Design, synthesis and anticancer evaluation of novel spirobenzo[h]chromene and spirochromane derivatives with dual EGFR and B-RAF inhibitory activities

Article abstract of DOI:10.1016/j.ejmech.2018.03.001

A novel series of spirobenzo[h]chromene and spirochromane derivatives was designed, synthesized and evaluated as potential anticancer agents against MCF-7 (human breast carcinoma), HT-29 (human colorectal adenocarcinoma) and A549 (human lung carcinoma) cell lines using MTT assay. Eight compounds 7, 8e, 13a-e and 16 showed a better anticancer activity than that of sorafenib, the multi-kinase inhibitor with IC₅₀ values between 1.78 and 5.47 μM or erlotinib with IC₅₀ values over 20 μM. Representative compounds 8e, 13c and 16 were selected for further mechanistic investigation via EGFR, B-RAF and tubulin polymerization assays. Compound 16 was the most potent EGFR inhibitor (IC₅₀ = 1.2 μM), yet compounds 8e, 13c and 16 displayed moderate tubulin polymerization inhibition effects. Molecular docking studies of those compounds revealed their possible binding modes into the active sites of both EGFR and B-RAF kinases. The newly developed compounds represent a therapeutically promising approach for the treatment of different types of cancer.

Full text of DOI:10.1016/j.ejmech.2018.03.001

Accepted Manuscript
Design, synthesis and anticancer evaluation of novel Spirobenzo[h]chromene and
spirochromane derivatives with dual EGFR and B-RAF inhibitory activities
Shaimaa A. Abdelatef, Mohammed T. El-Saadi, Noha H. Amin, Ahmed H.
Abdelazeem, Hany A. Omar, Khaled R.A. Abdellatif
PII:
S0223-5234(18)30236-8
10.1016/j.ejmech.2018.03.001
EJMECH 10266
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 December 2017
Revised Date: 28 February 2018
Accepted Date: 1 March 2018
Please cite this article as: S.A. Abdelatef, M.T. El-Saadi, N.H. Amin, A.H. Abdelazeem, H.A. Omar,
K.R.A. Abdellatif, Design, synthesis and anticancer evaluation of novel Spirobenzo[h]chromene and
spirochromane derivatives with dual EGFR and B-RAF inhibitory activities, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.001.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Anticancer Evaluation of Novel
Spirobenzo[h]chromene and Spirochromane Derivatives
with Dual EGFR and B-RAF Inhibitory Activities
a
a
a
Shaimaa A. Abdelatef , Mohammed T. El-Saadi , Noha H. Amin , Ahmed H.
a,
b,c
d,
Abdelazeem *, Hany A. Omar , Khaled R.A. Abdellatif *
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt;
b
Sharjah Institute for Medical Research and College of Pharmacy, University of Sharjah, Sharjah 27272,
c
United Arab of Emirates, Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, 62514,
d
Egypt; Department of Pharmaceutical Organic Chemistry, Beni-Suef University, Beni-Suef 62514, Egypt.
*To whom correspondence should be addressed.
Khaled R.A. Abdellatif, Ph.D. Department of Pharmaceutical Organic Chemistry, Faculty of
Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt.
Tel.: +201223662720
Fax: +20-0822317958.
E-mail address: khaled.ahmed@pharm.bsu.edu.eg
Ahmed H. Abdelazeem, Ph.D. Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-
Suef University, Beni-Suef 62514, Egypt.
Tel.: (002)-0100-2877405
E-mail address: ahmed.abdelazeem@pharm.bsu.edu.eg, ahmed.pharm@yahoo.com
1

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Abstract
A novel series of spirobenzo[h]chromene and spirochromane derivatives was designed, synthesized
and evaluated as potential anticancer agents against MCF-7 (human breast carcinoma), HT-29
(
human colorectal adenocarcinoma) and A549 (human lung carcinoma) cell lines using MTT assay.
Eight compounds 7, 8e, 13a-e and 16 showed a better anticancer activity than that of sorafenib, the
multi-kinase inhibitor with IC values between 1.78 and 5.47 µM or erlotinib with IC values over
5
0
50
2
0 µM. Representative compounds 8e, 13c and 16 were selected for further mechanistic
investigation via EGFR, B-RAF and tubulin polymerization assays. Compound 16 was the most
potent EGFR inhibitor (IC = 1.2 µM), yet compounds 8e, 13c and 16 displayed moderate tubulin
50
polymerization inhibition effects. Molecular docking studies of those compounds revealed their
possible binding modes into the active sites of both EGFR and B-RAF kinases. The newly
developed compounds represent a therapeutically promising approach for the treatment of different
types of cancer.
Keywords: Spirobenzo[h]chromene; Spirochromane; B-RAF; EGFR; Docking; Anticancer agents
GRAPHICAL ABSTRACT
2

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1. Introduction
Cancer has been a major public health problem worldwide with an increasing number of patients
being diagnosed with cancer every year [1]. Unfortunately, the effectiveness of chemotherapy, as a
principal mode of cancer treatment is limited by drug resistance, severe side effects and poor
selectivity [2, 3]. Thus, newly combined, multi-targeted therapies or recently, immunotherapy have
been advocated [4-6]. The activation of kinases in different cell signaling pathways has been
implicated in cancer cell survival, invasiveness and drug resistance [7, 8]. So, targeting kinases such
as epidermal growth factor receptor (EGFR) and serine/threonine kinases such as B-RAF have
become one of the most intensively pursued classes of anticancer agents [9, 10]. In addition, the
®
approval of multikinase inhibitors such as Sorafenib (Nexavar ), dual VEGFR and EGFR kinase
inhibitors such as vandetanib or RAF and antiangiogenic VEGFR-PDGFR
chemotherapeutics have been adopted to produce higher potency and selectivity [11-13].
β targeted
On the other hand, spirochromane has attracted significant interest as a privilege structure in the
development of several bioactive compounds with a wide of biological activities [14-18].
Compounds 1 and 2 possessing spirochromanone scaffold were reported to have potent anticancer
activity where they exert their action through inhibiting histone deacetylase (HDAC) enzyme, Fig. 1
[19, 20]. Nevertheless, various thiosemicarbazone (TSC) and semicarbazide structures, a large
group of thio/urea derivatives, have been evaluated as anticancer therapeutics over the last years,
such as compounds 3, 4, Fig. 1. The conjugated N-N-S or N-N-O tri-dentate ligand system of
thiosemicarbazides and semicarbazones, respectively, seemed essential for the anticancer activity
through the inhibition of DNA synthesis produced by the modification in the reductive conversion
of ribonucleotides to deoxyribonucleotides [14, 21] via reactive oxygen species generation [22-25].
3

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O
O
N
HO
H
N
HO
O
O
1
2
H
N
NH2
N
H
N
N
N
HO
O
N
R1
R2
S
O
3
4
Fig. 1. Some reported spiroheterocyclic (1 and 2), thiosemicarbazone (3) and semicarbazones (4)
derivatives with anticancer activity
Based on the aforementioned data regarding the biological significance of both spirochromane and
thio/semicarbazone moieties, it was conceptualized that the tethering of these pharmacophores in
one scaffold using fragment-based drug design approach would be of great interest to develop
highly potent anticancer agents. Thus, we described herein, for the first time, the design and
synthesis of new hybrid spiro-molecules and evaluate their anticancer potential and their dual
inhibitory activities against EGFR and B-RAF kinases as a possible and valid mechanism of action
for this novel class of anticancer agents.
2
. Results and discussion
.1. Chemistry
2
The general reactions used for the synthesis of the novel targeted spiro derivatives are outlined in
Schemes 1-3. The starting material 5, spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-one, was
synthesized via a thermal condensation of cyclohexanone, 1'-hydroxy-2'-acetonaphthone and
pyrrolidine in methanol using the reported Kabbe’s multi-component reaction [26]. The furnished
spirobenzo[h]chromenone compound 5 was subsequently coupled with methyl hydrazine-
carbodithioate 6, which was prepared by the reaction of hydrazine hydrate with carbon disulfide and
4

