Looking At Cinnamide Derivatives Biology Essay


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A series of cinnamide derivatives was designed using molecular hybridization approach using structural features of clinically used as well as molecules under clinical trials for anti-tubercular and key pharmacophoric features of compounds having synergistic properties with known antimycobacterials. The designed molecules were synthesized by reaction between various diamine and substituted cinnamic acids. Topliss operational method was used to optimize the potency of the molecules. The minimum inhibitory concentration (MIC) was determined against M. tuberculosis H37Rv in Middlebrook 7H9 medium using resazurin microtitre plate assay. Some of the synthesized molecules shows good activity against M. tuberculosis H37Rv with MIC in the range of 5.1-143.1μM. The most potent compound 1a having MIC of 5.1μM also shows synergistic action (FICI-0.31) with rifampicin.

Keywords: Diamine; Cinnamic acid; Mycobacterium tuberculosis (Mtb); Antimycobacterial activity; Synergy

Tuberculosis (TB), a contagious disease infects humans via the airways by Mycobacterium tuberculosis (Mtb). WHO reports nearly 8-9 million new cases of TB while 3 million deaths which represents the largest number of incidence of human deaths attributable to a single etiological agent [i] . Inadequacy of current TB therapy has resulted in growth of the disease. Even after elucidation of the genomic sequence of the Mtb and emergence of a multiplicity of targets, residence of Mtb within the macrophages attached with its cell wall barrier, remain the key issues to successful therapy. In particular the current concern is the massive problem resulting from extensively drug-resistant tuberculosis, XDR-TB, which is commonly known as strains resistant to all current first line drugs, fluoroquinolones and at least to one of the three injectable second-line drugs (capreomycin, kanamycin or amikacin) [ii] . Due to the importance of this disease, various initiatives have been undertaken by government research laboratories, academic institutions, pharmaceutical industries etc. with the aim of developing the new drugs and strategies to combat with tuberculosis. The present work focuses on a possible strategy to overcome these challenges.

1,2-ethylenediamine is a basic scaffold of ethambutol, a clinically used first line drug for tuberculosis. Based on this scaffold a molecular library of 63,238 compounds has been developed by Sequella Inc. in collaboration with NIH/NIAID. From this library a drug candidate SQ109 was identified as the most active compound possessing 35 fold improved activity as compared to that of ethambutol [iii] . SQ109 has recently advanced into clinical trials for the treatment of tuberculosis, though it appears that the target for SQ109 is not 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 also be changed either to homopiperazine or piperazine (Fig 1.).

Fig 1. Diamine based Antimycobacterials

On the other hand, cerulenin and trans-cinnamic acid (Fig 2.) are traditionally known to have antimycobacterial activity and also proven to have synergistic action when tested along with known clinically used drugs [iv] . Additionally, cinnamyl derivative of rifampicin shows decline in MIC of rifampicin by 2- to 8-fold for most of the susceptible and multi drug resistant Mtb strains. Also this derivative has better intracellular and in vivo activities compared to those of rifampicin [v] .

Fig 2. Traditionally known Antimycobacterials having synergistic actions

In our continuous program in the search of new potent and safe antimycobacterial agent, we decided to construct a new class of cinnamide derivatives as attractive antitubercular agents [vi] . The designing strategy is based on hybridization of basic pharmacophoric features of diamine analogs i.e. Ethambutol, SQ786, SQ109, SQ775 i.e. diamine linkage, amide of cerulenin along with its α, β-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 mode of action through different target. Further optimization of molecule for its potency has been carried out using conventional method of Topliss for substitution at aromatic centre in molecule.

Fig 3. Design concept of new cinnamide derivatives.

To recognize 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 coupling with trans-cinnamic acid as first set of molecules. The target compounds i.e. cinnamide derivatives were prepared according to Scheme 1. Compounds 1a-1d were obtained in high yield through a one-step reaction using trans-cinnamic acid, ethyl chloroformate, triethylamine and diamine as starting materials. [vii] All synthesized compounds were identified by spectral data. The antimycobacterial activity of all synthesized compounds was tested against M. tuberculosis H37Rv and the minimum inhibitor concentration (MIC) was determined using REMA (Resazurin Microtitre Assay) method [viii] . Ethambutol was used as reference drug.

