72,
2.1.2f 4-(4-((5-Chloro-3-(hydroxyimino)-2-oxoindolin-
1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxybutan-
32.51. HR-MS (ESI) m/z calculated for C14H13ClN6O4, [M
amide (5c):Yellowsolid;Yield:64%. M.p.:187.5–188.5◦C.
R f = 0.42 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr,
cm−1): 3417 (NH), 3219, 3138 (OH), 3044 (C-H, aren), 2874
+ H]+ 365.0760. Found, 365.0754.
1
2.1.2dN-Hydroxy-4-(4-((3-(hydroxyimino)-2-oxoindol-
in-1-yl)methyl)-1H-1,2,3-triazol-1-yl)butanamide (5a):
Yellow solid; Yield: 68%. M.p.: 181.0–182.5 ◦C. R f = 0.45
(DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm−1):
3135 (OH), 3022 (C-H, aren), 2918, 2856 (CH, CH2), 1721,
1640 (C=O), 1608 (C=C), 1463 (C-N). 1 H-NMR (500 MHz,
DMSO-d6 , ppm): δ 13.58 (1H, s, NH); 10.52 (1H, s, OH);
10.40 (1H, s, NH); 8.12 (1H, s, H-5’); 7.97 (1H, d, J = 7.5 Hz,
H-4”); 7.39 (1H, td, J = 8.0 Hz, Jꢀ = 1.0 Hz, H-6”); 7.11 (1H,
d, J = 7.5 Hz, H-7”); 7.07 (1H, t, J = 7.5 Hz, H-5”); 4.97 (2H,
s, H-6’a, H-6’b); 4.29 (2H, t, J = 6.5 Hz, H-4a, H-4b); 1.99–
1.95 (4H, m, H-3a, H-3b, H-2a, H-2b). 13C NMR (125 MHz,
DMSO-d6 , ppm): δ 168.16, 162.83, 143.36, 142.60, 141.87,
131.94, 126.83, 123.39, 122.77, 115.32, 109.68, 48.93, 34.67,
29.01, 25.85.MS (ESI) m/z343.12 [M-H]−. Anal. Calcd. For
C15H16N6O4 (344.33): C, 52.32; H, 4.68; N, 24.41. Found:
C, 52.37; H, 4.71; N, 24.38.
(CH, CH2), 1720, 1643 (C=O), 1610 (C=C), 1463 (C-N).
H-NMR (500 MHz, DMSO-d6 , ppm): δ 13.82 (1H, s, NH);
10.38 (1H, s, NH); 8.71 (1H, s, OH); 8.10 (1H, s, H-5’); 7.96
(1H, d, J = 1.75 Hz, H-4”); 7.47 (1H, dd, J = 7.5 Hz, Jꢀ =
1.75 Hz, H-6”); 7.14 (1H, d, J = 8.5 Hz, H-7”); 4.98 (2H, s,
H-6’a, H-6’b); 4.30 (2H, t, J = 6.5 Hz, H-4a, H-4b); 2.00–
1.92 (4H, m, H-3a, H-3b, H-2a, H-2b). 13C NMR (125 MHz,
DMSO-d6 , ppm): δ 168.05, 162.41, 142.71, 141.63, 141.38,
131.40, 126.56, 126.12, 123.33, 116.43, 111.28, 48.92, 34.82,
28.96, 25.76. MS (ESI) m/z401.07 [M + Na]+. Anal. Calcd.
For C15H15ClN6O4 (378.77): C, 47.56; H, 3.99; N, 22.19.
Found: C, 47.61; H, 3.95; N, 22.22.
2.1.2g 4-(4-((5-Bromo-3-(hydroxyimino)-2-oxoindolin-
1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxybutan-
amide (5d): Yellow solid; Yield: 68%. M.p.: 188.5–189.5
◦C. R f = 0.47 (DCM : MeOH : AcOH = 90 : 5 : 1). IR
(KBr, cm−1): 3407 (NH), 3138 (OH), 3023 (C-H, aren), 2968,
2.1.2e 4-(4-((5-Fluoro-3-(hydroxyimino)-2-oxoindolin- 2853 (CH, CH2), 1731, 1704 (C=O), 1606 (C=C), 1463 (C-
1
1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxybutan-
amide (5b): Yellow solid; Yield: 72%. M.p.: 180.0–182.0 s, NH); 8.72 (1H, s, OH); 8.10 (1H, s, H-5’); 8.08 (1H, d,
◦C. R f = 0.43 (DCM : MeOH : AcOH = 90 : 5 : 1). IR J = 1.5 Hz, H-4”); 7.60 (1H, dd, J = 8.25 Hz, Jꢀ = 1.5
N). H-NMR (500 MHz, DMSO-d6 , ppm): δ 10.42 (1H,
(KBr, cm−1): 3441 (NH), 3208, 3137 (OH), 3029 (C-H, aren), Hz, H-6”); 7.10 (1H, d, J = 8.25 Hz, H-7”); 4.98 (2H, s,
2872 (CH, CH2), 1722, 1640 (C=O), 1477 (C-N). 1 H-NMR H-6’a, H-6’b); 4.30 (2H, t, J = 6.5 Hz, H-4a, H-4b); 2.00–
(500 MHz, DMSO-d6 , ppm): δ 10.40 (1H, s, NH); 8.72 1.94 (4H, m, H-3a, H-3b, H-2a, H-2b). 13C NMR (125 MHz,
(1H, s, OH); 8.11 (1H, s, H-5’); 7.75 (1H, dd, J = 7.5 Hz, DMSO-d6 , ppm): δ 168.07, 162,34, 142.52, 141.72, 141.60,
Jꢀ = 2.5 Hz, H-4”); 7.28 (1H, td, J = 8.5 Hz, Jꢀ = 2.5 134.17, 128.76, 123.35, 116.88, 114.20, 111.78, 48.92, 34.79,
Hz, H-6”); 7.13 (1H, dd, J = 8.5 Hz, Jꢀ = 4.0 Hz, H-7”); 28.97, 25.78.MS (ESI) m/z421.10 [M-H]−. Anal. Calcd. For

J. Chem. Sci. (2018) 130:63
Page 5 of 13 63
C15H15BrN6O4 (422.23): C, 42.57; H, 3.57; N, 19.86. Found: 3399, 3312 (NH), 3196 (OH), 3059 (C-H, aren), 2876 (CH,
C, 42.59; H, 3.61; N, 19.85.
