Synthesis and evaluation of new benzodioxole-based dithiocarbamate derivatives as potential anticancer agents and hCA-I and hCA-II inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.09.035

In the current work, new benzodioxole-based dithiocarbamate derivatives were synthesized via the reaction of N-(1,3-benzodioxol-5-ylmethyl)-2-chloroacetamide with appropriate sodium salts of N,N-disubstituted dithiocarbamic acids. These derivatives were evaluated for their cytotoxic effects on A549 human lung adenocarcinoma and C6 rat glioma cell lines. N-(1,3-Benzodioxol-5-ylmethyl)-2-[4-(4-nitrophenyl)-1-piperazinylthiocarbamoylthio]acetamide (10) can be identified as the most promising anticancer agent against C6 cell line due to its notable inhibitory effect on C6 cells with an IC₅₀value of 23.33 ± 7.63 μg/mL when compared with cisplatin (IC₅₀= 19.00 ± 5.29 μg/mL). On the other hand, compound 10 did not show any significant cytotoxic activity against A549 cell line. The compounds were also tested for their in vitro inhibitory effects on hCA-I and hCA-II. Generally, the tested compounds were more effective on CAs than acetazolamide, the reference agent. Among these compounds, N-(1,3-benzodioxol-5-ylmethyl)-2-[(morpholinyl)thiocarbamoylthio]acetamide (3) and N-(1,3-benzodioxol-5-ylmethyl)-2-[(thiomorpholinyl)thiocarbamoylthio]acetamide (4) were found to be the most effective compounds on hCA-I with IC₅₀values of 0.346 nM and 0.288 nM, and hCA-II with IC₅₀values of 0.287 nM and 0.338 nM, respectively.

Full text of DOI:10.1016/j.ejmech.2016.09.035

Accepted Manuscript
Synthesis and evaluation of new benzodioxole-based dithiocarbamate derivatives as
potential anticancer agents and hCA-I and hCA-II inhibitors
Mehlika Dilek Altıntop, Belgin Sever, Gülşen Akalın Çiftçi, Kaan Kucukoglu, Ahmet
Özdemir, Seyedeh Sara Soleimani, Hayrunnisa Nadaroglu, Zafer Asım Kaplancıklı
PII:
S0223-5234(16)30765-6
10.1016/j.ejmech.2016.09.035
EJMECH 8901
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 June 2016
Revised Date: 25 August 2016
Accepted Date: 9 September 2016
Please cite this article as: M.D. Altıntop, B. Sever, G. Akalın Çiftçi, K. Kucukoglu, A. Özdemir, S.S.
Soleimani, H. Nadaroglu, Z.A. Kaplancıklı, Synthesis and evaluation of new benzodioxole-based
dithiocarbamate derivatives as potential anticancer agents and hCA-I and hCA-II inhibitors, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.035.
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ACCEPTED MANUSCRIPT
NO2
N
O
S
N
O
N
H
S
O
Compound 10
IC50 = 23.33±7.63 µg/mL
The most promising anticancer agent
O
S
O
O
N
S
N
S
O
N
H
O
N
S
H
S
O
O
Compound 3
IC50 = 0.287 nM for hCAꢀII
Compound 4
IC50 = 0.288 nM for hCAꢀI
The most effective CA inhibitors

ACCEPTED MANUSCRIPT
Synthesis and Evaluation of New Benzodioxole-Based Dithiocarbamate Derivatives as
Potential Anticancer Agents and hCA-I and hCA-II Inhibitors
Mehlika Dilek Altıntopa,*, Belgin Severa, Gülşen Akalın Çiftçib Kaan Kucukogluc, Ahmet
,
Özdemira, Seyedeh Sara Soleimanid, Hayrunnisa Nadaroglue, Zafer Asım Kaplancıklıa
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470
Eskişehir, Turkey
b
Department of Biochemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir,
Turkey
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, 25240,
Erzurum, Turkey
d
Department of Nano-Science and Nano-Engineering, Faculty of Engineering, Ataturk
University, 25240, Erzurum, Turkey
e Department of Food Technology, Erzurum Vocational Training School, Ataturk University,
25240, Erzurum, Turkey
* Corresponding author.
E-mail address: mdaltintop@anadolu.edu.tr (M.D. Altıntop).
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ABSTRACT
In the current work, new benzodioxole-based dithiocarbamate derivatives were synthesized
via the reaction of N-(1,3-benzodioxol-5-ylmethyl)-2-chloroacetamide with appropriate
sodium salts of N,N-disubstituted dithiocarbamic acids. These derivatives were evaluated for
their cytotoxic effects on A549 human lung adenocarcinoma and C6 rat glioma cell lines. N-
(1,3-Benzodioxol-5-ylmethyl)-2-[4-(4-nitrophenyl)-1-
piperazinylthiocarbamoylthio]acetamide (10) can be identified as the most promising
anticancer agent against C6 cell line due to its notable inhibitory effect on C6 cells with an
IC50 value of 23.33±7.63 µg/mL when compared with cisplatin (IC50= 19.00±5.29 µg/mL).
