Synthesis And Biological Evaluation of New S-Mannich Bases of 3-Methyl-4-Phenyl-3,4,5,6,7,8, Hexahydroquinazoline-2(1h)-Thione.

 

SenthilkumarN.*, Jayalakshmi B., Joicy Jose and SumathiK.

Department of Pharmaceutical Chemistry, JKKMMRF College of Pharmacy, B. Komarapalayam, Namakkal, Tamil Nadu – 638 183.

*Corresponding Author E-mail: senthilkumarjkkm@gmail.com

ABSTRACT:

The chalcone was synthesized from cyclohexanone and an aromatic aldehyde by aldol condensation reaction, followed by cyclisation with thiourea results in hexahydro quinazoline. The third position is methylated by using methyl iodide, finally the mannich reaction was carried out with five different amines at  second position of the hexahydro quinazoline to get five different S-Mannich bases. All the 5 synthesized new compounds were studied for their physiochemical characters. And their structures were confirmed by spectral studies (IR, NMR). These new derivatives were evaluated for analgesic and anti-inflammatory activity.

 

 

INTRODUCTION:

Quinazoline is a building block for approximately 150 naturally occurring alkaloids, such as glycosminine, echinozolinone, deoxyvasicinone, rutaecarpine and drugs like methaqualone isolated to date from a number of families of the plant kingdom, from animals and from microorganisms. The first quinazoline was synthesized in the late 1860s from anthranilic acid and cyanogens to give 2-cyanoquinazoline. Methaqualone was synthesized for the first time in 1951 and it is the most well-known synthetic quinazoline drug, famous for its sedative–hypnotic effects¹. Moreover, the quinazoline skeleton is very common in several naturally occurring alkaloids, displaying a wide range of biological activities useful in developing chemotherapeutic agents against many diseases and hence the exploration of this skeleton as privileged new chemical entities (NCE’s) in drug discovery research is of paramount importance². Quinazoline derivatives are pharmaceutically interesting compounds and many of them have been registered as drugs. Quinazoline-2-thiones, Quinazolin-2-ones and quinazoline-4-one can possess hypnotic, analgesic, antiallergic, anticonvulsant, antimalarial, and other effects.  In our previous study we found that some 3-phenylquinazolin-4 (3H)-ones were active against atypical strains of mycobacteria.

 

The conversion of the oxo group into the thioxo function leads, in general, to an increase in anti mycobacterial activity. Although the antimicrobial activity of some substituted quinazoline-2-thiones is known³.

 

Chalcone derivatives constitute an important group of secondary metabolites found ubiquitously in the plant kingdom. Chalcone are open-chain flavonoids with common skeleton of 1, 3-diaryl-2-propen-1-one. Their wide-range biological properties, including antimicrobial activities, are largely attributed to the β-unsaturated ketone moiety. Introduction of various substituent’s into the two aryl rings is also a subject of interest because it leads to useful SAR conclusions and thus helps to synthesize pharmacologically active chalcones⁴ Chalcone, which are considered to be precursors of flavonoids and isoflavonoids, are abundant in edible plants. Chalcones exhibit many pharmacological activities, including anti-leishmanial ,anti-inflammatory, antimitotic, anti-invasive, anti-tuberculosis, anti-fungal,CyLT1(LTD4)receptor antagonist, anti-malarial, anti-plasmodial, immunosupressive, cytotoxic, anti-tumor, and anti-oxidant properties⁵ .

 

The Mannich reaction is an organic reaction which consists of an amino alkylation of an acidic proton placed next to a carbonyl functional group with formaldehyde and ammonia or any primary or secondary amine. The final product is a β-amino-carbonyl compound also known as a Mannich base. Reactions between aldimines and α-methylene carbonyls are also considered Mannich reactions because these imines form between amines and aldehydes. The reaction is named after Chemist Carl Mannich^6s.Mannich bases possess comprehensive bioactivities like anticancer, analgesic, antibacterial and antifungal activities. Multi-component reactions (MCRs) constitute a major part in the present day organic synthesis with advantages ranging from lower reaction times, increased reaction rates to higher yields and reproducibility. Mannich reaction is a three-component condensation reaction involving active hydrogen containing compound, formaldehyde and a secondary amine. The aminoalkylation of aromatic substrates by Mannich reaction is of considerable importance for the synthesis and modification of biologically active compounds⁷.

 

EXPERIMENTAL WORK:

Materials and Methods: All chemicals used were of analytical reagent grade purchased from LOBA Chemie, Mumbai, India.

 

Instrument used:

Melting point was determined in open capillary method. Purity of the compounds was checked on Silica Gel TLC plates. Electronic Balance used was Type-BL 220H SHIMADZU. IR spectra were recorded on FT-IR-8400S SHIMADZU, the pellets were prepared in KBr press, model M-15 techno search Instruments. The NMR was measured in BRUKER 300 MHz FT –NMR for proton in CDCl₃ .

