Synthesis, anticonvulsant activity and molecular modeling study of some new hydrazinecarbothioamide, benzenesulfonohydrazide, and phenacylacetohydrazide analogues of 4(3H)-quinazolinone

Accepted Manuscript

Synthesis, Anticonvulsant Activity and Molecular Modeling Study of Some

New Hydrazinecarbothioamide, Benzenesulfonohydrazide, and Phenacylace-

tohydrazide Analogues of 4(3H)-quinazolinone

Huda S.A. Al-Salem, Gehan H. Hegazy, Kamal E.H. El-Taher, Shahenda M.

El-Messery, Abdulrahman M. Al-Obaid, Hussein I. El-Subbagh

PII:

S0960-894X(15)00132-8

DOI:

http://dx.doi.org/10.1016/j.bmcl.2015.02.025

BMCL 22437

Reference:

Bioorganic & Medicinal Chemistry Letters

To appear in:

Received Date:

Revised Date:

Accepted Date:

29 November 2014

6 February 2015

11 February 2015

Please cite this article as: Al-Salem, H.S.A., Hegazy, G.H., El-Taher, K.E.H., El-Messery, S.M., Al-Obaid, A.M.,

El-Subbagh, H.I., Synthesis, Anticonvulsant Activity and Molecular Modeling Study of Some New

Hydrazinecarbothioamide, Benzenesulfonohydrazide, and Phenacylacetohydrazide Analogues of 4(3H)-

quinazolinone, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.

2015.02.025

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.

Synthesis, Anticonvulsant Activity and Molecular Modeling Study of Some New

Hydrazinecarbothioamide, Benzenesulfonohydrazide, and Phenacylacetohydrazide Analogues

of 4(3H)-quinazolinone.^#

Huda S. A. Al-Salem^a, Gehan H. Hegazy^b, Kamal E. H. El-Taher^c, Shahenda M. El-Messery^d,

Abdulrahman M. Al-Obaid^a, Hussein I. El-Subbagh^e,f,*

^aDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box

2457, Riyadh 11451, Saudi Arabia

^bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo 11562,

Egypt

^cDepartment of Pharmacology, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi

Arabia

^dDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,

Mansoura 35516, Egypt

^eDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516,

Egypt

^fDepartment of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences & Pharmaceutical

Industries, Future University, 12311 Cairo, Egypt.

Correspondence:. Hussein I. El-Subbagh, Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura

University, Mansoura 35516, Egypt

E-mail: subbagh@yahoo.com.

Tel: +20-50-2247800, Fax: +20-50-2247900

Abstract

A new series of quinazoline analogues was designed and synthesized to get the target compounds 18-

21, 30-41, 46-53, and 57-76. The Obtained compounds were evaluated for their anticonvulsant

activity using PTZ and picrotoxin convulsive models. Compounds 47, 63, 68 and 73 proved to be the

most active compounds in this study with a remarkable 100% protection against PTZ induced

convulsions. Compounds 47, 63, 68 and 73 proved to be 10, 4, 4, and 5 fold more active, respectively

than the used positive control sodium valproate. Structure activity correlation concluded valuable

pharmacophoric information which confirmed by molecular modeling studies. Molecular docking

study of 68 suggested its agonistic behavior toward GABA_Areceptor. The studied quinazoline

analogues could be considered as useful templates for future development and further derivatization.

Keywords: Synthesis, 4(3H)-Quinazolinones, Anticonvulsant activity, molecular modeling study.

_________________

^#Part of the European Patent, EP 2 740 727 A1, June 2014; See reference 21

1

Despite the broad and growing array of antiepileptic drugs (AEDs) available for treatment,

approximately 30% of epileptic patients have inadequate seizure control and a further 25% suffer from

significant adverse effects [1]. Thus there is an ongoing need to develop more AEDs that are effective

and endowed with improved safety profile. Recently, a number of fused pyrimidine derivatives

became known as potential drug molecules against various types of diseases. One of the most

important compound families are quinazolinones which are the building blocks for approximately 150

naturally occurring alkaloids and drugs [2]. Literature survey reveals that natural quinazolinones and

their synthetic analogues possess a variety of pharmacological activities, including antitumor [3,5],

CNS depressant [6], antimicrobial [7,8], and muscle relaxant activities [9,11].

Methaqualone (1) is an important landmark in the field of synthetic anticonvulant [12], and its 6-

chloro analogue 2 which proved to possess marked anticonvulsant action, 1.5 times more potent than

phenytoin sodium (3) against electroshock induced convulsions and 10 times more potent than

troxidone (4) against pentylenetetrazol induced seizures [13,14]. Several quinazolinones related to 1

have been synthesized and tested; a persistent problem arises from the fact that nearly every derivative

exhibited neurotoxicity values (TD₅₀) that are less than or only slightly higher than the ED₅₀values.

Consequently, the protective index (PI) corresponding to TD₅₀/ED₅₀is too low [15,16]. Modification

of the 2-methyl group of 1 by some other chemical moiety yielded structural analogues with potent

anticonvulsant activity [17]. In continuation to our previous efforts, recently some new quinazoline

analogues that possess remarkable anticonvulsant activity were prepared in our laboratory such as 5,

(ED₅₀73.1 mg/kg), which showed a 100% protection against PTZ induced clonic convulsion (Chart

1), [18-20].

Based on the previous mentioned considerations, bearing in mind the inherited anticonvulsant potency

of quinazoline nuclei, the CNS activity enhancement of the thioacetic acid hydrazide, and the

potentiating effect of alkoxy functions and halogens to the anticonvulsant activity [2, 10], the

combination of all these features in one structure to produce derivatives 6-9 was rationalized, (Chart

2). A new series of quinazoline analogues were synthesized to explore the influence of both phenyl or

benzyl substituent at position 3- of the quinazoline nucleus, in addition to the electronic effect of the

electron withdrawing Cl group at position 6- and two adjacent electron donating OCH₃groups at

positions 6- and 7- on the anticonvulsant activity and hence the deduction of structure activity

relationship (SAR). Biological evaluation of the new synthesized anticonvulsant compounds 18-21,

30-41, 46-53, and 57-76 were performed using different convulsive models, as well as the

determination of the median effective (ED₅₀), median sedative (TD₅₀), and median lethal (LD₅₀)

doses; Protective index (PI), and Therapeutic index (TI) of the proven active compounds. Molecular

2

modeling study was employed to explain the reasons behind the obtained remarkable anticonvulsant

potency, which allowed the filling for a European Patent [21].