CH2), 1714, 1678 (C=O), 1608 (C=C), 1455 (C-N). 1 H-NMR
(500 MHz, DMSO-d6 , ppm): δ 13.45 (1H, s, NH); 10.35 (1H,
s, NH); 8.67 (1H, s, OH); 8.09 (1H, s, H-5’); 7.98 (1H, d, J =
7.5 Hz, H-4”); 7.40 (1H, t, J = 7.5 Hz, H-6”); 7.12-7.06 (2H,
m, H-5”, H-7”); 4.98 (2H, s, H-6’a, H-6’b); 4.29 (2H, t, J = 7.0
Hz, H-5a, H-5b); 1.95 (2H, t, J = 7.5 Hz, H-2a, H-2b); 1.79–
2.1.2hN-Hydroxy-4-(4-((3-(hydroxyimino)-5-methyl-2-
oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)butana-
mide (5e): Yellow solid; Yield: 71%. M.p.: 179.5–181.0 ◦C.
R f = 0.41 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr,
cm−1): 3280 (NH), 3184 (OH), 3068 (C-H, aren), 2908 (CH,
CH2), 1713, 1673 (C=O), 1624, 1552 (C=C), 1476 (C-N). 1
H-NMR (500 MHz, DMSO-d6 , ppm): δ 13.44 (1H, s, NH);
10.38 (1H, s, NH); 8.71 (1H, s, OH); 8.08 (1H, s, H-5’);
7.83 (1H, s, H-4”); 7.20 (1H, d, J = 8.0 Hz, H-7”); 6.99
(1H, d, J = 8.0 Hz, H-6”); 4.95 (2H, s, H-6’a, H-6’b);
4.30 (2H, t, J = 6.5 Hz, H-4a, H-4b); 2.27 (3H, s, -CH3);
1.98-1.93 (4H, m, H-3a, H-3b, H-2a, H-2b). 13C NMR (125
MHz, DMSO-d6 , ppm): δ 168.08, 162.77, 143.59, 141.93,
140.39, 132.14, 131.77, 127.38, 123.26, 115.35, 109.42,
48.89, 34.67, 28.97, 25.78, 20.51. HR-MS (ESI) m/z calcu-
lated for C16H18N6O4[M+Na]+ 381.1282. Found 381.1270.
1.71(2H, m, H-4a, H-4b);1.45–1.39(2H, m, H-3a, H-3b). 13
C
NMR (125 MHz, DMSO-d6 , ppm): δ 168.68, 162.74, 143.44,
142.61, 141.80, 131.95, 126.85, 123.26, 122.73, 115.28,
109.64, 49.05, 34.67, 29.22, 22.04. HR-MS (ESI) m/z cal-
culated for C16H18N6O4[M-H]− 357.1317. Found 357.1313.
2.1.2l 5-(4-((5-Fluoro-3-(hydroxyimino)-2-oxoindolin-
1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxypentan-
amide (6b):Yellowsolid;Yield:69%. M.p.:194.5–196.0◦C.
R f = 0.48 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr,
cm−1): 3396, 3320 (NH), 3172 (OH), 3058 (C-H, aren), 2876
1
(CH, CH2), 1715, 1680 (C=O), 1630 (C=C), 1477 (C-N).
H-NMR (500 MHz, DMSO-d6 , ppm): δ 13.76 (1H, s, NH);
10.34 (1H, s, NH); 8.67 (1H, s, OH); 8.08 (1H, s, H-5’); 7.75
(1H, dd, J = 8.0 Hz, Jꢀ = 2.5 Hz, H-4”); 7.28 (1H, td, J =
8.75 Hz, Jꢀ = 2.5 Hz, H-6”); 7.12 (1H, dd, J = 8.75 Hz, Jꢀ =
4.0 Hz, H-7”); 4.98 (2H, s, H-6’a, H-6’b); 4.29 (2H, t, J = 7.0
Hz, H-5a, H-5b); 1.95 (2H, t, J = 7.5 Hz, H-2a, H-2b); 1.76-
1.73 (2H, m, H-4a, H-4b); 1.45–1.40 (2H, m, H-3a, H-3b).
13C NMR (125 MHz, DMSO-d6 , ppm): δ 168.68, 162.61,
158.00, 143.16, 141.64, 138.93, 123.29, 118.15, 115.77,
113.83, 110.72, 49.06, 34.79, 31.51, 29.19, 22.03. HR-MS
(ESI) m/z calculated for C16H17FN6O4[M-H]− 375.1222.
Found 375.1229.
2.1.2i N-Hydroxy-4-(4-((3-(hydroxyimino)-5-methoxy-
2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)butan-
amide (5f):Yellowsolid;Yield:60%. M.p.:180.0–182.0◦C.
