Synthesis of new indirubin derivatives and their in vitro anticancer activity

Article abstract of DOI:10.1007/s11696-018-0659-4

The opening of epoxy rings from (2′Z)-N-1-(oxiran-2-ylmethyl)indirubin (2) and (2′Z-3′E)-indirubin-3?-[O-oxiran-2-ylmethyl)oxime] (6) with thiols gave 17 new derivatives of indirubin in good yields. Their structures were elucidated by 1D-, 2D-NMR and HRMS

Full text of DOI:10.1007/s11696-018-0659-4

Chemical Papers
https://doi.org/10.1007/s11696-018-0659-4
ORIGINAL PAPER
Synthesis of new indirubin derivatives and their in vitro anticancer
activity
1
1
1
2
2
Dan Trong Nguyen · Giang Nguyen Truong · Truong Van Vuong · Tai Nguyen Van · Cuong Nguyen Manh ·
2
2
2,3
4
Cuong To Dao · Thuy Dinh Thi Thuy · Chinh Luu Van · Vu Tran Khac
Received: 27 August 2018 / Accepted: 27 November 2018
©
Institute of Chemistry, Slovak Academy of Sciences 2018
Abstract
The opening of epoxy rings from (2′Z)-N-1-(oxiran-2-ylmethyl)indirubin (2) and (2′Z-3′E)-indirubin-3ʹ-[O-oxiran-2-ylmethyl)
oxime] (6) with thiols gave 17 new derivatives of indirubin in good yields. Their structures were elucidated by 1D-, 2D-NMR
and HRMS spectra. Screening for anticancer activity was performed with four human cancer cell lines: SW480, LU-1, HepG2
and HL-60 in comparison with indirubin, indirubin-3′-oxime and 6-mercaptopurine. The results showed that cytotoxic and
anti-proliferative activities of ꢀve derivatives were found in the range of 1.35–19.24 µM. Among synthesized derivatives,
4
f showed the strongest activity against all four tested cancer cell lines with IC values of 1.65, 2.21, 1.90 and 1.35 µM,
5
0
respectively.
Keywords Indirubin · Indirubin-3′-oxime · 6-mercaptopurine · Cytotoxic
Introduction
leukemia (CML) for decades (Xiao et al. 2002). Indirubin
1) could be easily synthesized from istatin and 3-acetox-
(
The well-known indole skeleton dyes including indigo,
isoindigo and (2′Z)-indirubin can be early found in a num-
ber of plants (Cuong et al. 2010a, b, c; Bektas et al. 2016),
and indigo, indirubin and their derivatives are the con-
stituents of a preparation called indigo naturalis (Stasiak
et al. 2014). In addition, indirubin was determined to be a
main active ingredient of a traditional Chinese medicinal
recipe including Indigofera tinctoria L. and Isatis tincto-
ria L, which were used for treatment of chronic myelocytic
yindole in alkaline medium (Riepl and Urmann 2012),
and it was also reported to inhibit several kinases such as
glycogen synthase kinase-3 (GSK-3) and cyclin-dependent
kinases (CDKs) by interaction with the ATP-binding site
of kinase through van der Waals interaction and hydrogen
bonds, and induce apoptosis in human cancer cells (Meijer
et al. 2003; Hoessel et al. 1999; Jung et al. 2016; Nam et al.
2005; Lectere et al. 2001). In 2011, Karapetyan reported the
synthesis of some indirubin-N-glycosides and their activity
against several human cancer cell lines such as A-427, Kyse-
7
0 and breast MCF-7. Among these glycosides, the activity
*
*
Chinh Luu Van
of indirubin-α-N-rhamnoside and indirubin-β-N-rhamnoside
chinhluuvan@gmail.com
against the human breast cancer cell line was much higher
Vu Tran Khac
(
10- to 100 fold) than that of the non-glycosylated indiru-
vu.trankhac@hust.edu.vn
bins tested before (Karapetyan et al. 2011). Indirubin-3′-
oxime, an indirubin derivative was considered as potential
candidate to inhibit the growth of lymphocytes in leukemia
patients. This compound also reduced CDK activity and
gene expression (Girard et al. 2007), and indirubin-3′-oxime
was found to causes arrest at G0/G1 phase accompanied
by a signiꢀcant decrease in the percentage of cell in S and
G2/M phases in cell cycle (Liao and Leung 2013). Lo et al.
also recognized that indirubin-3-oxime had anti-proliferative
activity and apoptosis in both Ca27 and SHC-3 oral cancer
1
2
Vietnam-Russia Tropical Center, Nguyen Van Huyen, Cau
Giay, Hanoi, Vietnam
Institute of Natural Products Chemistry, Vietnam Academy
of Science and Technology, 18-Hoang Quoc Viet, Cau Giay,
Hanoi, Vietnam
3
4
Graduate University of Science and Technology, 18-Hoang
Quoc Viet, Cau Giay, Hanoi, Vietnam
School of Chemical Engineering, Hanoi University
of Science and Technology, No 1, Dai Co Viet Hai Ba Trung,
Hanoi, Vietnam
Vol.:(0
1
123456789)
3

Chemical Papers
cell lines though the activation of cytochrome C (Lo and
Chang 2013). Additionally, indirubin-3′-oxime suppressed
migration and invasion in Ca27 by inhibiting the expres-
sion of local adhesion kinase, urokinase-type plasminogen
inhibitor and matrix metalloproteinase 9 (Lo and Chang
standard. Mass spectra were recorded on an Agilent 6530
Accurate-Mas Q-TOG LC/MS. Progress of the reaction was
monitored by thin-layer chromatography (TLC) using pre-
coated TLC sheets (Merck 60F254), and spots were visual-
ized by UV lamp at 254 nm. Multiplicities are shown as
follows: s (singlet), d (doublet), t (triplet), m (multiplet).
Column chromatography was carried out using silica gel 60
(0.04–0.06 mm) from Scharlau, Spain. Solvents were com-
mercially available materials of reagent grade.
2
013). In 2014, the activation of Wnt/b-catenin signaling
and the inhibition of adipocyte diꢁerentiation and obesity
were also reported (Choi et al. 2014). Recently, indirubin-3′-
oxime derivatives have also attracted the interest of chemists
due to their anticancer activity. Notably, a report of Cuong
and colleagues demonstrated that (2′Z-3′E)-indirubin-3′-[O-
(
(
3-bromoprop-1-yl)-oxime] and (2′Z-3′E)-indirubin-3′-[O-
Synthesis of (2′Z)‑indirubin derivatives 4a‑h
3-methoxycarrbonylmethyl)-oxime] revealed stimulative
eꢁects on MC3T3-E1 osteoblastic cell growth and diꢁer-
entiation (Cuong et al. 2010a, b, c). Another derivative of
indirubin, 5-methoxyindirubin 3′-oxime also expressed the
induction of cell death in pancreatic ductal adenocarcinoma
both in vitro and in vivo (Sano et al. 2017).
Synthesis of (Z)-1ʹ-(oxiran-2-ylmethyl)-[2,3ʹ-
biindolinylidene]-2ʹ,3-dione (2). A solution of compound
1
(786 mg, 3 mmol), epichlorohydrin (0.784 mL, 10 mmol),
K CO (1242 mg, 9 mmol), KI (50 mg, 0.3 mmol) and
2
3
(
1-butyl)triethylammonium bromide (71.5 mg, 0.3 mmol)
in dried DMF (50 mL) was stirred at 45 °C for 4 days. The
mixture was poured in ice water (200 ml) and the resulting
precipitate was ꢀltered and washed with water. Compound
2
was puriꢀed by chromatography on silica gel eluting with
CH Cl /EtOAc 10:1
2
(
2
Z)-1ʹ-(Oxiran-2-ylmethyl)-[2,3ʹ-biindolinylidene]-2ʹ,3-
1
dione (2). Yield 40%, dark violet solid, m.p. 178–179 °C. H
NMR (DMSO-d , 500 MHz), δ(ppm): 11.11 (s, 1H, H-1ʹ),
6
8
.83 (d, J = 7.50 Hz, 1H, H-4), 7.68 (d, J = 7.50 Hz, 1H,
H-4ʹ), 7.61 (m, 1H, H-6ʹ), 7.43 (d, J=8.0 Hz, 1H, H-7ʹ), 7.35
m, 1H, H-6), 7.21 (d, J= 8.0 Hz, 1H, H-7), 7.10 (m, 1H,
H-5), 7.06 (m, 1H, H-5ʹ), 4.20 (dd, J =15.0 Hz, J =3.75 Hz,
In a search for new agents for cytotoxic eꢁects, we sug-
gest that exploring new indirubin conjugates containing
nitrogen and sulfur heterocycles could achieve potential
compounds for pharmacological purpose. Therefore, in
present study, two series of conjugates from indirubin were
designed, synthesized and evaluated for in vitro anticancer
activity.