The synthetic strategy for the preparation of the target compounds 18-21, 30-41, 46-53, 57-76 were

illustrated in schemes 1 and 2. The 2-mercapto-3-(phenyl or benzyl)-quinazolin-4(3H)one derivatives

14-17 were prepared by the reaction of the substituted-anthranilic acid 10 and 11 with phenyl-

isothiocyanate (12) or benzyl-isothiocyanate (13) in ethanol in the presence of catalytic amount of

triethylamine to give the 3-phenyl (14, 15), and the 3-benzyl (16, 17) derivatives, respectively.

Compound 15 was previously reported [22]. The obtained products were methylated using

CH₃I/K₂CO₃to give the 2-(methylthio)- analogues 18-21, or treated with ethyl 2-chloroacetate to

yield the 3-substituted-quinazolin-2-yl-thioacetates 22-25. The latter products were reacted with

hydrazine hydrate in ethanol to afford the 2-acetohydrazide derivatives 26-29. Compounds 26-29 were

further reacted with the isothiocyanate derivatives 12 or 13 to obtain the target compounds 30-37. On

the other hand, the target compounds 38-41 were obtained by stirring the acetohydrazide derivatives

26-29 with 4-toluene sulphonylchloride in chloroform at room temperature (Scheme 1, Table 1). The

3-phenyl-acetohydrazide analogues 26 and 27 were acylated by the use of chloroformic solution of

aliphatic acid chlorides namely; acetyl, propionyl, butyryl, and 2-methylbutanoyl chlorides (42-45) to

afford the target compounds 46-53; or aromatic acid chlorides namely; benzoyl, 2-phenylacetyl, and

dihydrocinnamoyl chlorides (54-56) to give the target compounds 57-62 in presence of catalytic

amount of pyridine. Similarly, the 3-benzyl-acetohydrazide analogues 28 and 29 were also acylated

using the same aliphatic and aromatic acid chlorides to afford the targets 63-76, (Scheme 2, Table 2).

Structure elucidation of the synthesized compounds was determined using IR spectroscopy, Mass

1

13

1

spectrometry, H and C NMR spectroscopy. H-NMR spectra of compounds 14-16 showed singlet

proton of SH at 2 position of quinazolinone at around 4-5 ppm. Moreover, the mass spectrum of 16

showed a molecular ion peak at 303. The methylated products 18-21 showed a characteristic singlet at

1

2.5 for methl group. H-NMR spectra of compounds 22-25 adopted the appearance of significant

characteristic triplet and quartet peaks for ethyl group. The IR spectrum of compounds 24, revealed

1

the existence of stretching absorption of carbonyl ester at around 1730 cm^-1. H-NMR spectrum of

compounds 26-29 showed two singlets at 9-10 and 12 ppm due to two NH₂and NH protons

1

respectively . H-NMR spectrum of compounds 30-37 showed three deuterium oxide-exchangeable

singlets at expected chemical shifts attributed to three NH protons, while the toluenyl sulphonyl

derivatives 38-41 showed the peaks of two singlets at 7 and 10 ppm due to two NH protons, indicating

the existence of the compound in the hydrazono form in addition to 3 singlet protons at around 2 ppm

referred to methyl group. The mass spectrum of target compounds showed the molecular ion at m/z

3

(%) M⁺and M+2. The target compounds 46-53 showed characterstic singlets of 2 NH protons along

with phenyl multiplet protons and different alkyl chain at far upper field while their benzyl

counterparts' compounds 63-70 spectra were confirmed by the formation of additional CH₂Ph singlet

protons at around 5 ppm. The phenacylacetohydrazide derivatives 57-62 and their benzyl isosteres

13

showed characteristic peaks at their expected values. C NMR spectral analysis was in accordance

with proposed structures. ¹³C NMR spectra of 73 as an example revealed the presence of C=O,

quinazoline, N=CH, and SCH₂carbon atoms resonating as double singlet at approximately 170.5–

165.9 and 160.5–157.5, 156.81–156.40, 141.5–146.1 and 34.6–35.2 and 39.6–39.8 ppm.

A typical anticonvulsant agent can prevent the convulsion episodes caused by convulsion-inducing

material such as Pentylenetetrazole (PTZ) and picrotoxin which can be used to evaluate the potency

of any alleged anticonvulsant agents [23,24]. In the present investigation, test compounds were

injected and animals were monitored to register the onset of the first tonic convulsion; the frequency

of convulsions till death; and the time of death following the injection of the convulsant. During the

20 min pre-treating time, the animals were followed carefully and any change of behavior was noted.

Compounds 18-76 were injected at doses of 25-200 mg/kg, i.p. Pentylenetetrazole (PTZ) was used at

dose level of 100 mg/kg, i.p., 20 min after the test compounds. This dose was found to be the

minimum dose that induced 100% clonic convulsion. Sodium valproate was used as positive control

at dose level of 200 mg/kg (1.38 mmol/kg) i.p.

Pentylenetetrazole (PTZ) induced clonic convulsions within 1.6 min. frequency of convulsions was

every 2.0 min and animal died in 4.0 min. Any compound able to prevent such episodes or delayed

this time pattern in varying magnitude will be considered to possess an anticonvulsant activity [25-

27]. Compound 21 proved to be lethal at dose of 150 mg/kg i.p. and non-effective at lower doses

while compound 22 proved to devoid any anticonvulsant activity. Compound 63 protected the

animals prolonging the death time by 303%. Compounds 47 and 73 prolonged the convulsion onset

time by more than 600% (P< 0.001, N=4). The order of the test compounds in prolonging the

convulsion onset time was 47>73>68 = 35>75 = 46>71 (Table 3). The order of the test compounds in

reducing the frequency of convulsions by more than 80% were 49= 57>48>36 = 68 = 76 (P< 0.01,

N=4); while the order of compounds in prolonging the death time by more than 300% were 47>19>63

(P< 0.001, N=4). Compounds prolonged the death time by more than 200% found to be in the

following order 73> 46 = 68 (P< 0.001, N=4). Compounds 47, 63 and 68 (P< 0.001, N=4) succeeded

in prolonging the onset time, the death time and reduced the frequency of PTZ induced convulsions.

