Identification of (1S,4S)-2,5-diazabicyclo[2.2.1]heptane-dithiocarbamate-nitrostyrene hybrid as potent antiproliferative and apoptotic inducing agent against cervical cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2018.01.020

The present study seeks to describe the design and synthesis of six new Michael adducts of (1S,4S)-2,5-diazabicyclo[2.2.1]heptane-dithiocarbamate with nitrostyrenes and their in vitro antiproliferative activity against human cervical cancer cell lines [HeLa (HPV 18 positive), CaSki (HPV 16 positive) and ViBo (HPV negative) cervical cancer cell lines]. Virtual screening of the physicochemical properties of all compounds have also been presented. All the compounds exploited significant antiproliferative activity on the three cervical cancer cell lines. Compound 8a was found to be most potent, displaying in vitro antiproliferative activity against HeLa, CaSki and ViBo cervical cancer cell lines superior to Cisplatin and Paclitaxel with IC₅₀ values 0.99 ± 0.007, 2.36 ± 0.016 and 0.73 ± 0.002 μM respectively. In addition, compound 8a did not trigger the necrosis cell death to the test cancer cell lines. Further mechanistic study revealed that compound 8a could inhibit the cancer cell proliferation by inducing apoptosis through caspase-3 activation. Moreover, cell cycle analysis indicated that compound 8a could arrest the cell cycle at the G1 phase for HeLa and CaSki cancer cells. At the predetermined IC₅₀ values on cancer cells, compound 8a did not induce any necrotic (cytotoxic) death to the normal human lymphocytes. In the present design, (1S,4S)-2,5-diazabicyclo[2.2.1]heptane system was found to be superior than the piperazine counterpart 11.

Full text of DOI:10.1016/j.ejmech.2018.01.020

Accepted Manuscript
Identification of (1S,4S)-2,5-diazabicyclo[2.2.1]heptane-dithiocarbamate-nitrostyrene
hybrid as potent antiproliferative and apoptotic inducing agent against cervical cancer
cell lines
Sujay Laskar, Luis Sánchez-Sánchez, Sebastián M. Flores, Hugo López-Muñoz,
María L. Escobar-Sánchez, Manuel López-Ortiz, Marcos Hernández-Rodríguez,
Ignacio Regla
PII:
S0223-5234(18)30020-5
10.1016/j.ejmech.2018.01.020
EJMECH 10097
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 June 2017
Revised Date: 17 December 2017
Accepted Date: 7 January 2018
Please cite this article as: S. Laskar, L. Sánchez-Sánchez, Sebastiá.M. Flores, H. López-Muñoz, Marí.L.
Escobar-Sánchez, M. López-Ortiz, M. Hernández-Rodríguez, I. Regla, Identification of (1S,4S)-2,5-
diazabicyclo[2.2.1]heptane-dithiocarbamate-nitrostyrene hybrid as potent antiproliferative and apoptotic
inducing agent against cervical cancer cell lines, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.01.020.
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Preliminary communication
(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-dithiocarbamate-nitrostyrene
Identification
of
hybrid as potent antiproliferative and apoptotic inducing agent against cervical cancer cell
lines
Sujay Laskara , Luis Sánchez-Sánchezb , Sebastián M. Floresa, Hugo López-Muñozb, María L.
* *
Escobar-Sánchezc, Manuel López-Ortiza, Marcos Hernández-Rodríguezd, Ignacio Reglaa
*
a
Lab. de Síntesis de Fármacos, Laboratorio 9 UMIEZ, Facultad de Estudios Superiores Zaragoza,
Universidad Nacional Autónoma de México, Ejercito de Oriente, Iztapalapa 09230, Ciudad de México,
México.
b
Lab. Biología Molecular del Cáncer; Laboratorio 2 PB UMIEZ, Facultad de Estudios Superiores
Zaragoza, Universidad Nacional Autónoma de México, Ejercito de Oriente, Iztapalapa 09230, Ciudad de
México, México
.
c
Lab. Microscopía Electrónica, Depto. Biología Celular, Facultad de Ciencias, Coyoacán 04510, Cd.
Universitaria, Universidad Nacional Autónoma de México, Ciudad de México, México.
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad
Universitaria, 04510, Ciudad de México, México.
d
*Corresponding authors
Tel.: +52 (55) 56 23 07 95
sl.sujay@gmail.com (Dr. Sujay Laskar)
Tel.: +52 (55) 56 23 07 95
regla@unam.mx (Dr. Ignacio Regla)
Tel.: +52 (55) 56230723
luisss@unam.mx (Dr. Luis Sánchez-Sánchez)
Address:
Facultad de Estudios Superiores Zaragoza, UMIEZ, Universidad Nacional Autónoma de México,
Iztapalapa 09230, CDMX, México.

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Graphical Abstract
Highlights
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
New Michael adducts of (1S,4S)-2,5-diazabicyclo[2.2.1]heptane-dithiocarbamate with nitrostyrenes
were designed empirically and synthesized.
Compound 8a was found to be the most promising in vitro antiproliferative agent against cervical
cancer cell lines HeLa (HPV 18 positive), CaSki (HPV 16 positive) and ViBo (HPV negative).
In respect to IC50 values on cancer cells, compound 8a was found to be superior than Cisplatin and
Paclitaxel.
Compound 8a induced apoptosis on all cancer cells through caspase-3 activation without triggering
necrosis cell death and arrested the cell cycle at G1 phase for HeLa and CaSki cells.
Compound 8a did not exert any (necrotic) cytotoxic effect on human lymphocytes.

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Identification
of
(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-dithiocarbamate-nitrostyrene
hybrid as potent antiproliferative and apoptotic inducing agent against cervical cancer cell
lines
ABSTRACT
The present study seeks to describe the design and synthesis of six new Michael adducts of (1S,4S)-2,5-
diazabicyclo[2.2.1]heptane-dithiocarbamate with nitrostyrenes and their in vitro antiproliferative activity
against human cervical cancer cell lines [HeLa (HPV 18 positive), CaSki (HPV 16 positive) and ViBo
(HPV negative) cervical cancer cell lines]. Virtual screening of the physicochemical properties of all
compounds have also been presented. All the compounds exploited significant antiproliferative activity on
the three cervical cancer cell lines. Compound 8a was found to be most potent, displaying in vitro
antiproliferative activity against HeLa, CaSki and ViBo cervical cancer cell lines superior to Cisplatin and
Paclitaxel with IC50 values 0.99 ± 0.007, 2.36 ± 0.016 and 0.73 ± 0.002 µM respectively. In addition,
compound 8a did not trigger the necrosis cell death to the test cancer cell lines. Further mechanistic study
revealed that compound 8a could inhibit the cancer cell proliferation by inducing apoptosis through
caspase-3 activation. Moreover, cell cycle analysis indicated that compound 8a could arrest the cell cycle
at the G1 phase for HeLa and CaSki cancer cells. At the predetermined IC50 values on cancer cells,

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compound 8a did not induce any necrotic (cytotoxic) death to the normal human lymphocytes. In the
present design, (1S,4S)-2,5-diazabicyclo[2.2.1]heptane system was found to be superior than the
piperazine counterpart 11.
Keywords: Nitrostyrene, Dithiocarbamate, Antiproliferative, Pro-apoptotic, Necrosis
Preliminary communication
Identification
of
(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-dithiocarbamate-nitrostyrene
hybrid as potent antiproliferative and apoptotic inducing agent against cervical cancer cell
lines
1. Introduction
Despite of the advances in screening, diagnosis and treatment ‘cancer’ still continues as a dark
spot to the society. Cervical cancer is the most common gynecologic cancer worldwide among women in
developing countries and is associated with persistent Human Papillomavirus (HPV) infection [1]. Among
the different HPV types, type 16 and 18 are the most dangerous and responsible for about 70% of all cases
of cervical cancer [2-3]. According to the literature, each year cervical cancer accounts for 528,000 new
cases and 266,000 deaths worldwide [4]. Surgery and radiotherapy is considered to be the first option for
early-stage cervical cancer treatment [5-6]. However, metastatic/advanced cervical cancer is incurable due
to lack of an effective therapeutic regimen [7]. Currently, chemotherapy is the most practiced treatment
against advanced cervical cancer and often associated with serious challenges including chemoresistance

