α,?-Didehydrosuberoylanilide hydroxamic acid (DDSAHA) as precursor and possible analogue of the anticancer drug SAHA

Article abstract of DOI:10.3762/bjoc.15.245

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

Full text of DOI:10.3762/bjoc.15.245

α,ß-Didehydrosuberoylanilide hydroxamic acid (DDSAHA) as
precursor and possible analogue of the anticancer drug SAHA
Shital K. Chattopadhyay*1,§, Subhankar Ghosh1, Sarita Sarkar2 and Kakali Bhadra2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 2524–2533.
1
Department of Chemistry, University of Kalyani, Kalyani - 741235,
doi:10.3762/bjoc.15.245
West Bengal, India and 2Department of Zoology, University of
Kalyani, Kalyani - 741235, West Bengal, India
Received: 03 July 2019
Accepted: 08 October 2019
Published: 24 October 2019
Email:
Shital K. Chattopadhyay* - skchatto@yahoo.com
Associate Editor: D. Spring
*
§
Corresponding author
Fax: +91 33 25828282
© 2019 Chattopadhyay et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
anticancer drug; cross metathesis; HDAC inhibition; hydroxamates;
reactive oxygen species
Abstract
An 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 alkene
and N-benzyloxyacrylamide. Some of the didehydro derivatives of SAHA were preliminarily evaluated for anticancer activity
towards 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.
Introduction
Suberoylanilide hydroxamic acid (SAHA, 1, Figure 1, vorino- closely related hydroxamic acid derivative belinostat (2) is also
stat [1,2], has now emerged as a FDA approved drug for the approved for the treatment of peripheral T-cell lymphoma
treatment of relapsed and refractory cutaneous T-cell lymphoma (PTCL) [9]. On the other hand, trichostatin A (3), containing an
(
CTCL) [3]. Moreover, it also shows anticancer activity against α,ß-unsaturated hydroxamic acid unit is the best known HDAC
a large number of hematological and solid malignancies [4,5]. inhibitor which shows antifungal activities [10,11]. Because of
Its anticancer activity is related to inhibition of histone deacety- the impressive level of biological activity, SAHA has received
lase inhibitor (HDACi) at nanomolar concentrations considerable attention in terms of SAR studies in the quest for a
(
IC50 < 86 nM). Although, originally recognized as a pan-inhib- selective inhibitor and/or improved/altered biological activity
itor, recent studies have established that it is unable to inhibit [12,13]. Many of these studies have shown that minor varia-
Class IIA lysine deacetylases (HDAC/4/5/79), thus showing tions in the structure of SAHA have sometimes led to signifi-
some selectivity profile [6,7]. Moreover, pan-inhibition is also a cant changes in biological activity [14]. Three syntheses of this
cause of increased concern due to adverse side effects [8]. The important anticancer drug have been described [15-17]. Howev-
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er, need for the development of alternative synthetic routes rated carbonyl compound (or similar electron-deficient alkenes)
amenable for SAR studies does exist. Although a number of has been used to prepare functionalized alkenes in several occa-
α,ß-dehydrohydroxamates (e.g., 2 and 3) display an useful level sions [18-20]. We have recently reported the CM of a terminal
of bioactivity, α,ß-didehydro SAHA derivatives remain to be alkene with a hydroxamic acid derivative [21]. Thus, anilide 7a
explored, to the best of our knowledge. Herein, we disclose two was prepared by straightforward amide bond formation be-
alternative synthetic routes to SAHA and preliminary biologi- tween aniline and 6-heptenoic acid (5) to study its cross metath-
cal evaluation of some α,ß-didehydro derivatives of SAHA.
esis with the hydroxamate 8. Pleasingly, use of Grubbs’ second
generation catalyst [(1,3-bis(2,4,6-trimethylphenyl)-2-imidazo-
lidinylidene)dichloro(phenylmethylene)(tricyclohexylphos-
phine)ruthenium, 9a] in dichloromethane at room temperature
provided the CM-product 10a in a moderate yield of 41%
within 16 hours. However, raising the catalyst concentration to
5
mol % and employing elevated temperature increased the
yield 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 and
better yielding (77%). The product 10a was obtained as a single
geometric isomer identified as E. It may be mentioned that CM
with other unsaturated carbonyl compounds has occasionally
led to a mixture of isomers or even the Z-isomer as the predom-
inant one [22]. Hydrogenation of 10a led to saturation of the
double bond as well as concomitant removal of the O-benzyl
group leading to SAHA (1) in an acceptable overall yield of
61% over three steps. On the other hand, removal of the
Figure 1: Some hydroxamic acid-based anti-tumor drugs.