4

Picrotoxin is a chloride channel blocker producing hyperpolarization and clonic convulsions.

Compounds protect the animals against picrotoxin-induced convulsions; act as GABA_Areceptor

agonist by increasing chloride influx via brain chloride channels. Picrotoxin model will help to find

out the mode of action of an anticonvulsant agent [22]. Picrotoxin (10 mg/kg, IP) induced clonic

convulsions within 6.75 min; frequency of convulsions was 36 per 10 min and animal died in 12 min.

Any compound was able to prevent such episodes or delayed this time pattern in varying magnitude

will be considered to possess an anticonvulsant activity. Compounds 18, 19, 30, 31, 34, 38-41, 47-49,

57-59, 64, 66, 67, 69-72, 74, 76 proved to be inactive in this test indicating that they act through a

mechanism other than GABA_Areceptor agonism. Compounds 20, 37 and 63 significantly prolonged

the onset time for convulsions and the death time and decreased the frequency of picrotoxin induced

convulsions. The order in prolonging the onset time for convulsions was 37>63>20 (P< 0.01, N=4).

Three compounds (75>20 = 36) significantly decreased the frequency of convulsions by > 89%. Two

potent compounds (65>63) prolonged the death time significantly. In general, the order of the test

compounds in decreasing the frequency of picrotoxin induced convulsions was: 75>20 =

36>68>37>65 = 63> 35>24, (Table 4). Compound 63 (0.36 mmole/kg), proved to be active in both

pentylenetetrazole and picrotoxin test models suggesting that its anticonvulsant activity might be

through GABA_Areceptor agonist mode of action; while compound 65 (0.33 mmole/kg), was the only

active compound against picrotoxin-induced convulsion.

In general, compounds 47, 63, 68 and 73 proved to be the most active compounds in this study with

remarkable 100% protection against PTZ induced convulsions as compared with the standard drug

sodium valproate (Table 5). It is worth mentioning that compounds 47 (0.12 mmole/kg), 63 (0.36

mmole/kg), 68 (0.33 mmole/kg) and 73 (0.29 mmole/kg) proved to be almost 10, 4, 4, and 5 fold

more active, than the used positive control sodium valproate (1.38 mmole/kg), respectively.

Structure activity correlation of the obtained results allowed the withdrawal of some valuable

pharmacophoric information about the anticonvulsant drug design of this class of compounds. Four

series of compounds were studied in the present investigation, namely: 6-Chloro-2-(substituted-thio)-

3-phenyl-quinazolin-4(3H)-one series; 6-Chloro-2-(substituted-thio)-3-benzyl-quinazolin-4(3H)-one

series; 6,7-Dimethoxy-2-(substituted-thio)-3-phenyl-quinazolin-4(3H)-one series; and 6,7-

Dimethoxy-2-(substituted-thio)-3-benzyl-quinazolin-4(3H)-one series. Regarding the 6-chloro-3-

phenyl- series, methylation of the 2-mercapto function of 14 produced the methylthio analogue 18

which prolonged PTZ-induced convulsion onset time in experimental animal by 114%, while

alkylation of 14 with ethyl chloroacetate produced the biologically inactive ester 22. Hydrazinolysis

of 22 produced the inactive hydrazone analogue 26. Reacting 26 with benzyl isothiocyanate produced

5

the active compound 31 which regained the activity of 18 prolonging PTZ induced convulsion onset

time by 114%. Reaction of the hydrazone 26 with 4-toluene sulphonyl chloride produced 38 with

diminished activity into 43%. Acylation of the hydrazone moiety of 26 with aliphatic acid chlorides

of various alkyl lengths or aromatic acid chlorides produced active anticonvulsant agents of various

magnitudes. Acylation using acetyl chloride produced 46 with increased potency (186%), while

acylation using propionyl chloride produced 47, one of the most active compounds in this

investigation (1471%). Acylation with acid chloride of longer length such as butyryl chloride or acid

chloride of branched alkyl chain as 2-methylbutanoyl chloride produced 48 and 49 with reduced

activity (43 and 114%, respectively). Acylation with aryl-alkyl acids of various lengths as benzoic,

phenyl acetic and dihydrocinnamic acids produced compounds 57, 58 and 59 with anticonvulsant

activity (114, 43, 43%, respectively) which decreased by the increase of the acid alkyl chain length. It

is proved that short chain aliphatic acid acylation of the hydrazone 26 favors the activity rather than

the aryl-alkyl acids acylation. Regarding the 6-chloro-3-benzyl- series, replacement of the 3-phenyl

moiety of 31 by 3-benzyl group produced 35 with four fold increases in activity (471%). The same

was also applicable in case of 46 producing 63 which prevented the convulsion episodes by 100%; 57

producing 71 with modest increase in the anticonvulsant activity and 59 producing 73 with

remarkable increase of activity by almost 16 fold. The series of 6,7-dimethoxy-3-phenyl- showed the

lowest anticonvulsant activity in this study represented by 19 and 24. In the 6,7-dimethoxy-3-benzyl-

series, the presence of 3-benzyl moiety brought the inactive 3-phenyl counterpart into the active side

as exemplified by compounds 36, 68, 75 and 76 which prolonged PTZ induced convulsion onset time

in experimental animal by 150, 471, 186 and 114% , respectively. In general, 6-chloro function

proved to enhance the anticonvulsant activity of the synthesized quinazolin-4(3H)-one more than the

6,7-dimethoxy group. Also, 3-phenyl moiety at the synthesized quinazolin-4(3H)-one proved to

contribute to the anticonvulsant potency as well as safety more than the 3-benzyl function.

