Accepted Manuscript Synthesis and anticancer activity of some 8-substituted-7-methoxy-2H-chromen-2one derivatives toward hepatocellular carcinoma HepG2 cells Kamilia M. Amin , Sahar M. Abou-Seri , Fadi M. Awadallah , Amal A.M. Eissa , Ghaneya S. Hassan , Mohamed M. Abdulla PII:
S0223-5234(14)01059-9
DOI:
10.1016/j.ejmech.2014.11.027
Reference:
EJMECH 7520
To appear in:
European Journal of Medicinal Chemistry
Received Date: 1 October 2014 Revised Date:
4 November 2014
Accepted Date: 14 November 2014
Please cite this article as: K.M. Amin, S.M. Abou-Seri, F.M. Awadallah, A.A.M. Eissa, G.S. Hassan, M.M. Abdulla, Synthesis and anticancer activity of some 8-substituted-7-methoxy-2H-chromen-2-one derivatives toward hepatocellular carcinoma HepG2 cells, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.11.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical abstract:
H3CO
O
O
H3CO
O
N
N N R
O
NH
Ar NH2
O
Ar
SC
R= H, C6H5, COCH3, CONH2
RI PT
Four series of coumarin-pyrazoline hybrids, in addition to a series of coumarins bearing non-cyclic isosteres of pyrazolines, were synthesized. Cytotoxicity against HepG2 cell line was evaluated. Telomerase inhibition and apoptosis induction were examined for the most active compounds.
Ar= thiophen-2-yl, phenyl, p-(methyl)phenyl, p-(trifluoromethyl)phenyl, p-(methylthio)phenyl.
7a-e - 10a-e
AC C
EP
TE D
M AN U
2% G2 p. e6 H8 t7 s= n ie as gr a ae Mm no l e t f o . h n i
7e: IC50 = 10
11a-e
Synthesis and anticancer activity of some 8-substituted-7-methoxy-2HACCEPTED MANUSCRIPT chromen-2-one derivatives toward hepatocellular carcinoma HepG2 cells. Kamilia M. Amin,a Sahar M. Abou-Seri,a Fadi M. Awadallah,*a Amal A.M. Eissa,a Ghaneya S. Hassan,a Mohamed M. Abdullab a
Pharmaceutical Chemistry department, Faculty of Pharmacy, Cairo University, Kasr El-Aini street 11562,
b
Research Unit, Saco Pharm. Co., 6th October City, Egypt
SC
Abstract.
RI PT
Cairo Egypt.
Based on the reported anticancer activity of coumarin and pyrazoline derivatives, the present investigation dealt with the design and synthesis of coumarin derivatives bearing
M AN U
diversely substituted pyrazoline moieties 7-10. The non-cyclic isosteres 11a-e of compounds 10a-e were synthesized for comparative reasons. The target compounds were synthesized from 8-acetyl-7-methoxycoumarin that underwent Claisen-Schmidt condensation with various aldehydes to give the chalcones 6a-e, followed by reaction with hydrazine hydrate, phenyl hydrazine or semicarbazide under the appropriate conditions. Cytotoxicity of the synthesized
TE D
compounds was evaluated in vitro against liver HepG2 cell lines. Compounds were active in the nanomolar range. The most active compounds were investigated for their telomerase inhibition and proapoptotic activities.
AC C
EP
Keywords: Coumarin; pyrazoline; synthesis; anticancer activity; liver cancer.
*
Corresponding author: [email protected] Tel: 002-02-25353400, 002-012-23483941 Fax:002-02-23628426
1
ACCEPTED MANUSCRIPT 1. Introduction. It is observed that telomerase is up-regulated in 80−90% of all cancer cells isolated from primary human tumors but is absent in neighboring normal cells. This resulted in significant efforts to validate telomerase as an anticancer drug target and to develop effective approaches toward its inhibition. When considering anticancer drug targets, several
RI PT
advantages for targeting telomerase and telomere maintenance exist when compared to other targets and pathways. Most important is the apparently universal requirement for telomere maintenance in cancer cells including putative cancer stem cells [1]. Telomerase remains active in the early stages of life maintaining telomere length and the chromosomal integrity of
SC
frequently dividing cells. It turns dormant in most somatic cells during adulthood [2,3]. However telomerase gets reactivated in the vast majority of invasive cancers. Extension of
M AN U
telomeres by telomerase is required for un-limited proliferation and it is well accepted that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression [4,5]. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction [6]. Thus, the inhibition of telomerase activity is a potential target for cancer therapeutic discovery which might have minimal side effects.