Results and Discussion
Our synthesis of SAHA and α,ß-dehydro-SAHA derivatives O-benzyl group keeping the double bond intact proved to be
relied on the identification that a successful cross metathesis be- problematic under a range of conditions. Pleasingly, use of
tween anilide 7a (Scheme 1) containing a terminal double bond BCl3 in tetrahydrofuran solvent effected the desired transfor-
and N-benzyloxyacrylamide (8) may provide access to α,ß- mation but in a modest yield of 51%. Changing the solvent to
dehydro-SAHA derivative 10a, which may serve as precursor dichloromethane and using a lower temperature proved to be
of both SAHA and its dehydro derivative 11a [11]. Cross me- beneficial and an optimized yield of 89% was achieved after
tathesis (CM) between a terminal alkene and an α,ß-unsatu- experimentation. The dehydro-SAHA derivative 11a was ob-
Scheme 1: Synthesis of SAHA and DDSAHA.
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tained as a colourless solid. It also displayed a characteristic This was then hydrolyzed to get the corresponding acid 13. An
signal broadening in 1H NMR when recorded as dilute solution amide bond formation between this acid and aniline under usual
in CDCl3 indicating a conformational equilibrium. The use of conditions provided DDSAHA derivative 10a in an overall
DMSO-d6 resulted in a well resolved spectrum. The series of yield of 56% over three steps.
compounds 11b–f (Table 1) were similarly obtained using the
three-step sequence detailed for the conversion 4a→11a in Biological studies
comparable overall yields of 57–66%. Short chain SAHA deriv- Although approved for the treatment of CTCL, SAHA has been
atives have occasionally displayed biological activity of compa- shown to display anticancer activity over a large range of other
rable magnitude or even better than SAHA [23]. We therefore hematological and solid malignancies such as leukemia [24],
prepared the one-carbon shorter dehydro-SAHA derivative 11g lung cancer [25], cervical cancer (HeLa), breast cancer (MCF-
starting from aniline and 5-hexenoic acid (6) following the 7) [26], mesothelioma [27], B cell lymphoma (A20 cells) [28],
three-step sequence. Compound 11g displayed similar spectros- and head and neck squamous cell carcinoma (HNSCC cells)
copic behaviour to that of SAHA.
[29], among others. The mechanism of biological action of
SAHA in different types of cells is indeed plural in nature.
Some of the common cell death pathways are intrinsic and
extrinsic apoptosis [30], ROS-facilitated cell death [31,32], and
autophagic 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 HeLa
cells to determine the ability of the dehydro analogues to inhibit
cell growth, to induce apoptosis, and to induce generation of
ROS. Compounds 11b and 11f were arbitrarily chosen, while
the shorter chain analogue 11g was selected to compare the
effect of chain length, if any. Direct comparison with SAHA
was made to quantify the effects shown by these three com-
Table 1: Compounds 4–11.
Entry
R
n
Compound (yield %)
1
2
3
4
5
6
7
H
F
2
2
2
2
2
2
1
4a, 7a (89), 10a (77), 11a (89)
4b, 7b (82), 10b (80), 11b (87)
4c, 7c (93), 10c (78), 11c (82)
4d, 7d (94), 10d (77), 11d (80)
4e, 7e (91), 10e (74), 11e (88)
4f, 7f (97), 10f (78), 11f (84)
4g, 7g (95), 10g (75), 11g (93)
Cl
Br
I
OMe
H
In an alternative approach, cross metathesis of 8 was first done pounds in each of the experiments under identical conditions.
with methyl heptenoate to prepare compound 12 in good yield. The results are presented in Figure 2.