The binding study of compound 68 as an example representing the most active compounds into

GABA_Areceptor binding site where the diazepam bound GABA_Areceptor homology model was used

where only the extracellular domains of the structures with PDB codes 2BG9 and 2QC1 were

considered. [28], for possible revealing of the mode of action was quite interesting. The orientation of

compound 68 inside the benzodiazepine (BZD)-binding site of GABA_Areceptor was examined by a

flexible docking experiment using MOE software. The docking was performed using the Alpha

Triangle placement method [29-31]. Diazepam was used as a reference ligand for validation purpose.

The important amino acid interacting with 68 were displayed in Figure 1a, where hydrogen bonding

interaction between the carbonyl amide functions with Thr56 amino acid by 90%; the methoxy group

6

oxygen and Gln119 by 30% was observed. The aligned binding conformations of 68 at the binding

site revealed a clear quite well binding preference as seen in Figure 1b. The best conformation for 68

showed interaction binding energy of -15.46 kcal/mol possessing a similar binding mode of diazepam

in the homology model pocket of GABA_Areceptor (RMSD: 0.17 Å). This finding supports the

hypothesis that 68 exert its anticonvulsant activity as GABA_Aagonist.

To probe similarity between the 3D structures of the most active compounds 73 and diazepam,

MOE/MMFF94 flexible alignment using systematic conformational search [32-34] was employed to

automatically generate superposition of 73 with minimal user bias [35]. 200 conformers of the

compound were generated and minimized with a distance-dependant dielectric model. A low energy

set of 100 was selected for further analysis. The top scoring alignment of both the least strain energy

and the best alignment score is shown in Figure 2. Compound 73 and diazepam align fairly well

(Figure 2a), while the least active compound 22 showed a distinct different pattern with dramatic

alignment deviation from that of diazepam (Figure 2b).

The atomic-level details of the structure of pharmaceutically relevant receptors are not available; in

such cases, 3D superposition of putative ligands can be used to deduce specific structural

requirements for biological activity [35,36]. Structure superposition of the most active compounds 47,

63, and 73 with specified mark points (Figure 3) such as N-1 of the quinazoline nucleus, the phenyl

ring substituent, N-3 of the quinazoline nucleus, 2-S-linkage directly attached to quinazoline moiety,

NH at side chain and the π-system represented by aryl group of the quinazoline core, align fairly well

with almost complete superposition of the quinazoline rings (0.40 Å). This superposition provides

possible existence of a region on the receptor suited for specific recognition of the aforementioned

selected features. This hypothesis is ascertained by biological results which strongly suggest that

these chosen mark points could be used as a pharmacophore model for further anticonvulsant

compounds. The features responsible for the remarkable anticonvulsant activity of 47 were employed

for surface map of its lowest energy conformer to examine the similarity or the dissimilarity in the

conformational properties of its molecule surface in comparison to the surface mapping of the

reference compound diazepam. Compound 47 (Figure 4) showed hydrophilic region, hydrogen bond

acceptor–donor, region located on the 4-carbonyl, N-1 and non-polar area located on the aryl moiety

attached to the quinazoline core distributed on both sides of the aryl parts; which are generally having

a common feature, with the surface mapping distribution of diazepam (Figure 4b). Such results

indicated the structure mapping similarity and hence biological resemblance between 47 and

diazepam. Moreover the results provided further support that the investigated compounds might exert

their anticonvulsant action as GABA_Aagonist.

7

Oral bioavailability plays an important role in the development of bioactive molecules into

therapeutic agents. Many potential therapeutic agents fail to reach the clinic because of their

unfavorable absorption, distribution, metabolism, elimination and toxic (ADMET) factors [37].

Therefore, a computational study for the ADMET properties prediction of the most active

anticonvulsants 47, 63, 68, and 73 was performed for the determination of topological polar surface

area (TPSA), and the ‘‘rule of five’’ formulated by Lipinski for activity prediction of an orally

administered drug [38], if it has no more than one violation of the following rules: ClogP <5; number

of hydrogen bond donors sites ≤5; number of hydrogen bond acceptor sites ≤10; molecular weight

<500; number of rotatable bonds < 5. In addition, the total polar surface area (TPSA) is another key

property linked to drug bioavailability; the passively absorbed molecules with TPSA >140 have low

oral bioavailability [31]. All calculated descriptors were obtained using the MOE package, and the

results are listed in Table 6. The obtained results revealed that the molecular weight was <500, except

for compound 73 (507.014), CLogP <5, hydrogen bond acceptor <10 and hydrogen bond donors <5

which fulfill Lipinski’s rule. Also, the percent absorption of all tested compounds proposed a higher

bioavailability with total surface area with <140. Lip V molecules should have less than 2 violations;

all of the tested compounds applied no violations except compound 73 had only one violation. Since

compounds 47, 63, 68 and 73 has a highly anticonvulsant activity, and from the data above, it could

be suggested that they could be used as good orally absorbed agents with diminished toxicity.

In conclusion, compounds 47, 63 and 68 (P< 0.001, N=4) succeeded in prolonging the onset time, the

death time and reduced the frequency of PTZ induced convulsions. Compounds 37 and 63

significantly prolonged the onset time for convulsions and the death time and decreased the frequency

of picrotoxin induced convulsions. Compounds 47, 63, 68 and 73 (Figure 5) proved to be the most

active compounds in this study against PTZ induced convulsions. It is worth mentioning that

compound 47 (0.12 mmole/kg), 63 (0.36 mmole/kg), 68 (0.33 mmole/kg) and 73 (0.29 mmole/kg)

proved to be almost 10, 4, 4, and 5 fold more active, respectively than the used positive control

sodium valproate (1.38 mmole/kg). Compound 63 (0.36 mmole/kg), showed 100% protection against

PTZ- induced convulsions in addition to 63% protection against picrotoxin-induced convulsions

suggesting that it might act directly as GABA_Areceptor agonist or indirectly by increasing GABA

synthesis or its release as a brain inhibitory neurotransmitter. Structure activity correlation of the

obtained results revealed that, 6-chloro function proved to enhance the anticonvulsant activity of the

synthesized quinazolin-4(3H)-one more than the 6,7-dimethoxy group. Also, the 3-phenyl moiety at

quinazolin-4(3H)-one proved to contribute to the anticonvulsant potency as well as safety more than

the 3-benzyl function. Molecular modeling studies revealed that the investigated compounds bind to

8

the benzodiazepine (BZD)-binding site of GABA_Areceptor through hydrogen bonding with Thr56

and Gln119 amino acids. Superposition among the active compounds provides evidence that chosen

mark points: N-1 of the quinazoline nucleus, the phenyl ring substituent, N-3 of the quinazoline

nucleus, 2-S-linkage directly attached to quinazoline moiety, NH at side chain and the π-system

represented by aryl group of the quinazoline core could be used as a pharmacophore model for

anticonvulsant activity. ADMET study suggested that the obtained active compounds can be used as

good orally absorbed anticonvulsant agents with expected diminished toxicity. Further binding

affinity studies to GABA_Areceptor will be quite informative. The studied quinazoline analogues

could be considered as useful templates for future development and further derivatization or

modification to obtain more potent anticonvulsant compounds.

