A series of cinnamide derived functions was designed utilizing molecular hybridisation attack utilizing structural characteristics of clinically used every bit good as molecules under clinical tests for anti-tubercular and cardinal pharmacophoric characteristics of compounds holding interactive belongingss with known antimycobacterials. The designed molecules were synthesized by reaction between assorted diamine and substituted cinnamic acids. Topliss operational method was used to optimise the authority of the molecules. The minimal repressive concentration ( MIC ) was determined against M. TB H37Rv in Middlebrook 7H9 medium utilizing resazurin microtitre home base check. Some of the synthesized molecules shows good activity against M. TB H37Rv with MIC in the scope of 5.1-143.1I?M. The most powerful compound 1a holding MIC of 5.1I?M besides shows interactive action ( FICI-0.31 ) with rifampicin.
Keywords: Diamine ; Cinnamic acid ; Mycobacterium TB ( Mtb ) ; Antimycobacterial activity ; Synergy
Tuberculosis ( TB ) , a contagious disease infects humans via the air passages by Mycobacterium TB ( Mtb ) . WHO reports about 8-9 million new instances of TB while 3 million deceases which represents the largest figure of incidence of human deceases attributable to a individual aetiologic agent[ I ]. Insufficiency of current TB therapy has resulted in growing of the disease. Even after elucidation of the genomic sequence of the Mtb and outgrowth of a multiplicity of marks, abode of Mtb within the macrophages attached with its cell wall barrier, remain the cardinal issues to successful therapy. In peculiar the current concern is the monolithic job ensuing from extensively drug-resistant TB, XDR-TB, which is normally known as strains resistant to all current first line drugs, fluoroquinolones and at least to one of the three injectable second-line drugs ( capreomycin, Kantrex or amikacin )[ two ]. Due to the importance of this disease, assorted enterprises have been undertaken by authorities research research labs, academic establishments, pharmaceutical industries etc. with the purpose of developing the new drugs and schemes to battle with TB. The present work focuses on a possible scheme to get the better of these challenges.
1,2-ethylenediamine is a basic scaffold of ethambutol, a clinically used first line drug for TB. Based on this scaffold a molecular library of 63,238 compounds has been developed by Sequella Inc. in coaction with NIH/NIAID. From this library a drug campaigner SQ109 was identified as the most active compound possessing 35 fold improved activity as compared to that of ethambutol[ three ]. SQ109 has late advanced into clinical tests for the intervention of TB, though it appears that the mark for SQ109 is non same as that of ethambutol. In this library, the compounds SQ775 and SQ786 exhibiting activity about that of SQ109 show that the basic scaffold of 1,2-ethylenediamine can besides be changed either to homopiperazine or piperazine ( Fig 1. ) .
Fig 1. Diamine based Antimycobacterials
On the other manus, cerulenin and trans-cinnamic acid ( Fig 2. ) are traditionally known to hold antimycobacterial activity and besides proven to hold interactive action when tested along with known clinically used drugs[ four ]. Additionally, cinnamyl derived function of rifampicin shows decline in MIC of rifampicin by 2- to 8-fold for most of the susceptible and multi drug immune Mtb strains. Besides this derived function has better intracellular and in vivo activities compared to those of rifampicin[ V ].
Fig 2. Traditionally known Antimycobacterials holding interactive actions
In our uninterrupted plan in the hunt of new potent and safe antimycobacterial agent, we decided to build a new category of cinnamide derived functions as attractive antitubercular agents[ six ]. The designing scheme is based on hybridisation of basic pharmacophoric characteristics of diamine parallels i.e. Ethambutol, SQ786, SQ109, SQ775 i.e. diamine linkage, amide of cerulenin along with its I± , I?-unsaturated carbonyl character and to this attached an aromatic ring which give the molecule cinnamic acid like quality ( Fig 3. ) . Aim is to potencialize the activity of diamines, possible by the incorporation of a different manner of action through different mark. Further optimisation of molecule for its authority has been carried out utilizing conventional method of Topliss for permutation at aromatic Centre in molecule.
Fig 3. Design construct of new cinnamide derived functions.
To acknowledge the best diamine linker we have chosen four different diamines i.e. 1,2-ethylenediamine, piperazine, homopiperazine and 2- ( piperazin-1-yl ) ethanamine ( Fig. 4. ) for matching with trans-cinnamic acid as first set of molecules. The mark compounds i.e. cinnamide derived functions were prepared harmonizing to Scheme 1. Compounds 1a-1d were obtained in high output through a one-step reaction utilizing trans-cinnamic acid, ethyl chloroformate, triethylamine and diamine as get downing stuffs.[ seven ]All synthesized compounds were identified by spectral informations. The antimycobacterial activity of all synthesized compounds was tested against M. TB H37Rv and the minimal inhibitor concentration ( MIC ) was determined utilizing REMA ( Resazurin Microtitre Assay ) method[ eight ]. Ethambutol was used as reference drug.
