Synthesis and evaluation of novel hybrids β-carboline-4-thiazolidinones as potential antitumor and antiviral agents

Article abstract of DOI:10.1016/j.ejmech.2016.10.018

A series of novel hybrids β-carboline-4-thiazolidinones were synthesized and evaluated for their in vitro antitumor activity against human cancer cell lines and for antiviral activity towards Herpes simplex virus type-1 (HSV-1). From the N′-(2-ylidene-4-thiazolidinone)-β-carboline-3-carbohydrazide series (9–11), compounds 9c and 11d were the most active, showing growth inhibition 50% (GI₅₀) values less than 5 μM for all cell lines tested. Compound 9c, bearing the 4-dimethylaminophenyl group at C-1 of β-carboline was selected for further investigation concerning cell death and cell cycle profile, focusing on the human renal adenocarcinoma cell line 786-0. Treatments with 25 μM of compound 9c induced cell death after 15 h of treatment, characterized by phosphatidylserine exposure and loss of membrane integrity. Moreover, treatment with 12.5 μM promoted a sub-G1 arrest, which indicates cell death. Derivatives of the N-(2-substituted-aryl-4-thiazolidinone)-β-carboline-3-carboxamide series (18–23) showed a potent activity and high selectivity for glioma (U251) and ovarian (OVCAR-3) cancer cell lines. Also, some β-carboline-4-thiazolidinone hybrids showed potent antiviral activity against Herpes simplex virus type-1. The N-(2-substituted-aryl-4-thiazolidinone)-carboxamide moiety in 18, 19 and 22 confer a potent anti-HSV-1 activity for these derivatives, which presented EC₅₀values of 0.80, 2.15 and 2.02 μM, respectively. The assay results showed that the nature of 4-thiazolidinone moiety and of the substituent attached at the 3- and 1- position of β-carboline nucleus influenced the antitumor and antiviral activities.

Full text of DOI:10.1016/j.ejmech.2016.10.018

Accepted Manuscript
Synthesis and evaluation of novel hybrids β-carboline-4-thiazolidinones as potential
antitumor and antiviral agents
Valéria Aquilino Barbosa, Paula Baréa, Renata Sespede Mazia, Tania Ueda-
Nakamura, Willian Ferreira da Costa, Mary Ann Foglio, Ana Lucia T. Goes Ruiz, João
Ernesto de Carvalho, Débora Barbosa Vendramini Costa, Celso Vataru Nakamura,
Maria Helena Sarragiotto
PII:
S0223-5234(16)30880-7
10.1016/j.ejmech.2016.10.018
EJMECH 8983
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 June 2016
Revised Date: 6 October 2016
Accepted Date: 9 October 2016
Please cite this article as: V.A. Barbosa, P. Baréa, R.S. Mazia, T. Ueda-Nakamura, W. Ferreira da
Costa, M.A. Foglio, A.L.T. Goes Ruiz, J. Ernesto de Carvalho, D.B. Vendramini Costa, C.V. Nakamura,
M.H. Sarragiotto, Synthesis and evaluation of novel hybrids β-carboline-4-thiazolidinones as potential
antitumor and antiviral agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.10.018.
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ACCEPTED MANUSCRIPT
O
S
N
N
H
N
Ph
O
O
S
N
N
H
N
Annexin-V 18h
N
H
N
100
80
60
40
20
0
O
Compound 11d
GI50 = 0.19 to 3.91 µM
for all cell lines
2
N
Vehicle
9c (25 µM)
R
N
H
1
NO
Antitumor
assay
2
R
2
R
O
***
***
S
***
9a-f
10a-f
H
Et
Ph
N
N
H
N
H
O
N
11a, c-d, f
N
H
Compound 9c
GI50 = 1.60 µM; TGI = 13 µM;
LC50 = 105 µM for renal
786-0 cell line
N
O
O
S
N
1
2
N
H
R
R
R2
N
18
19
22
Ph
Ph
Ph
Ph
4-NM e -Ph
Anti-HSV-1 assay
EC50 0.80 to 2.15 µM
IS > 200
N
H
R1
2
3-NO -Ph
2
18-23

ACCEPTED MANUSCRIPT
Synthesis and evaluation of novel hybrids β-carboline-4-
thiazolidinones as potential antitumor and antiviral agents
Valéria Aquilino Barbosaa, Paula Baréaa, Renata Sespede Maziab, Tania Ueda-
Nakamurab, Willian Ferreira da Costaa, Mary Ann Foglioc, Ana Lucia T. Goes Ruizc,
João Ernesto de Carvalhoc, Débora Barbosa Vendramini Costac, Celso Vataru
Nakamurab and Maria Helena Sarragiottoa,*
a Universidade Estadual de Maringá (UEM), Departamento de Química, Av. Colombo, 5790,
CEP: 87020-900 Maringá, PR, Brazil
b
Universidade Estadual de Maringá (UEM), Departamento de Análises Clínicas, Universidade
Estadual de Maringá, Av. Colombo, 5790, CEP: 87020-900 Maringá, PR, Brazil
c
Universidade Estadual de Campinas (Unicamp), Centro Pluridisciplinar de Pesquisas
Químicas, Biológicas e Agrícolas (CPQBA), CEP: 13083-970 Campinas, SP, Brazil
*corresponding author. E-mail: mhsarragiotto@uem.br (M. H. Sarrragiotto)

ACCEPTED MANUSCRIPT
ABSTRACT
A series of novel hybrids β-carboline-4-thiazolidinones were synthesized and
evaluated for their in vitro antitumor activity against human cancer cell lines and for
antiviral activity towards Herpes Simplex virus type-1 (HSV-1). From the N’-(2-
ylidene-4-thiazolidinone)-β-carboline-3-carbohydrazide series (9-11), compounds 9c
and 11d were the most active, showing growth inhibition 50% (GI50) values less than 5
µM for all cell lines tested. Compound 9c, bearing the 4-dimethylaminophenyl group at
C-1 of β-carboline was selected for further investigation concerning cell death and cell
cycle profile, focusing on the human renal adenocarcinoma cell line 786-0. Treatments
with 25 µM of compound 9c induced cell death after 15 h of treatment, characterized by
phosphatidylserine exposure and loss of membrane integrity. Moreover, treatment with
12.5 µM promoted a sub-G1 arrest, which indicates cell death. Derivatives of the N-(2-
substituted-aryl-4-thiazolidinone)-β-carboline-3-carboxamide series (18-23) showed a
potent activity and high selectivity for glioma (U251) and ovarian (OVCAR-3) cancer
cell lines. Also, some β-carboline-4-thiazolidinone hybrids showed potent antiviral
activity against Herpes simplex virus type-1. The N-(2-substituted-aryl-4-
thiazolidinone)-carboxamide moiety in 18, 19 and 22 confer a potent anti-HSV-1
activity for these derivatives, which presented EC50 values of 0.80, 2.15 and 2.02 µM,
respectively. The assay results showed that the nature of 4-thiazolidinone moiety and of
the substituent attached at the 3- and 1- position of β-carboline nucleus, respectively,
influenced the antitumor and antiviral activities.
Keywords: β-carboline, 4-thiazolidinone, antitumor, antiviral, medicinal chemistry

ACCEPTED MANUSCRIPT
1. Introduction
Naturally occurring and synthetic β-carboline alkaloids are widely studied due to
their large spectrum of important pharmacological and biological properties, such as
antitrypanosomal and antileishmanial [1-4], anti-Alzheimer [5,6], anti-platelet
aggregation and anti-thrombotic [7,8], anti-Parkinson [9], and as DYRK1A inhibitors
[10,11]. It’s worth mentioning the potent antitumor activity of this class of compounds
that has been reported in several studies involving design, synthesis and structure-
activity relationship (SAR) of β-carboline derivatives [12-20]. These studies revealed
that the introduction of appropriate substituents into 1-, 3- and 9-positions of
β-
carboline ring can result in more potent drugs with reduced toxicity and neurotoxic
effects. Besides the antitumor activity, recent reports have been shown that β-carboline
derivatives can act as antiviral agents against Human Immunodeficiency Virus type 1
(HIV-1) and type 2 (HIV-2) [21-23], and Tobacco Mosaic Virus (TMV) [24]. Also,
recent works showed that several nitrogen heterocyclic compounds displayed anti-HSV
activity [25-28].
In our previous work we demonstrated that the presence of an appropriately
substituted phenyl group at 1-position, and substituents at 3-position of the
nucleus, enhanced the antitumor activity [29-33]. Also, our work indicated that hybrids
of -carboline with different heterocyclic nucleus at 3-position, as for compounds I, II
β-carboline
β
and III (Fig. 1), displayed potent antitumor properties [30, 32, 33]. In addition, the
interaction of compound I with DNA was investigated by using UV and fluorescence
spectroscopic analysis. Our studies showed that I interact with ctDNA by intercalation
binding [30].
The antiviral activity of 1-(substituted-phenyl)-N’-(substituted-benzylidene)-β-
carboline-3-carbohydrazides against HSV-1 and vaccinal poliovirus (VP) was also
demonstrated for our research group [34, 35].
4-Thiazolidinone derivatives have been extensively investigated due to their
gamma of biological activities, such as anti-inflammatory, anticonvulsant, anti-diabetic,
cardiovascular, anti-tubercular, antifungal, antibacterial, antiviral, as well as anticancer
activity against different cell lines [36-38].
Concerning to anticancer activity, recent reports have indicated that 4-
thiazolidinones can inhibit tumor cell proliferation through inducing cell cycle arrest
[39-48]. This was demonstrated for a series of 2,3-diaryl-4-thiazolidinone derivatives,