Acknowledgements

The authors would like to express their appreciation and thanks to King Abdulaziz City for Science

and Technology, Riyadh, Saudi Arabia for the grant number AT-18-6.

References and notes

1. Ben-Menachem, E.; Gabbai, A.; Hufnagel, A.; Maia, J.; Almeida, L.; Soares-da-Silva, P.

Epilepsy Res. 2010, 89, 278.

2. Laddha, S.; Bhatnagar, S. Novel fused quinazolines: Further studies on the anticonvulsant

activity of 1,2,9,11-tetrasubstituted-7H-thieno[2`,3`:4,5]pyrimido[6,1-b]-quinazolin-7-one and

1,3,10,12-tetrasubstituted-8H-pyrido[2`,3`:4,5]pyrimido[6,1-b]quinazolin-8-one.

International Conference on Synthetic Organic Chemistry (ECSOC-13) 2009.

The

13^th

3. Al-Rashood, S.; Aboldahab, I.; Nagi, M.; Abouzeid, L.; Abdel-aziz, A.; Abdel-Hamide, S.;

Youssef, K.; Al-Obaid, A.; El-Subbagh, H. Bioorg. Med. Chem. 2006, 14, 8608.

4. Al-Obaid, A.; Abdel-Hamide, S.; El-Kashef, H.; Abdel-Aziz, A.; El-Azab, A.; Al-Khamees, H.;

El-Subbagh, H. Eur. J. Med. Chem. 2009, 44(6), 2379.

5. Al-Omary, F.; Abou-Zeid, L.; Nagi, M.; Habib, E.; Abdel-Aziz, A.; El-Azab, A.; Abdel-Hamide,

S.; Al-Omar, M.; Al-Obaid, A.; El-Subbagh, H. Bioorg. Med. Chem. 2010, 18, 2849.

6. Kashaw. S.; Gupta, V.; Kashaw, V.; Mishra, P.; Stables, J.; Jain, N. Med. Chem. Res. 2010, 19,

250.

7. Al-Omar, M.; Abdel-Hamide, S.; Al-Khamees, H.; El-Subbagh, H. Saudi. Pharm. J. 2004, 12(2-

3), 63.

8. Mathew, J.; Dinakaran, V.; Kaur, N.; Srinivasan, K. Pharmacologyonline 2008, 2, 618.

9. Kashaw, S.; Kashaw, V.; Mishra, P.; Jain, N.; Stables, J. Med. Chem. Res. 2010, 20(6),738.

10. Kashaw, S.; Gupta, V.; Kashaw, V.; Mishra, P.; Stables, J.; Jain, N. Med. Chem. Res. 2010,19,

250.

11. Kadi, A.; El-Azab, A.; Alafeefy, A.; Abdel-Hamide, S. Az. J. Pharm. Sci. 2006, 34, 147.

12. Gujral, M.; Saeen, K.; Kohli, R. Ind. J. Med. Res. 1957, 45(2), 207.

13. Salimath, R.; Patel, S.; Shah, N. J. Ind. Chem. Soc. 1964, 33, 140.

14. Bhaduri, A.; Khanna, N.; Dhar, M. Ind. J. Chem. 1964, 2, 159.

9

15. Barthwal, J.; Tandon, S.; Agarwal, V.; Dixit, K.; Parmar, S. J. Pharm. Sci. 1973, 62(4), 613.

16. Wolfe, J.; Rathman, T.;Sleevi, M. J. Med. Chem. 1990, 33(1), 161- 6.

17. Jatava, V.; Mishra, P.; Kashaw, S.; Stables, J. Eur. J. Med. Chem. 2008, 43, 135.

18. Abdel-Hamide, S.; El-Obeid, E.; Al-Majed, A.; Al-Kashef, H.; El-Subbagh, H. Med. Chem. Res.

2001, 10(6), 378.

19. El-Subbagh, H.I; Hassan, G.S.; El-Azab, A.S.; Abdel-Aziz, A.A.-M.; Kadi, A.A.; Al-Obaid,

A.M.; Al-Shabanah, O.A.; Sayed-Ahmed M.M. Eur. J. Med. Chem. 2011, 46, 5567.

20. El-Azab, A.S.; Abdel-Hamide, S.G.; Sayed-Ahmed, M.M.; Hassan, G.S.; El-Hadiyah, T.M.;

Al-Shabanah, O.A.; Al-Deeb, O.A.; El-Subbagh. H.I. Med. Chem. Res. 2013, 22, 2815.

21. Huda S. A. Al-Salem, Hussein I. El-Subbagh, Kamal E. H. El-Taher, Gehan H. Hegazy,

Abdulrahman M. Al-Obaid. Quinazolinone analogues for use as anticonvulsant agents. European

Patent, EP 2 740 727 A1, June 2014.

22. Li, Z.; Huang, H.; Sun, H.; Jiang, H.; Liu, H. J. Comb. Chem. 2008, 10(3), 484

23. White, H.; Woodhead, J.; Franklin, M.; Swinyard, E.; Wolf, H. In Antiepileptic Drugs, 4th ed.;

Levy, R.H., Mattson, R.H. Meldrum, B.S., Eds, Raven: New York, 1995, pp 99-121

24. Ulrich, R.; Miller, J. Percept Psychophys. 2004, 66, 517.

25. Khanna, N.; Garg, A.; Sharma, K.; Khosta, R. Indian J. Clin. Biochem. 1997, 12, 86.

26. Ankica, V.; Joranovic, M.; Nincovic, M.; Maksimovic, M.; Boscovic, B. Acta. Veterinaria. 2003,

53, 103-112.