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o
potassium hydroxide followed by treating with methyl iodide at 10 C, affording intermediate 7 in a
quantitative yield [27]. The final targeted thiosemicarbazide compounds 8a-f were obtained upon
the displacement of the S-methyl group through the condensation of the intermediate 6 with
respective amines in isopropanol with reflux for 48 h till the evolution of methyl mercaptan ceased.
Meanwhile, various substituted phenyl isocyanates were reacted with hydrazine hydrate in ethanol
o
at 0 C to provide the phenylsemicarbazides 9a-c which in turn were coupled with the starting
spirobenzo[h]- chromenone material 5 in the presence of TFA to afford the final targeted
semicarbazide compounds 10a-c in good yields, Scheme 1. All Postulated structures of the final
1
spiro compounds were fully confirmed using NMR, HRMS and elemental analysis. The H NMR
spectrum of thiosemicarbazide derivative 8a, as a representative example of the first series, revealed
the absence of the S-CH singlet peak at
δ
2.68 (s, 3H) and appearance of a singlet one with two
2.96 assigned to methylene protons (CH C=N) of spirobenzo[h]chromenone
scaffold and a triplet signal at 3.84 with four protons integration corresponding to the two
methylene groups attached to the nitrogen atom of piperidine moiety. Moreover, the C NMR
spectrum of 8a showed the appearance of a characteristic peak at 182.55 assigned to thiocarbonyl
3
protons integration at
δ
2
δ
1
3
δ
group and another peak at 56.51 attributed to the two methylene groups attached to the nitrogen
atom of piperidine moiety. The structure of 8a was also confirmed by HRMS where a molecular ion
+
peak at m/z 408.1487 [M+H] was detected, which was consistent with the molecular formula
24 29 3
C H N OS of the targeted product.
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Scheme 1
Reagents and Reaction Conditions: a) Pyrrolidine, MeOH, reflux, 12 h; b) i. KOH, i-PrOH/H O,
2
o
o
0
4
C, 2h, ii. CH I, 0 C, 2h; c) Conc HCl, iPrOH, reflux, 2 h; d) Appropriate amine, iPrOH, reflux,
3
o
8 h; e) NH NH .H O, EtOH, 0 C, 30 min; f) TFA, iPrOH, reflux, overnight.
2
2
2
Furthermore, the synthesis of final semicarbazide derivatives 13a-e were carried out as depicted in
Scheme 2. the spirochromanone starting materials 11a,b were prepared using the same
methodology of Kabbe’s reaction through refluxing cyclohexanone or cycloheptanone with 2-
hydroxyacetophenone and pyrrolidine in methanol to give the intermediates 11a and 11b,
respectively. The spirochromanone starting materials 11a and 11b were reacted with hydrazine
hydrate in ethanol at room temperature affording the corresponding hydrazone derivatives 12a and
12b which were coupled directly, without separation, with various phenylisocyanates to furnish the
6

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final targeted semicarbazide compounds 13a-e. The structure of semicarbazide derivative 13b was
1
taken as an example for this series, which was verified by H NMR spectrum that showed two
1
3
singlet peaks at
NMR spectrum revealed the appearance of some characteristic peaks at
corresponding to carbonyl, methylene (CH -C=N) and methyl groups, respectively. HRMS showed
δ
2.80 and 2.27 assigned to the methylene and methyl groups, respectively. C
δ
154.40, 56.52 and 20.86
2
25 3 2
a molecular ion peak at 364.2045 which was in agreement with the molecular formula C22H N O
of compound 13b.
Scheme 2
2 2 2
Reagents and Reaction Conditions: a) Pyrrolidine, MeOH, reflux, 12 h; b) NH NH .H O, EtOH,
rt, 3 h; c) Appropriate phenylisocyanate derivative, EtOH, rt, overnight.
Finally, the final hydrazide compounds 15-17 were prepared using the synthetic pathway showed in
Scheme 3. The previous prepared spirobenzo[h]chromenone starting material 5 was allowed to
react with hydrazine hydrate, in a similar manner to 11a,b in ethanol with stirring at room
temperature to give the respective hydrazone intermediate 14 which in turn was refluxed directly in
glacial acetic acid with phthalic anhydride and isatin in presence of anhydrous sodium acetate to
afford the final compounds 15 and 17, respectively and with adamantyl carbonyl chloride in the
7

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presence of triethylamine to furnish the final compound 16 in a good yield. The structures of
1
compounds 15-17 were confirmed by H NMR and HRMS where molecular ion peaks at m/z
411.1680, 443.2698 and 410.1933 were obtained and they were found to be consistent with the
exact molecular weights of 15-17, respectively.
Scheme 3
Reagents and Reaction Conditions: a) NH NH .H O, EtOH, rt, 6 h; b) Phthalic anhydride, anhyd.
2
2
2
CH COONa, gl. AcOH, reflux, overnight; c) Adamantyl carbonyl chloride, TEA, THF, reflux, 2 h;
3
d) Isatin, anhyd. CH COONa, gl. AcOH, reflux, overnight.
3
2.2.
Pharmacological Screening
2.2.1. Anticancer activity
In vitro anticancer activity of the newly synthesized compounds was assessed against three cancer
cell lines; MCF-7 (human breast carcinoma), HT-29 (human colorectal adenocarcinoma) and A549
(human lung carcinoma) cell lines using MTT assay [28, 29]. The results were summarized in table
1
and expressed in terms of IC50 values, where sorafenib and erlotinib were used as positive
8

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controls. The tested compounds showed variable anticancer activities against the three cell lines.
Spirochromane derivatives 13 were noticed to be the most common anticancer agents against the
three cell lines, if compared to sorafenib (IC = 3.64, 6.21 and 3.97
µ
M against MCF-7, A549 and
M in A549 and IC50 > 20 M in the other cell lines).
M) and 13d (IC50 = 2.15 M) were the most active against MCF-7
and HT-29 cell lines, respectively. On the other hand, the spirobenzochromene 16 (IC50 = 1.78 M)
was the most potent anticancer agent against A549 cell line, followed by compound 13e (IC = 2.24
5
0
HT-29, respectively) or erlotinib (IC50 = 17.32
Compounds 13e (IC50 = 3.09
µ
µ
µ
µ
µ
5
0
µ
M). Accordingly, it could be assumed that the anticancer potency of spirochromane derivatives
3c-e (IC = 2.15-7.24 M) dominated over that of bulkier spirobenzochromene derivatives 8, 10,
4, 15 and 17 (IC = 3.24-15.07 M) against the three cell lines tested, except for the adamantyl
1
1
µ
5
0
µ
5
0
derivative 16. However, it could be observed that the semicarbazide derivatives of spirochromane
3 were generally more potent than the thiosemicarbazide or hydrazine derivatives of
spirobenzochromenes such as compounds 8f, 10c, 8b (IC50 = 15.07, 14.96, 14.18 M) on MCF-7,
4, 15 (IC = 12.87, 12.36 M) on A549, 14, 8c (IC = 9.97, 9.31 M) on HT-29 cell lines. These
1
µ
1
µ
µ
5
0
50
two observations gave the rise to the importance of bulkiness effects, in accordance with electronic
ones, on the anticancer activity of these compounds. The best anticancer activity amongst
compounds 8a-f was accredited to the n-hexyl derivative 8e (IC = 3.24 and 3.87 µM against A549
5
0
and HT-29, respectively). As for compounds 10a-c, the chlorophenyl derivative 10c the least
potency, against the three cell lines used. Compounds 13a-e demonstrated better potency in cases of
the cycloheptane substitution (13 d,e) over the cyclohexane ones (13a-c); where the chlorophenyl
substituent 13c gave the most active derivative amongst the cyclohexane ones. As for compounds
14-17, the highest anticancer activity was accredited to the adamantly hydrazine derivative of
spirobenzochromene 16 against the three cell lines used. The variation in activity amongst the
derivatives of each group could be clearly attributed to changeable electronic effects, but the steric
9

