Novel adamantane-pyrazole and hydrazone hybridized: Design, synthesis, cytotoxic evaluation, SAR study and molecular docking simulation as carbonic anhydrase inhibitors

Article abstract of DOI:10.1016/j.molstruc.2020.128966

A series of pyrazole derivatives 4, 5, 6, 12, 13, 14 as well as hydrazone derivatives 7, 10, 11 were synthesized starting from adamantane-1-carbohydrazide as the bioactive core. All newly designed adamantane derivates were established by full characterized using different spectroscopic methods. The novel derivatives were investigated for their antitumor activity against three cell line MCF-7, HepG-2 and A549. They displayed good IC₅₀ values ranged between 1.55 to 42.17 μM in comparison to Doxorubicin (IC50 =3.58–8.19 μM). Surprisingly, adamantine derivatives revealed more sensitivity and selectivity to lung cancer cells (A549) with eight compounds (4, 5, 9a, 9b, 9c, 12, 13a and 14c) having IC₅₀ less than or equal ten micromoles. The most promising three adamantane derivatives 9a, 12 and 13a with IC₅₀ values less than 5 μM were selected to study enzymatic assay for isoenzyme hCAIX and hCAXII. Also, pyrazole core 13a and 12 showed higher K_I values than hydrazone derivatives 9a with submicromolar between (0.085–0.527 μM), in comparison to Acetazolamide (0.041–0.068 μM). Compound 13a is the most promising derivatives with anti-proliferative (A549) (IC₅₀=1.55 ± 0.08 μM) which showed CAIX/XII inhibitory activity (K_I = 0.085 and 0.14 μM), respectively. Finally, molecular docking simulation was performed to determine the binding modes and possible interaction of the adamantane derivatives within the active site of 3IAI and 1JD0 for CAIX / XII respectively with low binding affinity.

Full text of DOI:10.1016/j.molstruc.2020.128966

Journal Pre-proof
Novel adamantane-pyrazole and hydrazone hybridized: design,
synthesis, cytotoxic evaluation, SAR study and molecular docking
simulation as carbonic anhydrase inhibitors
Mohammed M.S. Wassel , Ahmed Ragab , Gameel A.M. Elhag Ali ,
Ahmed B.M. Mehany , Yousry A. Ammar
PII:
S0022-2860(20)31290-4
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.128966
MOLSTR 128966
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
26 June 2020
23 July 2020
23 July 2020
Please cite this article as: Mohammed M.S. Wassel , Ahmed Ragab , Gameel A.M. Elhag Ali ,
Ahmed B.M. Mehany , Yousry A. Ammar , Novel adamantane-pyrazole and hydrazone hy-
bridized: design, synthesis, cytotoxic evaluation, SAR study and molecular docking sim-
ulation as carbonic anhydrase inhibitors, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.128966
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Highlights


A new pyrazole and hydrazone derivatives were synthetized.
The structure of the designed compound was confirmed by spectroscopic
methods.


Anti-proliferative activity and SAR study were evaluated and discussed.
The carbonic anhydrase (IX & XII) were determined for the most promising
compounds.

Docking study was carried out as well as some physicochemical properties.
1

Novel adamantane-pyrazole and hydrazone hybridized: design, synthesis, cytotoxic
evaluation, SAR study and molecular docking simulation as carbonic anhydrase inhibitors
1
2,*
2
3
Mohammed M. S. Wassel , Ahmed Ragab , Gameel A. M. Elhag Ali , Ahmed B. M. Mehany , Yousry
2,*
A. Ammar
1
Department of Foot and Mouth Disease, Veterinary Serum and Vaccine Research Institute
VSVRI), Abbasia, Cairo, Egypt
Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo 11884,
(
2
Egypt
3
Zoology Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo 11884, Egypt
Abstract
A series of pyrazole derivatives 4, 5, 6, 12, 13, 14 as well as hydrazone derivatives 7, 10, 11 were
synthesized starting from adamantane-1-carbohydrazide as the bioactive core. All newly designed
adamantane derivates were established by full characterized using different spectroscopic
methods. The novel derivatives were investigated for their antitumor activity against three cell
line MCF-7, HepG-2 and A549. They displayed good IC values ranged between 1.55 to 42.17
5
0
µM in comparison to Doxorubicin (IC50 =3.58-8.19 µM). Surprisingly, adamantine derivatives
revealed more sensitivity and selectivity to lung cancer cells (A549) with eight compounds (4, 5,
9
a, 9b, 9c, 12, 13a and 14c) having IC less than or equal ten micromoles. The most promising
50
three adamantane derivatives 9a, 12 and 13a with IC values less than 5 µM were selected to
5
0
study enzymatic assay for isoenzyme hCAIX and hCAXII. Also, pyrazole core 13a and 12
showed higher K values than hydrazone derivatives 9a with submicromolar between (0.085-
I
0
.527 µM), in comparison to Acetazolamide (0.041-0.068 µM). Compound 13a is the most
promising derivatives with anti-proliferative (A549) (IC =1.55 ± 0.08 µM) which showed
5
0
CAIX/XII inhibitory activity (K = 0.085 and 0.14 µM), respectively. Finally, molecular docking
I
simulation was performed to determine the binding modes and possible interaction of the
adamantane derivatives within the active site of 3IAI and 1JD0 for CAIX / XII respectively with
low binding affinity.
*
Corresponding author: Ahmed Ragab (Ph.D.) Tel.: +202 01009341359; E-mail:
ahmed_ragab@azhar.edu.eg & ahmed_ragab7@ymail.com.
Corresponding authors; Yousry Ahmed Ammar ; E-mail: yossry@yahoo.com &
yossry@azhar.edu.eg.
*
2

Keywords: Adamantane; Anti-proliferative activity; Carbonic anhydrase inhibitors; Molecular
docking
1
. Introduction
One of the most significant diseases responsible for worldwide deaths, cancer is a general term
used to describe the uncontrolled proliferation of cells resulting from disruptions or dysfunctions
of the regulatory signaling pathway [1,2]. Cancer is a complex disease caused by genetic and/or
epigenetic changes in one cell or a group of cells [3]. At present, it is the major public concerned
hotspot across the globe [4]. Deaths owing to cancer are predicted to continue increasing by 3
million deaths in 2030 [5]. Lung, breast, colon, and melanoma cancers are the most recorded
types in developing and underdeveloped countries [6]. The emergence and growing resistance to
the currently obtainable chemotherapeutic agents demonstrates a critical need for producing
novel, more powerful, and selective anticancer drugs [7]. Lung cancer is one of the most
frequently diagnosed malignancies in addition to that leads to cancer-associated mortality [8–10].
Due to the histopathological results, lung cancer is categorized into two main subgroups: small
cell lung carcinoma (SCLC) and non-SCLC (NSCLC). NSCLCs include 85% of all lung cancer
cases, that are classified into three types as adenocarcinoma, squamous cell carcinoma, and large
cell carcinoma [11]. Carbonic anhydrases (CAs) enzymes which undergo a physiological
reaction that hydrates CO reversibly into a proton and HCO [12–15]. The human carbonic
2
3
anhydrase (hCA) consists of fifteen different isoforms; it differs in sequences, mainly for
expression tissue, localization, and catalytic activity [16,17]. There are different physiological
processes containing electrolyte secretion in a variety of tissues and organs, transport of CO2 /
bicarbonate metabolizing tissues and lungs, pH and CO homeostasis, respiration, bone
2
resorption, biosynthetic reactions and tumorigenicity, calcification, and many other
Pathophysiological processes [18]. Nowadays, the current approaches to the treatment of cancer,
including surgery, radiotherapy, and chemotherapy either alone or in combination, but due to
metastasis (spreading of the disease to other parts of the body), only 40 percent of patients can be
cured and are associated with a high mortality rate [19]. Chemotherapy is the main weapon
against neoplastic diseases, and most of the anticancer drugs clinics are of synthetic origin [20].
These chemopreventive drugs act by various mechanisms that may involve inhibition of
3