TE D
In the course of identifying various chemical substances which may serve as leads for designing new anticancer agents, the present work particularly emphasizes on the coumarin and pyrazoline derivatives which have been identified as cores for chemotherapeutic agents
EP
with significant therapeutic efficacy against tumors. Coumarin comprised the nucleus of a wide variety of naturally occurring compounds.
AC C
They were extremely variable in structure, due to various types of substitutions in their basic structure, which could influence their biological activity. Literature survey revealed their wide spectrum and diversity of biological activities [7-16], particularly their cytotoxic activity against numerous types of cancers [17- 24]. Several studies revealed the mechanism of anticancer activity of coumarin derivatives which might include induction of apoptosis [2531]. Osthole I, a bioactive simple coumarin derivative extracted from Cnidium monnieri (L) Cusson, proved effective against human hepatocellular carcinoma (HCC) through induction of apoptosis [32]. Additionally, the coumarin-monastrol hybrid II demonstrated anticancer efficacy against many cancer cell lines mediated by apoptotic activity [33] (Figure 1). 2
On the other hand, pyrazoline-based compounds received considerable attention
ACCEPTED MANUSCRIPT
owing to their high chemotherapeutic potential. Diversely substituted pyrazoline derivatives showed good cytotoxic and antiproliferative activities against a wide range of human tumor cell lines [34-40]. This could be exemplified by the 3,5-diarylsubstituted pyrazolines III [41]
RI PT
and IV [42]. (Figure 2).
Moreover, it was pointed out that introducing the dihydropyrazole moiety in the coumarin skeleton, as illustrated by compounds V [43] and VI [44], gave highly potent
of apoptosis. (Figure 3).
M AN U
SC
cytotoxic compounds that were believed to act through inhibition of telomerase and induction
The current strategy in developing new anticancer drugs shifted toward the multiple mechanistic approach and several drugs have been validated and developed. Inspired by the inherent biological relevance of the coumarin and pyrazoline rings, the present investigation pertained to the hybridization of these two pharmacophoric moieties, in a single molecule. Expectedly, the additive effect of this combination might produce compounds of high
TE D
anticancer activity that might act by dual mechanism through induction of apoptosis and inhibition of telomerase. The target compounds had the general structure 7a-e - 9a-e, featuring a 7-methoxycoumarin nucleus substituted at position 8 with a 5-arylsubstitutedpyrazoline group. The pyrazoline ring was either unsubstituted at position 1, or carrying a
EP
phenyl group or an acetyl group. Isosteric replacement of the N-acetyl group with the carboxamide function gave compounds of general formula 10a-e. A cyclic versus noncyclic
AC C
bioisosteric modification of compounds 10a-e was also adopted to obtain the noncyclic bioisosteres 11a-e (Figure 4).
Antiproliferative activity of the synthesized compounds was evaluated in vitro against
human liver cell line HepG2. Telomerase inhibition and apoptosis induction of the most active compounds were also investigated. 2. Results and Discussions 2.1. Chemistry 3
The synthesis of the target compounds 7-11 was depicted in Schemes 1 and 2. 8-
ACCEPTED MANUSCRIPT
Acetyl-7-hydroxy-2H-chromen-2-one 4 was obtained together with its regioisomer 3 through a Fries rearrangement of the intermediate acetoxy derivative 2, which was obtained by acetylation of the commercially available 7-hydroxycoumarin 1 with acetic anhydride. The regioisomers 3 and 4 were separated by fractional crystallization from aqueous ethanol [46]. Methylation of 4 with methyl iodide in the presence of potassium carbonate in dry acetone gave the corresponding 8-acetyl-7-methoxy-2H-chromen-2-one 5 [47-49]. The chalcones 6
RI PT
were prepared by conventional Claisen-Schmidt condensation reaction of 5 with (un)substituted-arylaldehydes in the presence of 10 % sodium hydroxide in ethanol (Scheme 1) [50].
