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α,ß-Didehydrosuberoylanilide hydroxamic acid (DDSAHA) asprecursor and possible analogue of the anticancer drug SAHAShital K. Chattopadhyay*1,§, Subhankar Ghosh1, Sarita Sarkar2 and Kakali Bhadra2

Full Research Paper Open Access

Address:1Department of Chemistry, University of Kalyani, Kalyani - 741235,West Bengal, India and 2Department of Zoology, University ofKalyani, Kalyani - 741235, West Bengal, India

Email:Shital K. Chattopadhyay* - skchatto@yahoo.com

* Corresponding author§ Fax: +91 33 25828282

Keywords:anticancer drug; cross metathesis; HDAC inhibition; hydroxamates;reactive oxygen species

Beilstein J. Org. Chem. 2019, 15, 2524–2533.doi:10.3762/bjoc.15.245

Received: 03 July 2019Accepted: 08 October 2019Published: 24 October 2019

Associate Editor: D. Spring

© 2019 Chattopadhyay et al.; licensee Beilstein-Institut.License and terms: see end of document.

AbstractAn alternate synthetic route to the important anticancer drug suberoylanilide hydroxamic acid (SAHA) from its α,ß-didehydro de-rivative is described. The didehydro derivative is obtained through a cross metathesis reaction between a suitable terminal alkeneand N-benzyloxyacrylamide. Some of the didehydro derivatives of SAHA were preliminarily evaluated for anticancer activitytowards HeLa cells. The administration of the analogues caused a significant decrease in the proliferation of HeLa cells. Further-more, one of the analogues showed a maximum cytotoxicity with a minimum GI50 value of 2.5 µg/mL and the generation of reac-tive oxygen species (ROS) as some apoptotic features.

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IntroductionSuberoylanilide hydroxamic acid (SAHA, 1, Figure 1, vorino-stat [1,2], has now emerged as a FDA approved drug for thetreatment of relapsed and refractory cutaneous T-cell lymphoma(CTCL) [3]. Moreover, it also shows anticancer activity againsta large number of hematological and solid malignancies [4,5].Its anticancer activity is related to inhibition of histone deacety-lase inhibitor (HDACi) at nanomolar concentrations(IC50 < 86 nM). Although, originally recognized as a pan-inhib-itor, recent studies have established that it is unable to inhibitClass IIA lysine deacetylases (HDAC/4/5/79), thus showingsome selectivity profile [6,7]. Moreover, pan-inhibition is also acause of increased concern due to adverse side effects [8]. The

closely related hydroxamic acid derivative belinostat (2) is alsoapproved for the treatment of peripheral T-cell lymphoma(PTCL) [9]. On the other hand, trichostatin A (3), containing anα,ß-unsaturated hydroxamic acid unit is the best known HDACinhibitor which shows antifungal activities [10,11]. Because ofthe impressive level of biological activity, SAHA has receivedconsiderable attention in terms of SAR studies in the quest for aselective inhibitor and/or improved/altered biological activity[12,13]. Many of these studies have shown that minor varia-tions in the structure of SAHA have sometimes led to signifi-cant changes in biological activity [14]. Three syntheses of thisimportant anticancer drug have been described [15-17]. Howev-

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Scheme 1: Synthesis of SAHA and DDSAHA.

er, need for the development of alternative synthetic routesamenable for SAR studies does exist. Although a number ofα,ß-dehydrohydroxamates (e.g., 2 and 3) display an useful levelof bioactivity, α,ß-didehydro SAHA derivatives remain to beexplored, to the best of our knowledge. Herein, we disclose twoalternative synthetic routes to SAHA and preliminary biologi-cal evaluation of some α,ß-didehydro derivatives of SAHA.

Figure 1: Some hydroxamic acid-based anti-tumor drugs.

Results and DiscussionOur synthesis of SAHA and α,ß-dehydro-SAHA derivativesrelied on the identification that a successful cross metathesis be-tween anilide 7a (Scheme 1) containing a terminal double bondand N-benzyloxyacrylamide (8) may provide access to α,ß-dehydro-SAHA derivative 10a, which may serve as precursorof both SAHA and its dehydro derivative 11a [11]. Cross me-tathesis (CM) between a terminal alkene and an α,ß-unsatu-