Figure 2: Cell viability from MTT assay for SAHA, 11b, 11f and 11g on HeLa after 24 h treatment.
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The dose-dependent reduction in the viability of the cells was and showed 4.0 ± 0.8% necrotic, 11.0 ± 1.5% apoptotic and
observed and the GI50 values were calculated (Table 2). After 85.0 ± 1.5% viable cells in the control cell line, whereas the
2
4 h treatment, it was found that among the above analogues, SAHA treated cell line at GI50 dose showed 4.4 ± 1.1%,
1
1b showed the highest degree of concentration-dependent 50.6 ± 1.7% and 45.0 ± 1.3% necrotic, apoptotic and viable
increase in growth inhibition on the HeLa cell line, presenting a cells, respectively (Figure 3).
GI50 value of 8.9 μM (Figure 2) which was even less than
SAHA (12.8 μM). However, for compounds 11f and 11g, the Furthermore, treatment with compound 11b gave 4.3 ± 0.9%
GI50 values were 12.65 and 165 μM, respectively. The data for necrotic, 53.5 ± 1.2% apoptotic and 42.7 ± 1.8% viable cells,
compound 11f was similar to that of 11b but clearly the trun- while compound 11f showed 6.7 ± 1.1% necrotic, 49.8 ± 1.1%
cated analogue 11g is less effective.
apoptotic and 43.5 ± 1.4% viable cells and with compound 11g,
0.1 ± 1.4%, 30.7 ± 1.6% and 49.2 ± 1.5% necrotic, apoptotic
2
and viable cells, respectively. The values have been collected in
Table 3. The experimental protocol was repeated with the
human embryonic liver cell line WRL-68 with compound 11b.
It exhibited a minimal effect of cytotoxicity (≈9.1% necrotic as
well as apoptotic cells, respectively), by observing the morphol-
ogy of the cells under a microscope at a concentration of
Table 2: Percent growth inhibition valuesa at different concentration of
four compounds by MTT assay [34].
Name of sample
SAHA
GI50 (µM)
12.85
8.92
1
1
1
1b
1f
1
5 μg/mL.
12.65
165
1g
aData presented from the average of three successive experiments.
Table 3: Percent of cell death by LDH assay at GI50 dose after 24 h
incubation.
Types of cells
% Necrotic % Apoptotic % Viable
Loss of cell viability by LDH assay
control
SAHA
11b
4.0 ± 0.8
4.4 ± 1.1
4.3 ± 0.9
6.7 ± 1.1
20.1 ± 1.4
11.0 ± 1.5
50.6 ± 1.7
53.5 ± 1.2
49.8 ± 1.1
30.7 ± 1.6
85.0 ± 1.5
45.0 ± 1.3
42.7 ± 1.8
43.5 ± 1.4
49.2 ± 1.5
LDH 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 assay
emphasizing on apoptotic index parameters were compared in
both untreated (control) and with the compounds treated cells
11f
1
1g
aData presented from the average of three successive experiments.
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 of
apoptotic and necrotic cell death was also calculated. Data are representative of three independent experiments.
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ROS generation study by DCFDA (2′,7′-
dichlorodihydrofluorescein diacetate) by
FACS (fluorescence-activated cell sorting)
and GI75 concentrations for 24 h and analyzed in the presence
of ROS sensitive probe DCFH-DA (dichlorodihydrofluorescein
hydrate diacetate) by using FACS (Figure 4). Cellular ROS
The results were further complimented with ROS dependent levels were found to be increased with dose. The mean fluores-
cytotoxicity in the above cell line. ROS generation, induced by cent intensities for four compounds were presented in Table 4
various anticancer agents plays a key role in apoptosis [37]. and maximum changed intensity for 11b was observed from
Cells were treated with SAHA, 11b, 11f and 11g at GI25, GI50 20.6 ± 0.8 (control) to 90.5 ± 1.5 μM after 24 h treatment, re-
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 ROS
SAHA
11b
11f
11g
control
GI25
GI50
20.3 ± 0.9
32.8 ± 1.2
60.9 ± 1.4
83.4 ± 1.7
20.6 ± 0.8
38.9 ± 1.2
69.4 ± 1.1
90.5 ± 1.5
20.4 ± 0.8
28.7 ± 1.2
52.4 ± 1.5
86.9 ± 1.6
20.4 ± 0.7
30.6 ± 1.6
33.1 ± 1.2
34.5 ± 1.5
GI75
aAverage of three individual experiments at same conditions.