(
1
2
1
–
1
–
1
H, –N –CH –), 3.87 (dd, J =15.0 Hz, J =5.25 Hz, 1H,
1
2
1
2
N –CH –), 3.24 (m, 1H, CH
), 2.79 (t, J = 4.50 Hz,
1
2
epoxy
H, –CH
), 2.66 (dd, J = 5.0 Hz, J = 3.0 Hz, 1H,
2
epoxy
1 2
1
3
CH
). C NMR (DMSO-d , 125 MHz), δ(ppm):
2
epoxy
6
89.0 (C-3ʹ), 169.6 (C-2), 153.0 (C-7ʹa), 141.9 (C-7a), 139.3
(
C-2ʹ), 137.7 (C-6ʹ), 129.6 (C-6), 125.0 (C-4), 124.9 (C-4ʹ),
22.4 (C-3a), 121.9 (C-5), 121.1 (C-5ʹ), 119.5 (C-3ʹa), 114.0
C-7ʹ), 109.6 (C-7), 105.7 (C-3), 49.9 (–CH– ), 45.2
1
(
(
epoxy
Experimental
–CH –
), 41.6 (–N –CH –). HR–ESI–MS: calculated
2
epoxy
1
2
+
for C H N O [M] : 318.1004, found: 318.0998.
1
9
14
2
3
All chemicals were purchased from Sigma-Aldrich (USA),
Santa cruz (USA) except for 3a, 3b, 3c, 3d and 3f were pre-
pared according to the references (Raju et al. 2015; Gilani
et al. 2011; Vaughan 1957). Indirubin with (2′Z) confor-
mation was isolated from Strobilanthes cusia in Vietnam
as described by Cuong et al. (2007). Dry solvents were
prepared according to procedures of Perrin and Armarego
General procedure for the synthesis
of (2′Z)‑indirubin derivatives 4a‑h
A solution of compound 2 (47.7 mg, 0.15 mmol), triethyl-
amine (0.45 mmol, 63 µl) and corresponding thiols (3a-h)
(0.2 mmol) in dried DMF (3 ml) was stirred at room tem-
perature for 40 h. The mixture was then diluted with EtOAc
(50 ml) and washed with NaCl 3% solution (3 × 100 ml).
The combined EtOAc extract was dried on anhydrous
sodium sulfate, and solvent was removed under reduced
pressure to aꢁord crudes 4a-h that was chromatographed
(
1988). Melting points were measured in open capillary
tubes on a Buchi 530 (Switzerland) melting point appara-
tus and were uncorrected. NMR spectra were recorded on
1
a Bruker Advance 500 MHz operating at 500 MHz for H
1
3
and 125 MHz for C. Chemical shifts are reported in parts
per million (ppm) using tetramethylsilane (TMS) as internal
1
3

Chemical Papers
using chloroform: EtOAc 2:1 as solvent to aꢁord 4a-h in
7.03 (m, 1H, H-5ʹ), 5.74 (d, J=5.50 Hz, 1H, –CHOH-), 4.26
(m, 1H, –CHOH–), 4.01 (dd, J = 14.50 Hz, J = 5.50 Hz,
6
3.1–77.0.3% yields.
1
2
(
Z)-1ʹ-(2-Hydroxy-3-((5-phenyl-1,3,4-oxadiazol-2-yl)
1H, –N–CH -), 3.95 (dd, J = 14.50 Hz, J = 6.50 Hz,
2 1 2
thio)propyl)-[2,3ʹ-biindolinylidene]-2ʹ,3-dione (4a).
1H, –N–CH -), 3.61 (dd, J = 13.50 Hz, J = 4.50 Hz,
2 1 2
1
Yield 73.2%, dark violet solid, m.p. 222–223 °C. H NMR
1H, –CH –S–), 3.43 (dd, J =13.50 Hz, J =7.50 Hz, 1H,
2 1 2
1
3
(
DMSO-d , 500 MHz), δ(ppm): 11.1 (s, 1H, H-1ʹ), 8.82
–CH –S–). C NMR (DMSO-d , 125 MHz), δ(ppm):
6
6
2
(
d, J = 8.0 Hz, 1H, H-4), 7.93 (d, J = 7.0 Hz, 2H, H-2″,
189 (C-3ʹ), 169.9(C-2), 165.9 (phenyl–C
), 164.1
oxadiazole
H-6″), 7.67 (d, J = 7.0 Hz, 1H, H-4ʹ), 7.58 (m, 4H, H-3″,
H-5″, H-6ʹ, H-4″), 7.41 (d, J= 8.0 Hz, 1H, H-7ʹ), 7.33 (t,
J=7.50 Hz, 1H, H-6), 7.2 (d, J=7.50 Hz, 1H, H-7), 7.09 (t,
J=7.50 Hz, 1H, H-5), 7.04 (t, J=7.25 Hz, 1H, H-5ʹ), 5.71
(–S–C
), 152.9 (C-7ʹa), 149.5 (C-4″), 142.3 (C-7a),
oxadiazole
139.1 (C-2ʹ), 137.7 (C-6ʹ), 129.5 (C-6), 129 (C-1″), 128.1
(C-2″, C-6″), 125 (C-3″, C-5″), 124.9 (C-4), 124.8 (C-4ʹ),
122.3 (C-3a), 121.9 (C-5), 121.3 (C-5ʹ), 119.5 (C-3ʹa),
113.9 (C-7ʹ), 109.6 (C-7), 106 (C-3), 67.4(–CHOH–),
45.1(–N–CH -), 37.6(–CH –S–). HR-ESI–MS: Caculated
(
d, J=5.0 Hz, 1H, –CHOH–), 4.24 (m, 1H, –CHOH–), 3.98
(
m, 2H, –N–CH ), 3.58 (dd, J = 13.50 Hz, J = 3.50 Hz,
2
1
2
2
2
+
1
–
1
H, –CH –S–), 3.4 (dd, J = 13.50 Hz, J = 7.50 Hz, 1H,
for C H N O S: [M] : 541.1056, found: 541.1047.
2
1
2
27 19 5 6
1
3
CH –S). C NMR (DMSO-d , 125 MHz), δ(ppm):
(Z)-1ʹ-(2-Hydroxy-3-((5-(pyridin-4-yl)-1,3,4-oxadiazol-
2-yl)thio)propyl)-[2,3ʹ-biindolinylidene]-2ʹ,3-dione (4d).
2
6
89.1 (C-3ʹ), 169.9 (C-2), 165.5 (phenyl–C
), 164.5
oxadiazole
1
(
–S–C
), 152.9 (C-7ʹa), 142.3 (C-7a), 139.1 (C-2ʹ),
Yield 65.0%, dark violet solid, mp: 183–184 °C. H NMR
oxadiazole
1
1
1
37.7 (C-6ʹ), 132.4 (C-4″), 129.9 (C-3″, C-5″), 129.5 (C-6),
(DMSO-d , 500 MHz), δδ(ppm): 11.1 (s, 1H, H-1ʹ), 8.82
6
26.8 (C-2″, C-6″), 124.9 (C-4), 124.8 (C-4ʹ), 123.5 (C-1″),
22.2 (C-3a), 121.9 (C-5), 121.3 (C-5ʹ), 119.5 (C-3ʹa), 113.9
(d, J=7.0 Hz, 1H, H-4), 8.78 (dd, J =4.50 Hz, J =1.50 Hz,
1
2
2H, H-2″, H-6″), 7.84 (dd, J =4.50 Hz, J =1.50 Hz, 2H,
1
2
(
(
C-7ʹ), 109.6 (C-7), 106.1 (C-3), 67.5(–CHOH–), 45.2
H-3″, H-5″), 7.66 (d, J=7.50 Hz, 1H, H-4ʹ), 7.59 (m, 1H,
H-6ʹ), 7.4 (d, J=8.0 Hz, 1H, H-7ʹ), 7.32 (m, 1H, H-6), 7.19
(d, J = 8.0 Hz, 1H, H-7), 7.09 (m, 1H, H-5), 7.03 (m, 1H,
H-5ʹ), 5.73 (d, J = 5.50 Hz, 1H, –CHOH), 4.25 (m, 1H,
–CHOH–), 3.99 (m, 2H, –N–CH -), 3.61 (dd, J =13.50 Hz,
–N–CH –), 37.6(–CH –S–). HR–ESI–MS: Caculated for
2
2
+
C H N O S: [M] : 496.1205, found: 496.1195.
2
7
20
4
4
(
Z)-1ʹ-(2-Hydroxy-3-((5-(4-methoxyphenyl)-1,3,4-
oxadiazol-2-yl)thio)propyl)-[2,3ʹ-biindolinylidene]-2ʹ,3-
2
1
dione (4b). Yield 71.8%, dark violet solid, mp: 225–226 °C.