27. Chakrabarti, A.; Saini, H.; Garg, S. J. Pharm. Pharmacol. 1997, 49, 812.

28. Richter, L.; De Graaf, C.; Sieghart, W.; Varagic, Z.; Mrzinger, M.; Pde Esch, I.J.; Ecker, G. F.;

Ernst, M. Nat. Chem. Biol. 2012, 8, 455.

29. Kearsley, S.; Smith, G.M. Tetrahedron Comput. Methodol. 1990, 3, 615.

30. Profeta, S.; Allinger. N. L. J. Am. Chem. Soc. 1985, 107, 1907.

31. Allinger. N. L. J. Am. Chem. Soc. 1977, 99, 8127.

32. Profeta, S.; Allinger. N. L. J. Am. Chem. Soc. 1985, 107, 1907-1918.

33. Cornell, W.D.; Cieplak, P.; Bayly, C.I.; Gould, I.R.; Merz Jr., K.M.; Ferguson, D.M.;

Spellmeyer, D.C.; Fox, T.; Caldwell, J.W.; Kollman, P.A. J. Am. Chem. Soc. 1995, 117, 5179-

5197.

34. Dewar, M.J.S.; Zoebisch, E.G.; Healy, E.F.; Stewart, J.J.P. J. Am. Chem. Soc. 1985, 107, 3902-

3909.

35. Labute, P.; Williams, C.; Feher, M.; Sourial, E.; Schmidt, J.M. J. Med. Chem. 2001, 44, 1483.

36. Mihic, S. J.; Whiting, P. J.; Klein, R.L.; Wafford, K.A.; Harris, R.A. J. Biol. Chem. 1994, 269,

32768.

37. Zhao, Y.; Abraham, M.H.; Lee, J.; Hersey, A.; Luscombe, N.C.; Beck, G.; Sherborne, B.;

Cooper, I.; Pharm. Res. 2002, 19, 1446.

38. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Adv. Drug. Delivery Rev. 1997, 23,

3.

10

O

COOH

NH₂

n

N

C

S

N

C₂H₅OH

R¹

+

n

(C₂H₅)₃N

N

SH

12

: n = 0

13: n = 1

10: R₁= 5-Cl

11: R₁= 4,5-(OCH₃)₂

14-17

Cl

O

CH₃I

Acetone

O

n

N

R¹

O

N

S

N

SCH₃

O

18-21

22-25

NH₂NH₂

C₂H₅OH

O

N

O

N

n

S

N

R¹

12 or 13

H

N

R²

NH NH₂

S

C₂H₅OH

S

N

H

N

H

O

30-37

26-29

O

S Cl

O

CH₃

CHCl₃/ pyridine

O

N

R¹= 6-Cl, 6,7-(OCH₃)₂,

R²= C₆H₅, C₆H₅CH₂,

n = 0, 1

n

N

O

R¹

NH NH S

O

CH₃

S

O

38-41

Scheme 1: Synthesis of the target compounds 18-41.

11

O

N

O

N

O

pyridine/ CHCl₃

N

R¹

H

N

R²COCl

+

N

R²

S

NH₂

Cl

N

H

O

26,27

46-53

42-45

O

n

pyridine/ CHCl₃

54-56

O

N

R¹

H

N

S

N

n

H

O

57-62

O

N

O

N

pyridine/ CHCl₃

R¹

H

N

H

N

R²COCl

+

R²

N

S

NH₂

N

H

O

63-70

28,29

42-45

O

Cl

pyridine/ CHCl₃

n

54-56

O

R¹= 6-Cl, 6,7-(OCH₃)₂

R²= CH₃, C₂H₅, C₃H₇, C₄H₉

N

O

R¹

H

N

S

N

n

n = 0, 1, 2

H

O

71-76

Scheme 2: Synthesis of the target compounds 46-53 and 57-76.

12

O

H₃C

N

HN

O

N

H₃C

NH

O

N

R

O

CH₃

O

CH₃

4

3

1, R = H

2

, R = Cl

O

N

I

N

CH₂COOH

S

5

Chart 1: Structures of some literature anticonvulsants.

O

N

O

N

R²

S

N

S

R¹

H

N

R¹

R³

SCH₃

N

H

N

H

O

7

6

O

N

O

R²

S

R²

S

N

O

N

O

R³

R¹

H

N

R¹

H

N

S

N

H

R³

N

H

O

8

9

R¹= 6-Cl, 6,7-(OCH₃)₂

R²= C₆H₅, C₆H₅CH₂,

R³= Alkyl, Arylalkyl groups

Chart 2: Structures of the proposed target compounds

13

Figure 1: (a) The binding mode and interaction of 68 (orange) with amino acids residues in

diazepam-binding pocket of GABA_Areceptor. (b) The aligned conformation of the most active

compound 68 (space filled) occupying diazepam-binding pocket of GABA_Areceptor.

Figure 2: (a) Flexible alignment of the most active compound 73 (cyan), and diazepam (pink). (b)

Flexible alignment of the least active compound 22 (yellow) and diazepam (pink).

14

Figure 3: Superposition of the most active compounds 47 (in yellow), 63 (in red), and 73 (in cyan)

with specified mark points.

Figure 4: (a) Surface map for the most active compound 47 in pocket side. (b) Surface map for

Diazepam. Pink, hydrogen bond, blue: mild polar, green hydrophobic.

O

N

O

N

Cl

O

N

O

H

N

H

N

CH₂

CH₃

S

N

H

S

N

H

CH₃

O

47

63

O

N

O

N

CH₃O

Cl

N

O

N

O

H

N

H

N

S

N

H

CH₂

CH₃

S

N

H

O

68

73

Figure 5: Structures of the most active anticonvulsant agents 47, 63, 68 and 73.

15

Table 1: Physicochemical properties of the synthesized compounds 14-41.