On the other hand, compound 10 did not show any significant cytotoxic activity against A549
cell line. The compounds were also tested for their in vitro inhibitory effects on hCA-I and
hCA-II. Generally, the tested compounds were more effective on CAs than acetazolamide, the
reference
agent. Among
these
compounds,
N-(1,3-benzodioxol-5-ylmethyl)-2-
[(morpholinyl)thiocarbamoylthio]acetamide (3) and N-(1,3-benzodioxol-5-ylmethyl)-2-
[(thiomorpholinyl)thiocarbamoylthio]acetamide (4) were found to be the most effective
compounds on hCA-I with IC50 values of 0.346 nM and 0.288 nM, and hCA-II with IC50
values of 0.287 nM and 0.338 nM, respectively.
Keywords: Dithiocarbamate, Benzodioxole, Cancer, Human carbonic anhydrase.
2

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1. Introduction
The metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) takes an important role in carbon
-
dioxide hydration to bicarbonate (CO2 + H2O
HCO3 + H+). CAs are involved in
essential cellular processes such as respiration and pH homeostasis, electrolyte secretion, bone
resorption, calcification in diverse tissues and organs and biosynthetic processes such as
-
lipogenesis, gluconeogenesis and ureagenesis that require HCO3 as a substrate [1-4].
These abundant zinc enzymes have attracted a great deal of interest in medicinal chemistry as
important targets in the treatment or prevention of a variety of disorders such as glaucoma,
acid-base disequilibria, epilepsy, and other neuromuscular diseases, altitude sickness, edema
and obesity. One of the outstanding applications belonging to CA inhibitors is in the treatment
of hypoxic tumors. The involvement of CAs in tumor growth and metastasis has been
investigated for so long and lots of researchers have described these enzymes as the key
components for fighting acidosis during hypoxia [5,6]. Two CA isozymes (CA IX and CA
XII) have been reported to be overexpressed in many tumors, whereas they show moderate-
to-abundant expression in normal tissues. These two isozymes are associated with cancer
progression, metastasis, and impaired therapeutic response. In particular, CA IX is expressed
in various tumors such as carcinomas of cervix uteri, breast, lung, colon, brain, kidney and
other types of neoplasia and CA IX is found in very limited amount in normal tissues just as
gastrointestinal tract and it comprises epithelia of glandular stomach, small intestine and
gallbladder [7-9]. CA XII is also present in many tumors but it is more findable in some
healthy tissues [10]. CA I and II are the most abundant CAs in the body and they are also
substantial drug targets [11]. CA I is reported to be involved in retinal and cerebral edema so
its inhibition may be important to deal with these conditions [12]. Besides, CA II is associated
with some disorders such as glaucoma, edema, epilepsy, and probably altitude sickness [13].
Dithiocarbamates (DTCs) have received considerable attention due to their diverse biological
activities such as antifungal [14], antibacterial [15], and antioxidant activities [16]. In recent
years, DTC has been identified as a pharmacophore to show anticancer activity [17]. DTCs
are also capable of forming complexes with a variety of transition metal ions in diverse
-
oxidation modes due to the presence of the anionic CS2 moiety [18]. They have been reported
to act as efficient metalloenzyme inhibitors such as tyrosinase and CA inhibitors in various
organisms [18,19]. Innocenti et al. reported the interaction of CAs with N,N-
-
diethyldithiocarbamate containing a new zinc-binding group (CS2 ) as shown in Fig. 1. DTCs
have been found to inhibit several CA isoforms in the low micromolar or submicromolar
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-
range [20]. The discovery of CS2 as a new zinc-binding group has led to the design of new
DTC derivatives as potent human carbonic anhydrase (hCA) inhibitors [18-20].
On the other hand, 1,3-benzodioxole has been found in a number of anticancer agents such as
podophyllotoxin, steganacin and combretastatin A-2 (Fig. 2) due to its good bioavailability
and low toxicity [21].
On the basis of afore-mentioned findings, in the present investigation we reported the
synthesis and evaluation of a new series of benzodioxole-based dithiocarbamate derivatives as
anticancer agents against A549 human lung adenocarcinoma and C6 rat glioma cell lines. The
synthesized compounds were also tested for their in vitro inhibitory effects on hCA-I and
hCA-II.
2. Results and Discussion
The synthesis of dithiocarbamate derivatives (1-10) was carried out according to the steps
shown in Scheme 1. Sodium salts of N,N-disubstituted dithiocarbamic acids were obtained by
the reaction of secondary amine with carbon disulfide in the presence of sodium hydroxide
[22,23]. The reaction of N-(1,3-benzodioxol-5-ylmethyl)-2-chloroacetamide with sodium salts
of N,N-disubstituted dithiocarbamic acids afforded novel dithiocarbamate derivatives (1-10).
