Design, synthesis and biological evaluation of novel α-acyloxy carboxamides via Passerini reaction as caspase 3/7 activators

Article abstract of DOI:10.1016/j.ejmech.2019.02.051

Evasion of apoptosis is a hallmark of cancer. Caspases; the key executors of apoptotic cascade are attractive targets for selective induction of apoptosis in cancer cells. Within this approach, various caspase activators were introduced as lead anticancer agents. In the current study, a new series of multifunctional Passerini products was synthesized and evaluated as potent caspase-dependent apoptotic inducers. The synthetic strategy adopted this isocyanide-based multicomponent reaction to possibly mimic the pharmacophoric features of various lead apoptotic inducers, where a series of α-acyloxy carboxamides was prepared from p-nitrophenyl isonitrile, cyclohexanone and various carboxylic acids. Accordingly, the main amide-based scaffold was decorated by substituents with varying nature and size to gain more information about structure-activity relationship. All the synthesized compounds were screened for cytotoxicity against normal human fibroblasts and their potential anticancer activities against three human cancer cell lines; MCF-7 (breast), NFS-60 (myeloid leukemia), and HepG-2 (liver) utilizing MTT assay. Among the most active compounds, 13, 21 and 22 were more potent and safer than doxorubicin with nanomolar IC₅₀ values and promising selectivity indices. Mechanistically, 13, 21 and 22 induced apoptosis by significant caspase activation in all the screened cancer cell lines utilizing flow cytometric analysis and caspase 3/7 activation assay. Again, 13 and 21 recorded higher activation percentages than doxorubicin, while 22 showed comparable results. Apoptosis-inducing factor1 (AIF1) quantification assay declared that 13, 21 and 22 didn't mediate apoptosis through AIF1-dependent pathway (i.e. only by caspase activation). Physicochemical properties, pharmacokinetic profiles, ligand efficiency metrics and drug-likeness data of all the synthesized compounds were computationally predicted and showed that 13, 21 and 22 could be considered as drug-like candidates. Finally, selected compounds were preliminarily screened for possible antimicrobial activities searching for dual anticancer/antimicrobial agents as an advantageous approach for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2019.02.051

Accepted Manuscript
Design, synthesis and biological evaluation of novel α-acyloxy carboxamides via
Passerini reaction as caspase 3/7 activators
Mohammed Salah Ayoup, Yasmin Wahby, Hamida Abdel Hamid, El Sayed Ramadan,
Mohamed Teleb, Marwa M. Abu-Serie, Ahmed Noby
PII:
S0223-5234(19)30165-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.051
EJMECH 11143
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 December 2018
Revised Date: 16 February 2019
Accepted Date: 17 February 2019
Please cite this article as: M. Salah Ayoup, Y. Wahby, H. Abdel Hamid, E.S. Ramadan, M. Teleb, M.M.
Abu-Serie, A. Noby, Design, synthesis and biological evaluation of novel α-acyloxy carboxamides via
Passerini reaction as caspase 3/7 activators, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.02.051.
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ACCEPTED MANUSCRIPT
O
H
NO
O
2 H
N
H
N
N
N
N
N
N
N
H
H
O
O
O
HN
N
O
O
S
O
LCL-161
F
Lead caspase activators
Passerini
O
H
O
NC
N
O
R
O
HO
R
O
O2N
NO2
13; R= 4-NO2C6H4, 21; R= CH(OH)C6H5, 22; R= CH2C6H5
Good in silico drug-like properties

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel α-acyloxy
carboxamides via Passerini reaction as caspase 3/7 activators
Mohammed Salah Ayoup a *, Yasmin Wahbya, Hamida Abdel Hamid a, El Sayed Ramadan a,
Mohamed Teleb b, Marwa M. Abu-Serie c, Ahmed Noby d
a Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Alexandria, Ibrahimia, 21321, Egypt.
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt.
c Medical Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research
and Technological Applications (SRTA-City), Egypt
d Department of Microbiology and Immunology, Faculty of Pharmacy, Pharos University in Alexandria, Alexandria 21311,
Egypt.
* Corresponding author at: Chemistry Department, Faculty of Science, Alexandria University, P.O. Box
426, Alexandria, Ibrahimia, 21321, Egypt
Email address: mohammedsalahayoup@gmail.com
Mohamed.salah@alexu.edu.eg
______________________________________________________________
Abstract
Evasion of apoptosis is a hallmark of cancer. Caspases; the key executors of apoptotic
cascade are attractive targets for selective induction of apoptosis in cancer cells. Within this
approach, various caspase activators were introduced as lead anticancer agents. In the current
study, a new series of multifunctional Passerini products was synthesized and evaluated as
potent caspase-dependent apoptotic inducers. The synthetic strategy adopted this isocyanide-
based multicomponent reaction to possibly mimic the pharmacophoric features of various lead
apoptotic inducers, where a series of α-acyloxy carboxamides was prepared from p-nitrophenyl
isonitrile, cyclohexanone and various carboxylic acids. Accordingly, the main amide-based
scaffold was decorated by substituents with varying nature and size to gain more information
about structure-activity relationship. All the synthesized compounds were screened for cyto-
toxicity against normal human fibroblasts and their potential anticancer activities against three
human cancer cell lines; MCF-7 (breast), NFS-60 (myeloid leukemia), and HepG-2 (liver)
utilizing MTT assay. Among the most active compounds, 13, 21 and 22 were more potent and
safer than doxorubicin with nanomolar IC50 values and promising selectivity indices.
Mechanistically, 13, 21 and 22 induced apoptosis by significant caspase activation in all the
screened cancer cell lines utilizing flow cytometric analysis and caspase 3/7 activation assay.
Again, 13 and 21 recorded higher activation percentages than doxorubicin, while 22 showed
comparable results. Apoptosis-inducing factor1 (AIF1) quantification assay declared that 13,
21 and 22 didn’t mediate apoptosis through AIF1-dependent pathway (i.e. only by caspase
activation). Physicochemical properties, pharmacokinetic profiles, ligand efficiency metrics
and drug-likeness data of all the synthesized compounds were computationally predicted and
showed that 13, 21 and 22 could be considered as drug-like candidates. Finally, selected
compounds were preliminarily screened for possible antimicrobial activities searching for dual
anticancer/antimicrobial agents as an advantageous approach for cancer therapy.
Keywords: Passerini; caspase 3/7 activator; anticancer; apoptotic inducer
1

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1. Introduction
In 2017, the World Health Organization passed the resolution “Cancer Prevention and
Control through an Integrated Approach” recognizing cancer as a leading cause of morbidity
globally, accounting for about 9.6 million deaths in its up-to-date statistics [1]. The estimated
annual number of new cancer cases projected to increase from 14.1 million in 2012 to 21.6
million by 2030 [2]. In recognition of the burden posed by cancer as a growing public health
problem, extensive studies have been conducted to investigate various cancer mechanisms [3]
and possible treatment strategies [4]. Within this context, abnormal apoptosis is considered as a
hallmark in carcinogenesis and a popular target of many anticancer agents [5,6] Apoptosis is
the normal programmed cell death process that organisms use to maintain tissue homeostasis
[7]. The mechanism underlying apoptosis is essentially controlled by a cascade of initiator and
effector caspases that are sequentially activated. Caspases are a family of cysteinyl-aspartate-
specific proteases that cleave an aspartic acid residue from their respective peptide substrates in
the presence of a histidine residue [8,9]. This property irreversibly leads to cell death. Among
these caspases, caspases-3 and -7 are believed to be the key effectors of the apoptotic pathway
whose activities are critical for the execution of apoptosis [10–12]. It is worth mentioning that
in addition to the classical caspase-dependent apoptosis, cells can undergo caspase-independent
apoptosis by releasing the apoptosis-inducing factor1 (AIF1), which translocates to the nucleus
for disrupting the nuclear chromatin [13].
Along these lines, apoptotic caspases are considered as attractive targets for selective
induction of apoptosis in cancer cells [14]. Literature survey revealed various anticancer lead
compounds that efficiently trigger apoptosis by different caspase-mediated mechanisms [15]. A
series of substituted N-(2-nitrophenyl)nicotinamides I (Fig.1) has been evaluated as potent
inducers of apoptosis utilizing HTS caspase-based assay [16]. Interestingly, a mammalian
protein called secondary mitochondria-derived activator of caspases that triggers apoptosis was
identified. This endogenous protein abrogates the inhibitory effects on caspases thus induces
caspase-dependent apoptosis [17,18]. Despite these impressive studies, most of the therapeutic
applications of this peptide were hampered by its peptide characteristics, such as proteolytic
instability. A tetrapeptide motif of the secondary mitochondria-derived activator of caspases
was then envisioned as a good lead for designing peptidomimetic compounds as apoptotic
inducers in a similar fashion. Towards this approach, a series of capped tripeptides was
synthesized to reduce the peptide character of the lead and improve its binding affinity [19].
Pioneering research has led to various small-molecule analogues as efficient apoptotic
inducers, and some have shown promising preclinical activity [20,21]. Most recently,
optimized fragment-derived nonpeptidomimetic clinical candidates based on amide core
were synthesized and evaluated as potential apoptotic inducers [22]. The optimization
strategy identified the amide core as an essential part of the molecule that should not be
changed.
Taking all together, the current study aims at the design and synthesis of amide-based
multifunctional caspase-dependent apoptotic inducers V (Fig. 1) via Passerini reaction. The
designed compounds were based on the essential amide core. The substitution pattern was
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rationalized to mimic the structural features of lead compounds (I-IV) (Fig. 1). To gain more
information about the structure-activity relationship, various lipophilic and hydrophilic
moieties were introduced to the basic scaffold. Towards this goal, our design strategy utilized
Passerini multicomponent reaction [23] as a rapid and facial method to generate various
substituted amide-based compounds. As our main target compounds, different α-acyloxy
carboxamides were designed and synthesized via Passerini reaction utilizing combinations of
various aliphatic and aromatic carboxylic acids, cyclohexanone, and p-nitrophenyl isocyanide
in one-pot reaction protocol. It is worth mentioning that Passerini multicomponent reactions
involving p-nitrophenyl isocyanide 3 [24] are rare due to the presence of a nitro group (-I, -R),
which deactivates the isonitrile nucleophilicity. Herein, we report new efficient optimized
conditions to carry out Passerini reactions involving p-nitrophenyl isocyanide. Furthermore,
investigations of such reactions utilizing different halogenated acids or ninhydrin afforded new
unexpected products.
All the newly synthesized target compounds V (Fig. 1) were screened for cytotoxicity
against normal human fibroblasts and their potential anticancer activities against three human
cancer cell lines; MCF-7 (breast), NFS-60 (myeloid leukemia), and HepG-2 (liver) utilizing
MTT assay [25]. Hence, their respective selectivity indices against the screened cell lines were
calculated to assess their efficacy and safety profiles. Compounds exhibiting the most
promising anticancer activities were evaluated as caspase-dependent apoptotic inducers
utilizing the flow cytometric analysis and caspase 3/7 activation assay. In addition, possible
caspase-independent apoptotic pathway through AIF1 was investigated utilizing AIF1
quantification assay. Furthermore, the tested compounds were preliminarily screened for
possible antimicrobial activities searching for possible dual anticancer/antimicrobial agents.
This desirable dual activity is now viewed as an advantageous approach for cancer therapy
[26]. Finally, physicochemical properties, pharmacokinetic profiles, ligand efficiency metrics
and drug-likeness data of the tested compounds were computationally predicted as a useful tool
for selecting and optimizing lead compounds.
3