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effects still could not be neglected when observing activity variations between groups 8, 10, 13 and
14-17.
Table 1: IC50 values of the newly synthesized compounds on the cell viability of three cancer cell
lines; MCF-7, HT-29 and A549 cell lines
a
Anticancer activity IC (µM)
5
0
Comp.
MCF-7
A549
HT-29
5
7
10.65±1.25
5.10±1.31
13.35±1.74
14.18±1.61
11.92±0.97
11.66±1.03
4.97±0.42
15.07±1.29
11.96±1.18
9.07±1.10
14.96±1.27
12.92±1.71
7.24±1.36
4.12±0.98
3.32±0.77
3.09±0.11
9.74±0.33
12.06±1.25
4.09±0.21
5.11±0.75
3.64±0.78
10.55±1.07
3.99±0.65
11.36±1.97
12.01±1.06
10.07±1.25
10.56±1.73
3.24±0.89
9.14±0.36
8.15±1.06
8.35±0.78
9.54±0.92
5.47±1.23
4.08±0.71
3.65±1.06
3.81±0.95
2.24±0.07
12.87±1.30
12.36±1.87
1.78±0.24
11.12±0.39
6.21±0.55
6.15±0.71
4.13±1.08
6.77±1.19
8.34±1.31
9.31±1.35
8.17±1.23
3.87±0.37
7.44±1.51
4.74±0.32
7.09±0.74
7.37±0.85
3.11±0.31
4.71±0.68
2.75±0.12
2.15±0.72
2.44±0.32
9.97±1.24
8.34±1.63
4.45±0.54
7.67±1.07
3.97±0.14
>20
8
a
8
b
8
c
8
d
8
e
8
f
1
0a
0b
0c
3a
3b
3c
3d
3e
1
1
1
1
1
1
1
1
4
5
6
7
1
1
1
b
Sorafenib
b
Erlotinib
>20
17.32+3.11
1
0

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Three human cancer cell lines were used; MCF-7 (human breast carcinoma), HT-29 (human
colorectal adenocarcinoma) and A549 (human lung carcinoma) were treated with the test
compounds or vehicle in RPMI media containing 10% FBS for 96 h and cell viability was assessed
a
b
by MTT assay. Data are mean ± S.D. (n = 6); Positive controls.
.2.2. Effect on normal cells
2
Representative compounds 7, 8e, 13e,d, 16 and 17 together with sorafenib were further investigated
for their effects on the cell viability of normal fibroblasts (F180) using MTT assay, thus assaying
their safety and selectivity profiles. Compounds 7, 16, 17 and sorafenib demonstrated good safety
profiles with high IC values (IC >40
µM) on normal fibroblasts. Table 2
5
0
50
Table 2: IC values of the most active newly synthesized compounds on the cell viability of the
5
0
normal fibroblasts (F180)
a
Comp.
7
IC (
µ
M) F180
>40
5
0
1
3d
29.87±1.87
31.65±2.03
23.65±1.05
>40
8
e
1
3e
1
6
17
>40
b
Sorafenib
>40
Normal fibroblasts (F180 cells) were treated with the test compounds or vehicle in MEM media
containing 10% FBS for 96 h and cell viability was assessed by MTT assay.
a
b
Data are mean ± S.D. (n = 6); Positive control.
2.2.3. EGFR, B-RAF and Tubulin assays
Further mechanistic studies were conducted through investigating the binding affinity of
representative active spirochromane and spirobenzo[h]chromene derivatives (8e, 13c and 16) to
EGFR and B-RAF and assaying their effects on tubulin polymerization using suitable positive
controls in each assay. The results showed more or less close, yet significant IC values (IC = 1.4,
5
0
50
1
1

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1.9 and 1.2 µM on EGFR and 2.7, 2.9 and 2.6 on B-RAF kinase for compounds 8e, 13c and 16,
respectively), if compared to erlotinib (IC = 0.08 on EGFR and 0.06 µM on B-RAF). These values
5
0
showed expected higher EGFR affinity than B-RAF kinase. The hydrazine derivative of
spirobenzochromenes 16 showed the most potent inhibitory activity against EGFR, suggesting
preferential steric and electronic arrangements on EGFR, compared to compounds 8e and 13c.
Moreover, the three tested compounds showed moderate tubulin polymerization effects, if
compared to vincristine and docetaxel. Results were shown in table 3.
Table 3: Effects of compounds on EGFR, B-RAF, and Tubulin Polymerization.
a
IC50 ±SEM (µM)
(Tubulin)
Effect
Arbitrary units)
Comp.
EGFR
inhibition
B-RAF
inhibition
(
8
e
1.4±0.8
2.7±1.3
1875±217
1424±320
1820±290
-
1
3c
1.9±0.7
2.9±1.2
1
6
1.2±0.4
2.6±1.0
b
Erlotinib
0.08±0.04
0.06±0.02
b
Vincristine
-
-
-
-
-
747±205
4825±249
2872±214
b
Docetaxel
c
DPBS
-
a
b
c
Data are mean ± S.E. (n=3); Positive controls; Negative control
2.3. Molecular Docking Study
The present study was performed in an attempt to understand the potency of the newly synthesized
spiro compounds and gain some structural insights into their potential binding patterns and possible
interactions with both EGFR and B-RAF kinases. Accordingly, the most active spirochromane and
spirobenzo[h]chromene derivatives (8e, 13c and 16) were docked inside the active sites of both
EGFR and B-RAF using LIBDOCK embedded in the Discovery Studio software (San Diego, USA).
The 3D crystal structures of EGFR (PDB ID: 1M17) and B-RAF (PDB ID: 2FB8), in complex with
1
2

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erlotinib and SB-590885, respectively, were used for this docking simulation study [30-32]. The
amino acid residues within a distance of 9 Å around the EGFR and B-RAF co-crystallized ligands
in ATP binding pockets were isolated and the possible interactions and ligands orientation were
examined. The overlay of the top docking poses EGFR and B-RAF proteins binding pockets were
presented in Fig. 2 where the poses 8e, 13c and 16 showed good shape complementarity with the
ATP-binding sites of both kinases.
Fig. 2. (A) Overlay of the top docked poses 8e, 13c and 16 in addition to the co-crystallized ligand
erlotinib into the EGFR kinase binding pocket (PDB code: 1M17); (B) Overlay of the top docked
poses 8e, 13c, 16 and SB-590885 as a co-crystallized ligand into the B-RAF active site (PDB code:
2
FB8). EGFR and B-RAF proteins are represented as a solid surface colored according to atom
charges. The binding sites are depicted as transparent green solid surface.
Examination of the docking results revealed that the most active compound 8e, 13c and 16, fit
nicely inside the ATP-active site engaging in some H-bonds with Met769 and Thr830 residues. In
general, it was noticed that the top docked poses adopted a common binding mode where the
hydrophobic moieties in the three compounds; p-chlorophenyl, n-hexyl and adamantyl, aligned
1
3

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towards the DFG motif (Asp831, Phe832 and Gly833) with its well-known role in the regulation of
kinase activity. Meanwhile, the spirochromane and spirobenzo[h]chromene cores were extended
towards the hinge region, forming hydrophobic interactions with Leu694, Val702, Leu768, Cys773,
Asp776 and Leu820 amino acid residues, Fig. 3(A-C). Compound 13c was found to bind to EGFR
via another one
π-cation interaction between p-chlorophenyl ring and Lys721 residue with a
distance of 4.3 Å. In addition, the p-chlorophenyl ring in the same compound formed also some
other hydrophobic interactions near the DFG motif with Ala719, Met742 and Leu764. Similarly, n-
hexyl and adamantyl moieties in compounds 8e and 16, respectively formed the same hydrophobic
interactions like p-chlorophenyl in compound 13c. Moreover, compound 16 was found to protrude
towards the hinge region to some extent more than the other two compounds, although it showed
the most inhibitory activity against the EGFR kinase. Compound 8e was involved in another H-
bond of a distance of 2.2 Å with Thr830 residue. Interestingly, there is a remarkable similarity
between the binding pattern of the three docked ligands and that of erlotinib, Fig. 3(D). The 3-
ethynylphenyl moiety in erlotinib was located near DFG motif in a similar manner like p-
chlorophenyl, n-hexyl and adamantyl moieties forming mostly the same hydrophobic interactions.
This apparent similarity between the binding modes of the newly synthesized compounds and the
native ligand may greatly contribute to their remarkable potency against the EGFR kinase.
1
4