J = 4.0 Hz, 1H, –CH –S–), 3.42 (dd, J = 13.50 Hz,
2 2 1
1
13
H NMR (DMSO-d , 500 MHz), δ(ppm): 11.09 (s, 1H,
J = 7.50 Hz, 1H, –CH –S–). C NMR (DMSO-d ,
6
6
2
2
H-1ʹ), 8.83 (d, J = 7.5 Hz, 1H, H-4), 7.85 (d, J = 8.5 Hz,
125 MHz), δ(ppm): 189 (C-3ʹ), 169.9 (C-2), 166 (pyri-
2
H, H-2″, H-6″), 7.67 (d, J = 7.5 Hz, 1H, H-4ʹ), 7.59 (t,
dine–C ), 163.9 (–S–C ), 152.9 (C-7ʹa), 151.4
oxadiazole
oxadiazole
J=7.5 Hz, 1H, H-6ʹ), 7.41 (d, J=8.0 Hz, 1H, H-7ʹ), 7.33 (t,
J=7.75 Hz, 1H, H-6), 7.2 (d, J=8.0 Hz, 1H, H-7), 7.10 (m,
(C-2″, C-6″), 142.3 (C-7a), 139.1 (C-2ʹ), 137.7 (C-6ʹ), 130.5
(C-4″), 129.5 (C-6), 124.9 (C-4), 124.8 (C-4ʹ), 122.2 (C-3a),
121.9 (C-5), 121.3 (C-5ʹ), 120.4 (C-3″, C-5″), 119.5 (C-3ʹa),
113.9 (C-7″), 109.6 (C-7), 106.1 (C-3), 67.5(–CHOH–),
45,1(–N–CH -), 37,6(–CH –S–). HR-ESI–MS: Caculated
3
H, H-5, H-3″, H-5″), 7.04 (t, J=7.5 Hz, 1H, H-5ʹ), 5.71 (d,
J=5.0 Hz, 1H, –CHOH–), 4.22 (m, 1H, –CHOH–), 3.97 (m,
H, –N–CH –), 3.83 (s, 3H, CH O–), 3.56 (dd, J =13.5 Hz,
2
2
3
1
2
2
1
3
+
J = 4.0 Hz, 1H, –CH –S–), 3.38 (m, 1H,–CH –S–).
C
for C H N O S [M] : 497.1158, found: 497.1157.
2
2
2
26 19 5 4
NMR (DMSO-d , 125 MHz), δ(ppm): 189.1 (C-3ʹ), 169.9
(Z)-1ʹ-(3((7H-Purin-6-yl)thio)-2-hydroxypropyl)-
6
(
C-2), 165.4 (phenyl–C
), 163.6 (–S–C
),
oxadiazole
[2,3ʹ-biindolinylidene]-2ʹ,3-dione (4e). Yield 63.1%,
oxadiazole
1
1
1
1
62.4 (C-4″), 152.9 (C-7ʹa), 142.3 (C-7a), 139.1 (C-2ʹ),
dark violet solid, mp: 172–173 °C. H NMR (DMSO-d ,
6
37.7 (C-6ʹ), 129.5 (C-6), 128.6 (C-2″, C-6″), 124.9 (C-4),
24.8 (C-4ʹ), 122.2 (C-3a), 121.9 (C-5), 121.3 (C-5ʹ), 119.5
500 MHz), δ(ppm): 13.49 (s, 1H, NH
), 11.11 (s, 1H,
purine
H-1ʹ), 8.83 (d, J = 7.50 Hz, 1H, H-4), 8.49 (s, 1H, H-8″),
8.41 (s, 1H, H-2″), 7.67 (d, J = 7.50 Hz, 1H, H-4ʹ), 7.59
(t, J = 7.75 Hz, 1H, H-6ʹ), 7.41 (d, J = 8.0 Hz, 1H, H-7ʹ),
7.31 (t, J = 7.75 Hz, 1H, H-6), 7.16 (d, J = 8.0 Hz, 1H,
H-7), 7.08 (t, J = 7.75 Hz, 1H, H-5), 7.04 (t, J = 7.50 Hz,
1H, H-5ʹ), 5.56 (d, J=5.0 Hz, 1H, –CHOH-), 4.16 (m, 1H,
–CHOH–), 4.0 (dd, J =14.0 Hz, J =5.0 Hz, 1H, –N–CH -
(
C-3ʹa), 115.9 (C-1″), 115.3 (C-3″, C-5″), 113.9 (C-7ʹ),
1
4
09.6 (C-7), 106.1 (C-3), 67.6 (–CHOH–), 55.9 (CH O–),
3
5.2 (–N–CH –), 37.6 (–CH –S–). HR–ESI–MS: Caculated
2
2
+
for C H N O S: [M] : 526.1311, found: 526.1303.
2
8
22
4
5
(
Z)-1ʹ-(2-Hydroxy-3-((5-(4-nitrophenyl)-1,3,4-
oxadiazol-2-yl)thio)propyl)-[2,3ʹ-biindolinylidene]-2ʹ,3-
1
2
2
dione (4c). Yield 66.1%, dark violet solid, mp: 168–169 °C.
), 3.92 (dd, J = 14.0 Hz, J = 7.0 Hz, 1H, –N–CH -), 3.7
1 2 2
1
13
H NMR (DMSO-d , 500 MHz), δ(ppm): 11.1 (s, 1H,
(m, 1H, –CH –S–), 3.33 (m, 1H, –CH –S–). C NMR
6
2
2
H-1ʹ), 8.82 (d, J=7.0 Hz, 1H, H-4), 8.36 (dd, J =7.0 Hz,
(DMSO-d , 125 MHz), δ(ppm): 189.1 (C-3ʹ), 169.9 (C-2),
1
6
J = 2.0 Hz, 2H, H-3″, H-5″); 8.16 (dd, J = 7.0 Hz,
159.1 (C-6″), 153 (C-7ʹa), 151.7 (C-4″, C-8″), 149.6 (C-5″),
143.4 (C-2″), 142.5 (C-7a), 138.9 (C-2ʹ), 137.7 (C-6ʹ), 129.5
(C-6), 124.9 (C-4), 124.8 (C-4ʹ), 122.1 (C-3a), 121.9 (C-5),
121.3 (C-5ʹ), 119.5 (C-3ʹa), 113.9 (C-7ʹ), 109.7 (C-7), 106.2
2
1
J =2.0 Hz, 2H, H-2″, H-6″), 7.65 (d, J=7.50 Hz, 1H, H-4ʹ),
2
7
.58 (m, 1H, H-6ʹ), 7.39 (d, J=8.0 Hz, 1H, H-7ʹ), 7.33 (m,
1
H, H-6), 7.2 (d, J=7.5 Hz, 1H, H-7), 7.09 (m, 1H, H-5),
1
3

Chemical Papers
(
C-3), 68.2 (–CHOH–), 45.5 (–N–CH -), 33.2 (–CH –S–).
(4 h). Yield 67.4%, dark violet solid, mp: 241–242 °C
2
2
+
1
HR-ESI–MS: Caculated for C H N O S [M] : 470.1161,
(decomposition). H NMR (DMSO-d , 500 MHz),
2
4
18
6
3
6
found: 470.1159.
δ(ppm): 11.09 (s, 1H, H-1ʹ), 9.42 (s, 1H, NH-phenyl), 8.81
(d, J=7.50 Hz, 1H, H-4), 7.67 (d, J=7.50 Hz, 1H, H-4ʹ),
7.60 (m, 2H, H-6ʹ, H-5″), 7.41 (d, J = 8.0 Hz, 1H, H-7ʹ),
7.31 (1H, m, H-6), 7.16 (d, J= 8.0 Hz, 1H, H-7), 7.09 (d,
J = 7.50 Hz, 1H, H-5), 7.04 (m, 2H, H-5ʹ, H-3″), 6.98 (d,
J=8.0 Hz, 1H, H-6″), 5.58 (d, J=5.50 Hz, 1H, –CHOH-),
(
Z)-1ʹ-(2-Hydroxy-3-((5-(phenylamino)-1,3,4-
thiadiazol-2-yl)thio)propyl)-[2,3ʹ-biindolinylidene]-
2
ʹ,3-dione (4f). Yield 77.0%, dark violet solid, mp:
−
1
2
32–233 °C. IR (KBr, ν
(cm )): 3416 (O–H), 3316
max
(
N–H), 2923 (C–H), 1710 (C=O), 1643 (C=N), 1607 and
1
499 (C=C), 1319 (C–N), 1093 (N–N), 660 (C–S–C).