MTT assay, which is based on the ability of metabolically active cells to convert the pale
yellow MTT dye to a spectrophotometrically quantifiable blue formazan product, was carried
out to determine the cytotoxic effects of the compounds on A549 human lung
adenocarcinoma and C6 rat glioma cell lines [24]. According to the MTT assay, the tested
compounds showed more potent inhibitory effects on C6 cells than A549 cells (Table 1). The
effects of the heterocyclic rings on anticancer activity of the compounds against C6 cell line
revealed the following potency order: Piperazine > Morpholine > Thiomorpholine >
Piperidine.
Considering the anticancer activity of piperidine substituted compounds 1 and 2, it can be
concluded that the methyl substituent at position 4 of the piperidine ring increased the
cytotoxic effects on A549 and C6 cell lines.
Compound 3 showed more inhibitory activity against A549 and C6 cell lines than compound
4. This outcome indicated that morpholine ring increased the anticancer activity against A549
and C6 cell lines when compared with thiomorpholine ring.
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Piperazine derivatives were more effective than piperidine, morpholine and thiomorpholine
derivatives on C6 cells. Among piperazine derivatives, the most effective anticancer agents
were found as compound 10 (IC50= 23.33±7.63 µg/mL), compound 9 (IC50= 48.33±7.64
µg/mL) and compound 8 (IC50= 58.33±14.43 µg/mL) when compared with cisplatin (IC50=
19.00±5.29 µg/mL). This outcome pointed out the importance of the alkyl/aryl/heteroaryl
substituent at position 4 of the piperazine ring for the anticancer activity against C6 cell line.
The effects of the substituents at position 4 of the piperazine ring on anticancer activity of the
piperazine substituted compounds against C6 cell line revealed the following potency order:
4-Nitrophenyl > 2-Pyrimidinyl > 4-Methoxyphenyl > Ethyl > Phenyl > Methyl. Generally the
aryl substituted compounds except compound 7 were more effective than alkyl substituted
compounds on C6 cell line. Among aryl groups, electron withdrawing groups on benzene ring
such as nitro group increased the anticancer activity against C6 cell line. The elongation of the
alkyl chain at position 4 of the piperazine ring increased the anticancer activity of the alkyl
substituted compounds against A549 and C6 cell lines.
Compounds 1-10 were tested for their in vitro inhibitory effects on hCA-I and hCA-II (Table
2). Acetazolamide (AAZ) was used as the reference agent. Generally, the tested compounds
were more effective on CAs than AAZ (IC50= 5.8 nM). In particular, compounds 3 and 4
exhibited the most potent inhibitory effect on hCA-I with IC50 values of 0.346 and 0.288 nM,
respectively. It is noteworthy to indicate that morpholine and thiomorpholine rings
significantly increased the carbonic anhydrase inhibitory activity when compared with the
other chemical structures. Compounds 1, 5 and 7 revealed inhibitory effects on hCA-I with
the range of 0.518-0.556 nM. Similarly, compounds 6, 8 and 9 revealed inhibitory effects on
hCA-I with the range of 0.624-0.699 nM. Although compounds 2 and 10 were more effective
than AAZ on hCA-I, these compounds showed less inhibitory activity than other derivatives
against hCA-I.
Similarly, compounds 3 and 4 showed the most potent inhibitory effect on hCA-II isozyme
with IC50 values of 0.287 and 0.338 nM, respectively. Compounds 7 and 8 were found to be
ineffective on hCA-II, whereas other derivatives showed notable inhibitory effect on hCA-II
isozyme. The inhibitory effects of compounds 1 and 5 showed fairly close inhibition on hCA-
II with IC50 values of 0.651 nM and 0.622 nM, respectively. On the other hand, compounds 6,
9 and 10 inhibited hCA-II isozyme with the range of 0.799-0.855 nM when compared with
AAZ (IC50= 6.7 nM).
Compounds 1, 2, 4, 5, 6, 7, 8 and 9 exhibited more significant inhibitory effects on hCA-I
than hCA-II, whereas compounds 3 and 10 showed more significant inhibitory effects on
5

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hCA-II than hCA-I. The methyl substitution at position 4 of the piperidine ring significantly
decreased the CA inhibition on both hCA-I and hCA-II.
3. Conclusion
In the current research, new benzodioxole-based dithiocarbamate derivatives were
synthesized. All synthesized compounds (1-10) were evaluated for their cytotoxicity against
A549 human lung adenocarcinoma, and C6 rat glioma cell lines. Among these compounds,
compound 10 can be considered as the most promising anticancer agent against C6 cell line
(IC50= 23.33±7.63 ꢀg/mL) when compared with cisplatin (IC50= 19.00±5.29 µg/mL).
Moreover, compounds 1-10 were also investigated for their ability to inhibit human carbonic
anhydrase isozymes (hCA-I and hCA-II). Although compounds 1-10 do not carry a
sulfonamide group, an important pharmacophore for hCA inhibitory activity, all compounds
(1-10) showed significant inhibitory effect on hCA-I. In addition, except compounds 7 and 8,
they also notably inhibited hCA-II as well. Among the synthesized compounds, compounds 3
and 4 can be identified as potential hCA-I and hCA-II inhibitors.