 

SCHEME:

Compounds	R1	R2
CH IV a	-CH2-CH2-OH	-CH2-CH2-OH
CH IV b
CH IV c	- CH2-CH3	-H
CH IV d	-H
CH IV e	-H

 

Synthesis; Step-I: 2-Benzylidene Cyclohexanone (CH I):

Cyclohexanone (0.01 mole) and benzaldehyde (0.01 mole) were taken into a clean and dry reaction vessel and dissolved in methanol (50ml). To this solution alcoholic KOH (2%, 40ml) was added slowly drop wise while shaking the contents thoroughly. Then the reaction mixture was allowed to stand at room temperature for an hour while shaking periodically. A yellow crystalline solid that resulted was filtered and washed with small quantities of methanol and dried. The product was purified by recrystallization from methanol to get a yellow crystalline solid^8,10.

 

Step-II: 4-Phenyl-3,4,5,6,7,8- Hexahydroquinazoline-2(1H)-Thione (CH II):

A mixture of CH I (0.01 mole), thiourea (0.01mole) and an alcoholic solution of KOH (6g in 150ml alcohol) were taken into a reaction flask and heated under reflux for 3hr on a hot water bath. After defluxion the volume of the mixture was reduced to half by distilling off the alcohol .The concentrate was then added to cold water and precipitate obtained was filtered and washed with small portions of cold water and dried. It was purified by recrystallization from alcohol to get a colorless crystalline solid^8,10.

 

Step III: N-Methylation of 4-Phenyl-3,4, 5, 6, 7, 8- Hexahydroquinazoline-2(1H)-Thione (CH III):

CH II (0.01 mole) was weighed and dissolved in 1N alcoholic NaOH (100ml) in reaction vessel. To this solution methyl iodide (0.02mole) in ethanol was added and the reaction mixture was warmed with continuous stirring. The solid mass thus resulted was filtered and dried. Then it was purified by recrystallization from alcohol to get a dull white crystalline solid.

 

Step IV: S-Mannich Bases of 3-Methyl-4-Phenyl-3, 4, 5, 6, 7, 8- Hexahydro quinazoline-2(1H)-Thione (CH IV A – CH IV E)

An intimate mixture of CH III (0.01mole), formaldehyde (0.02mole) and amines (0.02mole) in DMSO was warmed on a water bath with stirring for 30mts, there after it was allowed to stand overnight at room temperature. Then the reaction mixture was added to cold water, the solid product thus resulted was filtered. Then the product was purified from alcohol chloroform mixture (1:1) to get a crystalline solid.

 

Acute Oral Toxicity Study:

Acute toxicological studies were done according to the OECD guide lines. Institutional ethical committee no:1158/ac/07/CPCSEA

 

Anti Bacterial study:

Anti Bacterial Study was carried out by using Gram Positive (Streptococcus pyogenes, ATCC49619) and Gram Negative Bacteria (Pseudomonas aeruginosa, ATCC27853) Standard Drug used was Amoxiclav. (1000 µg / ml), Solvent used is DMSO.

 

Figure 1.  Statstical Data of Analgesic Activity of synthesized Quinazoline derivatives

 

Figure 2. Statistical Data of Locomotors Activity of synthesized Quinazoline derivatives.

 

Analgesic Activity:

The Eddy’s hot plate is a hot surface of metal (10 ×10”) heated electrically to maintain a temperature of about 25ºC (preferably 55±1ºC) and covered from all the sides by per flex method. Animal is gently placed on the hot plate and time required to lick the paw is noted cut off time is 15-20 sec to avoid damage to the nerve endings of the paw that is the experiment is terminated at 15-20 sec and analgesia is considered as 100%⁹.

 

 

Table 1. Molecular formula, melting point, TLC-R_f values and percentage yield of the synthesized Quinazoline derivatives.

Code	Compounds	Mol. formula	Melting Point	Rf	% yield
CH IV a	3-methyl-4-phenyl 3,4,5,6,7,8 hexahydroquinazoline-2yl [N-bis(1-hydroxy ethyl)          methylamino]2-thione	C20 H 29N3 O2 S	58-60° C	0.704	70%
CH IV b	3- methyl-4-phenyl 3,4,5,6,7,8 hexahydroquinazoline -2yl[N,N-diphenyl methylamino]2-thione	C28H29 N3S	96-98° C	0.81	80%
CH IV c	3-methyl-4-phenyl 3,4,5,6,7,8 hexahydroquinazoline -2yl[N-ethyl methylamino]2-thione	C18 H 24 N 3 S	54-56 ° C	0.81	69%
CH IV d	3-methyl-4-phenyl 3,4,5,6,7,8  hexahydroquinazoline -2yl[N-phenyl methylamino]2-thione	C22 H25 N3 S	54-58° C	0.86	75%
CH IV e	3 -methyl-4-phenyl 3, 4, 5, 6, 7, 8  hexahydroquinazoline -2yl[ N-(4-NO2)-phenyl methylamino]2-thione	C22H19N4O2S	110-112° C	0.837	82%

Solvent system:  Chloroform: Benzene: Ethyl acetate (1:1:0.2)

 

Table 2. IR Data of the synthesized  quinazoline derivatives.