rated carbonyl compound (or similar electron-deficient alkenes)has been used to prepare functionalized alkenes in several occa-sions [18-20]. We have recently reported the CM of a terminalalkene with a hydroxamic acid derivative [21]. Thus, anilide 7awas prepared by straightforward amide bond formation be-tween aniline and 6-heptenoic acid (5) to study its cross metath-esis with the hydroxamate 8. Pleasingly, use of Grubbs’ secondgeneration catalyst [(1,3-bis(2,4,6-trimethylphenyl)-2-imidazo-lidinylidene)dichloro(phenylmethylene)(tricyclohexylphos-phine)ruthenium, 9a] in dichloromethane at room temperatureprovided the CM-product 10a in a moderate yield of 41%within 16 hours. However, raising the catalyst concentration to5 mol % and employing elevated temperature increased theyield to 56% within 6 hours. Contrarily, the reaction in the pres-ence of Hoveyda-Grubbs 2nd generation catalyst (HG-II, 9b,2 mol %) in refluxing DCM was found to be much faster andbetter yielding (77%). The product 10a was obtained as a singlegeometric isomer identified as E. It may be mentioned that CMwith other unsaturated carbonyl compounds has occasionallyled to a mixture of isomers or even the Z-isomer as the predom-inant one [22]. Hydrogenation of 10a led to saturation of thedouble bond as well as concomitant removal of the O-benzylgroup leading to SAHA (1) in an acceptable overall yield of61% over three steps. On the other hand, removal of theO-benzyl group keeping the double bond intact proved to beproblematic under a range of conditions. Pleasingly, use ofBCl3 in tetrahydrofuran solvent effected the desired transfor-mation but in a modest yield of 51%. Changing the solvent todichloromethane and using a lower temperature proved to bebeneficial and an optimized yield of 89% was achieved afterexperimentation. The dehydro-SAHA derivative 11a was ob-

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Figure 2: Cell viability from MTT assay for SAHA, 11b, 11f and 11g on HeLa after 24 h treatment.

tained as a colourless solid. It also displayed a characteristicsignal broadening in 1H NMR when recorded as dilute solutionin CDCl3 indicating a conformational equilibrium. The use ofDMSO-d6 resulted in a well resolved spectrum. The series ofcompounds 11b–f (Table 1) were similarly obtained using thethree-step sequence detailed for the conversion 4a→11a incomparable overall yields of 57–66%. Short chain SAHA deriv-atives have occasionally displayed biological activity of compa-rable magnitude or even better than SAHA [23]. We thereforeprepared the one-carbon shorter dehydro-SAHA derivative 11gstarting from aniline and 5-hexenoic acid (6) following thethree-step sequence. Compound 11g displayed similar spectros-copic behaviour to that of SAHA.

Table 1: Compounds 4–11.

Entry R n Compound (yield %)

1 H 2 4a, 7a (89), 10a (77), 11a (89)2 F 2 4b, 7b (82), 10b (80), 11b (87)3 Cl 2 4c, 7c (93), 10c (78), 11c (82)4 Br 2 4d, 7d (94), 10d (77), 11d (80)5 I 2 4e, 7e (91), 10e (74), 11e (88)6 OMe 2 4f, 7f (97), 10f (78), 11f (84)7 H 1 4g, 7g (95), 10g (75), 11g (93)

In an alternative approach, cross metathesis of 8 was first donewith methyl heptenoate to prepare compound 12 in good yield.

This was then hydrolyzed to get the corresponding acid 13. Anamide bond formation between this acid and aniline under usualconditions provided DDSAHA derivative 10a in an overallyield of 56% over three steps.

Biological studiesAlthough approved for the treatment of CTCL, SAHA has beenshown to display anticancer activity over a large range of otherhematological and solid malignancies such as leukemia [24],lung cancer [25], cervical cancer (HeLa), breast cancer (MCF-7) [26], mesothelioma [27], B cell lymphoma (A20 cells) [28],and head and neck squamous cell carcinoma (HNSCC cells)[29], among others. The mechanism of biological action ofSAHA in different types of cells is indeed plural in nature.Some of the common cell death pathways are intrinsic andextrinsic apoptosis [30], ROS-facilitated cell death [31,32], andautophagic cell death [33]. We opted to evaluate the possible bi-ological activity of some of the prepared dehydro-SAHA ana-logues along these lines. The studies were conducted with HeLacells to determine the ability of the dehydro analogues to inhibitcell growth, to induce apoptosis, and to induce generation ofROS. Compounds 11b and 11f were arbitrarily chosen, whilethe shorter chain analogue 11g was selected to compare theeffect of chain length, if any. Direct comparison with SAHAwas made to quantify the effects shown by these three com-pounds in each of the experiments under identical conditions.The results are presented in Figure 2.

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Figure 3: Percent of cell death by LDH assay at a GI50 dose of SAHA, 11b, 11f and 11g after 24 h incubation along with cytotoxicity, percentage ofapoptotic and necrotic cell death was also calculated. Data are representative of three independent experiments.

The dose-dependent reduction in the viability of the cells wasobserved and the GI50 values were calculated (Table 2). After24 h treatment, it was found that among the above analogues,11b showed the highest degree of concentration-dependentincrease in growth inhibition on the HeLa cell line, presenting aGI50 value of 8.9 μM (Figure 2) which was even less thanSAHA (12.8 μM). However, for compounds 11f and 11g, theGI50 values were 12.65 and 165 μM, respectively. The data forcompound 11f was similar to that of 11b but clearly the trun-cated analogue 11g is less effective.

Table 2: Percent growth inhibition valuesa at different concentration offour compounds by MTT assay [34].

