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Accepted Manuscript

Synthesis and biological evaluation of new C-12(α/β)-(N-) sulfomyl-phenyla-


mino-14-deoxy-andrographolide derivatives as potent anti-cancer agents

Sai Giridhar Sarma Kandanur, Srinivas Nanduri, Nageswara Rao Golakoti

PII: S0960-894X(17)30407-9
DOI: http://dx.doi.org/10.1016/j.bmcl.2017.04.033
Reference: BMCL 24886

To appear in: Bioorganic & Medicinal Chemistry Letters

Received Date: 11 December 2016


Revised Date: 8 February 2017
Accepted Date: 11 April 2017

Please cite this article as: Kandanur, S.G.S., Nanduri, S., Golakoti, N.R., Synthesis and biological evaluation of new
C-12(α/β)-(N-) sulfomyl-phenylamino-14-deoxy-andrographolide derivatives as potent anti-cancer agents,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.04.033

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Synthesis and biological evaluation of new C-12(α/β)-(N-) sulfomyl-phenylamino-14-deoxy-
andrographolide derivatives as potent anti-cancer agents.

Sai Giridhar Sarma Kandanur a, Srinivas Nandurib, Nageswara Rao Golakoti a*


a
Department of Chemistry, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam
Campus, Prasanthi Nilayam-515134, Andhra Pradesh, India.
b
Department of Process Chemistry, National Institute of Pharmaceutical Education and
Research, Balanagar-500037, Hyderabad, Telangana, India.

*Author for Correspondence:Email: gnageswararao@sssihl.edu.in, Tel: +91-9492595462

Sai Giridhar Sarma Kandanur: skandanur@gmail.com, Srinivas Nanduri:


nandurisrini92@gmail.com

1

Abstract

Andrographolide, the major diterpenoidal constituent of Andrographis paniculata (Acanthaceae)


and its derivatives have been reported to possess plethora of biological properties including
potent anti-cancer activity. In this work, synthesis and in-vitro anti-cancer evaluation of new C-
12-substituted aryl amino 14-deoxy-andrographolide derivatives (III a-f) are reported. The
substitutions include various sulfonamide moieties -SO2-NH-R1. The new derivatives (III a-e)
exhibited improved cytotoxicity (GI50,TGI and LC50) compared to andrographolide (I) and the
corresponding 3,14,19-O-triacetyl andrographolide (II) when evaluated against 60 NCI cell line
panel. Compounds III c and III e are found to be non-toxic to normal human dermal fibroblasts
(NHDF) cells compared to reference drug THZ-1.

Keywords: 12-amino-14-deoxy andrographolide; sulfonamide; anti-cancer activity.

[1-15]
Owing to the promising biological activity and structural flexibility, Andrographolide (I)
has been utilized as a starting material for generating a number of structurally diverse derivatives
with interesting biological properties.

Andrographolide has been the focus of research interest for many groups and a number of
derivatives with potent anti-cancer activity have been reported[16-34] apart from our earlier
findings.[21, 35-40] Detailed Structure Activity Relationship (SAR) studies arrived by modification
of the 3,14,19- hydroxyl groups, double bond across C-8,17 and the lactone moiety have been
reported. In our previous communication, we have reported the effect of introducing various
substituents at C-12 on 3,14,19-O-triacetyl andrographolide (II) via a tandem sequence of
Michael addition and rearrangement of the double bond on the cytotoxic activity.[41] Significant
cytotoxic activity exhibited by compound (III r)[41] prompted further synthesis of similar C-12-
substituted aryl amino 14-deoxy-andrographolide derivatives (III a-f) substituted with different
sulfonamide moieties.

2

 

(III r)

Fig 1: Potent compound in our previous report.

A number of derivatives have been synthesized (Scheme 1 and Table 1) and were evaluated for
their cytotoxic activity against a panel of 60 human cancer cell lines at National Cancer Institute
(NCI), U.S.A (complete specification IN-6104/CHE/2014, Filed on 3rd December 2015).

Scheme 1: Synthetic route for the synthesis of C-12-aryl sulfonamide-14-deoxy-andrographolide


derivatives. 

3

Table 1:


Entry R1 Yield m.p.