4. Experimental
4.1. Chemistry
N-(1,3-Benzodioxol-5-ylmethyl)-2-chloroacetamide (97%) was purchased from Maybridge,
whereas other reagents were purchased from Merck, Sigma-Aldrich and Acros. The melting
points (M.p.) of the compounds were determined on an Electrothermal 9100 melting point
apparatus (Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. IR spectra were
recorded on an IRPrestige-21 Fourier Transform Infrared spectrophotometer (Shimadzu,
1
Tokyo, Japan). H NMR and 13C NMR spectra were recorded on a Bruker spectrometer
(Bruker, Billerica, MA, USA). Liquid chromatography ion trap-time of flight tandem mass
spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization (ESI)
source, operating in both positive and negative ionization mode. Shimadzu's LCMS Solution
software was used for data analysis. Elemental analyses were performed on a Perkin Elmer
EAL 240 elemental analyzer (Perkin-Elmer, Norwalk, CT, USA) and the results were within
±0.4% of the theoretical values. Thin Layer Chromatography (TLC) was performed on TLC
Silica gel 60 F254 aluminium sheets (Merck, Darmstadt, Germany) to check the purity of the
compounds.
4.1.1. General procedure for the synthesis of the compounds
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4.1.1.1. Sodium salts of N,N-disubstituted dithiocarbamic acids
Sodium hydroxide (10 mmol) was dissolved in ethanol (80 mL) with constant stirring. After
addition of the secondary amine (10 mmol) the mixture was cooled in an ice bath and carbon
disulfide (100 mmol) was added dropwise with stirring. The reaction mixture was stirred for 2
h at room temperature. The products were afforded by filtration and washed with diethyl ether
[22,23].
4.1.1.2. N-(1,3-Benzodioxol-5-ylmethyl)-2-[(substituted)thiocarbamoylthio]acetamide (1-10)
A mixture of N-(1,3-benzodioxol-5-ylmethyl)-2-chloroacetamide (0.01 mol) and appropriate
sodium salt of N,N-disubstituted dithiocarbamic acid (0.01 mol) was treated in acetone at
room temperature for 6 h. The solvent was evaporated, the resulting solid was washed with
water and crystallized from ethanol [23].
4.1.1.2.1.
N-(1,3-Benzodioxol-5-ylmethyl)-2-[(piperidin-1-yl)thiocarbamoylthio]acetamide
(1)
Yield: 79 %. Mp 137.7 ºC.
IR νmax (cm-1): 3263 (N-H stretching), 3068 (aromatic C-H stretching), 2929 (aliphatic C-H
stretching), 2829 (O-CH2 stretching), 1641 (C=O stretching), 1548, 1504, 1489, 1473 (C=C
stretching and N-H bending).
1H NMR (500 MHz) (DMSO-d6) δ (ppm): 1.59-1.68 (m, 6H), 3.92 (s, 2H), 4.06 (s, 2H), 4.19
(d, J= 5.95 Hz, 4H), 5.97 (s, 2H), 6.72-6.74 (m, 1H), 6.82-6.84 (m, 2H), 8.52-8.55 (m, 1H).
13C NMR (125 MHz) (DMSO-d6) δ (ppm): 23.98 (3CH2), 52.00 (2CH2), 40.81 (CH2), 42.73
(CH2), 101.25 (CH2), 108.33 (CH), 108.40 (CH), 120.81 (CH), 133.57 (C), 146.48 (C),
147.68 (C), 166.98 (C), 193.58 (C).
For C16H20N2O3S2 Calculated: C, 54.52; H, 5.72; N, 7.95; O, 13.62; S, 18.19. Found: C,
54.10; H, 5.23; N, 7.34.
HRMS (ESI) (m/z): [M+Na]+ 375.08.
4.1.1.2.2.
N-(1,3-Benzodioxol-5-ylmethyl)-2-[(4-methylpiperidin-1-
yl)thiocarbamoylthio]acetamide (2)
Yield: 78 %. Mp 154.4 ºC.
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IR νmax (cm-1): 3273 (N-H stretching), 3041 (aromatic C-H stretching), 2910 (aliphatic C-H
stretching), 2833 (O-CH2 stretching), 1637 (C=O stretching), 1541, 1504, 1490, 1473 (C=C
stretching and N-H bending).
1H NMR (500 MHz) (DMSO-d6) δ (ppm): 0.92 (d, J= 6.25 Hz, 3H), 1.07-1.15 (m, 2H), 1.75-
1.79 (m, 3H), 3.19-3.25 (m, 2H), 4.05 (s, 2H), 4.19 (d, J= 5.90 Hz, 2H), 4.46-4.49 (m, 1H),
5.23-5.25 (m, 1H), 5.97 (s, 2H), 6.73 (d, J= 8.20 Hz, 1H), 6.82-6.85 (m, 2H), 8.52-8.55 (m,
1H).
13C NMR (100 MHz) (DMSO-d6) δ (ppm): 21.01 (CH3), 29.92 (CH), 33.21 (CH2), 33.72
(CH2), 40.37 (CH2), 42.24 (CH2), 50.13 (CH2), 51.83 (CH2), 100.74 (CH2), 107.82 (CH),
107.89 (CH), 120.31 (CH), 133.05 (C), 145.98 (C), 147.18 (C), 166.48 (C), 193.22 (C).