ACCEPTED MANUSCRIPT
especially for compound IV (Fig. 1), which displayed potent inhibitory effects on the
proliferation of A549 (human lung cancer) and MDA-MB-231 (human breast cancer)
[39]. Also, new hybrids acridine–thiazolidinone (compounds V, Fig. 1), displaying
strong cytotoxic activity in vitro against leukemic cells HL-60 and L1210, and human
epithelial ovarian cancer cell lines A2780, inhibited cells proliferation and induced an
arrest of the cell cycle and cell death. The effects on cells were associated with their
reactivity towards thiols and DNA binding interaction [40].
A compound from the 2-(thienothiazolylimino)-1,3-thiazolidin-4-ones series
synthesized by Huber-Villaume et al. [41] was found to be potential inhibitor of CDC25
protein, with an IC50 of 6.2 ± 1.0 µM. Treatment with the active 4-thiazolidinone
derivative led to MCF7 and MDA-MB-231 cell growth arrest. Studies developed by
Zhang et al. [48] shown that compounds bearing thiazolidinone nucleus act as either
microtubule polymerization inhibitors or histone deacetylase inhibitors. The derivatives
act synergistically, targeting multiple proteins and leading to the regulation of cell cycle
checkpoint proteins, which resulted in the G2/M cell cycle arrest and cell apoptosis
[42].
The properties of 4-thiazolidinones to inhibit proliferation through inducing cell
cycle arrest action, and through other action mechanisms, as well as the potent
anticancer exerted for 4-thiazolidinone derivatives, led us to explore this scaffold to
search for potential agents for treating multi-factorial diseases such cancer. The
molecular hybridization of β-carboline and 4-thiazolidinone pharmacophores was
chosen as a strategy to obtain more active compounds that impact multiple anticancer
targets [43]. Furthermore, as both β-carboline and 4-thiazolidinone nucleus exhibit
antiviral activity, it is expected that the proposed hybrids are also active against HSV-1.

ACCEPTED MANUSCRIPT
H
N
NH
N
N
O
N
O
O
S
O
N
H
N
N
N
H
N
N
N
H
H
N
N
OCH3
I
II
III
DNA intercalation binding
GI50 of 0.67 to 3.20 M
IC50 of 0.48 to 67.88 M
GI50 of 0.04 to 11.1 M
O
O
R
N
OMe
N
O
S
N
S
CH3
O
H
N
N
H
O
Br
IV
V
Anticancer activity
Inducing cell cycle arrest
O
O
O
S
N
S
N
N
N
H
R2
H
N
N
N
N
O
N
R2
H
H
R1
R1
Hybrids -carboline-thiazolidinone proposed
Figure 1: β-Carboline (I-III) and thiazolidinone (IV and V) derivatives with anticancer
activity.
More specifically, in this work we describe the synthesis of a series of N’-(2-
ylidene-4-thiazolidinone)-β-carboline-3-carbohydrazides and N-(2-substituted-aryl-4-
thiazolidinone)-β-carboline-3-carboxamides, as well as the antiviral activity against
Herpes simplex virus (HSV-1), and antitumor activity towards human cancer cell lines
of the new synthetized compounds. Additionally, studies evaluating cell death and
influence in the cell cycle profile were also carried out for the active compound 9c,
focusing on the human renal adenocarcinoma cell line 786-0.
2. Results and discussion
2.1. Chemistry

ACCEPTED MANUSCRIPT
The synthetic routes employed in the preparation of the hybrids N’-(2-ylidene-4-
thiazolidinone)-β-carboline-3-carbohydrazides (9a-f), (10a-f) and (11a, c-d, f), and of
N-(2-substituted-4-thiazolidinone)-β-carboline-3-carboxamides (18-23) are shown in
Scheme 1.
To obtain the hybrids β-carbolines-4-thiazolidinones proposed, firstly the β-
carboline-3-carbohydrazides (5a-f), the common intermediates for the synthetic routes,
were synthetized. For this, the commercial L-tryptophan (1) was esterified with
methanol in the presence of H2SO4 to afford L-tryptophan methyl ester (2), which was
subjected to Pictet Spengler condensation reaction with different aromatic aldehydes, in
acidic media, to provide the tetrahydro-β-carboline-3-carboxylates (3a-f). The oxidation
of 3a-f with sulfur in xylene under reflux afforded the β-carbolines 4a-f. The β-
carboline-3-carbohydrazides (5a-f) were obtained by nucleophilic substitution reaction
of the β-carbolines 4a-f with hydrazine hydrate in ethanol.
The synthesis of N’-(2-ylidene-4-thiazolidinone)-β-carboline-3-carbohydrazides
(9a-f) was carried out by the reaction of β-carboline-3-carbohydrazides (5a-f) with
potassium isothiocyanate, in ethanol, under reflux, followed by condensation reaction of
thiosemicarbazides 6a-f with ethyl bromoacetate, using sodium acetate and potassium
hydroxide in ethanol.
To evaluate the effect of a substituent at the nitrogen of thiazolidinone ring on
activity, the carbohydrazides 5a-f were treated with ethyl isothiocyanate and phenyl
isothiocyanate to give the respective thiosemicarbazides 7a- f and 8a, 8c-d and 8f,
which afforded the N-substituted derivatives 10a- f and 11a, 11c-d and 11f by using the
reaction conditions for preparation of derivatives 9a-f.
To
obtain
the
N-(2-substituted-aryl-4-thiazolidinone)-β-carboline-3-
carboxamides (18-23) the β-carboline-3-carbohydrazides 5(a, c, d, f) were subjected to
reaction with aromatic aldehydes in DMF under microwave irradiation to afford β-
carbolines-3-benzylidenecarbohydrazides (12-17). The formation of N-(2-substituted-
aryl-4-thiazolidinone)-β-carboline-3-carboxamides (18-23) was possible with addition
of the catalytic amount of p-toluenesulfonic acid to the reaction of imine intermediates
12-17 and thioglycolic acid, under toluene reflux.

ACCEPTED MANUSCRIPT
O
O
O
OR
OCH3
OCH3
b
d
c
NH2
NH
N
N
N
H
N
H
R1
R1
H
1:R = H
3a-f
a
4a-f
2:R = OCH3
O
1"
2" S
O
S
4
1
5
8
5"
4"
4b
4a
3
6
7
NHN
f
NHNH
NHR2
e
3" N
R2
N
O
N
8a N 9a
N
H
R1
H
R1
R2
H
Et
Ph
O
R2
H
Et
Ph
9a-f
10a-f
6a-f
7a-f
8a, c-d, f
NHNH2
N
11a, c-d, f
N
H
R1
O
5"
4"
O
O
3"N
4
5
S
5a-f
R2
4b
8a
4a
3
h
N
1"
g
6
7
N
N
H
2"
R2
N
H
N
N
H
9a
N
1
8
R1
H
R1
R1
R2
R1
Ph
4-NMe2-Ph
2-F-Ph
Ph
R2
18
Ph
Ph
Ph
12
Ph
Ph
Ph
Ph
19
20
21
22
23
13
14
15
16
17
4-NMe2-Ph
2-F-Ph
Ph
3-NO2-Ph
Ph
2-thienyl
Ph
2-Cl-Ph
2-thienyl
Ph
2-Cl-Ph
3-NO2-Ph
Ph
Cl
F
NO2
2'
3'
5'
1'
R1 = a)
4'
f)
b)
e)
OCH3 c)
N
d)
6'
Scheme 1. Reagents and conditions: (a) MeOH, H2SO4 (cat), reflux, 48 h, (b) R1CHO, TFA, DCM, rt; (c) S, xylene, reflux, 48 h
to 0 °C, 3 h; (d) NH2NH2.H2O, EtOH, reflux, 48 h, 77%: (e) KSCN, R2NCS, EtOH, reflux; (f) BrCH2CO2C2H5, AcONa, KOH,
EtOH reflux; (g) DMF, R2CHO, MW; (h) HSCH2COOH, p-TsOH (cat), toluene, reflux.