initiation, promotion, progression, and cancerous cells metastasis. However, during this process,
a normal cell can be exposed to toxic [21].
Figure 1: Drugs containing Adamantane nucleus or pyrazole moiety.
Many anticancer drugs have been approved, but most of them non-specific and have many side
effects during chemotherapy [22,23], so new medicines that are effective in drug-resistant cancers
need to be developed [24]. Moreover, there is an urgent need to design and synthesize new drugs
that have potency and highly selective to cancer cells with least or no toxicity to healthy cells.
Adamantane derivatives have been found to interfere with various enzymes and possess a variety
of therapeutic activities [25], such as anti-inflammatory [24,26,27], anti-viral as Tromantadine
[
28–31] and anti-anticancer activities [32,33]. Adamantyl moiety hybrid with several molecules to
enhance the biological availability of the designed compounds, and an example of that is
Adapalene that used as acne vulgaris therapy that containing adamantane in the main skeleton as
well as naphthalene derivatives that relatively high lipophilicity [34] (Fig. 1). Adamantane
nucleus present in many drugs as Vildagliptin (dipeptidyl peptidase-4 (DPP-4) inhibitor) [35],
Adatanserin (anti-depressant) [36], and Saxagliptin (hypoglycemic drug) [37], Arterolane
(antimalarial activity) [38] (Fig. 1). Furthermore, pyrazoles are an important class of heterocyclic
compounds that promising scaffolds in medicinal chemistry [39–42]. Crizotinib and Ruxolitinib
are examples of pyrazole-containing drugs used for treating myeloproliferative neoplasm and
non-small cell lung carcinoma (NSCLC), respectively [43] (Fig. 1). Also, hydrazones are an
essential class of compounds due to their flexibility and structural similarities with various natural
4

substances of biological importance [44,45]. Hydrazone derivatives have an imine (N=C) group,
that plays a significant role in the mechanism of transformation and racemization reaction in the
biological system [46–48]. Because of these facts and in continuation of our efforts in designing
new bioactive compounds [49–52]. We have designed and synthesized a novel pyrazole and
hydrazine derivatives bearing adamantane bioactive core as a promising heterocyclic scaffold and
screened for anticancer activity and tested the most promising to carbonic anhydrase and therefore
the molecular docking simulation and some drug-likeness parameter were evaluated to find
potential candidates.
2
.
Experimental
.1. Chemistry
2
Uncorrected melting points are recorded on digital Gallen Kamp MFB-595 instrument. The IR
-1
1
spectra (KBr) (cm ) were measured on a Shimadzu 440 spectrophotometer. H NMR spectra (δ,
ppm) were obtained in deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform
1
3
(
CDCl ) and C NMR at 100 MHz, spectra were obtained on a Bruker spectrometer (400 MHz)
3
spectrometer, using TMS as an internal standard; chemical shifts are reported as δ ppm units.
The data were presented as follows: chemical shift, multiplicity (s= singlet, d= doublet, t= triplet,
q= quartet, m= multiplet, br= broad, app= apparent), coupling constant(s) in Hertz (Hz), and
integration. Mass spectra were recorded on Thermo Scientific ISQLT mass spectrometer at the
Regional Center for Mycology and Biotechnology, Al-Azhar University. Elemental analyses
were carried out in Microanalytical Unit, Cairo University, Cairo, Egypt. Anticancer activity was
carried out in local strain identified in the Regional Center for genetic engineering, faculty of
Science (Boys), Al-Azhar University, and Carbonic Anhydrase IX and XII were carried out in
(VACSERA), Cairo, Egypt.
1
-(Adamantane-1-carbonyl)-5-amino-3-(methylthio)-1H-pyrazole-4-carbonitrile (4)
A mixture of adamantane-1-carbohydrazide (3) (0.01mol), 2-(bis(methylthio)methylene)malono-
nitrile (0.01mol), in absolute ethanol (20 mL) catalyzed with piperidine (0.5 mL) was heated
under reflux for 2h. The solid product formed was collected by filtration and crystallized from
ethanol.
5

-1
Orange powder, yield 72 %, M.p.= 238-240 ºC, IR (KBr, cm ): 3231, 3166 (NH ), 2911, 2849
2
1
(
CH aliph.) 2215, (CN) and 1693 (C=O); H NMR (400 MHz, DMSO) δ 1.63-1.70 (m, 6H,
3
CH ), 2.02 (s, 3H, 3CH), 2.18 (s, 6H, 3CH ), 2.55 (s, 3H, S-Me), 8.11 (s,2H, NH , exchangeable
2 2 2
1
3
by D O); C NMR (101 MHz, DMSO) δ 9.11 (S-Me), 27.70, 27.92, 28.03, 36.30, 36.57, 37.57,
2
3
1
8.97, 44.54, 46.21 (Adamant. Cs), 71.47 (C-CN), 113.42 (CN), 152.00 (C=N), 157.08 (C-NH2),
+
78.40 (C=O); MS (EI, 70 eV): m/z (%) = 316.67 [M ] (26.94%), 153 (100%); Anal. Calcd for
C H N OS (316.42): C; 60.73, H; 6.37; N; 17.71. Found: C; 60.43, H; 6.32, N; 18.04.
16
20
4
1
-(Adamantane-1-carbonyl)-5-amino-1H-pyrazole-4-carbonitrile (5)
To a solution of adamantane-1-carbohydrazide (3)(0.01 mol) in absolute ethanol (20 mL)
containing a catalytic amount of piperidine (0.5 mL) ethoxy methylene malononitrile (0.01 mol)
was added. The reaction mixture was heated under reflux for 3h. The solid product formed was
collected by filtration and recrystallized from ethanol.
-1
White crystals, yield 69 %, M.p.= 170-172 ºC. IR (KBr, cm ): 3416, 3229 (NH ), 2906 (CH
2
1
aliph.) 2222 (CN), and 1702 (C=O); H NMR (400 MHz, DMSO) δ 1.71 (s, 6H, 3CH ), 2.03 (s,
2
3
H, 3CH), 2.18 (s, 6H, 3CH ), 7.89 (s, 1H, pyrazole-H), 7.98 (s, 2H, NH , exchangeable with
2 2
1
3
D .O); C NMR (101 MHz, DMSO) δ 27.90, 36.43, 37.70, 44.75 (Adamant. Cs), 72.15 (C-CN),
2
1
14.32 (CN), 143.44 (C=N), 156.24 (C-NH ), 179.34 (C=O); MS (EI, 70 eV): m/z (%) = 270.47
2
+
[
M ] (10.34%), 115 (100%); Anal. Calcd for C H N O (270.34): C; 66.64, H; 6.71; N; 20.73.
1
5
18
4
Found: C; 66.98, H; 6.43, N; 20.51.
N-(1-(Adamantane-1-carbonyl)-4-cyano-1H-pyrazol-5-yl)acetamide (6)
To a solution of 5-amino-1H-pyrazole derivative 5 (0.01 mol) acetic anhydride (10 mL) was
added. The reaction mixture was heated under reflux for 1h. The solid product formed after
cooling was collected by filtration and crystallized from methanol.
-1
Yellow powder, yield 69 %, M.p. = 110-112 ºC. IR (KBr, cm ): 3258 (NH), 2910 (CH. aliph.)
1
2
3
230, (CN), and 1694 (br. C=O); H NMR (400 MHz, DMSO) δ 1.66 (s, 3H, CH ), 1.79 (s, 6H,
3
CH ), 1.96 (s, 3H, 3CH), 2.06 (s, 6H, 3CH ), 8.27 (s, 1H, pyrazole-H), 10.69 (s, 1H, NH,
2
2
1
3
exchangeable with D .O); C NMR (101 MHz, DMSO) δ 23.07 (CH ), 27.85, 36.50,38.96 ,
2
3
3
8.96 (Adamant. Cs), 75.27 (C-CN), 114.17 (CN), 160.17 (C=N), 162.39 (C-NH), 168.78, 178.98
6