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and toxicity [8-9]. As a consequence, patients with advanced cervical cancer have very poor survival rate
and unfortunately in the near future the incidence rate of cervical cancer is expected to expand [10]. Thus,
there is an urgent need to develop efficient compounds bearing new scaffolds for the treatment of cervical
cancer.
During the design of antiproliferative agents, it would be desirable to take into account the actual side
effects related with different cell death processes [10]. Apoptosis and necrosis represent two fundamental
types of cell death processes [11]. Apoptotic cell death is a regulated cellular mechanism; the plasma
membrane retains the integrity during the process [12]. Caspase-dependent apoptosis is the major form of
controlled cell death in normal and cancer cells [13]. In contrast, necrotic cells undergo plasma membrane
rupture, nuclear and cellular swelling [14]. Necrosis is usually elicits inflammatory response to the
released cellular contents, resulting in normal tissue damage [15]. Conventional chemotherapeutic agents
not only evoke apoptosis but also includes other form of death including necrosis in a dose dependent way
[16-19]. If a new molecule could precisely elicit apoptosis without triggering necrosis, we can succeed in
effective and less toxic chemotherapeutic regimens to combat against cancer.
During the past decade, dithiocarbamates (DTC) have received considerable attention owing to their
diverse biological activities such as antibacterial [20], antifungal [21], anti-HIV [22], platelet aggregation
inhibitor [23], anti-inflammatory [24], antihistaminic [25], antioxidant [26], anticholinesterase [27] etc.
[28]. Literature revealed that many dithiocarbamate derivatives displayed potent anticancer activity both
in in vitro and in vivo model and might act by induction of apoptosis (Fig. 1) [29]. Interestingly, natural
product brassinin, a phytoalexin first isolated from cabbage was capable of mediating chemopreventive
activity [30]. Structural modification of brassinin led to the development of potential cancer
chemopreventive agent sulforamate [31]. In recent times, molecular hybridization technique had been
widely adopted in designing new dithiocarbamate cytotoxic agents which includes tetrahydrocarbazole
[32], thalidomide [33], 1,2,3-triazoles [34-37], quinazolines [38-39], 4-anilinoquinazoline [40],
carboline [41], emetine [42], pyrrolobenzodiazepines [43], chalcone [44], benzimidazole [45], butenolide
[46], chromones [47], benzodioxole [48] dithiocarbamate derivatives etc. [49-52]. On the other hand,
β-
β
-
nitrostyrene compounds have been found to inhibit tumor cell growth and induce apoptosis even in some
multidrug-resistant tumor cells (Fig. 2) [53-54]. Steifelder et al. reported that nitrovinyl side chain
attached to an aromatic ring is the structure essential for the development of new pro-apoptotic agents
[55]. Moreover, Sreedhar et al. designed and synthesized nitrovinylstilbenes exhibiting significant anti-
proliferative activity with potent caspase-3 induction ability [56]. Recently, Shafiee et al. have reported a
series of hybrid β-nitrostyrene derivatives namely 2-aryl-3-nitro-2H-chromenes that induced apoptosis in
breast cancer cells by the induction of caspase-3 protein and DNA damage [57]. Furthermore, during the
design of small-molecule drugs nitrogen bearing heterocycles have gained extensive attention from the
researchers. Among the N-heterocycles, piperazine and the conformationally rigid homologue (1S,4S)-2,5-
diazabicyclo[2.2.1]heptane have gained significant importance in medicinal chemistry for synthesizing
potent drug candidates (Fig. 3) [58-59]. The most practical application of a diazabicyclo[2.2.1]heptane
containing molecule is the fluoroquinolone antibiotic danofloxacin. Surprisingly, only two literatures
(from Merck Research Laboratories and Wyeth Research) are currently available applying 2,5-
diazabicyclo[2.2.1]heptane to achieve antitumor agents [60-61]. In the context of current drug discovery
strategies, empirical approaches without particular target largely depends on the quality of the newly
synthesized molecules in respect to molecular complexity and diversity which can be built easily through
multi-component reactions [62]. Until now, there are no reports available for the functionalization of 2,5-

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diazabicyclo[2.2.1]heptanes through multicomponent reaction pathway to achieve rapid diversity in the
framework.
In the context of designing new drug candidates, the construction of hybrid molecules through the
combination of different pharmacophores in one frame lead to potential compounds with interesting
biological profiles [63-64]. Keeping in view of the above, the same idea could be valuable to design a new
potential antiproliferative agent with apoptosis inducing ability against cervical cancer cell line (Fig. 4).
To the best of our knowledge, molecular hybridization of piperazine/2,5-diazabicyclo[2.2.1]heptane with
dithiocarbamate and nitrostyrene to afford antiproliferative agents has not been reported before.
Therefore, based on this empirical design we synthesized some Michael adduct of piperazine/2,5-
diazabicyclo[2.2.1]heptane-dithiocarbamate with nitrostyrenes through
a
facile, catalyst free
multicomponent route in order to achieve new class of apoptotic inducer. The synthesized compounds
were evaluated for their possible in vitro antiproliferative activity against HeLa (HPV 18 positive), CaSki
(HPV 16 positive) and ViBo (HPV negative) human cervical cancer cell lines. Besides, necrotic toxicity
profile of the newly designed compounds on cancer cells as well as on normal cells, apoptosis inducing
ability, caspase-3 detection, cell cycle analysis, CFSE labeling assay on human lymphocytes and in-silico
ADME profiling have been presented.
Fig. 1. Structures of brassinin (I), sulforamate (II) and some dithiocarbamate derived hybrid molecules as
potent anticancer agents and apoptotic inducers.
Fig. 2. Structures of β-nitrostyrene derivatives as pro-apoptotic antitumor agents

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Fig. 3. Structures of danofloxacin (14) and 2,5-diazabicyclo[2.2.1]heptane bearing potent anticancer
compounds.
Fig. 4. Compounds designed in this work as new pro-apoptotic anti-proliferative agents.
2. Results and discussion
2.1. Chemistry
The synthetic pathway to achieve the dithiocarbamate hybrids 8a−8f and 11 have been outlined in Scheme
1. At the beginning, the
β-nitrostyrene derivatives 3a−3f were prepared by the base catalyzed
condensation between substituted benzaldehydes and nitromethane and obtained in good yields. The
starting compound 4 [(1S,4S)-2-benzyl-2,5-diazabicyclo[2.2.1]heptane dihydrobromide] was synthesized
according to the multistep synthetic procedure described by Regla (our group member) and Juaristi et al
[65]. Free base of the (1S,4S)-2-benzyl-2,5-diazabicyclo[2.2.1]heptane dihydrobromide salt was obtained
by the treatment with sodium methoxide (in methanol) solution and was used in the next stage without
storage. The free base 5 was then treated with di-tert-butyl dicarbonate followed by hydrogenolysis to
remove the benzyl group and afford (1S,4S)-2-Boc-2,5-diazabicyclo[2.2.1]heptane 7 in good yield. During
the optimization, we envisioned that such type of hybrid dithiocarbamate scaffold 8 might be constructed
through a one-pot procedure from its starting constituents 2,5-diazabicyclo[2.2.1]heptane, carbon
disulphide and nitrostyrenes. One-pot multicomponent reaction strategy was then successfully applied to
afford the corresponding final compounds 8a−8f in moderate yields using methanol as solvent in the
absence of any catalyst at 0oC to room temperature. The reaction profile was very clean, facile and energy
efficient. The products were isolated by simple filtration followed by crystallization. The analogous
dithiocarbamate 11 was prepared in a similar fashion. However, the mono tert-butyl piperazine-1-
carboxylate 10 was obtained by the reaction of piperazine with di-(tert-butyl)dicarbonate in the presence
of glacial acetic acid and tap water [66]. The structures of newly synthesized compounds 8a−8f and 11
1
were established by FT-IR, H-NMR, 13C-NMR and HRMS analysis as described in the experimental
section along with the detailed experimental procedure.