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Fluorescence microscopic images of Hela
cells using different staining techniques and
spectively. The ROS intensity increased nearly about 4.4-fold
relative to the control. Thus, it may be concluded that 11b in-
duced apoptosis in HeLa cells via a ROS-mediated pathway. DNA ladder formation
Henceforth detailed apoptotic induction ability of compound Hela cells with DAPI staining revealed a significant increase
1
1b has been studied since it was the most cytotoxic among all. in nucleosomal fragmentation and nuclear condensation in
1b treated cells with increasing doses (Figure 6 upper panel)
[39].
1
FITC (fluorescein-5-isothiocyanate)–annexin
V/PI flow cytometry of HepG2 cells
On HeLa cells, the efficacy of 11b was measured through the Furthermore, a cellular functional assay by JC-1 probe, a
application of other apoptotic parameters like phos- voltage sensitive fluorescent cationic dye, exhibits membrane
phatidylserine (PS) externalization, a known marker of an early potential dependent accumulation in mitochondria, indicated by
stage of apoptosis following our previously reported procedure a fluorescence emission shift from red to green. The exposure
[
37]. The quantitative results of bivariate FITC-Annexin V/PI of 11b to the cells caused remarkable loss of mitochondrial
FCM of HeLa cells after treatment with 11b at different con- membrane potential, hence the fluorescence gradually shifted
centrations (at 8.9, and 14.2 μM, respectively) are presented in from red to green as the membrane potential (Ψm) decreased
Figure 5 [38]. The lower right quadrant represents the apoptotic (Figure 6 middle panel).
cells, FITC–annexin V positive and PI negative, which depicts
Annexin V binding to PS and cytoplasmic membrane integrity. One of the later steps in apoptosis is ultimately the DNA frag-
The FITC+/PI− apoptotic cell population increased gradually mentation, a process which results from the endonuclease acti-
from 4.6 ± 0.8% of control to 76.8 ± 1.3% after 24 h drug treat- vation during apoptosis. Hence, we further characterized the
ment which is presented in Table 5. Thus, FITC–Annexin V/PI apoptotic phenomena in HeLa cells by the formation of comet
FCM and LDH assays (vide supra) both supported higher per- tails (Figure 6, lower panel) and DNA gel electrophoresis
centage of apoptosis in comparison to the negligible necrosis in (Figure 7). Apoptotic hallmark of chromosomal DNA laddering
HeLa cells treated with 11b.
was clearly evident in HeLa cells, treated with different doses
of the compound as compared to untreated cells. Thus these two
assays comet formation and DNA laddering indicated DNA
damage during the apoptotic induction.
Table 5: Percent apoptotic cellsa in presence and absence of 11b by
FITC-Annexin-V/PI staining by FACs analysis.
Concentration
control
Percentage of apoptotic cells
Conclusion
We have demonstrated two alternative syntheses of the impor-
tant anticancer drug SAHA through cross-metathesis reaction
with 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-
4.6 ± 0.8
47.3 ± 1.5
76.8 ± 1.3
8
1
.9 µM
4.2 µM
aData presented from average of three successive experiments.
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, and
4.2 µM, respectively).
1
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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 and reaction mixture was allowed to come to rt and stirred for 16 h.