(C=O); MS (EI, 70 eV): m/z (%) = 312.72 [M+] (55.22%), 94 (100%); Anal. Calcd. for
C H N O (312.37): C; 65.37, H; 6.45; N; 17.94. Found: C; 65.12, H; 6.74, N; 18.12.
17
20
4
2
Ethyl-3-(2-(adamantane-1-carbonyl)hydrazinyl)-2-cyanoacrylate (7)
A mixture of adamantane-1-carbohydrazide (3) in (20 mL) ethanol catalyzed with piperidine (0.5
mL) ethoxy methylene ethyl cyanoacetate (0.01 mol) was added. The reaction mixture was heated
under reflux for 3h. The solid product formed was collected by filtration and crystallized from
ethanol.
-1
Yellow powder, yield 72 %, M.p.= 198-200 ºC; IR (KBr, cm ): 3246 (NH), 2907 (CH aliph.)
1
2
213, (CN) and 1710,1671 (C=O); H NMR (400 MHz, CDCl ) δ 1.32 (t, J = 6.7 Hz, 3H, CH -
3
3
CH -), 1.74-1.77 (m, 6H, 3CH ), 1.91 (s, 5H, 3CH+CH ), 2.09 (s, 4H, 2CH ), 2.28 (s, 1H, vinylic-
2
2
2
2
H ), 4.26 (q, J = 6.7 Hz, 2H, CH -CH -), 7.31, 8.09 (2s, 2H, 2NH, exchangeable with D O); MS
3
2
2
+
(
EI, 70 eV): m/z (%) = 317.55 [M ] (25.51%), 190 (100%); Anal. Calcd for C H N O (317.39):
17 23 3 3
C, 64.33; H, 7.30; N, 13.24; Found: C, 64.67; H, 7.13; N, 13.07.
Synthesis of hydrazone derivatives 9a-c
To a solution of adamantane-1-carbohydrazide (3) in (20 mL) ethanol catalyzed with acetic acid
0.5 mL) 1,3-dicarbonyl derivatives (3-methylpentane-2,4-dione, benzoylacetone and ethyl
(
acetoacetate) (0.01 mol) was added. The reaction mixture was heated under reflux for 4-7h. The
solid product formed was collected by filtration and recrystallized from ethanol.
N'-(3-Methyl-4-oxopentan-2-ylidene)adamantane-1-carbohydrazide (9a)
-1
White crystals, yield 73%, M.p.= 83-85 ºC; IR (KBr, cm ): 3271 (NH), 2891, 2961, 2900, 2850
1
(
CH aliph.) and 1732, 1658 (C=O); H NMR (400 MHz, CDCl ) δ 1.24 – 1.27 (m, 3H, CH ),
3
3
1
2
.36–1.40 (m, 3H, CH ), 1.71-174 (m, 6H, 3CH ), 1.87 (s, 3H, CH ), 1.95 (s, 5H, 3CH+CH ),
3 2 3 2
1
3
.07 (s, 4H, 2CH ), 4.13-4.19 (m, 1H, -CH-), 8.36 (s, 1H, NH exchangeable with D O); C NMR
2
2
(
101 MHz, CDCl ) δ 14.17(CH ), 21.82 (CH ), 27.83, 27.89, 27.98, 28.44, 36.34, 36.40, 36.44,
3 3 3
3
8.67, 38.83, 39.09, 40.76, 48.49 (Adamant. Cs + CH ), 154.57 (C=N), 172.77, 203.81 (2C=O);
3
+
MS (EI, 70 eV): m/z (%) = 290.73 [M ] (21.41%), 227.45 (100%); Anal. Calcd for C H N O
1
7
26
2
2
(290.41): C, 70.31; H, 9.02; N, 9.65; Found: C, 70.51; H, 9.14; N, 9.47.
7

N'-(3-Oxo-1-phenylbutylidene)adamantane-1-carbohydrazide (9b)
-1
Colorless crystals, yield 73%, M.p.= 218-220 ºC; IR (KBr, cm ): 3294 (NH), 3062, 3020 (CH
1
Aro.), 2904, 2854 (CH aliph.) and 1693 (C=O); H NMR (400 MHz, CDCl ) δ 1.73 (s, 6H,
3
3
1
7
3
1
CH ), 1.90 (s, 3H, CH ), 2.02 (s, 5H, 3CH+CH ), 2.11 (s, 4H, 2CH ), 2.74-2.79 ( d, J= 18.4 Hz,
2 3 2 2
H), 3.11-3.16 (d, J= 18.8 Hz,1H), 7.31 (d, 3H, Ar-H), 7.32 (s, 1H, Ar-H), 7.33 (s, 1H, Ar-H),
1
3
.86 (s, 1H, NH exchangeable with D O), C NMR (101 MHz, CDCl ) δ 27.90, 28.28, 28.43,
2
3
6.34, 36.71, 37.41, 38.77, 38.93, 42.28 (Adamant. Cs), 52.52 (CH ), 123.53, 126.27, 126.99,
2
27.72, 128.22, 128.63 (Ar-Cs), 152.46 (C=N), 176.77, 193.83 (2C=O); MS (EI, 70 eV): m/z (%)
+
=
338 [M ] (38.86 %), 134.24 (100%); Anal. Calcd for C H N O (338.45): C, 74.53; H, 7.74;
2
1
26
2
2
N, 8.28; Found: C, 74.64; H, 7.58; N, 8.39.
Ethyl-3-(2-(adamantane-1-carbonyl)hydrazono)butanoate (9c)
-1
White powder, yield 73%, M.p = 92-94ºC. IR (KBr, cm ): 3236 (NH), 2908, 2850 (CH. aliph.)
1
and 1678, 1728 (C=O); H NMR (400 MHz, CDCl ) δ 1.25 (t, J = 7.0 Hz, 3H, CH -CH -), 1.72
3
3
2
(
s, 6H, 3CH ), 1.90 (s, 3H, CH ), 1.91 (s, 5H, 3CH+CH ), 2.06 (s, 4H, 2CH ), 2.33 (s, 2H, CH ),
2 3 2 2 2
1
3
3
.70 (q, J = 7.0 Hz, 2H, CH -CH -), 8.62 (s, 1H, NH exchangeable with D O); C NMR (101
3 2 2
MHz, CDCl ) δ 14.12 (CH -CH -), 27.66, 27.83, 27.98, 28.05, 36.34, 36.46, 38.66, 39.09, 40.05
3
3
2
(
Adamant. Cs), 58.45(CH ), 61.53, (CH -CH -), 147.17 (C=N), 165.03, 173.47(C=O); MS (EI, 70
2 3 2
+
eV): m/z (%) = 306.74 [M ] (19.00%), 288.23 (100%); Anal. Calcd for C H N O (306.41): C,
1
7
26
2
3
6
6.64; H, 8.55; N, 9.14; Found C, 66.35; H, 8.64; N, 9.02.
Synthesis of N'-((2-chloroquinolin-3-yl)methylene)adamantane-1-carbohydrazide (11)
A mixture of adamantane-1-carbohydrazide 3 (0.01mol), and 2-chloro-3-formyl-quinoline in
absolute ethanol (20 mL) catalyzed with acetic acid (0.5 mL), then the reaction heated under
reflux condition. The solid product formed when cooled then washed several time with ethanol
then collected by filtration and finally crystalized from a mixture of ethanol and with a few drops’
methanol.
-1
Yellow powder, yield 65.7%, M.p.= 250-252 ºC. IR (KBr, cm ): 3261 (NH), 2908 (CH. aliph.)
1
and 1663 (C=O), H NMR (400 MHz, CDCl ) δ 1.73 (s, 6H, 3CH ), 1.79 (s, 3H, 3CH), 2.12 (s,
3
2
6
H, 3CH ), 6.02 (s, 1H, NH; exchangeable with D O), 7.37 (t, J = 7.5 Hz, 1H, Ar-H), 7.66 (t, J =
2 2
8

7
.7 Hz, 1H, Ar-H), 7.77 (d, J = 8.0 Hz, 1H, Ar-H), 7.83 (d, J = 8.4 Hz, 1H, Ar-H), 8.45 (s, 1H,
CH-methylinic), 8.66 (s, 1H, Quinoline-H4); Anal. Calcd for C H ClN O (367.88): C, 68.56; H,
21
22
3
6
.03; N, 11.42; Found: C, 68.71; H, 5.96; N, 11.22.
Synthesis
of
N'-((5-phenyl-4-(p-tolyl)-1H-pyrazol-3-yl)methylene)adamantane-1-
carbohydrazide (12)
To a solution of adamantane-1-carbohydrazide 3 (0.01 mol) in ethanol (10 mL), 5-phenyl-4-(p-
tolyl)-1H-pyrazole-3-carbaldehyde was added. The reaction mixture was heated under reflux for
3
h, and the solution was allowed to be cooled, then the products were collected by filtration and
recrystallized from ethanol.
-1
White powder, yield 68%, M.p.= 210-212ºC; IR (KBr, cm ): 3240 (NH), 2904, 2850 (CH. aliph.)
1
and 1647 (C=O); H NMR (400 MHz, CDCl ) δ 1.73 (d, 6H, 3CH ), 1.95 (s, 6H, 3CH ), 2.07 (s,
3
2
2
3
H, 3CH-), 2.41 (s, 3H, CH ), 7.26 (s, 1H, Ar-H), 7.28 (s, 1H, Ar-H), 7.31 (d, J = 7.4 Hz, 1H, Ar-
3
H), 7.45 (t, J = 7.9 Hz, 2H, Ar-H), 7.51 (d, J = 8.0 Hz, 2H, Ar-H), 7.75 (d, J = 7.7 Hz, 2H, Ar-H),
1
3
8
.18 (s, 1H, CH methine), 8.60, 8.93 (s, 2H, 2NH; exchangeable with D O); C NMR (101 MHz,
2
CDCl ) δ 21.34 (CH ), 27.99, 36.39, 39.02, 40.39 (Adamant. Cs), 116.19, 119.16, 126.69, 127.02,
3
3
1
28.56, 129.35, 129.41, 129.50, 138.46 (Ar-Cs), 139.43, 140.96 (C=C), 153.06 (C=N), 173.91
+
(
C=O); MS (EI, 70 eV): m/z (%) = 438.34 [M ] (21.77%), 199.84 (100%); Anal. Calcd for
C H N O (438.58): C, 76.68; H, 6.90; N, 12.78, Found: C, 76.62; H, 6.96; N, 12.65.
28
30
4
Synthesis of (adamantan-1-yl)(3,5-diamino-4-(aryldiazenyl)-1H-pyrazol-1-yl)methanone
13a,b)
(
To a solution of adamantane-1-carbohydrazide 3 (0.01 mol), in absolute ethanol (20 mL),
arylhydrazonyl malononitrile derivatives (0.01 mol) were added. The reaction mixture was heated
under reflux for 2-5h. The solid product formed was collected by filtration and recrystallized from
ethanol to give (13a, b).
(Adamantan-1-yl)(3,5-diamino-4-(phenyldiazenyl)-1H-pyrazol-1-yl)methanone (13a)
-1
Yellow powder, yield 71.2 %, m.p.= 270-272ºC; IR (KBr, cm ): 3543, 3480, 3332, 3281 (NH ),
2
1
3
1
072 (CH arom.), 2908, 2895, 2849 (CH. aliph.) and 1663 (C=O); H NMR (400 MHz, DMSO)
.77 (s, 6H, 3CH ), 1.95 (s, 3H, 3CH-), 2.03 (s, 6H, 3CH ), 6.13 (s, 2H, NH , exchangeable with
2
2
2
9