III a 28.40 % 116-1200C

III b 22.90 % 119-1220C


 III c 16.80 % 118-1230C


III d 20.44 % 124-1280C


III e 20.77 % 117-1210C

III f 21.81 % 110-1160C

These analogues (III a-e) exhibited superior in-vitro cytotoxic activity compared to the
previously reported C-12 arylamino derivatives (12-NH-Ar) on most of the cell lines.[37, 41]

Improvement in cytotoxic activity could be attributed to the presence of sulfonamide substitution


on these analogues (C12-NH-Ar-SO2-NH-R1). Herein, we report the GI50, TGI and LC50 values of
the synthesized derivatives (III a –e). We have also included the previuosly reported results of
3,14,19-O-triacetyl andrographolide (II).[41] and mean values of three dose response parameters
(GI50, TGI and LC50) of andrographolide for comparison.[24] The synthesized derivatives were
purified by column chromatography using appropriate solvent system and characterized by 1H-
NMR,13C-NMR, 2D-NMR (HSQC and COSY), HRMS, IR, & UV spectroscopy.

The aryl sulfonamides were purchased from Sigma-Aldrich and used without further
purification. Solvent N,N,-dimethyl formamide (DMF) of analytical grade was used to carry out
the reactions. (Synthetic procedures are given in detail as supplementary information). All the
synthesized analogues were submitted to National Cancer Institute (NCI), USA for the
evaluation of their in-vitro cytotoxic activity. The selected six compounds (III a-f) were
subjected to preliminary in-vitro screening at a single dose (single dose study; 10 µM) against
NCI 60 cell line panel representing nine human cancer types such as leukemia, melanoma and

4

cancers of lung, colon, brain, breast, ovary, kidney and prostate. The compounds were added at a
concentration of 10 µM and the culture was incubated for 48 h. End point determinations were
determined with a protein binding dye, Sulforhodamine B[43] (Protocol given as supplementary
information). Results of the single dose study (supplementary information) are reported as a
mean graph of the percent growth of the treated cells when compared with untreated control
cells.[43, 44] Compounds II [41]
and III a -III e exhibited promising activity during preliminary
screening (10 µM) on most of the cell lines and hence were selected for further study at five
different concentrations 0.01, 0.1, 1, 10 and 100 µM (five dose study). The results of these tested
compounds are given by three response parameters (GI50: Table 2, TGI: Table 3 and LC50:
Table 4) for each cell line from log concentration versus % growth inhibition curves on nine
cancer disease.GI50 value (growth inhibitory activity) corresponds to the concentration of the
compound causing 50% decrease in net cell growth, TGI value (cytostatic activity) is the
concentration of the compound resulting in total growth inhibition and LC50 value (cytotoxic
activity) is the concentration of the compound causing net 50% loss of initial cells at the end of
the incubation period of 48 h. Compounds showing improved activity on a particular cell line
compared to II are highlighted and underlined in italics (Table 2-4). Furthermore, a mean graph
midpoint (MG-MID) was calculated giving an averaged activity parameter over all cell lines
(Table 5). Compound III f and Andrographolide was not selected for five dose study as it did
not satisfy the predetermined threshold growth inhibition criteria.
In general, IR spectra of all the compounds showed a peak near ~ 3450 cm-1 (νNH ) indicating the
presence of C-12 substitution. IR absorption around ~ 1375 cm-1 and ~ 1133cm-1 is accounted for
asymmetric and symmetric stretch vibration of S=O confirming the presence of sulfonyl group in
the molecule. In 13C-NMR, three peaks observed in the region ~ δ 165-170 ppm confirmed the
presence of carbonyl group (C=O) present in acetate (2 x –OCOCH3) and α,β-unsaturated
13
lactone (C-16). The presence of signals in the region ~δ 104-150 of C-NMR confirmed the
presence of aromatic ring (-NH-Ar-SO2NH-R1). These peaks in the proton NMR spectra were
seen overlapping with aromatic peaks arising out of R1 in the region ~δ 6.10-7.15. The presence
of fragment peaks at 417, 357, 297 in the HRMS can be identified as [M-(NH-Ar-SO2-NH-R1]+,
[M-(NH-Ar-SO2-NH-R1)-AcOH]+, [M-(NH-Ar-SO2-NH-R1-2AcOH]+. The presence of two
signals for most of the carbon peaks in the carbon nmr spectra is indicative that all the
compounds are diastereomers.

5

TABLE 2: Growth inhibition (50%) of the tested compounds against NCI human cancer cell line panel.