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Scheme 1. Synthesis of the hybrid dithiocarbamates (8a-8f and 11), designed in this present study
2.2. Biology
2.2.1. Theoretical predictions of physicochemical properties
The computed molecular properties of the newly synthesized derivatives (8a-8f and 11) have been
presented in Table 1. The lipophilicity (log P), total polar surface area (TPSA), molecular weight, number
of hydrogen bond acceptor, number of hydrogen bond donor, number of rotatable bonds, molecular
volume and number of violations had been calculated using Molinspiration online property calculation
software (version 2016.03) [67]. It is worthy to note that at the early stages of drug development program,
many active compounds failed due to their unfavorable properties such as absorption, distribution,
metabolism, and elimination (ADME). It is clear from Table 2 that all the synthesized hybrids have log P
values less than five. Therefore according to the Lipinski's rule, there may not be a problem for
penetration of this molecules into biomembrane. All the synthesized hybrids displayed molecular
properties in the acceptable range without any violation according to Lipinski's rule of five. The drug-
likeness model score had been calculated by MolSoft software and presented in Table 1 [68]. Also, the
predicted bioactivity score in terms of GPCR ligand activity, ion channel modulators, kinase inhibitors,
nuclear receptor ligand activity, protease inhibitors and other enzyme inhibitors had been calculated using
the same Molinspiration toolkit (version 2016.03). The results are summarized in Table 2. It is to mention
that most of the marketed drugs have negative values or very low positive values of score for above
mentioned enzyme targets. It has been revealed from Table 2 that all the synthesized compounds (8a-8f
and 11) have negative or low positive values of enzyme target scores.
Table 1. In silico physicochemical properties and drug-likeness of the synthesized dithiocarbamate
hybrids (8a-8f and 11).

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Compound miLogPa TPSAb
Mol.
wt.
n-
n-
n-
Molecular
volume
(Å3)
n-
Drug-
s
(Å2)
ONc OHNHd ROT
Be
violationsf likeness
model
score
3.67
4.12
3.84
4.35
4.57
3.73
3.57
78.61 423.56
78.61 437.59
78.61 441.55
78.61 458.00
78.61 491.56
87.84 453.59
78.61 411.55
7
7
7
7
7
8
7
0
0
0
0
0
0
0
8
9
8
8
9
9
8
369.10
385.66
374.03
382.63
400.39
394.64
363.08
0
0
0
0
0
0
0
.30
.45
.82
1.03
.53
.78
.38
8a
8b
8c
8d
8e
8f
11
b
c
d
a miLogP: Lipophilicity; TPSA: Total polar surface area; n-ON acceptors: number of hydrogen bond acceptors;
f
n-OHNH donors: number of hydrogen bonds donors; [e]n-ROTB: number of rotatable bonds; n- violations: number
of violations according to Lipinski’s rule
Table 2. In silico predicted bioactivity of the synthesized dithiocarbamate hybrids (8a−8f and 11).
Compounds
GPCR
0.07
0.03
0.07
0.06
0.10
0.02
-0.17
ICM
-0.07
-0.13
-0.08
-0.07
0.00
KI
NRL
-0.05
-0.07
-0.03
-0.07
0.07
PI
EI
-0.44
-0.46
-0.40
-0.44
-0.34
-0.44
-0.66
0.14
0.08
0.11
0.09
0.14
0.08
-0.09
0.12
0.07
0.10
0.09
0.11
0.07
-0.02
8a
8b
8c
8d
8e
8f
-0.13
-0.38
-0.06
-0.38
11
GPCRL: GPCR ligand; ICM: ion channel modulator; KI: kinase inhibitor; NRL: nuclear receptor ligand; PI: protease
inhibitor; EI: enzyme inhibitor.
2.2.2. In vitro antiproliferative activity study on cervical cancer cell lines
To determine whether the synthesized Michael adducts of (1S,4S)-2-Boc-2,5-diazabicyclo[2.2.1]heptane-
dithiocarbamate with nitrostyrenes were antitumor agents, antiproliferative activity of the synthesized
hybrids was evaluated against a panel of human cervical cancer cell lines employing ethanol as vehicle.
The antiproliferative activity (IC50) was determined after 24 h by crystal violet staining method. Among
the test cervicouterine cancer cell lines, HeLa cells are HPV 18 positive, CaSki cells are HPV 16 positive
and ViBo cells are HPV negative. The inhibitory effect of compounds 8a-8f on the proliferation of HeLa,
CaSki, and ViBo cells was observed to occur in a dose-dependent manner. The determined IC50 values for
the test compounds have been summarized in Table 3. The results indicated that all the synthesized
hybrids exhibited potent antiproliferative effect on all the cervical cancer cell lines with IC50 values less
than 8 µM. Interestingly, different substitution (at the 4- position) to the aromatic ring of compound 8a,

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had very little effect on the antiproliferative activity on the test cervical cancer cell lines. Therefore,
compound 8a was found to be the most potent antiproliferative agent among those designed hybrids. The
antiproliferative dose response curve of compound 8a against cervical cancer cell lines has been shown in
Figure 5. Comparing the antiproliferative activity (IC50 values) of 8a with commercial anticancer drugs
like Cisplatin, Camptothecin and Paclitaxel, compound 8a was found to be more potent antiproliferative
agent against all the cervical cancer cell line than Cisplatin and Paclitaxel. In the present study, we have
also compared the antiproliferative activity of the rigid 2,5-diazabicyclo[2.2.1]heptane system 8a with the
conformationally flexible piperazine analogue 11. The results indicated that the piperazine analogue 11
exhibited much less antiproliferative activity comparing with the compound 8a, implying the superiority
of using (1S,4S)-2-Boc-2,5-diazabicyclo[2.2.1]heptane system in the present study.
Eventually, the results summarized that such new type of Michael adducts could be very promising
candidates to achieve potent antiproliferative agent. It was crucial then, to investigate the primary cell
death processes induced by the most potent hybrid 8a.
Table 3. Antiproliferative activity of the synthesized dithiocarbamate derivatives (8a−8f and 11) against a
panel of human cervical cancer cell linesa.
IC50 ( µM)b
HeLa
CaSki
ViBo
Compounds
8a
0.989 ± 0.007
5.52 ± 0.043
7.92 ± 0.049
5.67 ± 0.089
6.10 ± 0.056
5.29 ± 0.074
31.58 ± 0.162
5.99 ± 0.063
0.94 ± 0.002
10.90 ± 0.044
2.36 ± 0.016
5.94 ± 0.091
6.79 ± 0.070
7.20 ± 0.126
6.10 ± 0.077
6.17 ± 0.099
29.40 ± 0.097
5.66 ± 0.109
1.80 ± 0.005
2.90 ± 0.026
0.73 ± 0.002
3.19 ± 0.025
6.56 ± 0.117
2.40 ± 0.124
7.32 ± 0.038
2.64 ± 0.057
19.19 ± 0.099
7.66 ± 0.059
1.92 ± 0.005
1.59 ± 0.013
8b
8c
8d
8e
8f
11
Cisplatinc
Camptothecinc
Paclitaxelc
a Antiproliferative activity of the compounds have been expressed as the required concentration of the compounds to
induce a 50 % decrement in the cell population (IC50). The concentrations were expressed in µM.
b Values are the mean ± SD. All the experiments had been performed at least three times.
c
The assay was performed with commercially available samples of Cisplatin, Camptothecin and Paclitaxel,
purchased from Sigma-Aldrich