Name of sample GI50 (µM)

SAHA 12.8511b 8.9211f 12.6511g 165

aData presented from the average of three successive experiments.

Loss of cell viability by LDH assayLDH assays quantitatively measures lactate dehydrogenase(LDH) released into the media from damaged cells as a bio-marker for cellular cytotoxicity [35,36]. Hence, to further char-acterize compound-induced HeLa cell death, the LDH assayemphasizing on apoptotic index parameters were compared inboth untreated (control) and with the compounds treated cells

Table 3: Percent of cell death by LDH assay at GI50 dose after 24 hincubation.

Types of cells % Necrotic % Apoptotic % Viable

control 4.0 ± 0.8 11.0 ± 1.5 85.0 ± 1.5SAHA 4.4 ± 1.1 50.6 ± 1.7 45.0 ± 1.311b 4.3 ± 0.9 53.5 ± 1.2 42.7 ± 1.811f 6.7 ± 1.1 49.8 ± 1.1 43.5 ± 1.411g 20.1 ± 1.4 30.7 ± 1.6 49.2 ± 1.5

aData presented from the average of three successive experiments.

and showed 4.0 ± 0.8% necrotic, 11.0 ± 1.5% apoptotic and85.0 ± 1.5% viable cells in the control cell line, whereas theSAHA treated cell line at GI50 dose showed 4.4 ± 1.1%,50.6 ± 1.7% and 45.0 ± 1.3% necrotic, apoptotic and viablecells, respectively (Figure 3).

Furthermore, treatment with compound 11b gave 4.3 ± 0.9%necrotic, 53.5 ± 1.2% apoptotic and 42.7 ± 1.8% viable cells,while compound 11f showed 6.7 ± 1.1% necrotic, 49.8 ± 1.1%apoptotic and 43.5 ± 1.4% viable cells and with compound 11g,20.1 ± 1.4%, 30.7 ± 1.6% and 49.2 ± 1.5% necrotic, apoptoticand viable cells, respectively. The values have been collected inTable 3. The experimental protocol was repeated with thehuman embryonic liver cell line WRL-68 with compound 11b.It exhibited a minimal effect of cytotoxicity (≈9.1% necrotic aswell as apoptotic cells, respectively), by observing the morphol-ogy of the cells under a microscope at a concentration of15 μg/mL.

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Figure 4: ROS generation by DCFDA.

Table 4: Comparison between quantitative assays of ROSa generation induced by four compounds by DCFH-DA in HeLa cells through flow cyto-metric analysis.

Added concentrations of compounds (μM) Percent ROSSAHA 11b 11f 11g

control 20.3 ± 0.9 20.6 ± 0.8 20.4 ± 0.8 20.4 ± 0.7GI25 32.8 ± 1.2 38.9 ± 1.2 28.7 ± 1.2 30.6 ± 1.6GI50 60.9 ± 1.4 69.4 ± 1.1 52.4 ± 1.5 33.1 ± 1.2GI75 83.4 ± 1.7 90.5 ± 1.5 86.9 ± 1.6 34.5 ± 1.5

aAverage of three individual experiments at same conditions.

ROS generation study by DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) byFACS (fluorescence-activated cell sorting)The results were further complimented with ROS dependentcytotoxicity in the above cell line. ROS generation, induced byvarious anticancer agents plays a key role in apoptosis [37].Cells were treated with SAHA, 11b, 11f and 11g at GI25, GI50

and GI75 concentrations for 24 h and analyzed in the presenceof ROS sensitive probe DCFH-DA (dichlorodihydrofluoresceinhydrate diacetate) by using FACS (Figure 4). Cellular ROSlevels were found to be increased with dose. The mean fluores-cent intensities for four compounds were presented in Table 4and maximum changed intensity for 11b was observed from20.6 ± 0.8 (control) to 90.5 ± 1.5 μM after 24 h treatment, re-

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Figure 5: The quantitative results of bivariate FITC-Annexin V/PI FCM of HeLa cells after treatment with 11b for different concentrations (at 8.9, and14.2 µM, respectively).

spectively. The ROS intensity increased nearly about 4.4-foldrelative to the control. Thus, it may be concluded that 11b in-duced apoptosis in HeLa cells via a ROS-mediated pathway.Henceforth detailed apoptotic induction ability of compound11b has been studied since it was the most cytotoxic among all.

FITC (fluorescein-5-isothiocyanate)–annexinV/PI flow cytometry of HepG2 cellsOn HeLa cells, the efficacy of 11b was measured through theapplication of other apoptotic parameters like phos-phatidylserine (PS) externalization, a known marker of an earlystage of apoptosis following our previously reported procedure[37]. The quantitative results of bivariate FITC-Annexin V/PIFCM of HeLa cells after treatment with 11b at different con-centrations (at 8.9, and 14.2 μM, respectively) are presented inFigure 5 [38]. The lower right quadrant represents the apoptoticcells, FITC–annexin V positive and PI negative, which depictsAnnexin V binding to PS and cytoplasmic membrane integrity.The FITC+/PI− apoptotic cell population increased graduallyfrom 4.6 ± 0.8% of control to 76.8 ± 1.3% after 24 h drug treat-ment which is presented in Table 5. Thus, FITC–Annexin V/PIFCM and LDH assays (vide supra) both supported higher per-centage of apoptosis in comparison to the negligible necrosis inHeLa cells treated with 11b.