GI 50(µM)
Cancer Subpanel II III a III b III c III d III e
CCRF-CEM 1.12 0.47 2.28 0.73 0.0073 0.46
HL-60(TB) 2.61 1.39 3.01 1.30 2.10 1.32
K-562 2.63 1.57 3.60 1.73 2.97 0.52
Leukemia
MOLT-4 3.80 2.12 3.68 2.27 3.22 1.96
RPMI-8226 2.07 1.54 2.49 0.54 1.95 0.44
SR 2.59 0.37 1.33 0.31 0.75 1.35
A549/ATCC 11.90 4.75 3.67 3.18 8.19 2.96
EKVX 12.90 11.00 14.00 8.18 13.90 4.53
HOP-62 4.40 8.88 13.80 3.02 10.30 1.46
HOP-92 13.20 3.07 12.40 3.10 6.24 2.70
NSCLC NCI-H226 2.14 1.81 2.36 1.63 2.33 1.67
NCI-H23 6.22 2.31 10.00 2.11 3.78 2.06
NCI-H322M 16.00 4.68 12.00 3.12 11.30 2.97
NCI-H460 18.60 2.35 10.70 1.98 4.88 2.03
NCI-H522 1.52 1.04 1.72 0.49 1.26 0.75
COLO 205 3.37 1.97 3.14 1.78 2.09 1.46
HCC-2998 12.20 2.14 7.78 2.10 4.98 1.84
HCT-116 1.45 1.12 1.75 0.87 1.60 1.08
Colon HCT-15 1.58 1.50 1.73 1.21 1.79 0.88
HT29 2.58 1.58 2.17 1.03 2.02 1.18
KM 12 10.50 1.83 7.57 2.33 4.59 1.70
SW-620 2.29 1.32 1.85 0.86 1.60 1.07
SF-268 8.56 2.35 11.30 1.93 6.19 1.80
SF-295 13.30 8.13 13.80 6.07 12.40 3.28
SF-539 1.70 1.59 2.35 1.50 2.07 1.23
CNS SNB-19 10.80 3.12 4.36 1.63 6.30 1.91
SNB-75 2.83 2.03 2.83* 1.27 2.69 1.44
U251 6.09 1.89 1.86 1.74 3.17 1.68
LOX IMVI 1.42 1.18 1.59 0.89 1.53 0.29
MALME-3M 2.97 1.89 n.t. n.t. n.t. 1.72
M14 2.22 1.69 2.83 1.44 2.81 1.45
MDA-MB-
Melanoma 2.63 1.74 3.29 1.58 2.92 1.40
435
SK-MEL-2 10.70 2.04 10.60 2.36 6.01 1.77
SK-MEL-28 1.75 2.02 3.95 1.69 2.83 1.68
SK-MEL-5 9.50 2.11 4.79 1.90 3.79 1.60
UACC-257 6.20 2.95 2.55 2.34 4.80 1.67
UACC-62 3.37 2.13 1.76 1.11 2.38 1.49
IGROV1 2.41 2.36 4.54 2.04 3.62 2.08
OVCAR-3 2.17 1.66 1.88 1.21 1.76 1.22
OVCAR-4 3.57 2.91 4.53 1.85 4.37 1.47
Ovarian OVCAR-5 1.93 2.16 4.82 1.98 3.86 2.01
OVCAR-8 3.38 1.88 3.14 1.48 2.61 1.35
NCI/ADR-
4.22 2.41 2.97 1.83 2.94 1.87
RES
SK-OV-3 19.10 11.00 14.40 6.27 12.70 4.05
n.t.- not tested * no improvement in activity

The tested compounds showing improved activity are highlighted and underlined here. The experiment was carried
out using sulphorhodamine B procedure.

6

GI 50(µM) TABLE 2 contd..
Cancer Subpanel II III a III b III c III d III e
786-0 2.02 1.96 2.54 1.77 2.27 1.69
A498 14.40 7.69 18.20 12.30 14.80 1.40
ACHN 1.71 1.71 5.99 1.72 3.64 1.68

Renal CAKI-1 6.67 4.14 3.80 1.74 3.56 2.28


RXF 393 1.95 2.05 2.36 1.95* 2.33 1.77
SN 12C 3.07 1.73 1.87 0.43 2.64 1.25
TK-10 3.81 3.04 9.45 2.77 4.94 2.13
UO-31 1.91 1.69 4.07 1.51 2.91 1.45
PC-3 6.07 1.98 7.74 2.11 4.46 2.24
Prostate
DU-145 4.47 2.43 4.47* 1.62 3.36 1.70
MCF7 1.83 0.59 2.59 1.07 2.27 0.78
MDA-MB-
12.50 2.22 3.30 1.95 3.62 1.90
231/ATCC

Breast HS 578T 12.30 3.92 10.80 3.54 6.65 6.17


BT-549 2.29 1.79 2.43 1.64 2.30 1.58
T-47D 2.86 2.80 3.39 2.00 2.77 1.51
MDA-MB-
2.04 1.49 1.83 1.33 1.99 1.32
468
n.t.- not tested * no improvement in activity

The tested compounds showing improved activity are highlighted and underlined here. The experiment was carried
out using sulphorhodamine B procedure.