ACCEPTED MANUSCRIPT
The sythetized compounds were characterized by their spectral data (HRMS-
ESI, 1H NMR and 13C NMR). The characterization of the N’-(2-ylidene-4-
thiazolidinone)-β-carboline-3-carbohydrazides without substituent at the nitrogen of
thiazolidinone ring (9a-f) was mainly supported by signals at δH 3.52 – 4.05 (s, 2H,
1
CH2, H-5”) in H NMR spectra, and signals at δC 34.2 – 38.0 (CH2, C-5”), δC 172.1 –
182.4 (C=O, C-4”), δC 162.7 – 167.3 (C=O, carbohydrazide) and δC 150.8 – 162.7
(C=N, C-2”) in the 13C NMR spectra.
The
N’-(3-ethyl-2-ylidene-4-thiazolidinone)-β-carboline-3-carbohydrazides
(10a-f) were characterized by signals at δH 3.86 – 4.19 (s or m, 2H CH2, H-5”), δH 3.76 –
4.00 (m or quart. J = 6.1 – 7.0 Hz; 2H CH3CH2), δH 1.20 – 1.33 (t, J = 6.3 – 7.2 Hz; 3H
CH3CH2) in the 1H NMR spectra. Signals at δC 29.6 – 38.7 (CH2, C-5”), δC 32.9 – 38.9
(CH3CH2), δC12.1 – 14.5 (CH3CH2), δC 170.7 – 171.5 (C=O, C-4”); δC 159.6 – 162.7
(C=O, carbohydrazide) and δC 151.1 – 161.9 (C=N, C-2”) in the 13C NMR spectra
confirmed the formation of these products.
The formation of N’-(3-phenyl-2-ylidene-4-thiazolidinone)-β-carboline-3-
carbohydrazides 11(a, c-d, f) was evidenced by the signals at δH 3.04 – 4.33 (s, 2H,
CH2, H-5”) and by the hydrogens signals of the phenyl group attached to the nitrogen
13
atom. In the C NMR spectra were observed signals at δC 33.0 – 33.2 (CH2, C-5”), δC
170.5 – 171.2 (C=O, C-4”), δC 158.3 –160.5 (C=O, carbohydrazide), and δC 159.1 –
160.3 (C=N, C-2”).
The N-(2-substituted-aryl-4-thiazolidinone)-β-carboline-3-carboxamides (18-23)
were characterized by the signals at δH 3.78 – 3.84 (dd, J = 1.5 – 1.8 and 15.0 Hz, 1H
CH2, Hb-5”) coupled in W with hydrogen (CH, H-2”) of thiazolidinone ring, δH 3.92 –
1
4.00 (d, J = 15.0 – 15.9, 1H, CH2, Ha-5”), δH 6.01 – 6.57 (s, 1H, CH, H-2”) in the H
NMR spectra. In the 13C NMR spectra were observed signals at δC 29.5 – 30.5 (CH2, C-
5”), δC 60.2 – 63.7 (CH, C-2”), δC 169.2 – 170.9 (C=O, C-4”), and δC 162.9 – 165.1
(C=O, carboxamide).
2.2. Biological activities
2.2.1. Antitumor activity
The in vitro anticancer activities of the synthesized β-carboline-4-
thiazolidinones were evaluated against nine human tumor cell lines – U251 (Glioma),
UACC-62 (melanoma), MCF-7 (Breast), NCI/ADR-RES (ovarian expressing multiple-

ACCEPTED MANUSCRIPT
drug-resistance phenotype), 786-0 (Renal), NCI-H460 (Lung), PC-3 (Prostate),
OVCAR-3 (Ovarian) and HT-29 (Colon). The response parameter GI50 was calculated
for each compound and cell line tested, and the results were summarized in Table 1 and
2. The GI50 values (growth inhibitory activity) refer to the drug concentration that
inhibits in 50% the cellular growth when compared to untreated control cells.
Compounds with GI50 values ≥ 100 µM were considered not active.
The analysis of GI50 values (Table 1) showed that for the N’-(2-ylidene-4-
thiazolidinone)-β-carboline-3-carbohydrazides (9a-f), the compound 9a containing
phenyl group in the 1-position of the β-carboline ring was active for all human tumor
cell lines tested, with GI50 values less than 30 µM.
In order to verify the influence of electron-withdrawing and -donating groups in
antitumor activity, the phenyl ring with different groups (OCH3, NMe2, NO2, Cl and F)
was introduced in the 1-position the β-carboline skeleton.
The substitution of the phenyl group at C-1 in 9a for a 4-methoxyphenyl group
(9b) or 4-dimethylaminophenyl group (9c) led to an improvement of activity for most of
cell lines tested. On the other hand, the presence of the withdrawing nitro substituent at
the phenyl group resulted in loss of antitumor activity, being the derivative 9d inactive
towards lung (NCI-H460), ovarian (OVCAR-3), and colon (HT-29) cell lines. However,
a potent activity (GI50 = 0.62 µM) and selectivity was observed for this derivative
against NCI/ADR-RES (ovarian expressing multiple-drug-resistance phenotype).
Selectivity was also observed for the derivative 9e, which contains 2-chlorophenyl
group in C-1, presenting a GI50 value of 0.01 µM against ovarian (OVCAR-3) cells.
To investigate the effect of substituents at the nitrogen atom of 4-thiazolidinone
ring, N-substituted derivatives 10a- f and 11(a, c-d, f), bearing the N3-ethyl and N3-
phenyl groups, respectively, were also evaluated for their antitumor activity.
In general, the introduction of the N3-ethyl group did not contribute to the
overall increase in activity, but enhanced the selectivity of some of the derivatives
against specific tumor cells. The derivative 10a was selective for glioma (U251) with
GI50 value of 0.46 µM. The derivative 10e was selective for glioma and ovarian
expressing multiple-drug-resistance phenotype (NCI/ADR-RES), with GI50 values of
0.74 and 0.25 µM, respectively. Strong selectivity was also observed for the derivative
10d against NCI/ADR-RES, which presented GI50 value of 3.27 µM for this cancer cell
lines, and GI50 values greater than 100 µM for all other cells tested.

ACCEPTED MANUSCRIPT
On the other hand, the presence of a phenyl group at the 3-position of
thiazolidinone ring provide an enhancement of activity for the derivative containing the
4-nitrophenyl at C-1 (11d), compared to their analogs 9d and 10d. Compound 11d was
the most active of the N’-(2-ylidene-4-thiazolidinone)-β-carboline-3-carbohydrazide
series, showing GI50 values in the range of 0.83 to 3.81 µM for all cell lines tested.
Comparison of GI50 values (Table 1) of the most active derivatives 9c and 11d
with those of compound I (Fig. 1) described previously for our group [30] reveled that
these compounds displayed similar activity. On the other hand, the incorporation of 4-
thiazolidinone nucleus into β-carboline skeleton resulted in an enhancement of
antitumor activity for 9c and 11d compared to compounds II and III [31, 33] (Fig 1),
as expected.

ACCEPTED MANUSCRIPT
Table 1. In vitro antitumor activity (GI50 in µM) of 1-(substituted-phenyl)-β-carboline 3-(substituted-4-thiazolidinone) derivatives 9-11.
Cell lines
Glioma
(U251)
Breast
(MCF-7)
Ovarian Resistant
(NCI/ADR-RES)
Renal
(786-0)
Lung
(NCI-H460)
Ovarian
(OVCAR-3)
Colon
(HT-29)
R1
R2
H
Comp
9a
Ph
15.75
NT
11.40
0.49
28.12
>100
0.48
16.33
9.98
1.60
16.11
6.10
20.33
nt
18.18
7.15
9.88
1.12
1.27
>100
0.01
12.30
>100
6.52
2.42
>100
3.97
>100
19.54
16.52
5.04
4-MeO-Ph
4-NMe2-Ph
3-NO2-Ph
2-Cl-Ph
2-F-Ph
H
9b
H
NT
2.93
1.44
9c
H
NT
40.07
1.42
0.62
>100
3.85
>100
10.81
32.41
>100
9.62
9d
H
NT
>100
66.42
8.89
9e
H
9.57
0.46
1.95
63.33
>100
0.74
>100
11.87
17.21
6.52
21.07
>100
8.56
9f
Ph
Et
Et
Et
Et
Et
Et
10a
10b
10c
10d
10e
10f
4-MeO-Ph
4-NMe2-Ph
3-NO2-Ph
2-Cl-Ph
2-F-Ph
12.16
2.60
nt
54.87
>100
6.84
6.61
>100
nt
58.95
>100
4.12
65.65
>100
88.87
>100
3.27
0.25
>100
>100
>100
>100

ACCEPTED MANUSCRIPT
Table 1. In vitro antitumor activity (GI50 in µM)……………………………………………………………..continued
Ph
Ph
Ph
Ph
Ph
17.54
12.51
1.59
27.96
14.97
2.36
3.13
35.42
0.19
13.64
3.97
37.25
17.92
3.91
17.54
>100
0.83
>100
38.06
2.28
11a
11c
11d
11f
4-NMe2-Ph
3-NO2-Ph
2-F-Ph
1.53
>100
>100
>100
>100
>100
>100
>100
NT = not tested. GI50: concentration of compound necessary to promote 50% of growth inhibition after 48h of treatment, comparing to cells
growing without treatment.

ACCEPTED MANUSCRIPT
The in vitro antitumor activity of derivatives 18-23 are shown in Table 2. The
changes in the nature of 4-thiazolidinone nucleus led to a decrease of activity for the
derivatives of the N-(2-substituted-aryl-4-thiazolidinone)-β-carboline-3-carboxamide
series compared to those from the N’-(2-ylidene-4-thiazolidinone)-β-carboline-3-
carbohydrazides (9-11) series. However, a potent activity and high selectivity for
glioma (U251) and ovarian (OVCAR-3) cancer cell lines was observed for some
derivatives. Compound 18, which contains a phenyl group in the 1-position of the β-
carboline ring, as well as in the nitrogen atom of 4-thiazolidinone ring that and
displayed GI50 values of 0.25 µM and 1.58 µM for glioma (U251) and ovarian
(OVCAR-3) cell lines, respectively.
The compounds 20 and 22, having the phenyl group at the 4-thiazolidinone
moiety, and the 2-fluorophenyl and 3-nitrophenyl group at the β-carboline nucleus,
showed potent activity and selectivity for ovarian (OVCAR) tumor cell lines, with GI50
values of 0.73 and 1.25 µM, respectively.
Furthermore, it was observed that the derivatives 22 and 23, containing a
substituent at the 2-position of the 4-thiazolidinone different of phenyl group, were no
active against all tumor cell lines tested. This indicated the influence of nature of the
substituent at 2-position of 4-thiazolidinone on antitumor activity.