O

N

O

N

n

N

NH NH₂

O

S

N

SCH₃

S

N

SH

R¹

O

18 21

-

14-17

26-29

22-25

O

N

O

n

S

N

O

R¹

H

N

R¹

R²

NH NH S

O

CH₃

S

N

H

N

H

N

S

O

30-37

38-41

Compound

R¹

R²

n

Solvent

Yield %

MP ^°C Molecular formula^a

-

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

6-Cl

6,7-OCH₃

6-Cl

6,7-OCH₃

6-Cl

6,7-OCH₃

6-Cl

6,7-OCH₃

6-Cl

6,7-OCH₃

6-Cl

6,7-OCH₃

6-Cl

0

1

0

1

0

1

0

1

0

1

0

1

EtOH

-

EtOH

38

-

261-3

-

C₁₄H₉ClN₂OS

Reference [32]

C₁₅H₁₁ClN₂OS

C₁₇H₁₆N₂O₃S

C₁₅H₁₁ClN₂OS

C₁₇H₁₆N₂O₃S

C₁₆H₁₃ClN₂OS

C₁₈H₁₈N₂O3S

C₁₈H₁₅ClN₂O₃S

C₂₀H₂₀N₂O₅S

C₁₉H₁₇ClN₂O₃S

C₂₁H₂₂N₂O₅S

C₁₆H₁₃ClN₄O₂S

C₁₈H₁₈N₄O₄S

C₁₇H₁₅ClN₄O₂S

C₁₉H₂₀N₄O₄S

C₂₃H₁₈ClN₅O₂S₂

C₂₄H₂₀ClN₅O₂S₂

C₂₅H₂₃N₅O₄S₂

C₂₆H₂₅N₅O₄S₂

C₂₄H₂₀ClN₅O₂S₂

C₂₅H₂₂ClN₅O₂S₂

C₂₆H₂₅N₅O₄S₂

C₂₇H₂₇N₅O₄S₂

C₂₃H₁₉ClN₄O₄S₂

C₂₅H₂₄N₄O₆S₂

C₂₄H₂₁ClN₄O₄S₂

C₂₆H₂₆N₄O₆S₂

48

64

83

98

45

42

70

87

90

98

81

10

89

79

98

44

25

68

19

57

55

29

30

36

71

190-2

222-4

148-50

173-5

96-8

194-6

85-7

EtOH/CHCl₃

CH₃COCH₃

CHCl₃

EtOH/CHCl₃

MeOH/CHCl₃

145-6

73-5

104-5

181-3

166-8

151-2

170-2

170-1

155-6

175-7

180-1

150-1

164-6

152-4

210-1

200-2

197-8

196-8

6,7-OCH₃

6-Cl

6,7-OCH₃

6-Cl

-

Ph

Bn

Ph

Bn

Ph

Bn

Ph

Bn

-

6-Cl

6,7-OCH₃

6-Cl

6,7-OCH₃

6-Cl

6,7-OCH₃

6-Cl

-

6,7-OCH₃

^aCompounds analyzed for C,H,N,; results were within 0.4 % of the theoretical values for the given formulae

16

Table 2: Physicochemical properties of the synthesized target compounds 46-53, 57-76

O

N

O

N

R¹

H

N

R¹

H

N

R²

N

S

N

H

N

S

N

H

n

O

57-62

46-53

O

N

O

N

O

N

O

R¹

H

N

R¹

H

N

R²

S

N

H

N

H

n

O

63-70

71-76

Compound R¹

R²

n

Solvent

Yield %

MP C^°

87-9

Molecular formulae^a

-

46

47

48

49

50

51

52

53

57

58

59

60

6-Cl

CH₃

MeOH

CHCl₃

EtOH/CHCl₃

MeOH

EtOH

MeOH

DMF/CHCl₃

MeOH/CHCl₃

EtOH/CHCl₃

MeOH/CHCl₃

CHCl₃

77

55

38

51

43

68

25

33

98

69

30

37

C₁₈H₁₅ClN₄O₃S

C₁₉H₁₇ClN₄O₃S

C₂₀H₁₉ClN₄O₃S

C₂₁H₂₁ClN₄O₃S

C₂₀H₂₀N₄O₅S

C₂₁H₂₂N₄O₅S

C₂₂H₂₄N₄O₅S

C₂₃H₂₆N₄O₅S

C₂₃H1₇ClN₄O₃S

6-Cl

C₂H₅

C₃H₇

C₄H₉

-

128-30

164-6

175-7

209-10

211-2

205-6

218-9

131-3

199-201 C₂₄H₁₉ClN₄O₃S

206-8

221-2

206-7

204-5

206-7

188-90

185-7

214-5

210-1

197-9

203-5

214-6

196-8

200-2

199-1

116-8

147-9

212-4

6,7-OCH₃CH₃

6,7-OCH₃C₂H₅

6,7-OCH₃C₃H₇

6,7-OCH₃C₄H₉

6-Cl

-

0

1

2

0

1

2

6-Cl

6,7-OCH₃

C₂₅H₂₁ClN₄O₃S

C₂₅H₂₂N₄O₅S

C₂₆H₂₄N₄O₅S

C₂₇H₂₆N₄O₅S

C₁₉H₁₇ClN₄O₃S

C₂₀H₁₉ClN₄O₃S

C₂₁H₂₁ClN₄O₃S

C₂₂H₂₃ClN₄O₃S

C₂₁H₂₂N₄O₅S

C₂₂H₂₄N₄O₅S

C₂₃H₂₆N₄O₅S

C₂₄H₂₈N₄O₅S

C₂₄H₁₉ClN₄O₃S

C₂₅H₂₁ClN₄O₃S

C₂₆H₂₃ClN₄O₃S

C₂₆H₂₄N₄O₅S

C₂₇H₂₆N₄O₅S

C₂₈H₂₈N₄O₅S

61

62

63

64

6,7-OCH₃

6-Cl

24

28

73

87

CH₃

C₂H₅

C₃H₇

C₄H₉

-

6-Cl

-

CHCl₃

65

66

67

68

69

70

71

72

73

74

75

76

6-Cl

MeOH/CHCl₃

DMF/CHCl₃

MeOH/CHCl₃

EtOH/CHCl₃

MeOH/CHCl₃

CHCl₃

66

58

85

96

98

62

74

67

68

88

86

78

6,7-OCH₃CH₃

6,7-OCH₃C₂H₅

6,7-OCH₃C₃H₇

6,7-OCH₃C₄H₉

6-Cl

-

0

1

2

0

1

2

6-Cl

CHCl₃

6,7-OCH₃

EtOH/CHCl₃

MeOH/CHCl₃

MeOH

^aCompounds analyzed for C,H,N,; results were within 0.4 % of the theoretical values for the given formulae