For C17H22N2O3S2 Calculated: C, 55.71; H, 6.05; N, 7.64. Found: C, 55.10; H, 6.23; N, 7.34.
HRMS (ESI) (m/z): [M+Na]+ 389.10.
4.1.1.2.3. N-(1,3-Benzodioxol-5-ylmethyl)-2-[(morpholinyl)thiocarbamoylthio]acetamide (3)
Yield: 83 %. Mp 131.5 ºC.
IR νmax (cm-1): 3273 (N-H stretching), 3076 (aromatic C-H stretching), 2985 (aliphatic C-H
stretching), 2910, 2854 (O-CH2 stretching), 1643 (C=O stretching), 1552, 1489, 1454 (C=C
stretching and N-H bending).
1H NMR (500 MHz) (DMSO-d6) δ (ppm): 3.67-3.68 (m, 4H), 3.94 (s, 2H), 4.09 (s, 2H), 4.19
(d, J= 5.95 Hz, 4H), 5.97 (s, 2H), 6.72-6.74 (m, 1H), 6.83-6.84 (m, 2H), 8.59 (s, 1H).
13C NMR (125 MHz) (DMSO-d6) δ (ppm): 40.58 (CH2), 42.75 (CH2), 50.67 (2CH2), 66.06
(2CH2), 101.26 (CH2), 108.33 (CH), 108.41 (CH), 120.82 (CH), 133.53 (C), 146.49 (C),
147.68 (C), 166.79 (C), 195.54 (C).
For C15H18N2O4S2 Calculated: C, 50.83; H, 5.12; N, 7.90. Found: C, 50.10; H, 5.23; N, 7.94.
HRMS (ESI) (m/z): [M+H]+ 355.08.
4.1.1.2.4. N-(1,3-Benzodioxol-5-ylmethyl)-2-[(thiomorpholinyl)thiocarbamoylthio]acetamide
(4)
Yield: 82 %. Mp 160.4 ºC.
IR νmax (cm-1): 3267 (N-H stretching), 3074 (aromatic C-H stretching), 2970, 2902 (aliphatic
C-H stretching), 2837 (O-CH2 stretching), 1635 (C=O stretching), 1552, 1504, 1489, 1460
(C=C stretching and N-H bending).
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1H NMR (500 MHz) (DMSO-d6) δ (ppm): 2.74 (s, 4H), 4.09 (s, 2H), 4.19 (d, J= 5.95 Hz,
2H), 4.25 (br, 2H), 4.50 (br, 2H), 5.97 (s, 2H), 6.72-6.74 (m, 1H), 6.83-6.84 (m, 2H), 8.56-
8.58 (m, 1H).
13C NMR (125 MHz) (DMSO-d6) δ (ppm): 26.90 (2CH2), 40.79 (CH2), 42.75 (CH2), 53.46
(2CH2), 101.26 (CH2), 108.33 (CH), 108.40 (CH), 120.80 (CH), 133.53 (C), 146.49 (C),
147.68 (C), 166.76 (C), 195.02 (C).
For C15H18N2O3S3 Calculated: C, 48.63; H, 4.90; N, 7.56. Found: C, 48.10; H, 4.23; N, 7.54.
HRMS (ESI) (m/z): [M+H]+ 371.06.
4.1.1.2.5.
N-(1,3-Benzodioxol-5-ylmethyl)-2-[(4-methyl-1-
piperazinyl)thiocarbamoylthio]acetamide (5)
Yield: 80 %. Mp 163.2 ºC.
IR νmax (cm-1): 3255 (N-H stretching), 3057 (aromatic C-H stretching), 2972, 2937, 2902
(aliphatic C-H stretching), 2833, 2785 (O-CH2 stretching), 1635 (C=O stretching), 1546,
1504, 1463, 1460 (C=C stretching and N-H bending).
1H NMR (500 MHz) (DMSO-d6) δ (ppm): 2.21 (s, 3H), 2.39-2.41 (m, 4H), 3.93-3.99 (m, 2H),
4.07 (s, 2H), 4.19 (d, J= 5.90 Hz, 4H), 5.97 (s, 2H), 6.73 (d, J= 8.0 Hz, 1H), 6.82-6.84 (m,
2H), 8.55-8.57 (m, 1H).
13C NMR (125 MHz) (DMSO-d6) δ (ppm): 40.76 (CH2), 42.75 (CH2), 45.57 (CH3), 50.07
(CH2), 51.57 (CH2), 54.44 (2CH2), 101.26 (CH2), 108.34 (CH), 108.40 (CH), 120.82 (CH),
133.54 (C), 146.49 (C), 147.68 (C), 166.85 (C), 195.01 (C).
For C16H21N3O3S2 Calculated: C, 52.30; H, 5.76; N, 11.43. Found: C, 52.10; H, 5.23; N,
11.54.
HRMS (ESI) (m/z): [M+H]+ 368.11.
4.1.1.2.6.