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Fig. 3. (A) Docking and binding pattern of compound 13c (yellow) into ATP-active site of EGFR
kinase (PDB code: 1M17); (B) Docking and binding pattern of compound 8e (orange) into ATP-
active site of EGFR kinase; (C) Docking and binding pattern of compound 16 (magenta red) into
ATP-active site of EGFR kinase; (D) Docking and binding pattern of erlotinib (cyan) into ATP-
active site of EGFR kinase. The superimposition of all docked poses and erlotinib within the ATP-
active site of the EGFR protein secondary structure was presented; The poses were rendered as
green line models. π-Interactions were represented as a yellow solid line. Hydrogen bonds were
represented as dashed blue lines. All hydrogens were removed for the purposes of clarity.
On the other hand, the analysis of the docking results of 8e, 13c and 16 into the B-RAF ATP-active
pocket indicated that the three shared a similar orientation and disposition inside the active site of
B-RAF kinase where the spirochromane and spirobenzo[h]chromene cores were located near the
DFG motif (Asp594, Phe595 and Gly596) making several hydrophobic interactions with Ala481,
Val482, Ile527, Val528 and Phe595 residues. Meanwhile, the hydrophobic moieties in the three
compounds; p-chlorophenyl, n-hexyl and adamantyl, were extended towards the hinge region,
1
5

ACCEPTED MANUSCRIPT
forming hydrophobic interactions with Ile463, Cys532, Ser535 and Asn580 amino acid residues,
Fig. 4(A-C). In particular, compound 13c formed a network of three H-bonds with Asn581 and
Asp594 residues with distances of 2.6, 2.3 and 2.3 Å, respectively. It was noticed also that the
phenyl ring of the spirochromane core was directed towards DFG motif and making a favorable
stacking interaction with the phenyl ring of Phe583 amino acid. Furthermore, it binds to the active
site of B-RAF via one more -cation interaction between the p-chlorophenyl ring and Lys578
π-
π
residue a distance of 4.5 Å. Regarding compound 8e, the n-hexyl moiety was leaning more towards
the bottom of the active site owing to the bulkiness of spirobenzo[h]chromene, which pushed this
moiety in a slightly different direction comparing to the p-chlorophenyl moiety in 13c within the
kinase hinge region, Fig. 4(B). Additionally, the cyclohexyl ring in compound 8e was involved in π-
stacking interaction with the phenyl ring of Phe583 amino acid residue. Due to the common
spirobenzo[h]chromene core present in both compounds 8e and 16, they adopted a completely
similar binding patterns. Compound 16 was involved in the same hydrophobic interactions like the
other docked compounds in addition to one π-stacking interaction with the phenyl ring of Phe583
amino acid residue Fig. 4(c). Comparing the binding patterns of the three docked poses with the co-
crystallized ligand, SB-590885, it could be observed the remarkable similarity and complementary
between them inside the ATP-binding pocket, Fig. 4(D). It can be concluded that the active site of
B-RAF can accommodate both spirochromane and bulkier spirobenzo[h]chromene of the docked
poses, however, the bulkier compounds 8e and 16 showed higher potency than spirochromane
derivative 13c.
1
6

ACCEPTED MANUSCRIPT
Fig. 4. (A) Docking and binding pattern of compound 13c (yellow) into ATP-active site of B-RAF
kinase (PDB code: 2FB8); (B) Docking and binding pattern of compound 8e (orange) into ATP-
active site of B-RAF kinase; (C) Docking and binding pattern of compound 16 (magenta red) into
ATP-active site of B-RAF kinase; (D) Docking and binding pattern of SB-590885 (cyan) into ATP-
active site of B-RAF kinase. The superimposition of all docked poses and erlotinib within the ATP-
active site of the EGFR protein secondary structure was presented; The poses were rendered as
green line models. Hydrogen bonds were represented as dashed blue lines.
π-Interactions were
represented as a yellow solid line. All hydrogens were removed for the purposes of clarity.
3
. Conclusion
In the present study, a novel series of spirobenzo[h]chromene and spirochromane derivatives was
synthesized and screened for their anticancer potential against three cancer cell lines. Eight
compounds 7, 8e, 13a-e and 16 exhibited better anticancer activity range (IC50 = 1.78-5.47
that of sorafenib (IC = 3.64-6.21 M) or erlotinib (IC > 20 M). The observed resistance of these
cell lines to erlotinib was also reported by many previous studies [33-37]. Thus, the appreciable
µM) than
µ
µ
5
0
50
1
7

ACCEPTED MANUSCRIPT
anticancer activity of our novel compounds in these resistant cell lines is another advantage and
promotes their translational promise. Mechanistic study of some representative compounds 8e, 13c
and 16 was carried out against three anticancer targets; EGFR kinase, B-RAF kinase and Tubulin.
The three tested compounds showed better affinity against EGFR than B-RAF kinase. The results
showed more or less a close, yet significant IC50 values (IC50 = 1.4, 1.9 and 1.2 µM on EGFR and
2
.7, 2.9 and 2.6 on B-RAF kinase for compounds 8e, 13c and 16, respectively), if compared to
erlotinib. The hydrazine derivative of spirobenzochromenes 16 showed the most potent inhibitory
activity (IC = 1.2 M) against EGFR, suggesting preferential steric and electronic arrangements on
µ
5
0
EGFR kinase, compared to compounds 8e and 13c. Moreover, compounds 8e, 13c and 16 showed
moderate tubulin polymerization inhibition effects. Molecular docking studies were performed to
predict the possible binding patterns of the newly synthesized compounds into the ATP-active sites
of EGFR and B-RAF kinases. Together, the docking simulation study, along with the in vitro assay
results, demonstrated that this class of spiro compounds is promising EGFR and B-RAF dual
inhibitors with potential anticancer activity and good leads for further optimization.
4
. Experimental Protocols
.1. Chemistry
4
Chemical reagents and solvents were obtained from commercial sources. Solvents were dried
by standard methods when necessary. Elemental analyses were carried out at the
1
13
microanalytical center in the Faculty of Science, Cairo University. H NMR and C NMR
spectra were recorded with Bruker APX400 spectrometer at 400 MHz and 101 MHz,
6
respectively in DMSO-d . Chemical shifts were reported on the δ scale and J values were
given in Hz. The high-resolution mass spectra (HRMS) were recorded on Agilent 6230 Series
Accurate-Mass Time-Of-Flight (TOF) LC/MS. Thin layer chromatography (TLC) was done by
1
8

ACCEPTED MANUSCRIPT
silica gel plates 60 GF254, cellulose plates (20×20 cm) from Sigma-Aldrich company for
chemicals.
Spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-one (5) [38, 39]. A mixture of cyclohexanone
(4.89 g, 0.049 mol), 1'-Hydroxy-2'-Acetonaphthone (9.4 g, 0.049 mol) and pyrrolidine (8.4 mL,
0
.10 mol) in anhydrous CH OH (40 mL) was refluxed overnight. The reaction mixture was then
3
concentrated in vacuo and ethyl acetate (40 mL) was added. The whole mixture was washed with 1
N HCl, brine and dried over Na SO . The organic layer was evaporated and hexane (30 mL) was
2
4
added to the mixture. The resulting precipitate was filtered off and washed with hexane to give pale
1
yellow crystals of compound 5 (75%), mp 162–164°C. H NMR (400 MHz, DMSO-d )
δ 8.33 (d, J
6
=
8.3 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.71 (m, 2H), 7.64 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 8.6 Hz,
1H), 2.87 (s, 2H), 2.07 (d, J = 14.3 Hz, 2H), 1.78 – 1.66 (m, 3H), 1.62 – 1.52 (m, 3H), 1.39 – 1.22
1
3
(
m, 2H). C NMR (101 MHz, DMSO-d )
δ
191.96 (C=O), 157.19, 137.61, 130.16, 128.47, 127.12,
6
125.39, 123.38, 121.45, 120.62, 115.06, 81.63, 47.72, 34.28, 25.17, 21.77. HRMS Calcd. for
+
C H O [M+H] 266.1307, Found 267.1398. Anal. Calcd. for: C H O : C, 81.17; H, 6.81.
18
18
2
18 18
2
Found: C, 81.35; H, 6.51.
Methyl hydrazinecarbodithioate (6) [40-42]. A mixture of hydrazine hydrate (6 ml, 0.1 mol) in
isopropanol (10 ml) was added slowly with stirring to a cooled solution of KOH (6.6 g, 0.1 M)
water (7 ml). Subsequently, CS (10 mL, 0.1 mol) was added dropwise over 1 h to the above ice-
2
cooled solution while the temperature was maintained below 10 °C. The reaction mixture was
allowed to stir for another 1 h where a bright yellow color was obtained. At this point, an ice-cooled
iodomethane (7 ml, 0.1 mol) was added dropwise over 2 h and the stirring was continued for an
additional 1.5 h until the reaction furnished a white precipitate of methyl hydrazinecarbodithioate 6.
This precipitate was filtered off, washed with cold water, and recrystallized from dichloromethane
1
9