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Fig. 5. Antiproliferative dose response curve of compound 8a on three cervical cancer cell lines
2.2.3 Cytotoxic (Necrotic) activity determination on the human cervical cancer cell lines
In order to determine whether necrosis was induced during cell death process, the necrotic effect of all the
designed hybrids was evaluated on HeLa, CaSki and ViBo cervical cancer cell lines using the lactate
dehydrogenase (LDH) cytotoxic assay. HeLa, CaSki, and ViBo cultures were stimulated with the
compounds at the level of their previously determined IC50 values (IC50 values Table 3). The amount of
lactate dehydrogenase (LDH) released in the culture supernatant was used as a measure of loss of plasma-
membrane integrity. The three cancer cell lines were treated with the nonionic surfactant Triton X-100 and
the released LDH was adjusted to 100% as a control. The results have been summarized in Figure 6 and
Table 4. Interestingly, the results demonstrated that the test compound 8a did not induce the tumor cells to
release LDH in the supernatants. Therefore, the compound 8a did not induce necrosis cell death to the
cervical cancer cells. During the study, it was observed that all the test compounds exhibited null or low
necrotic effect on the tumor cell lines. It is worthy to mention that necrosis usually elicits pro-
inflammatory response, resulting further tissue damage. We envisioned that the side effects directly
related to necrotic activity would be lower in case of compound 8a than those generated by cisplatin.
Hence, the decrease in cancer cell population during the antiproliferative study was caused by other type
of cell death process different to the necrosis.
Table 4. Amount of lactate dehydrogenase (LDH) released by the treated cervical cancer cells with the
test compounds (8a−8f and 11)a
Necrotic effect (% LDH released)b
Compounds
HeLa
%LDH
3±0.12
0±0.56
CaSki
%LDH
2±0.35
2±0.64
ViBo
%LDH
0±0.66
5±0.47
8a
8b

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2±0.49
4±0.85
5±0.53
0±0.38
0±0.32
57±3.82
3±0.45
5±0.45
15±0.12
20±1.75
0±0.27
0±0.19
26±2.11
2±0.53
8±0.35
12±0.42
0±0.21
5±0.57
0±0.28
17±2.14
0±0.38
8c
8d
8e
8f
11
Cisplatin
Camptothecin
a
HeLa, CaSki, and ViBo cultures were stimulated with the compounds at the level of their previously determined
IC50 values (IC50 Table 3).
b Values are the mean ± SD. All the experiments had been performed at least three times.
Fig. 6. Evaluation of necrotic-cytotoxicity for dithiocarbamate derivatives 8a-8f on cervicouterine cancer cell lines
[CPT: Camptothecin and CACP: Cisplatin]. 7500 cells/well were seeded in 96-well tissue culture plates. After 24 h
the medium was removed and cells were exposed to compounds 8a-8f at the respective IC50 values or with EtOH (10
µL/mL) and the amount of LDH released in the culture supernatant evaluated after 24 h. Experimental data are
presented as mean ± S.D. for three independent experiments with three repetitions. *P<0.05 versus Et-OH (ANOVA
followed by Tukey's test).
2.2.4. Cell cycle analysis
After obtaining the results from the necrosis experiment, we envisioned that the compound 8a might
inhibited the tumor cell proliferation during the antiproliferative study by interfering with cell cycle or
inducing cell death. Cell-cycle regulation ensures the fidelity of genomic replication and cell divisions to
prevent any impaired transmission of genetic information [69-70]. The cell cycle is regulated by two
major checkpoints located at the G1/S and G2/M transitions. These checkpoints allow the cells to control
any modifications of their DNA content [71]. Checkpoint failure results in genomic instability and has
been implicated in carcinogenesis [71-72]. Inducing cells to leave the cycle, also named as cell-cycle
arrest, in cancer cell lines constitutes one of the most prevalent strategies which used to stop or limit the
spread of cancer. Therefore, the cultures of HeLa, CaSki and ViBo cervical cancer cells were treated with
the most potent compound 8a at the respective IC50 values (IC50 values of 8a, Table 3) and the DNA
distribution was determined during several phases of the cell cycle (Fig. 7). The results indicate that the
compound 8a induced increment of cells in the phase G1 and decrease of cells in the G2/M phase which
implied that compound 8a induced these cells to exit G2/M to be arrested in the phase G1 of the cell cycle
(for HeLa and CaSki cultures). Here, it need to mention truly that the increased or decreased percentage of
cells were not too high. However, in ViBo cells, the compound 8a did not affect the cell cycle. Hence, the

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overall cell cycle analysis indicated that the antiproliferative activity of compound 8a was quite dependent
on the cell cycle for HeLa and CaSki cultures and independent for ViBo cells.
A
8a
Control
EtOH
G1= 69.25%
S= 10.37%
G1= 63.91%
S= 8.85%
G1= 70.26%
S= 10.68%
G2/M= 17.19%
G2/M= 21.18%
G2/M= 16.01%
B
8a
Control
EtOH
G1= 69.31%
S= 10.12%
G1= 72.29%
S= 10.49%
G1= 67.95%
S= 9.67%
G2/M= 16.59%
G2/M= 14.35%
G2/M= 17.97%
C
EtOH
Control
8a
G1= 64.38%
S= 10.05%
G1= 66.69%
S= 11.67%
G1= 60.20%
S= 12.62%
G2/M= 20.48%
G2/M= 16.20%
G2/M= 19.87%
Fig. 7. Effect of compound 8a on the cell cycle distribution of HeLa (A), CaSki (B) and ViBo (C) cervical cancer
cells. Cells were treated with compound 8a for 24 h, stained with PI and then cell cycle was analyzed by flow
cytometry. Values are expressed in distribution of % DNA.
2.2.5. Apoptosis induction
In the necrosis experiment, we observed that the compound 8a did not induce necrosis to the tumor cells.
Moreover, flow cytometric analysis of cell cycle revealed that cells were arrested in the G1 phase of the
cell cycle. Hence, we envisioned that the compound 8a could induce cell apoptosis. Apoptosis is
characterized by morphological changes and chromatin condensation that produces smaller, more compact
nuclei and/or the formation of apoptotic bodies. HeLa, CaSki, and ViBo cell cultures were stimulated with
compound 8a at the IC50 values (IC50 values of 8a, Table 3) and morphological changes, chromatin
condensation including the formation of apoptotic bodies were identified by staining with fluorochrome
4,6-diamidino-2-phenylindole (DAPI) and viewed under epifluorescence microscopy (Fig. 8A, Fig. 8B).

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The study revealed that the morphology of control nucleus was conserved with a normal shape. Under that
condition chromatin was distributed throughout the nucleoplasm (Fig. 8B-DAPI staining). In contrast,
after treatment with compound 8a, the nuclear morphology of the treated cells was seen to be altered.
Compact nuclei and apoptotic bodies were detected in the cultures treated with compound 8a (Fig. 8A,
Fig. 8B), suggesting apoptosis. Despite these results, it was still necessary to detect the activation of
effector caspases such as active caspase-3 to establish that the apoptotic process had been activated and
was dependent on caspases. Thus, more conclusive experiments were performed as described below.
Fig. 8A. Morphology view of HeLa, CaSki and ViBo cultures treated with compound 8a at the respective IC50 values
(IC50 values of 8a, Table 3). The phase contrast images evidence cellular shapes under the different experimental

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conditions. The control cells are polyhedral in shape and have extended cytoplasm with cytoplasmic projections and
normal nucleus. The treated cells are spherical in shape without cytoplasmic projections and both the cytoplasm and
the nucleus are contracted (arrows).
Fig. 8B. Nuclear Morphology (DAPI staining) view of HeLa, CaSki and ViBo cultures treated with compound 8a at
the respective IC50 values (IC50 values of 8a, Table 3). The DAPI staining images evidence nucleus shapes under the
different experimental conditions. The nucleus of the control cells show chromatin distributed in the nuclear space

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evidenced by the fluorescent distribution of DAPI. After treatment, the nuclei are contracted and several apoptotic
bodies could be seen (arrows).
2.2.5.1. Detection of active caspase-3
The caspases or cysteine-aspartic proteases are a family of cysteine proteases that are crucial mediators of
apoptosis. Caspase-3 is probably the best understood of the mammalian caspases in terms of its specificity
and role in apoptosis. Caspase-3 is required for some typical hallmarks of apoptosis and is indispensable
for apoptotic chromatin condensation and DNA fragmentation. It is essential for certain processes
associated with the formation of apoptotic bodies [73]. In the present study, we detected active caspase-3
using immunodetection to evaluate the presence of the enzyme by means of optical microscopy
(epifluorescence) and flow cytometry in order to obtain qualitative and quantitative results (Fig. 9, Fig.
10). Microscopic study demonstrated that the morphological alterations in several cells induced by
compound 8a corresponded to the presence of active caspase-3 (Fig. 9). Therefore, compound 8a induced
the caspase-3 activation, implying that apoptosis could be triggered through a caspase-3 dependent
process. Meanwhile, the quantitative data obtained from the flow cytometry assays evidenced that the
compound 8a induced different percentages of positive cells to active caspase-3 (Fig. 10). HeLa, CaSki
and ViBo cells presented 36%, 42% and 48% of positive cells respectively.
Therefore, all the above results reinforced the presence of apoptosis during the elimination of the cancer
cells.