SC
The synthesis of the novel coumarin-pyrazoline hybrids 7-10 was accomplished
M AN U
through 1,4-addition of hydrazine hydrate, phenyl hydrazine or semicarbazide to the α,βunsaturated carbonyl system of the precursor chalcones 6, followed by dehydration and rearrangement (Scheme 2). The N1 unsubstituted and N1 phenyl pyrazolines 7 and 8 were produced by cyclocondensation of the chalcones 6 with hydrazine hydrate 99% or phenyl hydrazine in absolute ethanol, respectively. Alternatively, condensation of 6 with hydrazine hydrate in absolute ethanol in the presence of glacial acetic acid furnished the N1 acetyl
TE D
pyrazoline derivatives 9 as a result of in situ acetylation of the initially formed pyrazolines 7. In addition, base catalyzed cyclization of chalcones 6 into the corresponding pyrazoline-1carboxamide 10 was achieved through reaction with semicarbazide HCl in ethanolic sodium hydroxide. On the other hand, reacting 6 with semicarbazide HCl in absolute ethanol yielded
AC C
EP
the uncyclic allylidene semicarbazide derivatives 11.
The structure elucidation of the newly synthesized compounds was based on the
analytical and spectral data (IR, 1H NMR,
13
C NMR, and mass spectrometry). 1H NMR
spectra of most pyrazolines 7-10 exhibited an AMX pattern for the presence of two diastereotopic protons at C-4 (HA and HM) and one single proton at the C-5 positions (HX). These protons appeared as three doublets of doublets, respectively, at δ 2.90-3.35, 3.46-3.89, 4.91-5.63 ppm regions, each integrating for one proton. In case of compound 8a simple splitting patterns was observed with pyrazoline protons, which appeared as two signals, one doublet equivalent to 2H at δ 3.73 ppm and a multiplet equivalent to 1H at δ 5.75 ppm. Moreover, the 1H NMR spectra of compounds 7 and 10 displayed singlet exchangeable signal at δ 4.20-5.37 or δ 10.20-11.05 ppm corresponding to the pyrazoline (NH) or the N1 4
carboxamide (NH2), respectively. Meanwhile, the analogs 9 showed singlet signal derived
ACCEPTED MANUSCRIPT
from the N1 acetyl (CH3) at δ 2.18-2.37 ppm. On the other hand, the 1H NMR spectra of the allylidene semicarbazide derivatives 11 revealed the presence of two exchangeable singlet signals resonating at δ 6.47-6.54 and 8.88-9.10 ppm for the semicarbazide (NH2 and NH) protons and two doublets at δ 6.05-6.74 and 6.85-7.29 ppm (J= 15.0-16.9 Hz) representing the allylidene (CH=CH) protons, which asserted the production of the uncyclized derivatives 11. The protons belonging to the aromatic system and phenyl substituents were observed at the
RI PT
expected chemical shifts and integral values. 2.2. Anticancer activity. 2.2.1. Antiproliferative activity study.
SC
The in vitro anti-hepatocellular carcinoma (anti-HCC) activity of the newly synthesized compounds was examined against HepG2 cell line using the MTT method [51].
M AN U
Doxorubicin which is one of the most effective anticancer agents was used as positive control. The activity was expressed by median growth inhibitory concentration (IC50) and was provided in Table 1. The reported antiproliferative activity of Osthole I [32] and the lead compound VI [44] was included for comparison. The results revealed that, all the new coumarin-pyrazoline hybrids exhibited potent antiproliferative activity with IC50 values ranging from 10 ̶ 99 nM, which were far more potent than Osthole I (IC50= 161.4µM) and
TE D
compound VI (IC50= 6.64µM). In particular compounds 7a, 7d, 7e, 8a and 10e possessed superior activity at low nanomolar levels (IC50 = 11, 13, 10, 15 and 18 nM, respectively) The N1-unsubstituted pyrazoline derivatives 7a-e displayed excellent growth
EP
inhibitory activity (IC50 = 10 ̶ 48 nM). Compound 7b having phenyl substituent at position 5 of the pyrazoline ring was the least active member of this group (IC50 = 48 nM). Appending a
AC C
lipophilic substituent to the p-position of the phenyl in 7d-e contributed to an increase in potency, where the highest activity was associated with the 4-trifluoromethylphenyl 7d and 4methylthiophenyl 7e congeners (IC50 = 13 and 10 nM, respectively). Similarly, isosteric replacement of the phenyl ring with the thienyl rest in 7a resulted in about 4 fold increase in growth inhibition.