ROS-mediated apoptosis as possible mechanism of action of It was then diluted with DCM (20 mL) and the combined
these analogues, have shown that the data obtained for one organic solution was washed successively with a saturated
compound (11b) are very close to those of SAHA for the cell aqueous solution of NaHCO3 (2 × 15 mL), HCl (2 N,
line studied. Further studies are required to fully explore its ac- 2 × 10 mL), H2O (1 × 20 mL), brine (1 × 20 mL), and then
tivity profile. Moreover, their HDAC inhibitory effects remain dried (Na2SO4).It was then concentrated under reduced pres-
to be evaluated. Studies will be continued along these direc- sure and the residue was purified by column chromatography on
tions [40-44].
silica gel using a mixture of hexane/ethyl acetate (85:15) as
eluent to provide the product 7a (98 mg, 89%) as colourless
viscous 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,
Experimental
Procedure for the synthesis of anilides 7a–g
These were prepared following the procedure described for 7a 2H), 7.29 (t, J = 7.6 Hz, 2H), 7.10 (t, J = 7.6 Hz, 1H), 5.84–5.74
[
45]. DCC (206 mg, 0.7 mmol) was added portionwise to a (m, 1H), 5.03–4.95 (m, 2H), 2.36 (t, J = 7.6 Hz, 2H), 2.06 (q,
stirred solution of the acid 5 (83 mg, 0.65 mmol) in dry DCM J = 6.8 Hz, 2H), 1.72 (quin, J = 7.2 Hz, 2H), 1.44 (quin, J =
(
(
5 mL) at 0 °C during 15 minutes. Then a solution of aniline 7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 172.1, 138.4,
50 mg, 0.54 mmol) and N-methylmorpholine (55 mg, 60 μL, 138.2, 128.9, 124.2, 120.2, 114.8, 37.4, 33.5, 28.3, 25.2; anal.
0
1
.54 mmol) in dry DCM (3 mL) was added dropwise over calcd for C13H17NO; C, 76.81; H, 8.43; N, 6.89; found: C,
0 min to the solution of the acid at the same temperature. The 76.99; H, 8.68; N, 6.72.
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degassed several times. A hydrogen-filled balloon was attached
and the heterogeneous mixture was vigorously stirred at rt for
2
h. It was filtered through Celite, the filter cake was washed
with 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
(
3
34 mg, 91%) as colorless solid. Mp 158–159 °C; IR (neat):
267, 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,
1
H), 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);
3C NMR (100 MHz, DMSO-d6) δ 171.7, 169.6, 139.8, 129.1,
23.4, 119.5, 36.8, 32.7, 28.9, 25.5.
1
1
General procedure for the synthesis of α,ß-
didehydro-SAHA derivatives 11a–g
A BCl3 solution (0.30 mL, 1 M in heptane, 1.2 equiv) was
added drop wise to a solution of 10a (90 mg, 0.25 mmol) in
DCM (4 mL) at 0 °C under argon and was stirred for 5 minutes.
The reaction mixture was allowed to come to rt while stirring
and it was further stirred for an additional 2 h at room tempera-
ture. The mixture was then quenched with saturated NaHCO3
solution (2.5 mL) and extracted with DCM (2 × 25 mL). The
organic layer was separated, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified by
Figure 7: DNA Ladder formation in a gel electrophoresis study of 11b
at different concentrations (at 8.9, and 14.2 µM, respectively) on HeLa
after 24 h treatment.