D O), 7.27-7.30 (m, 1H, Ar-H), 7.42 (t, 1H, J = 7.2 Hz, Ar-H), 7.66 (t, 1H, J = 7.2 Hz, Ar-H),
2
7
.78 (d, 1H, J = 7.4 Hz, Ar-H), 8.00 (d, 1H, J = 7.2 Hz, Ar-H), 8.82 (s, 2H, NH , exchangeable
2
13
with D O); C NMR (101 MHz, DMSO) δ 27.85, 28.01, 28.03, 28.17, 36.56, 36.59, 37.40, 44.08
2
(Adamant. Cs), 112.98, 119.37, 121.48, 124.40, 128.41, 128.40, 129.11, 129.35 (Ar-Cs+ pyrazole
+
C), 153.45 (2C-NH ), 176.78(C=O); MS (EI, 70 eV): m/z (%) = 364.54 [M ] (23.49%), 306.15
2
(
100%), Anal. Calcd for C H N O (364.45): C, 65.91; H, 6.64; N, 23.06, Found: C, 65.87; H,
20 24 6
6
.58; N, 23.21.
(Adamantan-1-yl)(3,5-diamino-4-((p-methoxyphenyl)diazenyl)-1H-pyrazol-1-yl)methanone
(13b)
-1
Red powder, yield 75%, m.p.= 280-282 ºC; IR (KBr, cm ): 3462, 3442, 3378, 3348 (NH ), 3062
2
1
(
CH arom.), 2903, 2849 (CH. aliph.) and 1666 (C=O); H NMR (400 MHz, DMSO) 1.73-1.76
m, 6H, 3CH ), 2.02 (s, 3H, 3CH-), 2.24 (s, 6H, 3CH ), 3.81 (s, 3H, OCH ), 6.07 (s, 2H, NH ,
(
2
2
3
2
exchangeable with D O), 6.98 (d, 2H, Ar-H), 7.76 (d, 2H, Ar-H), 8.09 (s, 2H, NH , exchangeable
2
2
1
3
with D O); C NMR (101 MHz, DMSO) δ 28.05, 36.37, 37.45, 39.06, 39.37 (Adamant. Cs),
2
5
5.85 (OCH ), 112.30, 114.57, 122.41, 147.64 (2C-NH ), 159.86 (C-OCH ), 178.77 (C=O); MS
3 2 3
+
(
EI, 70 eV): m/z (%)= 394.49 [M ] (24.70%), 93.40 (100%); Anal. Calcd for C H N O
21 26 6 2
(394.48): C, 63.94; H, 6.64; N, 21.30, Found: C, 64.07; H, 6.52; N, 21.45.
Synthesis of 1-((adamantan-1-yl)methyl)-4,5-dihydro-1H-pyrazole derivatives 14a-c
A mixture of adamantane-1-carbohydrazide 3 (0.01 mol), in absolute ethanol (20 mL) and
chalcone derivatives (0.01 mol) were heated under reflux for 4-6h. The solid product formed was
collected by filtration and recrystallized from ethanol to give (14a-c).
(Adamantan-1-yl)(5-(p-methoxyphenyl)-3-phenyl-1H-pyrazol-1-yl)methanone (14a)
-1
Faint yellow powder, yield 64%, M.p.= 102-104 ºC; IR (KBr, cm ): 3069, 3032 (CH arom.),
1
2
3
7
969, 2908, 2843 (CH aliph.) and 1655 (C=O); H NMR (400 MHz, DMSO) δ 1.64-1.66 (m, 6H,
CH ), 1.76 (s, 3H, 3CH), 1.94 (s, 6H, 3CH ), 3.83 (s, 3H, OCH ), 7.03 (d, J = 8.7 Hz, 2H, Ar-H),
2
2
3
.57 (t, J = 7.5 Hz, 2H, Ar-H), 7.67 (t, J = 7.3 Hz, 1H, Ar-H), 7.87 (d, J = 8.7 Hz, 2H, Ar-H), 8.14
13
(
d, J = 7.2 Hz, 2H, Ar-H), 8.69 (s, 1H, CH-pyrazole); C NMR (101 MHz, DMSO) δ 27.96,
2
1
8.04, 36.61 (Adamant. Cs), 54.90 (OCH ), 123.26, 128.92, 129.18, 129.32, 129.39, 129.68,
3
29.83, 130.30, 131.01, 134.17, 135.39 (Ar-Cs), 142.61, 144.18 (C=C), 176.77 (C-OCH ), 188.95
3
1
0

(
C=O); Anal. Calcd for C H N O (412.53): C; 78.61, H; 6.84, N; 6.79, Found: C; 78.23, H;
27 28 2 2
6
.27, N; 7.01.
(Adamantan-1-yl)(3,5-di-p-tolyl-1H-pyrazol-1-yl)methanone (14b)
-1
Yellow powder, yield 72 %, M.p.= 108-110 ºC. IR (KBr, cm ): 3032 (CH arom.), 2290, 2851
1
(
(
(
CH. aliph.) and 1655 (C=O); H NMR (400 MHz, DMSO) δ 1.64 (d, J = 7.3 Hz, 6H, 3CH ), 1.76
2
s, 6H, 3CH ), 1.94 (s, 3H, 3CH), 2.37 and 2.41 (2s, 6H, 2CH ), 7.27-7.33 (m, 1H, Ar-H), 7.39
2
3
d, J = 8.0 Hz, 2H, Ar-H), 7.53 (d, J = 8.4 Hz, 1H, Ar-H), 7.65 – 7.75 (m, 1H, Ar-H), 7.94 (d, J =
8
.4 Hz, 2H, Ar-H), 8.08 (d, J = 8.0 Hz, 1H, Ar-H), 8.69 (s, 1H, CH-pyrazole); MS (EI, 70 eV):
+
m/z (%)= 410.67 [M ] (22.26%), 392.45 (100%); Anal. Calcd for C H N O (410.56): C, 81.91;
2
8
30
2
H, 7.37; N, 6.82, Found: C, 81.71; H, 7.48; N, 6.75.
Adamantan-1-yl)(5-(p-chlorophenyl)-3-phenyl-1H-pyrazol-1-yl)methanone (14c)
(
-1
Yellow powder, yield 69.4 %, M.p.=118-120ºC; IR (KBr, cm ): 3056 (CH arom.), 2989, 2850
1
(
CH.aliph.) and 1657 (C=O); H NMR (400 MHz, DMSO) δ 1.64 (s, 6H, 3CH ), 1.77 (s, 6H,
2
3
7
CH ), 1.93 (s, 3H, 3CH), 7.02 (d, J = 8.4 Hz, 2H, Ar-H), 7.36 (d, J = 8.0 Hz, 2H, Ar-H), 7.73-
2
.77 (m, 1H, Ar-H), 7.84 (d, J = 8.8 Hz, 2H, Ar-H), 8.06 (d, J = 8.0 Hz, 2H, Ar-H), 8.70 (s, 1H, ,
1
3
CH-pyrazole); C NMR (101 MHz, DMSO) δ 25.77, 28.05, 36.60 (Adamant. Cs), 114.84,
1
1
19.98, 127.82, 128.99, 129.74, 131.17, 135.78, 143.73, 144.08 (C=C), 176.79 (C=N),
+
88.91(C=O); MS (EI, 70 eV): m/z (%) = 416.10 [M ] (16.89%), 293.63 (100%), Anal. Calcd for
C H ClN O (416.95): C, 74.90; H, 6.04; N, 6.72, Found: C, 74.75; H, 5.84; N, 6.57.
26
25
2
2
2
.2.
Biological evaluation
.2.1. Anti-proliferative activity
In vitro cytotoxicity of all the newly designed and synthetized adamantane nucleus were tested
against three human tumours cell lines including human breast adenocarcinoma cell line (MCF-
7
), human hepatocellular carcinoma cell line (HepG-2) as well as human lung adenocarcinoma
epithelial cells (A549), they were obtained from VACSERA- Cell Culture Unit, Cairo, Egypt, by
using colorimetric assay method (SRB) under standard conditions according to our reported
methods [45, 52].
1
1