Table 5: Percent apoptotic cellsa in presence and absence of 11b byFITC-Annexin-V/PI staining by FACs analysis.

Concentration Percentage of apoptotic cells

control 4.6 ± 0.88.9 µM 47.3 ± 1.514.2 µM 76.8 ± 1.3

aData presented from average of three successive experiments.

Fluorescence microscopic images of Helacells using different staining techniques andDNA ladder formationHela cells with DAPI staining revealed a significant increasein nucleosomal fragmentation and nuclear condensation in11b treated cells with increasing doses (Figure 6 upper panel)[39].

Furthermore, a cellular functional assay by JC-1 probe, avoltage sensitive fluorescent cationic dye, exhibits membranepotential dependent accumulation in mitochondria, indicated bya fluorescence emission shift from red to green. The exposureof 11b to the cells caused remarkable loss of mitochondrialmembrane potential, hence the fluorescence gradually shiftedfrom red to green as the membrane potential (Ψm) decreased(Figure 6 middle panel).

One of the later steps in apoptosis is ultimately the DNA frag-mentation, a process which results from the endonuclease acti-vation during apoptosis. Hence, we further characterized theapoptotic phenomena in HeLa cells by the formation of comettails (Figure 6, lower panel) and DNA gel electrophoresis(Figure 7). Apoptotic hallmark of chromosomal DNA ladderingwas clearly evident in HeLa cells, treated with different dosesof the compound as compared to untreated cells. Thus these twoassays comet formation and DNA laddering indicated DNAdamage during the apoptotic induction.

ConclusionWe have demonstrated two alternative syntheses of the impor-tant anticancer drug SAHA through cross-metathesis reactionwith a hitherto unexplored α,ß-didehydrohydroxamate deriva-tive. This has also led to the synthesis of a series of α,ß-dide-hydo-SAHA derivatives as potential analogues of SAHA. Pre-liminary biological evaluation of three such analogues involv-

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Figure 6: Fluorescence microscopic images of 11b at different concentrations (8.9, and 14.2 µM, respectively) of the HeLa cells after 24 h treatment.HeLa cells with (A) DAPI staining, (B) JC-1 probe and (C) comet assay revealed significant change in control and treated conditions.

ing the measurement of GI50 values against Hela cells andROS-mediated apoptosis as possible mechanism of action ofthese analogues, have shown that the data obtained for onecompound (11b) are very close to those of SAHA for the cellline studied. Further studies are required to fully explore its ac-tivity profile. Moreover, their HDAC inhibitory effects remainto be evaluated. Studies will be continued along these direc-tions [40-44].

ExperimentalProcedure for the synthesis of anilides 7a–gThese were prepared following the procedure described for 7a[45]. DCC (206 mg, 0.7 mmol) was added portionwise to astirred solution of the acid 5 (83 mg, 0.65 mmol) in dry DCM(5 mL) at 0 °C during 15 minutes. Then a solution of aniline(50 mg, 0.54 mmol) and N-methylmorpholine (55 mg, 60 μL,0.54 mmol) in dry DCM (3 mL) was added dropwise over10 min to the solution of the acid at the same temperature. The

reaction mixture was allowed to come to rt and stirred for 16 h.It was then diluted with DCM (20 mL) and the combinedorganic solution was washed successively with a saturatedaqueous solution of NaHCO3 (2 × 15 mL), HCl (2 N,2 × 10 mL), H2O (1 × 20 mL), brine (1 × 20 mL), and thendried (Na2SO4).It was then concentrated under reduced pres-sure and the residue was purified by column chromatography onsilica gel using a mixture of hexane/ethyl acetate (85:15) aseluent to provide the product 7a (98 mg, 89%) as colourlessviscous liquid [45]. IR (neat): 3267, 3076, 1666, 1579 cm−1;1HNMR (400 MHz, CDCl3) δ 8.33 (brs, 1H), 7.56 (d, J = 8 Hz,2H), 7.29 (t, J = 7.6 Hz, 2H), 7.10 (t, J = 7.6 Hz, 1H), 5.84–5.74(m, 1H), 5.03–4.95 (m, 2H), 2.36 (t, J = 7.6 Hz, 2H), 2.06 (q,J = 6.8 Hz, 2H), 1.72 (quin, J = 7.2 Hz, 2H), 1.44 (quin, J =7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 172.1, 138.4,138.2, 128.9, 124.2, 120.2, 114.8, 37.4, 33.5, 28.3, 25.2; anal.calcd for C13H17NO; C, 76.81; H, 8.43; N, 6.89; found: C,76.99; H, 8.68; N, 6.72.