N-(1,3-Benzodioxol-5-ylmethyl)-2-[(4-ethyl-1-
piperazinyl)thiocarbamoylthio]acetamide (6)
Yield: 82 %. Mp 144.9 ºC.
IR νmax (cm-1): 3250 (N-H stretching), 3066 (aromatic C-H stretching), 2972, 2895 (aliphatic
C-H stretching), 2816, 2779 (O-CH2 stretching), 1637 (C=O stretching), 1548, 1502, 1456,
1427 (C=C stretching and N-H bending).
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1H NMR (500 MHz) (DMSO-d6) δ (ppm): 1.01 (t, J= 7.2 Hz, 3H), 2.36 (q, J= 7.2 Hz, 2H),
2.45 (t, J= 5.15 Hz, 4H), 3.93 (s, 2H), 4.07 (s, 2H), 4.19 (d, J= 5.95 Hz, 4H), 5.97 (s, 2H),
6.72-6.74 (m, 1H), 6.82-6.84 (m, 2H), 8.53-8.55 (m, 1H).
13C NMR (125 MHz) (DMSO-d6) δ (ppm): 11.90 (CH3), 40.20 (CH2), 42.30 (CH2), 49.70
(CH2), 51.00 (2CH2), 51.70 (2CH2), 100.80 (CH2), 107.80 (CH), 107.90 (CH), 120.30 (CH),
133.00 (C), 146.00 (C), 147.20 (C), 166.40 (C), 194.40 (C).
For C17H23N3O3S2 Calculated: C, 53.52; H, 6.08; N, 11.01. Found: C, 53.10; H, 6.23; N,
11.04.
HRMS (ESI) (m/z): [M+H]+ 382.13.
4.1.1.2.7.
N-(1,3-Benzodioxol-5-ylmethyl)-2-[(4-phenyl-1-
piperazinyl)thiocarbamoylthio]acetamide (7)
Yield: 86 %. Mp 137.2 ºC.
IR νmax (cm-1): 3259 (N-H stretching), 3080 (aromatic C-H stretching), 2897 (aliphatic C-H
stretching), 2819 (O-CH2 stretching), 1672 (C=O stretching), 1647, 1600, 1560, 1500, 1452,
1429 (C=C stretching and N-H bending).
1H NMR (500 MHz) (DMSO-d6) δ (ppm): 3.29-3.31 (m, 4H), 4.11 (s, 4H), 4.19 (d, J= 5.90
Hz, 2H), 4.35 (br, 2H), 5.96 (s, 2H), 6.73-6.74 (m, 1H), 6.80-6.84 (m, 3H), 6.94-6.96 (m, 2H),
7.23-7.26 (m, 2H), 8.56 (s, 1H).
13C NMR (125 MHz) (DMSO-d6) δ (ppm): 40.20 (CH2), 42.30 (CH2), 47.50 (2CH2), 51.00
(2CH2), 100.70 (CH2), 107.80 (CH), 107.90 (CH), 115.40 (2CH), 119.20 (CH), 120.30 (CH),
129.00 (2CH), 133.00 (C), 146.00 (C), 147.20 (C), 150.00 (C), 166.30 (C), 194.60 (C).
For C21H23N3O3S2 Calculated: C, 58.72; H, 5.40; N, 9.78. Found: C, 58.10; H, 5.23; N, 9.74.
HRMS (ESI) (m/z): [M+H]+ 430.13.
4.1.1.2.8.
N-(1,3-Benzodioxol-5-ylmethyl)-2-[4-(4-methoxyphenyl)-1-
piperazinylthiocarbamoylthio]acetamide (8)
Yield: 84 %. Mp 158.7 ºC.
IR νmax (cm-1): 3302 (N-H stretching), 3074, 3003 (aromatic C-H stretching), 2902 (aliphatic
C-H stretching), 2816 (O-CH2 stretching), 1672 (C=O stretching), 1654, 1546, 1500, 1458,
1429 (C=C stretching and N-H bending).
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1H NMR (500 MHz) (DMSO-d6) δ (ppm): 3.13 (s, 4H), 3.69 (s, 3H), 4.12 (s, 4H), 4.20 (d, J=
5.55 Hz, 2H), 4.36 (s, 2H), 5.96 (s, 2H), 6.74 (d, J= 7.8 Hz, 1H), 6.82-6.85 (m, 4H), 6.92 (d,
J= 8.5 Hz, 2H), 8.57 (s, 1H).
13C NMR (125 MHz) (DMSO-d6) δ (ppm): 40.77 (CH2), 42.79 (CH2), 49.82 (2CH2), 51.00
(2CH2), 55.67 (CH3), 101.26 (CH2), 108.34 (CH), 108.41 (CH), 114.84 (2CH), 118.36 (2CH),
120.83 (CH), 133.54 (C), 144.89 (C), 146.50 (C), 147.69 (C), 153.89 (C), 166.86 (C), 195.14
(C).
For C22H25N3O4S2 Calculated: C, 57.50; H, 5.48; N, 9.14. Found: C, 57.10; H, 5.43; N, 9.74.
