INTRODUCTION:

Tuberculosis (TB) is the world’s serious disease in health problem caused by the rod-shaped, non-spore-forming, aerobic bacterium Mycobacterium tuberculosis¹. It was first isolated in 1882 by a German physician named Robert Koch who received the Nobel Prize for this discovery of Mycobacterium². TB spreads through the air like the spreads of common cold^3,4. Tuberculosis is caused by bacteria that spread from person to person through microscopic droplets released into the air. This can happen when someone with the untreated, active form of tuberculosis coughs, speaks, sneezes, spits, or laughs⁵. TB is characterized by tubercle lesion in the lungs and also can affects other body organ such as skin, nodes, brain and lymph^6,7.

In the developing countries suffering from high burden, the impact of TB can be devastating, TB case are increasing every year². In 2015, approximately there was more than ten million new cases, causing more than two million human infected was death over this disease⁸. Often, tuberculosis is accompanied by HIV-AIDSthat increase the secondary infection in immunocompromised patients^9,10,11,causing another serious healthproblem throughout the world¹², and followed by the spread of resistant strains of M. tuberculosis such as multidrugs-resistant TB strains (MDR-TB) and Extensively Drug Resistance TB strains (XDR-TB)^6,7. Nowadays, the existing TB drugs are incapable to cure the patients due to several limitations such as MDR-TB, XDR-TB and other side effects¹³. The current treatment routine for tuberculosis requires the combination of first-line drugs which include ethambutol (EMB), isoniazid (INH), pyrazinamide (PZA) and rifampin (RMP) for two months then followed by an additional four months of daily intake of INH and RMP¹⁴. However several side effect like patient non-compliance due to the prolonged period of treatment, inconsistent of treatment, and the unavailability of these drugs during the period of treatment have contributed to the treatment failure⁶. The concerted efforts of all members of society for ensuring the continued efficiency of antibiotic can minimized this antibiotic resistance problem, such as the development of new antituberculosis¹⁵.

Despite the terrible effect in health problem of TB disease, the discovery of new anti-tuberculosis drugs proceeded slowly after the discovery of Rifampicin in 1965. Even though a number of compounds have been reported to enter the phase I of clinical trial, phase II of clinical trial and some others compound candidates have entered the pre-clinical trial, the TB problem is increasingly become complex due to emergence of M. tuberculosis strains which are resistant to first-line and second-line drugs, meanwhile the discovery of new tuberculosis drugs is very limited¹⁶. Therefore, it is an urgent need in the scientific focus to discover new antituberculosis agent. The design of new TB drugs has been an important challenge for chemists and pharmacists in recent years. Research on the discovery of new anti-TB drugs has emerged, including using Multicomponent Reaction Methods to discover new anti-TB drugs that are potent and have lower side effects than anti-TB drugs currently used¹⁷. The demands for novel anti-microbial especially in anti-tuberculosis agents are now increasing significantly^{18, 19}.

One of the promising method in synthesis field is Multicomponent Reactions (MCRs). It is one-pot reactions employing more than two starting materials that suitable for rapid and low-cost synthesis method²⁰. This method constitute an especially attractive synthetic strategy for efficient library compound due to the fact that the products are formed in a single step and diversity could be achieved simply by varying the reacting components²¹. MCRs provide a new approach regarding to the efficient synthesis of diverse compounds, are evolving among researcher in recent years²². One of the reactions of many MCRs that have been developed is Ugi or U-4CR reactions, which involve isocyanides²¹. MCRs of isocyanides became the most variable way of forming chemical compounds. The great potential of isocyanides for development of MCRs lies in the diversity of bond forming processes that are available, their functional group tolerance and also the high levels of chemo, regio, and stereo selectivity often observed²¹. The main framework of the U-4CR products are basically determined by the type of amine and acid components²³. The challenge of MCR is to conduct a reaction in such a way that the network of pre-equilibrated reactions channel into the main product and do not yield side products produced by the reactions. The result is clearly dependent on the reaction conditions: solvent, temperature, catalyst, concentration, the kind of starting materials and functional groups that is used²⁴.