R f = 0.45 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm−1):
3281 (NH), 3187 (OH), 3071 (C-H, aren), 2916 (CH, CH2),
1711, 1673 (C=O), 1630, 1596 (C=C), 1552, 1480 (C-N). 1 H-
NMR (500 MHz, DMSO-d6 , ppm): δ 13.53 (1H, s, NH); 10.40
(1H, s, NH); 8.09 (1H, s, H-5’); 7.58 (1H, d, J = 2.5 Hz, H-4”);
7.04–6.98 (2H, m„ H-6”,H-7”); 4.94 (2H, s, H-6’a, H-6’b);
4.30 (2H, t, J = 7.0 Hz, H-4a, H-4b); 3.72 (3H, s, -OCH3);
2.00–1.92 (4H, m, H-3a, H-3b, H-2a, H-2b). 13C NMR (125
MHz, DMSO-d6 , ppm): δ 168.05, 162.62, 155.21, 143.68,
141.94, 136.24, 123.28, 116.86, 115.85, 113.09, 110.26,
55.65, 48.90, 34.70, 28.97, 25.78. HR-MS (ESI) m/z cal-
culated for C16H18N6O5[M-H]− 373.1266. Found 373.1241.
2.1.2m 5-(4-((5-Chloro-3-(hydroxyimino)-2-oxoindoli-
n-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxypenta-
namide (6c): Yellow solid; Yield: 69%. M.p.: 194.5–196.0
◦C. R f = 0.48 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr,
cm−1): 3286 (NH), 3143 (OH), 3042 (C-H, aren), 2851 (CH,
CH2), 1708, 1647 (C=O), 1608 (C=C), 1461 (C-N). 1 H-NMR
(500 MHz, DMSO-d6 , ppm): δ 10.36 (1H, s, NH); 8.68 (1H,
s, OH); 8.08 (1H, s, H-5’); 7.95 (1H, s, H-4”); 7.48 (1H, d,
J = 8.0 Hz, H-6”); 7.14 (1H, d, J = 8.0 Hz, H-7”); 4.98
(2H, s, H-6’a, H-6’b); 4.29 (2H, t, J = 7.0 Hz, H-5a, H-5b);
1.95 (2H, t, J = 7.5 Hz, H-2a, H-2b); 1.77–1.72 (2H, m, H-
4a, H-4b); 1.45–1.40 (2H, m, H-3a, H-3b). 13C NMR (125
MHz, DMSO-d6 , ppm): δ 168.73, 162.44, 142.71, 141.57,
141.38, 131.42, 126.59, 126.14, 123.31, 116.44, 111.29,
49.09, 34.84, 31.53, 29.22, 22.06. HR-MS (ESI) m/z calcu-
lated for C16H17ClN6O4[M-H]− 391.0927. Found 391.0938.
2.1.2j 4-(4-((7-Chloro-3-(hydroxyimino)-2-oxoindolin-
1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxybutan-
amide (5g):Yellowsolid;Yield:70%. M.p.:187.5–188.5◦C.
R f = 0.43 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr,
cm−1): 3517, 3378 (NH), 3248 (OH), 3030 (C-H, aren), 2897
(CH, CH2), 1717, 1664 (C=O), 1634, 1608 (C=C), 1441 (C-
1
N). H-NMR (500 MHz, DMSO-d6 , ppm): δ 10.41 (1H, s,
NH); 8.72 (1H, s, OH); 8.07 (1H, dd, J = 7.75 Hz, Jꢀ = 1.0
Hz, H-4”); 8.06 (1H, s, H-5’); 7.39 (1H, dd, J = 7.75 Hz,
Jꢀ = 1.0 Hz, H-6”); 7.11 (1H, t, J = 7.75 Hz, H-5”); 5.31
(2H, s, H-6’a, H-6’b); 4.29 (2H, t, J = 6.5 Hz, H-4a, H-4b);
2.00–1.92 (4H, m, H-3a, H-3b, H-2a, H-2b). 13C NMR (125
MHz, DMSO-d6 , ppm): δ 168.07, 163.64, 143.38, 142.19,
138.46, 133.78, 125.84, 124.23, 122.49, 118.30, 114.80,
48.88, 37.06, 28.96, 25.84. HR-MS (ESI) m/z calculated for
C15H15ClN6O4[M-H]− 377.0770. Found 377.0748.
2.1.2n 5-(4-((5-Bromo-3-(hydroxyimino)-2-oxoindolin-
1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxypentan-
amide (6d):Yellowsolid;Yield:74%. M.p.:201.5–203.0◦C.
R f = 0.51 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm−1):
2.1.2k N-Hydroxy-5-(4-((3-(hydroxyimino)-2-oxoindo- 3420, 3289 (NH), 3147 (OH), 3031 (C-H, aren), 2862 (CH,
lin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)pentanam◦ide
CH2), 1708, 1646 (C=O), 1606 (C=C), 1462 (C-N). 1 H-NMR
(500 MHz, DMSO-d6 , ppm): δ 10.34 (1H, s, NH); 8.67 (1H,
(6a): Yellow solid; Yield: 68%. M.p.: 189.0–191.5 C. R f
= 0.48 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm−1): s, OH); 8.08 (2H, s, H-5’, H-4”); 7.60 (1H, d, J = 7.75 Hz, H-

63 Page 6 of 13
J. Chem. Sci. (2018) 130:63
6”); 7.09 (1H, d, J = 7.75 Hz, H-7”); 4.98 (2H, s, H-6’a, 143.26, 142.20, 138.45, 133.77, 125.84, 124.22, 122.44,
H-6’b); 4.29 (2H, t, J = 6.0 Hz, H-5a, H-5b); 1.95 (2H, 118.29, 114.80, 49.01, 37.06, 31.50, 29.24, 22.01. HR-MS
t, J = 6.5 Hz, H-2a, H-2b); 1.75–1.73 (2H, m, H-4a, H- (ESI) m/z calculated for C16H17ClN6O4[M-H]− 391.0927.