17

Table 3: Effect of compounds 18-20, 24, 31, 34-38, 40, 41, 46-49, 57-59 and

63-76 on PTZ-induced convulsions in mice

% Prolongation

of Convulsion

Onset Time

% Decrease

in Convulsion

Frequency

Dose

% Delay of

Death Time

Compound

mmole/Kg mg/Kg

18

19

20

22

24

31

34

35

36

37

38

40

41

46

47

48

49

57

58

59

63

64

65

66

67

68

69

70

71

72

73

74

75

76

0.50

0.45

0.47

0.40

0.38

0.30

0.29

0.28

0.27

0.29

0.28

0.27

0.37

0.12

0.34

0.32

0.10

0.30

0.36

0.35

0.33

0.32

0.34

0.33

0.32

0.30

0.31

0.30

0.29

0.30

0.29

0.28

150

50

114 6

35 4

114 4

0.0

114 9

0.0

471 15

150 6

43 5

43 4

43 6

0.0

61 3

0.0

61 5

81 4

38 6

48 6

74 3

0.0

48 3

74 5

82 6

87 6

0.0

-----

74 6

61 6

0.0

81 9

0.0

23 3

61 5

74 4

0.0

68 7

81 6

44 5

461 11

5 1

0.0

145 8

0.0

101 7

5 1

32 4

40 3

47 4

0.0

216 12

636 12

75 4

47 4

0.0

303 17

0.0

14 3

0.0

43 4

186 11

1471 29

43 4

114 5

114 2

43 3

43 6

No Convulsions

43 5

78 5

0.0

471 13

114 6

0.0

185 11

43 4

685 21

0.0

150

50

150

0.0

216 10

58 8

0.0

75 4

47 2

254 9

0.0

186 11

114 5

58 6

0.0

18

Table 4: Effect of compounds 20, 24, 35-37, 63, 65, 68 and 75 on picrotoxin-

induced convulsions in mice

% Prolongation

of Convulsion

% Decrease

in Convulsion

Frequency

Dose

% Delay of

Death Time

Compound

Onset Time

mmole/Kg mg/Kg

20

24

35

36

37

63

65

68

75

0.47

0.38

0.29

0.28

0.27

0.36

0.33

0.29

150

36

5 1

15 2

4 1

78 6

63 6

0.0

4

91 6

25 4

39 4

89 6

66 4

60 4

61 5

79 9

97 11

0.0

17 3

25

22 3

50 3

61 4

8 2

17 2

11 2

18 3

Table 5: ED₅₀, TD₅₀, PI, LD₅₀and TI for 47, 63, 68 and 73

b

d

%

Protection

Dose

mmole/Kg

ED₅₀

mg/Kg

TD₅₀

mg/Kg

LD₅₀

mg/Kg

Compound^a

PI^c

TI^e

mg/Kg

47

63

68

73

0.12

0.36

0.33

0.29

1.13

50

150

50

100

11.79

24.75

32.7

29.5

189

100.3

215.3

134.1

135.7

470

8.5

8.7

4.1

4.6

2.4

290.3

239.6

167.5

290.3

985

24.6

9.7

5.1

9.8

5.2

Sodium Valproate

a

Each dose of selected compounds was tested using 4 animals and the percentage of animals protected was

recorded and the anticonvulsant activity was calculated.

^bED₅₀and TD₅₀: Median effective and median sedative doses, respectively.

^cPI: Protective index = TD₅₀/ED₅₀

^dLD₅₀: Median lethal dose

^eTI: Therapeutic index = LD₅₀/ED₅₀.

Table 6: ADMET properties prediction parameters of compounds 47, 63,

68 and 73

Compound

Mwt

TPSA

Log P

Lip. don Lip. acc

Lip. V

47

63

68

73

416.889 90.87

456.523 109.33

507.014 90.83

3.2785

3.1504

2.9043

4.76327

2

7

9

7

0

1

Mwt: Molecular weight, TPSA: Polar surface area, Log P: Calculated lipophilicity,

Lip.don: Number of hydrogen bond donors, Lip.acc: Number of hydrogen bond

acceptors, Lip.V: Number of violations of Lipinski rule.

19

Graphical Abstract

Synthesis, Anticonvulsant Activity and Molecular Modeling Study of Some New Hydrazine-carbothioamide,

Benzenesulfonohydrazide, and Phenacylacetohydrazide Analogues of 4(3H)-quinazolinone. #

Huda S. A. Al-Salem^a, Gehan H. Hegazy^b, Kamal E. H. El-Taher^c, Shahenda M. El-Messery^d, Abdulrahman M. Al-

Obaid^a, Hussein I. El-Subbagh^e,f,*

O

R¹

R²

S

O

N

H

N

R³

N

H

O

1

2

3

47:

R = Cl; R = Ph; R = C₂H₅

63: R¹= Cl; R²= PhCH₂-; R³= CH₃

68: R¹= 6,7-(CH₃O)-; R²= PhCH₂-; R³= C₂H₅

1

2

3

47:

R = Cl; R = PhCH₂-; R = PhCH₂CH₂-

Structures of compounds 47, 63, 68 and 73, the most active anticonvulsants with a

remarkable 100% protection against PTZ induced convulsions.

20

Article Doi

Synthesis, anticonvulsant activity and molecular modeling study of some new hydrazinecarbothioamide, benzenesulfonohydrazide, and phenacylacetohydrazide analogues of 4(3H)-quinazolinone

DOI: 10.1016/j.bmcl.2015.02.025

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmcl.2015.02.025

Full text of DOI:10.1016/j.bmcl.2015.02.025

Products guided by the article

R&D Labs maybe for 1612898-58-4

Relevant to this article

Hot Product