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Figure 7: DNA Ladder formation in a gel electrophoresis study of 11bat different concentrations (at 8.9, and 14.2 µM, respectively) on HeLaafter 24 h treatment.

General procedure for cross metathesisThis was done following our earlier procedure [21]. Grubbs’second generation catalyst HG-II (9b, 5 mg, 2 mol %), wasadded to a stirred solution of the olefin 7a (149 mg, 0.73 mmol)and olefin 8 (66 mg, 0.37 mmol) in anhydrous and degassedDCM (3 mL) at rt and the reaction mixture was heated to refluxfor 3 h under an argon atmosphere. It was allowed to cool toroom temperature and then concentrated in vacuo. The residualmass was purified by column chromatography on silica gel(CHCl3/MeOH 98:2) to provide the coupled product 10a(101 mg, 77%) as colourless solid. Mp 148–150 °C; IR (neat):3296, 3211, 2926, 2858, 1663, 1639 cm−1; 1H NMR (400 MHz,DMSO-d6) δ 11.04 (s, 1H), 9.82 (s, 1H), 7.52 (d, J = 7.6 Hz,2H), 7.31 (brs, 5H), 7.21 (t, J = 8 Hz, 2H), 6.95 (t, J = 7.2 Hz,1H), 6.65 (dt, J = 15.2, 8 Hz, 1H), 5.66 (d, J = 15.2 Hz, 1H),4.75 (s, 2H), 2.24 (t, J = 7.2 Hz, 2H), 2.10 (q, J = 7.6 Hz, 2H),1.52 (quin, J = 7.2 Hz, 2H), 1.35 (quin, J = 7.2 Hz, 2H);13C NMR (100 MHz, DMSO-d6) δ 171.6, 163.3, 144.1, 139.7,136.5, 129.2, 129.1, 128.8, 123.4, 121.4, 119.5, 77.4, 36.6, 31.6,27.8, 25.1; anal. calcd for C21H24N2O3; C, 71.57; H, 6.86; N,7.95; found: C, 71.79; H, 7.01; N, 8.22.

Synthesis of SAHA from DDSAHAFollowing our earlier procedure [21], CM product 10a (50 mg,0.14 mmol) was taken up in MeOH (3 mL) containing 1 drop ofTFA. Then Pd(OH)2-C (10 mg) was added and the solution was

degassed several times. A hydrogen-filled balloon was attachedand the heterogeneous mixture was vigorously stirred at rt for2 h. It was filtered through Celite, the filter cake was washedwith methanol (5 mL) and the combined filtrate was concen-trated in vacuo. The residue was purified by column chromatog-raphy on silica gel (CHCl3/MeOH 9:1) to provide SAHA(34 mg, 91%) as colorless solid. Mp 158–159 °C; IR (neat):3267, 2927, 2854, 1667, 1645 cm–1; 1H NMR (400 MHz,DMSO-d6) δ 10.29 (s, 1H), 9.80 (s, 1H), 8.63 (brs, 1H), 7.51 (d,J = 7.6 Hz, 2H), 7.21 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 7.6 Hz,1H), 2.22 (t, J = 7.6 Hz, 2H), 1.87 (t, J = 7.6 Hz, 2H), 1.51 (q,J = 7.6 Hz, 2H), 1.42 (q, J = 6.8 Hz, 2H), 1.23–1.16 (m, 4H);13C NMR (100 MHz, DMSO-d6) δ 171.7, 169.6, 139.8, 129.1,123.4, 119.5, 36.8, 32.7, 28.9, 25.5.

General procedure for the synthesis of α,ß-didehydro-SAHA derivatives 11a–gA BCl3 solution (0.30 mL, 1 M in heptane, 1.2 equiv) wasadded drop wise to a solution of 10a (90 mg, 0.25 mmol) inDCM (4 mL) at 0 °C under argon and was stirred for 5 minutes.The reaction mixture was allowed to come to rt while stirringand it was further stirred for an additional 2 h at room tempera-ture. The mixture was then quenched with saturated NaHCO3solution (2.5 mL) and extracted with DCM (2 × 25 mL). Theorganic layer was separated, dried over Na2SO4, and concen-trated under reduced pressure. The residue was purified bycolumn chromatography on silica gel using a mixture of CHCl3/MeOH (19:1) to provide the product dehydro-SAHA 11a(59 mg, 89%) as a colourless solid. Mp 154–158 °C; IR (neat):3287, 3193, 2939, 2854, 1670, 1633 cm−1; 1H NMR (400 MHz,DMSO-d6) δ 10.49 (s, 1H), 9.81 (s, 1H), 8.81 (brs, 1H), 7.51 (d,J = 8 Hz, 2H), 7.21 (t, J = 7.6 Hz, 2H), 6.95 (t, J = 7.6 Hz, 1H),6.57 (dt, J = 15.6, 5.6 Hz, 1H), 5.68 (d, J = 15.2 Hz, 1H), 2.24(t, J = 7.2 Hz, 2H), 2.09 (q, J = 7.2 Hz, 2H), 1.52 (quin, J =7.2 Hz, 2H), 1.40–1.32 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 171.6, 163.2, 142.5, 139.7, 129.1, 123.4, 121.8, 119.5,36.6, 31.5, 27.9, 25.1; anal. calcd for C14H18N2O3; C, 64.10; H,6.92; N, 10.68; found: C, 64.24; H, 7.08; N, 10.54.