4b); 1.45–1.41 (2H, m, H-3a, H-3b). 13C NMR (125 MHz, Found 391.0918.
DMSO-d6 , ppm): δ 168.68, 162.31, 142.57, 141.73, 141.54,
134.20, 128.79, 123.27, 116.86, 114.21, 111.76, 49.07,
2.2 Biological evaluation
34.80, 31.51, 29.20, 22.04. HR-MS (ESI) m/z calculated for
C16H17BrN6O4, [M-H]− 437.0422. Found 437.0421.
2.2a Cytotoxicity assay: The cytotoxicity of the synthe-
sized 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
2.1.2oN-Hydroxy-5-(4-((3-(hydroxyimino)-5-methyl-2-
oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)pentan-
amide (6e):Yellowsolid;Yield:75%. M.p.:199.5–201.6◦C.
R f = 0.46 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr,
cm−1): 3317 (NH), 3188 (OH), 3061 (C-H, aren), 2879 (CH,
CH2), 1713, 1680 (C=O), 1622 (C=C), 1460 (C-N). 1 H-NMR
(500 MHz, DMSO-d6 , ppm): δ 13.43 (1H, s, NH); 10.34 (1H,
s, NH); 8.66 (1H, s, OH); 8.06 (1H, s, H-5’); 7.83 (1H, s,
(Dulbecco’s Modified Eagle Medium) until confluence. The
H-4”); 7.21 (1H, s, H-6”); 6.99 (1H, d, J = 6.0 Hz, H-7”);
cells were then trypsinized and suspended at 3 × 104 cells/mL
4.94 (2H, s, H-6’a, H-6’b); 4.29 (2H, s, H-5a, H-5b); 2.27
of cell culture medium. On day 0, each well of the 96-well
(3H, s, -CH3); 1.95 (2H, s, H-2a, H-2b); 1.74 (2H, s, H-
plates was seeded with 180 µL of cell suspension. The plates
4a, H-4b); 1.42 (2H, s, H-3a, H-3b). 13C NMR (125 MHz,
DMSO-d6 , ppm): δ 168.25, 163.25, 144.08, 142.45, 140.86,
were then incubated in a 5% CO2 incubator at 37 ◦C for 24
h. Compounds were initially dissolved in dimethyl sulfoxide
132.61, 132.25, 127.86, 123.74, 115.83, 109.89, 49.54, 35.17,
(DMSO) and diluted to appropriate concentrations by culture
32.00, 29.69, 22.52, 20.97. HR-MS (ESI) m/z calculated for
medium. Then 20 µL of each sample of compounds, which
were prepared as described above, were added to each well
C17H20N6O4, [M-H]− 371.1473. Found 371.1484.
of the 96-well plates, which had been seeded with cell sus-
2.1.2p N-Hydroxy-5-(4-((3-(hydroxyimino)-5-methoxy-
2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)penta-
namide (6f): Yellow solid; Yield: 64%. M.p.: 196.0–197.5
◦C. R f = 0.48 (DCM : MeOH : AcOH = 90 : 5 : 1). IR
(KBr, cm−1): 3421 (NH), 3134 (OH), 3047 (C-H, aren), 2929,
2838 (CH, CH2), 1718, 1628 (C=O), 1598 (C=C), 1480 (C-
pension 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.21 with slight modifications.22–24 The
IC50 values were calculated using a Probits method,25 taking
average values from three independent determinations (SD ≤
10%).
1
N). H-NMR (500 MHz, DMSO-d6 , ppm): δ 10.35 (1H, s,
NH); 8.67 (1H, s, OH); 8.05 (1H, s, H-5’); 7.57 (1H, d, J
= 2.5 Hz, H-4”); 7.03–6.98 (2H, m, H-6”, H-7”); 4.94 (2H,
s, H-6’a, H-6’b); 4.29 (2H, t, J = 7.0 Hz, H-5a, H-5b); 3.72
(3H, s, -OCH3); 1.95 (2H, t, J = 7.0 Hz, H-2a, H-2b); 1.75–
1.72 (2H, m, H-4a, H-4b); 1.45–1.40 (2H, m, H-3a, H-3b).
13C NMR (125 MHz, DMSO-d6 , ppm): δ 168.74, 162.66,
155.25, 143.71, 141.87, 136.25, 123.24, 116.90, 115.87,
113.14, 110.28, 55.68, 49.07, 34.73, 31.54, 29.22, 22.06. HR-
MS (ESI) m/z calculated for C17H20N6O5[M-H]− 387.1422.
Found 387.1440.
2.2b HDAC2 enzyme assay: The HDAC2 enzyme was
purchased from BPS Bioscience (San Diego, CA, USA).
The HDAC enzymatic assay was performed using a Flu-
orogenic 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 ◦C in the presence
of an HDAC fluorimetric substrate. The HDAC assay devel-
oper (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 sub-
tracted by the vehicle-treated control enzyme activities and
IC50values were calculated using GraphPad Prism (Graph-
Pad Software, San Diego, CA, USA).
2.1.2q5-(4-((7-Chloro-3-(hydroxyimino)-2-oxoindolin-
1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-hydroxypentan-
amide (6g):Yellowsolid;Yield:73%. M.p.:198.5–201.0◦C.