Substitution at N1 of the pyrazoline ring with a phenyl group, 8a-e, led to significant drop in activity (IC50 = 15 ̶ 78 nM), with the 5-thienylpyrazoline derivative, 8a, having the highest activity in this series. Likewise, compounds bearing the N1-acetyl group, 9a-e and the N1-carboxamide derivatives 10a-e revealed a decrease in activity compared to the N1unsubstituted pyrazolines 7a-e. Moreover, the allylidene semicarbazides 11a-e, which could 5
be considered as the non-cyclic isosteres of 10a-e, showed reduced growth inhibitory effect
ACCEPTED MANUSCRIPT
(IC50 = 75 ̶ 99 nM). All compounds in this series demonstrated the lowest activity compared to their N1-un/substituted pyrazoline counterparts 7-10. In summary, the antiproliferative activity of the novel coumarin-pyrazoline hybrids was influenced by the substitution pattern on the pyrazoline ring. Mostly, the introduction of a more lipophilic substituent to position 5 of the pyrazoline ring produced compounds with enhanced potency as shown by the 4-trifluoromethylphenyl 7d and 4-methylthiophenyl
RI PT
analogs 7e and 10e. Also, isosteric replacement of the phenyl ring with the thienyl group as in 7a and 8a enhanced the activity. However, the activity was not favored by the presence of N1substitutions as in 8a-e, 9a-e and 10a-e, which was less active than the N1-unsubstituted derivatives, 7a-e. Furthermore, a noticeable drop in potency was observed with the coumarin
2.2.2. Telomerase inhibitory activity.
M AN U
S0223-5234(14)01059-9
DOI:
10.1016/j.ejmech.2014.11.027
Reference:
EJMECH 7520
To appear in:
European Journal of Medicinal Chemistry
Received Date: 1 October 2014 Revised Date:
4 November 2014
Accepted Date: 14 November 2014
Please cite this article as: K.M. Amin, S.M. Abou-Seri, F.M. Awadallah, A.A.M. Eissa, G.S. Hassan, M.M. Abdulla, Synthesis and anticancer activity of some 8-substituted-7-methoxy-2H-chromen-2-one derivatives toward hepatocellular carcinoma HepG2 cells, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.11.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical abstract:
H3CO
O
O
H3CO
O
N
N N R
O
NH
Ar NH2
O
Ar
SC
R= H, C6H5, COCH3, CONH2
RI PT
Four series of coumarin-pyrazoline hybrids, in addition to a series of coumarins bearing non-cyclic isosteres of pyrazolines, were synthesized. Cytotoxicity against HepG2 cell line was evaluated. Telomerase inhibition and apoptosis induction were examined for the most active compounds.
Ar= thiophen-2-yl, phenyl, p-(methyl)phenyl, p-(trifluoromethyl)phenyl, p-(methylthio)phenyl.
7a-e - 10a-e
AC C
EP
TE D
M AN U
2% G2 p. e6 H8 t7 s= n ie as gr a ae Mm no l e t f o . h n i
7e: IC50 = 10
11a-e
Synthesis and anticancer activity of some 8-substituted-7-methoxy-2HACCEPTED MANUSCRIPT chromen-2-one derivatives toward hepatocellular carcinoma HepG2 cells. Kamilia M. Amin,a Sahar M. Abou-Seri,a Fadi M. Awadallah,*a Amal A.M. Eissa,a Ghaneya S. Hassan,a Mohamed M. Abdullab a
Pharmaceutical Chemistry department, Faculty of Pharmacy, Cairo University, Kasr El-Aini street 11562,
b
Research Unit, Saco Pharm. Co., 6th October City, Egypt
SC
Abstract.