Percent growth inhibition: MTT assayThe percentage of cell viability and the GI50 (50% growth inhi-bition) values of SAHA, 11b, 11f, 11g for HeLa cells (cervicalcarcinoma) were calculated by MTT (1 mg/mL of the tetra-zolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide dissolved in phosphate-buffered saline, pH 7.4)assay [34]. Cells without any drug treatment were considered ascontrol. The GI50 was calculated by using the following equa-tion.

(1)

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Where, T is the optical density of the test well after 24 hours ofdrug exposure, To is the optical density at time zero and C is theoptical density of control. The experiment was repeated threetimes and the average value was considered.

Loss of cell viability by LDH assayLDH assays quantitatively measures lactate dehydrogenase(LDH) released into the media from damaged cells as a bio-marker for cellular cytotoxicity [35,36]. LDH or lactate dehy-drogenase activity was measured quantitatively for both controland most active 11b treated cells according to a literatureprotocol. The percent apoptotic cells as well as necrotic celldeaths were determined as follows:

(2)

(3)

The floating cells were collected from the culture media bycentrifugation (3000 rpm) at 4 °C for 4 min, and here the LDHcontent from the pellets was determined as an index of apop-totic cell death (LDHp). LDHe was marked as an index ofnecrotic cell death from the released extracellular LDH in theculture supernatant, and the in the adherent viable count LDHiwas marked as intracellular LDH. HeLa cells were seeded in6-well plates at a density of 2 × 104 cells/well and treated with11b of 2.5 and 4.0 μg/mL concentrations at 37 °C for 24 h.

Quantification of ROS by FACSCells (2 × 105) were treated with SAHA, 11b, 11f, 11g at GI25,GI50 and GI75 concentrations for 24 h and the levels of intracel-lular ROS were assessed by flow cytometry after incubatingwith DCFH-DA (25 μM) (2′,7′-dichlorodihydrofluoresceindiacetate) for 30 min at 37 °C as described previously [38].

Estimation of DNA damage (comet assayand DNA gel electrophoresis)Comet assay was performed on HeLa cells by single cell gelelectrophoresis as reported by Liao et al. and the examinationwas performed with a fluorescence microscope [46]. Cometassays consist of mainly the following steps: slide preparationand cell lysis to liberate the DNA, DNA unwinding, electropho-resis, neutralization of the alkali, DNA staining. Dye used forelectrophoresis: From 10 mg/mL stock solution of ethidiumbromide 5 μL was added per 100 mL gel solution for a finalconcentration of 0.5 μg/mL. Comet assays for detection ofDNA damage of both control and 11b treated HeLa cells weredetected by single cell gel electrophoresis.

We isolated DNA of HeLa cells by a standardized salting outmethod according to Miller et al. [47] and performed DNA gelelectrophoresis in 1.2% agarose gel.

Supporting InformationSupporting Information File 1Analytical data of all new compounds as well as copies oftheir 1H and 13C NMR spectra.[https://www.beilstein-journals.org/bjoc/content/supplementary/1860-5397-15-245-S1.pdf]

AcknowledgementsWe are thankful to DST New Delhi, for funds (EMR/2017/001336), and University of Kalyani for assistance.

ORCID® iDsShital K. Chattopadhyay - https://orcid.org/0000-0001-5972-6891

References1. Marks, P. A.; Breslow, R. Nat. Biotechnol. 2007, 25, 84–90.

doi:10.1038/nbt12722. Spratlin, J. L.; Pitts, T. M.; Kulikowski, G. N.; Morelli, M. P.;

Tentler, J. J.; Serkova, N. J.; Eckhardt, S. G. Anticancer Res. 2011, 31,1093–1103.

3. Kavanaugh, S. A.; White, L. A.; Kolesar, J. M.Am. J. Health-Syst. Pharm. 2010, 67, 793–797.doi:10.2146/ajhp090247

4. Kelly, W. K.; O'Connor, O. A.; Krug, L. M.; Chiao, J. H.; Heaney, M.;Curley, T.; MacGregore-Cortelli, B.; Tong, W.; Secrist, J. P.;Schwartz, L.; Richardson, S.; Chu, E.; Olgac, S.; Marks, P. A.;Scher, H.; Richon, V. M. J. Clin. Oncol. 2005, 23, 3923–3931.doi:10.1200/jco.2005.14.167