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Table 2. In vitro antitumor activity (GI50 in µM) of 1-(substituted-phenyl)-β-carboline 3-(N-2-substituted-aryl-4-thiazolidinone) derivatives 18-23.
Cell lines
Ovarian
Breast
(MCF-
7)
Lung
(NCI-
H460)
Ovarian
(OVCAR-
3)
Resistant
(NCI/ADR
-RES)
Glioma
(U251)
Melanoma
(UACC-62)
Renal
(786-0)
Prostate
PC-3
Colon
(HT-29)
R1
R2
Comp
Ph
4-NMe2-Ph
2-F-Ph
Ph
Ph
Ph
0.25
64.49
84.13
>100
>100
>100
80.42
nt
19.10
>100
63.15
>100
>100
>100
18.37
64.49
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
7.23
nt
1.58
>100
0.73
>100
>100
>100
>100
>100
>100
18
19
20
21
22
23
Ph
Nt
nt
2-thienyl
Ph
>100
Nt
>100
nt
>100
1.25
3-NO2-Ph
Ph
2-Cl-Ph
>100
>100
>100
nt = not tested. GI50: concentration of compound necessary to promote 50% of growth inhibition after 48h of treatment, comparing to cells growing without
treatment.

ACCEPTED MANUSCRIPT
2.2.2. Cell death and cell cycle profile in 786-0 cell line for compound 9c
In view of the potency of 9c, this compound was chosen for further studies
concerning cell death and cell cycle profile by flow cytometry. The studies were
focused on the human renal adenocarcinoma cell line 786-0 since the derivative 9c was
able to inhibiting totally the growth and for killing 50% of this cell lines, at
concentrations of 13 µM and 105 µM, respectively.
The exposure of phosphatidylserine (PS) on cell membrane surface signals for
recognition and engulfment of dying cells, because cell membrane of viable cells
exhibits substantial phospholipid asymmetry, with most of the PS residing on the inner
leaflet of the plasma membrane [45]. One of the methods used for assessment of
exposure of PS is the double staining for Annexin-V and 7-amino-actinomycin D (7-
AAD) by flow cytometry analyses. Annexin-V binds to PS translocated to the outer face
of the cell membrane during the initial process of apoptosis (early apoptosis). 7-AAD
binds to the DNA of the cell and acts as an indicator of membrane structural integrity,
since it is not able to enter viable cells and early apoptotic cells, thus indicating late
apoptosis or necrosis. Double staining for annexin-V and 7-AAD without the annexin-V
positive only stage can be a characteristic of necrosis or necroptosis (programmed
necrosis). For cell death evaluation, we performed the Annexin-V/7-AAD double
staining assay by flow cytometry.
Treatment with 25 µM of compound 9c for 24 hours led to membrane integrity
loss, since cells were double stained for annexin-V and 7-AAD or only stained for 7-
AAD (Figure 2A). Cells treated with 25 µM of compound 9c presented PS
externalization, being 38.7 ± 0.3% of cells double stained for annexin-V and 7-AAD
and 36.6 ± 2.3% of the cells stained with 7-AAD only (Figure 2A). To evaluate when
cells were committed to cell death, we evaluated the influence of the treatment with
compound 9c for 18 and 15h. After 18h of treatment, 34.4 ± 1.5% of the cells were
double stained for annexin-V and 7-AAD and 34.7 ± 0.2% of the cells stained with 7-
AAD only (Figure 2B), meaning that there was no difference between 18 and 24h of
treatments and that signalling for cell death may occur before 18 hours. In fact, after
15h of treatments, only 3.6 ± 0.8% of the cells were double stained for annexin-V and
7-AAD and 3.9 ± 0.1% of the cells stained with 7-AAD only, which was not different
from the cells treated with vehicle, suggesting that compound 9c at 25 µM induces cell
death between 15 and 18 hours of treatment (Figure 2C). The fact that even in the

ACCEPTED MANUSCRIPT
beginning there is no staining for annexin-V only suggest that the compound 9c may
induce necrotic or necroptotic cell death.
A
B
Annexin V - 24 h
Annexin-V 18h
100
80
60
40
20
0
100
80
60
40
20
0
Vehicle
Vehicle
9c (25 µM)
9c (25µM)
***
***
***
***
***
***
e
l
+
V
+
+
D
b
a
D
A
-
A
i
n
i
A
-
V
A
-
x
e
7
7
/
n
V
-
n
n
A
i
x
e
n
n
A
Annexin-V 15h
C
100
80
60
40
20
0
Vehicle
9c (25µM)
Figure 2. Compound 9c (25 µM) induced cell death in 786-0 cells, with externalization of
phosphatidylserine (Annexin-V positive cells – NEX-V+) and cell membrane degradation (7-
AAD positive cells – 7AAD+). Cells were exposed to vehicle (DMSO) and Compound 9c (25
µM) for 24 (A), 18 (B) and 15 hours (C) and analysed by flow cytometry, using Annexin V/7-
AAD double staining assay. Results represented by mean ± standard error, in percentage of
cells. Experiments were conducted three times, in triplicate.***p<0.001, Tukey’s Multiple
Comparison Test. ANOVA, statistically different from vehicle.
Alterations in the cell cycle profile are closely linked to cell death, either by
being a consequence or preceding this phenomenon. Many of cancer cells treatment
with anticancer agents usually result in cell cycle arrest, which subsequently leads the
cells to enter apoptosis [44]. To evaluate 786-0 cell cycle profile after treatment with

ACCEPTED MANUSCRIPT
compound 9c, we performed cell cycle analysis by flow cytometry. For this study, cells
were treated with 12.5 µM and 6.25 µM for 24 hours, non-cytotoxic concentrations. As
a positive control, colchicine (1.25 nM) was employed, as it promoted cell cycle arrest
on G2/M phase. The cell cycle distribution of treated 780-6 cells is presented in Table
3
. As expected, colchicine (1.25 nM) increased the percentage of cells in G2/M phase
(61.7 ± 2.1%), which was accompanied by a decreased percentage of cells in the G1
phase. No interference on the cell cycle profile was observed with compound 9c at the
concentration of 6.25
which indicates DNA fragmentation, typical of cell death.
Treatments with compound 9c induced cell death in the renal human tumor cell
line 786-0 between 15 and 18 hours after treatment with 25 M. These cells were
µM, but the treatment with 12.5 µM promoted a sub-G1 arrest,
µ
stained with annexin-V and 7-AAD, meaning exposure of phosphatidylserine and cell
membrane degradation respectively, which could suggest death through necrosis or
necroptosis. Further experiments will confirm this hypothesis. Treatments with lower
concentrations (6.25 and 12.5 µM) did not affect the cell cycle profile of 786-0 cells.
Table 3. Cell cycle profile of 786-0 cells treated with vehicle (DMSO), colchicine (1.25
nM) and compound 9c (6.25 and 12.5 µM) for 24 hours.
Treatment
Vehicle
Sub-G1
3.7 ± 0.9
9.0 ± 0.6
2.3 ± 0.5
G1
S
G2/M
47.1 ± 1.0
12.6 ± 0.7
36.5 ± 1.1
Colchicine (1.25 µM)
18.3 ± 0.9*** 11.0 ± 1.1 61.7 ± 2.1***
Compound 9c (6.25µM)
48.7 ± 0.8
40.5 ± 2.9
11.9 ± 0.4
12.4 ± 1.4
37.0 ± 1.7
31.8 ± 6.5
Compound 9c (12.5µM) 15.3 ± 5.0**
Results represented by mean ± standard error, in percentage of cells. Experiments were
conducted three times, in triplicate **p<0.01, ***p<0.001, Tukey’s Multiple Comparison Test.
ANOVA.
2.2.3. Antiviral activity
Some of the
for their activity towards the Herpes simplex virus type-1(HSV-1). Compounds with
EC50 > 100 M were considered inactive. The cytotoxicity to Vero cells and the
β-carboline-4-thiazolidinone derivatives synthesized were evaluated
µ
selectivity index for the active derivatives were also determined.
The antiviral assay results (Table 4) showed that regarding N’-(2-ylidene-4-
thiazolidinone)-β-carboline-3-carbohydrazide series (9-11), the derivative 9f with 2-

ACCEPTED MANUSCRIPT
fluorophenyl group at 1-position was the most active with EC50 value of 19.57 µM. The
introduction of the ethyl or phenyl groups at 3-position of the thiazolidinone ring of 9f
led to a loss of activity, resulting in the inactive derivatives 10f and 11f, respectively.
Contrarily, the presence of ethyl group resulted in an important increment in the activity
of derivative containing the 4-dimetylaminophenyl substituent at 1-position of the
β-
carboline. The EC50 values changed of 96.82 for 9c to 6.78 M for their N-ethyl
µ
analogue 10c. Concerning to derivatives containing the phenyl group at the 3-position
of the thiazolidinone ring (11a, c, d, f) only the compound 11d, with the 3-nitrophenyl
group attached to the 1-position of the
with EC50 value of 17.24 M.
From the comparison of EC50 data of derivatives 9-11 with those of
substituted-aryl-4-thiazolidinone)- -carboline-3-carboxamides (18-20 and 22, Table 4),
β-carboline, showed significant antiviral activity
µ
N
-(2-
β
it was observed that nature of thiazolidinone moiety exerts significant influence in the
antiviral potency. The N-(2-substituted-aryl-4-thiazolidinone)-carboxamide moiety in
18
EC50 values of 0.80, 2.15 and 2.02
fluorophenyl group at 1-position, showed moderate activity with EC50 value of 30.28
M. Based on the above information, it is possible to suggest that both groups
,
19 and 22 confer a potent anti-HSV-1 activity for these derivatives, which presented
µ
M, respectively. The derivative 20, with 2-
µ
appropriate in the 1-position and the 3-position of the
antiviral activity.
β-carboline ring influence the