RI PT
Cairo Egypt.
Based on the reported anticancer activity of coumarin and pyrazoline derivatives, the present investigation dealt with the design and synthesis of coumarin derivatives bearing
M AN U
diversely substituted pyrazoline moieties 7-10. The non-cyclic isosteres 11a-e of compounds 10a-e were synthesized for comparative reasons. The target compounds were synthesized from 8-acetyl-7-methoxycoumarin that underwent Claisen-Schmidt condensation with various aldehydes to give the chalcones 6a-e, followed by reaction with hydrazine hydrate, phenyl hydrazine or semicarbazide under the appropriate conditions. Cytotoxicity of the synthesized
TE D
compounds was evaluated in vitro against liver HepG2 cell lines. Compounds were active in the nanomolar range. The most active compounds were investigated for their telomerase inhibition and proapoptotic activities.
AC C
EP
Keywords: Coumarin; pyrazoline; synthesis; anticancer activity; liver cancer.
*
Corresponding author: [email protected] Tel: 002-02-25353400, 002-012-23483941 Fax:002-02-23628426
1
ACCEPTED MANUSCRIPT 1. Introduction. It is observed that telomerase is up-regulated in 80−90% of all cancer cells isolated from primary human tumors but is absent in neighboring normal cells. This resulted in significant efforts to validate telomerase as an anticancer drug target and to develop effective approaches toward its inhibition. When considering anticancer drug targets, several
RI PT
advantages for targeting telomerase and telomere maintenance exist when compared to other targets and pathways. Most important is the apparently universal requirement for telomere maintenance in cancer cells including putative cancer stem cells [1]. Telomerase remains active in the early stages of life maintaining telomere length and the chromosomal integrity of
SC
frequently dividing cells. It turns dormant in most somatic cells during adulthood [2,3]. However telomerase gets reactivated in the vast majority of invasive cancers. Extension of
M AN U
telomeres by telomerase is required for un-limited proliferation and it is well accepted that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression [4,5]. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction [6]. Thus, the inhibition of telomerase activity is a potential target for cancer therapeutic discovery which might have minimal side effects.
TE D
In the course of identifying various chemical substances which may serve as leads for designing new anticancer agents, the present work particularly emphasizes on the coumarin and pyrazoline derivatives which have been identified as cores for chemotherapeutic agents
EP
with significant therapeutic efficacy against tumors. Coumarin comprised the nucleus of a wide variety of naturally occurring compounds.
AC C
They were extremely variable in structure, due to various types of substitutions in their basic structure, which could influence their biological activity. Literature survey revealed their wide spectrum and diversity of biological activities [7-16], particularly their cytotoxic activity against numerous types of cancers [17- 24]. Several studies revealed the mechanism of anticancer activity of coumarin derivatives which might include induction of apoptosis [2531]. Osthole I, a bioactive simple coumarin derivative extracted from Cnidium monnieri (L) Cusson, proved effective against human hepatocellular carcinoma (HCC) through induction of apoptosis [32]. Additionally, the coumarin-monastrol hybrid II demonstrated anticancer efficacy against many cancer cell lines mediated by apoptotic activity [33] (Figure 1). 2
On the other hand, pyrazoline-based compounds received considerable attention
ACCEPTED MANUSCRIPT
owing to their high chemotherapeutic potential. Diversely substituted pyrazoline derivatives showed good cytotoxic and antiproliferative activities against a wide range of human tumor cell lines [34-40]. This could be exemplified by the 3,5-diarylsubstituted pyrazolines III [41]
RI PT
and IV [42]. (Figure 2).
Moreover, it was pointed out that introducing the dihydropyrazole moiety in the coumarin skeleton, as illustrated by compounds V [43] and VI [44], gave highly potent
of apoptosis. (Figure 3).