R f = 0.47 (DCM : MeOH : AcOH = 90 : 5 : 1). IR (KBr, cm−1):
3422 (NH), 3165 (OH), 3057 (C-H, aren), 2959, 2866 (CH,
CH2), 1724, 1618 (C=O), 1608 (C=C), 1469 (C-N). 1 H-NMR
(500 MHz, DMSO-d6 , ppm): δ 13.83 (1H, s, NH); 10.34 (1H,
s, NH); 8.66 (1H, s, OH); 8.07 (1H, d, J = 7.0 Hz, H-4”); 8.03
(1H, s, H-5’); 7.39 (1H, d, J = 8.0 Hz, H-6”); 7.11 (1H, t, J
2.3 Docking studies
= 7.5 Hz, H-5”); 5.31 (2H, s, H-6’a, H-6’b); 4.29 (2H, t, J = An AutoDockVina program (The Scripps Research Insti-
7.0 Hz, H-5a, H-5b); 1.95 (2H, t, J = 7.25 Hz, H-2a, H-2b); tute, CA, USA)was used for docking.26 The structure of
1.77–1.71 (2H, m, H-4a, H-4b); 145–1.39 (2H, m, H-3a, H- HDAC2 protein in complex with SAHA was obtained from
3b). 13C NMR (125 MHz, DMSO-d6 , ppm): δ 168.67, 163.63, the Protein Data Bank (PDB) (PDB ID: 4LXZ).27 The

J. Chem. Sci. (2018) 130:63
Page 7 of 13 63
coordinates of the compounds were generated by using the This reaction proceeded smoothly in acetonitrile as a
GlycoBioChem PRODRG2 Server (http://davapc1.bioch.dun
dee.ac.uk/prodrg/).28 For the docking studies, the grid maps
were centered on the SAHA binding site and comprised
26 × 26 × 22 points with 1.0 Å spacing after SAHA was
removed from the complex structure, as described in previous
works.17,18,20 The AutoDockVina program was performed
using eight-way multithreading and the other parameters were
default settings.
solvent and copper iodide as a catalyst. In most cases,
the ester intermediates were precipitated by titration of
the reaction residues, which were obtained by evapo-
ration of acetonitrile, with cold water. The final step in
the pathway involved a nucleophilic acyl substitution
of hydroxylamine hydrochloride with the esters 3. This
reaction occurred under alkaline conditions in a solvent
mixture which included tetrahydrofuran and methanol
◦
at 0–5 C. The overall yields of compounds 4-6 were
moderate.
2.4 Prediction of absorption, distribution,
metabolism, elimination, and toxicity (ADMET)
The structures of the synthesized compounds were
determined straightforwardly based on analysis of spec-
In this work ADMET-related properties were computed using
Volsurf+ v.1.0.4 and the Chemistry Development Kit (CDK)
software (http://cdk.sourceforge.net/). Compound topology
was saved as SMILES format (simplified molecular-input
line-entry system) using MarvinSketch and further submitted
to Volsurf and CDK for molecular descriptor computation.
ADMET descriptors comprise Blood-Brain Barrier distribu-
tion (logBB), percentage of protein binding (PB, %), volume
of distribution (VD, L/kg) and metabolic stability against
human CYP3A4 enzyme (MetStab, %). The ability to interact
with cytochrome enzyme isoforms (substrate and/or inhibi-
tion of CYP3A4, 1A2, 2C9, 2C19, and 2D6) was also checked
using admetSAR server developed by Cheng et al.29 Physic-
ochemical descriptors including molecular weight (MW),
partition coefficients (clogP, clogDpH=7.4), total polar surface
1
troscopic data, including IR, MS, H and 13C NMR
(Supporting Information). The concurrent condensation
of the hydroxylamine with the oxo group at position 3
on the indoline ring under the above conditions occurred
in all cases. This condensation has been observed and
fully explained previously.19
3.2 Bioactivity
Due to limited funding resource, in this investigation the
compounds synthesized were preliminarily evaluated
for histone deacetylase inhibition using HDAC2. We
chose HDAC2 because it has been shown that deacety-
lation of a number of key histone proteins is regulated
principally by HDAC2 and HDAC3. HDAC2 has been
demonstrated to play very important role in promoting
cellular proliferation, while inhibiting normal cell apop-
tosis.34 In addition, the compounds were also evaluated
for cytotoxicity against three human cancer cell lines,
including SW620 (colon cancer), PC3 (prostate can-
cer), and AsPC-1 (pancreatic cancer). The results are
presented in Table 1.
When designing these compounds, we initially envi-
sioned that the triazole moiety would add more benefits
in hydrogen bonding with the amino acid regions in
the active binding sites of HDAC, while still serving
as a part of a linker between the 2-oxoindoline and
hydroxamic acid moieties. Due to shorter length of
the C=N and C-N bonds compared to C-C bond, we
area (PSA, Å2), number of hydrogen bond donors and accep-
tors, thermodynamic and pH-dependent aqueous solubility
(mg/ml) were calculated. In addition, 3PRule was applied to
classify permeability across adenocarcinoma (Caco-2) cell
membrane30 - a model commonly used for intestinal absorp-
tion estimation. The P-glycoprotein (P-gp, MDR1) substrate
states were identified using chemoinformatic approaches
developed by Levatic et al.31 (available at http://pgp.biozyne.
com). Lipinski’s Rule of Five (Ro5) was also applied to reveal
drug-likeness of designed compounds.32 Finally, in vivo tox-
icity was predicted by analyzing two risk factors (clogP and
PSA) following the same criteria described by Hughes et al.33
3. Results and Discussion
3.1 Chemistry
The targeted hydroxamic acids 4–6 were synthesized via estimated that if there were only 2C between the tri-
three-step pathway, as illustrated in Scheme 1. The first azole and hydroxamic acids, the linker between the
step was a nucleophilic substitution between isatins 1 2-oxoindoline and hydroxamic acid moieties (in com-
and propargyl bromide under basic conditions (K2CO3) pounds 4a-c) would be shorter than 6C linker in SAHA.
in dimethylformamide (DMF) with a catalytic amount Therefore we decided to design series 5a-g with n =
of KI to furnish 1-propargylisatins 2. In the second 1 as the target compounds. Series 6a-g with one extra
step, a Click reaction between propargyl compounds C was also synthesized to compare with series 5a-g.