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(A)

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(B)

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(C)
Fig. 9. Determination of active caspase-3 in HeLa (A), CaSki (B) and ViBo (C) cervical cancer cell lines treated with
compound 8a at the respective IC50 values (IC50 values of 8a, Table 3).

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HeLa
EtOH
HeLa
HeLa
HeLa
8a
Camptothecin
Control
3%
2%
29%
36%
CaSki
CaSki
CaSki
EtOH
CaSki
8a
Camptothecin
Control
42%
62%
5%
7%
ViBo
8a
ViBo
ViBo
ViBo
Control
EtOH
Camptothecin
5%
8%
48%
35%
Fig. 10. Flow cytometric evaluation of active caspase-3 of HeLa, CaSki and ViBo cultures treated with compound 8a
at the respective IC50 values (IC50 values of 8a, Table 3). After 24 h, the amount of positive cells to active caspase-3
was evaluated. Cells were analyzed by FACS from CellQuest software.
2.2.6. Effect of the compound on non-tumor cells
2.2.6.1. Effect of the compound on Lymphocytes proliferation
To determine the proliferative potential from nontumor cells, human lymphocyte cells cultures were
treated with compound 8a at the respective IC50 values (IC50 values of 8a, Table 3) and the proliferation of
lymphocyte cells were evaluated after 72 h by carboxifluorescein technique followed by flow cytometric
quantification (Fig. 11). Results indicated that after 72 h of treatment, compound 8a did not affect the
proliferation of lymphocyte cells significantly at 0.73 and 0.99 µM concentrations. However, at
concentration 2.36 µM, the proliferation of lymphocyte cells is strongly inhibited. Therefore, at 2.36 µM
concentration, compound 8a might have immunosuppressive effect.

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Activated
EtOH
Non Activated
68.36%
73.36%
0.92%
8a (0.73 µM)
8a (2.36 µM )
8a (0.99 µM)
62.38%
64.02%
7.44%
Fig. 11. Effect of compound 8a on the proliferation of enriched lymphocyte population (ELP) using CFSE labeling
assay after 72 h.
2.2.6.2. Cytotoxic (Necrotic) activity determination on non-tumor cells
Since the previous study had established a strong inhibition of lymphocyte cell proliferation, it was
necessary to determine whether this inhibition was due to a cytotoxic effect of compound 8a on
lymphocytes. Therefore, lymphocyte cell cultures were treated with compound 8a at 0.73, 0.99 and 2.36
µM concentrations and the activity of the cytoplasmic LDH enzyme was determined in the supernatants of
the lymphocyte cultures (Fig. 12). Results indicated that such concentrations did not induce necrotic death
to human lymphocytes and the decrease in the number of lymphocyte cells in the CFSE labeling assay was
due to an event different to the necrotic cell death. So, it could be concluded that after 72 h treatment in
the LDH assay, the compound 8a did not induce any cytotoxicity to human lymphocytes.

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Fig. 12. Evaluation of the cytotoxicity of compound 8a on human lymphocyte cultures. Lymphocytes were seeded in
96-well tissue culture plates, exposed to 0.73, 0.99 and 2.36 µM of compound 8a, or with EtOH (10 µL/mL) and
evaluated after 72 h by the amount of LDH released into the culture supernatant. Activated lymphocytes with PHA
(15 µL/mL) were used as a control. Experimental data are presented as the mean ± S.D. of three independent
experiments with three repetitions (Student’s t test).
3. Conclusion
In summary, a new type of Michael adduct of (1S,4S)-2,5-diazabicyclo[2.2.1]heptanes-dithiocarbamate
with nitrostyrenes were designed and synthesized using an energy efficient multicomponent approach. All
compounds exploited significant in vitro antiproliferative activity in HeLa (HPV 18 positive), CaSki
(HPV 16 positive) and ViBo (HPV negative) cervical cancer cell lines with null or low necrotic
(cytotoxic) effect. The most potent compound 8a in this study exhibited in vitro antiproliferative activity
superior to Cisplatin and Paclitaxel. Compound 8a induced apoptosis in all the cervical cancer cells
through caspase-3 activation without triggering necrosis cell death. Moreover, compound 8a did not have
any cytotoxic (necrotic) effect on human lymphocytes as revealed from the LDH cytotoxic assay.
However during the CFSE labeling assay, at higher concentration compound 8a strongly affected the
proliferation of peripheral blood lymphocytes. In the present design, (1S,4S)-2,5-
diazabicyclo[2.2.1]heptane system was found to be superior than the piperazine counterpart. These in vitro
results certainly augur well for deeper assays on mechanistic effects in the next stage of the research
including the in vivo models. Therefore, we believe that such hybrid could provide promising new lead
derivatives for more potent anticancer drug development.
4. Experimental
4.1. Chemistry
Melting points were determined in open capillaries in a KRUSS melting point apparatus and uncorrected.
1H and 13C- NMR spectra were obtained at 300 MHz and 75 MHz respectively, using Jeol Eclipse 300
NMR Spectrometer and CDCl3 as the solvent; chemical shifts were recorded in parts per million (ppm)
with TMS as the internal reference. High resolution mass spectra were measured on a JEOL JMS-700
MStation and Jeol JMS-SX 102A Mass Spectrometer and signals were given in m/z. Thin-layer
chromatography was performed using silica gel 60 F254 plates (Merck, A.G., Germany). All the solvents
were distilled prior to use unless otherwise stated. Optical rotations were determined in a Perkin-Elmer
341 polarimeter using a 1-dm cell path length (sodium D-line 589 nm), at 20°C sample compartment
temperature.
4.1.2. Synthesis of (1S,4S)-2-Boc-2,5-diazabicyclo[2.2.1]heptane (7)
Free base of the (1S,4S)-2-Benzyl-2,5-diazabicyclo[2.2.1]heptane dihydrobromide salt was obtained by
the treatment of sodium methoxide (in methanol) solution and was used in the next stage without storage.
In a 250 mL round bottom flask, 10.75g (57.12 mmol) of (1S,4S)-2-benzyl-2,5-diazabicyclo[2.2.1]heptane
in 250 mL of dichloromethane was added and flask was placed in an ice-bath. 13.71 g (62.83 mmol) of di-
tert-butyl dicarbonate was added in portions into the reaction flask followed by the addition of 8.82 mL
(62.83 mmol) triethylamine. The reaction flask was slowly warmed to room temperature and the stirring
was continued. After completion of the reaction (as monitored by TLC), the reaction mixture was washed
with distilled water (3X200 mL), dried over sodium sulfate and concentrated under vacuum to get white