ACCEPTED MANUSCRIPT
Table 4. Antiviral activity against Herpes simplex virus type-1, cytotoxicity and selectivity
index (SI) data for β-carboline-4-thiazolidinones 9-11 and 18-20, and 22
a
b
CC50 ± DP
EC50 ± DP
R1
R2
H
Compounds
9a
ISc
24.39
18.02
n d
(µM)
(µM)
Ph
2493 ± 70.71
102.22 ± 1.732
4-NMe2-Ph
2-Cl-Ph
2-F-Ph
H
1744 ± 23.54
766 ± 6.35
96.82 ± 2.000
>100
9c
H
9e
H
1073 ± 53.03
1731 ± 5.77
1649 ± 11.02
1792 ± 47.16
1404 ± 88.0
2980 ± 17.68
1478 ± 14.14
817 ± 14.14
1168 ± 56.57
339 ± 17.68
506 ± 6.429
467 ± 14.142
637 ± 26.501
1080 ± 37.859
19.57 ± 1.170
26.57 ± 0.600
6.78 ± 0.517
86.48 ± 1.040
>100
54.88
65.20
243.40
20.73
n d
9f
4-MeO-Ph
4-NMe2-Ph
3-NO2-Ph
2-Cl-Ph
2-F-Ph
Et
Et
Et
Et
Et
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
10b
10c
10d
10e
10f
11a
11c
11d
11f
18
>100
n d
Ph
78.87 ± 1.607
49.98 ± 0.913
17.24 ± 0.985
>100
18.75
16.35
67.78
n d
4-NMe2-Ph
3-NO2-Ph
2-F-Ph
Ph
2.15 ± 1.732
0.80 ± 2.000
30.28 ± 1.628
2.02 ± 0.494
235.0
592.5
21.0
4-NMe2-Ph
2-F-Ph
19
20
3-NO2-Ph
533.9
22
a Concentration at which 50% cytotoxicity is observed. b Concentration at which 50% efficacy
in antiviral assay is observed. c Selectivity index (CC50/EC50). nd not determined

ACCEPTED MANUSCRIPT
3. Conclusions
In this work, novel hybrids N’-(2-ylidene-4-thiazolidinone)-
β
-carboline-3-
-carboline-3-
carbohydrazides (9-11) and -(2-substituted-4-thiazolidinone)-
N
β
carboxamides (18-23) have been synthesized and evaluated for their in vitro antitumor
and anti-HSV-1 activities. The assay results showed that the nature of 4-thiazolidinone
moiety and of the substituent attached at the 3- and 1- position of
respectively, influenced the antitumor and antiviral activities.
β-carboline nucleus,
Compound 11d, containing the 2-nitrophenyl at C-1 of
-phenyl substituent at 4-thiazolidinone ring, was the most active for the N’-(2-
ylidene-4-thiazolidinone)- -carboline-3-carbohydrazides (9-11), showing GI50 values
in the range of 0.83 to 3.81 µM for all cell lines tested. Compound 9c, bearing the 4
dimethylaminophenyl group at C-1 of -carboline, also presented high potency and
β-carboline nucleus, and
the
N
β
-
β
induced cell death characterized by exposure of phosphatidylserine and cell membrane
degradation in the 786-0 renal cancer cell line, with no interference in the cell cycle
profile.
The changes in the nature of 4-thiazolidinone nucleus led to a decrease of
activity for derivatives of the
N-(2-substituted-aryl-4-thiazolidinone)-β-carboline-3-
carboxamide series (18-23); however, a potent activity and high selectivity for glioma
(U251) and ovarian (OVCAR) cancer cell lines was observed for some compounds of
this series.
Furthermore,
activity against Herpes simplex virus type-1. The N-(2-substituted-aryl-4-
thiazolidinone)-carboxamide moiety in 18 19 and 22 confer a potent anti-HSV-1
β-carboline-4-thiazolidinone hybrids showed potent antiviral
,
activity for these derivatives, which presented EC50 values of 0.80, 2.15 and 2.02
respectively.
µM,
In summary, from our results it was possible to obtain new hybrids 4-
thiazolidinone- -carbolines with remarkable antitumor and antiviral activities, which
β
can be considered as potential compounds for future studies in view to development of
new anticancer and antiviral agents.
4. Experimental
4.1. General methods

ACCEPTED MANUSCRIPT
All reagents were purchased from commercial suppliers. The reactions were
monitored by thin layer chromatography conducted on Merk TLC plates (Silica Gel 60
F254). 1H and 13C spectra were recorded in Varian spectrometer model Mercury plus BB
300 MHz and 75 MHz com deuterated solvents and TMS as internal standard,
respectively, High Resolution Mass Spectra (HRMS) were recorded on a TOF Bruker
Daltonics model IMPACT II spectrometer in a positive mode.
4.2. Synthesis
4.2.1. General procedure for the synthesis of L-tryptophan methyl ester (2)
To a suspension of 2.04 g (10 mmol) of commercial L-tryptophan (1) in 50 mL
of MeOH was added dropwise concentrated sulfuric acid until complete solubilization.
The mixture was kept under reflux, stirring for 48 hours and then cooled, neutralized
with a solution of 5% sodium carbonate, and extracted with ethyl acetate (3 x 35 mL).
The organic phase was dried with anhydrous sodium sulfate and after filtration, the
solvent was removed in a rotary evaporator. The pure product was obtained in 95%
yield.
4.2.2. General procedure for the synthesis of cis + trans 1-(substituted-phenyl)-3-
carbomethoxy-tetrahydro-
β
-carbolines (3a-f)
To a solution of 1.09 g (5 mmol) of
L
-tryptophan methyl ester (2) in CH2Cl2 was
added 5 mmol of the appropriate aldehyde and 2 equivalents of trifluoroacetic acid. The
reaction mixture was kept at room temperature for 48 hours and after evaporation of the
solvent and the remaining trifluoroacetic acid, the crude product was neutralized with a
solution of 5% sodium carbonate and then extracted with ethyl acetate (3 x 35 mL). The
organic phase was dried with anhydrous sodium sulfate and after filtration, the solvent
was removed in a rotary evaporator. The solid obtained was washed thoroughly with
methanol, providing a mixture of cis and trans 1-(substituted-phenyl)-3-carbomethoxy-
tetrahydro-β-carboline.
4.2.3. General procedure for synthesis of 1-(substituted-phenyl)-3-carbometoxy-β-
carbolines (4a-f)
A suspension of the corresponding cis and trans 1-(phenyl-substituted)-3-
carbomethoxy-tetrahydro- -carbolines (3a-f) (5 mmol) and sulfur (15 mmol) in xylene
β

ACCEPTED MANUSCRIPT
(50 ml) was refluxed for 48 hours and subsequently at 0 °C for 3 hours. The precipitate
formed was then filtered and washed with petroleum ether providing the
β
-carbolines.
4.2.4. General procedure for synthesis of 1-(substituted-phenyl)-
β-carboline-3-
carbohydrazides (5a-f)
To a suspension of 4.0 mmol of β-carbolines (4a-f) in ethanol (50 mL) were
added 53 mmol of hydrazine hydrate 51%. This mixture was refluxed for 72 hours and
at 0 °C for 3 hours to complete precipitation. The precipitate formed was filtered and
washed with ethanol, which gave the corresponding pure products.
4.2.5
. General procedure for the synthesis of 1-(susbtituted-phenyl)-β-carboline-3-
thiosemicarbazides (6a-f)
To a suspension of 1-(substituted-phenyl)-β-carboline-3-carbohydrazide (1
mmol) (5a-f) in distilled water (30 ml) was added potassium thiocyanate (4 mmol) and
drops of concentrated hydrochloric acid. The reaction was stirred and refluxed for a
period of 48h. The precipitate formed was filtered on a sintered glass funnel and washed
with distilled water.
4.2.6. General procedure for the synthesis of 1-(substituted-phenyl)-β-carboline-3-
thiosemicarbazides (7a-f) and (8a, c-d, f)
To a solution of 1-(substituted-phenyl)-β-carboline-3-carbohydrazide (5a-f) (1
mmol) in ethanol (20 mL) were added ethyltioisocyanate and / or phenyl isothiocyanate
(1 mmol). The reaction was stirred and refluxed for a period of 48h. The precipitate
formed was filtered and washed with ethanol.
4.2.7. General procedure for the synthesis of 1-(substituted-phenyl)-N’-(3-substituted-4-
thiazolidinona-2-ylidene)-β-carbolina-3-carbohydrazides (9a-f), (10a- f) and (11a, c-d,
f)
To a solution of the β-carboline-3-thiosemicarbazides (6a-f), (7a-f) and (8a, c-d, f) (1.0
mmol) in ethanol (5 mL) was added ethyl bromoacetate (1.0 mmol), sodium acetate (4.0
mmol) and potassium hydroxide (1 0 mmol). The reaction mixture was stirred and
refluxed for 48 hours. The precipitate formed was filtered and washed with ethanol.
4.2.7.1.
(Z)-1-(4-methoxyphenyl)-N’-(4-oxothiazolidin-2-ylidene)-9H-pyrido[3,4-
1
b]indole-3-carbohydrazide (9b). Yield: 62%; mp 256-257 °C. H NMR (300 MHz,