5. Roche, J.; Bertrand, P. Eur. J. Med. Chem. 2016, 121, 451–483.doi:10.1016/j.ejmech.2016.05.047

6. Bantscheff, M.; Hopf, C.; Savitski, M. M.; Dittmann, A.; Grandi, P.;Michon, A.-M.; Schlegl, J.; Abraham, Y.; Becher, I.; Bergamini, G.;Boesche, M.; Delling, M.; Dümpelfeld, B.; Eberhard, D.;Huthmacher, C.; Mathieson, T.; Poeckel, D.; Reader, V.; Strunk, K.;Sweetman, G.; Kruse, U.; Neubauer, G.; Ramsden, N. G.; Drewes, G.Nat. Biotechnol. 2011, 29, 255–265. doi:10.1038/nbt.1759

7. Bradner, J. E.; West, N.; Grachan, M. L.; Greenberg, E. F.;Haggarty, S. J.; Warnow, T.; Mazitschek, R. Nat. Chem. Biol. 2010, 6,238–243. doi:10.1038/nchembio.313

8. Hornig, E.; Heppt, M. V.; Graf, S. A.; Ruzicka, T.; Berking, C.Exp. Dermatol. 2016, 25, 831–838. doi:10.1111/exd.13089

9. Plumb, J. A.; Finn, P. W.; Williams, R. J.; Bandara, M. J.;Romero, M. R.; Watkins, C. J.; La Thangue, N. B.; Brown, R.Mol. Cancer Ther. 2003, 2, 721–728.

10. Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A.Curr. Opin. Oncol. 2001, 13, 477–483.doi:10.1097/00001622-200111000-00010

11. Sanaei, M.; Kavoosi, F.; Roustazadeh, A.; Golestan, F.J. Clin. Transl. Hepatol. 2018, 6, 141–146.doi:10.14218/jcth.2018.00002

Beilstein J. Org. Chem. 2019, 15, 2524–2533.

2533

12. Walton, J. W.; Cross, J. M.; Riedel, T.; Dyson, P. J.Org. Biomol. Chem. 2017, 15, 9186–9190. doi:10.1039/c7ob02339a

13. Negmeldin, A. T.; Padige, G.; Bieliauskas, A. V.; Pflum, M. K. H.ACS Med. Chem. Lett. 2017, 8, 281–286.doi:10.1021/acsmedchemlett.6b00124

14. Oger, F.; Lecorgne, A.; Sala, E.; Nardese, V.; Demay, F.;Chevance, S.; Desravines, D. C.; Aleksandrova, N.; Guevel, R. L.;Lorenzi, S.; Beccari, A. R.; Barath, P.; Hart, D. J.; Bondon, A.;Carettoni, D.; Simonneaux, G.; Salbert, G. J. Med. Chem. 2010, 53,1937–1950. doi:10.1021/jm901561u

15. Gore, V. G.; Patil, M. S.; Bhalerao, R. A.; Mande, H. M.; Mekde, S. G.Process for the preparation of Vorinostat. U.S. Patent US9,162,974B2,Oct 20, 2015.

16. Stowell, J. C.; Huot, R. I.; Van Voast, L. J. Med. Chem. 1995, 38,1411–1413. doi:10.1021/jm00008a020

17. Gediya, L. K.; Chopra, P.; Purushottamachar, P.; Maheshwari, N.;Njar, V. C. O. J. Med. Chem. 2005, 48, 5047–5051.doi:10.1021/jm058214k

18. O'Leary, D. J.; O'Neil, G. W. Cross-Metathesis. In Handbook ofMetathesis; Grubbs, R. H.; Wenzel, A. G.; O'Leary, D. J.; Khosravi, E.,Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany,2015; Vol. 2, pp 171–294.

19.Żukowska, K.; Grela, K. Cross Metathesis. In Comprehensive OrganicSynthesis II; Knochel, P.; Molander, G. A., Eds.; Elsevier: Amsterdam,Netherlands, 2014; Vol. 5, pp 1257–1301.doi:10.1016/b978-0-08-097742-3.00527-9

20. Zukowska, K.; Grela, K. Cross Metathesis. In Olefin Metathesis-Theoryand Practice; Grela, K., Ed.; Wiley: Hoboken, NJ,, 2014; pp 37–83.doi:10.1002/9781118711613.ch2

21. Chattopadhyay, S. K.; Ghosh, S.; Sil, S. Beilstein J. Org. Chem. 2018,14, 3070–3075. doi:10.3762/bjoc.14.285

22. Bidange, J.; Fischmeister, C.; Bruneau, C.; Dubois, J.-L.;Couturier, J.-L. Monatsh. Chem. 2015, 146, 1107–1113.doi:10.1007/s00706-015-1480-1

23. Fass, D. M.; Shah, R.; Ghosh, B.; Hennig, K.; Norton, S.; Zhao, W.-N.;Reis, S. A.; Klein, P. S.; Mazitschek, R.; Maglathlin, R. L.; Lewis, T. A.;Haggarty, S. J. ACS Med. Chem. Lett. 2011, 2, 39–42.doi:10.1021/ml1001954