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O
H
NO2
O
H
H
N
N
N
N
N
N
N
N
H
N
N
H
O
O
NH
O
O
HN
N
O
O
S
O
I
III
LCL-161
IV
II
Fragment hit
F
Lead caspase dependant apoptotic inducers
Merging pharmacophoric features
of lead compounds via Passerini
multicomponent reactions
O
NC
O
HO
R
O2N
O
H
N
O
R
O
O2N
V
Target Compounds
Fig. 1. Rationale for designing the target compounds
2. Results and Discussion
2.1. Chemistry
Multicomponent reactions are reported as efficient synthetic methods to construct
polyfunctional molecules with high diversity and complexity during few steps. Among
isocyanide-based multicomponent reactions (IMCR), one-pot combinations of carboxylic acid,
ketone and isocyanide were discovered by Passerini in 1921 affording α-acyloxy carboxamides
[23]. Herein, our strategy involved utilizing p-nitrophenyl isocyanide 3, which was synthesized
by formylation of p-nitroaniline 1 with formic acid in presence of iodine as a catalyst to give
N-formyl-p-nitroaniline 2 [27] followed by dehydration using POCl3/ Et3N [28] to afford 3 in
good yield (Scheme 1). IR of 3 showed the characteristic strong isocyanide band at 2127 cm-1.
4

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NHCHO
NH2
NC
i
ii
O2N
O2N
O2N
2
1
3
Reaction conditions:
i. HCOOH, I2, Reflux (8 hrs), 86 %;
ii. TEA, DCM/CHCl3 (2:1), POCl3, 0 C to Reflux (15 min.), 79 %
Scheme 1. Synthesis of p-nitrophenyl isonitrile 3
Optimization of Passerini reaction conditions of 3 with cyclohexanone 4 [29] and o-
nitrobenzoic acid 5i was accomplished utilizing method A (Reflux in mixture of 2,2,2
trifluoroethanol (TFE) and ethanol co-solvent (1:1)) or method B (stirring in excess
cyclohexanone at room temperature) (supplementary data). Passerini reaction in other
solvents such as DCM, THF, Benzene, Toluene, and t-BuOH didn’t afford the corresponding
α-acyloxy carboxamide product in the reported time and temperature (supplementary data).
Regarding method A, the efficiency of TFE/EtOH mixture may be due to combining both, the
acidic properties of TFE and the ability to effectively solvate the reaction mixture by EtOH. It is
worth mentioning that TFE is a very useful solvent in synthetic chemistry as it possesses an
interesting set of properties compared with ethanol; It is more acidic (pKa 12.5) than ethanol
(pKa 16.0) and has lower nucleophilicity due to the electron-withdrawing effect of the three fluorine
atoms [30].
In Scheme 2, α-acyloxy carboxamide derivatives 6-17 were prepared via Passerini
reaction of 3, 4 and miscellaneous carboxylic acids 5a-l incorporating electron withdrawing (-
CF3, -Cl, -Br, -I, -NO2) or electron donating groups (-CH3, -OCH3) utilizing method A.
Passerini reaction utilizing method A didn’t afford the expected bis-α-acyloxy carboxamides
derivatives in case of phthalic acid 5j with 3 and 4. Instead, it afforded ethyl [1-[(4-
nitrophenyl)carbamoyl]cyclohexyl] phthalate 15. This may be attributed to the planarity of
phenyl ring preventing the second Passerini reaction due to steric hindrance. The structures of
1
compounds 6-17 were established on the basis of their spectral data. H NMR spectra of 6-17
showed NH signals (δH: 10.1 - 10.6 ppm) and cyclohexylidene protons (δH: 1.2-2.6 ppm) at
their expected chemical shifts. The characteristics trans olefinic protons of the cinnamate
derivative 16 appeared at δH: 7.68 and 6.72 ppm with J coupling = 15.5 Hz. 13C NMR spectra
of this series also showed the expected amido-ester C=O groups in the range of δC: 163.0 to
172.0 ppm. The characteristic cyclohexylidene carbon (O=C-C-O) signals appeared in the
range of δC: 81.0 to 84.5 ppm, while other carbons of the cyclohexyl moiety appeared as three
13
signals ranging from δC: 20.9 to 32 ppm. C NMR spectrum of 12 showed the characteristic
C-I at δC: 94.7 ppm.
5

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Scheme 2. Synthesis of compounds 6-17 via Passerini reaction by method A.
Method A wasn’t convenient in case of the utilized aliphatic acids, mandelic acid,
phenylacetic acid, and aspirin where all trials led to hydrolysis of 3 to 1. However, such
Passerini reactions carried out by method B [31] were excellent regarding yield and time
(Scheme 3). The obtained α-acyloxy carboxamide derivatives 18-24 were confirmed based on
spectral data. 1H NMR spectra of this series showed NH signals ranging from δH: 10.0 to 10.3
ppm. Protons of cyclohexylidene moiety also appeared in the expected range (δH: 1.2-2.3 ppm).
6

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13C NMR spectra showed signals ranging from δC: 161.0 to 173.0 ppm corresponding to the
two amido-ester C=O groups. Cyclohyxylidene carbons (O=C-C-O) appeared at the expected
range (δC: 80.0-82.0 ppm). In addition, carbons of the cyclohexyl moiety appeared as three
signals ranging from δC: 20 to 32 ppm.
Literature review revealed that synthesis of bis α-acyloxy carboxamides could be
accomplished involving succinic acid [32]. Herein, the target bis α-acyloxy carboxamide 24
was synthesized by stirring a mixture of succinic acid 5s, 3, and 4 utilizing method B. It was
proposed that the free rotation of the C-C bond of the two methylene groups may enhance the
flexibility to overcome the steric hindrance around the -COOH. EI-MS of 24 showed m/z
[M]+= 610.47 (Calcd. = 610.22).
Scheme 3. Synthesis of compounds 18-24 via Passerini reaction by method B.
7

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Interestingly
,
Passerini reactions utilizing trihaloacetic acids (Scheme 4) gave
unexpected products, where trifluoroacetic acid (TFA) afforded 1-hydroxy-N-(4-
nitrophenyl)cyclohexanecarboxamide 25b in a good yield. It was proposed that the expected
inseparable Passerini product 25a was completely hydrolyzed [33]. The structure of 25b was
1
confirmed by H NMR, where NH and OH protons appeared at δH: 10.28 and 5.58 ppm,
respectively. In addition, its 13C NMR spectrum showed signals at δc: 177.1 and 74.0 ppm
corresponding to amide carbonyl and tertiary alcohol carbons, respectively. On the other hand,
reaction of trichloroacetic acid (TCA) with 4 and 3 proceeded in different manner, where the
Passerini
product;
1-[(4-nitrophenyl)carbamoyl]cyclohexyl-2,2,2-trichloroacetate
26a
tautomerized to the iminol 26b. Then internal nucleophilic attack of nitrogen atom on the
trichloro acetyl group led to release of the -CCl3 and subsequent cyclization to afford the
unexpected spiro compound; 3-(4-nitrophenyl)-1-oxa-3-azaspiro[4.5]decane-2,4-dione 26c
[34] (Scheme 4). The structure of 26c was confirmed by 1H NMR, where the aromatic protons
appeared as two doublets at δH: 8.39 and 7.81 ppm. In addition, the signals corresponding to
13
the cyclohexylidene protons appeared as expected (δH: 2.1-1.30 ppm). C NMR showed the
characteristic spiro C signal at δC: 85.1 ppm and the oxazolinedione ring C=O groups [35] at
δC: 173.7 and 152.5 ppm.
Scheme 4. Synthesis of 26c and 25b via Passerini reaction of 3, 4 and trihaloacetic acids.
Furthermore, Passerini reaction of 3 with different carbonyl compounds was studied by
method A. The reaction of 3 with dimedone 27 and 5i didn’t afford the expected Passerini
product. Instead, this reaction led to acidic hydrolysis of 3 to the corresponding amine 1, which
condensed with dimedone to form imine 28 that tautomerized to 29 then to the more stable
8

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isomer α, β-unsaturated ketone 30 [36]. Trials utilizing other carboxylic acids also led to the
same result (Scheme 5).
Scheme 5. One-pot reaction of 3 with dimedone and 5i.
Moreover, Passerini reactions of 3 and ninhydrin 31 utilizing p-trifluoromethyl benzoic
acid 5a afforded the expected Passerini product 32 [37] (Scheme 6). The structure of 32 was
1
confirmed by H NMR that showed NH at δH: 11.42 ppm and aromatic protons at 8.42-7.90
ppm. 13C NMR spectrum of 32 showed the characteristic indanedione sp3 C at δC: 84.7 ppm as
well as the C=O groups at δC: 190.7 (2C=O), 163.2 (NH-C=O), and 161.5 ppm (O-C=O).
NO2
O
O
O
O
O
HO OH
i
N
31
H
O
NC
HOOC
O
O2N
CF3
CF3
3
32 (46%)
5a
Reaction conditions: i. TFE/EtOH, Reflux (3 hrs).
Scheme 6. Synthesis of 32 by Passerini reaction of 3 with Ninhydrin 31 and 5a
On the other hand, boiling a mixture of cinnamic acid 5k, 3 and ninhydrin in TFE/EtOH (1:1)
gave an inseparable mixture of products. Meanwhile, stirring the reaction mixture at room
temperature for 16 h led to the unexpected ethyl azaketal 34 in good yield. Replacing EtOH
with MeOH afforded the corresponding methyl azaketal 35. Explanation of this reaction may
be illustrated by hydrolysis of 3 to the corresponding amine 1, which condensed with 31 to
9