2 and respective methyl azidoalkanoates (including The results presented in Table 1 demonstrate that in
methyl 3-azidopropanoate, methyl 4-azidobutanoate, series 5a-g and 6a-g, with the exception of a 5-fluorine
methyl 5-azidovalerate) gave the intermediate esters 3. substituent, other substituents at either position 5 or 7

63 Page 8 of 13
J. Chem. Sci. (2018) 130:63
Table 1. Inhibition of HDAC activity and cytotoxicity of the compounds synthesized against several human
cancer cell lines.
Cpd code
R
n
LogP1 HDAC2 inhibition (IC50,2µM) Cytotoxicity (IC50,2 µM)/Cell lines3
SW620
PC3
AsPC-1
4a
4b
4c
5a
5b
5c
5d
5e
5f
5g
6a
H
5-F
5-Cl
H
0
0
0
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0.41
0.61
1.05
0.90
1.10
1.54
1.79
1.44
0.98
1.54
1.39
1.59
2.03
2.28
1.94
1.47
2.03
1.44
1.70
6.24
2.80
6.16
8.27
1.72
3.53
1.28
0.91
5.08
4.87
26.64
2.65
2.16
3.52
4.15
4.77
1.06
29.0
>30
>30
26.26
23.64
13.46
2.93
0.73
1.61
10.58
>30
>30
9.16
5.64
>30
>30
>30
3.20
>30
>30
>30
>30
>30
16.28
6.08
0.76
1.74
9.27
>30
>30
4.69
3.42
>30
>30
>30
3.70
>30
>30
>30
26.87
>30
11.60
3.01
0.49
1.49
12.90
>30
>30
4.51
4.43
>30
>30
>30
3.75
5-F
5-Cl
5-Br
5-CH3
5-OCH3
7-Cl
-H
6b
6c
6d
6e
5-F
5-Cl
5-Br
5-CH3
5-OCH3
7-Cl
6f
6g
SAHA4
1Calculated by ChemDraw 9.0 software; 2The concentration (µM) of compounds that produces a 50% reduction
in enzyme activity or cell growth, the numbers represent the averaged results from triplicate experiments with
deviation of less than 10%.; 3Cell lines: SW620, colon cancer; PC3, prostate cancer; AsPC-1, pancreatic cancer;
4SAHA, suberoylanilide acid, a positive control.
on the 2-oxoindoline ring generally enhanced HDAC2 inhibitioncomparedtoSAHA, meanwhilecompound5e
inhibition. Also, in overall, series 5a-g inhibited the emerged as the most potential candidate with cytotoxi-
activity of HDAC2 more potently compared to series city or up to 8-fold more potent than SAHA in all three
6a-g. A similar trend was also observed with cytotox- cancer cell lines tested. It could be seen that in these two
icity when comparing series 5a-g and series 6a-g. In compounds the correlation between HDAC2 inhibitory
series 5a-g, it was found that the relationships between potency and cytotoxicity was not finely observed. Some
HDAC2 inhibition and cytotoxicity of the compounds reasons might explain for this discrepancy. First, com-
were relatively well correlated. Compounds 5d and5e, pound 5e could be more potently inhibit other types of
which were the most potent HDAC2 inhibitors in the HDACs of class I and class II, which are also important
series, were also found to be the most cytotoxic against in promoting cell proliferation and inhibiting cellular
all three cancer cell lines tested. In contrast, compounds apoptosis. Second, from logP values, it would be pos-
5b and5g, which were the least potent HDAC2 inhibitors sible that compound 5f (with lower logP value of 0.98)
in the series, were also among the least cytotoxic ones. was not as good as compound 5e (with higher logP value
From the results obtained, it is suggested that, when of 1.44, similar to that of SAHA) in penetrating through
considering the 4-methyl-1H-1,2,3-triazole moiety as cellular membrane.
a part of a linker between the 3-oxoindoline system
and hydroxamic acid group, an alkyl length of three
carbon connecting hydroxamic acid and triazole 1H-
1,2,3-triazole moieties would be most favorable for
bioactivities.
3.3 Docking studies and structure-activity
relationships
Among the compounds synthesized, compound 5f
was found to exhibit similar potency in term of HDAC2
It has been shown that histone-H3 and histone-H4
deacetylation is regulated principally by HDAC2 and

J. Chem. Sci. (2018) 130:63
Page 9 of 13 63
HDAC3.34 and the crystal structure of HDAC2 in Interestingly, methylandmethoxysubstituentsenhanced
complex with SAHA (PDB ID: 4LXZ) has been the interactions between oxoindoline groups and hydro-
reported by Lauffer and co-workers.27 So we decided phobic residues at the rim of the pocket, such as Pro34
to select the structure of SAHA-HDAC2 complex a and Leu276. In these docking experiments, however,
template in docking experiments to study the structure- the indoline part was found to insignificantly interact
activity relationships of these hydroxamic acids and with the enzyme. Subsequently, very little variance in
HDAC. Firstly, a control re-docking with co-crystal the binding affinities among the compounds with differ-
SAHA to the crystal structures of HDAC2 was executed ent substituted groups was observed. The most potent
following the procedures reported previously.17,19,20 The HDAC2 inhibitor compound 5e and 5f, exhibited exten-
results showed very similar interaction pattern between sive hydrogen bonds with His146, Asp181, Glu208 and
re-docked and the original SAHA in the crystal struc- Tyr308.