Until now, MCRs are still being developed using three or four component compounds^17,20,25,26. The most developed MCRs is U-4CR which leads to the formation of α-acylaminocarboamides²⁷. In recent years, there have been many new discoveries about MCRs that modify Ugi reactions. One of them is the amide-aldehyde condensation reaction or it can be called amidoalkylation, which in this reaction does not use isocyanide as a reactive species but involves formic acid as a reducing agents²⁸. The amidoalkylation used carbonyl compounds such as aldehydes and ketones, which will produce compounds with carbon amide bonds –CONH– that are expected to have high biological activity. Therefore this methods are suitable for their use in infectious tropical disease drug discovery lies in the fact that they are extremely powerful to introduce in only one step both molecular complexity and especially, structural diversity²⁹.

Various studies in the discovery of new drugs using MCRs synthesis methods, producing compounds that have various biological activities^30,31, such as anti-tumor³², antibacterial applications³³, antifungal³⁴, anticancer³⁵, anti-tuberculosis^17,36. Our approach to the discovery of novel antituberculosis agent because of the structural similarity of the target compound to one of the first-line antituberculosis drugs, namely isoniazid. This fact inspired us to synthesize novel class of amide derivative and screen their anti-tubercular activity against M. tuberculosis H₃₇Rv. In the present work, we report the synthesis of newly designed N-(chlorobenzyl) formamide 3(a-d) and study the influenced of substituent groups in the benzene ring of the synthesized compound, also evaluated these compounds for the antituberculosis activity.

MATERIALS AND METHODS:

Chemicals and reagents:

All reagents and solvent were pro analysis grade. 2-chlorobenzaldehyde, 4-chlorobenzaldehyde, 2,6-dichlorobenzaldehyde (Sigma Aldrich), Formamide (Merck), formic acid 97% (Alfa Aesar), ethyl acetate (Merck), dichloromethane (Merck), chloroform (Merck), ethanol (Merck), n-hexane (Merck), toluene (Alfa Aesar), aquadest, DMSO (Merck).

Antituberculosis evaluation:

M. Tuberculosis strain H37Rv, OADC (Becton Dickinson), Middlebrook 7H9 broth medium, Middlebrook 7H10 agar medium, Alamar Blue reagent (Invitrogen), PANTA Antibiotic Mixture (Becton Dickinson), Tween 80, deionized water sterile, aquadest sterile and isoniazid (Sigma Aldrich).

Instrumentations:

Three neck flask, glassware, boiling stone, mantle heater (Labmaster Isopad LMM/ER/ 500ML), reflux apparatus, TLC chamber and silica gel F254 (Merck), UV lamp 254 nm and 366 nm (DESAGA), parafilm, capillary pipes (NESCO), analytical scales (Ohaus), chromatography columns (Pyrex Iwaki), melting point test equipment (BUCHI melting point B-540), rotary evaporator (BUCHI), GC-MS (QP2010S Shimadzu), FTIR Perkin Elmer spectrometers (Shimadzu), ¹H-NMR (JOEL) spectrometers, ¹³C-NMR (Joel), Incubators, Biosafety Cabinets (BSC) type 2 plus, sterile volumetric pipettes (Pyrex Iwaki), vortex tool, 96-well microplate (Pyrex Iwaki), sterile screw-covered tube, sterile petridish.

General Procedure of Synthesis:

10 mmol of aromatic-aldehyde compound (2-chlorobenzaldehyde; 4-chlorobenzaldehyde; 2,6-dichlorobenzaldehyde; or 2,4-dichlorobenzaldehyde) was mixed together with 150 mmol formamide and 80 mmol formic acid into three neck flask in 150^oC using heating mantel filled with boiling stones. The stirring reaction is carried out until the starting material disappeared, the reaction result are analyzed using Thin Layer Chromatography (TLC) with Ethyl Acetate and Chloroform (1:2) as an eluent. Then the reaction results were added with saturated NaHCO₃ and extracted with dichloromethane (3x10 mL). Dichloromethane phase was collected, rinsed using saturated NaCl. The organic phase (DCM) is taken and dried with MgSO₄ anhydrous. Then filtered and evaporated using a rotary evaporator so that the synthesis product is obtained²⁸.