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solid product 6 with 15.5 g yield. After that, 15.5 g (53.7 mmol) of 6 was dissolved in 200 mL dry
methanol and placed in a 500 mL hydrogenation flask (60psi) in presence of 10% by weight of Pearlman’s
catalyst. After completion of the reaction, the catalyst was filtered off and the filtrate containing the
product was concentrated to obtain a white solid as N-Boc-DBH (10.4 g). The analytical data were well
accordance with the reported literature values [74].
4.1.3. General method for the synthesis of nitrostyrenes (3a−3f)
Aromatic aldehyde (1 mmol) and nitromethane (1.3 mmol) were dissolved in methanol and the reaction
mixture was cooled to -10 to 5 °C. Ice cold aqueous solution of NaOH (2.5 mmol) was added very slowly
into the reaction mixture. Subsequently, the reaction mixture was stirred for about 1-3 h. The progress of
the reaction was monitored by thin-layer chromatography. On completion of the reaction, the reaction
mixture was neutralized with 4N HCl. After neutralization, the pale yellow colored solid was formed
which was filtered off and washed thoroughly with water followed by recrystallization to obtain the pure
nitrostyrenes.
4.1.4. Multicomponent synthesis of the designed hybrids (8a−8f)
An oven-dried screw cap reaction flask was charged with a magnetic stir bar, (1S,4S)-2-Boc-2,5-
diazabicyclo[2.2.1]heptane (3 mmol) and 6 mL methanol. After stirring for 15 minutes, the reaction flask
was placed in an ice-bath. Carbon disulfide (4.5 mmol) was added drop wise to the stirred mixture at 0oC
temperature. Stirring was continued for 30 minutes. Subsequently, nitrostyrenes (2.7 mmol) was added in
small portions to the stirred reaction mixture, and the stirring was continued for overnight at ambient
temperature. The progress of the reaction was monitored by thin-layer chromatography. On completion of
the reaction, a solid mass precipitated out, which was filtered off followed by washing with cold methanol
to obtain the crude product 8 in almost pure form which was further purified by recrystallization from
suitable solvents.
N-Boc piperazine 9 was prepared according to a literature described procedure [66]. The hybrid 11 was
synthesized by the above mentioned multicomponent procedure.
4.1.5. Analytical & Spectral data of the newly synthesized compounds (8a−8f) and 11.
4.1.5.1. (1S,4S)-tert-butyl 5-((2-nitro-1-phenylethylthio)(thiocarbonyl))-2,5-diaza-bicyclo[2.2.1]heptane-
o
1
2-carboxylate (8a). Yield: 60%; White solid (mp 100.3 C); [
(300 MHz, CDCl3, ppm): =7.39-7.33 (5H, m), 5.77 (1H, dd, J = 10.1 Hz, 4.8 Hz), 5.64 (0.5H, s), 5.29
(1H, dd, J = 13.3 Hz, 5.0 Hz), 4.95-4.88 (1.5 H, m), 4.68-4.53 (1H, m), 3.95-3.35 (4H, m), 2.00-1.87 (2H,
α]
20: 300.558 (c = 1.05, CHCl3); H-NMR
D
δ
13
m), 1.47-1.43 (9H, m); C-NMR (75 MHz, CDCl3):
δ = 190.34, 153.96, 134.85, 129.41, 129.20, 129.16,
129.02, 128.93, 128.32, 128.28, 80.47, 77.68, 64.61, 62.37, 61.76, 58.35, 57.38, 56.44, 55.98, 55.02,
52.21, 51.89, 51.01, 50.87, 50.77, 37.99, 37.52, 36.73, 36.37, 28.43; IR υmax (cm-1): 2979.95, 2949.4,
2883.7, 1691.96, 1549.70, 1442.00, 1407.95, 1368.46, 1322.47, 1115.31, 1141.91, 1107.21, 1040.86,
963.90, 865.81, 747.26, 696.64, 548.49 cm-1; HRMS (FAB+): m/z calculated for C19H25N3O4S2 [M+H]+:
424.1365, found: 424.1375.
4.1.5.2. (1S,4S)-tert-butyl 5-((2-nitro-1-p-tolylethylthio)(thiocarbonyl))-2,5-diaza-bicyclo[2.2.1]heptane-
o
1
2-carboxylate (8b). Yield 53%; White solid (mp 98.4 C); [
(300 MHz, CDCl3, ppm): 7.30-7.25 (2H, m), 7.17 (2H, brs), 5.71-5.61 (1.5H, m), 5.32-5.28 (1H, m), 4.93-
α]
20: -260.991 (c = 1.02, CHCl3); H-NMR
D

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4.85 (1.5H, m), 4.68-4.53 (1H, m), 3.95-3.34 (4H, m), 2.33 (3H, s), 2.00-1.90 (2H, m), 1.47-1.42 (9H, m);
13C-NMR (75 MHz, CDCl3):
δ = 190.52, 153.97, 138.92, 131.70, 129.90, 129.86, 128.18, 128.14, 80.46,
77.70, 64.53, 62.33, 58.34, 56.00, 56.47, 52.20, 51.89, 50.80, 50.66, 37.98, 37.52, 36.37, 28.44, 21.18; IR
υmax (cm-1): 2978.15, 2923, 2888, 1688.61, 1548.75, 1440.98, 1406.89, 1419.7, 1378.06, 1366.90,
1322.12, 1298.48, 1184.86, 1150.4, 1142.10, 1106.16, 1040.69, 964.63, 849.8, 792.21, 548.85; HRMS
(ESI): m/z calculated for C20H27N3O4S2 [M+H]+: 438.1521, found: 438.1528.
4.1.5.3.
(1S,4S)-tert-butyl
5-((1-(4-fluorophenyl)-2-nitroethylthio)(thiocarbonyl))-2,5-diaza-
o
bicyclo[2.2.1]heptane-2-carboxylate (8c). Yield: 51%; White solid (mp 63.5 C); [
α]
D
20: -260.209 (c =
1
1.002, CHCl3); H-NMR (300 MHz, CDCl3, ppm):
(1H, m), 5.63-5.61 (0.5H, m), 5.34-5.24 (1H, m), 4.91-4.80 (1.5H, m), 4.69-4.54 (1H, m), 3.94-3.30 (4H,
m), 2.02-1.89 (2H, m), 1.47-1.43 (9H, m); 13C-NMR (75 MHz, CDCl3, ppm):
= 189.97, 188.92, 164.40,
δ = 7.39-7.35 (2H, m), 7.08-7.03 (2H, m), 5.80-5.72
δ
153.93, 130.62, 130.21, 130.18, 130.10, 130.07, 116.36, 116.31, 116.07, 116.02, 80.44, 77.77, 64.70,
64.04, 62.48, 61.83, 58.39, 57.38, 56.44, 55.97, 55.00, 52.17, 51.95, 50.30, 50.14, 50.05, 37.99, 37.50,
36.73, 36.39, 28.41; IR υmax (cm-1): 2975.96, 2953.7, 2888, 1695.86, 1604.77, 1550.45, 1510.04 , 1639.46,
1405.41, 1368.14, 1321.55, 1233.73, 1160.00, 1140.42, 1103.53, 1041.23, 962.20, 853.35, 783.65,
684.95, 531.43; HRMS (ESI): m/z calculated for C19H24FN3O4S2 [M+H]+: 442.1271, found: 442.1286.
4.1.5.4.
(1S,4S)-tert-butyl
5-((1-(4-chlorophenyl)-2-nitroethylthio)(thiocarbonyl))-2,5-diaza-
o
bicyclo[2.2.1]heptane-2-carboxylate (8d). Yield 40%; White solid (mp 75.5 C); [
α]
D
20: -266.118 (c =
1
1.002, CHCl3); H-NMR (300 MHz, CDCl3, ppm):
Hz), 5.63 (0.5H, s), 5.25 (1H, dd, J = 13.4 Hz, 5.0 Hz), 4.91-4.83 (1.5H, m), 4.69-4.54 (1H, m), 3.94-3.30
(4H, m), 2.02-1.89 (2H, m), 1.47-1.43 (9H, m); 13C-NMR (75 MHz, CDCl3, ppm):
= 189.83, 188.75,
δ = 7.35-7.33 (4H, m), 5.77 (1H, dd, J = 10.0 Hz, 4.9
δ
153.93, 134.97, 134.86, 133.62, 129.67, 129.40, 129.35, 80.51, 77.57, 64.73, 64.07, 62.53, 61.80, 58.39,
57.35, 56.44, 55.98, 55.00, 52.17, 51.87, 50.35, 50.19, 38.00, 37.53, 36.71, 36.37, 28.42.; IR υmax (cm-1):
2971.2, 2936.2, 2888, 1689.63, 1552.33, 1435.40, 1401.23, 1367.1, 1328.98, 1143.78, 1104.30, 1041.68,
967.57, 849.05, 784.56, 545; HRMS (ESI): m/z calculated for C19H24ClN3O4S2 [M+H]+: 458.1339, found:
458.1279.
4.1.5.5. (1S,4S)-tert-butyl 5-((1-(4-(trifluoromethyl)phenyl)-2-nitroethylthio)(thiocarbonyl))-2,5-diaza-
o
bicyclo[2.2.1]heptane-2-carboxylate (8e). Yield: 55%; White solid (mp 128.5 C); [
α
]
D
20: -263.713 (c =
1
1.024, CHCl3); H-NMR (300 MHz, CDCl3, ppm):
dd, J = 9.8 Hz, 5.0 Hz), 5.63 (0.5H, s), 5.27 (1H, dd, J = 13.7 Hz, 5.1 Hz), 4.97-4.89 (1.5H, m), 4.70-4.54
(1H, m), 3.95-3.30 (4H, m), 2.02-1.89 (2H, m), 1.47-1.43 (9H, m); 13C-NMR (75 MHz, CDCl3, ppm):
δ = 7.66-7.61 (2H, m), 7.56-7.51 (2H, m), 5.89 (1H,
δ
=
189.48, 188.40, 153.93, 139.35, 139.01, 131.18, 130.75, 128.82, 128.79, 126.17, 126.11, 126.06, 125.52,
121.91, 80.52, 77.41, 64.88, 64.23, 62.64, 61.85, 58.41, 57.33, 56.44, 55.96, 55.01, 52.13, 51.87, 50.44,
50.29, 38.01, 37.54, 36.71, 36.37, 28.40; IR υmax (cm-1): 2978.00, 2896.8, 1698.41, 1549.83, 1447.25,
1408.16, 1324.91, 1372.37, 1165.09, 1152.95, 1134.49, 1107.02, 170.42, 1039.52, 961.29, 862.89,
768.23, 692.21, 548.81; HRMS (ESI): m/z calculated for C19H24F3N3O4S2 [M+H]+: 492.1239, found:
492.1263.
4.1.5.6.
(1S,4S)-tert-butyl
5-((1-(4-methoxyphenyl)-2-nitroethylthio)(thiocarbonyl))-2,5-diaza-
o
bicyclo[2.2.1]heptane-2-carboxylate (8f). Yield: 45%; White solid (mp 111.1 C); [
1.002, CHCl3); H-NMR (300 MHz, CDCl3, ppm):
α]
D
20: -311.061 (c =
1
δ = 7.34-7.27 (2H, m), 6.90-6.85 (2H, m), 5.73-5.64
(1.5H, m), 5.29 (1H, dd, J = 13.1 Hz, 5.1 Hz), 4.91-4.83 (1.5H, m), 4.68-4.53 (1H, m), 3.95-3.75 (4H, m),
13
3.64-3.30 (3H, m), 2.00-1.87 (2H, m), 1.47-1.42 (9H, m); C-NMR (75 MHz, CDCl3, ppm):
δ = 190.52,

