An Exquisite Design Process and Impacting Synthesis of 2-methyl Nicotinamide Derivatives and their Anti-bacterial Activity Closet to Fab I Inhibitors

Introduction

Over the past decade, thousands of infectious diseases[1,2] have been reported around the world, making the development of anti-infective chemotherapeutics more important than ever before. Currently, drug discovery is developing inhibitors of Fab I [3]. The NADH enoyl reductases are manufactured by bacteria using the TYPE-II fatty acid biosynthesis pathway (FAS-II). Bacterial fatty acid biosynthesis (FAS) enzymes are more desirable anti-microbial targets because they generate phospholipid membrane precursors, which are essential for the survival of both Gram-positive and Gram-negative bacteria. Furthermore, there is an inadequate amount of homology and basic structural differences between the FAS-I system in mammals and the FAS-II method in bacteria. Antibacterial drugs including triclosan, isoniazid, and diazaborines aim their inhibitory efforts at NAD(P)H-dependent enoyl-ACP reductase (ENR), the enzyme responsible for the last stage of the FAS-II elongation cycle.

Figure 1: Various synthetic strategies for the generation of 2,5-difluoro benzaldehyde and 2,6-difluoro benzaldehydes via reductive amination (previous and current). Click here to View Figure

Figure 2: Schematic representation of various functional based novel molecules showing activity against Fab I Click here to View Figure

Figure 3: The well-known FabI inhibitors and anti-infective drugs available in the literature 17 Click here to View Figure

An established method for inhibiting the biosynthetic process of type II fatty acids produced by bacteria, Fab-I inhibitors are NADH-dependent enoyl reductases (FAS-II). However, it is the underexploited target for drug discovery [18].     

On the available literature and the works done in medicinal chemistry and crystallographic studies of Fab K from Streptococcus pneumoniae, Kitagawa et al., [19] explored the dual inhibitory properties of phenyl imidazole derivatives of 4-pyridone[20,21]. Fab K showed good activity against S pneumoniae. Additionally, the obtained material did not show any significant cytotoxicity (IC50 > 69 μM). In the process of obtaining better outcomes, a novel agent is used for treating bacterial infections and the results of the research support the development of the scaffolds.

According to Yong-Mei Zhang et al., vital steps in the production of bacterial membrane lipids should be reviewed. When it comes to producing cellular membrane lipids, dissociated type II fatty acid synthase is the most energetic component. Controlling the regulatory biochemistry that dictates the speed of cellular lipids, the module of initiation contains the enzymes needed to commence creation of type II fatty acid. According to a 2014 study by Ben C. L. van Schaijk et al., falciparum sporozoites must undergo Type II Fatty Acid Biosynthesis in the midguts of Anopheles mosquitoes. These results demonstrate that the FAS-II pathway is necessary for P. berghei and P. yoelii parasite development in the liver stage of rodents but is not necessary for oocyst formation in the midguts of Anopheles mosquitoes. Mycobacterium TB resistance mechanisms linked to suppression of type II fatty acid synthase were described by Grzegorzewicz et al. Inhibiting FAS-II dehydratase, an enzyme necessary for mycolic acid production, isoxyl (ISO) and thiacetazone (TAC) kill Mycobacterium tuberculosis (Mtb). Many more instant mutations are associated with high-end resilience to ISO and TAC preventing either their active form from being converted or their target from being covalently modified [33-34]. The molecular mechanisms responsible for M.tb spreads strains around are related to mutations in stage II and the non-essential FAS-II dehydratase HadBC, are still unknown [35]. The development of NADH enoyl reductase inhibitors using type-II biosynthetic bacterial fatty acid routes occurs in its infancy [36–38]. Therefore, it is necessary to develop bio-synthetic pathway technologies to discover new key interventions that inhibit a variety of diseases that affect humans.  

Figure 4: Binding mode of MUT056399 with enzyme Click here to View Figure

Based on the previous literature of Fab I inhibitors there should be some important binding centers to the enzyme. The ethereal oxygen was binding with the ribose moiety of NADPH. The LHS halogen atom is bound with Tyr 156. The LHS alkyl chain is bound tothe triad of Ala, Tyr, and Ly as well and π-stacking with the benzamide moiety of NADPH cofactor. The RHS amide part is combined with Ala 140 [39-40]. 

A fluoroquinolone antibiotic called ciprofloxacin is used to treat a variety of infections. Bone and joint problems, infections of the urinary and respiratory systems, skin infections, typhoid fever, and infectious diarrhea are all included in this.The MIC of developed compounds was computed using this as a reference compound.    