7

TABLE 3: Total Growth inhibition by the tested compounds against NCI human cancer cell line panel.

TGI (µM)
Cancer Subpanel II III a III b III c III d III e
CCRF-CEM 5.32 2.71 6.76 3.41 3.09 2.60
HL-60(TB) 6.78 3.69 13.30 3.64 5.63 4.44
K-562 32.10 8.42 29.00 11.70 21.70 5.19
Leukemia
MOLT-4 16.10 6.39 73.20 8.37 17.90 >100.00
RPMI-8226 7.68 5.42 7.62 3.12 6.84 3.04
SR 10.10 3.36 >100.00 17.20 >100.00 8.10
A549/ATCC 30.70 15.40 11.20 9.98 21.90 10.70
EKVX 25.60 25.90 27.00 20.60 27.10 17.00
HOP-62 21.30 22.90 27.50 6.44 22.50 2.99
HOP-92 26.40 8.00 25.30 8.85 18.90 7.89
NSCLC NCI-H226 5.42 3.85 5.37 3.57 5.91 3.30
NCI-H23 23.00 5.90 24.80 5.38 16.00 5.35
NCI-H322M 29.70 16.10 24.90 11.10 23.90 11.60
NCI-H460 37.70 5.70 25.30 4.06 18.30 4.31
NCI-H522 3.63 2.42 4.32 2.07 3.10 2.10
COLO 205 11.40 4.34 10.50 3.80 4.66 3.16
HCC-2998 27.30 4.05 21.30 3.81 17.90 3.86
HCT-116 2.98 2.39 3.75 2.49 3.56 2.32
Colon HCT-15 4.33 3.83 4.12 3.32 4.24 2.30
HT29 n.t. 3.09 4.70 2.79 4.24 2.68
KM 12 27.70 3.69 21.70 5.54 16.40 3.49
SW-620 5.85 2.91 3.98 2.25 3.68 2.80
SF-268 22.70 6.11 25.90 4.16 20.90 3.67
SF-295 26.80 21.00 27.20 18.70 25.60 12.60
SF-539 3.22 3.06 6.31 2.94 4.73 2.63
CNS
SNB-19 24.50 12.20 16.10 3.76 20.70 3.91
SNB-75 14.20 11.50 15.60 3.23 14.90 5.90
U251 19.30 3.40 3.50 3.28 10.70 3.05
LOX IMVI 2.85 2.54 3.31 2.21 3.24 1.19
MALME-3M 7.95 4.00 n.t. n.t. n.t. 3.90
M14 6.48 4.17 10.50 3.98 10.80 3.41
MDA-MB-
10.10 4.37 12.70 3.77 10.40 3.17
435
Melanoma
SK-MEL-2 26.30 4.40 24.30 7.20 20.00 3.99
SK-MEL-28 3.38 4.31 15.10 3.24 10.10 3.46
SK-MEL-5 22.40 4.65 17.10 3.85 14.00 3.13
UACC-257 19.90 6.19 5.61 6.07 16.40 3.89
UACC-62 13.50 7.06 4.42 2.56 7.12 3.86
IGROV1 6.23 6.51 17.10 4.41 13.60 5.00
OVCAR-3 4.21 3.09 3.64 2.54 3.36 2.51
OVCAR-4 15.40 10.50 17.70 5.26 15.80 3.17
OVCAR-5 3.92 4.05 17.60 3.85 12.80 4.16
Ovarian
OVCAR-8 22.00 4.30 10.80 3.55 8.56 3.43
NCI/ADR-
19.80 12.00 12.70 5.28 13.50 6.20
RES
SK-OV-3 36.30 23.10 27.80 18.30 25.30 12.30
n.t.- not tested

The tested compounds showing improved activity are highlighted and underlined here. The experiment was carried
out using sulphorhodamine B procedure.