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give the imine 33. The planarity of 33 was proposed to support the addition of EtOH or MeOH
to the imine π bond to give the novel azaketals 34 and 35, respectively in good yields (Scheme
7). This proposed mechanism was confirmed by reacting 1 directly with 31 and 5k in ethanol
under the same conditions to afford the same product 34, which was confirmed by 1H NMR
showing D2O exchangeable NH signal at δH: 8.01 ppm, in addition to the characteristic ethyl
protons as quartet (δH: 3.74 ppm) and triplet (δH: 1.09 ppm). 13C NMR spectrum of 34
showed 2 C=O and NH-C-CO at δC: 193.1 ppm and 82.7 ppm, respectively. Compound 35 was
1
confirmed by H NMR showing D2O exchangeable NH signal at δH: 8.03 ppm, in addition to
the methyl protons at δH: 3.42 ppm.
Scheme 7. One-pot reaction of 3 with ninhydrin 31 and 5k.
2.2. Biological evaluation
2.2.1. Cytotoxicity screening
All the newly synthesized compounds were evaluated for their cytotoxic effects on
three human cancer cell lines (MCF-7, NFS-60, HepG-2) and normal human lung fibroblasts
(Wi-38) compared to the standard anticancer drug doxorubicin utilizing microculture MTT
method [25,38,39] (Table 1). Most of the screened compounds showed promising anticancer
activities against the tested cell lines, especially MCF-7 (breast cancer). The most potent
anticancer activities against MCF-7 cell line were exhibited by compounds 7, 13, 21, 22 and
26c with single-digit nanomolar IC50 values. Hence, these compounds were 2-4 folds more
active than doxorubicin. Compounds 8, 14, 20 and 23 were also more potent than doxorubicin
against MCF-7 with IC50 values in the range of 0.0141-0.0169 µM. IC50 values of compounds
6 and 35 were comparable to that of doxorubicin, while other screened compounds were less
active against MCF-7. Regarding NFS-60 (myeloid leukemia) cell line, compounds 13, 20, 21,
10

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22 and 26c were more potent than doxorubicin with single-digit nanomolar IC50 values. In
addition, two other compounds (8 and 23) showed slightly higher activities (IC50 = 0.0103 and
0.0113 µM, respectively) in comparison to the reference. Compounds 6, 14, and 34 showed
moderate anticancer activities ranging from 0.0188 to 0.0278 µM, while the remainders were
less active. The antitumor activities of the tested compounds against HepG-2 (liver cancer)
revealed two promising compounds (21 and 26c) with single-digit nanomolar IC50 values, thus
more potent than doxorubicin. In addition, compounds 13 and 22 exhibited comparable
anticancer activities to the reference. Other compounds were less active.
Interestingly, results showed that the tested compounds exerted remarkable high safety
profiles on normal human lung fibroblasts with IC50 values ranging from 0.032 to 1.964 µM,
therefore less toxic than doxorubicin (IC50= 0.0266 µM). Compounds 25b and 32 recorded the
highest IC50 values (1.221 and 1.964 µM, respectively). Moreover, EC100 (The concentration of
the test compound that allows 100% cell viability) values [39] of all the test compounds were
higher than doxorubicin, except for compounds 7, 11, 14 and 26c.
While the potency of the screened compounds is an important consideration, assessing
their selectivity towards cancer cells is key to the real evaluation of both effectiveness and
safety [40]. Such a measure of the drug candidate selectivity towards cancer cells rather than
normal cells is known as selectivity index (SI). It is the ratio between the compound IC50 on
normal cells and its IC50 on cancer cells. Generally, a drug with SI ≥3 is considered highly
selective [41]. Accordingly, the tested compounds were evaluated based on their selectivity
index (SI) values. Herein, all the tested compounds were more selective than doxorubicin
against MCF-7 cell lines, except compounds 9, 10, 12, 15, 16, 17, 19 and 30. Furthermore,
compounds 6, 8, 13, 21, 22, 23, 26c and 32 showed higher selectivity index values than the
reference against NFS-60 and HepG-2 cell lines. Interestingly, compounds 8, 13 and 21
showed the highest selectivity profiles in comparison with other investigated compounds and
doxorubicin against all the screened cancer cell lines. Compounds 6, 22, 26c and 32 also
showed promising SI values against the three cancer cell lines. Compound 20 showed high SI
(6.968) against NFS-60 cell line as well as moderate SI (3.163) against MCF-7 and lacked
selectivity against HepG-2 cells. Moreover, compound 7 was selective against MCF-7 without
considerable selectivity against NFS-60 and HepG-2cells.
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Table 1: In vitro cytotoxic activities and selectivity index values (SI) of the tested
compounds
Wi-38
EC100 (µM)
MCF-7
IC50 (µM)
NFS-60
IC50 (µM)
HepG-2
IC50 (µM)
Code
IC50 (µM)*
0.185±0.01
0.032±0.002
0.355±0.001
SI
SI
SI
0.0398±0.04
0.0064±0.001
0.1128±0.007
0.0271±0.004
0.0098±0.0003
0.015±0.005
0.0412±0.004
0.516±0.03
6.826
3.265
23.666
0.970
0.682
2.206
0.639
87.471
2.353
0.869
0.711
0.272
1.307
0.957
3.163
0.026±0.004
0.0454±0.003
0.0103±0.001
0.0388±0.003
-
7.115
0.704
0.0291±0.001
0.0612±0.006
6.357
0.522
6
7
34.466 0.0218±0.003 16.284
8
0.04002±0.002 0.0178±0.001
1.031
0.0571±0.004
0.700
9
0.352±0.006
0.523±0.062
0.396±0.01
0.018±0.006
0.0095±0.001
0.0247±0.016
0.149±0.002
0.0067±0.001
0.122±0.011
-
2.216
-
-
-
1.841
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25b
26c
30
32
34
35
DOX
0.237±0.004
0.619±0.005
0.0087±0.003
0.0147±0.002
0.444±0.045
0.0576±0.003
0.903±0.01
0.236±0.023
-
0.284±0.014
-
0.761±0.048
0.0346±0.003
0.386±0.005
0.0077±0.002
0.0188±0.002
0.488±0.004
0.0505±0.0003
0.766±0.081
0.0338±0.0001
0.517±0.01
0.0064±0.0001
98.831 0.0141±0.006 53.971
1.840
0.790
0.044±0.005
0.517±0.017
0.786
0.746
0.041±0.0001 0.0197±0.0001
0.811 0.05291±0.001 0.774
0.246±0.011
0.0463±0.001
0.386±0.005
0.0446±0.006
0.0844±0.001
0.0439±0.001
0.0484±0.002
0.203±0.012
1.221±0.0001
0.035±0.001
0.319±0.004
1.964±0.158
0.047±0.002
0.0537±0.001
0.0266±0.005
0.148±0.014
0.0271±0.001
0.122±0.011
0.0169±0.002
0.0507±0.001
0.032±0.0001
0.0355±0.002
0.020±0.007
0.53±0.002
0.322
1.369
0.746
6.968
0.734±0.047
0.0341±0.002
0.519±0.008
0.335
1.357
0.743
0.0354±0.001
0.403±0.008
0.0141±0.003
0.0215±0.002 2.0744
0.00501±0.001 16.846 0.0078±0.0016
10.820 0.0077±0.001 10.961
0.0077±0.001
0.0169±0.003
-
5.701
2.863
-
0.0081±0.0008
0.0113±0.004
-
5.419
4.283
-
0.0142±0.004
0.0177±0.003
-
3.091
2.734
-
0.664±0.011
0.0065±0.001
0.597±0.013
0.393±0.015
0.0349±0.0004
0.0254±0.004
0.0234±0.002
1.838
0.716±0.016
1.705
0.648±0.023
1.884
0.006±0.0001
0.157±0.015
0.635±0.06
5.384 0.00898±0.0008
3.897 0.0094±0.0001 3.723
0.534
4.997
1.346
2.114
1.136
0.645±0.006
0.484±0.043
0.0278±0.0009
0.0328±0.003
0.013±0.002
0.494
4.057
1.690
1.637
2.046
0.667±0.003
0.428±0.004
0.0389±0.004
0.036±0.004
0.01±0.001
0.478
4.588
1.208
1.491
2.66
0.0285±0.002
0.043±0.001
0.0121±0.001
*Values are expressed as mean ± SEM
2.2.2. Morphological examination of the induced apoptosis
The three cancer cell lines (MCF-7, NFS-60 and HepG-2) were examined for
morphological changes when treated with the most active and safe compounds 13, 21 and 22 in
comparison with the untreated cancer cells and cells treated with the reference doxorubicin
(Fig. 2). As illustrated, all the treated cells obviously lost their normal shapes. Additionally,
their characteristic severe shrinkage indicated potent anticancer activities of the tested
compounds 13, 21 and 22 (particularly compound 21) in comparison to doxorubicin.
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Fig. 2. Morphological alterations of the most effective compounds-treated cancer cell
lines, untreated cancer cells and doxorubicin-treated cells.
2.2.3. Flow cytometric analysis of apoptosis
Compounds 13, 21 and 22 exhibiting the most promising anticancer activities were
tested for their apoptotic effects utilizing flow cytometric annexin V/propidium iodide analysis.
In this assay, the cancer cell lines (MCF-7, NFS-60 and HepG-2) were treated with IC50 of the
most active compounds for 72 h to study the mode of cell death. The apoptosis-dependent
anticancer effects were then determined by quantification of annexin-stained apoptotic cells.
Results (Table 2 and Fig. 3) showed high percentages of annexin-stained population cells in
cancer cell lines treated with compounds 13, 21 and 22 compared to the control untreated
cancer cells. Compounds 13 and 21 induced apoptosis-dependent death by above 41% in all the
treated cancer cell lines compared to less than 37% in case of doxorubicin-treated cells,
whereas 22 was comparable to doxorubicin. Interestingly, compound 21 exhibited the highest
significant potential for induction of apoptosis (44.185-53.129%), particularly in the late stage
of apoptosis (23.6-42.85%) in MCF-7, NFS-60 and HepG-2 cell lines. Compound 13 induced
apoptosis by approximately similar percentages (18.43-24.1%) in early and late stages.
Meanwhile, no significant difference was observed between the total apoptotic populations
(early apoptosis; 6.1-13.6% and late apoptosis; 20.1-27.8%) of 22- and doxorubicin-treated
cancer cells.
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Table 2: Percentages of the apoptotic cell populations in the most effective compounds-
treated cancer cells lines.
MCF-7
Late
apoptosis
%
NFS-60
Late
apoptosis
%
HepG-2
Late
apoptosis
%
Early
apoptosis
%
Total
apoptosis
%
Early
apoptosis
%
Total
apoptosis
%
Early
apoptosis
%
Total
apoptosis
%
Code
Negative
control
13
21
22
Dox
0.47±0.01
0.01±0.001
0.48±0.01d
0.02±0.01
0.015±0.01
18.965±1.41 41.795±0.34b
23.645±0.75 44.185±0.45a
20.09±1.88
23.215±1.6
0.495±0.01d
0.78±0.09
0.1±0.001
23.385±2.61 41.815±1.14b
42.85±0.97
27.77±1.19
25.49±1.58
0.169±0.02d
24.065±0.96 20.439±2.13 44.505±1.18b
22.345±1.08 30.785±1.89 53.129±0.81a
22.83±1.75
21.04±0.7
13.64±1.16
18.43±1.47
6.055±1.06
48.905±0.09a
33.485±0.48c
36.08±0.23c
11.885±1.26 22.09±1.52
33.98±0.26c
34.129±0.42c 6.0649±1.07
13.53±1.35 20.445±1.67 33.974±0.32c 11.379±1.38
34.595±0.22c
10.59±1.35
*All values are expressed as mean ± SEM. Different letters are significantly different in the same
column at P < 0.05.
Fig. 3. Flow charts of annexin-PI analysis of the most effective compounds-treated cancer
cells lines, untreated cancer cells and doxorubicin-treated cells.
2.2.4. Caspase 3/7 activation assay
The mechanism of apoptotic induction exhibited by the most active compounds 13, 21
and 22 was investigated by determining the percentages of caspase 3/7 activation in cancerous
cell lines when treated with IC50 of these compounds. Results (Table 3) revealed that
compounds 13 and 21 significantly induced caspase 3/7 activation (more than 49%) higher
than the reference; doxorubicin (42.94-48.09%) in all tested human cancer cells. Interestingly,
14