ACCEPTED MANUSCRIPT
+
to give colorless prism. Yield 51 %. m.p. 90-92 °C. HRMS Calcd. for C H N S [M+H] 121.9972,
2
6
2 2
Found 123.0070.
Methyl 2-(spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidene)hydrazinecarbodithioate (7)
.
Methyl hydrazinecarbodithioate 6 (0.65 g, 5.39 mmol) was added portionwise to a solution of
spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-one 5 (2.0 g, 5.39 mmol) in 40 ml of isopropanol
in the presence of a catalytic amount of conc. HCl. The reaction mixture was refluxed for 2 h until a
yellow solid began to precipitate. The solid was filtered off and washed with cold isopropanol and
1
air-dried to afford creamy yellow crystals of 3 (77%). H NMR (400 MHz, DMSO-d )
δ
8.31 (d, J =
6
8
1
1
.2 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.69 (m, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.47 (d, J = 8.5 Hz,
H), 6.89 (s, 1H, NH), 2.68 (s, 3H, SCH ), 2.32 (s, 2H), 1.77 – 1.64 (m, 4H), 1.59 – 1.51 (m, 4H),
3
+
.21 – 1.13 (m, 2H). HRMS Calcd. for C20
OS
General Procedure A for synthesis of compounds (8a-f)
The appropriate amine (0.02 mol) was added to a solution of methyl 2-(spiro[benzo[h]chromene-
,1'-cyclohexan]-4(3H)-ylidene)hydrazinecarbodithioate 7 (2.4 g, 0.02 mol) in 30 ml isopropanol
and the mixture was refluxed for 48 h until the complete evolution of methyl mercaptan which can
be detected by the yellow color produced with moistened Pb(OAc) paper. The reaction was also
H N
22 2
OS
2
[M+H] 370.1174, Found 371.1143. Anal.
Calcd. for: C20
H
22
N
2
2
: C, 64.83; H, 5.98; N, 7.56. Found: C, 65.03; H, 6.21; N, 7.84.
.
2
2
monitored by TLC to confirm its completion. The reaction mixture was then evaporated in vacuo
and the resulted precipitate was collected, washed with cold methanol and recrystallized from
ethanol to furnish the final compounds 8a-f in good yields.
N'-(Spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidene)piperidine-1-carbothiohydrazide
1
(
8a). General Procedure A, yellow solid (63 %). H NMR (400 MHz, DMSO-d )
δ
8.24 – 8.17 (m,
6
1
H), 7.91 (d, J = 8.8 Hz, 1H), 7.88 – 7.83 (m, 1H), 7.59 – 7.54 (m, 2H), 7.46 (d, J = 8.8 Hz, 1H),
.42 (s, 1H, NH), 3.84 (t, J = 7.5 Hz, 4H), 2.96 (s, 2H), 1.97 – 1.83 (m, 2H), 1.74 – 1.45 (m, 12H),
4
2
0

ACCEPTED MANUSCRIPT
1
3
1
.37 – 1.18 (m, 2H). C NMR (101 MHz, DMSO-d ) δ 182.55 (C=S), 135.23, 128.22, 128.09,
6
126.69, 126.48, 126.42, 125.75, 123.37, 122.36, 122.30, 120.71, 77.55, 56.51, 34.58, 26.32, 25.40,
+
2
4.40, 21.85, 18.99. HRMS Calcd. for C H N OS [M+H] 407.2031, Found 408.1487. Anal.
2
4
29
3
29 3
Calcd. for: C24H N OS: C, 70.73; H, 7.17; N, 10.31. Found: C, 70.61; H, 6.99; N, 10.57.
N'-(spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidene)piperazine-1-carbothiohydrazide
1
(
8b). General Procedure A, yellow solid (65 %). H NMR (400 MHz, DMSO-d )
δ
8.26 – 8.19 (m,
6
1H), 7.95 – 7.84 (m, 2H), 7.59 – 7.52 (m, 2H), 7.47 (d, J = 8.8 Hz, 1H), 4.05 (s, 1H, NH), 3.86 –
3.75 (m, 4H), 2.97 (s, 1H, NH), 2.83 – 2.76 (m, 4H), 2.37 (s, 2H), 1.98 – 1.83 (m, 2H), 1.75 – 1.44
+
(m, 6H), 1.40 – 1.20 (m, 2H). HRMS Calcd. for C23
H
28
N
4
OS [M+H] 408.1984, Found 409.2132.
28 4
Anal. Calcd. for: C23H N OS: C, 67.61; H, 6.91; N, 13.71. Found: C, 67.84; H, 6.53; N, 13.73.
N'-(spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidene)morpholine-4-carbothiohydrazide
1
(
8c). General Procedure A, dark yellow solid (67 %). H NMR (400 MHz, DMSO-d )
δ
8.22 (d, J =
6
8.2 Hz, 1H), 7.88 (m, 2H), 7.57 (m, 2H), 7.48 (d, J = 8.5 Hz, 1H), 4.40 (s, 1H, NH), 3.90 (t, J = 8.6
Hz, 2H), 3.69 (t, J = 8.6 Hz, 2H), 2.98 (s, 2H), 1.95 – 1.92 (m, 2H), 1.69 – 1.67 (m, 4H), 1.56 – 1.53
1
3
(
m, 4H), 1.39 – 1.22 (m, 2H). C NMR (101 MHz, DMSO-d
6
)
δ
183.12 (C=S), 135.32, 133.78,
1
2
28.25, 127.76, 126.73, 125.73, 124.90, 122.35, 121.51, 120.61, 78.74, 66.56, 35.69, 34.58, 25.41,
+
1.84, 19.00. HRMS Calcd. for C H N O S [M+H] 409.1824, Found 410.1932. Anal. Calcd. for:
2
3
27
3
2
C H N O S: C, 67.45; H, 6.65; N, 10.26. Found: C, 67.77; H, 6.31; N, 10.49.
23
27
3
2
4-Methyl-N'-(spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidene)piperazine-1-
1
carbothiohydrazide (8d). General Procedure A, yellow solid (68 %). H NMR (400 MHz, DMSO-
d
1
1
1
6
)
δ
8.22 (d, J = 9.4 Hz, 1H), 7.92 – 7.85 (m, 2H), 7.56 (m, 2H), 7.47 (d, J = 8.7 Hz, 1H), 4.42 (s,
H, NH), 3.88 (t, J = 8.4 Hz, 4H), 2.47 (s, 3H), 2.42 (t, J = 8.4 Hz, 4H), 1.91 (s, 2H), 1.61 (m, 8H),
1
3
.36 – 1.18 (m, 2H). C NMR (101 MHz, DMSO-d )
δ
182.92 (C=S), 135.56, 128.24, 128.16,
6
27.43, 126.74, 125.74, 123.84, 123.38, 122.41, 122.33, 118.42, 77.59, 56.51, 52.25, 45.98, 43.60,
2
1