8

TGI (µM) TABLE 3 contd..
Cancer Subpanel II III a III b III c III d III e
786-0 4.34 3.66 6.74 3.39 5.06 3.14
A498 27.50 20.30 32.60 24.70 28.40 4.26
ACHN 3.22 3.48 18.90 3.27 13.00 3.32

Renal CAKI-1 19.60 14.70 14.20 4.04 12.50 100.00


RXF 393 4.13 3.84 4.74 3.58 4.18 3.29
SN 12C 13.30 4.52 4.39 1.96 6.17 3.66
TK-10 13.30 7.11 21.20 6.03 15.80 4.19
UO-31 3.89 3.52 16.10 2.97 10.60 2.88
PC-3 19.90 4.31 21.50 5.07 15.70 5.25
Prostate
DU-145 14.70 6.53 15.80 3.07 11.80 3.45
MCF7 6.79 2.76 13.60 4.54 12.50 2.90
MDA-MB-
27.10 5.11 8.25 3.93 10.20 3.96
231/ATCC

Breast HS 578T 55.40 26.70 60.40 24.40 43.00 >100.00


BT-549 5.63 3.47 6.50 3.39 5.61 3.14
T-47D 13.80 7.37 20.80 5.46 8.33 4.25
MDA-MB-
5.69 3.29 4.43 3.57 4.30 3.37
468
n.t.- not tested

The tested compounds showing improved activity are highlighted and underlined here. The experiment was carried
out using sulphorhodamine B procedure.

9

TABLE 4: Lethality (50%) by the tested compounds against NCI human cancer cell line panel.

LC 50(µM)
Cancer Subpanel II III a III b III c III d III e
CCRF-CEM > 100.00 > 100.00 > 100.00 > 100.00 > 100.00 > 100.00
HL-60(TB) 78.50 9.82 > 100.00 > 100.00 > 100.00 > 100.00
K-562 > 100.00 > 100.00 > 100.00 > 100.00 > 100.00 > 100.00
Leukemia MOLT-4 > 100.00 > 100.00 > 100.00 > 100.00 > 100.00 > 100.00
RPMI-8226 > 100.00 > 100.00 > 100.00 > 100.00 > 100.00 > 100.00
SR > 100.00 > 100.00 > 100.00 > 100.00 > 100.00 > 100.00
A549/ATCC 79.30 42.20 40.40 34.60 52.20 40.90
EKVX 50.90 60.80 52.00 45.60 52.70 41.30
HOP-62 > 100.00 56.90 54.70 21.50 49.30 6.12
HOP-92 52.70 28.00 52.00 31.10 44.50 27.50
NCI-H226 19.90 n.t. >100.00 n.t. >100.00 n.t.
NSCLC NCI-H23 66.90 31.40 61.60 26.70 50.60 43.20
NCI-H322M 55.30 44.60 51.60 35.60 50.70 36.10
NCI-H460 76.20 27.70 59.90 8.32 53.90 9.15
NCI-H522 8.670 5.61 21.20 6.45 7.62 4.82
COLO 205 > 100.00 9.55 48.90 8.11 11.90 6.86
HCC-2998 61.20 7.64 50.30 6.90 48.10 8.08
HCT-116 6.15 5.10 8.00 6.51 7.96 4.96
Colon HCT-15 65.50 9.74 9.84 9.15 10.20 5.51
HT29 > 100.00 6.04 15.80 n.t. 8.93 n.t.
KM 12 73.40 7.42 53.30 17.80 44.40 7.16
SW-620 41.10 6.44 8.55 5.36 8.48 7.34
SF-268 55.50 32.90 59.10 8.97 54.20 7.51
SF-295 54.20 47.70 53.80 44.60 52.90 35.60
SF-539 6.08 5.89 23.50 5.78 12.40 5.62
CNS SNB-19 55.60 46.40 46.00 8.65 52.70 8.00
SNB-75 37.60 33.90 39.50 8.20 38.60 24.30
U251 48.60 6.12 6.57 6.17 33.3 5.52
LOX IMVI 5.73 5.46 6.92 5.06 6.89 4.17
MALME-3M 29.90 n.t. n.t. n.t. n.t. 8.84
M14 26.50 10.80 42.20 15.90 72.60 8.04
MDA-MB-
Melanoma 32.20 12.50 38.00 9.04 34.20 7.16
435
SK-MEL-2 64.30 9.49 56.10 30.40 52.10 8.97
SK-MEL-28 6.51 9.20 41.80 6.20 36.30 7.13
SK-MEL-5 51.00 11.70 41.30 7.81 41.20 6.13
UACC-257 53.20 18.70 17.20 21.90 42.80 9.03
UACC-62 40.70 30.50 13.50 5.91 30.30 10.00
IGROV1 32.00 76.20 47.50 9.53 45.50 18.30
OVCAR-3 8.15 5.75 7.06 5.31 6.43 5.18
OVCAR-4 41.80 35.10 43.90 19.30 41.40 6.82
Ovarian OVCAR-5 7.97 7.61 46.90 7.47 42.20 8.63
OVCAR-8 > 100.00 9.83 47.70 8.53 47.60 n.t.
NCI/ADR-
> 100.00 > 100.00 > 100.00 > 100.00 > 100.00 >100.00
RES
SK-OV-3 68.80 48.60 53.50 42.80 50.30 35.00
n.t.- not tested

The tested compounds showing improved activity are highlighted and underlined here. The experiment was carried
out using sulphorhodamine B procedure.