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189.52, 160.01, 159.93, 153.96, 129.54, 129.49, 126.53, 114.61, 114.56, 80.45, 77.81, 64.52, 63.85,
62.32, 61.74, 58.34, 57.38, 56.46, 56.00, 55.31, 55.02, 52.20, 51.86, 50.61, 50.46, 37.97, 37.50, 36.73,
36.35, 28.43; IR υmax (cm-1): 3010.6, 2977.45, 2879, 1686.36, 1606.08, 1549.72, 1510.54, 1405.09,
1368.18, 1249.57, 1173.18, 1151.89, 1142.18, 1106.21, 1402.53, 1025.25, 964.37, 850.24, 769.18,
684.89, 549.41; HRMS (ESI): m/z calculated for C20H27N3O5S2 [M+H]+: 454.1470, found: 454.1457.
4.1.5.7. tert-butyl 4-((2-nitro-1-phenylethylthio)(thiocarbonyl))piperazine-1-carboxylate (11). Yield 49%;
1
White solid (mp: 96.1 oC); H-NMR (300 MHz, CDCl3, ppm):
δ =7.38-7.34 (5H, m), 5.78 (1H, dd, J = 9
Hz, 6 Hz), 5.33-5.27 (1H, m), 4.90 (1H, dd, J = 13.3 Hz, 10.6 Hz), 4.32-3.86 (4H, m), 3.54 (4H, brs), 1.47
(9H, s); 13C-NMR (75 MHz, CDCl3):
δ = 193.59, 154.35, 134.69, 129.19, 129.02, 128.38, 80.76, 77.62,
51.66, 42.79, 28.34; IR υmax (cm-1): 3006.3, 2980.52, 2918.7, 2870.6, 1683.20, 1554.09, 1475.49, 1454.63,
1413.42, 1372.36, 1282.81, 1225.42, 1165.04, 1126.33, 1020.80, 993.84, 932.02, 858.48, 746.27, 697.99,
534.99 1; HRMS (ESI): m/z calculated for C18H25N3O4S2 [M+H]+: 412.1365, found: 412.1357.
4.2 Biology
4.2.1 Cell cultures
HeLa (HPV 18 positive), CaSki (HPV 16 positive) and ViBo (HPV negative) human cervical cancer cell
lines were purchased from the American Type Culture Collection (ATCC Rockville, MD) and cultured in
RPMI-1640 medium (GIBCO USA) containing 5% Newborn Calf Serum (NCS, GIBCO, USA) with red
phenol supplemented by benzylpenicillin. Cultures were maintained in a humidified atmosphere with 5%
CO2 at 37 °C. All cell-based assays were performed using cells in the exponential growth phase.
4.2.2 Cell proliferation assay
Assays were performed by seeding 7500 cells/well in 96-well tissue culture plates at a volume of 100 µL
of RPMI-1640 medium supplemented with 5% NCS per well. Cells were allowed to grow for 24h in the
culture medium prior to exposure to the compounds. Also, 1% of vehicle (EtOH) was added to the control
cells. Antiproliferative activity (IC50) was determined after 24 h by crystal violet staining. Cell counts
were determined by measuring absorbance at 590 nm in an enzyme-linked immunosorbent assay (ELISA)
plate reader (Tecan, USA).
4.2.3 Determination of cytotoxicity (Necrotic effect)
Cytotoxic activity (necrotic) was determined by means of the LDH Cytotoxicity Assay Kit (BioVision
USA) following the instructions of manufacturer. LDH oxidizes lactate to pyruvate, which then reacted
with the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitro phenyl)-5-phenyl tetrazolium (INT) to produce
formazan. The increase in the amount of formazan generated in the culture supernatant directly correlated
to the increase in the number of lysed cells. Formazan dye was water-soluble and could be easily detected
with a spectrophotometer at 500 nm.
4.2.4. Cell cycle analysis