Compound	IR Data
CH I	2929.97(Ar-CH), 1659.80(-C=O),   1573.97 (-C=C-)
CH II	3450.77(NH), 3359.14(NH),2924.18 (Ar-CH), 1636.65 (-C=N- ring),
CH III	3450.77(NH), 2927.08(Ar-CH), 2857.64(N-CH3), 1637.62(-C=N-ring)
CH IV a	3448.84 (NH), 2924.18 ( Ar-CH),  2854.74 (N-CH3),  2340 (-S-C), 1624.12 (-C═N ring), 1522.85(-C═C), 1022.33(-C-O-alcohols), 691(C-S-C)
CH IV b	3426.66(NH), 2923(Ar-CH), 2855.71(N-CH3),  2343.59(-S-C), 1520.92(-C═C-), 696.33(C-S-C),1621(C═N ring)
CH IV c	3421.83(NH), 2925.15(Ar-CH), 2331.05 (-S-C), 1525.75-(C=C-),     698.25(-C-S-C), 2855.71(N-CH3), 1627.97(-C=N-ring)
CH IV d	3418.94(NH), 2924.18(Ar-CH), 2855.71(N-CH3),2327.19(-S-C),  1514.17(-C=C-), 696.33(-C-S-C), 1621.22(-C=N-ring).
CH IV e	3449.80(NH), 2924.18(Ar-CH), 2375.45(N-CH3),   695.36(-C-S-C-), 1320.32(NO2), 1525.75(-C=C-), 1642.44(-C=N- ring)

 

 

Loco motor Activity:

The actophotometer is also known as activity cage or activity monitor. The instrument is used to measure the motor activity of animal. Both horizontal and perpendicular movements (rearing) can be measured. About 1cm from the base of cage 5 beams of UV light are made incident on the photomultiplier tube located at the other end of the cage. The interruption of light beam activates a counter.

 

RESULTS AND DISCUSSION:

All the five synthesized compounds were characterized by Melting Point Analysis, Thin Layer Chromatography and Spectral Analysis. Melting points were found in an open end capillary tube. The purity of the synthesized compounds was checked by TLC and the spot was visualized in iodine vapor. The structures of the compounds were elucidated by Ultraviolet, Infrared and NMR Spectroscopy. The antibacterial activities of the synthesized compounds have been performed against Gram +ve and Gram –ve bacteria. Among all the five compounds CH IVa was more active and that was selected for the Acute Oral Toxicity studies.

 

¹H NMR Data:

The PMR spectroscopic values were measured is δppm in CDCl₃. 1.202-1.249(t, 2H, CH₂- aliphatic), (1.688-1.769 p, 4H, CH₂- CH₂-cyclic), (2.759-2.799 t, 2H, CH₂ cyclic), (2.881-2.91 t, 2H, CH₂ cyclic), (2.643 s, 3H, N-CH₃), (7.2891-7.6090 m, 15H Ar H) and (8.27 s, H).

 

Acute oral toxicity studies:

Acute toxicity of the compound was evaluated in mice. From these observations the lethal dose was concluded as >1000mg/kg. Testing dose was fixed at 100mg/kg for all the compounds.

Analgesic activity:

Analgesic activity is done by using Eddy’s hot plate. All the compounds tested for analgesic activity and CH IVa was found to be more active.

 

Locomotor activity:

Locomotor activity was done by actophotometer using male albino rats. All the prepared compounds were showing Locomotor Activity, by CNS depression and was more for CH IVa and CH IVe.

 

CONCLUTION:

The present study was carried out with the aim of Synthesis and Biological Evaluation of New S-Mannich bases of 3-Methyl-4-phenyl-3,4,5,6,7,8-Hexahydroquinazoline-2-thione. All the compounds were purified by recrystallization and column chromatography. The synthesized compounds were confirmed by Physicochemical and Spectral analysis (UV, IR, and NMR). The results revealed from the anti bacterial study, that the compound CH IVa has shown comparatively more zone of inhibition. So the acute oral toxicity study was carried out with CH IVa. The Analgesic and Locomotor activity of all the compounds were screened by in vivo methods. Analgesic activity was found to be more with CH IV a. All the prepared compounds showed Locomotor activity, by CNS depression and was more in CH IV a  and CH IV e. In short all the derivatives showing the pharmacological activity mainly due to the presence of basic nucleus and was modified with the various substituent’s used for Mannich Base formation on thio group.

 

ACKNOWLEDGEMENT:

The authors are thankful to Dr. J.K.K. Munirajah, the Managing trustee of JKKMMRF, College of Pharmacy Komarapalayam, Namakkal, who have provided the facilities to carry out the research work.

 

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Received on 16.06.2011        Modified on 21.07.2011

Accepted on 14.08.2011        © AJRC All right reserved

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