4.13 (m, 1H, –CHOH–), 3.92 (m, 2H, –N–CH -), 3.37 (m,
2
1
H NMR (DMSO-d , 500 MHz), δ(ppm): 11.1 (s, 1H,
1H, –CH –S–), 3.21 (dd, J = 13.50 Hz, J = 7.0 Hz, 1H,
6
2
1
2
H-1ʹ), 10.36 (s, 1H, NH-phenyl), 8.82 (d, J = 8.0 Hz, 1H,
H-4), 7.67 (d, J = 7.50 Hz, 1H, H-4ʹ), 7.6 (m, 1H, H-6ʹ),
–CH –S–), 2.24 (s, 3H, CH –C-4″), 2.19 (s, 3H, CH –C-
2 3 3
1
3
2″). C NMR (DMSO-d , 125 MHz), δ(ppm): 189 (C-3ʹ),
6
7
1
.55 (d, J=7.50 Hz, 2H, H-2″, H-6″), 7.42 (d, J=8.0 Hz,
H, H-7ʹ), 7.33–7.31 (m, 3H, H-3″, H-5″, H-6), 7.18 (d,
169.9 (C-2), 168.1 (–NH–C
), 152.9 (C-7ʹa), 152.8
thiadiazole-
(–CH –S–C
), 142.4 (C-7a), 139 (C-2ʹ), 137.7 (C-6ʹ),
2
thiadiazole
J = 8.0 Hz, 1H, H-7), 7.08 (t, J = 7.50 Hz, 1H, H-5), 7.04
137.1 (C-1″), 133.9 (C-4″), 131.8 (C-5″), 130.1 (C-2″),
129.5 (C-6), 127.6 (C-3″), 124.9 (C-4), 124.8 (C-4ʹ),
122.4 (C-6″), 122.2 (C-3a), 121.9 (C-5), 121.3 (C-5ʹ),
119.5 (C-3ʹa), 113.9 (C-7ʹ), 109.6 (C-7), 106.1 (C-3),
67.9 (–CHOH–), 45.3 (–N–CH -), 40 (–CH –S–), 20.9
(
m, 1H, H-5ʹ), 7.0 (m, 1H, H-4″), 5.61 (d, J=5.50 Hz, 1H,
CHOH-), 4.16 (m, 1H, –CHOH–), 3.94 (m, 2H, –N–CH -
–
2
)
, 3.43 (dd, J = 13.25 Hz, J = 4.50 Hz, 1H, –CH –S–),
1 2 2
1
3
3
.26 (dd, J = 13.25 Hz, J = 7.25 Hz, 1H, –CH –S–).
C
1
2
2
2
2
NMR (DMSO-d , 125 MHz), δ(ppm): 189.1 (C-3ʹ), 169.9
(CH –C-4″), 18.3 (CH –C-2″). HR-ESI–MS: Caculated
6
3
3
+
(
C-2), 165.1 (–NH–C
), 153.7 (–CH –S–C
),
for C H N O S [M] : 555.1399, found: 555.1393.
thiadiazole-
2
thiadiazole
29 25 5 3 2
1
52.9 (C-7ʹa), 142.4 (C-7a), 140.9 (C-1″), 139 (C-2ʹ), 137.7
(
(
(
(
(
[
C-6ʹ), 129.6 (C-3″, C-5″), 129.4 (C-6), 124.9 (C-4), 124.8
C-4ʹ), 122.4 (C-4″), 122.2 (C-3a), 121.9 (C-5), 121.3
C-5ʹ), 119.5(C-3ʹa), 117.8 (C-2″, C-6″), 113.9 (C-7ʹ), 109.7
Synthesis of (2′Z‑3′E)‑indirubin‑3′‑oxime
derivatives 7a‑i
C-7), 106.2 (C-3), 67.9 (–CHOH–), 45.4 (–N–CH -), 39.4
2
–CH –S–). HR-ESI–MS: Caculated for C H N O S
Synthesis of (2Z,3E)‑3‑((Oxiran‑2‑ylmethoxy)imino)‑
[2,3ʹ‑biindolinylidene]‑2ʹ‑one (6)
2
27 21
5
3 2
+
M] : 527.1086, found: 527.1082.
(
Z)-1ʹ-(3-((5-((2,3-Dimethylphenyl)amino)-1,3,4-thia-
diazol-2-yl)thio)propyl)-[2,3ʹ-biindolinylidene]-2ʹ,3-dione
Indirubin-3′-oxime 5 with (2′Z-3′E) conformation was ꢀrst
obtained by the reaction of indirubin (1) with hydroxy-
lamine in pyridine according to the procedure as described
by Cuong and et al. (2010a, b, c), indirubin-3′-oxime (5)
was then O-alkylated with epibromohydrin using a proce-
dure described by Ichimaru (Ichimaru et al. 2015) to give
1
(
4 g). Yield 69.5%, dark violet solid, mp: 247–248 °C. H
NMR (DMSO-d , 500 MHz), δ(ppm): 11.09 (s, 1H, H-1ʹ),
6
9
7
.54 (s, 1H, NH-phenyl), 8.81 (d, J = 8.0 Hz, 1H, H-4),
.67 (d, J = 7.50 Hz, 1H, H-4ʹ), 7.59 (m, 1H, H-6ʹ), 7.44
(
d, J = 8.0 Hz, 1H, H-4″), 7.41 (d, J = 8.0 Hz, 1H, H-7ʹ),
′
7
2
.31 (m, 1H, H-6), 7.16 (d, J=8.0 Hz, 1H, H-7), 7.08 (m,
H, H-5, H-5″), 7.04 (t, J = 7.50 Hz, 1H, H-5ʹ), 6.99 (d,
((2′Z-3′E)-Indirubin-3 -(O-oxiran-2-ylmethyl)oxime) 6 in
70% yield.
J=7.50 Hz, 1H, H-6″), 5.58 (d, J=5.50 Hz, 1H, –CHOH-),
4
1
–
.12 (m, 1H, –CHOH–), 3.92 (m, 2H, –N–CH -), 3.37 (m,
General procedure for the synthesis
of indirubin‑3′‑oxime derivatives 7a‑i
2
H, –CH –S–), 3.21 (dd, J = 13.50 Hz, J = 7.0 Hz, 1H,
2
1
2
CH –S–), 2.26 (s, 3H, CH –C-3″), 2.13 (s, 3H,CH –C-2″).
2
3
3
1
3
C NMR (DMSO-d , 125 MHz), δ(ppm): 189 (C-3ʹ),
A solution of compound 6 (50 mg, 0.15 mmol), triethyl-
amine (0.45 mmol, 63 µl) and the corresponding thiols 3a-3i
(0.2 mmol) in dried DMF (3 ml) was stirred at room tem-
perature for 40 h. The mixture was then diluted with EtOAc
(50 ml) and washed with NaCl 3% solution (3×100 ml). The
combined EtOAc extract was dried on anhydrous sodium
sulfate and solvent was removed under reduced pressure.
Crude 7a-i was chromatographed using chloroform: EtOAc
as solvent to aꢁord 7a-i in 59.2–75.0% yields.
6
1
69.9 (C-2), 168.6 (–NH–C
), 152.9 (C-7ʹa), 152.7
thiadiazole-
(
–CH –S–C
), 142.4(C-7a), 139.5 (C-1″), 139(C-
thiadiazole
2
2
1
ʹ), 137.9 (C-3″), 137.7 (C-6ʹ), 129.7 (C-2″), 129.5 (C-6),
26.9 (C-4″), 126.4 (C-5″), 124.9 (C-4), 124.8 (C-4ʹ), 122.2
(
C-3a), 121.9 (C-5), 121.3 (C-5ʹ), 121 (C-6″), 119.5 (C-3ʹa),
1
4
13.9 (C-7ʹ), 109.6 (C-7), 106.1 (C-3), 67.9 (–CHOH),
5.3(–N–CH -), 40(–CH –S–), 20.7 (CH –C-3″), 14.3
2
2
3
(
[
CH –C-2″). HR-ESI–MS: Caculated for C H N O S
3
29 25
5
3 2
+
M] : 555.1399, found: 555.1396.