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compound 21 recorded the highest caspase 3/7 activation percentages (52.83-55.04%).
Accordingly, results of the caspase 3/7 activation assay were concordant with that of the flow
cytometric analysis of apoptosis. Meanwhile, no significant difference was recorded between
the percentages of caspase 3/7 activation in compound 22 and doxorubicin-treated cancer cells.
Table 3: Percentages of caspase 3/7 activation in the most effective compounds-treated
cancer cells lines
Percentage of caspase 3/7 activation
Code
MCF-7
50.94±0.69a
NFS-60
49.59±0.28a
HepG-2
50.68±0.31a
13
21
55.04±0.055a
43.04±0.29b
42.94±0.27b
52.835±0.295a
45.41±0.59b
45.39±0.52b
53.64±0.425a
47.64±0.77b
48.09±0.43b
22
Dox
*All values are expressed as mean ± SEM. Different letters are significantly different in the same
column at P < 0.05.
2.2.5. Quantification of the apoptosis-inducing factor1 (AIF1)
Based on the fact that cells can undergo caspase-independent apoptosis through AIF1-
mediated pathway [12,13], the compounds under investigation (13, 21 and 22) were evaluated
utilizing AIF1 quantification assay. Results (Table 4) declared that no significant difference in
the AIF1 levels was recorded between the untreated cancer cells (MCF-7, NFS-60 and HepG-2
cells) and the most effective anticancer compounds (13, 21 and 22)-treated cell lines.
Accordingly, these compounds mediated apoptosis mainly by caspase activation through AIF1-
independent pathway.
Table 4: AIF1 levels (ng/mL) in the most effective compounds-treated cancer cells lines
AIF1 (ng/mL)
MCF-7
NFS-60
HepG-2
1.18±0.06a
1.28±0.03a
1.305±0.01a
1.23±0.04a
2.28±0.06a
2.34±0.04a
2.36±0.015a
2.33±0.025a
2.37±0.035a
2.44±0.015a
2.47±0.01a
2.41±0.03a
Untreated cells
13
21
22
All values are expressed as mean±SEM. Different letters are significantly different in the same
column at P < 0.05.
2.3. Antimicrobial evaluation
Selected compounds were evaluated for their in vitro antibacterial activities against
Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212) as
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representatives of Gram-positive bacteria, as well as Escherichia coli (ATCC 25922) and
Pseudomonas aeruginosa (ATCC 27853) as representatives of Gram-negative bacteria.
Additionally, their antifungal activities were evaluated against Candida albicans (ATCC
90028). The antimicrobial evaluation protocol is rationalized to screen the tested compounds
for possible antimicrobial activities utilizing the agar well diffusion assay [42]
(supplementary data). The compounds showing antimicrobial activities were then evaluated
for their minimum inhibitory concentrations through the standard broth microdilution method
[43] (Table 5).
All the screened compounds showed detectable inhibition zones except 6, 7 and 16.
Accordingly, minimum inhibitory concentrations of the selected compounds were measured
against their respective sensitive microorganisms. Generally, all the tested compounds showed
weak antimicrobial activities relative to the standards. Compound 34 showed antibacterial
activity only against S. aureus with MIC = 1.569 µM/mL, while compounds 8, 9, 14, 18, 21,
22, 23, 26c and 35 were slightly active against E. coli with compound 8 showing the lowest
MIC (1.271 µM/mL) among the group. All compounds showed no detectable antifungal
activity except for compound 25 which exhibited weak activity against C. albicans with MIC =
1.040 µM/mL.
Table 5: Antimicrobial evaluation of the tested compounds by microdilution assay.
MIC (µM/mL)
Code
E. coli.
1.271
2.570
2.477
3.503
2.570
2.677
2.401
NA
S. aureus
NA*
NA
C. albicans
NA
8
NA
9
NA
NA
14
NA
NA
18
NA
NA
21
NA
NA
22
NA
NA
23
NA
1.040
NA
25
26c
NA
3.527
NA
1.569
NA
NA
34
3.279
0.003
-
NA
35
0.006
-
-
Ciprofloxacin
Fluconazole
*NA: No activity
0.001
2.4. In silico prediction of physicochemical properties, drug-likeness data and ligand
efficiency metrics
The drug discovery sector now utilizes the computational prediction of
physicochemical properties and drug-likeness data as a useful tool to identify drug candidates.
In the current study, we applied various computational methods to assess whether the most
active compounds possess the optimum drug-likeness parameters or not. Molinspiration [44]
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software was employed to predict the descriptors formulating Lipinski’s rule of five [45],
which states that oral bioavailability and cell permeability are possible if at least three rules of
the following five are obeyed: n-octanol-water partition coefficient (log P) ≤5, molecular
weight (M.Wt) ≤500 Da, number of hydrogen bond donors (HBD) ≤5 and number of hydrogen
bond acceptors (HBA) ≤10. Accordingly, Lipinski’s violation could be predicted.
Molinspiration also calculated the number of rotatable bonds (NROTB) and topological polar
surface area (TPSA) as useful descriptors of oral bioavailability of drug candidates [46]. As
estimated, TPSA values for most known drugs are below 140–150 A2 [47,48]. The percentage
of absorption could be also predicted based on TPSA applying the following equation:
%ABS=109–0.345TPSA. Accordingly, in silico physicochemical properties of all the
synthesized compounds were calculated (Table 6).
Table 6: In silico physicochemical properties of the tested compounds as predicted by
Molinspiration
Lipinski’s
Violation
Volumes
(A)3
Code LogPa M.Wtb HBAc HBDd
TPSAe %ABSf
NROTBg
5.74 436.39
5.29 382.42
5.52 402.83
4.90 398.42
4.91 382.42
5.66 447.29
5.54 494.29
4.80 413.39
4.94 413.39
4.67 440.45
5.26 394.43
4.85 368.39
2.84 292.29
3.12 306.32
3.48 320.35
3.73 398.42
4.72 382.42
4.43 426.43
5.44 610.62
2.42 264.28
3.01 290.27
3.11 260.29
4.31 498.37
2.64 326.31
2.27 312.28
7
7
7
8
7
7
7
10
10
9
7
7
7
7
7
8
7
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
2
2
0
1
1
1
1
1
1
1
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
3
0
0
0
0
0
0
101.23 74.07
101.23 74.07
101.23 74.07
110.46 70.89
101.23 74.07
101.23 74.07
101.23 74.07
147.05 58.26
147.05 58.26
127.53 65.00
101.23 74.07
101.23 74.07
101.23 74.07
101.23 74.07
101.23 74.07
121.45 67.09
101.23 74.07
127.53 65.00
202.45 39.15
359.11
344.37
341.35
353.36
344.37
345.70
351.80
351.14
351.14
389.14
355.23
327.81
256.40
272.96
289.76
352.66
344.61
372.34
533.77
236.45
245.94
238.57
384.48
277.17
260.36
7
6
6
7
6
6
6
7
7
9
7
6
5
5
6
7
7
8
13
3
2
3
7
5
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25b
26c
30
32
34
35
14
6
7
5
9
95.15
92.44
74.92
76.17
77.10
83.15
135.37 62.29
101.23 74.07
101.23 74.07
7
7
aLog P: logarithm of compound partition coefficient between n-octanol and water.
bM.Wt: molecular weight.
cHBA: number of hydrogen bond acceptors.
dHBD: number of hydrogen bond donors.
eTPSA: polar surface area.
f%ABS: percentage of absorption.
gNROTB: number of rotatable bonds.
17