24. Garcia-Manero, G.; Yang, H.; Bueso-Ramos, C.; Ferrajoli, A.;Cortes, J.; Wierda, W. G.; Faderl, S.; Koller, C.; Morris, G.; Rosner, G.;Loboda, A.; Fantin, V. R.; Randolph, S. S.; Hardwick, J. S.; Reilly, J. F.;Chen, C.; Ricker, J. L.; Secrist, J. P.; Richon, V. M.; Frankel, S. R.;Kantarjian, H. M. Blood 2008, 111, 1060–1066.doi:10.1182/blood-2007-06-098061

25. Karelia, N.; Desai, D.; Hengst, J. A.; Amin, S.; Rudrabhatla, S. V.;Yun, J. Bioorg. Med. Chem. Lett. 2010, 20, 6816–6819.doi:10.1016/j.bmcl.2010.08.113

26. Vickers, C. J.; Olsen, C. A.; Leman, L. J.; Ghadiri, M. R.ACS Med. Chem. Lett. 2012, 3, 505–508. doi:10.1021/ml300081u

27. Hurwitz, J. L.; Stasik, I.; Kerr, E. M.; Holohan, C.; Redmond, K. M.;McLaughlin, K. M.; Busacca, S.; Barbone, D.; Broaddus, V. C.;Gray, S. G.; O’Byrne, K. J.; Johnston, P. G.; Fennell, D. A.;Longley, D. B. Eur. J. Cancer 2012, 48, 1096–1107.doi:10.1016/j.ejca.2011.11.009

28. Yang, B.; Yu, D.; Liu, J.; Yang, K.; Wu, G.; Liu, H. Tumor Biol. 2015,36, 5051–5061. doi:10.1007/s13277-015-3156-1

29. Gillenwater, A. M.; Zhong, M.; Lotan, R. Mol. Cancer Ther. 2007, 6,2967–2975. doi:10.1158/1535-7163.mct-04-0344

30. Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug Discovery2006, 5, 769–784. doi:10.1038/nrd2133

31. Rosato, R. R.; Grant, S. Expert Opin. Ther. Targets 2005, 9, 809–824.doi:10.1517/14728222.9.4.809

32. Gillenwater, A. M.; Zhong, M.; Lotan, R. Mol. Cancer Ther. 2007, 6,2967–2975. doi:10.1158/1535-7163.mct-04-0344

33. Shao, Y.; Gao, Z.; Marks, P. A.; Jiang, X.Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 18030–18035.doi:10.1073/pnas.0408345102

34. Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.doi:10.1016/0022-1759(83)90303-4

35. Kim, Y.-M.; Talanian, R. V.; Billiar, T. R. J. Biol. Chem. 1997, 272,31138–31148. doi:10.1074/jbc.272.49.31138

36. Smith, S. M.; Wunder, M. B.; Norris, D. A.; Shellman, Y. G. PLoS One2011, 6, e26908. doi:10.1371/journal.pone.0026908

37. Sarkar, S.; Bhattacharjee, P.; Bhadra, K. Chem.-Biol. Interact. 2016,258, 142–152. doi:10.1016/j.cbi.2016.08.024

38. Böhmer, R. H.; Trinkle, L. S.; Staneck, J. L. Cytometry 1992, 13,525–531. doi:10.1002/cyto.990130512

39. Chatterjee, S.; Mallick, S.; Buzzetti, F.; Fiorillo, G.; Syeda, T. M.;Lombardi, P.; Saha, K. D.; Kumar, G. S. RSC Adv. 2015, 5,90632–90644. doi:10.1039/c5ra17214d

40. Pahari, A. K.; Mukherjee, J. P.; Chattopadhyay, S. K. Tetrahedron2014, 70, 7185–7191. doi:10.1016/j.tet.2014.07.045

41. Mukherjee, J. P.; Sil, S.; Chattopadhyay, S. K. Tetrahedron Lett. 2016,57, 739–742. doi:10.1016/j.tetlet.2016.01.005

42. Mukherjee, J. P.; Sil, S.; Pahari, A. K.; Chattopadhyay, S. K. Synthesis2016, 48, 1181–1190. doi:10.1055/s-0035-1561561

43. Mukherjee, J.; Sil, S.; Chattopadhyay, S. K. Beilstein J. Org. Chem.2015, 11, 2487–2492. doi:10.3762/bjoc.11.270

44. Chattopadhyay, S. K.; Sil, S.; Mukherjee, J. P. Beilstein J. Org. Chem.2017, 13, 2153–2156. doi:10.3762/bjoc.13.214

45. Hernandez, E.; Hoberg, H. J. Organomet. Chem. 1987, 328, 403–412.doi:10.1016/0022-328x(87)80256-5

46. Liao, W.; McNutt, M. A.; Zhu, W.-G. Methods 2009, 48, 46–53.doi:10.1016/j.ymeth.2009.02.016

47. Miller, S. A.; Dykes, D. D.; Polesky, H. F. Nucleic Acids Res. 1988, 16,1215. doi:10.1093/nar/16.3.1215

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