(a) N-(2-chlorobenzyl) formamide:

Recrystallization from toluene:n-hexane. C₈H₈ClNO. M. wt 169 g/mol. Yield 36,5%. m.p74,5 – 75,0°C. white needle-shaped crystal. IR spectrum (KBr) cm^-1: 3263,56 (N-H stretch), 3055,24 (C-H aromatic stretch), 2885,51 (C-H stretch), 1658,78 (C=O stretch amide), 1543,05 (N-H bend), 1381,03 and 1357,89 (C-N stretch, amida), 1249,87 (benzene substituted Cl), 1049,28 and 756,10 (C-H bend oop orthosubtituted aromatic). Mass spectrum (EI-MS): m/z M⁺ 169 (100%), 134; 125; 107; 89; 77; 63; 51; 29; 28. ¹H-NMR (500 MHz, CDCl₃) δ 4.563 (d, J=6,5 Hz, 2H), 8.231 (s, 1H), 6.104 (s, 1H), 7.341-7.395 (m, 2H), 7.213-7.295 (m, 1H). ¹³C-NMR (125 MHz, CDCl₃) δ 40.343, 127.412, 129.399, 129.783, 130.494, 133.834, 135.207, 161.190.

(b) N-(4-chlorobenzyl) formamide:

Recrystallization from toluene:n-hexane. C₈H₈ClNO. M. wt 169 g/mol. Yield 41%. m.p108,3 – 109,0°C. white needle-shaped crystal. IR spectrum (KBr) cm^-1: 3278,99 (N-H stretch), 3032,10 (C-H aromatic stretch), 2893,22 (C-H stretch), 1913,39 (overtone parasubstituted aromatic), 1651,07 (C=O stretch amide), 1535,34 (N-H bend), 1465,90 (C=C aromatic stretch), 1381,03 and 1350,17 (C-N stretch, amide), 1226,73 (benzene substituted Cl), 1087,85 and 825,53 (C-H bend oop parasubtituted aromatic. Mass spectrum (EI-MS): m/z M⁺ 169 (100%); 169; 134; 125; 106; 89; 77; 63; 51; 29; 28. ¹H-NMR (500 MHz, CDCl₃) δ 4.441 (d, J= 6 Hz, 2H), 5.870 (s, broad peak, 1H), 7.213 (dd, J1=8,5 Hz, J2= 2 Hz, 2H), 7.297 (dd, J1= 8 Hz, J2= 1,5 Hz, 2H), 8.252 (s, 1H). ¹³C-NMR (125 MHz, CDCl₃) δ 41.686, 129.341, 129.130, 133.747, 136.300, 161.161.

(c) N-(2,6-dichlorobenzyl) formamide:

Recrystallization from toluene:n-hexane. C₈H₇Cl₂NO. M. wt 203 g/mol. Yield 14%. m.p146,6 – 147,2°C. white needle-shaped crystal. IR spectrum (KBr) cm^-1: 3271,27 (N-H stretch), 3024,38 (C-H aromatic stretch), 2893,22 (C-H stretch), 1843,95 and 1774,51 (overtone, trisubstituted aromatic), 1651,07 (C=O stretch amide), 1535,34 (N-H bend), 1442,75 (C=C aromatic stretch), 1381,03 and 1342,46 (C-N stretch, amide), 1219,01 (benzene substituted Cl), 779,24 and 694,37 (C-H bend oop trisubstituted aromatic). Mass spectrum (EI-MS): m/z M⁺²170; M⁺ 168; 140; 111; 89; 75; 63; 50; 29; 28. ¹H-NMR (500 MHz, CDCl₃) δ 4.801 (d, J= 6 Hz, 2H), 5.781 (s, broad peak, 1H), 7.167 (d, J= 7,5 Hz, 1H), 7.308 (dd, J1= 8 Hz, J2= 1 Hz, 2H), 8.184 (s, 1H). ¹³C-NMR (125 MHz, CDCl₃) δ 41.196, 128.756, 128.900, 130.061, 136.339, 160.691.