ture (the all-atom root-mean-square deviation of 0.609
Å). It was found from docking experiments that all of
the hydroxamic acids synthesized were well located in
the active site of the enzyme with binding affinities
3.4 ADMET prediction
(ꢀG) between −6.7 and −8.1 kcal/mol, comparable or Today, it is of great interest for modern medicinal chem-
lower than that of SAHA (Table 2). It is noted that, in istry to use computational tools to predict the ADMET
some instances the predicted binding affinities of the properties in parallel with optimizing the bioactive
compounds were not readily correlated to the experi- potency of novel chemical entities.35 Based on the
mental data obtained from HDAC2 inhibition assay. For cytotoxicity as well as HDAC2 inhibition potency, we
example, the stabilization energies of predicted bind- decided to further investigate the ADMET profiles of
ing modes on HDAC2 of compounds 5b and 5c were six derivatives (5a-f and 6c-d) from synthesized com-
found to be quite similar (−7.7 and −7.8 kcal/mol, pounds using computational approaches.
respectively). These results were not explainable for
Firstly, accomplishment of Lipinski’s Ro5 was
the 5-fold difference in the HDAC2 inhibitory effects checked in order to roundly predict the drug-likeness of
of compounds 5b and 5c. Especially in case of com- chemicals.32 All the compounds exceeded the number
pound 5f, which has the same calculated stabilization of five hydrogen bond acceptors, and predicted as pos-
energies as SAHA (−7.4 kcal/mol) was found to exhibit sible non-oral drug candidates since recent studies have
similar potency compared to SAHA in HDAC2 inhibi- suggested extending the chemical space beyond Ro5 to
tion (IC50 value was 0.91 µM vs. 1.06 µM of SAHA). avoid lost opportunities, especially for new anticancer
Compounds 5e (ꢀG = −8.1 kcal/mol) and5f were two agents.36
most potent in the series in term of HDAC2 inhibi-
As can be appreciated from Table 3, there is no
tion. Interestingly however, the length of aliphatic chain significant difference of ADME properties calculated.
linking hydroxamic group with triazole ring appeared Considering the chemicals as drug-like candidates, we
to be an important factor. There was a remarkable are now discussing some details about the predicted
drop in the docking affinity ranges from butanamide values. Compounds 5e and 5f are very slightly solu-
to pentanamide moieties, which suggested the negative ble in water (solubility ≥0.01mg/mL) while others are
effect of lengthening the aliphatic chain beyond four- identified as insoluble ones.37 Additionally, based on
carbons.
3PRule developed by Pham-The et al.,30 all the com-
From the docking experiments, it was also found that pounds were classified as low intestinal permeability.
a zinc ion (grey sphere), which was coordinated by three As stated previously, low permeable compounds are
residues of HDAC2, including Asp181, His183 and generally absorbed via gastrointestinal tract in a wide
Asp269, interacted with all the hydroxamic acid groups range of extent.37 Interestingly, the poor relationship
in a similar manner as SAHA did. This zinc-chelating between permeability classes and lipophilicity (logP)
functionality has been identified as the dominant fac- suggests that the transport mechanisms of these ana-
tor for inhibitor potency of synthesized hydroxamic logues are others than passive diffusion.30 Other ADME
derivatives. For all the compounds, it was found that a parameters of interest were those related to the first-pass
1-alkyl-4-methyl-1H-1,2,3-triazole linker between the metabolism such as P-gp substrate and metabolic sta-
indoline and hydroxamic acid moieties was tightly bility against cytochrome P450 enzymes. Accordingly,
stacked between Phe155 and Phe210 residues of the all the compounds were identified to be highly resis-
enzymes (Figures 3, 4) and this pi-stacking interac- tant to both P-gp and CYP3A4 metabolism factors. A
tion could be the key factor attributing to the good further inspection of metabolism interactions towards
binding affinities of the compounds with HDAC2. different CYP isoforms revealed that all compounds are

63 Page 10 of 13
J. Chem. Sci. (2018) 130:63
Table 2. Binding affinities of compounds 4-6 towards HDAC2.
Cpd. code Binding
Affinity
HDAC2
inhibition
(IC50, µM)
Cpd. code
Binding
Affinity
(kcal/mol)
HDAC2
inhibition
(IC50, µM)
Cpd. code
Binding
Affinity
(kcal/mol)
HDAC2
inhibition
(IC50, µM)
(kcal/mol)
4a
4b
4c
5a
5b
5c
− 7.4
− 7.6
− 7.7
− 7.5
− 7.7
− 7.8
1.70
6.24
2.80
6.16
8.27
1.72
5d
5e
5f
5g
6a
6b
− 7.8
− 8.1
− 7.4
− 7.9
− 6.7
− 7.0
3.53
1.28
0.91
5.08
4.87
26.64
6c
6d
6e
6f
6g
− 7.1
− 7.0
− 7.4
− 7.4
− 7.2
− 7.4
2.65
2.16
3.52
4.15
4.77
1.06
SAHA
Table 3. Physicochemical and pharmacokinetic profiles of the synthesized compounds.