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Most of the screened compounds were in full accordance to Lipinski’s rule of five. Compounds
6, 7, 8, 11, 12 and 16 showed slight violation regarding log P, while compound 24 didn’t obey
Lipinski’s rule regarding log P, M.Wt and HBA. TPSA values of all compounds were in the
acceptable range except for 24. Hence, most of the tested compounds displayed reasonable
%ABS in the range of 58.26–83.15% indicating promising predicted oral bioavailability.
Molsoft software [49] was employed to predict solubility and drug-likeness model scores of the
tested compounds (Table 7). About 80% of marketed drugs have an estimated solubility above
0.0001 mg/L. Interestingly; all tested compounds fulfill this solubility requirement, except 24
and 32. Compounds 18, 19, 20, 25b and 26c recorded remarkable predicted solubility profiles.
Finally, the drug-likeness model score; a collective descriptor of the predicted physicochemical
properties, pharmacokinetics and pharmacodynamics parameters, was predicted for each
compound [50]. Compounds with zero or negative values should not be considered as drug-
like. As illustrated (Table 7), compounds 10, 12, 15, 21 and 23 recorded positive drug-likeness
values indicating good predicted drug-likeness potential. Taking all together, it could be
concluded that the most active compounds exhibited reasonable physicochemical properties
and drug-likeness values, which might raise them to be drug-like candidates.
Table 7: In silico drug-likeness data of the tested compounds as predicted by Molsoft
Sa
(mg/L) model score
Drug-likeness
Code
0.04
0.19
0.06
0.32
0.24
0.28
0.08
0.08
0.31
1.76
0.09
0.53
71.66
25.54
14.19
1.31
1.33
0.61
0.00
25.83
25.33
3.22
0.00
1.42
2.36
-0.63
-0.43
-0.08
-0.35
0.05
-0.41
0.18
-0.48
-0.35
0.17
-0.68
-0.59
-0.63
-0.63
-0.32
0.25
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25b
26c
30
32
34
35
-0.53
0.60
-0.46
-0.47
-0.57
-1.13
-0.65
-0.87
-1.07
Sa aqueous solubility
18

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Recently, medicinal chemists utilize ligand efficiency (LE) as a useful parameter for
selecting and optimizing lead compounds. This metric represents the balance of potency and
molecular size, which is related to many pharmacokinetic and toxicological parameters [51–
53]. It measures the average binding energy contribution per non-hydrogen atom instead of
considering the binding affinity or potency of the whole molecule. Accordingly, this approach
allows comparing and prioritizing the screened ligands corrected for their sizes [54]
.
LE can be calculated from following equations [54]
.
LE = ∆ G / NHA = 1.37 (pIC50) / NHA
Where:
∆G = Gibb's free energy
NHA = non-hydrogen atom.
pIC50 = half-maximal inhibitory concentration (in term of molar concentration).
The lower acceptable limit of LE is 0.3 [55].
The concept of ligand efficiency has been extended to consider other important
physicochemical properties, such as lipophilicity. This allows the introduction of the new
metric Liphophilic Ligand Efficiency (LLE). LLE is defined as a measure of how efficiently a
ligand exploits its lipophilicity to bind to a given target [56]. Monitoring LLE can thus
highlight the price paid in lipophilicity on the expense of potency. This parameter can be
calculated as the difference between log P and the negative logarithm of a potency measure
(pIC50)
LLE = pIC50 – cLog P
LLE values ≥ 3 are considered acceptable for lead compounds, while values ≥ 5 are
recommended for drug-like candidates [55,57]
In the current study, LE and LLE values of all the synthesized compounds were
calculated based on their respective IC50 values against the screened cancer cell lines utilizing
the previously mentioned equations. Results (Table 8) indicated that all the tested compounds
had acceptable LE values (> 0.3) against MCF-7, NFS-60, and HepG-2 cell lines except 15, 24
and 32. Compound 26c showed the highest LE values against MCF-7 and HEpG-2 cells (0.534
and 0.523, respectively), while compound 20 showed the highest LE value against NFS-60
(0.547). Concerning LLE, compounds 26c and 35 showed the highest drug-like LLE values
(>5) against the three screened cancer cell lines. Moreover, compound 20 showed remarkable
LLE (5.713) against NFS-60 cell lines and lead like value against other cell lines. Compounds
13, 18, 19, 21, 22, 23, 25b, 30 and 34 exhibited LLE values above the recommended limit for
lead compounds (>3) against all the screened cancer cells. Other compounds were beyond the
acceptable limit (0.668-2.892).
19

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Table 8: Ligand Efficiency (LE) and Ligand Lipophilic Efficiency (LLE) of the tested
compounds.
MCF-7
LE
NFS-60
LE
HepG-2
LE LLE
Code NHAa LogPb
c
pIC50
LLE pIC50
LLE pIC50
31
28
28
29
28
28
28
30
30
32
29
27
21
22
23
29
28
31
44
19
21
19
36
24
23
5.74
5.29
5.52
4.90
4.91
5.66
5.54
4.80
4.94
4.67
5.26
4.85
2.84
3.12
3.48
3.73
4.72
4.43
5.44
2.42
3.01
7.567 0.334 1.827 7.585 0.335 1.845 7.536 0.333 1.79
8.008 0.391 2.718 7.342 0.359 2.052 7.213 0.352 1.923
7.823 0.382 2.303 7.987 0.390 2.467 7.661 0.374 2.141
7.385 0.348 2.485 7.411 0.350 2.511 7.243 0.342 2.343
6
7
8
9
6.287 0.307 1.377
6.625 0.324 0.965 6.627 0.324 0.967 6.546 0.320 0.886
6.208 0.303 0.668
-
-
-
-
-
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25b
26c
30
32
34
35
-
-
-
-
-
-
8.060 0.368 3.26 8.113 0.370 3.313 7.850 0.358 3.05
7.832 0.357 2.892 7.725 0.352 2.785 7.356 0.335 2.866
6.352 0.271 1.68 6.311 0.270 1.641 6.286 0.269 1.616
7.239 0.341 1.979 7.296 0.344 2.036 7.276 0.343 2.016
6.044 0.306 1.194 6.115 0.310 1.265 6.134 0.311 1.284
7.450 0.486 4.610 7.471 0.487 4.631 7.467 0.487 4.627
6.394 0.398 3.274 6.286 0.391 3.166 6.284 0.391 3.164
7.850 0.467 4.370 9.193 0.547 5.713 7.667 0.456 4.187
8.300 0.392 4.570 8.107 0.382 4.377 8.113 0.383 4.383
8.113 0.396 3.393 8.091 0.395 3.371 7.847 0.383 3.127
7.772 0.313 3.342 7.946 0.351 3.516 7.752 0.342 3.322
-
-
-
-
-
-
-
-
-
6.177 0.445 3.757 6.145 0.443 3.725 6.188 0.446 3.768
8.187 0.534 5.177 8.046 0.524 5.036 8.026 0.523 5.016
6.224 0.448 3.114 6.190 0.446 3.080 6.175 0.445 3.065
6.405 0.243 2.095 6.315 0.240 2.005 6.368 0.242 2.058
7.457 0.425 4.817 7.555 0.431 4.915 7.410 0.422 4.77
7.595 0.452 5.325 7.484 0.445 5.214 7.443 0.443 5.173
3.11
4.31
2.64
2.27
aLogP: logarithm of compound partition coefficient between n-octanol and water.
bNHA = non-hydrogen atom.
cpIC50= -log (IC50)
3. Structure activity relationship
The general activity pattern suggests that the designed amide-based scaffold conserved
the intrinsic anticancer effect of the reported lead apoptotic inducers (Fig. 1). However,
activity and selectivity were found to be a function of the substitution nature and size.
Seemingly, most of the target amide esters showed relatively better anticancer activities
than the unexpected azaketals 34 and 35, hydroxy derivative 25b and α, β-unsaturated ketone
30. However, the unexpected spiro compound 26c was among the most active compounds
against the three screened cell lines with single-digit nanomolar IC50 values.
Within the evaluated esters, the mandelate ester 21 conferred the highest activities
against MCF-7and HepG-2 cells. In addition, it showed promising activity against NFS-60 cell
20

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lines. The absence of the α-hydroxy group led to slightly less active derivative 22. It is worth
mentioning that both 21 and 22 were more potent than the reference against MCF-7and NFS-
60 cells. Moreover, α-vinylic moiety of the cinnamate ester 16 dramatically decreased the
anticancer activities against the three cell lines. Obviously, benzoate esters showed variable
anticancer activities according to the aromatic ring substitution pattern. In this series, the p-
nitro derivative 13 was among the three most active compounds of the current study. Shifting
the nitro group to ortho- position (14) slightly decreased the antitumor activities against the
screened cell lines. On the other hand, p-methyl group (7) conferred high potency against
MCF-7 cells, moderate activity against NFS-60 and weak activity against HepG-2 cells.
Oxygen insertion (p-methoxy derivative 9) critically decreased the anticancer activity against
MCF-7 cells, while allowing a slight increase in activity regarding NFS-60 and HepG-2 cells.
Shifting the methyl group to ortho- position (10) also decreased the anticancer activity against
MCF-7 cells, but abolished activity against both NFS-60 and HepG-2 cell lines. Interestingly,
replacing p-methyl group (7) with p-trifluoromethyl (6) endowed remarkable potency against
the three cancer cell lines, especially NFS-60 and HepG-2 cell lines. Concerning halogenated
derivatives, the p-chloro derivative 8 displayed promising activities against MCF-7 and NFS-
60 cells (higher than the reference) and moderate effect against HepG-2, while the p-bromo
derivative 11 was 10-20 folds less active against the three cell lines. Furthermore, the o-iodo
derivative 12 was the least active among the group with no considerable activity against NFS-
60 and HepG-2 cell lines. Results also showed that derivatives with o-esters (15 and 23) were
active against the screened cell lines. However, the methyl ester (23) conferred much more
potency to the molecule than the ethyl ester (15) did. In comparison to the previously
mentioned compounds, the unsubstituted benzoate derivative 17 was less active towards the
screened cell lines. Furthermore, replacing the aromatic ring with aliphatic moieties also
controlled both activity and selectivity. Among this series, the propionate ester 20 showed
higher potency than the standard drug against both MCF-7 and NFS-60, as well as acceptable
activity against HepG-2 cells. The formate ester 18 was slightly less active than 20 against the
three cell lines with no detected selectivity, while the acetate ester 19 was the least active.
Obviously, the bis α-acyloxy carboxamide derivative 24 was inactive against the screened cell
lines. Also, the indenyl benzoate derivative 32 showed much lower activity the corresponding
cyclohexyl derivative 6.
4. Conclusion
This study portrays the design and synthesis of new multifunctional Passerini
compounds. In vitro cytotoxicity screening against three cancerous and one normal human cell
lines revealed that most of these compounds showed promising anticancer activities against all
the screened cancer cell lines with acceptable safety and selectivity profiles. Among tested
compounds, 7, 8, 13, 14, 20, 21, 22, 23 and 26c were more potent than doxorubicin against
MCF7 cell line, while IC50 values of compounds 6 and 35 were comparable to that of the
reference. Regarding NFS-60 cell line, compounds 8, 13, 20, 21, 22, 23 and 26c showed higher
IC50 values in comparison to doxorubicin. Furthermore, compounds 13, 21, 22, 23 and 26c
exhibited higher or comparable anticancer activity to the reference against HepG-2 cancer cell
line. The tested compounds were also evaluated for their safety based on their selectivity index
21