(d) N-(2,4-dichlorobenzyl) formamide:

Recrystallization from toluene:n-hexane. C₈H₇Cl₂NO. M. wt 203 g/mol. Yield 5%. m.p132,6 – 133,4°C. white needle-shaped crystal. IR spectrum (KBr) cm^-1: 3255,84 (N-H stretch), 3062,96 (C-H aromatic stretch), 2885,51 (C-H stretch), 1851,66 (overtone, trisubstituted aromatic), 1658,78 (C=O stretch amide), 1550,77 (N-H bend), 1473,62 (C=C aromatic stretch), 1388,75 and 1357,89 (C-N stretch, amide), 1249,87 (benzene substituted Cl), 887, 26 and 825,53 (C-H bend oop trisubstituted aromatic). Mass spectrum (EI-MS): m/z M⁺²170; M⁺ 168; 140; 123; 111; 89; 75; 63; 50; 29; 28. ¹H-NMR (500 MHz, CDCl₃) δ 4.514 (d, J= 6 Hz, 4H), 6.043 (s, broad peak, 2H), 7.221 (dd, J1= 8,5 Hz, J2= 2,5 Hz, 2H), 7.332 (d, J1= 8,5 Hz, 2H), 8.228 (s, 2H). ¹³C-NMR (125 MHz, CDCl₃) δ 39.795, 127.681, 129.600, 131.328, 134.429, 134.496, 161.199.

Antituberculosis activity assay:

All of the compounds were screened in MABA³⁷ and Middlebrook 7H9-7H11 medium. The solid culture of M. Tuberculosis strain H37Rv was obtained from Microbiology Laboratory, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada. Preparation of M. tuberculosis suspension stock solution using a weighing colony. The method of weighing the colony is carried out by weighing the weight of the bacterial colony taken by ose a certain amount in mg units which is equal to the volume of aquadest in mL units added to dissolve the bacterial colony, so that it will produce a concentration of 1 mg / mL of bacterial suspension equivalent to 0.5 Mc Farland, where 0.5 Mc Farland is equivalent to 10⁸ CFU / mL. The freshly prepared M. Tuberculosis strain H37Rv cultures then diluted in Middlebrook 7H9 broth supplemented with 10% OADC, then adjusted to 10⁷ CFU/mL.

All the synthesis compound solved in dimethyl sulfoxide (DMSO) to obtain 4000 µg/mL stock solution, then sterilized with a 0.2 μm filter membrane and stored at -20°C as a stock solution. DMSO was used as negative control and Isoniazid (2 L/mL) were used as positive control in all experiments reported in the literature by Franzblau (1997). In this study 10 concentrations series were used; 1000; 500; 250; 125; 62.5; 31.25; 15,625; 7,813; 3,907 and 1,954 μg/mL to get Minimum Inhibitory Concentration (MIC) from MABA method. In MABA method, after the each well in the microplate was filled, then the microplates were wrapped in parafilm and incubated at 37°C for 5 days. After 5 days, a total of 50 µL mixture of Alamar Blue reagent and 10% Tween 80 in a 1: 1 ratio were freshly prepared, then added to the bacterial control well and re-incubated for 24 hours to observe whether the cultures were grow in the condition used, identified with a changing colour in the well from blue to pink. Then all the well in the microplate were added with 50 µL mixture of Alamar Blue reagent and 10% Tween 80 (1:1). In Middlebrook 7H9-7H11 medium, the M. Tuberculosis strain H37Rv cultures was freshly prepared in Middlebrook 7H9 broth supplemented with 10% OADC, and adjusted to concentration series of the synthesis compound. After that, all the test tube were then incubated at 37°C for 48 hours. Then, they were subcultured in the agar medium Middlebrook 7H11 to obtain Minimum Bactericidal Concentration (MBC).

RESULTS AND DISCUSSION:

Chemistry:

Considering our interest in discovering new antituberculosis agent from multicomponent reaction using formic acid, in this study we used chloro-substituted aromatics aldehyde which expected to enhance the inhibitory activity of the molecule as an antituberculosis. The results of the synthesis using formic acid evaluated by TLC show that synthesis using this method gives results in the form of a mixture of compounds, in accordance with the results obtained by Ivar Ugi (2005) that synthesis of MCRs can produce several variations of compounds marked with the formation of many spots in the crude product.

The study of multicomponent reactions between substituted aromatic-aldehydes of chloro and formamide atoms using formic acid was based on the amidoalkylation reaction. Amidoalkylation reaction is a reaction that leads to the formation of carbon-carbon bonds with the replacement of group X in the electrophile reagent, where the X group can be halogen, –OH, -OR, –OCOR, –NHCOR, or –NR2. In this study, we used acid condition that will obtained imine intermediates, which are reacted with a nucleophile to form new bonds on C and N atoms which then form α-amidoalkylation products by releasing H₂O³⁸.