HRMS (ESI) (m/z): [M+H]+ 460.14.
4.1.1.2.9.
N-(1,3-Benzodioxol-5-ylmethyl)-2-[4-(2-pyrimidinyl)-1-
piperazinylthiocarbamoylthio]acetamide (9)
Yield: 87 %. Mp 179.1 ºC.
IR νmax (cm-1): 3257 (N-H stretching), 3051 (aromatic C-H stretching), 2997 (aliphatic C-H
stretching), 2893 (O-CH2 stretching), 1645 (C=O stretching), 1585, 1550, 1489, 1462, 1440,
1421 (C=N, C=C stretching and N-H bending).
1H NMR (500 MHz) (DMSO-d6) δ (ppm): 3.88-3.97 (m, 4H), 4.08-4.11 (m, 4H), 4.19 (d, J=
5.85 Hz, 2H), 4.25-4.33 (m, 2H), 5.97 (s, 2H), 6.70 (t, J= 4.7 Hz, 1H), 6.74 (d, J= 8.0 Hz,
1H), 6.82-6.85 (m, 2H), 8.41 (d, J= 4.7 Hz, 2H), 8.57-8.59 (m, 1H).
13C NMR (125 MHz) (DMSO-d6) δ (ppm): 40.74 (CH2), 42.98 (CH2), 49.78 (2CH2), 51.19
(2CH2), 101.25 (CH2), 108.35 (CH), 108.41 (CH), 111.11 (CH), 120.84 (CH), 133.54 (C),
146.49 (C), 147.68 (C), 158.48 (2CH), 161.32 (C), 166.82 (C), 195.29 (C).
For C19H21N5O3S2 Calculated: C, 52.88; H, 4.91; N, 16.23. Found: C, 52.10; H, 4.93; N,
16.24.
HRMS (ESI) (m/z): [M+H]+ 432.12.
4.1.1.2.10.
N-(1,3-Benzodioxol-5-ylmethyl)-2-[4-(4-nitrophenyl)-1-
piperazinylthiocarbamoylthio]acetamide (10)
Yield: 90 %. Mp 197.2 ºC.
IR νmax (cm-1): 3257 (N-H stretching), 3066 (aromatic C-H stretching), 2980 (aliphatic C-H
stretching), 2875 (O-CH2 stretching), 1643 (C=O stretching), 1597 (NO2 stretching), 1552,
1496, 1442, 1423 (C=C stretching and N-H bending).
11

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1H NMR (500 MHz) (DMSO-d6) δ (ppm): 3.72 (s, 4H), 4.12 (s, 4H), 4.19 (d, J= 5.90 Hz,
2H), 4.36 (s, 2H), 5.97 (s, 2H), 6.74 (d, J= 8.0 Hz, 1H), 6.83-6.84 (m, 2H), 6.95 (d, J= 9.35
Hz, 2H), 8.10 (d, J= 9.25 Hz, 2H), 8.58-8.60 (m, 1H).
13C NMR (125 MHz) (DMSO-d6) δ (ppm): 40.66 (CH2), 42.78 (CH2), 45.18 (2CH2), 48.84
(CH2), 50.58 (CH2), 101.26 (CH2), 108.34 (CH), 108.41 (CH), 112.31 (2CH), 120.83 (CH),
126.26 (2CH), 133.53 (C), 137.34 (C), 146.50 (C), 147.69 (C), 154.18 (C), 166.78 (C),
195.30 (C).
For C21H22N4O5S2 Calculated: C, 53.15; H, 4.67; N, 11.81. Found: C, 53.10; H, 4.63; N,
11.84.
HRMS (ESI) (m/z): [M+H]+ 475.11.
4.2. Biochemistry
4.2.1. Cell culture and drug treatment
C6 Rat glioma cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM)
(Sigma, Deisenhofen, Germany) supplemented with 10% fetal calf serum (Gibco, Paisley,
Scotland). A549 Human lung adenocarcinoma cells were incubated in 90% RPMI
supplemented with 10% fetal bovine serum (Gibco, Paisley, Scotland). All media were
supplemented with 100 IU/mL penicillin-streptomycin (Gibco, Paisley, Scotland) and cells
were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. Exponentially
growing cells were plated at 2x104 cells/mL into 96-well microtiter tissue culture plates
(Nunc, Denmark) and incubated for 24 h before the addition of the drugs (the optimum cell
number for cytotoxicity assays was determined in preliminary experiments). The stock
solutions of the compounds were prepared in dimethyl sulfoxide (DMSO; Sigma Aldrich,
Poole, UK) and further dilutions were made with fresh culture medium (the concentration of
DMSO in the final culture medium was <0.1% which had no effect on the cell viability).
4.2.2. MTT assay
The level of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Sigma) reduction was quantified as previously described in the literature with small
modifications [24,25].
After 24 h of preincubation, the tested compounds and cisplatin (positive control) were added
to give final concentration in the range 3.9-1000 µg/mL and the cells were incubated for 24 h.