2
.2.2. Carbonic anhydrase assay
Carbonic anhydrase (CA) IX and XII inhibition activities were evaluated using Recombinant
Human Carbonic Anhydrase IX / XII Protein, CF Kit, that obtained from R&D Systems
(Minneapolis, MN, USA), following the same instructions from the manufacturer protocol that
measured by its esterase activity according to reported methods [53]. The Assay procedure
involved material as (1) Assay Buffer: 12.5 mM Tris, 75 mM NaCl, pH 7.5, (2)Recombinant
Human Carbonic Anhydrase IX/CA9 (rhCA9) (Catalog # 2188-CA) or Carbonic Anhydrase
XII/CA12 (rhCA12) (Catalog # 2190-CA), (3) Substrate: 4-Nitrophenyl Acetate (4-NPA)
(Sigma, Catalog # N8130), 100 mM stock in acetone. The following steps involving the producer
as; firstly, both rhCAIX or rhCAXII was diluted to 20 ng/µL and substrate (4-Nitrophenyl
acetate) also diluted to 2 mM by using a buffer. In 96-well Clear Plate (Costar, Catalog # 92592),
load 50 µL of 20 ng/µL rhCA12, and start the reaction by adding 50 µL of 2 mM Substrate to
wells. Include a substrate blank containing 50 µL assay Buffer and 50 µL of 2 mM 4-
Nitrophenyl acetate. Inhibitory effect of the three compounds were compared with
Acetazolamide were obtained by different inhibitor concentrations where the tested compound
and standard dissolved in DMSO (0.1 mM) with dilution from (10 nM) up to (0.01 nM) by using
distilled water and all compounds were tested in triplicate at each concentration used to form the
enzyme-inhibitor complex. Read absorbance at a wavelength of 400 nm (bottom read) in kinetic
mode for 5 minutes. The calculations were performed as per the kit guidelines, where a specific
activity calculated by the following equation:
Adjusted Vmax* (OD/min) x Conversion Factor** (pmol/OD)
Specific Activity (pmol/min/µg) =
amount of enzyme (µg)
Control cuvette activity was acknowledged as 100% in the absence of inhibitor, and an activity
%
– [inhibitor] graph was drawn for each inhibitor then the inhibitory efficacy was calculated by
using classic Michaels Menten kinetics according to reported method [54].
2
.2.3. Molecular docking
Study of molecular docking of the adamantane derivatives and the standard drug was performed
according to the described reported method under standard protocol and methods [55], using
Molecular Operating Environment (MOE) software version 2008.10. The X-ray crystallography
1
2

structure of both CA IX (PDB: 3IAI) and CA XII (PDB: 1JD0) with original ligand 5-acetamido-
,3,4-thiadiazole-2-sulfonamide (AZM) inhibitor [56] downloaded from protein data bank [57].
1
Protein was prepared by protonated 3D and removed the water molecule and ligand that not
implicated in the active site. The active site then generated with the default protocol. Trigonal
+
2
matcher was selected as placement method and London dG as docking score energy using Zn
ion chelate as a constrain for molecular docking. Docking process was firstly performed by self-
docking of original ligand AZM in the active site, and the evaluation of the RMSD values for CA
IX (PDB: 3IAI) and CA XII (PDB: 1JD0) were 1.02 and 1.06 Å respectively. (binding of co-
crystallized ligand and docking score energy with the figure in supplementary material file). The
newly designed compounds were generated from Chemdraw14.0 then subjected to protonate 3D
and minimize energy and finally washed the structure and saved as mdb file as ligand atom that
then used in docking protocol after replaced the co-crystalized AZM and under the same
methods.
3
. Results and discussion
3
.1. Chemistry
The synthetic strategy to achieve the target compounds pyrazole or hydrazone containing
adamantane derivatives 4-14 are shown in schemes 1 & 2. Adamnatan-1-carbohyrazide (3) that
used as key starting material prepared according to the reported method [58]. The reaction of
adamantane-1-carbohydrazide 3 with bis(thiomethyl)methylene malononitrile and ethoxy
methylene malononitrile to afforded 2-amino pyrazole derivatives 4 and 5. The reaction proceeds
via an addition-elimination mechanism where the amino group of hydrazide derivatives act as
nucleophilic that attack the β-carbon of ethylene dinitrile derivatives followed by elimination of
methanethiol (CH SH), followed by cyclization (that involve addition and as well as proton shift)
3
to afford the pyrazole enaminonitriles derivatives 4, 5.
IR spectrum of compound 4 showed absorption bands at υ 3404, 3304, 3231, 3166, for two amino
−
1
groups besides, 2911, 2849, 2215 and 1693 cm corresponding to adamantane CH, CN, and
1
carbonyl group respectively. Its H NMR spectrum of compound 4 showed a significant three
peaks at δ 1.63-1.70, 2.02 and 2.18 ppm related to protons of adamantyl that appear as multiplet
and two singlet signals. Furthermore, two-singlet signals were appearing at δ 2.55 and 8.11 ppm
1
3

for thiomethyl and the amino groups, respectively, and the amino group was exchangeable by
13
D O. C NMR spectrum of compound 5 displayed signals for adamantyl carbons between δ 27.9-
2
4
4.75 ppm besides, five signals at δ 72.15, 114.32, 143.44, 156.24, 179.34 for the carbon of
pyrazole attached to cyano, C=N, C-NH and carbonyl group. Enaminonitrile derivative 5
2
underwent acetylation when heated with acetic anhydride for one hour under reflux condition to
obtain acetanilide pyrazole derivative containing adamantane moiety 6. The IR spectrum of
-1
acetanilide pyrazole derivative 6 reveled signals at υ 3258 and broad 1694 cm related to NH and
1
two carbonyl. H NMR spectrum showed significant two signals at δ 8.27 and 10.69 ppm for
1
3
pyrazole-H and NH of acetanilide exchangeable by D O, while C NMR displayed two singlet
2
signals at δ 168.78 and 178.98 ppm for two carbonyl groups as well as signal for (C-NH) at δ
1
62.39 and adamantane carbons.
1
4

Scheme 1: Synthesis of 2-amino pyrazole and hydrazone derivatives containing adamantane
pharmacophore
Similarity, the reaction of hydrazide derivative 3 with ethoxy methylene ethyl-cyanoacetate not
demonstrate pyrazole derivatives as previous compounds 4, 5 but the mechanism stops in addition
elimination step and not underwent cyclization to give hydrazine derivative containing 2-
cyanoacrylate and adamantane moiety 7. The IR spectra of compound 7 showed absorption bands
−1
1
at υ 3246, 2213, 1710 and 1671 cm corresponding to NH, CN and carbonyl group. While, H
NMR spectrum observed triplet and quartet signals at δ 1.34 and 4.26 with coupling constant (J =
6
.7 Hz) besides, three singlet signals at δ 2.28, 7.31 and 8.09 ppm for CH-vinylic and 2NH that
1
5

exchangeable by D O added to three signals at δ 1.74-1.77, 1.91 and 2.09 ppm belonged to
2
adamantane protons.
In the same way, the reactivity of hydrazide derivative 3 toward some electrophiles were checked
as 1,3-dicarbonyl derivatives, and some heterocyclic bioactive core that is containing formyl
derivatives as 2-chloro-3-formyl-quinoline and 5-phenyl-4-(p-tolyl)-1H-pyrazole-3-carbaldehyde
and both don’t undergo cyclization and produce acyclic hydrazone derivatives 9a-c, 11 and 12
depending on characterization data. The IR spectrum of compound 9c reveled stretching signals
-1
1
for NH and two carbonyl groups at υ 3236, 1678 and 1728 cm . The H NMR spectrum of
compound 9c distinguished triplet and quartet signals, at δ 1.25, 3.70 ppm for ethyl group in
addition to, five singlet signals at δ 1.72, 1.90, 1.91, 2.06 and 8.62 ppm related to adamantane
1
protons, methyl and NH proton that exchangeable by D O respectively. While H NMR spectrum
2
of compound 11 exhibited three singlet signals at δ 6.02, 8.45 and 8.66 ppm for NH, CH-
methylinic and quinoline hydrogen, as well as two triplets and two doublet signals at δ 7.37, 7.66,
7
.77 and 7.82 ppm associated to aromatic protons with coupling constant 7.5, 7.7, 8.0 and 8.4 Hz
1
3
respectively. C NMR spectrum of compound 9c displayed signals for adamantane ranged
between δ 27.66 to 40.05 ppm. Besides, three singlet signals two of them for the ethoxy group at δ
1
4.12, 61.53 ppm and the third signal at δ 58.45 ppm for methylene group as well as, three signals
at δ 147.17, 165.03 and 173.47 for C=N and two carbonyl groups.
1
6