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(SI) values. Compounds 6, 8, 13, 21, 22, 26c and 32 were the safest ones, showing the highest
selectivity profiles in comparison with other investigated compounds and doxorubicin against
NFS-60, MCF-7 and HepG-2 cell lines. Compounds 13, 21 and 22 were selected for further
mechanistic studies. The three compounds significantly induced apoptosis by caspase
activation in all the screened cancer cell lines utilizing flow cytometric analysis and caspase
3/7 activation assay. Both 13 and 21 were more potent caspase 3/7 activators than
doxorubicin, while 22 showed comparable results. Furthermore, evaluation utilizing
quantification of AIF1 assay excluded possible AIF1-mediated apoptotic induction and
clarified that 13, 21 and 22 were caspase-dependent apoptotic inducers. Physicochemical
parameters, ligand efficiency metrics and drug-likeness data of the all the evaluated
compounds were computationally predicted. In silico results showed that compounds 13, 21
and 22 may be considered as drug-like candidates. Finally, selected compounds were
preliminarily screened for possible antimicrobial activities searching for possible dual
anticancer/ antimicrobial agents as an advantageous approach for cancer therapy. Although,
results revealed weak antimicrobial activities, compounds 21 and 22 were slightly active
against E. coli. Therefore, it could be concluded that Passerini based amides may hold promise
for a new scaffold of caspase activators that deserve further studies. Compounds 13, 21 and 22
may be considered as selective anticancer leads inducing apoptosis via caspase activation
mechanism with promising predicted drug likeness data.
5. Experimental
5.1. Chemistry
5.1.1. General experimental information
All reactions were carried out in dried glassware. Triethylamine and Trifluoroethanol
were purchased and used without further purification. NMR spectra were measured using a
JEOLJNM ECA 500 or 400 MHz. The deuterated solvent was used as an internal deuterium
lock. 13C NMR spectra were recorded using the UDEFT pulse sequence and broad band proton
decoupling at either 100 or 125 MHz. All chemical shifts (δ) are stated in units of parts per
million (ppm) and presented using TMS as the standard reference point. Melting points were
recorded using Thermo Scientific, Model NO: 1002D, 220-240v; 200watts; 50/60Hz and are
uncorrected. Mass spectra were carried out on direct probe controller inlet part to single
quadropole mass analyzer in (Thermo Scientific EIMS), Model: ISQ LT, using thermo x-
calibur software. Values are reported as a ratio of mass to charge (m/z) in Daltons. IR [ν/cm-1]
data were recorded using Perkin Elmer; FT-IR Spectrum BX and Bruker tensor 37 FT-IR.
Visualization of the TLC during monitoring of the reaction was done by UV VILBER
LOURMAT 4w-365nm or 254nm tube.
Synthesis of p-nitrophenyl formamide (2)
Iodine (0.46 g, 0.003 mol) was added to a solution of p-nitroaniline 1 (10 g, 0.07 mol) in
formic acid (50 ml). The mixture was stirred under Reflux for 8 hours. After completion of the
reaction, the reaction was kept overnight at room temperature. The precipitate of the desired
22

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product was collected by filtration, washed by a solution of Na2S2O3, and water to give 2 [27],
a pale yellow crystals, (10 gm, 86%) Mp= 196-199 °C, [lit.[58] Mp =197.5-198 °C]
Synthesis of p-nitrophenyl isocyanide (3)
In a three neck round-bottomed flask compound 2 (10 g, 0.06 mol) was added to solution of
Et3N (41.9 mL, 0.3 mol) in a mixture of CHCl3/DCM (1:2, 50 mL). The mixture was stirred in
an ice bath for 10 minutes. POCl3 (6.1 ml, 0.066 mol) was added to the stirred mixture [28] till
the color of mixture turned to the dark brown. Then the mixture was stirred under Reflux for
15 minutes. The reaction was quenched by adding cold water (50 mL), then, the mixture was
washed with CHCl3 (3 × 50 mL). The organic layers were combined, dried over anhydrous
sodium sulfate, filtered and evaporated under reduced pressure to afford 3 (7.2 gm, 79 %) as
orange solid; Mp=116-118 °C; [lit.[59] MP =119-120 °C]; IR: νmax/cm-1 2127 (-NC), 1572
(NO2), 1346 (NO2); 1H NMR (500 MHz, DMSO-d6) δH: 7.92 (d, J = 9 Hz, 2H), 7.55 (d, J = 9
Hz, 2H).
5.1.2. General method A for Passerini reactions
p-Nitrophenyl isocyanide 3 (50 mg, 0.337 mmol, 1.1 eq) was added to a solution of
cyclohexanone (0.337 mmol, 1.1 eq), and the appropriate carboxylic acid (0.307 mmol, 1.0 eq)
in a mixture of 2,2,2-trifluoroethanol (TFE) and ethanol (1:1, 2 mL). The mixture was stirred
under Reflux. The reaction was monitored by TLC. After the reaction completion, the mixture
was cooled to precipitate the desired product. The product was filtered, washed with a
saturated solution of NaHCO3, dried and collected without further purification.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl-4-(trifluoromethyl)benzoate (6)
Yield 48%; off white powder; Mp= 232-237°C; Rf 0.76 (EtOAc - petroleium ether, 1:2); IR:
1
νmax/cm-1 3297 (NH), 1729 (CO), 1681 (OCN); H NMR (500 MHz, DMSO-d6) δH: 10.22 (s,
1H, NH), 8.21 (d, J = 8.4 Hz, 2H, Ar-H), 8.16 (d, J = 9.1 Hz, 2H, Ar-H), 7.92 (d, J = 7.6 Hz,
2H, Ar-H), 7.85 (d, J = 9.1 Hz, 2H, Ar-H), 2.29 (d, J = 13.7 Hz, 2H, Cyclohex-H), 1.90 (dist.t,
J = 12.2, 11.4 Hz, 2H, Cyclohex-H), 1.66 (bs, 3H, Cyclohex-H), 1.60 - 1.52 (m, 2H,
Cyclohex-H), 1.35 - 1.28 (m, 1H, Cyclohex-H); 13C NMR (125 MHz, DMSO-d6) δC: 171.8
(NH-C=O), 163.9 (O-C=O), 145.6, 142.9, 134.0, 131.0, 130.9, 126.3, 125.2, (Ar-C), 125.1
(CF3), 120.3 (Ar-C), 82.9 (O=C-C-O), 31.8, 25.0, 21.6 (Cyclohex-C); EI-MS, m/z [M]+ calcd
for [C21H19F3N2O5]+: 436.12, found 436.52; Anal. calcd. for C21H19F3N2O5: C, 57.80; H, 4.39;
N, 6.42; found C, 57.72; H, 4.28; N, 6.55.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl-4-methylbenzoate (7)
Yield 68%; off white powder; Mp= 201-203°C; Rf 0.72 (EtOAc - petroleium ether, 1:2); IR:
νmax/cm-1 3285 (NH), 1704 (br, CO, OCN); 1H NMR (500 MHz, DMSO-d6) δH: 10.21 (s, 1H,
NH), 8.18 (d, J = 9 Hz, 2H, Ar-H), 7.92 (d, J = 7.5 Hz, 2H, Ar-H), 7.88 (d, J = 10 Hz, 2H, Ar-
H), 7.36 (d, J = 8 Hz, 2H, Ar-H), 2.38 (s, 3H, CH3), 2.29 (d, J = 14 Hz, 2H, Cyclohex-H),
1.87 (td, J = 13, 2.5 Hz, 2H, Cyclohex-H), 1.67 (d, J = 10.5 Hz, 3H, Cyclohex-H), 1.60 – 1.53
(m, 2H, Cyclohex-H), 1.35 – 1.29 (m, 1H, Cyclohex-H); 13C NMR (125 MHz, DMSO-d6) δC:
171.7(NH-C=O), 164.4 (O-C=O), 145.2, 144.0, 142.2, 129.6, 129.3, 127.0, 124.6, 119.7 (Ar-
23