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HeLa, CaSki, and ViBo cells were seeded at 5 X 105 cells/mL in 50 mm tissue culture plates and allowed
to grow for 24 h in culture medium prior to exposure to the IC50 determined values for compounds 8a.
Cells were harvested with versene solution. For DNA content analysis, cells were fixed and permeabilized
in 50% methanol in phosphate-buffered saline (PBS), washed in PBS, treated with RNase (2.5 U/mL) and
stained with propidium iodide (200 mg/L) for 2 min, washed twice with PBS and immediately analyzed
by flow cytometry. The percentage of cells in each phase of the cell cycle was obtained using a FACSAria
II apparatus (Becton Dickinson, USA).
4.2.5 Immunolocalization of active caspase-3 by fluorescence microscopy
Cells were cultured in glass coverslips and were treated with the IC50 of glucolaxogenin during 24 h. The
cells that adhered to the glass were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) at
pH 7.2 for 20 min. Cells were permeated with 0.05% Triton X-100 for 5 min at 4 °C. After rinsing in the
same buffer, they were blocked with PBS Bovine Serum Albumin (0.1%)−Tween (0.05%) 20 for30 min,
incubated with anti-active caspase-3 antibody (Novous Biologicals) and diluted in PBS (1:100) for 18 h.
Next, the samples were washed and incubated with a secondary goat anti-rabbit antibody with fluorescein
isothiocyanate (FITC) diluted to 1:200 in PBS for 2 h. Finally, they were counterstained with 4,6-
diamidino-2-phenylindole (DAPI). Immunoassays were evaluated under a Nikon Eclipse E600
Microscope and images were recorded with a Nikon Digital DXM1 200F Camera.
4.2.6 Immunolocalization of active caspase-3 by flow Cytometry
HeLa, CaSki and ViBo cells were seeded at 105 cells/mL in 60-mm tissue culture plates and allowed to
grow for 24 h in culture medium before treatment with compound 8a at pre-determined IC50 values. Cells
were harvested with versene solution. For active caspase-3 immunodetection, they were fixed and
permeabilized in 50 % methanol in PBS, washed in PBS, incubated with primary anti-body anti-active-
caspase-3 (Novous Biologicals) and diluted in PBS (1:500) for 18 h. Next, the cells were washed and
incubated with the secondary goat anti-rabbit antibody with FITC diluted to 1:200 in PBS for 2 h. The
samples were analyzed by flow cytometry, acquiring a minimum of 10,000 events from each sample. Data
analysis was performed using CellQuest software (Becton–Dickinson).
4.2.7 CFSE-labeling assay
Heparinized blood samples were obtained from healthy human volunteers. Peripheral blood mononuclear
cells (PBMCs) were isolated using standard Hypaque (Sigma-Aldrich USA) density gradient
centrifugation. The PBMCs were washed twice with RPMI 1640 (GIBCO USA) medium containing 10%
NCS, penicillin (100 U/mL), and streptomycin (100 U/mL). The lymphocyte population was further
enriched (ELP) by eliminating adherent cells (cells were incubated at 37 °C, 5% CO2 for 1 h, and non-
adherent cells were harvested). ELPs were re-suspended in RPMI-1640 medium at a concentration of
1x106 cells/mL. 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma-Aldrich, USA)
was added to the cell suspension at a final concentration of 2 mM and then incubated for 15 min at room
temperature in the dark. Labeling was completed by adding the same volume of NCS during 5 min at
room temperature to quench the free CFSE. Labeled cells were washed 5 times with sterile PBS
containing 10% NCS, counted, and then re-suspended in RPMI-1640 medium at 1x106 cells/mL [75].

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Unstimulated, phytohaemagglutinin (PHA)-stimulated, or treated, cells were plated at 2x105 cells/well in
96-well, flat-bottomed cell culture plates, and five replicate samples for each treated amount were
prepared. Cells were incubated in a 5% CO2 incubator at 37 °C for 72 h. Cultured cells were harvested,
washed twice with PBS, fixed with 1% formaldehyde, then analyzed by flow cytometry, acquiring a
minimum of 20,000 events from each sample. Data analysis was performed using CellQuest software
(Becton-Dickinson).
Acknowledgements
The authors acknowledge instrumental and infrastructural facility from UMIEZ-FES Zaragoza, UNAM.
LSS and HLM would like to thank the financial supports from CONACyT (Grants 255881, 253979) and
PAPIIT (IN216718, IN220916). SL acknowledges a postdoctoral fellowship from DGAPA/UNAM.
Declaration of interest
The authors report no conflicts of interest.
References
[1] M.A. Kane, B. Serrano, S. de Sanjose, S. Wittet, Implementation of human papillomavirus
immunization in the developing world, Vacc. 30 (2012) F192–200.
[2] H. Chen, X. Zeng, C. Gao, P. Ming, J. Zhang, C. Guo, L. Zhou, Y. Lu, L. Wang, L. Huang, X. He,
L. Mei, A new arylbenzofuran derivative functions as an anti-tumour agent by inducing DNA
damage and inhibiting PARP activity, Sci. Rep. 5 (2015) 10893.
[3] P. Jiang, Y. Yue, Human papillomavirus oncoproteins and apoptosis, Exp. Ther. Med. 7 (2014) 3–7.
[4] R.D. Burk, Integrated genomic and molecular characterization of cervical cancer, Nature 543 (2017)
378-384.
[5] M.C. Macdonald, J.A. Tidy, Can We Be Less Radical with Surgery for Early Cervical Cancer?,
Curr. Oncol. Rep. 18, (2016) 16.
[6] F. Landoni, A. Maneo, A. Colombo, F. Placa, R. Milani, P. Perego, G. Favini, L. Ferri, C.
Mangioni, Randomised study of radical surgery versus radiotherapy for stage Ib-IIa cervical cancer,
Lancet. 350 (1997) 535-40.
[7] K.S. Pfaendler, K.S. Tewari, Changing paradigms in the systemic treatment of advanced cervical
cancer, Am. J. Obstet. Gynecol. 214 (2016) 22-30.
[8] L.M. Elit, H. Hirte, Management of advanced or recurrent cervical cancer: chemotherapy and
beyond. Expert Rev. Anticancer Ther. 14 (2014) 319-32.
[9] L. Huang, L.M. Liao, A.W. Liu, J.B. Wu, X.L. Cheng, J.X. Lin, M. Zheng, Analysis of the impact
of platinum-based combination chemotherapy in small cell cervical carcinoma: a multicenter
retrospective study in Chinese patients, BMC Cancer 14 (2014) 140.

ACCEPTED MANUSCRIPT
[10] Orrenius S, Nicotera P, Zhivotovsky B. Cell death mechanisms and their implications in toxicology.
Toxicol. Sci. 119 (2011) 3−19.
[11] Y. Fuchs, H. Steller, Live to die another way: modes of programmed cell death and the signals
emanating from dying cells, Nat. Rev. Mol. Cell Biol. 16 (2015) 329-344.
[12] R.C. Taylor, S.P. Cullen, S.J. Martin, Apoptosis: controlled demolition at the cellular level, Nat.
Rev. Mol. Cell Biol. 9 (2008) 231-241.
[13] C. Pop, G.S. Salvesen. Human caspases: activation, specificity, and regulation, J. Biol. Chem. 284
(2009) 21777-21781.
[14] A.L. Edinger, C.B. Thompson, Death by design: apoptosis, necrosis and autophagy, Curr. Opin.
Cell Biol. 16 (2004) 663-669.
[15] K.L. Rock, H. Kono, The inflammatory response to cell death, Annu. Rev. Pathol. 3 (2008) 99-126.
[16] M.S. Ricci, W.X. Zong. Chemotherapeutic approaches for targeting cell death pathways, Oncologist
11 (2006) 342-357.
[17] T.K. Yeung, C. Germond, X. Chen, Z. Wang, The mode of action of taxol: apoptosis at low
concentration and necrosis at high concentration. Biochem. Biophys. Res. Commun. 263 (1999)
398-404.
[18] S.M. Sancho-Martínez, F.J. Piedrafita, J.B. Cannata-Andía, J.M. López-Novoa, F.J. López-
Hernández, Necrotic concentrations of cisplatin activate the apoptotic machinery but inhibit effector
caspases and interfere with the execution of apoptosis, Toxicol. Sci. 122 (2011) 73-85.
[19] V.M. Gonzalez, M.A. Fuertes, C. Alonso, J.M. Perez, Is cisplatin-induced cell death always
produced by apoptosis?, Mol. Pharmacol. 59 (2001) 657-63.
[20] H. Nabipour, S. Ghammamy, S. Ashuri, Z.S. Aghbolagh, Synthesis of a new dithiocarbamate
compound and study of its biological properties, Org. Chem. J. 2 (2010) 75-80.
[21] S.M. Monti, A. Maresca, F. Viparelli, F. Carta, G. De Simone, F.A. Mühlschlegel, A. Scozzafava,
C.T. Supuran, Dithiocarbamates are strong inhibitors of the beta-class fungal carbonic anhydrases
from Cryptococcus neoformans, Candida albicans and Candida glabrata, Bioorg. Med. Chem. Lett.
22 (2012) 859-862.
[22] X.Y. He, L. Lu, J. Qiu, P. Zou, F. Yu, X.K. Jiang, L. Li, S. Jiang, S. Liu, L. Xie, Small molecule
fusion inhibitors: design, synthesis and biological evaluation of (Z)-3-(5-(3-benzyl-4-oxo-2-
thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related
derivatives targeting HIV-1 gp41, Bioorg. Med. Chem. 21 (2013) 7539-7548.
[23] R. Rossi, D. Giustarini, I. Dalle-Donne, A. Milzani, Protein S-glutathionylation and platelet anti-
aggregating activity of disulfiram, Biochem. Pharmacol. 72 (2006) 608-615.