In this work, the prepared molecules were designed as per the structure of previous Fab I inhibitors and with essential binding sites like piperazine moiety as present in ciprofloxacin[41–42]. It is also noted that One of the most sought-after heterocyclics to develop novel pharmaceutical components with a wide range of applications is piperazine [43–44]. Many molecules emerge with variousbioactivities, like antitumor, antibacterial, anti-inflammatory, antioxidant, and other activities.

Figure 5: The biosynthetic pathway of Fab I inhibitors Click here to View Figure

All the solvents and the reagents were purchased from Aldrich, BLD Pharma, Rankem India, and Spectrochem. Merck alumina plates coated with silica gel 60 F₂₅₄ were used for thin-layer chromatography.5% Methanol/DCM was used as eluent for the spot visibility achieved through UV or iodine vapor exposure. The purification of crude compounds was done on silica gel using a comb flash. Fisher-Johns melting point apparatus was used to determine the melting points in open capillaries.¹HNMR and ¹³C NMR spectra were recorded using JEOL 600 MHz and 100 MHz systems. CDCl₃was used as solvent with TMS as the internal standard. FT-IR-8400S instrument with KBr pelletswas used to record the FT-IR spectra.  HRMS spectra of the samples were recorded on the Waters TQF system.The results of the FT-IR, HRMS, ¹HNMR, and ¹³C NMR spectra were represented and interpreted in the supplementary section.

Scheme 1 Click here to View Scheme

The experimental section of scheme-1 was performed with intermediate 5 synthesis by treating n-methyl piperazine with 4-bromo-2,5 -difluoro benzaldehyde via reductive amination in the presence of Titanium isopropoxide. This step was tried with different reducing agents and with different solvents which are listed in Table 1.

Table 1: Screening of reducing agents for reductive amination for the preparation of intermediate (5) in Scheme 1

S. No	Reducing agent	Solvents	Volume	Time (h)	Temp 0C	Yield (%)Intermediate (5)
1.	NaBH4	DCM	20.0	16	25-30	39 %
2.	NaHB(OAc)3	DCM	20.0	16	25-30	48 %
3	H2/Pd	Methanol	10.0	16	25-30	30 %
4	NaCNBH3	Methanol	10.0	4	25-30	40 %
5.	Ti{OCH(CH3)2}4,NaBH4	THF	20.0	4	25-30	90 %

Note: Volume* = Weight of input (W) x Solvent ratio (R) (Ex: 1.0 g Compound in 20 mL of Solvent)

Various reducing agents, protic,and aprotic solvents were used for the reductive amination of intermediate 5. At the beginning, the schematic reaction was conducted by using sodium borohydride as a reducing agent and dichloro methane (acting as aprotic solvent), methanol (protic solvent) under room temperature yielded 39 % in 16 h.  But this step was successfully done with Ti[OCH(CH₃)₂]₄ in the presence of THF as solvent within 4h. And it is very affordable. Then intermediate 6 was successfully prepared by making a Suzuki reaction with bromo intermediate 5on a reaction with pyridine boronated esters 2. The final amides 9A-9Ehave been prepared with different amines in moderate to high yields in the presence of a coupling reagent HATU.

Scheme 2 Click here to View Scheme

Initially, reductive amination of intermediate 10 was tried with n-methyl piperazine in the presence of titanium isopropoxide. But surprisingly SNAr coupling happened with the replacement of fluorine. This is the novelty of the present work. According to the literature, titanium isopropoxide would be used as a catalyst for the asymmetric allylation of ketones, the diastereoselective reduction of alpha flour ketones, and the production of acyclic and allylic epoxy alcoholsand also for intra molecular formal [3+2] cyclo addition. So intermediate 11 was obtained through SNAr coupling of n-methyl piperazine to intermediate 10 in the presence of titanium isopropoxide at room temperature. After that different boranate esters were attached to intermediate 11 via Suzuki coupling to prepare the compounds 13A-13G. This route is very short, affordable, and can be utilized to synthesize various biologically active molecules.

Results and Discussion      

Novel molecules were prepared with novel synthetic routes. The synthetic route proposed was affordable, cost-effective, and well-yielded. The antibacterial activity of the synthesized molecules was established on two types of bacteria.