ACCEPTED MANUSCRIPT
DMSO-
d
6):
δ
3.90 (s, 3H, OCH3), 4.05 (s, 2H, CH2, H-5”), 7.21 (d,
J
= 8.7 Hz, 2H, H-
= 7.3
= 8.1 Hz, 1H, H-5), 8.83
(s, 1H, H-4), 10.66 (s, 1H, NH), 11.91 (s, 1H, NH), 11.99 (s, 1H, NH). 13C NMR (75.45
MHz), DMSO- 6): 34.6 (CH2, C-5”), 55.4 (OCH3), 112.7 (CH, C-8), 112.9 (CH, C-4),
3’, H-5’), 7.32 (t,
J
= 7.3 Hz, 1H, H-6), 7.51 (t,
J = 7.6 Hz, 1H, H-7), 7.71 (d, J
Hz, 1H, H-8), 8.16 (d,
J
= 8.7 Hz, 2H, H-2’, H-6’), 8.43 (d,
J
d
114.3 (2CH, C-3’, C-5’), 120.3 (CH, C-6), 121.2 (C0, C-4b), 122.1 (CH, C-5), 128.6
(C0, C-4a), 129.7 (CH, C-7), 129.9 (C0, C-1’), 130.1 (2CH, C-2’, C-6’), 134.1 (C0, C-
9a), 138.5 (C0, C-3), 140.4 (C0, C-8a), 141.5 (C0, C-1), 160.1 (C0, C-4’); HRMS-ESI:
calcd for C22H18N5O3S [M+H]+ 432,1130, found: 432.1099.
4.2.7.2.
(Z)-1-(4-(dimethylamino)phenyl)-N’-(4-oxothiazolidin-2-ylidene)-9H-
1
pyrido[3,4-b]indole-3-carbohydrazide (9c). Yield: 75%; mp > 270 °C (decomp.). H
NMR (300 MHz, DMSO-d6): δ 3.10 (s, 6H, N(Me)2), 3.97 (s, 2H, CH2), 6.96 (d, J = 9.0
Hz, 2H, H-3’, H-5’), 7.32 – 7.34 (m, 1H, H-6), 7.55 – 7.64 (m, 2H, H-7, H-8), 7.98 (d,
J
13
= 9.0 Hz, 2H, H-2’, H-6’), 8.22, (d,
(75.45 MHz), DMSO-
J
= 8.1 Hz, 1H, H-5), 8.76 (s, 1H, H-4). C NMR
d
6): 34.2 (CH2, C-5”),, 39.8 (2CH3), 112.1 (CH, C-4), 112.2
(2CH, C-3’, C-5’), 112.7 (CH, C-8), 119.8 (C0, C-4b), 120.6 (CH, C-6), 121.8 (CH, C-
5), 128.3 (CH, C-7), 129.3 (C0, C-4a), 129.4 (2CH, C-2’, C-6’), 133.5 (C0, C-1’), 133.8
(C0, C-9a), 138.3 (C0, C-1), 141.3 (2C0, C-3, C-8a), 150.7 (C0, C-4’), 150.8 (C=N, C-
2”); HRMS-ESI: calcd for C23H20N6O2S [M+H]+ 445.1447, found 445.1162:
4.2.7.3. (Z)-1-(3-nitrophenyl)-N’-(4-oxothiazolidin-2-ylidene)-9H-pyrido[3,4-b]indole-
1
3-carbohydrazide (9d). Yield: 81%; mp 212-214 °C. H NMR (300 MHz, DMSO-
d
6):
= 7.5 Hz, 1H, H-7), 7.71 (d,
= 7.8 Hz, 1H, H-5’), 8.42 (d,
= 7.8 Hz, 1H, H-5), 8.59 (s, 1H, H-2’), 8.64 (d, = 7.8 Hz, 1H, H-4’), 9.00 (s, 1H,
H-4), 10.80 (s, 1H, NH), 12.00 (s, 1H, NH), 12.10 (s, 1H, NH). 13C NMR (75.45 MHz),
DMSO- 6): 34.6 (CH2, C-5”), 112.7 (CH, C-8), 114.4 (CH, C-8), 120.6 (CH, C-6),
δ
4.04 (s, 2H, CH2), 7.36 (t,
= 8.1 Hz, 1H, H-8), 7.95 (t,
(d,
J
= 7.5 Hz, 1H, H-6), 7.65 (t,
J
J
J
J = 7.8 Hz, 1H, H-6’), 8.49
J
J
d
121.2 (C0, C-4b), 122.3 (CH, C-5), 123.6 (CH, C-6’), 123.7 (CH, C-2’), 129.1 (CH, C-
7), 130.4 (CH, C-5’), 130.5 (C0, C-4a), 134.5 (C0, C-1’), 135.3 (CH, C-4’), 138.3 (C0,
C-9a), 138.8 (C0, C-3), 139.0 (C0, C-8a), 141.7 (C0, C-1), 148.3 (C0, C-3’), 161.4 (C=N,
C-2”), 172.1 (C=O, C-4”); HRMS-ESI: calcd for C21H15N6O4S [M+H]+ 447.0875,
found: 447.0570.

ACCEPTED MANUSCRIPT
4.2.7.4. (Z)-1-(2-chlorophenyl)-N’-(4-oxothiazolidin-2-ylidene)-9H-pyrido[3,4-b]indole-
1
3-carbohydrazide (9e). Yield: 72%; mp 236-241 °C. H NMR (300 MHz, DMSO-
d
6):
δ
4.01 (s, 2H, CH2), 7.34 (t,
– 7.79 (m, 2H, H-7, H-8, H-6’), 8.46 (d,
(s, 1H, NH), 11.81 (s, 1H, NH), 11.93 (s, 2H, NH, NH). 13C NMR (75.45 MHz),
DMSO- 6): 34.5 (CH2, C-5”), 112.4 (CH, C-8), 114.1 (CH, C-4), 120.3 (CH, C-6),
J
= 7.3 Hz, H-6), 7.60 – 7.65 (m, 3H, H-3’, H-4’, H-5’), 7.72
J
= 7.8 Hz, 1H, H-5), 8.95 (s, 1H, H-4), 10.46
d
121.0 (C0, C-4b), 122.3 (CH, C-5), 128.9 (CH, C-7), 129.2 (CH, C-6’), 130.0 (C0, C-
4a), 130.0 (CH, C-4’), 130.7 (CH, C-5’), 132.1 (C0, C-1’), 132.5 (CH, C-3’), 135.3 (C0,
C-2’), 138.2 (C0, C-3), 139.7 (C0, C-9a), 141.3 (C0, C-1), 141.4 (C0, C-8a), 161.2 (C=N,
C-2”); HRMS-ESI: calcd for C21H15ClN5O2S [M+H]+ 436.0635, found: 436.0334.
4.2.7.5. (Z)-1-(2-fluorophenyl)-N’-(4-oxothiazolidin-2-ylidene)-9H-pyrido[3,4-b]indole-
1
3-carbohydrazide (9f). Yield: 65%; mp > 250 °C (decomp.). H NMR (300 MHz,
DMSO-
H-7, H-8), 7.54 – 7.68 (m, 3H, H-3’, H-4’, H-6’), 7.75 (t,
(d,
NMR (75.45 MHz), DMSO-
d6): δ 3.52 (s, 2H, CH2), 7.29 (td, J =7.1, 1.8 Hz, 1H, H-6), 7.44 – 7.50 (m, 2H,
J
= 6.9 Hz, 1H, H-5’), 8.45.
13
J
= 7.8 Hz, 1H, H-5), 8.88 (s, 1H, H-4), 11.09 (s, 1H, NH), 11.68 (s, 1H, NH). C
d
6): 38.0 (CH2, C-5”), 112.3 (CH, C-6’), 113.3 (CH, C-4),
116.4 (CH, C-8), 119.9 (CH, C-6), 120.9 (C0, C-4b), 122.1 (CH, C-5), 124.9 (CH, C-7),
128.6 (C0, C-4a), 129.1 (CH, C-4’), 131.0 (CH, C-3’), 131.8 (CH, C-5’), 135.0 (C0, C-
1), 136.4 (C0, C-3), 140.4 (C0, C-8a), 141.2 (C0, C-9a), 157.1 (C0, C-2’), 158.1 (C=N,
C-2”), 167.3 (C=O, carbohydrazide), 182.4 (C=O, C-4”); HRMS-ESI calcd for
C21H15FN5O2S [M+H]+ 420.0930, found: 420.0624.
4.2.7.6. (Z)-N’-(3-ethyl-4-oxothiazolidin-2-ylidene)-1-phenyl-9H-pyrido[3,4-b]indole-3-
carbohydrazide (10a). Yield: 72%; mp 257-258 °C. 1H NMR (300 MHz,
CDCl3/CD3OD):
δ
1,28 (t,
J = 7.2 Hz, 3H, CH3), 3.89-3.96 (m, 4H, CH2, CH2), 7.31 (t,
J
= 7.3 Hz, 1H, H-6), 7.48-7.54 (m, 2H, H-7, H-8), 7.57-7.61 (m, 3H, H-3’, H-4’, H-5’),
8.02 (d,
J = 7.9 Hz, 2H, H-2’, H-6’), 8.20 (d, J = 8,1 Hz, 1H, H-5), 8.84 (s, 1H, H-4).
13C NMR (75.45 MHz, CDCl3/CD3OD): 12.1 (CH2CH3), 33.1 (CH2, C-5”), 38.5
(CH2CH3), 112.2 (CH, C-8), 113.6 (CH, C-4), 120.6 (CH, C-6), 121.7 (CH, C-5), 128.2
(2CH, C-2’, C-6’), 128.7 (CH, C-7), 128.9 (2CH, C-3’, C-5’), 129.1 (CH, C-4’), 130.4
(C0, C-1’), 137.7 (2C0, C-1, C-9a), 138.0 (C0, C-3), 141.4 (C0, C-8a), 154.3 (C=N, C-
2”), 162.1 (C=O, carbohydrazide), 170.8 (C=O, C-4”); HRMS-ESI: calcd for
C23H20N5O2S [M+H]+ 430.1338, found: 430.1322.