10

LC 50(µM) TABLE 4 contd...
Cancer Subpanel II III a III b III c III d III e
786-0 9.29 6.82 23.60 6.51 13.80 5.83
A498 52.60 45.00 58.50 49.90 54.40 15.60
ACHN 6.06 7.05 45.00 6.20 38.40 6.57
Renal
CAKI-1 44.30 38.60 37.70 9.39 35.50 31.70
RXF 393 8.75 7.22 9.53 6.60 7.49 6.14
SN 12C 72.00 20.70 14.30 5.82 81.20 12.70
TK-10 38.20 23.80 46.10 18.30 40.40 8.25
UO-31 7.92 7.33 41.80 5.85 33.30 5.73
PC-3 48.90 9.40 52.20 17.50 43.30 15.80
Prostate
DU-145 38.40 23.00 39.80 5.83 34.90 7.02
MCF7 > 100.00 n.t. > 100.00 > 100.00 > 100.00 n.t.
MDA-MB-
58.70 17.10 37.60 7.91 40.50 8.27
231/ATCC
HS 578T > 100.00 > 100.00 > 100.00 > 100.00 > 100.00 >100.00
Breast BT-549 18.80 6.73 23.10 7.04 18.70 6.28
T-47D > 100.00 > 100.00 > 100.00 > 100.00 > 100.00 >100.00
MDA-MB-
74.40 7.27 14.10 n.t. 9.30 8.62
468
n.t.- not tested

The tested compounds showing improved activity are highlighted and underlined here. The experiment was carried
out using sulphorhodamine B procedure.

11

GI50 of III c in NHDF cells GI50 of III e in NHDF cells

GI50 1536.252
GI50 LC50 3496.358
LC50

nM nM

GI50 of THZ-1in NHDF cells

GI50 46.632
LC50 250.743

nM

Fig 2: Cytotoxicity of III c and III e on Normal Human Dermal Fibroblasts cells.

In tables 2-4, each value represents average activity of the compound across five different
concentrations. All the C-12 derivatives (III a-e) are found to be more potent than II (3,14,19-O-
triacetyl andrographolide) on majority of the cell lines (highlighted and underlined in italics,
Table 2-4). The common structural moiety 12-NH-Ar-SO2-NH-R1 present in all these
derivatives (III a –e) could be attributed to the potent activity exhibited by these derivatives
against many of the cancer cell lines. Among these compounds, variation in activity on specific
cell lines can be accounted due to the structural variant (R1). Following are some of the
significant observations from the above study. Analysis on leukemia cell lines indicate that
compound III d is found to be the most potent compound with a GI50 of 0.0073 µM against
CCRF-CEM cell line. The significant anti-cancer activity exhibited could be attributed to the
presence of merazine unit in III d. This compound is also found to exhibit improved activity on
other leukemia cell lines except K-562. Most of the other compounds III a (GI50: 0.37 to