Invitro antibacterial testing

The synthesized two series of molecules were evaluated on “two gram-positive bacteria (S.aureus, B.subtilis) and also on two gram-negative bacteria (E.Coli, P.aeruginosa).” The compounds 9A-9F & 13A-13G were tested in DMSO at 8,16,32,64, 128 & 256 µg/ml concentrations and their MICs were evaluated. Compound 9B has shown the MIC at 32 µg/ml. The compounds 9C and 9D are shown the MIC at 64 µg/ml, against both gram-positive bacteria. At 32 µg/ml, the aldehyde derivatives 13C and 13D demonstrated resistance to the gram-positive bacteria S. aureus and B. subtilis.

Table 2: MIC results of synthesized molecules Click here to View Table

Docking studies

The molecular docking for the compounds with the highest MIC values was corroborated theoretically using molecular docking studies. The protein used for the docking of molecules is 7ap6. The software used for the molecular docking is AutodockVina 1.5.7. As shown in Table 3, the result shows that the synthesized novel molecules showedhigher binding energy than the standard drug Trichlosan. In the docking picture, the dotted lines indicate the hydrogen bonding donor and acceptors. The various types of interactions were mentioned at the bottom of the picture in the square box. The number of hydrogen bonds that the molecule’s protein forms (9B) is 8 with a binding energy of 7.67 kcal/mol, which is more in comparison with the standard molecule Triclosan. Molecule 13 (D) is also the other best molecule with a binding energy of -8.43 kcal/mol and best fitsinto the protein pocket by binding with 10 hydrogen bonds.This is the best molecule among the synthesized molecules. The benzyloxy groupisresponsible for additional hydrogen bond formation with the protein.

Table 3: Docking studies of the synthesized molecules with protein Click here to View Table

Table 3 illustrates the interaction between compounds 9B & 13Dwith the target enzyme. The Ligand-Protein complex’s 2D structure is displayed in the second column, its 2D structure is displayed in the first column, and its 3D structure is displayed in the third column. The binding active residues in the 2D complex are denoted by three-letter codes, whilst the docked compounds are shown as a grey stick color. Yellow lines represent the π-stacking interactions while pink dotted lines represent the Hydrogen Bond (HB) interactions. The 3D representation shows the docked compounds as cyan stick models and the binding active residues as grey stick models. The figure displays interactions with HB on the left side and π-stacking on the right side, shown by the dotted lines, respectively.

General Procedure

Scheme-1: Procedure for the Synthesis of 1-(4-Bromo-2,5-diflourobenzyl)-4−methyl piperazine (5)

The reaction was initiated by mixing 4-bromo-2,5 -difluoro benzaldehyde (5 g, 22,624 mmol, 1eq.), N-methyl piperazine (2.26 g, 22,624 mmol, 1eq.) in THF (100 mL, 20 vol.) to 0°C, which was followed by adding titanium isopropoxide (13.72 mL, 45.248 mmoL). One hour was spent stirring the reaction mixture at room temperature. A further hour of stirring at RT was continued by the addition of NaBH₄ (0.85g, 22.624 mmol,1 eq.). Filtering the reaction blend through a celite bed was the next step. Researchers diluted the celite bed filtrate with “ethyl acetate” and then cleaned it with water, saturated with brine, evaporated over Na2SO4, and then condensed at a minimised compression. A light-yellow solid (3.25g, 90 % yield) was obtained from the crude material using silica gel. LC−MS: m/z 306.16 [M + 2].

Procedure for the Synthesis of Ethyl 5-(2,5-diflouro-4-((4-methyl piperazine-1-yl) methyl) phenyl)-phenyl)-2-methyl nicotinate (6)

The compound 1-(4-Bromo-2,5-diflourobenzyl)-4-methylpiperazine (3 g, 9.830 mmol, 1 equivalent) was dissolved in a mixture of 1,4 dioxane/water (4:1).To this solution, ethyl 2-methyl-5-(4,4,5,5-tetra methyl-1,3,2-dioxaboralan-2-yl) nicotinate (3.43, 11.796 mmol, 1.2 equivalents) and K2CO3 (2.71 g,19.66 mmol, 2 equivalents) were added.

The reaction mixture was purged with N₂ gas for 5 minutes to remove any dissolved gases, and then Pd₂Cl₂(dppf) was added. The amount of dichloromethane adduct used was 1.80 grammes, which corresponds to 0.983 millimoles and is equivalent to 0.1 equivalents. Upon reaching a temperature of 100 ^oC, the mixture was kept at that temperature for a duration of 16 hours. TLC oversaw the conclusion of the retreat. A light brown semi-solid, consisting of ethyl “5-(2,5-difluoro-4-((4-methyl piperazine-1-yl) methyl)-phenyl)-2-methyl nicotinate”, was produced with a yield of 84% (3.20 g). The resulting product was concentrated under decreased pressure after being diluted in ethyl acetate, cleaned with water and brine solution, and evaporated with Na2SO4. Liquid chromatography-mass spectrometry: mass-to-charge ratio of 390.16 with a molecular ion peak at [M + 1].