Cpd. Ro5∗ Solubility1 Caco-2 per- BBB
P-gp substrate4
Metabolism Plasma
Volume of
distribution
binding (%) (l/kg)
(mg/ml)
meability
distribution3
stability5
(%)
protein
class2
5c
5d
5e
5f
6c
6d
1
1
1
1
1
1
5.62 × 10−3
2.88 × 10−3
9.12 × 10−3
2.95 × 10−2
3.02 × 10−3
1.58 × 10−3
Low
Low
Low
Low
Low
Low
Very low
Very low
Very low
Very low
Very low
Very low
No
No
No
No
No
No
100.00
100.00
100.00
100.00
93.78
51.66
55.86
44.10
39.62
55.27
59.42
0.68
0.74
0.67
0.57
0.82
0.85
88.29
1
◦
∗Number of Lipinski Rule of five violations; intrinsic solubility at 25 C calculated by Volsurf+1.0.4 (VolSurf+,
version 1.0.4 edn. available from Molecular Discovery Ltd., London, U.K. (http://www.moldiscovery.com)); 2Caco-2
cell permeability classification using 3PRule:30 High class if Papp ≥ 16 × 106cm/s, Moderate class if 0.7×10−6
≤
Papp <16 × 10−6cm/s; 3Blood-Brain Barrier distribution classes based on Volsurf+1.0.4 logBB descriptor: Moderate
class if 0 ≤ logBB< 0.5, Low class if −0.3 ≤ logBB< 0, and Very low class if logBB< −0.3; 4P-glycoprotein efflux
5
substrate state identified via online server http://pgp.biozyne.com; Metabolic stability in human CYP3A4 enzyme
estimated by Volsurf+1.0.4: a value greater than 50% corresponds to metabolic stable compound.
Figure 3. Stereo-view of the overlapping of the compounds 5a-g and SAHA’s binding modes at the binding
site of HDAC2. Compounds are represented as a stick model. SAHA presented as bold magenta stick.

J. Chem. Sci. (2018) 130:63
Page 11 of 13 63
Figure 4. Stereo-view presentations of the actual binding poses of SAHA and simulated docking poses of
compound 5f to HDAC2. A ligand is represented as a stick model shown in dark-orange and yellow-orange
color, respectively. The most important parts for the enzyme for interaction of these compounds were shown
as a stick model with carbon, nitrogen, and oxygen colored as grey, blue and red, respectively. Zn2+ ion is
shown as a bright gray sphere.
both substrate and inhibitor of CYP3A4. They were also linker showing strong HDAC2 inhibitory effects and
predicted to be inhibitors of CYP2C9 while did not potent cytotoxicity against several human cancer cell
interact with other isoforms. On the other hand, the lines, including SW620 (human colon cancer), PC-
information of plasma protein (mainly albumin) bind- 3 (prostate cancer) and AsPC-1 (pancreas cancer).
ing (PB) and volume of distribution (VD) is helpful for The results we obtained from this study again con-
establishing safety margins and dose regimen of drug firm that 3-hydroxyimino-2-oxoindolines could well
candidates.38 In this aspect, compounds 5e and 5f (with serve as a cap group for hydroxamic acid HDAC
the lowest PB value) could be considered safer than the inhibitors. Also, different substituents on the ben-
others. With respect to the drug distribution parameter, zene ring of the 3-hydroxyimino-2-oxoindoline moi-
all analogues could be moderately distributed in body ety substantially influence both HDAC inhibition and
tissue rather than the plasma since VD > 0.57 l/kg, cytotoxicity of the resulting compounds. Especially,
which is estimated to be total body water volume (> 1-alkyl-4-methyl-1H-1,2,3-triazole linker is tolerable
60% body weight).39 They also displayed a very low for HDAC inhibitory activity. Computational predic-
disposition profile in the central nervous system.40
tions suggested that compounds 5e and 5f, while
Lastly, in vivo toxicity of all the compounds was pre- showing potent HDAC2 inhibitory bioactivity, hold
dicted based on two risk factor approaches developed by desirable ADMET characteristics for anticancer com-
Hughes and colleagues.33 The results indicated that the pounds. From this study, more extensive bioevaluation
calculated properties are within the drug-like range and and ADMET study are being continued to investi-
any compound concurrently presented both risk factors, gate the potentials of these compounds as anticancer
such as logP > 3 and PSA < 75 Å2. Especially, 5e agents.
and 5f have desirable characteristics for anticancer drug
candidate.
Supplementary information (SI)
1
All H NMR, 13C NMR and MS spectra can be found in
Supplementary Information which is available free of charge
at www.ias.ac.in/chemsci.
4. Conclusions
Acknowledgements
In conclusion, we have reported a series of 3-
hydroxyimino-2-oxoindoline-based hydroxamic acids
We acknowledge the principal financial supports from the
incorporated with 1-alkyl-4-methyl-1H-1,2,3-triazole National Foundation for Science and Technology of Vietnam

63 Page 12 of 13
J. Chem. Sci. (2018) 130:63
(NAFOSTED, Grant number 104.01-2017.301). The work 12. Jiyang L, Guangqiang L and Wenqing X 2013 Histone
deacetylase inhibitors: An attractive strategy for cancer
therapy Curr. Med. Chem. 20 1858
13. Ververis K, Hiong A, Karagiannis T C, Licciardi P V
2013 Histone deacetylase inhibitors (HDACIs): Multi-
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was also partly supported by the project No. 2017R1A5A201
5541 (from NRF, Korea, for extra-biology evaluation) and
project No. QG.16.14 (from VNU for L-T-T. Huong to
perform ADMET prediction which was not supported by
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Competing interests
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JSK performed the bioactivity testing assays. P-T.H, L-T-T.H,
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