(2′Z-3′E)-Indirubin-3ʹ-[O-2-hydroxy-3-(5-phenyl-
(
Z)-1ʹ-(3-((5-((2,4-Dimethylphenyl)amino)-1,3,4-thia-
1,3,4-oxadiazole-2-thio)prop-1-yl]oxime (7a). Yield
1
diazol-2-yl)thio)propyl)-[2,3ʹ-biindolinylidene]-2ʹ,3-dione
67.1%, red solid, m.p. 196–197 °C. H NMR (DMSO-d ,
6
1
3

Chemical Papers
1
3
5
00 MHz), δ(ppm): 11.68 (s, 1H, H-1ʹ), 10.75 (s, 1H, H-1),
.58 (d, J = 7.50 Hz, 1H, H-4), 8.22 (d, J = 7.50 Hz, 1H,
J = 13.50 Hz, J = 6.50 Hz, 1H, –CH –S–). C NMR
1
2
2
8
(DMSO-d , 125 MHz), δ(ppm): 171.3 (C-2), 165.7 (phe-
6
H-4ʹ), 7.89 (m, 2H, H-2″, H-6″), 7.6 (m, 1H, H-4″), 7.54 (t,
J=7.50 Hz, 2H, H-3″, H-5″), 7.43 (m, 2H, H-6ʹ, H-7ʹ), 7.11
nyl–C
), 164.1 (–S–C
), 152.2 (C-3ʹ), 149.4
oxadiazole
oxadiazole-
(C-4″), 146 (C-2ʹ), 144.1 (C-7ʹa), 139.1 (C-7a), 133.4 (C-6ʹ),
129.2 (C-4ʹ), 128.9 (C-1″), 127.9 (C-2″, C-6″), 126.9 (C-6),
124.8 (C-3″, C-5″), 123.7 (C-4), 122.7 (C-3a), 121.9 (C-5ʹ),
121 (C-5), 116.6 (C-3ʹa), 112.2 (C-7ʹ), 109.4 (C-7), 100.8
(C-3), 79.1 (–O–CH -), 67.8 (–CHOH), 36.7 (–CH –S–).
(
m, 1H, H-6), 7.03 (m, 1H, H-5ʹ), 6.96 (t, J=7.75 Hz, 1H,
H-5), 6.87 (d, J=7.50 Hz, 1H, H-7), 5.86 (d, J=5.50 Hz, 1H,
–
CHOH-), 4.7 (dd, J =11.0 Hz, J =5.0 Hz, 1H, –O–CH -
1
2
2
)
, 4.65 (dd, J =11.0 Hz, J =6.0 Hz, 1H, –O–CH -), 4.42
1
2
2
2
2
+
(
m, 1H, –CHOH–), 3.69 (dd, J = 13.50 Hz, J = 4.5 Hz,
HR-ESI–MS: calculated for C H O N S [M] : 556.1165,
1
2
27 20
6
6
1
–
1
1
H, –CH –S–), 3.53 (dd, J = 13.5 Hz, J = 7.0 Hz, 1H,
found: 556.1155.
2
1
2
1
3
CH –S–). C NMR (DMSO-d , 125 MHz), δ(ppm):
(2′Z-3′E)-Indirubin-3ʹ-[O-2-hydroxy-3-(5-(pyridin-4-
2
6
71.3 (C-2), 165.5(phenyl–C
), 164.4(–S–C
),
yl)-1,3,4-oxadiazole-2-thio)prop-1-yl]oxime (7d). Yield
oxadiazole-
oxadiazole
1
52.2 (C-3ʹ), 146 (C-2ʹ), 144.3 (C-7ʹa), 139.2 (C-7a), 133.4
61.7%, red solid, m.p. 216–217 °C. H NMR (DMSO-d ,
6
(
(
(
(
(
C-6ʹ), 132.4 (C-4″), 129.8 (C-3″, C-5″), 129.2 (C-4ʹ), 126.9
C-6), 126.7 (C-2″, C-6″), 123.8 (C-4), 123.5 (C-1″), 122.7
C-3a), 121.9 (C-5ʹ), 121.1 (C-5), 116.7 (C-3ʹa), 112.2
500 MHz), δ(ppm): 11.67 (s, 1H, H-1ʹ), 10.74 (s, 1H, H-1),
8.75 (dd, J =4.50 Hz, J =1.50 Hz, 2H, H-2″, H-6″), 8.56
1
2
(d, J = 8.0 Hz, 1H, H-4), 8.22 (d, J = 7.50 Hz, 1H, H-4ʹ),
7.78 (dd, J = 4.25 Hz, J = 1.75 Hz, 2H, H-3″, H-5″),
C-7ʹ), 109.4 (C-7), 100.8 (C-3), 79.5 (–O–CH -), 67.9
2
1
2
–CHOH–), 36.6(–CH –S–). HR-ESI–MS: calculated for
7.43 (m, 2H, H-6ʹ, H-7ʹ), 7.1 (t, J = 7.50 Hz, 1H, H-6),
7.03 (m, 1H, H-5ʹ), 6.96 (t, J=7.75 Hz, 1H, H-5), 6.85 (d,
J= 7.50 Hz, 1H, H-7), 5.88 (d, J =5.50 Hz, 1H, –CHOH-
), 4.7 (dd, J = 11.25 Hz, J = 5.25 Hz, 1H, –O–CH -),
2
+
C H O N S [M] : 511.1314, found: 511.1305.
2
7
(
21
4
5
2′Z-3′E)-Indirubin-3ʹ-[O-2-hydroxy-3-(5-(4-
methoxyphenyl)-1,3,4-oxadiazole-2-thio)prop-1-yl]
1
2
2
1
oxime (7b). Yield 69.4%, red solid, m.p. 207–208 °C. H
4.65 (dd, J =11.25 Hz, J =5.75 Hz, 1H, –O–CH -), 4.43
1
2
2
NMR (DMSO-d , 500 MHz), δ(ppm): 11.67 (s, 1H, H-1ʹ),
(m, 1H, –CHOH–), 3.72 (dd, J = 13.50 Hz, J = 5.0 Hz,
6
1
2
1
0.75 (s, 1H, H-1), 8.58 (d, J=8.0 Hz, 1H, H-4), 8.21 (d,
1H, –CH –S–), 3.56 (dd, J = 13.50 Hz, J = 7.0 Hz, 1H,
2
1
2
13
J = 8.0 Hz, 1H, H-4ʹ), 7.8 (dd, J = 6.75 Hz, J = 2.25 Hz,
–CH –S–). C NMR (DMSO-d , 125 MHz), δ(ppm): 171.3
1
2
2
6
2
H, H-2″, H-6″), 7.43 (m, 2H, H-6ʹ, H-7ʹ), 7.12 (m, 1H,
(C-2), 165.8 (pyridine–C
), 164 (–S–C
),
oxadiazole
oxadiazole-
H-6), 7.04 (m, 3H, H-3″, H-5″, H-5ʹ), 6.96 (m, 1H, H-5),
152.2 (C-3ʹ), 151.3 (C-2″, C-6″), 146 (C-2ʹ), 144.2 (C-7ʹa),
139.1 (C-7a), 133.4 (C-6ʹ), 130.5 (C-4″), 129.2 (C-4ʹ),
126.9 (C-6), 123.8 (C-4), 122,7 (C-3a), 121.9 (C-5ʹ), 121.1
(C-5), 120.3 (C-3″, C-5″), 116.7 (C-3ʹa), 112.2 (C-7ʹ),
6
–
3
1
–
1
1
.88 (d, J = 7.50 Hz, 1H, H-7), 5.86 (d, J = 5.50 Hz, 1H,
CHOH-), 4.67 (m, 2H, –O–CH -), 4.42 (m, 1H, –CHOH–),
2
.84 (s, 3H, CH O-), 3.66 (dd, J =13.25 Hz, J =4.75 Hz,
3
1
2
H, –CH –S–), 3.5 (dd, J = 13.75 Hz, J = 7.25 Hz,1H,
109.4 (C-7), 100.8 (C-3), 79,3(–O–CH -), 67.8 (–CHOH–),
2
1
2
2
1
3
CH –S–). C NMR (DMSO-d , 125 MHz), δ(ppm):
36.7(–CH –S–). HR-ESI–MS: calculated for C H O N S
2
6
2
26 20
4
6
+
71.3(C-2), 165.5(phenyl–C
), 163.5 (–S–C
),
[M] : 512.1267, found: 512.1259.
oxadiazole-
oxadiazole
62.4 (C-4″), 152.2 (C-3ʹ), 146 (C-2ʹ), 144.2 (C-7ʹa), 139.2
(2′Z-3′E)-Indirubin-3ʹ-[O-2-hydroxy-3-(purine-6-
(
(
C-7a), 133.4 (C-6ʹ), 129.1(C-4ʹ), 128.6 (C-2″, C-6″), 126.9
C-6), 123.8 (C-4), 122.7 (C-3a), 121.9 (C-5ʹ), 121.1 (C-5),
thio)prop-1-yl]oxime (7e). Yield 59.2%, red solid, m.p.