Procedure for the Synthesis of Ethyl 5-(2,5-diflouro-4-((4-methyl piperazine-1-yl) methyl) phenyl)-phenyl)-2-methyl nicotinic acid (7)

A solution of ethyl “5- (2,5-difluoro-4-[(4-methyl piperazine-1-yl)methyl)phenyl)-2-methyl nicotinate” (3.20 g, 8.221 mmol, 1 equivalent) in a mixture of tetrahydrofuran (THF), methanol, and water (20 mL THF, 5 mL methanol, and 5 mL water) was adjusted to room temperature before adding lithium hydroxide monohydrate (LiOH.H2O). The mixture is swirled continuously for over 16 hours. Thin-layer chromatography (TLC) was used to monitor the advancement of the reaction. Citric acid was used as an acidifier to counterbalance the alkalinity of the organic solvent. The system is kept at reduced pressure, and the resulting solids are filtered and dried to produce the compound ethyl “5-(2,5-difluoro-4-((4-methyl piperazine-1-yl) methyl)-phenyl)-2-methyl nicotinic” acid was obtained with a yield of 2.80 grammes, which corresponds to a 94% yield.

1H NMR (400 MHz, DMSO-d6): 8.76 (s, 1H), 8.28 (s, 1H), 7.51-7.49 (m,1H)7.42-7.38 (m.1H),3.67 (s,2H),3.19 (s,3H),3.06-2.60 (m,8H),2.58 (s,3H). LC−MS: m/z 362.33 [M+1].

General procedure for the synthesis of Ethyl 5-(2,5-diflouro-4-((4-methyl piperazine-1-yl)methyl) phenyl)-phenyl)-2-methyl nicotinamide derivatives (9A-9F)

“A solution of ethyl 5-(2,5-difluoro-4-((4-methyl piperazine-1-yl) methyl)-phenyl)-2-methyl nicotinic acid(7) (0.1 g, 0.28 mmol, 1 eq.) was combined with a solution of DMF (10 mL), along with DIPEA (0.09 mL, 0.552 mmol, 2 eq.) and HATU (0.12 g, 0.331 mmol, 1.2 eq.).” The 2-methoxy benzyl amine (0.037 g, 0.276 mmol, 1 eq.) was added. The reaction mixture was once again agitated for a duration of sixteen hours at the ambient temperature.The TLC measuring instrument was used to ascertain the completeness of the reaction. After being diluted with distilled water, the recovered mixtures were subjected to ethyl acetate extraction. The procedure included a freshwater rinse, immersion in a brine solution, drying with Na₂SO₄, and concentration at reduced pressure. The crude chemical was refined using silica gel as the stationary phase and a 5% Methanol/DCM mixture as the luent. This process resulted in the isolation of “(5-(2,5-diflouro-4-(4-methyl piperazin-1-yl) methyl) phenyl) -N- (2 -methoxybenzyl) -2-methyl nicotinamide (9D)” as a light brown solid, with a yield of 0.1 g (75%).

m.p.: 322-324^4c; FT-IR (KBr): 1673.48 cm^-1 -C=O stretching, 3125.35 cm^-1: -N-H stretching; ¹H NMR (400 MHz, DMSO-d6): 8.80 (s, 1H), 8.79 (s, 1H), 7.58-7.56 (m,1H),7.29-7.26 (m.1H) ,7.27- 6.94 (m, 4H), 4.44-4.42 (m,2H), 3.80 (s, 3H), 3.54-3.48 (m, 2H), 2.58 (s, 3H), 2.44−2.30 (m, 8H), 2.14 (s, 3H). ¹³ C NMR (100 MHz, DMSO-d6): d 167.38, 156.20, 148.87, 135.00, 131.62, 128.23, 127.83, 127.11, 126.85, 126.32, 124.47,120.19, 118.39, 118.13, 116,.92, 110.52, 55.32 54.66, 52.37, 45.73,40.13, 39.92, 39.08, 38.87 & 37.80. HRMS found: ESI m/z 481.21918 (M+H)+, [M.F: C27H30F2N4O2, requires 480.23]

Scheme-2

Procedure for the synthesis of 4-bromo-2-flouro-6-(4-methylpiperazine-1-yl) benzaldehyde (11)