Fig 4. Different diamine linkers used for synthesis
Scheme 1. Man-made strategy for synthesis of cinnamide derived functions
The biological consequences ( Table 1. ) show that the compound holding diamine linkage of 1,2-ethylenediamine ( 1a ) is most active amongst the four synthesized molecules piperazine ( 1b ) , homopiperazine ( 1c ) and 2- ( piperazin-1-yl ) ethanamine ( 1d ) analogs all thenceforth with regard to anti-mycobacterial activity, which is in understanding with the activity of reported molecules. Further to optimise the authority of the compound derived from 1,2-ethylenediamine i.e. 1a, we had followed the conventional Topliss operational attack of modulating the hydrophobic and electronic parametric quantities on the phenyl pealing Fig 5.
Table 1. Cinnamide derivatives with their antimycobacterial activity and toxicity
Compound No
MIC ( AµM ) *
CC50 ( AµM ) **
Silicon
1a
5.1
618
121
1b
9.0
855
95
1c
17.3
708
41
1d
16.7
480
29
*Compounds were tested against Mtb H37Rv strains utilizing REMA method
**Cytotoxicity surveies were done on Vero Cell line C1008 utilizing MTT check.
SI- selective index
Fig 5. Topliss operational tree
As per the batchwise method of Topliss attack, the 2nd set of parallels of the cinnamide derivative 1a includes substituent ‘s 4-methyl 1e, 4-methoxy 1f, 4-chloro 1g, and the 3,4-dichloro 1h to the aromatic ring. The synthesis was carried out utilizing the same man-made Scheme 1 used above with substituted trans-cinnamic acids. Assay consequences of this initial set of parallels were expected to uncover the favourable electronic ( R ) and hydrophobic ( P ) demands of the pharmacophore. Unfortunately from the biological consequences it can be seen that none of the molecule showed better activity so parent molecule i.e. 1a ( Table 2, entry 2-5 ) . Based on these consequences it can be concluded that there may be unfavourable steric consequence of parity substitutent. Compound 1h shows activity better than other parity substituted derived functions likely be due to substitutent at meta place besides, which later directed us to synthesis the derived functions holding permutation other so para place in the optimisation procedure. Traveling in front with these consequences, we planned to synthesise the compounds holding permutation at ortho and meta place i.e. 2-chloro 1i, 3-nitro 1j, 3-methoxy 1k and 2,6-dichloro 1l. Biological consequences of these derived functions besides do non exhibit better activity than unsubstituted cinnamide derivative 1a ( Table 2, entry 6-9 ) .
Table 2. Cinnamide derivatives with their antimycobacterial activity and toxicity
Entry No.
Compound No.
Roentgen
MIC ( AµM ) *
CC50 ( AµM ) **
Silicon
1
1a
Hydrogen
5.1
618
121
2
1e
4-CH3
143.7
565
4
3
1f
4-OCH3
131.6
680
5
4
1g
4-Cl
128.9
788
6
5
1h
3,4-diCl
54.8
653
12
6
1i
2-Cl
64.4
725
11
7
1j
3-NO2
68.5
615
9
8
1k
3-OCH3
65.8
695
11
9
1l
2,6-diCl
54.8
653
12
10
Ethambutol
–
15.3
1470
96
*Compounds were tested against Mtb H37Rv strains utilizing REMA method
**Cytotoxicity surveies were done on Vero Cell line C1008 utilizing MTT check.
SI- selective index
To turn out our hypothesis of piecing intercrossed molecule possessing pharmacophoric characteristics of both cerulenin and cinnamic acid which are known to hold interactive action, we had subjected our most powerful hit compound 1a from the series for its interactive activity with rifampicin against Mtb. One of the best known and really simple signifiers of such trials is the ‘chequerboard ‘ experiment[ nine ]in which a two dimensional array of consecutive concentrations of trial compounds is used as the footing for computation of a fractional inhibitory concentration index ( FICI ) to show that mated combinations of agents can exercise repressive effects that are more than the amount of their effects entirely ( synergy ; FICI & lt ; 1.0 ) , or less than the amount of their effects entirely ( hostility ; FICI & gt ; 1.0 )[ x ].
The expression for finding FICI is as follows:
The synergism surveies reveal that the MIC of rifampicin entirely is 0.25Aµg/mL and that of compound 1a is 1.62Aµg/mL, while in combination MIC of rifampicin reduced to 0.0078 and that of compound 1a to 0.41. These consequences indicate that add-on of sub-MIC of compound 1a resulted in 16 fold decrease of MIC of rifampicin with fractional inhibitory concentration index ( FICI ) of 0.31 ( Table 3 ) , proposing a synergic interaction against M. TB H37Rv.
Table 3. Synergy results* .
MIC of Rifampicin
0.125
FICI – 0.31
Combination MIC of rifampicin
0.0078
MIC of 1a
1.6
Combination MIC of 1a
0.41
*Compounds were tested against Mtb H37Rv strains utilizing chequre board method
In drumhead, a little library of cinnamide derived functions has been synthesized consistently utilizing molecular hybridisation attack to club the two different structural characteristics every bit good as biological belongingss. The usage of Topliss operational method for optimising the authority of molecules reduced the clip, cost and manual attempts to acquire the hit. Among the synthesized molecules compound 1 shows assuring consequence as an antimycobacterial agent. The purpose of clubbing the molecule to integrate the interactive activity has besides been proved successfully. Furthermore, attempts for synthesising asymmetric molecules with better authority every bit good as interactive activity are under advancement.