1
255–256 °C. H NMR (DMSO-d , 500 MHz), δ(ppm):
6
1
1
5
16.7 (C-3ʹa), 115.8 (C-1″), 115.2 (C-3″, C-5″), 112.2 (C-7ʹ),
13.52 (s, 1H, NH
), 11.69 (s, 1H, H-1ʹ), 10.76 (s, 1H,
purine
09.4 (C-7), 100.8 (C-3), 79.4 (–O–CH -), 67.9 (–CHOH–),
H-1), 8.56 (s, 1H, H-8″), 8.55 (d, J = 8.0 Hz, 1H, H-4),
8.44 (s, 1H, H-2″), 8.24 (d, J = 7.50 Hz, 1H, H-4ʹ), 7.44
(m, 2H, H-6ʹ, H-7ʹ), 7.08 (t, J=7.50 Hz, 1H, H-6), 7.03 (m,
1H, H-5ʹ), 6.86 (m, 2H, H-5, H-7), 5.74 (d, J=5.0 Hz, 1H,
–CHOH-), 4.69 (dd, J =11.0 Hz, J =5.0 Hz, 1H, –O–CH -
2
5.9 (CH O-), 36.7 (–CH –S–). HR-ESI–MS: calculated for
3
2
+
C H N O S [M] : 541.1420, found: 541.1414.
2
8
(
23
5
5
2′Z-3′E)-Indirubin-3ʹ-[O-2-hydroxy-3-(5-(4-
nitrophenyl)-1,3,4-oxadiazole-2-thio)prop-1-yl]
1
2
2
oxime (7c). Yield 75.0%, red solid, m.p. 213–214 °C.
), 4.64 (dd, J =11.0 Hz, J =6.0 Hz, 1H, –O–CH -), 4.35
1
2
2
1
H NMR (DMSO-d , 500 MHz), δ(ppm): 11.64 (s, 1H,
(m, 1H,–CHOH), 3.79 (dd, J = 13.50 Hz, J = 5.50 Hz,
6
1
2
H-1ʹ), 10.71 (s, 1H, H-1), 8.53 (d, J = 8.0 Hz, 1H, H-4),
1H, –CH –S–), 3.56 (dd, J =13.75 Hz, J =6.75 Hz, 1H,
2
1
2
1
3
8
8
.3 (m, 2H, H-3″, H-5″), 8.21 (d, J = 7.50 Hz, 1H, H-4ʹ),
.06 (m, 2H, H-2″, H-6″), 7.45 (m, 1H, H-6ʹ), 7.4 (d,
–CH –S–). C NMR (DMSO-d , 125 MHz), δ(ppm):
2
6
171.3 (C-2), 159.1 (C-6″), 152 (C-3ʹ), 151.7 (C-4″, C-8″),
149.7 (C-5″), 145.9 (C-2ʹ), 144.3 (C-7ʹa), 143.5 (C-2″),
139.1 (C-7a), 133.4 (C-6ʹ), 129.1 (C-4ʹ), 126.8 (C-6), 123.8
(C-4), 122.7 (C-3a), 121.9 (C-5ʹ), 121 (C-5), 116.7 (C-3ʹa),
J = 7.50 Hz, 1H, H-7ʹ), 7.1 (m, 1H, H-6), 7.03 (m, 1H,
H-5ʹ), 6.94 (m, 1H, H-5), 6.83 (d, J = 7.50 Hz, 1H, H-7),
5
.89 (d, J=5.50 Hz, 1H, –CHOH-), 4.7 (dd, J =11.50 Hz,
1
J = 5.50 Hz, 1H, –O–CH -), 4.65 (dd, J = 11.25 Hz,
112.2 (C-7ʹ), 109.4 (C-7), 100.7 (C-3), 79.9 (–O–CH -), 68.5
2
2
1
2
J =5.75 Hz, 1H, –O–CH -), 4.44 (m, 1H, –CHOH–), 3.72
(–CHOH–), 31.9 (–CH –S–). HR-ESI–MS: calculated for
2
2
2
+
(
dd, J = 13.50 Hz, J = 5.0 Hz, 1H, –CH –S–), 3.57 (dd,
C H N O S [M] : 485.1270, found: 485.1268.
1
2
2
24 19
7
3
1
3

Chemical Papers
(
2′Z-3′E)-Indirubin-3ʹ-[O-2-hydroxy-3-(5-
J=7.50 Hz, 1H, H-4), 8.28 (d, J=8.0 Hz, 1H, H-4ʹ), 7.65
(d, J = 8.0 Hz, 1H, H-5″), 7.53 (m, 2H, H-6ʹ, H-7ʹ), 7.23
(m, 1H, H-6), 7.10 (m, 4H, H-5ʹ, H-3″, H-6″, H-5), 7.01 (d,
J=8.0 Hz, 1H, H-7), 5.85 (d, J=5.50 Hz, 1H, –CHOH-),
4.76 (dd, J = 11.0 Hz, J = 4.50 Hz, 1H, –O–CH -), 4.69
phenylamino)-1,3,4-thiadiazole-2-thio)prop-1-yl]oxime
(
7f). Yield 69.6%, red solid, m.p. 236–237 °C. IR (KBr,
−1
ν
(cm )): 3416 (O–H overlap N–H), 1735 (C=O), 1616
max
(
C=N), 1600 and 1524 (C=C), 1338 (C–N), 1086 (N–N),
1
2
2
1
6
1
42 (C–S–C). H NMR (DMSO-d , 500 MHz), δ(ppm):
(dd, J = 11.0 Hz, J = 6.0 Hz, 1H, –O–CH -), 4.42 (m,
6
1
2
2
1.67 (s, 1H, H-1ʹ), 10.72 (s, 1H, H-1), 10.32 (s, 1H, NH-
1H, –CHOH–), 3.58 (dd, J =13.50 Hz, J =5.50 Hz, 1H,
1
2
phenyl), 8.58 (d, J=7.50 Hz, 1H, H-4), 8.19 (d, J=7.50 Hz,
–CH –S–), 3.44 (1H, –CH –S–), 2.35 (s, 3H, CH –C-4″),
2 2 3
1
3
1
2
H, H-4ʹ), 7.53 (d, J = 8.0 Hz, 2H, H-2″, H-6″), 7.41 (m,
H, H-6ʹ, H-7ʹ), 7.32 (t, J=7.75 Hz, 2H, H-3″, H-5″), 7.1 (t,
2.12 (s, 3H, CH –C-2″). C NMR (DMSO-d , 125 MHz),
3
6
δ(ppm): 171.3 (C-2), 168.2 (–NH–C
), 152.6
thiadiazole-
J=7.50 Hz, 1H, H-6), 7.01 (m, 3H, H-5ʹ, H-4″, H-5), 6.89
(–CH –S–C
), 152.1 (C-3ʹ), 146 (C-2ʹ), 144.3 (C-7ʹa),
2
thiadiazole
(
d, J=7.50 Hz, 1H, H-7), 5.76 (d, J=5.50 Hz, 1H, –CHOH-
139.1 (C-7a), 137 (C-1″), 133.9 (C-4″), 133.3 (C-6ʹ), 131.8
(C-5″), 130.1 (C-2″), 129.1 (C-4ʹ), 127.6 (C-3″), 126.9
(C-6), 123.7 (C-4), 122.8 (C-3a), 122.4 (C-6″), 121.9 (C-5ʹ),
121.2 (C-5), 116.7 (C-3ʹa), 112.2 (C-7ʹ), 109.4 (C-7), 100.8
(C-3), 79.6 (–O–CH -), 68.3 (–CHOH–), 38.4 (–CH –S–),
)
, 4.66 (dd, J =11.0 Hz, J =4.50 Hz, 1H, –O–CH -), 4.59
1
2
2
(
dd, J = 11.0 Hz, J = 6.0 Hz, 1H, –O–CH -), 4.34 (m,
1
2
2
1
–
1
1
H, –CHOH), 3.53 (dd, J = 13.50 Hz, J = 5.0 Hz, 1H,
1
2
1
3
CH –S–), 3.37 (1H, –CH –S–). C NMR (DMSO-d ,
2
2
6
2
2
25 MHz), δ(ppm): 171.4 (C-2), 165.2 (–NH–C
),
20.9 (CH –C-4″), 18.2 (CH –C-2″). HR-ESI–MS: calcu-
thiadiazole-
3
3
+
53.5 (–CH –S–C
), 152.1 (C-3ʹ), 145.9 (C-2ʹ), 144.3
lated for C H O N S [M] : 570.1508, found: 570.1502.