12

2.12µM), III c (GI50: 0.31 to 2.27µM), III e ( GI50: 0.44 to 1.96 µM) are seen exhibiting potent
activity on all the cell lines of leukemia in comparison with compound II [41] (GI50: 1.12 to 3.80
µM).The LC50 values (Table 4) reveal that compound III a is found to be more potent
(cytocidal) than other derivatives with a LC50 of 9.82 µM against HL-60 (TB) cell line. This
indicates that methoxazole group in III a is responsible for causing 50 % termination (LC50) of
HL-60(TB) cells at concentration <10 µM. Among the 5-membered heterocyclic units,
methoxazole moiety in III a is found to exhibit siginificant activity (GI50 and TGI) when
compared with thiazole moiety in III b. Among the 6-membered heterocycles, chloropyridazine
unit in III e is found to exhibit significant acitivity (GI50 and TGI) when compared with diazine
(III c) and merazine units (III d).Overall, the screening results of compounds on leukemia cell
lines indicate potent growth inhibitory activity (GI50) on SR cell line and cytostatic effect (TGI)
on CCRF-CEM and RPMI-8226 cell lines. Analyzing the results on NSCLC cell lines, all the
tested 6-membered heterocycles (III c, III d , and III e) are observed with good GI50 (0.49 to
1.26 µM), TGI (2.07 to 3.10 µM) and LC50 (4.82 to 7.62 µM) values on NCI-H522 cell line. The
5-memebered methoxazole (III a) has also favoured potent activity on this cell line. The
aforementioned four compounds also showed significant activity on the remaining subpanels
The aryl sulfonamide inclusive of thiazole unit (III b), is seen to be active only on few of the
subpanels. Cell line SW-620 is found to be the most sensitive cell line among colon cancer
subpanels as all the 5-membered and 6-membered units are found to be extremely active.The
SAR on the HCT-15 colon cell line suggests that GI50, TGI and LC50 activity is favoured in the
order of chloropyridazine (III e), > diazine(III c) . methoxazole (III a) units. Among the CNS
subpanels, all the compounds are seen exhibiting promising inhibitory and cytocidal activity on
U-251 cell line. Cell line LOXIMVI of melanoma is seen to be most affected among other
subpanels as III a, III c (except LC50) III e showed good activity(GI50:0.29 to 1.18 µM; and
TGI: 1.19 to 2.54 µM; and LC50: 4.17 to 5.46 µM) on this cell line. Among these derivatives,
potent activity is observed for III e. The Chloropyridazine unit in III e is found to be most
favorable structural moiety among other units for achieving best GI50 and TGI on LOXIMVI
(Melanoma) cell line. For ovarian cancer, all the compounds are seen showing promising activity
(GI50: 1.21 to 1.88 µM; TGI: 2.51 to 3.64 µM and LC50: 5.18 to 7.06 µM) on OVCAR-3 cell line.
The SAR on OVCAR-3 cell line demonstrates that the presence of chloropyridazine group is
responsible for III e showing potent inhibitory activity (LC50, TGI) when compared with other
molecules. On careful analysis of this cell line, SAR reveals that the TGI activity is favoured in
the order chloropyridazine (III e) >diazine (III c) > methoxazole (III a) > merazine (III d) >

13

thiazole (III b) where as LC50 is favoured in the order chloropyridazine (III e) >diazine (III c) >
methoxazole (III a) > merazine (III d) > thiazole (III b). Excellent activity is displayed by
thiazole (III b), diazine (III c) and chloropyridazine (III e) rings on SN12C Renal cell line (TGI:
1.96 to 4.52 µM and LC50: 5.82 to 14.3 µM) among which compound III c is found to be the
most potent. The SAR on this cell line indicates that the activity is favoured in the order diazine
(III c) > chloropyridazine( III e) > thiazole (III b) units. Compounds III c, III d and III e are
found to be active against prostate DU-145 cell line (GI50: 1.62 to3.36 µM, TGI: 3.07 to 3.45 µM
and LC50: 5.83 to 34.9). Compound III b did not exhibit significant activity on prostate cells
whereas III a is seen showing potent GI50 and TGI activity on PC-3 (1.98 to 4.31µM) cell line
and promising activity on DU-145 (23 µM) cell line. Our analysis on prostate cancer concluded
that 12-NH-Ar-SO2-NH-R1-14-deoxy-andrographolide structural unit is more favorable for DU-
145 subpanel as compared to PC-3 cell line. The SAR of DU-145 subpanel indicates that the
activity is highly favored in the order of diazine (III c) >chloropyridazine (III e) > merazine
units (III d). Analyzing the screening results against breast cancer cell lines, good activity is
exhibited by III b and III d on MDA-MB-468 and MDA-MB-231/ATCC cell lines (GI50: 1.83
to 3.62; TGI: 4.30 to 10.20 µM and LC50: 9.30 to 40.50 µM) which indicates that thiazole and
merazine rings are suitable inhibitors of these cell lines. Remaining compounds are found to be
potent inhibitors of MCF-7 and BT-549 cell lines). The overall in-vitro results indicates III c and
III e are found to be the most promising compounds as they exhibited potent activity (GI50, TGI
& LC50) on more than 40 subpanels of different cancer cells. Additionally, compounds III c and
III e were evaluated for their cytotoxicity of on Normal Human Dermal Fibroblast cells
(Protocol given as supplementary information). Compound III c was found to be inactive on this
cell line whereas for compound III e 50% cell death was not observed up to 1536 nM (Fig 2).
The reference drug used for this study THZ-1(covalent CDK7 inhibitor) displayed a GI50 of
46.63 nM.

14

Table 5: Mean values (MG-MID) of dose response parameters of the tested compounds in
the NCI In-vitro screen.