Scheme (2): Reaction of adamantane-1-carbohydrazide with some hetero aldehyde, azo arylidine
and chalcone derivatives
As shown in Scheme 2, Pyrazole derivatives having an azo moiety were synthesized by the
reaction of hydrazide derivatives 3 that coupled with N-phenyl hydrazonoyl malononitrile in the
presence of ethanol and produced the corresponding diamino-pyrazole 13a,b. The structure of
compound 13a,b pyrazole derivatives were confirmed with the help of analytical and
spectroscopic data. Thus, the IR spectrum of 13a exhibited stretching significance absorption
-1
-1
bands at υ 3543, 3480, 3332 and 3281 cm for two amino groups and 1663 cm for carbonyl
1
group. Its H NMR spectrum of compound 13a revealed three singlet signals between δ 1.77-2.03
ppm for adamantyl protons, two singlet signals at δ 6.13, 8.82 ppm for two amino groups
1
3
exchangeable with D O as well as five aromatic protons between δ 7.27-8.00 ppm. C NMR
2
spectrum of compound 13b exhibited signals for adamantane ranged between δ 28.05 to 39.37
ppm, in addition to, two singlet signals at δ 55.85 and δ 178.77 related to methoxy and carbonyl
groups respectively. Moreover, signals for carbon attached to methoxy and amino groups at δ
1
59.86 and δ 147.64 as well as aromatic signals between δ 112.30-122.41 ppm.
1
7

Finally, carbohydrazide derivative 3 was heated under reflux condition with α,β-unsaturated
carbonyl compound (chalcone) and furnished the corresponding pyrazole containing two arylidine
groups at position three and five 14a-c. The postulated structures of the newly designed pyrazole
derivatives were confirmed based on elemental analysis and spectral data. IR spectrum of
-1
1
compound 14a showed stretching characteristic bands at υ 1655 cm for carbonyl groups. H
NMR spectrum of compound 14c demonstrated singlet signal at δ 8.70 ppm for CH-pyrazole
1
3
proton as well as adamantane and aromatic protons. C NMR spectrum of 14c exhibited signals
for adamantane in region δ 25.77-36.60 ppm, aromatic carbon between δ 114.84-143.73 ppm and
three singlet signals at δ 144.08, 176.79, 188.91 ppm for C=C, C=N and carbonyl groups
respectively.
3
.2. Biological evaluation
3
.2.1. Anti-proliferative activity
The in vitro cytotoxic activity of the new fourteen compounds that containing adamantane as an
important pharmacophore backbone on different three cell lines, namely human breast
adenocarcinoma cell line (MCF-7), human hepatocellular carcinoma cell line (HepG-2), as well
as human lung adenocarcinoma epithelial cells (A549), were evaluated by using sulforhodamine
B (SRB) assay according to reported methods [54,55]. Doxorubicin was used as a positive
control and the obtained data represented in table 1 as IC values expressed in µM.
5
0
The newly designed compounds in scheme 1 and 2 observed two mainly products as pyrazole
derivatives (4, 5, 6, 12, 13a-b and 14a-c) and hydrazone derivatives (9a-c and 11) with only one
hydrazine derivative compound 7 as well as the main bioactive core adamantane nucleus. Most
of the designed and synthesized derivatives exhibited moderate to good activity against the tested
cancer cell lines. In general, the tested compounds exhibited more sensitive and selectivity to
lung cancer cells (A549) with IC values ranged between 1.55 ± 0.08 to 15.42 ± 1.4 µM, with
5
0
eight compounds (4, 5, 9a, 9b, 9, 12, 13a and 14c) having IC50 less than or equal ten
micromoles, except compound 14a that showed IC50 (27.18 ± 1.95µM) in comparison to
Doxorubicin (IC = 2.58 ± 0.03µM) rather than breast cancer (MCF-7) and liver cancer cells
5
0
(HepG-2). At the same time, the tested compounds reveled activity against HepG-2 with IC50
between (2.7 ± 0.15 to 38.12 ± 2.3µM) with five compounds less than ≤ 10 µM and (MCF-7)
displayed IC between (4.68 ± 0.25 to 42.17 ± 2.58 µM) with only four compound ≤ 10 µM
5
0
1
8

compared to Doxorubicin for both cancer cells ( 11.46 ± 0.95 and 15.29 ± 1.2 µM) respectively.
From table 1, it’s observed that four compounds (9a, 9c, 12 and 13a) exhibited promising and
broad activity to all cell lines.
Table 1: IC values expressed in (µM) of the newly designed compounds against three cell lines
5
0
Cpd.
IC %(µM) ± S.E*
50
No.
4
MCF-7
HepG-2
A549
12.45 ± 0.97
15.54 ± 1.46
23.45 ± 1.75
35.13 ± 2.45
8.35 ± 0.74
20.64± 1.8
11.28 ± 0.92
24.19 ± 1.85
7.46 ± 0.54
4.68 ± 0.25
13.77 ± 1.1
42.17 ± 2.58
17.32 ± 1.5
15.29 ± 1.2
8.19 ± 0.72
9.31 ± 0.82
13.89 ± 1.2
18.21 ± 1.61
28.17 ± 1.9
7.82 ± 0.64
12.48 ± 1.1
8.72 ± 0.74
22.71 ± 1.74
5.11 ± 0.45
2.7 ± 0.15
8.4 ± 0.75
5
10.57 ± 0.86
13.46 ± 1.08
15.42 ± 1.4
4.39 ± 0.35
10.78 ± 0.86
5.06 ± 0.43
14.5 ± 1.2
6
7
9a
9b
9C
1
1
1
2
3.75 ± 0.26
1.55 ± 0.08
11.5 ± 0.95
27.18 ± 1.95
11.73 ± 0.95
8.52 ± 0.65
3.58 ± 0.03
1
3a
3b
4a
4b
4c
Dox.
1
14.95 ± 1.25
38.12 ± 2.3
13.55 ± 1.1
11.46 ± 0.95
7.46 ±0.12
1
1
1

Each concentration was performed three times.
3
.2.2. Structure activity relationship study
The activity of the synthesized compounds varies with respect to substitutions where the
presence of pyrazole derivatives containing enaminonitrile as compounds 4 and 5 displayed
activity to all cell lines, but the presence of (S-Me) in the ring of pyrazole in compound 4
observed higher activity than its analogue 5 with IC ( 12.45 ± 0.97, 9.31± 0.82 and 8.4 ± 0.75
5
0
µM) and (15.54 ± 1.46, 13.89 ± 1.2 and 10.57 ± 0.86 µM) against MCF-7, HepG-2 and A549
respectively. Also, acetylation of the amino group in pyrazole enaminonitrile derivatives 5 failed
to increase activity with IC (23.45 ± 1.75, 18.21 ± 1.61 and 13.46 ± 1.08 µM) compared to
5
0
1
9