2
thiadiazole
29 26
3
6 2
(
(
(
C-7ʹa), 140.9 (C-1″), 139.1 (C-7a), 133.3 (C-6ʹ), 129.6
C-3″, C-5″), 129.2 (C-4ʹ), 126.9 (C-6), 123.9 (C-4), 122.8
C-3a), 122.5 (C-4″), 121.9 (C-5ʹ), 121.2 (C-5), 117.9 (C-2″,
(2′Z-3′E)-Indirubin-3ʹ-[O-2-hydroxy-3-(5-((4-
ethylphenyl)amino)-1,3,4-thiadiazole-2-thio)prop-1-yl]
1
oxime (7i). Yield 65.6%, red solid, m.p. 221–222 °C. H
C-6″), 116.7 (C-3ʹa), 112.1 (C-7ʹ), 109.4 (C-7), 100.8 (C-3),
NMR (DMSO-d , 500 MHz), δ(ppm): 11.68 (s, 1H, H-1ʹ),
6
7
9.6 (–O–CH -), 68.3 (–CHOH–), 38.3 (–CH –S–). HR-
10.74 (s, 1H, H-1), 10.24 (s, 1H, NH-phenyl), 8.58 (d,
J=7.50 Hz, 1H, H-4), 8.19 (d, J=8.0 Hz, 1H, H-4ʹ), 7.43 (d,
J=8.50 Hz, 2H, H-2″, H-6″), 7.40 (m, 2H, H-6ʹ, H-7ʹ), 7.15
(d, J=8.50 Hz, 2H, H-3″, H-5″), 7.1 (m, 1H, H-6), 7.03 (m,
1H, H-5ʹ), 6.98 (m, 1H, H-5), 6.88 (d, J=8.0 Hz, 1H, H-7),
2
2
+
ESI–MS: calculated for C H O N S [M] : 542.1195,
2
7
22
3
6 2
found: 542.1190.
(
2′Z-3′E)-Indirubin-3ʹ-[O-2-hydroxy-3-(5-((2,3-
dimethylphenyl)amino)-1,3,4-thiadiazole-2-thio)prop-
1
-yl]oxime (7 g). Yield 72.3%, red solid, m.p. 231–232 °C.
5.75 (d, J=5.50 Hz, 1H, –CHOH-), 4.66 (dd, J =11.0 Hz,
1
1
H NMR (DMSO-d , 500 MHz), δ(ppm): 11.7 (s, 1H,
J = 4.50 Hz, 1H, –O–CH -), 4.59 (dd, J = 11.0 Hz,
6
2
2
1
H-1ʹ), 10.77 (s, 1H, H-1), 9.55 (s, 1H, NH-phenyl), 8.58
J = 6.50 Hz, 1H, –O–CH -), 4.34 (m, 1H, –CHOH–),
2
2
(
d, J = 8.0 Hz, 1H, H-4), 8.18 (d, J = 7.50 Hz, 1H, H-4ʹ),
3.52 (dd, J = 13.50 Hz, J = 5.0 Hz, 1H, –CH –S–), 3.34
1
2
2
7
.43 (m, 3H, H-6ʹ, H-7ʹ, H-4″), 7.13 (m, 1H, H-6), 7.05
m, 1H, H-5″), 7.00 (m, 3H, H-5ʹ, H-6″, H-5), 6.9 (d,
J=7.50 Hz, 1H, H-7), 5.75 (d, J=5.50 Hz, 1H, –CHOH-),
(1H, –CH –S–), 2.55 (q, J=7.50 Hz, 2H, CH –CH -), 1.16
2
3
2
1
3
(
(t, J = 7.50 Hz, 3H, CH –CH -). C NMR (DMSO-d ,
3
2
6
),
125 MHz), δ(ppm): 170.9 (C-2), 165.0 (–NH–C
thiadiazole-
4
.66 (dd, J = 11.0 Hz, J = 4.50 Hz, 1H, –O–CH -), 4.59
152.5(–CH –S–C
), 151.6 (C-3ʹ), 145.5 (C-2ʹ), 143.8
1
2
2
2
thiadiazole
(
dd, J = 11.0 Hz, J = 6.50 Hz, 1H, –O–CH -), 4.31 (m,
(C-7ʹa), 138.7 (C-7a), 138.2 (C-1″), 137.5 (C-4″), 132.9
(C-6ʹ), 128.7 (C-4ʹ), 128.3 (C-3″, C-5″), 126.4 (C-6), 123.4
(C-4), 122.3 (C-3a), 121.5 (C-5ʹ), 120.8 (C-5), 117.6 (C-2″,
C-6″), 116.2 (C-3ʹa), 111.7 (C-7ʹ), 108.9 (C-7), 100.3 (C-3),
79.2 (–O–CH -), 67.8 (–CHOH–), 37.9 (–CH –S–), 27.5
1
2
2
1
–
2
H, –CHOH–), 3.47 (dd, J = 13.50 Hz, J = 5.0 Hz, 1H,
1
2
CH –S–), 3.34 (1H, –CH –S–), 2.26 (s, 3H, CH –C-3″),
2
2
3
1
3
.11 (s, 3H, CH –C-2″). C NMR (DMSO-d , 125 MHz),
3
6
δ(ppm): 171.3 (C-2), 168.8 (–NH–C
), 152.6
thiadiazole-
2
2
(
–CH –S–C
), 152.1 (C-3ʹ), 146 (C-2ʹ), 144.3 (C-7ʹa),
(CH –CH -), 15.7 (CH –CH -). HR-ESI–MS: calculated
2
thiadiazole
3
2
3
2
+
1
39.5 (C-7a), 139.1 (C-1″), 138 (C-3″), 133.3 (C-6ʹ), 129.7
C-2″), 129.1 (C-4ʹ), 127 (C-4″), 126,9 (C-6), 126.4 (C-5″),
23.9 (C-4), 122.8 (C-3a), 121.9 (C-5ʹ), 121.2 (C-5), 121.1
C-6″), 116.6 (C-3ʹa), 112.2 (C-7ʹ), 109.4 (C-7), 100.7 (C-3),
9.6 (–O–CH ), 68.3 (–CHOH–), 38.5 (–CH –S–), 20.7
for C H O N S [M] : 570.1508, found: 570.1502.
29 26 3 6 2
(
1
(
Results and discussion
7
2
2
(
CH –C-3″), 14.2 (CH –C-2″). HR-ESI–MS: calculated
(2′Z)-Indirubin derivatives 4a-h (Table 1) were synthesized
via a two-step procedure (Scheme 1). Indirubin was ꢀrst con-
verted into the key intermediate 2 by the nucleophilic sub-
stitution at N1 position with epichlorohydrin using K CO
3
3
+
for C H O N S [M] : 570.1508, found: 570.1501.
2
9
26
3
6 2
(
2′Z-3′E)-Indirubin-3ʹ-[O-2-hydroxy-3-(5-((2,4-
dimethylphenyl)amino)-1,3,4-thiadiazole-2-thio)prop-
2
3
1
-yl]oxime (7 h). Yield 66.8%, red solid, m.p. 233–234 °C.
as a catalyst in the presence of a transfer calalyst (1-butyl)
triethylammonium bromide) to give 2 in 40% yield. The
N1-alkylation of indirubin was conꢀrmed by the interactions
1
H NMR (DMSO-d , 500 MHz), δ(ppm): 11.78 (s, 1H,
6
H-1ʹ), 10.87 (s, 1H, H-1), 9.53 (s, 1H, NH-phenyl), 8.69 (d,
1
3

Chemical Papers
Table 1 Structures and numbering of 4a-h and 7a-i
No Compound
s
R
No Compounds
R
1
2
3
4a
4b
4c
1
2
3
7a
7b
7c
4
5
4d
4e
4
5
7d
7e
6
4f
6
7f
7
4g
7
7g
8
9
4h
8
9
7h
7i
Scheme 1 Preparation of new derivatives 4a-h: Reagents and conditions: (i) epichlorohydrin, DMF, K2CO3, KI, (1-butyl)triethylammonium
bromide; (ii) thiols 3a-h, DMF, rt, 40 h
1
3

Chemical Papers
of protons in –N –CH - at 4.20 ppm (dd, J = 15.0 Hz,
attributed to H-4 and H-4′ protons, and the overlapped dou-
blet signal (δ: 7.53 ppm, J=8.0 Hz) was assigned to protons
H-2′’ and H-6′’. Protons H-3′’ and H-5′’ were recognized
by a triplet at 7.32 ppm (J = 7.75 Hz) and other triplet at
7.10 ppm (J=7.50 Hz) arose from H-6. Besides a multiplet
at 7.41 ppm arising from H-6′ and H-7′ of indirubin moiety,
signal of the remaining thiol proton H-4′’ overlapped by pro-
tons H-5 and H-5′ of indirubin component was found due to
a multiplet at (7.01 ppm). The ꢀnal proton H-7 in indirubin
1
2
1
J =3.75 Hz) and 3.88 ppm (dd, J =15.0 Hz, J =5.25 Hz)
2
1
2
with C-2 (169.6 ppm) and C-7a (141.9 ppm) of indirubin
in HMBC spectrum. Epoxy 2 was then opened with thiols
3
a-h to obtain target compounds 4a-h in 63.1–77.0% yields
(
Scheme 1).