At the end of this period, MTT was added to a final concentration of 0.5 mg/mL and the cells
12

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were incubated for 4 h at 37 °C. After the medium was removed, the formazan crystals
formed by MTT metabolism were solubilized by addition of 200 µL DMSO to each well and
absorbance was read at 540 nm with a microtiter plate spectrophotometer (Bio-Tek plate
reader). Every concentration was repeated in three wells. IC50 values were defined as the drug
concentrations that reduced absorbance to 50% of control values.
4.2.3. Purification of human carbonic anhydrase isozymes (hCA-I and hCA-II) from human
erythrocytes by affinity chromatography
Fresh human blood was obtained from the blood center, Ataturk University, it was stored at
4oC used within 2-3 days at most. The blood samples were centrifuged to separate
erythrocytes at 2500 rpm for 15 min and plasma and buffy coat were carefully removed.
Then, underlying erythrocytes were washed with 0.9% NaCl solution twice and upper
portions were also discarded. The erythrocytes were hemolyzed with distilled water at 0oC, it
was stirred for half an hour at 4oC. The hemolysate was centrifuged at 20000 rpm for 30 min
and cell membranes were separated. pH was adjusted to 8.7 with solid Tris. So, the
hemolysate was recovered to be applied to the column [26,27].
The affinity gel was prepared on Sepharose-4B matrix. After Sepharose-4B was activated
with CNBr, L-tyrosine was covalently fitted. Then sulfanilamide was coupled to tyrosine with
diazotization reaction as a ligand. The hemolysate was applied to the prepared Sepharose-4B-
L-tyrosine-sulfanilamide affinity column equilibrated with 25 mM Tris-HCl/0.1 M Na2SO4
(pH 8.7). The affinity gel was washed with 25 mM Tris-HCl/22 mM Na2SO4 (pH 8.7). The
human carbonic anhydrase (hCA I and hCA II) isozymes were eluted with 1 M NaCl/25 mM
Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4 (pH 5.6), respectively. All
procedures were performed at 4oC [28].
4.2.4. Determination CA activity
4.2.4.1. Hydratase activity
Carbonic anhydrase activity was determined using the Wilbur-Anderson Method which was
modified by Rickli et al. [28-29]. This method, as a result hydration of CO2 is released H+
ions and the pH changes were determined by means of bromine thymol blue indicator, based
on measuring the elapsed time. Enzyme Unit (EU) was calculated using the equation (to-tc/tc)
where to and tc are the times for pH change of the nonenzymatic and the enzymatic reactions,
respectively.
4.2.4.2. Inhibition assays
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The inhibitory effects of compounds 1-10 and AAZ on the hydratase activity of hCA-I and
hCA-II enzymes were investigated. IC50 values were calculated for the compounds at different
concentrations while maintaining a constant substrate concentration. The activities of
enzymes in the medium without inhibitors were used as 100% activity. The activity % values
of enzymes were calculated by measuring the hydratase activity in the presence of different
concentrations of inhibitors. The IC50 value was calculated by utilizing graphs of activity%-[I]
for each inhibitor [29-31].
Declaration of interest
The authors report no conflicts of interest.
Acknowledgements
This study was supported by Anadolu University Scientific Research Projects Commission
under the grant no: 1404S123.
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Table 1
IC50 values of the compounds against A549 and C6 cells for 24 h
IC50 (µg/mL)
Compound
A549
875.00±106.07
810.00±14.14
700.00±278.39
>1000
C6
633.33±208.17
566.67±76.38
206.67±11.55
220.00±26.46
166.67±23.09
156.67±20.82
163.33±15.28
58.33±14.43
48.33±7.64
1
2
3
4
>1000
5
850.00±212.13
>1000
6
7
>1000
8
833.33±175.59
>1000
9
10
23.33±7.63
27.00±3.06
19.00±5.29
Cisplatin

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Table 2
The results obtained from regression analysis graphs for hCA-I and hCA-II in the presence of
the tested compounds.
hCA-I Inhibition*
IC50
hCA-II Inhibition*
IC50
Compound
hCA-I / hCA-II
0.556
1.69
0.651
1.875
0.287
0.338
0.622
0.802
-
0.854
0.901
1.206
0.852
0.860
0.844
-
1
2
0.346
0.288
0.535
0.677
0.518
0.624
0.699
0.903
5.8
3
4
5
6
7
-
-
8
0.855
0.799
6.7
0.818
1.129
0.87
9
10
Acetazolamide (AAZ)
* They were determined as nM.

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Fig. 1. N,N-diethyldithiocarbamate structurally important for the interaction with CAs [20].
Fig. 2. Benzodioxole-based anticancer agents [21].

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Scheme 1. The synthetic route for the preparation of the benzodioxole-based dithiocarbamate
derivatives (1-10).

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Highlights
► New dithiocarbamate derivatives were investigated for their anticancer effects.
► Compound 10 was found to be the most promising anticancer agent against C6 cell line.
► New dithiocarbamate derivatives were investigated for their CA inhibitory effects.
► Generally, the tested compounds were more effective on CAs than acetazolamide.
► Compounds 3 and 4 were found to be the most effective compounds on hCA-I and hCA-II.