ACCEPTED MANUSCRIPT
C), 81.3 (O-C-C=O), 31.3, 24.5, 21.2, 21.0 (CH3, Cyclohex-C); EI-MS, m/z [M]+ calcd for
[C21H22N2O5]+: 382.15, found 382.21; Anal. calcd. for C21H22N2O5: C, 65.96; H, 5.80; N, 7.33;
found C, 65.81; H, 5.68; N, 7.32.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl-4-chlorobenzoate (8)
Yield 50%; off white powder; Mp= 239-241°C; Rf 0.77 (EtOAc - petroleium ether, 1:2); IR:
1
νmax/cm-1 3284 (NH), 1714 (CO), 1692 (OCN); H NMR (500 MHz, DMSO-d6) δH: 10.21 (s,
1H, NH), 8.16 (d, J = 9.2 Hz, 2H, Ar-H), 8.01 (d, J = 8.4 Hz, 2H, Ar-H), 7.86 (d, J = 9.1 Hz,
2H, Ar-H), 7.61 (d, J = 8.4 Hz, 2H, Ar-H), 2.27 (d, J = 13 Hz, 2H, Cyclohex-H), 1.87 (dist.t, J
= 13, 11.4 Hz, 2H, Cyclohex-H), 1.64 (s, 3H, Cyclohex-H), 1.58 – 1.51 (m, 2H, Cyclohex-H),
1.34 – 1.27 (m, 1H, Cyclohex-H); 13C NMR (125 MHz, DMSO-d6) δC: 172.0 (NH-C=O),
164.2 (O-C=O), 145.6, 142.8, 139.1 (C-Cl), 132.0, 131.8, 129.0, 125.3, 120.2 (Ar-C), 82.5
(O=C-C-O), 31.9, 25.1, 21.6 (Cyclohex-C); EI-MS, m/z [M]+ calcd for [C20H19ClN2O5]+:
402.09, found 402.87; Anal. calcd. for C20H19ClN2O5: C, 59.63; H, 4.75; N, 6.95; found C,
59.71; H, 4.66; N, 6.86.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl-4-methoxybenzoate (9)
Yield 43%; pale yellow powder; Mp= 186-189°C; Rf 0.66 (EtOAc - petroleium ether, 1:2); IR:
νmax/cm-1 3328 (NH), 1703 (br, CO, OCN); 1H NMR (500 MHz, DMSO-d6) δH: 10.20 (s, 1H,
NH), 8.17 (d, J = 10 Hz, 2H, Ar-H), 7.98 (d, J = 9 Hz, 2H, Ar-H), 7.88 (d, J = 10 Hz, 2H, Ar-
H), 7.07 (d, J = 9 Hz, 2H, Ar-H), 3.83 (s, 3H, OCH3), 2.28 (d, J = 15 Hz, 2H, Cyclohex-H),
1.85 (td, J = 13.5, 3 Hz, 2H, Cyclohex-H), 1.67 (bd, J = 10.5 Hz, 3H, Cyclohex-H), 1.60 –
13
1.52 (m, 2H, Cyclohex-H), 1.35 – 1.28 (m, 1H, Cyclohex-H); C NMR (125 MHz, DMSO-
d6) δC: 171.9 (NH-C=O), 164.1 (O-C=O), 163.4 (C-OCH3), 145.3, 142.2, 131.7, 124.6, 121.9,
119.6, 114.0 (Ar-C), 81.1 (O-C-C=O), 55.5 (OCH3), 31.3, 24.5, 21.1 (Cyclohex-C); EI-MS,
m/z [M]+ calcd for [C21H22N2O6]+: 398.14, found 398.59; Anal. calcd. for C21H22N2O6: C,
63.31; H, 5.57; N, 7.03; found C, 63.40; H, 5.47; N, 7.22.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl-2-methylbenzoate (10)
Yield 67%; off white powder; Mp= 162-165°C; Rf 0.74 (EtOAc - petroleium ether, 1:2); IR:
νmax/cm-1 3375 (NH), 1713 (br, CO, OCN); 1H NMR (500 MHz, DMSO-d6) δH: 10.28 (bs, 1H,
NH), 8.18 (d, J = 10 Hz, 2H, Ar-H), 7.95 (d, J = 8 Hz, 1H, Ar-H), 7.90 (d, J = 9 Hz, 2H, Ar-
H), 7.50 (t, J = 7.5 Hz, 1H, Ar-H), 7.38 – 7.32 (m, 2H, Ar-H), 2.41 (s, 3H, CH3), 2.28 (d, J =
14 Hz, 2H, Cyclohex-H), 1.89 (td, J = 14.2, 3.5 Hz, 2H, Cyclohex-H), 1.69 (d, J = 10.5 Hz,
13
3H, Cyclohex-H), 1.64 – 1.56 (m, 2H, Cyclohex-H), 1.36 – 1.29 (m, 1H, Cyclohex-H); C
NMR (125 MHz, DMSO-d6) δC: 171.9 (NH-C=O), 165.7 (O-C=O), 142.1, 139.2, 132.3,
131.5, 130.1, 129.8, 126.0, 124.7, 119.7 (Ar-C), 81.8 (O-C-C=O), 31.3, 24.5, 21.1 (Cyclohex-
C, CH3), 20.8; EI-MS, m/z [M]+ calcd for [C21H22N2O5]+: 382.15, found 382.47; Anal. calcd.
for C21H22N2O5: C, 65.96; H, 5.80; N, 7.33; found C, 65.87; H, 5.78; N, 7.32.
24

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1-[(4-Nitrophenyl)carbamoyl]cyclohexyl-4-bromobenzoate (11)
Yield 62%; off white powder; Mp= 243-245°C; Rf 0.61 (EtOAc - petroleium ether, 1:2); IR:
1
νmax/cm-1 3282 (NH), 1713 (CO), 1686 (OCN); H NMR (500 MHz, DMSO-d6) δH: 10.24 (s,
1H, NH), 8.18 (d, J = 9 Hz, 2H, Ar-H), 7.95 (d, J = 8 Hz, 2H, Ar-H), 7.87 (d, J = 9 Hz, 2H,
Ar-H), 7.78 (d, J = 8.5 Hz, 2H, Ar-H), 2.29 (d, J = 14 Hz, 2H, Cyclohex-H), 1.89 (td, J =
13.5, 3 Hz, 2H, Cyclohex-H), 1.67 (d, J = 10 Hz, 3H, Cyclohex-H), 1.59 – 1.54 (m, 2H,
13
Cyclohex-H), 1.36 – 1.28 (m, 1H, Cyclohex-H). C NMR (125 MHz, DMSO-d6) δC: 171.4
(NH-C=O), 163.8 (O-C=O), 145.1, 142.3, 131.9, 131.5, 128.9, 127.7, 124.6, 119.8 (Ar-C),
82.0 (O-C-C=O), 31.2, 24.4, 21.0 (Cyclohex-C); EI-MS, m/z [M]+ calcd for [C20H19BrN2O5]+:
446.04, found 446.12; Anal. calcd. for C20H19BrN2O5: C, 53.71; H, 4.28; N, 6.26; found C,
53.65; H, 4.13; N, 6.19.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl-2-iodobenzoate (12)
Yield 42%; off white powder; Mp= 136-139°C; Rf 0.70 (EtOAc - petroleium ether, 1:2); IR:
νmax/cm-1 3352 (NH), 1717 (br, CO, OCN); 1H NMR (400 MHz, DMSO-d6) δH: 10.58 (s, 1H,
NH), 8.49 (d, J = 9.6 Hz, 2H, Ar-H), 8.31 (d, J = 8 Hz, 1H, Ar-H), 8.23-8.19 (m, 3H, Ar-H),
7.86 (t, J = 8 Hz, 1H, Ar-H), 7.60 (td, J = 8.4, 1.2 Hz, 1H, Ar-H), 2.59 (d, J = 13.2 Hz, 2H,
Cyclohex-H), 2.20 (td, J = 12.8, 4 Hz, 2H, Cyclohex-H), 1.96 – 1.91 (bs, 5H, Cyclohex-H),
13
1.67-1.63 (m, 1H, Cyclohex-H); C NMR (100 MHz, DMSO-d6) δC: 171.3 (O-C=O), 165.2
(NH-C=O), 145.2, 142.3, 140.8, 135.2, 133.2, 130.7, 128.3, 124.6, 119.8 (Ar-C), 94.7 (C-I),
82.7 (O=C-C-O), 31.2 , 24.4 , 21.0 (Cyclohex-C); EI-MS, m/z [M]+ calcd for [C20H19IN2O5]+:
494.03, found 494.35; Anal. calcd. for C20H19IN2O5: C, 48.60; H, 3.87; N, 5.67; found C,
48.57; H, 3.65; N, 5.55.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl-4-nitrobenzoate (13)
Yield 71%; pale yellow powder; Mp= 234-236°C; Rf 0.72 (EtOAc - petroleium ether, 1:2); IR:
νmax/cm-1 3379 (NH), 1722 (br, CO, OCN); 1H NMR (500 MHz, DMSO-d6) δH: 10.37 (bs, 1H,
NH), 8.38 (d, J = 9 Hz, 2H, Ar-H), 8.27 (d, J = 9 Hz, 2H, Ar-H), 8.18 (d, J = 9 Hz, 2H, Ar-
H), 7.86 (d, J = 9.5 Hz, 2H, Ar-H), 2.31 (d, J = 13.5 Hz, 2H, Cyclohex-H), 1.93 (td, J = 13.5,
2.5 Hz, 2H, Cyclohex-H), 1.68 (bs, 3H, Cyclohex-H), 1.62 – 1.54 (m, 2H, Cyclohex-H), 1.38 –
13
1.30 (m, 1H, Cyclohex-H); C NMR (125 MHz, DMSO-d6) δC: 171.1 (NH-C=O), 163.0 (O-
C=O), 150.4, 145.0, 142.4, 135.1, 131.0, 124.6, 123.9, 119.8 (Ar-C), 82.7 (O=C-C-O), 31.2,
24.4, 21.0 (Cyclohex-C); EI-MS, m/z [M]+ calcd for [C20H19N3O7]+: 413.12, found 413.16;
Anal. calcd. for C20H19N3O7: C, 58.11; H, 4.63; N, 10.16; found C, 58.06; H, 4.69; N, 9.87.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl-2-nitrobenzoate (14)
Yield 57%; white powder; Mp= 200-204°C; Rf 0.65 (EtOAc - petroleium ether, 1:2); IR:
1
νmax/cm-1 3379 (NH), 1731 (CO), 1696 (OCN); H NMR (500 MHz, DMSO-d6) δH: 10.25 (s,
1H, NH), 8.19 (d, J = 8.4 Hz, 2H, Ar-H), 8.03 (d, J = 7.6 Hz, 1H, Ar-H), 7.97 (d, J = 7.6 Hz,
1H, Ar-H), 7.90 – 7.81 (m, 4H, Ar-H), 2.27 (d, J = 13.7 Hz, 2H, Cyclohex-H), 1.92 (t, J =
12.2 Hz, 2H, Cyclohex-H), 1.64 - 1.53 (m, 5H, Cyclohex-H), 1.33 - 1.31 (m, J = 9.1 Hz, 1H,
25