Starting Material

Product

a. R1 = Cl

b. R3 = Cl

c. R1 and R5 = Cl

a. R1 = Cl

b. R3 = Cl

c. R1 and R5 = Cl

Scheme 1: Synthesis reaction of N-(chlorobenzyl)formamide

In this study, the reaction occurred between three kinds of compounds (aromatic-aldehyde, formamide and formic acid) mixed at the same time in the presence of heat, without the isolation process of intermediate products being formed as shown in Scheme 1. Formic acid has a dual role in amidoalkylation and reductive amination reactions, namely as a catalyst and reductant. The results of synthesis using formic acid provide a mixture of compounds in the synthesis product. Products synthesized using formic acid were separated to obtain a single spot from each product using a column chromatography method with an optimum eluent. Based on the results of the separation, the 4^th spot of the synthesis results showed a dominant amount so that it was thought to be a target compound from the synthesis results.

The mechanism of amidoalkylation in multicomponent reaction using formic acid is shown in Scheme 2. The first stage is Protonation and Addition. Formic acid as a catalyst plays a role in the protonation stage of oxygen (O) at the carbonyl aldehyde group, in which the donor proton at this stage causes reactivity of C carbonyl to become a strong electrophile so that it will be easily attacked by weak nucleophiles such as amides. At the addition stage, the lone pair of electrons from the N amide atom (nucleophile) will attack the C carbonyl atom on the positively charged aldehyde (electrophile), so that the amide will bind to the carbonyl carbon position of the aldehyde, followed by the release of protons from the N atom to neutralize the positive charge on the N atom.

The second step is Elimination. The release of a water molecule in the presence of a proton released at the addition stage causes a free electron pair from the O atom to pull the proton so that it binds to H2O with a positive charge on the O atom, the movement of the free electron pair of N atoms forms a double C = N bond, then the release of water molecules results in iminium ions.

The third step is Reduction. Formic acid in the form of hydride will reduce the iminium ion, becoming a target compound by releasing CO₂ molecules, at this stage formic acid acts as a reducing agent that can reduce imin intermediate compounds formed from the condensation of aldehydes with amides.

Step 1. Protonation and Addition

Step 2: Elimination

Step 3: Reduction

Scheme 2: Mechanism of Amidoalkylation in MCRs using formic acid:

The structure of all the synthesized compounds are presented in Table 1. All novel compounds were characterized using, 1H and 13C NMR, mass spectra, IR spectrophotometer.

Table 1: Structure of N-(Chlorobenzyl)Formamide

Compound	Structure
a	N-(2-chlorobenzyl) formamide
b	N-(4-chlorobenzyl) formamide
c	N-(2,6-dichlorobenzyl) formamide
d	N-(2,4-dichlorobenzyl) formamide

Antituberculosis Activity:

The four products then were evaluated their antituberculosis activity to determine the Minimum Inhibitory Concentration (MIC) through Microplate Alamar Blue Assay. MABA method is based on the mechanism of M. tuberculosis redox reaction that can reduce the blue resazurin to the pink resofurin. The wells that retain the blue are the MIC value of the test compound, which indicates that M. tuberculosis bacteria have died as their metabolism cannot convert resazurin to resofurin in pink³⁹. Meanwhile Middlebrook 7H9-7H11 Medium is used to determine the Minimum Bactericidal Concentration and to identify the inhibitory effect (%inhibition) of the compound. Isoniazid was used as standard drug for comparison, it is mostly used in the treatment and prevention of tuberculosis. It acts by inhibiting the synthesis of mycolic acid, required for mycobacerial cell wall synthesis in Mycobacterium tuberculosis⁴⁰.

Antituberculosis activity test using these two methods aims to strengthen the results obtained, in which the acquisition of MIC value from the MABA method, will be confirmed using Middlebrook 7H9-7H11 media to obtain the MBC value. The use of the MABA method has advantages including a relatively short time to obtain test results (5-14 days), the need for test samples and media that are used less, have high accuracy and sensitivity, and can be done in a large number of samples in a short time so that it is suitable for rapid screening of many new compounds to determine their biological activity in a microorganism^41-45. MABA method required just 7 days producing results (6 days to observe full growth in the corresponding controls and 1 day to develop remaining wells. Furthermore, this method requires only 200 µL per well to perform the entire assay, where as others use 9-10 mL of solid medium^46,47,48.