Fig 4. Different diamine linkers used for synthesis

Scheme 1. Synthetic scheme for synthesis of cinnamide derivatives

The biological results (Table 1.) show that the compound having 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 thereafter with respect to anti-mycobacterial activity, which is in agreement with the activity of reported molecules. Further to optimized the potency of the compound derived from 1,2-ethylenediamine i.e. 1a, we had followed the conventional Topliss operational approach of modulating the hydrophobic and electronic parameters on the phenyl ring Fig 5.

Table 1. Cinnamide derivatives with their antimycobacterial activity and toxicity

Compound No

MIC (µM)*

CC50 (µM)**


















*Compounds were tested against Mtb H37Rv strains using REMA method

**Cytotoxicity studies were done on Vero Cell line C1008 using MTT assay.

SI- selective index

Fig 5. Topliss operational tree

As per the batchwise method of Topliss approach, the second set of analogs 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 using the same synthetic Scheme 1 used above with substituted trans-cinnamic acids. Assay results of this initial set of analogs were expected to reveal the favorable electronic (r) and hydrophobic (p) requirements of the pharmacophore. Unfortunately from the biological results it can be seen that none of the molecule showed better activity then parent molecule i.e. 1a (Table 2, entry 2-5). Based on these results it can be concluded that there may be unfavorable steric effect of para substitutent. Compound 1h shows activity better than other para substituted derivatives probably be due to substitutent at meta position also, which subsequently directed us to synthesis the derivatives having substitution other then para position in the optimization process. Moving ahead with these results, we planned to synthesize the compounds having substitution at ortho and meta position i.e. 2-chloro 1i, 3-nitro 1j, 3-methoxy 1k and 2,6-dichloro 1l. Biological results of these derivatives also do not 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.


MIC (µM)*

CC50 (µM)**






























































*Compounds were tested against Mtb H37Rv strains using REMA method

**Cytotoxicity studies were done on Vero Cell line C1008 using MTT assay.

SI- selective index

To prove our hypothesis of assembling hybrid molecule possessing pharmacophoric features of both cerulenin and cinnamic acid which are known to have synergistic action, we had subjected our most potent hit compound 1a from the series for its synergistic activity with rifampicin against Mtb. One of the best known and very simple forms of such tests is the 'chequerboard' experiment [ix] in which a two dimensional array of serial concentrations of test compounds is used as the basis for calculation of a fractional inhibitory concentration index (FICI) to demonstrate that paired combinations of agents can exert inhibitory effects that are more than the sum of their effects alone (synergy; FICI < 1.0), or less than the sum of their effects alone (antagonism; FICI > 1.0) [x] .

The formula for determining FICI is as follows:

The synergy studies reveal that the MIC of rifampicin alone is 0.25µg/mL and that of compound 1a is 1.62µg/mL, while in combination MIC of rifampicin reduced to 0.0078 and that of compound 1a to 0.41. These results indicate that addition of sub-MIC of compound 1a resulted in 16 fold reduction of MIC of rifampicin with fractional inhibitory concentration index (FICI) of 0.31 (Table 3), suggesting a synergic interaction against M. tuberculosis H37Rv.

Table 3. Synergy results*.

MIC of Rifampicin


FICI - 0.31

Combination MIC of rifampicin


MIC of 1a


Combination MIC of 1a


*Compounds were tested against Mtb H37Rv strains using chequre board method

In summary, a small library of cinnamide derivatives has been synthesized systematically using molecular hybridization approach to club the two different structural features as well as biological properties. The use of Topliss operational method for optimizing the potency of molecules reduced the time, cost and manual efforts to get the hit. Among the synthesized molecules compound 1 shows promising result as an antimycobacterial agent. The aim of clubbing the molecule to incorporate the synergistic activity has also been proved successfully. Furthermore, efforts for synthesizing asymmetric molecules with better potency as well as synergistic activity are under progress.

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