The structure of 4a-h was determined by 1D, 2D and
HRMS spectra. The spectral signal distinction among
derivatives 4a-h was found due to the resonance of protons
and carbons in thiol moieties. Compound 4f was selected to
assign structure supported extensively by HSQC and HMBC
resonates as a doublet (J =7.50 Hz) at 6.89 ppm. In 2-ol-
1
1,3-propylen linker, a doublet (J=5.50 Hz) at 5.76 ppm was
assigned to proton –OH and methine proton attached to this
group appeared as a multiplet at 4.34 ppm. In addition, dou-
blet–doublet signals at 4.66 ppm (J =11.0 Hz, J =4.50 Hz)
1
13
correlations. The data given from H- and C-NMR spectra
of 4f as well as the support from HSQC and HMBC spec-
tra agreed well with the structure 4f. The structure of the
remaining was in good agreement with NMR and HRMS
data.
1
2
and 4.59 ppm (J =11.0 Hz, J =6.0 Hz) were determined
1
2
to be resonance signals of protons O–CH -, and the sig-
2
Series of new (2′Z-3′E)-indirubin-3′-oxime derivatives
nals at 3.53 ppm and 3.37 ppm were characteristic of two
(
7a-i) were produced by opening epoxy ring at oxime posi-
protons, which were neighbor to sulfur (S–CH ). Based on
2
tion of the key intermediate 6 that was synthesized from
indirubin (1) in two steps. Compound 5 with (2E,3Z) con-
formation was ꢀrst obtained in 93% yield by a condensation
reaction of indirubin with hydroxylamine hydrochloride
in pyridine (Cuong et al. 2010a, b, c). Compound 5 was
then O-alkylated using epibromohydrin catalyzed by trieth-
ylamine according to a known procedure (Ichimaru et al.
HSQC spectrum, the chemical shifts of the corresponding
carbons were elucidated as: C-4: 123.9, C-4′: 129.2, (C-2′’,
C-6′’): 117.9, (C-3′’, C-5′’): 129.6; C
: 68.3, C–O
:
CH2-
CHOH
79.6, C
: 38.3 ppm. In addition, the key cross-
S–CH2–CHOH–CH2-
picks of C
(153.5 ppm) with H
in HMBC
thiadiazole
S–CH2–CHOH–
approved the correct structure of 7f (Scheme 2).
2
015) to aꢁord 6 in 70% yield (Scheme 2). In the last step,
was opened with the corresponding thiols 3a-i in DMF
6
in the presence of triethylamine as a catalyst to give 7a-i in
Bioactivity
5
9.2–75.0% yields (Table 1).
The structure of 7a-i was confirmed by 1D, 2D and
In the present study, indirubin (1), indirubin-3′-oxime (5)
and 6-mercaptopurine (6-MP), a medicine for treatment of
blood cancer were also evaluated for cytotoxicity against
SW480, LU-1, HepG2 and HL-60 cell lines along with
indirubin and indirubin-3′-oxime derivatives (4a-h and 7a-
i), and ellipticine was used as positive control for SAR evalu-
ation (Table 1). Cytotoxic activity against SW480, LU-1,
HepG2 cell lines and anti-proliferative activity of HL-60
cell line were undertaken using the method as described by
Monks (Monks et al. 1991). The derivatives 4a-h, 7a-i were
judged to have no activity when their IC values>20 µM.
HRMS spectra. The similarity of the multiplicity and signal
1
position of indirubin moiety was easily observed in H- and
1
3
C-NMR spectra of compounds 7a-i and the distinction
among those was also found due to the diꢁerent structure
of thiol components. Compound 7f was selected to assign
structure supported extensively by HSQC and HMBC cor-
1
relations. In H-NMR spectra, protons H-1, H-1′ of indiru-
bin and NH of thiol moiety appeared as singlet signals at
1
0.72, 11.67 and 10.32 ppm respectively. Two doublets
at 8.58 ppm (J = 7.50 Hz) and 8.19 ppm (J = 7.50 Hz)
5
0
Scheme 2 Preparation of new derivatives 7a-i: Reagents and conditions: (i) hydroxylamine, pyridine, 120 °C; (ii) epibromohydrin, TEA, DMF;
iii) thiols 3a-i, TEA, DMF, rt, 40 h
(
1
3

Chemical Papers
Table 2 In vitro cytotoxic activity of indirubin 4a-h and indirubin-
chemical intervention in this group resulted in the decreased
activity, and the synthesized derivatives showed no activity
against SW480, LU-1, HepG2, and HL-60 cell lines except
derivatives 7a, 7f, 7 g and 7 h containing phenylaminothia-
diazole, phenylthiazole and 6-mecarptopurin exhibited weak
activity with IC values in range of 15.34–19.24 µM and
3
-oxime derivatives 7a-i
No
Compounds
IC50 (µM)
SW480
LU-1
HepG2
HL60
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
4a
>20
>20
>20
>20
>20
1.65
>20
4.19
19.24
>20
>20
>20
>20
16.38
15.96
>20
>20
19.10
>20
14.26
2.19
>20
>20
>20
>20
>20
2.21
>20
4.53
19
>20
>20
>20
>20
16.90
19.11
19.20
>20
18.75
>20
13.11
1.91
>20
>20
>20
>20
>20
1.90
>20
4.47
>20
>20
>20
>20
>20
17.67
15.34
>20
>20
19.72
>20
15.87
1.79
>20
>20
>20
>20
>20
1.35
>20
4.28
>20
>20
>20
>20
>20
9.52
12.03
16.40
>20
16.04
>20
14.90
2.40
5
0
4b
not so prominent. An expected result was also found with
derivative 7e, a conjugate of indirubin-3′-oxime and a medi-
cine for treatment of blood cancer, 6-mecarptopurine was
negative with tested cell lines. Evidently, the evaluation of
cytotoxic and anti-proliferative activities of indirubin and
indirubin-3′-oxime derivatives resulted in conclusion that
the particular structure of parent compound, (2′Z)-indirubin
and 3′-oxime group were main activity centers of (2′Z-3′E)-
indirubin-3′-oxime. We suggest this be an important ori-
entation for the design, synthesis of indirubin-3-oxime
derivatives in a search for new cytotoxic agents in our future
research.
4c
4d
4e
4f
4 g
4 h
7a
0
1
2
3
4
5
6
7
8
9
0
1
7b
7c
7d
7e
7f
7 g
7 h
Conclusions
7i
6-MP
The design, synthesis and screening for cytotoxic and anti-
proliferative activities of indirubin derivatives containing
thiols were successfully performed. The cytotoxic and anti-
proliferative activities of ꢀve in 17 new derivatives were also
carried out. Data on cytotoxic and anti-proliferative activi-
ties of two series 4a-h and 7a-i could lead to conclusion that
the combination of speciꢀc conꢀguration of indirubin and
indirubin
Indirubin-3ʹ-oxim
Ellipticine
The reference substance, ellipticine, exhibited cytotoxic activity
against SW 480 (ATCC-CCL-228), LU-1 (ATCC-HTB-57), HepG2
(
ATCC-HB-8065) and HL-60 (ATCC-CCL-240) with IC values
50
of 2.19, 1.91, 1.79 and 2.40 μM, respectively. The values shown for
these compounds are the average of three determinations
3
′-oxime group in its derivative was necessary for antican-
cer activity. Therefore, chemical transformation at oxime
group did not improve anticancer activity while chemical
intervention at N1 position could produce new derivatives
that showed strong cytotoxic and anti-proliferative activi-
ties in some cases. This may be an important orientation in
In screening for cytotoxic and anti-proliferative activities
against four cancer cell lines: SW480, LU-1, HepG2 and
HL 60, indirubin did not show direct activity. This was also
observed in some its derivative 4a-h excluding 4f and 4 h
ꢀ
nding candidates from indirubin that can be used in cancer
(
Table 2). Obviously, the derivatives derived from N1 posi-
treatment in the future.
tion did not give the clear advantages to parent compound,
indirubin, even derivative 4e formed by the opening epoxy
with 6-mercaptopurine (6-MP). However, in some cases,
the structure of thiol component has a signiꢀcant eꢁect on
cytotoxic and anti-proliferative activities. Compounds 4f,
Acknowledgements The authors gratefully acknowledge Viet-
nam-Russia Tropical Center and Vietnam Academy of Science and
Technology (VAST) for ꢀnancial support via project VAST.HTQT.
BELARUS.04/15-16.
4
h derived from indirubin and 5-(phenylamino)-1,3,4-thi-
Compliance with ethical standards
adiazole-2-thiol or 5-((2,4-dimethylphenyl)amino)-1,3,4-
thiadiazole-2-thiol showed strong activity against SW480,
LU-1, HepG2 and HL-60 cell lines in which compound 4f
exhibited the strongest activity with IC values of 1.65,
Conflict of interest The authors declare no conꢂicts of interest.
5
0
2
.21, 1.90 and 1.35 µM, respectively, much stronger than
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