other derivatives 4, 5 and Doxorubicin. Replacement enaminonitrile pyrazole derivatives 4-6
with 3,5-diaminopyrazole 13a,b or 3,5-diarylpyrazole derivatives 14a-c exhibited that the
presence of diamino as well as azo derivatives in position four in pyrazole enhance activity
against all cell lines and among them azo-phenyl derivative 13a demonstrated higher activity
than azo-anisidine derivative 13b with IC values for 13a,b ( 4.68 ± 0.25, 2.7 ± 0.15, 1.55 ±
5
0
0
.08 µM) and ( 13.77 ± 1.1, 14.95 ± 1.25, 11.5 ± 0.95 µM) against MCF-7, HepG-2 and A549
and in comparison to Doxorubicin ( 8.19 ± 0.72, 7.46 ± 0.12, 3.58 ± 0.03 µM) respectively.
Furthermore, the structure-activity relationships of a series of hydrazone derivatives were
designed and tested on the same conditions. Adamantane-1-carbohydrazone derivatives 9a-c
showed that hydrazone of 3-methylacetylacetone 9a and ethyl acetoacetate derivative 9c
displayed inhibitory activity higher than benzoyl acetone derivative 9b. The presence of three
methyl groups in hydrazono-3-methylpentan-2-one derivatives 9a explored promising activity
than one methyl and one phenyl as 9b as well as compound 9c that have one methyl and one
ethoxy group and therefor IC values of hydrazone derivative 9a (8.35 ± 0.74, 7.82 ± 0.64, and
5
0
4
.39 ± 0.35µM) closely near to Doxorubicin. Additionally, hydrazine derivative 7 with
adamantane as the backbone and acrylate derivative that poses two functional groups as ethyl
ester and cyano groups displayed weak activity with almost IC values ranged between (15.42 ±
5
0
1
.4 to 35.13 ± 2.45µM). To our delight, hydrazone containing pyrazole moiety (hybridization of
two active core), compound 12 exhibited broad activity with IC values (7.46 ± 0.54, 5.11 ±
5
0
0
.45 and 3.75 ± 0.26 µM) with IC values higher than Doxorubicin with 1.1 and 1.45 folds
50
against MCF-7 and HepG-2.
Finally, based on the above analysis of SAR study, we can conclude that presence of pyrazole
with diamino, and the azo-phenyl group as compound 13a, hydrazone derivative by reaction with
formyl pyrazole as compound 12 or with 3-methyl acetylacetone 9a enhance the activity among
all the designed compounds. As well as, the most promising synthesis 3,5-diaminopyrazole
derivatives 13a demonstrated IC values (4.68 ± 0.25, 2.7 ± 0.15, 1.55 ± 0.08 µM) with 1.75,
5
0
2
.75 and 2.31 folds in comparison to all synthetized compounds and Doxorubicin as a positive
control.
2
0

3
.2.3. Carbonic anhydrase inhibitors
Carbonic anhydrase (CA) involved many isozymes that distributed to almost all organelle of the
human body, and they are necessary for diversities of cellular mechanisms [62]. Inhibition of
these isozymes is utilized to address an extensive range of disease situations containing
glaucoma to cancer [63,64]. Both CA IX and CA XII become an important target for lung cancer
drugs because it played a vital role in hypoxic condition by controlling intracellular and
extracellular pH as well as, they are expressed in a limited number of normal tissues, so
inhibition these two isoforms may have interesting clinical implications [56]. The modification
of new CA inhibitors has been required to develop as therapeutic agents by introducing several
groups as sulfonamide [65], thiourea derivatives [66], pyrazole derivatives [67], and
bromophenols [68].
Table 2: Inhibitory activity of adamantane derivatives 9a, 12 and 13a on tumor associated
carbonic anhydrase CAIX and CAXII
Cpd.
Carbonic anhydrase
No.
K (nM)*
I
CA IX
CA XII
9
1
a
2
256.32 ± 3.3
217.78 ± 4.2
85.75 ± 1.4
41.53 ±0.7
527.86 ± 7.8
252.25 ± 5.4
144.16 ± 2.5
68.75 ± 1.2
13a
Acetazolamide
*
Three independent measurements were performed for each used concentration of the tested
compounds.
Depending on the antiproliferative activity results, it found that the most adamantane derivatives
exhibited selectivity to lung cancer cell line A549. Among them, we selected the most potent
three compounds 9a, 12 and 13a (IC values less than 5µM) to further evaluation against in
5
0
vitro CA IX and CAXII. The data obtained was represented in table 2, by inhibitory constants
K s in (nM) according to 4-nitrophenyl acetate (4-NPA) esterase assay and Acetazolamide used
I
as a standard inhibitor drug.
From the inhibitory efficiency that represented in table 2, it found that the three adamantane
derivatives exhibited good inhibitory activity against two isoenzymes CAIX and CA XII by
2
1

displaying activity in small micromole between (0.085-0.527µM). Firstly, 3,5-
diaminoazopyrazole derivative 13a was the most active derivative and had a significant
inhibitory effect against both two isoenzymes CAIX and CA XII with K values 85.75 ± 1.4 and
I
1
6
1
44.16 ± 2.5 (nM), in comparison to Acetazolamide that revealed IC values (41.53 ± 0.7 and
50
8.75 ± 1.2 nM) respectively. Furthermore, the combination between pyrazole and adamantane-
-carbohydrazide to produce hydrazone derivative 12 with adamantane core showed moderate
activity on CAIX with reduce K values 217.78 ± 4.2 (nM) and 252.25 ± 5.4 (nM) for CAXII and
I
that illustrate that combination of the formyl pyrazole with hydrazine derivative decrease
activity. On the other hand, testing of adamantane hydrazone derivative that obtained by
incorporation of adamantane hydrazide moiety with 3-methyl acetylacetone without any pyrazole
core as adamantane derivative 9a arose as the weakest CA derivative in this study with K values
I
2
56.32 ± 3.3 and 527.85 ± 7.8 (nM) against CAIX and CA XII respectively (Fig.2).
Figure 2: Diagram illustrates the inhibitory efficiency K (nM) of adamantane derivative 9a, 12,
I
13a and Acetazolamide against transmembrane CA IX and CA XII
Finally, it can conclude that the adamantane derivatives exhibited potency activity to lung
adenocarcinoma A549 with IC50 values in low micromoles in general, but only adamantane
derivatives that having pyrazole moiety without any hydrazone part in the main skeleton
exhibited a good activity to carbonic anhydrase IX and XII, and its noteworthy to mention the
order of activity within pyrazole (13a) ˃ pyrazole-hydrazone derivative (12) ˃ hydrazone
derivatives (9a) and all containing bioactive adamantane core
2
2

3
3
.2.4. In silico Computational studies
.2.4.1. Predication of the Drug likeness and physicochemical properties
Physicochemical properties can be defined as the interaction of a new compound or drug with
the physical environment and used to determine both the proper formulation and delivery method
of a drug [69]. The three adamantane derivatives 9a, 12 and 13a that tested against CA IX and
CA XII were evaluated by using Swiss ADME (http://swissadme.ch/index.php) for
physicochemical properties and two drug-likeness rule (Lipinski’s and Veber rule) [70]. The
adamantane derivative that having hydrazone 9a, and diamino-pyrazole derivative 13a showed
oral bioavailability without any violations for both Lipinski’s Rule according to reported
methods [47,49,51] that discussed in details and summarized in table 3 and Veber filter [71] that
include two-parameter that can be known as (i) number of rotatable bonds less than or equal 10
2
and (ii) topological polar surface area (TPSA) less than or equal 140 Å . Except hydrazono-
pyrazole derivative 12 that displayed only one violation MLOGP > 4.15, but all the tested
compounds, as well as two standard drugs, meet the criteria of drug-likeness. while the two
standard drugs failed in Veber filter where TPSA > 140.
Table 3: In silico the physicochemical properties and Lipinski's rule of five and Veber filter for
the adamantane derivatives 9a, 12, 13a and Acetazolamide (AZM ) as a positive control
Cpd.
No.
Violations
from
Violations
from Veber
filter
MW
MLogP
nHBA
nHBD
nRB
TPSA
Lipinski’s
≤
140
2
Yes; 0 or 1
violation
Rule
<500
≤4.15
≤10
≤5
≤10
Yes; 0 violation
Å
9
1
a
2
290.40
2.52
3
1
5
58.53
Yes; 0
violation
Yes; 1
Yes; 0 violation
438.56
4.27
7
2
6
70.14
Yes; 0 violation
Yes; 0 violation
violation:
MLogP
Yes; 0
13a
364.44
222.25
3.50
4
6
2
2
4
3
111.65
151.66
violation
Yes; 0
AZM
-2.34
No; 1 violation:
TPSA>140
violation
2
3