M AN U
SC
cytotoxic compounds that were believed to act through inhibition of telomerase and induction
The current strategy in developing new anticancer drugs shifted toward the multiple mechanistic approach and several drugs have been validated and developed. Inspired by the inherent biological relevance of the coumarin and pyrazoline rings, the present investigation pertained to the hybridization of these two pharmacophoric moieties, in a single molecule. Expectedly, the additive effect of this combination might produce compounds of high
TE D
anticancer activity that might act by dual mechanism through induction of apoptosis and inhibition of telomerase. The target compounds had the general structure 7a-e - 9a-e, featuring a 7-methoxycoumarin nucleus substituted at position 8 with a 5-arylsubstitutedpyrazoline group. The pyrazoline ring was either unsubstituted at position 1, or carrying a
EP
phenyl group or an acetyl group. Isosteric replacement of the N-acetyl group with the carboxamide function gave compounds of general formula 10a-e. A cyclic versus noncyclic
AC C
bioisosteric modification of compounds 10a-e was also adopted to obtain the noncyclic bioisosteres 11a-e (Figure 4).
Antiproliferative activity of the synthesized compounds was evaluated in vitro against
human liver cell line HepG2. Telomerase inhibition and apoptosis induction of the most active compounds were also investigated. 2. Results and Discussions 2.1. Chemistry 3
The synthesis of the target compounds 7-11 was depicted in Schemes 1 and 2. 8-
ACCEPTED MANUSCRIPT
Acetyl-7-hydroxy-2H-chromen-2-one 4 was obtained together with its regioisomer 3 through a Fries rearrangement of the intermediate acetoxy derivative 2, which was obtained by acetylation of the commercially available 7-hydroxycoumarin 1 with acetic anhydride. The regioisomers 3 and 4 were separated by fractional crystallization from aqueous ethanol [46]. Methylation of 4 with methyl iodide in the presence of potassium carbonate in dry acetone gave the corresponding 8-acetyl-7-methoxy-2H-chromen-2-one 5 [47-49]. The chalcones 6
RI PT
were prepared by conventional Claisen-Schmidt condensation reaction of 5 with (un)substituted-arylaldehydes in the presence of 10 % sodium hydroxide in ethanol (Scheme 1) [50].
SC
The synthesis of the novel coumarin-pyrazoline hybrids 7-10 was accomplished
M AN U
through 1,4-addition of hydrazine hydrate, phenyl hydrazine or semicarbazide to the α,βunsaturated carbonyl system of the precursor chalcones 6, followed by dehydration and rearrangement (Scheme 2). The N1 unsubstituted and N1 phenyl pyrazolines 7 and 8 were produced by cyclocondensation of the chalcones 6 with hydrazine hydrate 99% or phenyl hydrazine in absolute ethanol, respectively. Alternatively, condensation of 6 with hydrazine hydrate in absolute ethanol in the presence of glacial acetic acid furnished the N1 acetyl
TE D
pyrazoline derivatives 9 as a result of in situ acetylation of the initially formed pyrazolines 7. In addition, base catalyzed cyclization of chalcones 6 into the corresponding pyrazoline-1carboxamide 10 was achieved through reaction with semicarbazide HCl in ethanolic sodium hydroxide. On the other hand, reacting 6 with semicarbazide HCl in absolute ethanol yielded
AC C
EP
the uncyclic allylidene semicarbazide derivatives 11.