Compound GI50 (µM) TGI (µM) LC50 (µM)


I[24] 16.20 49.70 80.90
II 4.07 11.74 39.80
III a 2.18 5.75 19.90
III b 4.07 12.88 38.01
III c 1.73 4.78 16.59
III d 3.23 10.96 37.15
III e 1.54 4.46 14.45
III r[41] 3.54 10.71 34.67

The above values (Mean Values across all cell lines) supplement our five dose analysis that all
the new C-12 derivatives are observed to have better activity than I[24] and II.[41] Among these
compounds, III c and III e are found to be the most potent.

Mean values (MG-MID; Table 5) represent the overall in-vitro activity across 60 cell lines.
Analysis of Table 5 reveals that III c and III e are the most potent and promising compounds.
This finding is found to be consistent with our earlier analysis on individual cancer cell lines.
These compounds have exhibited similar activity profile which suggests that presence of two or
more similar electronegative atoms in the ring R1 is responsible for favorable activity. The latter
is found to edge out on former and this can be attributed to the presence of electron withdrawing
chlorine. On comparing the compounds containing two different electronegative atoms, the
presence of highly electron-withdrawing oxygen in III a could be a factor in the compound
showing better activity than III b where the presence of sulphur (less electronegative than N,O,
& Cl) in the latter could be responsible for the lowered activity. Compound III b is also found to
be the least active among the tested compounds. On Comparing III c and III d it is observed that
presence of additional lipophilic group/ electron pumping group CH3 in III d (R1) is responsible
for lowered activity as compared to III c barring Leukemia CCRF-CEM cell line where the
former has shown better activity (0.073µM). The overall results (Table 1-4) conclude favorable
activity in the order of chloropyridazine (III e) >diazine (III c) > methoxazole (III a) >
merazine (III d) > thiazole (III b).

The interesting findings of the current investigation prompted us in comparing the activity with
the existing molecules in the C-12 library (in respective cell lines). The GI50 values (0.59-2.59
µM) of these new derivatives (III a-e) on MCF-7 cell line (Breast cancer) when compared with

15

the ED50 (drug concentration causing 50 % growth inhibition) values (4.16-28.91 µM) of the
[42]
derivatives prepared by Kasemsuk et.al. led to the conclusion that the substitution of
sulfonamide moiety (-SO2-NH-R1) to the aniline ring could be responsible for the superior
activity exhibited by all these compounds (III a-e) on breast cancer cell line (MCF-7). This
structural group was also seen playing a crucial role as most of the compounds exhibited better
and potent activity compared to mono/di substituted aryl moieties reported in our earlier
contribution. Potent activity by all the compounds was exhibited on SW-620 & HT-29 (Colon)
and M-14 (Melanoma) cell lines. On other cell lines, most of the compounds exhibited improved
activity.[37] In comparison to our earlier reported work,[41] all the compounds (III a-e) exhibited
superior activity compared to derivatives containing alkyl and benzyl moiety at 10µM. The best
compounds in this present work III c and III e are found to be more potent than previously
reported III q (Thiophenol) and III r[41](aryl sulfonamide).

In conclusion, addition of sulfonamide moiety in C-12 aryl amino andrographolide analogues


resulted in derivatives III a-e showing potent activity at 10 µM and superior activity (five dose
study) on many cancer cell lines compared to andrographolide and triacetyl andrographolide.
The R1 attached to sulfonamide played a very significant role as incorporation of different rings
led to varying activity. On comparison among the derivatives, III e is observed to be the most
potent compound which is slightly better than III c. Compounds III c exhibited no toxicity
towards normal human dermal fibroblasts(NHDF) cells, whereas III e exhibited low toxicity to
NHDF when compared with reference drug THZ-1 . The results from this study will further aid
in development of novel anti-cancer agents based on andrographolide scaffold.

Acknowledgements

We are thankful to Dr. Mohammed Nayel (Project Manager), Developmental Therapeutics


Program (DTP), National Cancer Institute (NCI)/NIH, Chemotherapeutic Agents Repository,
Fisher Bio Services, Rockville, MD, USA, and the team for in-vitro anticancer screening of the
compounds. We also thank Dr.Murali Ramchandra and Dr. Wesley Roy Balasubramanian,
Aurigene Discovery Technologies Limited, for the screening on Normal Human Dermal
Fibroblasts cells. Authors would like to express their deep sense of gratitude to the Founder
Chancellor, Bhagawan Sri Sathya Sai Baba for His constant inspiration and guidance throughout
the work.
Supplementary Information

16

The synthetic procedures, spectral characterization, 1H-NMR and 13
C-NMR spectra, In-vitro
screening protocols and results (One dose data) are given as supplementary information.

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