3
.2.4.2. Molecular docking study
Molecular docking simulation considered the simplified form of molecular dynamic (MD)
simulation that safe time and money spent and common component of the drug discovery
because traditional experimental methods for drug discovery take a long time [72,73]. Docking
study can also be defined as a computational procedure that studies how ligand and protein fit
both energetically and geometrically to give us a complete figure to predict the binding-
conformation of small drug-like molecules to target proteins [74,75]. To provide a rationale for
the cytotoxic activity and carbonic anhydrase values of the newly adamantane derivatives 9a, 12
and 13a, the molecular docking simulation was performed using Molecular Operating
Environment software 10.2008 (MOE) to predict the possible binding mode as well as the active
conformation of these derivatives inside the target enzyme. The three compounds were docked in
two proteins that retrieved from protein data bank (https://www.rcsb.org/) as CA IX (PDB: 3IAI)
and CA XII (PDB: 1JD0) [56,57].
Firstly, the validation process inside the active site of CA IX (PDB: 3IAI) showed that the
original ligand 5-acetamido-1,3,4-thiadiazole-2-sulfonamide that known as Acetazolamide
(AZM) inhibitor fitted deeply inside the active side with (RMDS = 1.02 Å) and exhibited energy
score S= -9.94 Kcal/mol, and three hydrogen bond that can be described as one hydrogen bond
acceptor between Thr200 between the oxygen of the sulphonamide with bond length 2.66 Å
(strength= 96%). Besides, two hydrogen bonds donor with the nitrogen of sulfonamide group
through a bond length of 2.77 Å (68%) and 3.24 Å (12%). The Zn ion that contacts one nitrogen
of sulphonamide and three amino acids residue as His 119, 96, 94 (supplementary information).
(Fig. 3).
Then the promising derivatives 9a, 12, and 13a were docked inside the active site of CA IX
PDB: 3IAI). Docking of hydrazone-adamantane derivatives 9a showed binding energy S = -
(
1
2
0.96 Kcal/mol and formed only one hydrogen bond acceptor with His 64 with bond length
+
2
.80Å (14%). Besides, the carbonyl of hydrazone side chain bound to Zn ion by coordinate
+
2
bond as well as Zn ion surrounded by three coordinated bonds with His 119, 96 and 94 as the
original ligand (AZM) (Fig. 4). By the same way, pyrazolo-hydrazone derivatives 12 ( Fig. 5)
demonstrate one side chain hydrogen bond donor between Gln92 and NH of pyrazole with
2
4

strength (12%) and bond length (2.95 Å) (See table 4). Furthermore, arene-cation interaction
between tolyl of pyrazole derivative and His64 added to arene-arene cation interaction between
His94 and phenyl ring at position five in pyrazole derivatives and these binding exhibited
binding energy S= -12.63 Kcal/mol. Compound 13a with K values against CA IX ( 85.75 ± 1.4
I
nM) advertised binding energy S = - 9.94 Kcal/mol with one hydrogen bond acceptor through
His64 and carbonyl of methanone adamantane derivative with bond length 2.51 Å (40%), beside
+
2
Zn ion form arene-cation interaction with phenyl of the azo-pyrazole derivative. Adamantane
moiety in all previous pose showed lipophilic interaction with the carbonic anhydrase binding
site.
Table 4: Docking results of the promising adamantane derivatives 9a, 12, and 13a inside 3IAI
and 1JD0 active site
Cpd.
No.
(S)
Interacting residues
Type of interaction
(Kcal/mol)
For CAIX (3IAI)
AZM
-9.94
Thr199 Thr200, His94 and [His94, His96,
His119(Zn)*]
H-bond
9
1
a
2
-10.96
-12.63
His64, His96 and [His94, His96, His119 (Zn)*]
Gln92, His64 and His94
H-bond
H-bond &aren-cation
aren-arene
1
3a
-9.94
His96
H-bond &aren-cation
For CAXII (1JD0)
AZM
-11.55
Thr199, Thr200, His94, His119 and [His94,
His96, His119(Zn)*]
H-bond
9
1
a
2
-14.31
-12.81
-13.87
His96 and [His94, His96, His119(Zn)*]
Lys 67
H-bond
H-bond & aren-cation
H-bond & aren-cation
aren-arene
1
3a
His94, Thr91
(*) meaning that Zn ion bind to ligand and the residue amino acid.
Self-docking of Acetazolamide inside the active site of (PDB: 1JD0) to perform the validation
process displayed binding energy S= -11.55 Kcal/mol and formed many binding bonds as amino
2
5

acid residue Thr199 formed one Hydrogen bond backbone acceptor with the oxygen of
sulfonamide with bond length 2.66 Å (79%) as well as another hydrogen bond side chain donor
with a nitrogen of sulfonamide group with bond length 2.59 Å (83%). In the same way, both His
1
19 and His94 formed two hydrogen bond side chain donors with the nitrogen of sulfonamide
with bond length 2.74 Å (20%) and 3.37 Å (11%), respectively. Also, Thr200 bind with two
nitrogen of thiadiazole of acetazolamide with two hydrogen bonds acceptor with a bond length of
3
.20 Å (15%) and 2.48 Å (27%). The Zn ions bound to the nitrogen of sulfonamide and formed
three coordinate bonds with amino acids (His94, His96, and His119) beside vital water molecule
that contact with the nitrogen of acetanilide of acetazolamide and nitrogen of thiadiazole as well
as two amino acids Thr200 and Pro201. (Fig. 6).
Furthermore, compound 9a that have hydrazone derivatives, His96 (hydrogen bond acceptor
3
.15Å) and Zn ion bind with oxygen of the carbonyl by coordinate bond and the same three
histidine amino acid as a ligand with binding energy S = -14.31Kcal/mol as well as hydrophobic
interaction between adamantane moiety and two methyl groups with the active site of pocket
(Fig. 7). Also, pyrazole derivative 12 with inhibitory efficiency 0.252 µM showed the lowest
binding energy S= -12.81Kcal/mol, where His 94 form one hydrogen bond acceptor (2.87 Å)
with the carbonyl of the adamantane carbohydrazide derivatives and one arene-cation interaction
with tolyl of pyrazole derivatives. Pyrazole derivative 13a with two amino (in position 3 and 5)
and azo-phenyl (position 4) derivative observed a lower docking score energy binding energy S=
-13.87 Kcal/mol. Moreover, it formed hydrogen bond acceptor (2.57Å and 42%) between the
carbonyl of methanone derivative and Thr91 and one arene-arene interaction between phenyl and
His94 as well as one arene-cation interaction through the pyrazole ring and Lys 67 (Fig. 8).
(supplementary data containing all figure).
Finally, the presence of pyrazole derivative enhances binding affinity because it can form many
different interactions, as well as the presence of carbonyl at a side chain or tightly direct to
pyrazole ring also, increase binding inside the pock in addition, it can bind to Zn ion, and
presence of adamantane core (lipophilic properties) exhibited hydrophobic interaction with the
active site in a pocket.
2
6

Figure 3: 2D &3D interactions of Acetazolamide(AZM) inhibitor in the active site of 3IaI (CA
IX)
Figure 4: 2D &3D interactions of compound 9a in the active site of 3IaI (CA IX)
Figure 5: 2D &3D interactions of compound 12 in the active site of 3IaI (CA IX).
2
7

Figure 6: 2D &3D interactions Acetazolamide (AZM) in the active site of 1JD0 (CA XII)
Figure 7: 2D &3D interactions of compound 12 in the active site of 1JD0 (CA XII).
Figure 8: 2D &3D interactions of compound 13a in the active site of 1JD0 (CA XII)
2
8

4
. Conclusion
Generally, we successfully designed and synthetized a small library of adamantane nucleus
lipophilic part) bearing pyrazole 4, 5, 6, 12, 13, 14, and hydrazone 7, 9, 11 derivatives at
(
position one and all the chemical reaction involve an only one-step reaction to obtain the desired
products. The cytotoxic activity against three cell line MCF-7, HepG-2 and A549 were evaluated
and displayed good to moderated activity with IC50 values (1.55-42.17 µM). The newly
synthesized derivatives revealed sensitive and selectivity to lung cancer cells (A549) with IC50
values ranged between 1.55 ± 0.08 to 15.42 ± 1.4 µM, with eight compounds (4, 5, 9a, 9b, 9c,
1
2, 13a and 14c) having IC less than or equal ten micromole except for compound 14a that
50
showed IC (27.18 ± 1.95 µM). The most promising three adamantane derivatives 9a, 12 and
5
0
1
3a with IC50 values less than 5 µM were elected to evaluate their inhibitory action against
isoenzyme hCAIX and hCAXII for the first time. Additionally, 3,5-diamino-pyrazole core 13a
showed higher K values than hydrazo-pyrazole 12 and hydrazone derivatives 9a that hybrid with
I
adamantane and exhibited inhibitory effect with submicromolar between (0.0.085-0.527 µM), in
comparison to Acetazolamide (0.041-0.068 µM). Among them, compound 13a is considered the
most promising derivative with anti-proliferative (A549) (IC =1.55 ± 0.08 µM) and CAIX/XII
5
0
inhibitors (K = 0.085 and 0.144 µM), respectively. Finally, some drug-likeness model as
I
Lipinski and Verber were predicted. Molecular docking simulation was performed inside the
active site of CA IX (PDB: 3IAI) and CA XII (PDB: 1JD0) to evaluate the binding modes of the
adamantane derivatives as well as Acetazolamide. Docking score of the promising compounds
showed lower values (-9.94 to -14.31 Kcal/mol) in comparison to Acetazolamide (-9.94 to -11.55
Kcal/mol) and different type of interaction as H-bond, arene-arene and arene-cation interaction
were present beside in some cases ligand coordinated to Zn ion. Moreover, due to lipophilic
characters of adamantane core the hydrophobic interaction appear with both active sites.
2
9