The structure elucidation of the newly synthesized compounds was based on the
analytical and spectral data (IR, 1H NMR,
13
C NMR, and mass spectrometry). 1H NMR
spectra of most pyrazolines 7-10 exhibited an AMX pattern for the presence of two diastereotopic protons at C-4 (HA and HM) and one single proton at the C-5 positions (HX). These protons appeared as three doublets of doublets, respectively, at δ 2.90-3.35, 3.46-3.89, 4.91-5.63 ppm regions, each integrating for one proton. In case of compound 8a simple splitting patterns was observed with pyrazoline protons, which appeared as two signals, one doublet equivalent to 2H at δ 3.73 ppm and a multiplet equivalent to 1H at δ 5.75 ppm. Moreover, the 1H NMR spectra of compounds 7 and 10 displayed singlet exchangeable signal at δ 4.20-5.37 or δ 10.20-11.05 ppm corresponding to the pyrazoline (NH) or the N1 4
carboxamide (NH2), respectively. Meanwhile, the analogs 9 showed singlet signal derived
ACCEPTED MANUSCRIPT
from the N1 acetyl (CH3) at δ 2.18-2.37 ppm. On the other hand, the 1H NMR spectra of the allylidene semicarbazide derivatives 11 revealed the presence of two exchangeable singlet signals resonating at δ 6.47-6.54 and 8.88-9.10 ppm for the semicarbazide (NH2 and NH) protons and two doublets at δ 6.05-6.74 and 6.85-7.29 ppm (J= 15.0-16.9 Hz) representing the allylidene (CH=CH) protons, which asserted the production of the uncyclized derivatives 11. The protons belonging to the aromatic system and phenyl substituents were observed at the
RI PT
expected chemical shifts and integral values. 2.2. Anticancer activity. 2.2.1. Antiproliferative activity study.
SC
The in vitro anti-hepatocellular carcinoma (anti-HCC) activity of the newly synthesized compounds was examined against HepG2 cell line using the MTT method [51].
M AN U
Doxorubicin which is one of the most effective anticancer agents was used as positive control. The activity was expressed by median growth inhibitory concentration (IC50) and was provided in Table 1. The reported antiproliferative activity of Osthole I [32] and the lead compound VI [44] was included for comparison. The results revealed that, all the new coumarin-pyrazoline hybrids exhibited potent antiproliferative activity with IC50 values ranging from 10 ̶ 99 nM, which were far more potent than Osthole I (IC50= 161.4µM) and
TE D
compound VI (IC50= 6.64µM). In particular compounds 7a, 7d, 7e, 8a and 10e possessed superior activity at low nanomolar levels (IC50 = 11, 13, 10, 15 and 18 nM, respectively) The N1-unsubstituted pyrazoline derivatives 7a-e displayed excellent growth
EP
inhibitory activity (IC50 = 10 ̶ 48 nM). Compound 7b having phenyl substituent at position 5 of the pyrazoline ring was the least active member of this group (IC50 = 48 nM). Appending a
AC C
lipophilic substituent to the p-position of the phenyl in 7d-e contributed to an increase in potency, where the highest activity was associated with the 4-trifluoromethylphenyl 7d and 4methylthiophenyl 7e congeners (IC50 = 13 and 10 nM, respectively). Similarly, isosteric replacement of the phenyl ring with the thienyl rest in 7a resulted in about 4 fold increase in growth inhibition.
Substitution at N1 of the pyrazoline ring with a phenyl group, 8a-e, led to significant drop in activity (IC50 = 15 ̶ 78 nM), with the 5-thienylpyrazoline derivative, 8a, having the highest activity in this series. Likewise, compounds bearing the N1-acetyl group, 9a-e and the N1-carboxamide derivatives 10a-e revealed a decrease in activity compared to the N1unsubstituted pyrazolines 7a-e. Moreover, the allylidene semicarbazides 11a-e, which could 5
be considered as the non-cyclic isosteres of 10a-e, showed reduced growth inhibitory effect
ACCEPTED MANUSCRIPT
(IC50 = 75 ̶ 99 nM). All compounds in this series demonstrated the lowest activity compared to their N1-un/substituted pyrazoline counterparts 7-10. In summary, the antiproliferative activity of the novel coumarin-pyrazoline hybrids was influenced by the substitution pattern on the pyrazoline ring. Mostly, the introduction of a more lipophilic substituent to position 5 of the pyrazoline ring produced compounds with enhanced potency as shown by the 4-trifluoromethylphenyl 7d and 4-methylthiophenyl
RI PT
analogs 7e and 10e. Also, isosteric replacement of the phenyl ring with the thienyl group as in 7a and 8a enhanced the activity. However, the activity was not favored by the presence of N1substitutions as in 8a-e, 9a-e and 10a-e, which was less active than the N1-unsubstituted derivatives, 7a-e. Furthermore, a noticeable drop in potency was observed with the coumarin
2.2.2. Telomerase inhibitory activity.
M AN U