Table 2 summarizes the antituberculosis results of the compounds using MABA methods determined in MIC values in the concentration (µg/mL) used. All the compounds were active against M. tuberculosis whereas the c compound indicate that it has the lowest MIC which is 500.0 µg/mL, meanwhile all the other compound give the same MIC, which is 1000.0 µg/mL. Determination of the MIC based on the smallest concentration that is able to prevent the change in blue to pink in the well is determined as the MIC value.

Table 2. Antituberculosis activity of novel synthetized compound using MABA method

Sample	MIC value (µg/mL)
Sample	Replication I	Replication II	Replication III
A	1000,0	1000,0	1000,0
B	1000,0	1000,0	1000,0
C	500,0	500,0	500,0
D	1000,0	1000,0	1000,0

Figure 1. The result of changes in the color of the wells after the addition of the Alamar Blue reagent to the a compound (N-(2-chlorobenzyl) formamide) (7th day)

Note: (1A) 1000.0μg / mL; (2A) 500,0μg / mL; (3A) 250.0μg / mL; (4A) 125.0 μg / mL; (5A) 62.5μg / mL; (6A) 31.2μg / mL; (7A) 15.6μg / mL; (8A) 7.8μg / mL; (9A) 3.9μg / mL; (10A) 1.9μg / mL. Number 1B-10B is a control compound (compound without bacteria). Number 1C-3C is the control drug INH 2 μg / mL. Number 4C-6C is a 1.25% DMSO solvent control. Number 7C-10C is media control. Number 1D-10D is bacterial control.

Figure 2. Results of changes in the color of the wells after the addition of the Alamar Blue reagent to the b compound (N-(4-chlorobenzyl) formamide (7th day)

Note: (1A) 1000.0μg / mL; (2A) 500,0μg / mL; (3A) 250.0μg / mL; (4A) 125.0 μg / mL; (5A) 62.5μg / mL; (6A) 31.2μg / mL; (7A) 15.6μg / mL; (8A) 7.8μg / mL; (9A) 3.9μg / mL; (10A) 1.9μg / mL. Number 1B-10B is a control compound (compound without bacteria). Number 1C-3C is the control drug INH 2 μg / mL. Number 4C-6C is a 5% DMSO solvent control. Number 7C-10C is media control. Number 1D-10D is bacterial control.

Figure 3. Results of changes in the color of the wells after the addition of the Alamar Blue reagent to the c compound (N-(2,6-dichlorobenzyl) formamide) (7th day)

Note: (1A) 1000.0μg / mL; (2A) 500,0μg / mL; (3A) 250.0μg / mL; (4A) 125.0 μg / mL; (5A) 62.5μg / mL; (6A) 31.2μg / mL; (7A) 15.6μg / mL; (8A) 7.8μg / mL; (9A) 3.9μg / mL; (10A) 1.9μg / mL. Number 1B-10B is a control compound (compound without bacteria). Number 1C-3C is the control drug INH 2 μg / mL. Number 4C-6C is a 5% DMSO solvent control. Number 7C-10C is media control. Number 1D-10D is bacterial control.

Figure 4. Results of changes in the color of the wells after the addition of the Alamar Blue reagent to the d compound (N-(2,4-dichlorobenzyl) formamide) (7th day)

Note: (1A) 1000.0μg / mL; (2A) 500,0μg / mL; (3A) 250.0μg / mL; (4A) 125.0 μg / mL; (5A) 62.5μg / mL; (6A) 31.2μg / mL; (7A) 15.6μg / mL; (8A) 7.8μg / mL; (9A) 3.9μg / mL; (10A) 1.9μg / mL. Number 1B-10B is a control compound (compound without bacteria). Number 1C-3C is the control drug INH 2 μg / mL. Number 4C-6C is a 5% DMSO solvent control. Number 7C-10C is media control. Number 1D-10D is bacterial control.

Based on the results of the study there are differences in the MIC of the two methods, the c compound, MIC on the MABA method shows the value of 500.0 μg / mL while the Middlebrook method 7H9-7H11 media shows the value of 1000.0 μg / mL. This difference is due to the difficulty of making visual observations of turbidity in the treatment group. The characteristics of M. tuberculosis bacteria are different from other bacteria, where bacterial suspensions in general will provide uniform turbidity to the liquid media used, while the colonies of M. tuberculosis bacteria will form sediment like white crumbs at the base of the liquid media. However, the% inhibition on the test results with Middlebrook 7H9-7H11 media obtained similar results, the c compound has a higher% inhibition than other compounds.