ACCEPTED MANUSCRIPT
+
3
4.58, 25.41, 21.84, 18.99. HRMS Calcd. for C H N OS [M+H] 422.2140, Found 423.2288.
24 30 4
Anal. Calcd. for: C H N OS: C, 68.21; H, 7.16; N, 13.26. Found: C, 68.61; H, 6.97; N, 13.35.
2
4
30
4
(N-hexyl-2-(spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidene)hydrazinecarbothioamide
1
(
8e). General Procedure A, off-white crystals (63 %). H NMR (400 MHz, DMSO-d )
δ
8.64 (s, 1H,
6
NH), 8.29 (d, J = 8.7 Hz, 1H), 8.21 (d, J = 6.9 Hz, 1H), 7.87 (m, 1H), 7.58 – 7.51 (m, 2H), 7.44 (d,
J = 8.8 Hz, 1H), 4.40 (s, 1H, NH), 3.59 (t, J = 8.4 Hz, 2H), 1.88 (s, 2H), 1.72 – 1.49 (m, 10H), 1.29
1
3
(
m, 11H). C NMR (101 MHz, DMSO-d6)
δ
177.99 (C=S), 150.74, 142.26, 135.26, 128.15,
1
2
4
9
27.94, 126.49, 125.69, 122.51, 122.27, 120.26, 114.56, 77.41, 56.51, 44.15, 34.68, 31.52, 29.26,
+
33 3
6.54, 22.54, 21.85, 18.99, 14.40. HRMS Calcd. for C25H N OS [M-H] 423.2344, Found
22.4500. Anal. Calcd. for: C25
.99.
33 3
H N OS: 70.88; H, 7.85; N, 9.92. Found: C, 71.05; H, 7.61; N,
N-cyclohexyl-2-(spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-
ylidene)hydrazinecarbothioamide (8f). General Procedure A, pale yellow solid (61 %). H NMR
1
6
(400 MHz, DMSO-d ) δ 10.28 (s, 1H, NH), 8.19 (d, J = 8.8 Hz, 1H), 8.13 (d, J = 8.6 Hz, 1H), 7.86
(d, J = 6.8 Hz, 1H), 7.59 – 7.51 (m, 2H), 7.45 (d, J = 8.8 Hz, 1H), 4.47 (s, 1H, NH), 4.29 – 4.13 (m,
1
3
1
H), 2.97 (s, 2H), 1.94 – 1.84 (m, 4H), 1.79 – 1.42 (m, 12H), 1.34 – 1.16 (m, 4H). C NMR (101
176.84 (C=S), 150.85, 142.69, 135.28, 128.19, 128.05, 126.57, 125.77, 125.67,
22.29, 120.42, 114.38, 77.44, 56.54, 53.27, 34.65, 32.17, 25.55, 25.37, 21.83, 18.93. HRMS Calcd.
MHz, DMSO-d6)
δ
1
+
for C H N OS [M+H] 421.2188, Found 422.2091. Anal. Calcd. for: C H N OS: C, 71.22; H,
2
5
31
3
25 31
3
7.41; N, 9.97. Found: C, 71.05; H, 7.61; N, 9.81.
General procedure B for preparing N-phenylhydrazinecarboxamide (4-phenylsemicarbazide)
derivatives (9a-c) [43].
The appropriate phenylisocyanate was added slowly to a cold solution of equimolar amount of
hydrazine hydrate in ethanol. The mixture was allowed to stir in ethanol in ice-bath for 1 h. The
2
2

ACCEPTED MANUSCRIPT
obtained white precipitate was collected and washed with ethanol. The product was used directly for
the next step without any further purification.
1
N-phenylhydrazinecarboxamide (9a). General Procedure B, white solid (81 %). H NMR (400
MHz, DMSO-d6) δ 8.78 (s, 1H, NH), 7.98 (s, 1H, NH), 7.50 (d, J = 8.0 Hz, 2H), 7.26 (t, J = 7.9 Hz,
1
3
2
H), 6.96 (t, J = 7.4 Hz, 1H). C NMR (101 MHz, DMSO-d )
δ
156.50 (C=O), 140.15, 129.08,
6
122.30, 118.93.
1
N-(p-tolyl)hydrazinecarboxamide (9b). General Procedure B, white solid (79 %). H NMR (400
6
MHz, DMSO-d ) δ 8.66 (s, 1H, NH), 7.89 (s, 1H, NH), 7.36 (d, J = 8.2 Hz, 2H), 7.05 (d, J = 8.3 Hz,
1
3
2
H). C NMR (101 MHz, DMSO-d6)
δ
156.56 (C=O), 137.58, 131.05, 129.47, 118.99, 20.79
(CH3).
1
N-(4-Chlorophenyl)hydrazinecarboxamide (9b). General Procedure B, white solid (77 %). H NMR
(400 MHz, DMSO-d6)
δ
8.97 (s, 1H, NH), 8.31 (s, 1H, NH), 7.89 (d, J = 8.3 Hz, 2H), 7.26 (d, J =
1
3
8
.3 Hz, 2H). C NMR (101 MHz, DMSO-d ) 157.11 (C=O), 137.99, 132.13, 130.65, 119.46.
δ
6
General procedure C for preparing N-phenyl-2-(spiro[benzo[h]chromene-2,1'-cyclohexan]-
(3H)-ylidene)hydrazinecarboxamide derivatives (10a-c). N-phenylhydrazinecarboxamide
derivative 9 (1.88 mmol) was added to a solution of spiro[benzo[h]chromene-2,1'-cyclohexan]-
(3H)-one 5 (0.5 g, 1.88 mmol) in 40 ml of isopropanol in the presence of a catalytic amount of
4
4
TFA. The reaction mixture was refluxed for overnight and the organic mixture was concentrated
under reduced pressure. The formed solid was filtered off, washed with cold isopropanol and air-
dried to afford the final targeted compounds 10a-c (67-71%).
N-phenyl-2-(spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidene)hydrazinecarboxamide
1
(
10a). General Procedure C, yellow solid (69 %). H NMR (400 MHz, DMSO-d )
δ
9.89 (s, 1H,
6
NH), 8.33 (d, J = 8.8 Hz, 1H), 7.97 (s, 1H, NH), 7.71 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 7.7 Hz, 3H),
.57 – 7.52 (m, 2H), 7.32 (t, J = 7.9 Hz, 2H), 7.04 (t, J = 7.4 Hz, 1H), 2.37 (s, 2H), 1.97 – 1.88 (m,
7
2
3

ACCEPTED MANUSCRIPT
1
3
1
1
1
3
7
H), 1.75 – 1.49 (m, 10H), 1.42 – 1.17 (m, 1H). C NMR (101 MHz, DMSO-d ) δ 156.52 (C=O),
6
46.20, 145.85, 145.75, 140.08, 132.34, 129.11, 128.98, 128.15, 122.39, 121.43, 120.64, 119.00,
+
18.96, 114.83, 113.39, 73.94, 56.53, 21.91, 18.95. HRMS calcd. for C H N O [M+H]
25
25
3
2
25 3 2
99.1947, Found 400.2048. Anal. Calcd. for: C25H N O : C, 75.16; H, 6.31; N, 10.52. Found: C,
4.93; H, 6.77; N, 10.37.
2-(Spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidene)-N-(p-tolyl)hydrazinecarboxamide
1
(
10b). General Procedure C, yellow solid (70 %). H NMR (400 MHz, DMSO-d
6
)
δ
10.13 (s, 1H,
NH), 8.30 (d, J = 8.7 Hz, 1H), 7.87 (s, 1H, NH), 7.69 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 7.6 Hz, 2H),
7
.58 – 7.53 (m, 2H), 7.32 – 7.23 (m, 2H), 7.11 (d, J = 7.6 Hz, 2H), 2.60 (s, 3H, CH ), 2.31 (s, 2H),
3
1
.93 – 1.84 (m, 2H), 1.73 – 1.51 (m, 6H), 1.44 – 1.10 (m, 2H). HRMS Calcd. for C H N O
2
2
6
27
3
+
[
M+H] 413.2103, Found 414.2586. Anal. Calcd. for: C H N O : C, 75.52; H, 6.58; N, 10.16.
26 27 3 2
Found: C, 75.78; H, 6.38; N, 9.83.
N-(4-chlorophenyl)-2-(spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-
ylidene)hydrazinecarboxamide (10c). General Procedure C, Off-white solid (62 %). H NMR (400
MHz, DMSO-d 9.88 (s, 1H, NH), 8.31 (d, J = 8.6 Hz, 1H), 7.93 (s, 1H, NH), 7.59 (d, J = 8.6 Hz,
H), 7.67 (d, J = 7.7 Hz, 2H), 7.60 – 7.56 (m, 2H), 7.35 – 7.24 (m, 2H), 7.17 (d, J = 7.7 Hz, 2H),
1
6
) δ
1
+
2.38 (s, 2H), 1.89 – 1.53 (m, 8H), 1.44 – 1.10 (m, 2H). HRMS Calcd. for C25
3 2
H24ClN O [M+H]
4
33.1557, Found 434.1577. Anal. Calcd. for: C H ClN O : C, 69.20; H, 5.57; Cl, 8.17; N, 9.68.
2
5
24
3
2
Found: C, 69.31; H, 5.90; N, 9.37.
Spiro[chroman-2,1'-cyclohexan]-4-one (11a) [34, 41]. A mixture of cyclohexanone (4.89 g, 49
mmol), 2-hydroxyacetophenone (6.8 g, 49.8 mmol) and pyrrolidine (8.4 ml, 100 mmol) in methanol
(40 ml) was refluxed overnight. The reaction mixture was then concentrated in vacuo and ethyl
acetate (40 ml) was added. The whole mixture was washed with 1 N HCl, 1 N NaOH, brine and
2
4