General procedure for cross metathesis
This was done following our earlier procedure [21]. Grubbs’ column chromatography on silica gel using a mixture of CHCl3/
second generation catalyst HG-II (9b, 5 mg, 2 mol %), was MeOH (19:1) to provide the product dehydro-SAHA 11a
added to a stirred solution of the olefin 7a (149 mg, 0.73 mmol) (59 mg, 89%) as a colourless solid. Mp 154–158 °C; IR (neat):
and olefin 8 (66 mg, 0.37 mmol) in anhydrous and degassed 3287, 3193, 2939, 2854, 1670, 1633 cm−1; 1H NMR (400 MHz,
DCM (3 mL) at rt and the reaction mixture was heated to reflux DMSO-d6) δ 10.49 (s, 1H), 9.81 (s, 1H), 8.81 (brs, 1H), 7.51 (d,
for 3 h under an argon atmosphere. It was allowed to cool to J = 8 Hz, 2H), 7.21 (t, J = 7.6 Hz, 2H), 6.95 (t, J = 7.6 Hz, 1H),
room temperature and then concentrated in vacuo. The residual 6.57 (dt, J = 15.6, 5.6 Hz, 1H), 5.68 (d, J = 15.2 Hz, 1H), 2.24
mass was purified by column chromatography on silica gel (t, J = 7.2 Hz, 2H), 2.09 (q, J = 7.2 Hz, 2H), 1.52 (quin, J =
(
(
CHCl3/MeOH 98:2) to provide the coupled product 10a 7.2 Hz, 2H), 1.40–1.32 (m, 2H); 13C NMR (100 MHz, DMSO-
101 mg, 77%) as colourless solid. Mp 148–150 °C; IR (neat): d6) δ 171.6, 163.2, 142.5, 139.7, 129.1, 123.4, 121.8, 119.5,
3296, 3211, 2926, 2858, 1663, 1639 cm−1; 1H NMR (400 MHz, 36.6, 31.5, 27.9, 25.1; anal. calcd for C14H18N2O3; C, 64.10; H,
DMSO-d6) δ 11.04 (s, 1H), 9.82 (s, 1H), 7.52 (d, J = 7.6 Hz, 6.92; N, 10.68; found: C, 64.24; H, 7.08; N, 10.54.
2
1
4
1
H), 7.31 (brs, 5H), 7.21 (t, J = 8 Hz, 2H), 6.95 (t, J = 7.2 Hz,
H), 6.65 (dt, J = 15.2, 8 Hz, 1H), 5.66 (d, J = 15.2 Hz, 1H), Percent growth inhibition: MTT assay
.75 (s, 2H), 2.24 (t, J = 7.2 Hz, 2H), 2.10 (q, J = 7.6 Hz, 2H), The percentage of cell viability and the GI50 (50% growth inhi-
.52 (quin, J = 7.2 Hz, 2H), 1.35 (quin, J = 7.2 Hz, 2H); bition) values of SAHA, 11b, 11f, 11g for HeLa cells (cervical
1
3C NMR (100 MHz, DMSO-d6) δ 171.6, 163.3, 144.1, 139.7, carcinoma) were calculated by MTT (1 mg/mL of the tetra-
1
2
7
36.5, 129.2, 129.1, 128.8, 123.4, 121.4, 119.5, 77.4, 36.6, 31.6, zolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
7.8, 25.1; anal. calcd for C21H24N2O3; C, 71.57; H, 6.86; N, zolium bromide dissolved in phosphate-buffered saline, pH 7.4)
.95; found: C, 71.79; H, 7.01; N, 8.22.
assay [34]. Cells without any drug treatment were considered as
control. The GI50 was calculated by using the following equa-
tion.
Synthesis of SAHA from DDSAHA
Following our earlier procedure [21], CM product 10a (50 mg,
0
.14 mmol) was taken up in MeOH (3 mL) containing 1 drop of
(
1)
TFA. Then Pd(OH)2-C (10 mg) was added and the solution was
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Beilstein J. Org. Chem. 2019, 15, 2524–2533.
Where, T is the optical density of the test well after 24 hours of We isolated DNA of HeLa cells by a standardized salting out
drug exposure, To is the optical density at time zero and C is the method according to Miller et al. [47] and performed DNA gel
optical density of control. The experiment was repeated three electrophoresis in 1.2% agarose gel.
times and the average value was considered.
Supporting Information
Loss of cell viability by LDH assay
LDH assays quantitatively measures lactate dehydrogenase
Supporting Information File 1
Analytical data of all new compounds as well as copies of
their 1H and 13C NMR spectra.
(
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 control
and most active 11b treated cells according to a literature
protocol. The percent apoptotic cells as well as necrotic cell
deaths were determined as follows:
[
https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-15-245-S1.pdf]
Acknowledgements
We are thankful to DST New Delhi, for funds (EMR/2017/
(
(
2)
3)
0
01336), and University of Kalyani for assistance.
ORCID® iDs
Shital K. Chattopadhyay - https://orcid.org/0000-0001-5972-6891
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