Based on the results of the study on MABA method, it can be seen the MIC value of the four target compounds is 1000.0μg / mL (a); 1000.0 μg / mL (b); 500.0 μg / mL (c) and 1000.0 μg / mL (d). The difference in MIC values can be based on the structural framework of each target compound. Where c and d compounds both have the substitution of two Cl atoms. However, c compound has 2 Cl atoms in the ortho position (at 2.6 carbon atoms) with respect to the benzene ring, while d compound has 2 Cl atoms in the ortho position and para on the benzene ring towards the N atoms. The aromatic ring has the effect of positive resonance and negative induction on the ring, this is because the Cl atom has three pairs of free electrons which can give a negative induction effect to the outside of the ring and positive resonance into the ring, considering that the Cl atom has 3.0 electronegativity. In c compound Cl atoms are more difficult to provide positive resonance into the ring because both Cl atoms are in an ortho position which is blocked by steric factors. Negative induction out of the ring causes the benzene ring to be more positive, while to increase the electron density through the resonance of free electrons Cl atom toward the ring becomes difficult due to steric factors, consequently the ring is more positively charged so it is easier to bind to the negative side (anion) of the cell wall M. tuberculosis bacteria. This makes the MIC value of c compound lower, that is 500.0 μg / mL compared to d compound which have Cl atoms in ortho and para substituents (do not have a high steric factor).

Chloro substitution can significantly increase biological activity, this is related to lipophilicity of a compound which is an important parameter in membrane permeation in biological systems. This is consistent with this research, namely the low biological activity of analogues N-(R) formamide with MIC = 1000.0 µg / mL (5,917µM) in the target a compound (% inhibition = 13.30%) and b (% inhibition = 12.91%), which is substituted for 1 (one) Cl atom. The target compounds c and d have higher inhibition than a and b because this compound has two Cl atoms substituted in the benzyl group. The c target compound has greater inhibition (% inhibition = 47.24%) with a MIC value = 500.0 µg / mL (2,463µM) than the d target compound (% inhibition = 34.82%) with MIC value = 1000.0 µg / mL (5,917 µM), due to the structure of the c target compound, has the substitution of two Cl atoms in the ortho position towards the amide group. The presence of two chloro substituent on the benzylformamide ring enhance the inhibition activity of the molecule. This is because in the structure of compound c which has 2 chlorine atoms, each of which has 3 free electron pairs so that it can form more hydrogen bonds with amino acids in bacterial cells compared to a-b compounds which only have 1 chlorine atoms. Based on this research, the target compound N- (R) formamide has antituberculosis activity, but is still in a high range.

CONCLUSION:

Chlorobenzyl formamide compounds, a novel compounds produced by using multicomponent reaction and give moderate to excellent yields. The synthesized compound were evaluated for their in vitro antituberculosis activity using MABA method. The result indicated that all of the compounds have inhibitory activity of M. tuberculosis at concentrations of 1000 g/mL with compound c (N-(2,6-dichlorobenzyl) formamide) showed the most potent antituberculosis activity up to a concentration of 500 g/mL.

CONFLICT OF INTEREST:

The authors have declared “no conflicts of interest with respect to the research, authorship, and/or publication of this article.”

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Received on 22.12.2019 Modified on 12.06.2020

Research J. Pharm. and Tech. 2021; 14(6):3253-3261.

DOI: 10.52711/0974-360X.2021.00566

Sample	Colony Forming Unit (CFU)			Average	% Inhibition	MIC (µg/mL)	MBC (µg/mL)
Sample	Replication I	Replication II	Replication III	Average	% Inhibition	MIC (µg/mL)	MBC (µg/mL)
A	523	526	522	523,67	13,30	1000	-
B	523	510	545	526,00	12,91	1000	-
C	302	358	296	318,67	47,24	1000	-
D	378	437	366	393,67	34,82	1000	-
DMSO 5% (Solvent Control)	596	566	528	563,33
Bacterial control 10⁷CFU/mL	*	*	604	604