ACCEPTED MANUSCRIPT
dried over Na SO . The organic layer was evaporated under reduced pressure to give a colorless oil
2
4
+
of compound 11a (83%). HRMS Calcd. for C H O [M+H] 216.1150, Found 217.1612.
1
4
16
2
Spiro[chroman-2,1'-cycloheptan]-4-one (11b) [26, 44]. A mixture of cycloheptanone (5.58 g, 49.8
mmol), 2-hydroxyacetophenone (6.8 g, 49.8 mmol) and pyrrolidine (8.4 ml, 100 mmol) in methanol
(40 ml) was refluxed overnight. The reaction mixture was then concentrated in vacuo and ethyl
acetate (40 ml) was added. The whole mixture was washed with 1 N HCl, 1 N NaOH, brine and
dried over Na
2
SO
4
. The organic layer was evaporated under reduced pressure to give a dark yellow
+
oil of compound 11b (69%). HRMS Calcd. for C15
H
18
O
2
[M+H] 230.1307, Found 231.1792.
General procedure D for preparing spiro[chroman-2,1'-cycloalkan]-4-ylidenehydrazine (12a,b).
Hydrazine hydrate (3.6 mmol) was added to a solution of spiro[chroman-2,1'-cycloalkan]-4-one 11
(3 mmol) in ethanol (20 ml) and the mixture was left to stir at room temperature for 3 h. The
reaction mixture was concentrated in vacuo, water was added and extracted with ethyl acetate (30
ml) and then evaporated under reduced pressure to give a colorless oil from the final intermediates
spiro[chroman-2,1'-cycloalkan]-4-ylidenehydrazine 12a,b in good yields which were used directly
for the next step without any further purification.
General procedure E for preparing spiro[chroman-2,1'-cycloalkan]-4-ylidenehydrazine (13a-c)
The appropriate phenyl isocyanate derivative (2 mmol) was added to a solution of spiro[chroman-
,1'-cycloalkan]-4-ylidenehydrazine 12a,b (2 mmol) in ethanol (20 ml). The reaction mixture was
.
2
allowed to stir at room temperature for overnight. The organic volatiles were removed in vacuo and
then water was added. The resulted mixture was extracted using ethyl acetate (40ml). The organic
layer was separated and dried over sodium sulfate and then evaporated under reduced pressure.
Petroleum ether was added to the residue to furnish the final compounds 13a-c in good yields as
off-white solids.
2
5

ACCEPTED MANUSCRIPT
N-phenyl-2-(spiro[chroman-2,1'-cyclohexan]-4-ylidene)hydrazinecarboxamide (13a)
.
General
1
Procedure E, Off-white solid (62 %). H NMR (400 MHz, DMSO-d )
δ 9.92 (s, 1H, NH), 8.25 (d, J
6
=
7.9 Hz, 1H), 7.65 (d, J = 7.7 Hz, 2H), 7.34 – 7.21 (m, 3H), 7.03 (t, J = 7.3 Hz, 1H), 6.96 (t, J =
7
1
1
1
3
1
.5 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 4.35 (s, 1H, NH), 2.83 (s, 2H), 1.80 – 1.67 (m, 2H), 1.65 –
1
3
6
.55 (m, 2H), 1.51 (t, J = 9.8 Hz, 5H), 1.41 – 1.32 (m, 1H). C NMR (101 MHz, DMSO-d ) δ
54.44 (C=O), 153.99, 140.60, 139.45, 131.27, 128.93, 125.32, 123.03, 121.00, 120.58, 120.53,
+
18.06, 76.47, 56.50, 34.92, 25.41, 21.66. HRMS Calcd. for C H N O [M+H] 349.1790, Found
2
1
23
3
2
50.1892. Anal. Calcd. for: C H N O : C, 72.18; H, 6.63; N, 12.03. Found: C, 71.93; H, 6.89; N,
2
1
23
3
2
1.73.
2
-(Spiro[chroman-2,1'-cyclohexan]-4-ylidene)-N-(p-tolyl)hydrazinecarboxamide (13b)
.
General
1
Procedure E, Off-white solid (59 %). H NMR (400 MHz, DMSO-d )
δ 9.84 (s, 1H, NH), 7.51 (d, J
6
=
=
8.3 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H), 7.06 (t, J = 6.7 Hz, 1H), 6.95 (t, J = 7.5 Hz, 1H), 6.87 (d, J
8.2 Hz, 1H), 4.41 (s, 1H, NH), 2.80 (s, 1H), 2.27 (s, 3H, CH ), 1.74 – 1.70 (m, 2H), 1.60 – 1.56
3
1
3
(
6
m, 2H), 1.51 – 1.48 (m, 5H), 1.39 – 1.21 (m, 1H). C NMR (101 MHz, DMSO-d ) δ 154.40
(C=O), 140.49, 136.82, 131.95, 131.26, 129.75, 129.36, 125.27, 121.02, 120.61, 118.89, 118.06,
+
7
3
1
6.45, 56.52, 34.89, 25.39, 21.64, 20.86. HRMS Calcd. for C H N O [M+H] 363.1947, Found
2
2
25
3
2
64.2045. Anal. Calcd. for: C H N O : C, 72.70; H, 6.93; N, 11.56. Found: C, 73.03; H, 6.77; N,
2
2
25
3
2
1.47.
N-(4-chlorophenyl)-2-(spiro[chroman-2,1'-cyclohexan]-4-ylidene)hydrazinecarboxamide
(13c).
1
General Procedure E, Off-white solid (61 %). H NMR (400 MHz, DMSO-d )
δ 9.99 (s, 1H, NH),
6
7
6
1
1
.72 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 8.8 Hz, 2H),
.96 (t, J = 7.5 Hz, 1H), 6.90 – 6.85 (m, 1H), 4.36 (s, 1H, NH), 2.83 (s, 2H), 1.77 – 1.68 (m, 2H),
1
3
.65 – 1.44 (m, 7H), 1.43 – 1.29 (m, 1H). C NMR (101 MHz, DMSO-d
6
) δ 154.48 (C=O), 140.94,
38.55, 131.35, 129.09, 128.75, 126.62, 125.43, 122.05, 120.96, 120.50, 118.06, 76.49, 56.50,
2
6