ACCEPTED MANUSCRIPT
13
Cyclohex-H); C NMR (125 MHz, DMSO-d6) δC: 171.2 (NH-C=O), 163.9 (O-C=O), 148.8,
145.6, 143.0, 133.9, 130.9, 126.4, 125.2, 124.7, 120.4, 120.2 (Ar-C), 84.5 (O=C-C-O), 31.7,
24.9, 21.4 (Cyclohex-C); EI-MS, m/z [M]+ calcd for [C20H19N3O7]+: 413.12, found 413.05;
Anal. calcd. for C20H19N3O7: C, 58.11; H, 4.63; N, 10.16; found C, 57.98; H, 4.55; N, 10.19.
Ethyl [1-[(4-nitrophenyl)carbamoyl]cyclohexyl] phthalate (15)
Yield 48%; Off white powder; Mp= 179-181°C; Rf 0.62 (EtOAc - petroleium ether, 1:2); IR:
1
νmax/cm-1 3358 (NH), 1740 (CO), 1698 (OCN); H NMR (400 MHz, DMSO-d6) δH: 10.14 (s,
1H, NH), 8.21 (d, J = 8.8 Hz, 2H, Ar-H), 7.98 – 7.90 (m, 3H, Ar-H), 7.71 (s, 3H, Ar-H), 4.14
(q, J = 7.2 Hz, 2H, CH2), 2.29 (d, J = 14 Hz, 2H, Cyclohex-H), 1.91 (td, J = 13, 2.8 Hz, 2H,
Cyclohex-H), 1.67-1.55 (m, 5H, Cyclohex-H), 1.34-1.31 (m, 1H, Cyclohex-H), 1.10 (t, J = 7.2
13
Hz, 2H, CH3); C NMR (100 MHz, DMSO-d6) δC: 171.1 (NH-C=O), 167.3 (CH2-O-C=O),
165.1 (O-C=O), 145.1, 142.3, 132.5, 132.1, 131.4, 130.7, 129.4, 128.7, 124.7, 119.6, (Ar-C),
82.7 (O-C-C=O), 61.5 (CH2), 31.2, 24.0, 20.9, 13.6 (Cyclohex-C, CH3); EI-MS, m/z [M]+
calcd for [C23H24N2O7]+: 440.15, found 440.86; Anal. calcd. for C23H24N2O7: C, 62.72; H,
5.49; N, 6.36; found C, 62.66; H, 5.36; N, 6.12.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl cinnamate (16)
Yield 54%; off white powder; Mp= 184-186°C; Rf 0.59 (EtOAc - petroleium ether, 1:2); IR:
1
νmax/cm-1 3285 (NH), 1700 (CO), 1632 (OCN); H NMR (500 MHz, DMSO-d6) δH: 10.23 (s,
1H, NH), 8.17 (d, J = 9 Hz, 2H, Ar-H), 7.89 (d, J = 8.5 Hz, 2H, Ar-H), 7.74 – 7.73 (m, 2H,
Ar-H), 7.68 (d, J = 15.5 Hz, 1H, OCO-CH=CH), 7.44 – 7.43 (m, 3H, Ar-H), 6.72 (d, J = 15.5
Hz, 1H, OCO-CH=CH), 2.22 (d, J = 13 Hz, 2H, Cyclohex-H), 1.84 (t, J = 12 Hz, 2H,
13
Cyclohex-H), 1.65 – 1.56 (m, 5H, Cyclohex-H), 1.32 – 1.30 (m, 1H, Cyclohex-H); C NMR
(125 MHz, DMSO-d6) δC: 171.9 (NH-C=O), 165.0 (O-C=O), 145.4 (Ar-C), 145.1 (OCO-
CH=CH), 142.2, 133.9, 130.6, 129.0, 128.4, 124.6, 119.6 (Ar-C), 118.1 (OCO-CH=CH), 81.1
(O-C-C=O), 31.3, 24.5, 20.9 (Cyclohex-C); EI-MS, m/z [M]+ calcd for [C22H22N2O5]+: 394.15,
found 394.93; Anal. calcd. for C22H22N2O5: C, 66.99; H, 5.62; N, 7.10; found C, 66.96; H,
5.54; N, 6.97.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl benzoate (17)
Yield 65%; off white powder; Mp= 186-189°C; Rf 0.65 (EtOAc - petroleium ether, 1:2); IR:
νmax/cm-1 3285 (NH), 1704 (br, CO, OCN); 1H NMR (500 MHz, DMSO-d6) δH: 10.23 (s, 1H,
NH), 8.18 (d, J = 10 Hz, 2H, Ar-H), 8.03 (d, J = 8 Hz, 2H, Ar-H), 7.88 (d, J = 9.5 Hz, 2H, Ar-
H), 7.69 (t, J = 7.5 Hz, 1H, Ar-H), 7.56 (t, J = 7.5 Hz, 2H, Ar-H), 2.30 (d, J = 14 Hz, 2H,
Cyclohex-H), 1.88 (td, J = 13.5, 3.5 Hz, 2H, Cyclohex-H), 1.68 (bd, J = 10.5 Hz, 3H,
13
Cyclohex-H), 1.62 – 1.54 (m, 2H, Cyclohex-H), 1.36 – 1.29 (m, 1H, Cyclohex-H); C NMR
(125 MHz, DMSO-d6) δC: 171.6 (NH-C=O), 164.5 (O-C=O), 145.2, 142.3, 133.6, 129.7,
129.5, 128.8, 124.6, 119.7 (Ar-C), 81.6 (O-C-C=O), 31.3, 24.5, 21.0 (Cyclohex-C); EI-MS,
m/z [M]+ calcd for [C20H20N2O5]+: 368.13, found 368.27; Anal. calcd. for C20H20N2O5: C,
65.21; H, 5.47; N, 7.60; found C, 64.99; H, 5.39; N, 7.55.
26

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5.1.3. General method B for Passerini reactions
p-Nitrophenyl isocyanide (50 mg, 0.337 mmol, 0.5 eq) was added to a mixture of
cyclohexanone (3.37 mmol, 5 eq), and the appropriate carboxylic acid (0.674 mmol, 1 eq). The
mixture was stirred at room temperature for 24 hours. The reaction was monitored by TLC.
After the reaction completion, the reaction was quenched by adding DCM (5 mL) and
neutralized with saturated solution of NaHCO3, then the mixture was washed with DCM (3 ×
10 mL). The organic layers were combined, dried over anhydrous sodium sulfate and
evaporated under reduced pressure [31]. The crude product was recrystallized from a mixture
of DCM/petroleum ether (1:1).
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl formate (18)
Yield 89%; pale yellow powder; Mp= 162-165°C; Rf 0.60 (EtOAc - petroleium ether, 1:2); IR:
νmax/cm-1 3383 (NH), 1705 (br, CO, OCN); 1H NMR (500 MHz, DMSO-d6) δH: 10.26 (s, 1H,
NH), 8.30 (s, 1H, O=CH), 8.19 (d, J = 9.5 Hz, 2H, Ar-H), 7.90 (d, J = 9.5 Hz, 2H, Ar-H),
2.15 (d, J = 16 Hz, 2H, Cyclohex-H), 1.84 (dist.t, J = 12.5, 11 Hz, 2H, Cyclohex-H), 1.62 (d, J
= 10 Hz, 3H, Cyclohex-H), 1.53 – 1.46 (m, 2H, Cyclohex-H), 1.33 – 1.25 (m, 1H, Cyclohex-
H); 13C NMR (125 MHz, DMSO-d6) δC: 171.3 (NH-C=O), 161.1 (O-C=O), 145.1, 142.4,
124.6, 119.7 (Ar-C) 81.5 (O-C-C=O), 31.3, 24.3, 20.6 (Cyclohex-C); EI-MS, m/z [M]+ calcd
for [C14H16N2O5]+: 292.10, found 292.15; Anal. calcd. for C14H16N2O5: C, 57.53; H, 5.52; N,
9.58; found C, 57.65; H, 5.45; N, 9.60.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl acetate (19)
Yield 77%; pale yellow powder; Mp= 143-145°C; Rf 0.58 (EtOAc - petroleium ether, 1:2); IR:
1
νmax/cm-1 3446 (NH), 1720 (CO), 1629 (OCN); H NMR (500 MHz, DMSO-d6) δH: 10.15 (s,
1H, NH), 8.18 (d, J = 9 Hz, 2H, Ar-H), 7.89 (d, J = 9 Hz, 2H, Ar-H), 2.14 (bs, 1H, Cyclohex-
H), 2.11 (s, 4H, Cyclohex-H, CH3), 1.76 (td, J = 13.5, 3 Hz, 2H, Cyclohex-H), 1.60 (d, J = 9.5
Hz, 3H, Cyclohex-H), 1.53 – 1.45 (m, 2H, Cyclohex-H), 1.31 – 1.22 (m, 1H, Cyclohex-H);
13C NMR (125 MHz, DMSO-d6) δC: 171.8 (NH-C=O), 169.6 (O-C=O), 145.2, 142.2, 124.6,
119.6 (Ar-C), 80.9 (O-C-C=O), 31.2, 24.5, 21.2, 20.8 (Cyclohex-C, CH3); EI-MS, m/z [M]+
calcd for [C15H18N2O5]+: 306.12, found 306.22; Anal. calcd. for C15H18N2O5: C, 58.82; H,
5.92; N, 9.15; found C, 58.72; H, 5.83; N, 8.88.
1-[(4-Nitrophenyl)carbamoyl]cyclohexyl propionate (20)
Yield 74%; Yellow powder; Mp= 147-150°C; Rf 0.66 (EtOAc - petroleium ether, 1:2); IR:
νmax/cm-1 3396 (NH), 1719 (br, CO, OCN); 1H NMR (500 MHz, DMSO-d6) δH: 10.13 (s, 1H,
NH), 8.18 (d, J = 9 Hz, 2H, Ar-H), 7.89 (d, J = 9.5 Hz, 2H, Ar-H), 2.43 (q, J = 7.5, 8 Hz, 2H,
OCOCH2), 2.13 (d, J = 13.5 Hz, 2H, Cyclohex-H), 1.77 (td, J = 13.5, 2.5 Hz, 2H, Cyclohex-
H), 1.60 (d, J = 10 Hz, 3H, Cyclohex-H), 1.52 – 1.44 (m, 2H, Cyclohex-H), 1.29 – 1.22 (m,
1H, Cyclohex-H), 1.03 (t, J = 7.5 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δC: 172.7
(NH-C=O), 171.9 (O-C=O), 145.3, 142.2, 124.6, 119.6 (Ar-C), 80.7 (O-C-C=O), 31.2, 27.2,
24.5, 20.8, 9.0 (Cyclohex-C, CH2, CH3); EI-MS, m/z [M]+ calcd for [C16H20N2O5]+: 320.13,
27