ACCEPTED MANUSCRIPT
4.2.7.7. (Z)-N’-(3-ethyl-4-oxothiazolidin-2-ylidene)-1-(4-methoxyphenyl)-9H-pyrido[3,4-
1
b]indole-3-carbohydrazide (10b). Yield: 63%; mp 158-160 °C. H NMR (300 MHz,
CDCl3/CD3OD):
3H, OCH3), 7.16 (d,
H-8), 8.02 (d, = 8.7 Hz, 2H, H-2’, H-6’), 8.22 (d,
δ
1,32 (t, J = 7.2 Hz, 3H, CH3), 3.93 – 4.00 (m, 4H, CH2 CH2), 3.93 (s,
= 9.0 Hz, 2H, H-3’, H-5’), 7.35 (H-6), 7.56 – 7.64 (m, 2H, H-7,
= 8.1 Hz, 1H, H-5), 8.84 (s, 1H, H-
J
J
J
4). 13C NMR (75.45 MHz, CDCl3/CD3OD): 12.3 (CH3CH2), 33.2 (CH2, C-5”), 38.7
(CH2CH3), 55.4 (OCH3), 112.2 (CH, C-8), 113.3 (CH, C-4), 114.4 (2CH, C-2’, C-6’),
120.7 (CH, C-6), 121.9 (CH, C-5), 128.8 (CH, C-7), 129.6 (2CH, C-3’, C-5’), 130.3
(C0, C-1’), 130.4 (C0, C-9a), 134.9 (C0, C-1), 138.2 (C0, C-3), 141.2 (C0, C-8a), 154.2
(C=N, C-2”), 162.1 (C=O, carbohydrazide), 170.8 (C=O, C-4”); HRMS-ESI: calcd for
C24H22N5O3S [M+H]+ 460.1443, found: 460.1430.
4.2.7.8.
pyrido[3,4-b]indole-3-carbohydrazide (10c). Yield: 73%; 1H NMR (300 MHz,
CDCl3/CD3OD): 1.33 (t, = 7.0 Hz, 3H, CH3), 3.07 (s, 6H, N(Me)2), 3.92 (s, 2H,
CH2), 3.98 (q, J = 7.0 Hz, 2H, CH2), 6.94 (d, = 9.0 Hz, 2H, H-3’, H-5’), 7.36 (dt,
15.0, 7.0 Hz, 1H, H-6), 7.53 – 7.61 (m, 2H, H-7, H-8), 7.94 (d, = 9.0 Hz, 1H, H-2’, H-
6’), 8.23 (d,
= 7.8 Hz, 1H, H-5), 8.85 (s, 1H, H-4). 13C NMR (75.45 MHz,
(Z)-N’-(3-ethyl-4-oxothiazolidin-2-ylidene)-1-(4-dimethylaminophenyl)-9H-
δ
J
J
J =
J
J
CDCl3/CD3OD): 12.2 (CH3CH2), 29.6 (CH2, C-5”), 38.6 (CH2CH3), 40.3 (CH3,
N(CH3)2), 112.1 (CH, C-8), 112.5 (2CH, C-3’, C-5’), 112.7 (CH, C-4), 120.6 (CH, C-6),
120.8 (CH, C-5), 122.0 (C0, C-1’), 125.5 (C0, C-4b), 128.5 (CH, C-7), 129.1 (2CH, C-
2’, C-6’), 130.0 (C0, C-1), 134.9 (C0, C-9a), 141.1 (C0, C-3), 151.1(C=N, C-2”).
4.2.7.9. (Z)-N’-(3-ethyl-4-oxothiazolidin-2-ylidene)-1-(3-nitrophenyl)-9H-pyrido[3,4-b]
1
indole-3-carbohydrazide (10d). Yield: 71%; mp 284-286 °C. H NMR (300 MHz,
DMSO-
CH2), 7.34 (t,
1H, H-5’), 8.40 (d,
d
6):
δ
1,21 (t,
J = 6.6 Hz, 3H, CH3), 3.78 (q, J = 6.6 Hz, 2H, CH2), 4.10 (s, 2H,
J
= 7.0 Hz, 1H, H-6), 7.60 – 7.71 (m, 2H, H-7, H-8), 7.93 (t, J = 7.6 Hz,
J
= 7.5 Hz, 1H, H-6’), 8.64 (d,
J
= 6.9 Hz, 1H, H-5), 8.92 (s, 1H, H-
13
4), 8.96 (s, 1H, H-2’), 10.82 (s, 1H, NH), 12.13 (s, 1H, NH). C NMR (75.45 MHz,
DMSO-d6): 12.2 (CH3CH2), 32.7 (CH2, C-5”), 37.6 (CH2CH3), 112.7 (CH, C-8), 114.1
(CH, C-4), 120.5 (CH, C-6), 121.1 (CH, C-4’), 122.3 (CH, C-6’), 123.6 (CH, C-2’),
129.0 (CH, C-7), 130.3 (C0, C-1’), 130.5 (CH, C-5’), 135.2 (CH, C-5), 138.1 (C0, C-3),
138.8 (C0, C-1), 141.1 (C0, C-9a), 145.9 (C0, C-8a), 148.3 (C0, C-3’), 158.7 (C=N, C-
2”), 160.0 (C=O, carbohydrazide), 171.1 (C=O, C-4”); HRMS-ESI: calcd for
C23H19N6O4S [M+H]+ 475.1188, found: 475.1166.

ACCEPTED MANUSCRIPT
4.2.7.10. (Z)-N’-(3-ethyl-4-oxothiazolidin-2-ylidene)-1-(2-chlorophenyl)-9H-pyrido[3,4-
1
b]indole-3-carbohydrazide (10e). Yield: 82%; mp 174-176 °C. H NMR (300 MHz,
CDCl3/CD3OD):
δ
1,31 (t,
J
= 7.05 Hz, 3H, CH3), 3.92 – 3.99 (m, 4H, 2CH2), 7.48 –
7.53 (m, 2H, H-7, H-8), 7.58 – 7.72 (m, 4H, H-3’, H-4’, H-5’, H-6’), 8.27 (d,
J
= 8.1
Hz, 1H, H-5), 8.94 (s, 1H, H-4), 8.95 (s, 1H, NH). 13C NMR (75.45 MHz,
CDCl3/CD3OD): 12.4 (CH3CH2), 33.5 (CH2, C-5”), 38.9 (CH2CH3), 112.6 (CH, C-8),
114.6 (CH, C-4), 120.9 (CH, C-6), 122.0 (C0, C-4b), 122.1 (CH, C-5), 127.5 (CH, C-7),
129.2 (CH, C-6’), 130.4 (C0, C-4a), 130.7 (CH, C-4’), 131.8 (CH, C-3’), 133.7 (C0, C-
1’), 136.3 (C0, C-2’), 136.5 (C0, C-3), 137.9 (C0, C-9a), 140.3 (C0, C-1), 141.9 (C0, C-
8a), 155.5 (C=N, C-2”), 162.7 (C=O, carbohydrazide), 171.5 (C=O, C-4”); HRMS-ESI:
calcd for C23H19ClN5O2S [M+H]+ 464.0948, found: 464.0896.
4.2.7.11. (Z)-N’-(3-ethyl-4-oxothiazolidin-2-ylidene)-1-(2-fluorophenyl)-9H-pyrido[3,4-
b]indole-3-carbohydrazide (10f). Yield: 67%; mp 150-153 °C. 1H NMR (300 MHz,
DMSO-
CH2), 7.31 – 7.36 (m, 3H, H-6, H-3’, H-6’), 7.41 (td, J = 7.6, 1.2 Hz, 1H, H-4’), 7.53 –
7.61 (m, 2H, H-7, H-8), 7.88 (td, J = 7.5, 1.8 Hz, 1H, H-5’), 8.26 (d, = 7.8 Hz, 1H, H-
5), 8.95 (s, 1H, H-4). 13C NMR (75.45 MHz, DMSO-
6): 12.3 (CH3CH2), 33.2 (CH2, C-
5”), 38.7 (CH2CH3), 112.6 (CH, C-8), 114.3 (CH, C-4), 116.3 (
120.8 (CH, C-6), 121.6 (C0, C-4b), 121.9 (CH, C-5), 124.9 (
125.4 (
CH, C-4’), 131.8 (
d6): δ 1,33 (t, J = 7.0 Hz, 3H, CH3), 3.95 (s, 2H, CH2), 3.97 (q, J = 6.1 Hz, 2H,
J
d
J
= 21.6 Hz, CH, C-3’),
= 3.3 Hz, CH, C-6’),
J
J
= 14.8 Hz, C0, C-1’), 129.1 (CH, C-7), 130.3 (C0, C-4a), 131.1 (
J = 8.2 Hz,
J
= 3.9 Hz, CH, C-5’), 136.0 (C0, C-1), 136.9 (C0, C-3), 138.2 (C0, C-
8a), 141.3 (C0, C-9a), 156.8 (J = 283.0 Hz, C0, C-2’), 161.9 (C=N, C-2”), 162.1 (C=O,
carbohydrazide), 171.0 (C=O, C-4”); HRMS-ESI: calcd for C23H19FN5O2S [M+H]+
448.1243, found: 448.1153.
4.2.7.12.
(Z)-N’-(4-oxo-3-phenylthiazolidin-2-ylidene)-1-phenyl-9H-pyrido[3,4-
1
b]indole-3-carbohydrazide (11a). Yield: 65%; mp 283-285 °C. H NMR (300 MHz,
DMSO-
H-4’’’, H-5’’’, H-6’’’), 7.32 (t,
7.69 (m, 1H, H-8), 8.19 (d, = 6.3 Hz, 2H, H-2’, H-6’), 8.44 (d,
8.83 (s, 1H, H-4), 10.60 (s, 1H, NH), 12.90 (s, 1H, NH). 13C NMR (75.45 MHz,
DMSO- 6): 33.1 (CH2, C-5”), 112.7 (CH, C-8), 113.2 (CH, C-4), 120.3 (CH, C-6),
d
6):
δ
4.27 (s, 2H, CH2), 7.30 – 7.54 (m, 8H, H-3’, H-4’, H-5’, H-2’’’, H-3’’’,
= 6.6 Hz, 1H, H-6), 7.57 – 7.61 (m, 1H, H-7), 7.65 –
= 7.2 Hz, 1H, H-5),
J
J
J
d
121.1 (C0, C-4b), 122.2 (CH, C-5), 128.3 (2CH, C-3”’, C-5’”), 128.6 (2CH, C-3’, C-5’),
128.7 (CH, C-7), 128.9 (2CH, C-2’”, C-6’”), 129.1 (2CH, C-2’, C-6’), 130.0 (C0, C-4a),