During the experiment, titanium isopropoxide (13.72 mL, 45.248 mmol, 2 eq.) was added to the solution of 4-bromo-2, 6-diflouro benzaldehyde (5 g, 22.624 mmol, 1 eq.) and N-methyl piperazine (4) (2.2 g, 22.624mmol, 1eq) in THF.For two hours, the reaction mixture was stirred constantly at room temperature. Monitoring of the reaction was carried out using TLC. Afterward, an ammonium hydroxide solution was utilized to quench the reaction. The celite bed is used to filter the solid. After cleaning in the brine solution, it was dried using Na₂SO₄ and concentrated under reduced pressure. As a yellow liquid, 4-bromo-2-fluoro-6-(4-methylpiperazine-1-yl) benzaldehyde was purified on silica gel in the presence of 50% ethyl acetate/hexane as the eluent to yield 6.20g (90 %) crude material.

¹H NMR (400 MHz, DMSO-d⁶): 10.18 (s, 1H), 6.95-6.94 (m, 1H), 6.92-6.91(m, 1H), 3.15-3.12 (m, 4H), 2.63-2.61 (m.4H), 2.26 (s, 3H). LC−MS:m/z 303.1 [M+2].

General procedure for the synthesis of 4-bromo-2-flouro-6-(4-methylpiperazine-1-yl) benzaldehyde derivatives (13A-13G)

4-Flouro-2-(4-methylpiperazine-1-yl) benzaldehyde (0.3 g, 0.996 mmol, 1 equivalent) was dissolved in a mixture of 1, 4 dioxane and water (4:1). To this solution, 2-Flouro-6 (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl) pyridine (0.22 g, 0.996 mmol,1.0 equivalent) and K₂CO₃ (0.27 g, 1.993 mmol,2 equivalents) were added.

After undergoing a degassing process for a duration of 5 minutes using nitrogen gas, I introduced Pd₂Cl₂(dppf) and DCM (0.08g, 0.099mmol, 0.1 equivalent). The reaction process was sustained at a temperature of 100 degrees Celsius for a duration of 16 hours. Thin-layer chromatography (TLC) was used to observe the progress of the reaction. The combination underwent a water wash, followed by dilution with ethyl acetate. It was then submerged in a “brine solution, dried using Na₂SO₄, and concentrated under reduced pressure.” 2-fluoro-4-(6-fluoropyridin-2-yl)The compound -6-(4-methylpiperazine-1-yl) benzaldehyde (13A) was produced with a yield of 0.20 g, which corresponds to a 63% yield.”

m.p.: 322-324^4c; FT-IR (KBr): 1673.48 cm^-1 -C=O stretching, 3125.35 cm^-1: -N-H stretching; ¹H NMR (400 MHz, DMSO-d⁶):10.28 (s, 1H), 8.44 (s,1H), 8.00-7.98 (m,1H) 7.05-7.00 (m.1H), 6.95-6.90 (m, 2H), 3.21-3.20 (m, 4H), 2.68-2.66 (s, 4H), 2.21 (s,3H). ¹³C NMR (100 MHz, DMSO-d⁶): d 151.91, 150.62, 144.26, 130.99, 129.84, 129.43, 129.18, 128.16, 127.11, 122.54, 120.84, 117.17 & 116.06; HRMS found: ESI m/z 318.2 (M+H)+, [ M.F C₁₇H₁₇F₂N₃O requires 317.13]

Conclusion

The prepared molecules are novel with simple structure and prepared in a very simple synthetic way. The methodology adopted for thepreparation was repeatable and the materials used were very much affordable.“At 32 (µg/ml), the antibacterial activity of molecules 9B, 13C, and 13D was shown to be the strongest against the gram-positive bacteria Staphylococcus aureus and B. subtilis.”Docking studies of these molecules show that they are the best fit in the enzyme and the binding energies of these molecules are also more than the standard molecule Triclosan. In the future in vivo studies of the molecules were planned on FabI enzymes.

Supporting Information

The supplemental information file contains the characterization of synthesized compounds, including ¹H NMR, ¹³C NMR spectra, IR spectra, and HRMS data.

Acknowledgment

The authors are extremely thankful to Aurigene Pharmaceutical Services Pvt Ltd for the execution of this research work. My special thanks to Dr. Amit Murlidhar Jabgunde, Dr. Ravikrishnababu, and APSL analytical team for helping us whenever required.

Conflict of Interest

The authors have no conflict of interest to declare. All co-authors have seen with the contents of manuscript, and there is no financial interest to report. We certify that the submission is original work and it is not under review at any other publication.

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