ACCEPTED MANUSCRIPT
+
3
4.90, 25.40, 21.65. HRMS Calcd. for C H ClN O [M+H] 383.1401, Found 384.1486. Anal.
2
1
22
3
2
Calcd. for: C H ClN O : C, 65.71; H, 5.78; N, 10.95. Found: C, 65.99; H, 6.01; N, 11.13.
2
1
22
3
2
N-phenyl-2-(spiro[chroman-2,1'-cycloheptan]-4-ylidene)hydrazinecarboxamide (13d)
.
General
1
Procedure E, Off-white solid (59 %). H NMR (400 MHz, DMSO-d )
δ 8.93 (s, 1H, NH), 7.56 (dd,
6
J = 8.1, 1.4 Hz, 1H), 7.51 (d, J = 7.7 Hz, 2H), 7.31 (t, J = 7.9 Hz, 2H), 7.25 (t, J = 7.7 Hz, 1H), 7.01
(t, J = 7.4 Hz, 1H), 6.91 – 6.85 (m, 2H), 4.37 (s, 1H, NH), 2.84 (s, 2H), 2.40 – 2.37 (m, 4H), 1.89 –
1
3
1
6
.84 (m, 2H), 1.76 – 1.49 (m, 4H), 1.47 – 1.18 (m, 2H). C NMR (101 MHz, DMSO-d ) δ 158.37
(C=O), 152.42, 150.84, 139.65, 130.74, 129.37, 128.33, 122.73, 120.71, 118.99, 118.81, 117.46,
+
8
3
1
0.64, 56.50, 29.33, 21.92, 19.02. HRMS Calcd. for C H N O [M+H] 363.1947, Found
2
2
25
3
2
64.2046. Anal. Calcd. for: C H N O : C, 72.70; H, 6.93; N, 11.56. Found: C, 72.58; H, 7.10; N,
2
2
25
3
2
1.47.
2-(Spiro[chroman-2,1'-cycloheptan]-4-ylidene)-N-(p-tolyl)hydrazinecarboxamide (13e). General
1
Procedure E, Off-white solid (57 %). H NMR (400 MHz, DMSO-d )
δ 9.61 (s, 1H, NH), 7.49 (d, J
6
=
8.1 Hz, 1H), 7.40 (d, J = 7.2 Hz, 4H), 7.18 – 7.13 (m, 2H), 6.90 – 6.88 (m, 1H), 4.45 (s, 1H, NH),
1
3
2.80 (s, 2H), 2.37 (s, 3H, CH
3
), 2.33 – 2.22 (m, 6H), 1.84 – 1.19 (m, 6H). C NMR (101 MHz,
DMSO-d
6
)
δ
156.83 (C=O), 134.56, 131.24, 129.36, 129.11, 126.18, 125.27, 124.81, 124.49,
27 3 2
124.43, 119.17, 117.34, 85.84, 56.52, 37.82, 29.09, 20.99, 18.96. HRMS Calcd. for C23H N O
+
[
M+H] 377.2103, Found 378.2440. Anal. Calcd. for: C H N O : C, 73.18; H, 7.21; N, 11.13.
23 27 3 2
Found: C, 72.90; H, 6.99; N, 10.88.
Spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidenehydrazine (14). A mixture of hydrazine
hydrate (3.6 mmol) and spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-one 5 (3 mmol) in ethanol
(20 ml) and a catalytic amount of acetic acid was refluxed for 6 h. The reaction mixture was then
concentrated in vacuo and the separated precipitate was filtered off and washed with water to afford
2
7

ACCEPTED MANUSCRIPT
the spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidenehydrazine 14 as a yellow solid in good
yield. This compound was used directly in the next reaction without any further purification.
2-(Spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylideneamino)isoindoline-1,3-dione (15). A
mixture of spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidenehydrazine 14 (0.5 g, 1.78 mmol)
and phthalic anhydride (0.26 g, 1.78 mmol) and anhydrous sodium acetate (0.19 g, 2.30 mmol) in
gl. acetic acid (20 ml) was heated under reflux for overnight. The produced precipitate was
collected and washed with water several times to give 2-(spiro[benzo[h]chromene-2,1'-cyclohexan]-
1
4
(3H)-ylideneamino)isoindoline-1,3-dione 15 as yellow solid (77%). H NMR (400 MHz, DMSO-
d )
6
δ
8.28 – 8.16 (m, 4H), 7.91 (d, J = 9.0 Hz, 2H), 7.73 – 7.36 (m, 4H), 2.90 (s, 2H), 2.04 – 1.99
+
(
m, 2H), 1.71 – 1.42 (m, 6H), 1.34 – 1.17 (m, 2H). HRMS Calcd. for C H N O [M+H]
26 22 2 3
410.1630, Found 411.1680. Anal. Calcd. for: C H N O : C, 76.08; H, 5.40; N, 6.82. Found: C,
26 22 2 3
76.57; H, 5.81; N, 7.03.
N'-(Spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidene)adamantane-1-carbohydrazide (16)
.
Adamantyl carbonyl chloride (0.46 g, 2.31 mmol) was added to a solution of
spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidenehydrazine 14 (0.5 g, 1.78 mmol) in THF
(20 ml) in the presence of catalytic amount of triethyl amine. The reaction mixture was heated under
reflux for 2 h then it was concentrated under reduced pressure. Water (40 ml) was added and the
resulted precipitated was collected. The yellow solid was then crystallized from ethanol to afford
1
compound 16 as yellow solid (63%). H NMR (400 MHz, DMSO-d )
δ 8.34 (d, J = 8.6 Hz, 1H),
6
8.25 (d, J = 6.9 Hz, 1H), 7.85 (m, 1H), 7.63 – 7.51 (m, 2H), 7.44 (d, J = 8.6 Hz, 1H), 2.83 (s, 2H),
2.31 – 2.15 (m, 4H), 1.79 – 1.62 (m, 10H), 1.59 – 1.47 (m, 8H), 1.31 – 1.16 (m, 3H). HRMS Calcd.
+
for C H N O [M+H] 442.2620, Found 443.2698. Anal. Calcd. for: C H N O : C, 78.70; H,
2
9
34
2
2
29 34
2
2
7.74; N, 6.33. Found: C, 78.62; H, 8.05; N, 6.47.
2
8

ACCEPTED MANUSCRIPT
3-((E)-Spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidenehydrazono)indolin-2-one (17). A
mixture of spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidenehydrazine 14 (0.5 g, 1.78 mmol)
and isatin (0.26 g, 1.78 mmol) and anhydrous sodium acetate (0.19 g, 2.30 mmol) in gl. acetic acid
(20 ml) was heated under reflux for overnight. The reaction mixture was allowed to cool and then
poured into ice- water (100 ml). The formed precipitate was collected and washed with water to
afford 3-((E)-Spiro[benzo[h]chromene-2,1'-cyclohexan]-4(3H)-ylidenehydrazono)indolin-2-one 17
1
as dark yellow solid (73%). H NMR (400 MHz, DMSO-d )
δ 8.81 (s, 1H, NH), 8.35 – 8.13 (m,
6
4H), 7.91 – 7.65 (m, 2H), 7.45 – 7.21 (m, 4H), 2.87 (s, 2H), 1.98 – 1.90 (m, 2H), 1.82 – 1.51 (m,
+
6
H), 1.39 – 1.08 (m, 2H). HRMS Calcd. for C H N O [M+H] 409.1790, Found 410.1933. Anal.
2
6
23
3
2
Calcd. for: C H N O : C, 76.26; H, 5.66; N, 10.26. Found: C, 75.97; H, 5.31; N, 10.45.
2
6
23
3
2
4
.2. Biological Screening
.2.1. Cell Culture
4
The used cancer cell lines in this study; MCF-7 (human breast carcinoma), HT-29 (human
colorectal adenocarcinoma) and A549 (human lung carcinoma) were obtained from European
Collection of Cell Cultures (ECACC, UK). The normal fibroblasts (F180) were kindly provided by
professor Ekkehard Dikomey (University Cancer Center, Hamburg University, Hamburg,
Germany). Cancer cell lines were cultured in Roswell Park Memorial Institute medium (RPMI,
Sigma-Aldrich, St. Louis, MO, USA) while the fibroblast cells were maintained in Minimum
Essential Media (MEM). All media were supplemented with 10% fetal bovine serum (FBS, Sigma-
o
Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich). All incubations were done at 37 C in a
humidified atmosphere of 5 % CO
.2.2. Cell viability analysis
Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTT) assay as described before assay [28, 29]. In brief, cancer cells or normal fibroblast cells
2
.
4
(
2
9