ACCEPTED MANUSCRIPT
134.3 (C0, C-1’), 134.9 (C0, C-1’”), 137.3 (C0, C-9a), 138.6 (C0, C-3), 140.6 (C0, C-1),
141.5 (C0, C-8a), 158.5 (C=N, C-2”), 160.5 (C=O, carbohydrazide), 171.1 (C=O, C-4”);
HRMS-ESI: calcd for C27H20N5O2S [M+H]+ 478.1338, found: 478.1115.
4.2.7.13. (Z)-1-(4-dimethylaminophenyl)-N’-(4-oxo-3-phenylthiazolidin-2-ylidene)-9H-
1
pyrido[3,4-b]indole-3-carbohydrazide (11c). Yield: 68%; mp 278-279 °C. H NMR
(300 MHz, DMSO-
1H, H-6), 6.96 (d,
5’)7.47 – 7.50 (m, 1H, H-4’’’), 7.54 (d, J = 7.5 Hz, 2H, H-2’’’, H-6’’’), 7.47 – 7.70 (m,
2H, H-7, H-8), 8.05 (d, = 8.7 Hz, 2H, H-2’, H-6’), 8.38 (d, = 7.8 Hz, 1H, H-5), 8.68
(s, 1H, H-4), 10.64 (s, 1H, NH), 11.82 (s, 1H, NH). 13C NMR (75.45 MHz, DMSO-
6):
d
6):
δ
3.04 (s, 6H, N(Me)2), 4.28 (s, 2H, CH2), 7.28 (t,
J = 7.5 Hz,
J
= 8.7 Hz, 2H, H-3’’’, H-5’’’), 7.41 (d,
J
= 7.5 Hz, 2H, H-3’, H-
J
J
d
33.2 (CH2, C-5”), 111.8 (CH, C-4), 112.1 (2CH, C-3’”, C-5’”), 112.7 (CH, C-8), 120.2
(CH, C-6), 121.2 (C0, C-4b), 122.0 (CH, C-5), 124.7 (C0, C-1’), 128.4 (2CH, C-3’, C-
5’), 128.7 (CH, C-7), 129.2 (2CH, C-2”’, C-6’”), 129.4 (CH, C-4’”), 133.9 (C0, C-1’”),
134.9 (C0, C-9a), 138.3 (C0, C-1), 141.4 (2C0, C-8a, C-3), 150.9 (C0, C-4’), 157.3 (C=N,
C-2”), 160.5 (C=O, carbohydrazide), 171.1 (C=O, C-4”); HRMS-ESI: calcd for
C29H25N6O2S [M+H]+ 521.1760, found: 521.1594.
4.2.7.14. (Z)-1-(3-nitrophenyl)-N’-(4-oxo-3-phenylthiazolidin-2-ylidene)-9H-pyrido[3,4-
1
b]indole-3-carbohydrazide (11d). Yield: 75%; mp 256-258 °C. H NMR (300 MHz,
DMSO-
3’’’, H-5’’’), 7.47 – 7.49 (m, 1H, H-4’’’), 7.54 (d,
– 7.70 (m, 2H, H-7, H-8), 8.40 (d, = 7.5 Hz, 1H, H-4’), 8.47 (d,
8.63 (d, = 7.8 Hz, 1H, H-6’), 8.87 (s, 1H, H-4), 8.96 (s, 1H, H-2’), 10.81 (s, 1H, NH),
12.13 (s, 1H, NH). 13C NMR (75.45 MHz, DMSO-
6): 33.0 (CH2, C-5”), 112.6 (CH, C-
d
6):
δ
4.26 (s, 2H, CH2), 7.30 – 7.37 (m, 1H, H-6), 7.41 (d,
= 7.6 Hz, 2H, H-2’’’, H-6’’’), 7.62
= 7.5 Hz, 1H, H-5),
J = 7.6 Hz, 2H, H-
J
J
J
J
d
8), 114.1 (CH, C-4), 120.1 (CH, C-6), 120.5 (C0, C-4b), 122.3 (CH, C-5), 123.5 (CH, C-
2’), 123.6 (CH, C-4’), 128.3 (2CH, C-3’”, C-5’”), 128.7 (CH, C-7), 129.0 (CH, C-4’”),
129.1 (2CH, C-2’”, C-6’”), 130.3 (CH, C-5’), 130.5 (C0, C-1’), 134.4 (C0, C-4a), 135.2
(C0, C-1’”), 138.1 (C0, C-1), 138.7 (C0, C-9a), 139.0 (C0, C-3), 141.6 (C0, C-8a), 148.3
(C0, C-3’), 159.7 (C=N, C-2”), 160.4 (C=O, carbohydrazide), 171.2 (C=O, C-4”);
HRMS-ESI: calcd for C27H19N6O4S [M+H]+ 523.1188, found: 523.0923.
4.2.7.15.
pyrido[3,4-b]indole-3-carbohydrazide (11f). Yield: 63%; mp 193-195 °C. 1H NMR
(300 MHz, DMSO- 6): 4.23 (s, 2H, CH2), 7.31 (td, J = 7.2, 1.5 Hz, 1H, H-6), 7.40 (d,
(Z)-1-(2-fluorophenyl)-N’-(4-oxo-3-phenylthiazolidin-2-ylidene)-9H-
d
δ

ACCEPTED MANUSCRIPT
J = 7.6 Hz, 2H, H-3’’’, H-5’’’), 7.45 – 7.50 (m, 3H, H-7, H-3’, H-6’), 7.53 (d, J = 7.5
Hz, 2H, H-2’’’, H-6’’’), 7.62 – 7.69 (m, 3H, H-8, H-5`, H-4’’’), 7.93 (td, J = 7.5, 1.5
Hz, 1H, H-4’), 8.45 (d, J = 7.8 Hz, 1H, H-5), 8.87 (s, 1H, H-4), 10.55 (s, 1H, NH),
11.85 (s, 1H, NH). 13C NMR (75.45 MHz, DMSO-
d6): 33.1 (CH2, C-5”), 112.4 (CH, C-
8), 113.7 (CH, C-4), 116.3 (J = 209.0 Hz, CH, C-3’), 120.3 (CH, C-6), 120.8 (C0, C-4b),
122.3 (CH, C-5), 124.9 (J = 3.15 Hz, CH, C-6’), 125.1 (C0, C-4a), 128.3 (2CH, C-3’”,
C-5’”), 128.7 (CH, C-7), 128.9 (CH, C-4’”), 129.1 (2CH, C-2’”, C-6’”), 131.3 (J = 8.4
Hz, CH, C-5’), 132.0 (J = 3.3 Hz, CH, C-4’), 133.2 (C0, C-1’”), 134.8 (C0, C-1’), 135.3
(C0, C-9a), 136.4 (C0, C-3), 138.6 (C0, C-1), 141.3 (C0, C-8a), 158.3 (C=O,
carbohydrazide), 159.2 (J = 247.8 Hz, C0, C-2’), 160.3 (C=N, C-2”), 171.1 (C=O, C-
4”); HRMS-ESI: calcd for C27H19FN5O2S [M+H]+ 496.1243, found: 496.1029.
4.2.8. General procedure for the synthesis of N’-arylidene-(1-substituted-phenyl)-β-
carboline-3-carbohydrazides (12-17)
To a solution of β-carboline-3-carbohydrazide (5a, c, d, f) (1.0 mmol) in DMF
(3.0 mL) were added the appropriate aromatic aldehydes (1.2 mmol). The reaction
mixture was irradiated in the domestic microwave oven at 60% power level for 1-3 min.
The reaction was monitored by TLC. After all the starting material was consumed the
reaction mixture was poured into water and the precipitate formed was collected by
filtration and washed successively with water.
4.2.9. General procedure for the synthesis of β-carboline-4-thiazolidinone (18-23)
The emulsion of arylidene-β-carboline-carbohydrazide (12-17) (1.0 mmol) in toluene
(10.0 mL) was added slowly mercapto acetic acid (2.0 mmol) and catalytic p-toluene
sulfonic acid. The reaction medium was kept under stirring and reflux and TLC
monitored the development of the reactions. After consumption of the starting material,
the reaction was treated with a 5% solution of potassium carbonate until pH 8.
Precipitate formed was collected by filtration on a sintered glass funnel and washed
with water. For the derivatives, 19 and 23 no precipitate formation after treatment with
base. In these cases, the reaction mixture was extracted with ethyl (3 x 20 mL). The
organic phase was dried with anhydrous sodium sulfate, filtered and the solvent
evaporated in vacuum.