Dehydrozingerone Inspired Styryl Hydrazine Thiazole Hybrids as Promising Class of Antimycobacterial Agents

Abstract

Series of styryl hydrazine thiazole hybrids inspired from dehydrozingerone (DZG) scaffold were designed and synthesized by molecular hybridization approach. In vitro antimycobacterial activity of synthesized compounds was evaluated against Mycobacterium tuberculosis H₃₇Rv strain. Among the series, compound 6o exhibited significant activity (MIC = 1.5 μM; IC₅₀ = 0.48 μM) along with bactericidal (MBC = 12 μM) and intracellular antimycobacterial activities (IC₅₀ = <0.098 μM). Furthermore, 6o displayed prominent antimycobacterial activity under hypoxic (MIC = 46 μM) and normal oxygen (MIC = 0.28 μM) conditions along with antimycobacterial efficiency against isoniazid (MIC = 3.2 μM for INH-R1; 1.5 μM for INH-R2) and rifampicin (MIC = 2.2 μM for RIF-R1; 6.3 μM for RIF-R2) resistant strains of Mtb. Presence of electron donating groups on the phenyl ring of thiazole moiety had positive correlation for biological activity, suggesting the importance of molecular hybridization approach for the development of newer DZG clubbed hydrazine thiazole hybrids as potential antimycobacterial agents.

Tuberculosis (TB) is a chronic necrotizing bacterial infection caused by Mycobacterium tuberculosis (Mtb), which has been a bane of humanity for thousands of years and remains as one of the rampant health problems in the world. TB is an ancient enemy, and current threat that has been ranked among the foremost killers of the 21st century.¹ According to a World Health Organization (WHO) report, around 9 million people were found infected and around 1.5 million casualties occurred because of TB. Besides, the life threatening strains of MDR-TB (Multi Drug Resistance Tuberculosis) are appearing, some of which can lead to high mortality rate (e.g., 72–89%) with death occurring in short period (4–16 weeks).² In 2013 around 480,000 affirmative cases of MDR-TB were witnessed.³ India, China, the Russian Federation, and South Africa have almost 60% of the world’s cases of MDR-TB. In addition, the risk becomes even greater if the person is coinfected with the HIV (human immunodeficiency virus).⁴ The global resurgence of TB and development of drug resistance necessitates for an imperative attention of medicinal chemists to develop innovative antimycobacterial agents as no new classes of anti-TB agents have been developed since the introduction of rifampin in to clinical practice in 1960s.

It is well-known fact that trans-cinnamic acid analogues have recently drawn back the intentness of medicinal chemists due to their admirable pharmacological properties like antioxidant,⁵ antibacterial,⁶ and antitumor.⁷ Rastogi et al. have demonstrated the synergistic activity of trans-cinnamic acid in amalgamation with INH, rifamycin, and other recognized antimicrobial agents against Mtb.⁸ Further, Reddy et al. have reported the superior intracellular and in vivo activity of a cinnamoyl–rifamycin derivative (Figure 1) in contrast with rifamycin when tested against susceptible and MDR strains of Mtb along with M. avium complex (MAC).⁹ Several compounds resembling cinnamic acid and bearing styryl group or α,β-unsaturated carbonyl groups are reported for antimycobacterial activities (Figure 2).¹⁰

Figure 1.

Cinnamoyl–rifamycin derivative.

Figure 2.

Compounds with styryl portion reported against M. tuberculosis H₃₇RV: (I, MIC 6.49 μM);¹¹ (II, MIC 12.5 μg/mL);¹² (III, MIC 6.25 μM).¹³

From the literature, it was also found that derivatives resulting by combining cinnamoyl portion with various chemical classes of compounds have been reported to possess promising antimycobacterial activity.¹⁴⁻¹⁶ Besides, various drug-like heterocycles, namely, benzimidazoles¹⁷ and quinazolinones,¹⁸ integrated with cinnamoyl or aryl styryl groups have also been reported to augment the antimycobacterial properties.

Dehydrozingerone (DZG), also known as feruloylmethane, a half structural analogue of curcumin, is isolated from Curcuma longa. Chemically DZG is (E)-4-(4-hydroxy-3-methoxy phenyl)but-3-en-2-one and possess an α,β-unsaturated carbonyl (styryl ketone) group that resembles the trans-cinnamic acid structure. DZG analogues have been reported to possess a broad range of biological activities like antioxidant, anticancer, anti-inflammatory, antidepressant, antimalarial, antifungal, etc.¹⁹

The thiazole nucleus is a common motif presently found in several FDA-approved drugs, such as the nonsteroidal anti-inflammatory drug meloxicam²⁰ and the tyrosine kinase inhibitor dasatinib.²¹ Recently, Meissner et al. have demonstrated the structure–activity relationships (SAR) of novel series of 2-aminothiazole analogues as effective antimycobacterial agents,²² and Carradori et al. have reported microwave-assisted method for the synthesis of substituted-thiazolyl hydrazines.²³ Therefore, thiazole is an essential scaffold in drug discovery since its derivatives known to possess wide spectrum of activities such as antihypertensive, anti-inflammatory, anti-HIV, antibacterial, and antimycobacterial,^24,25 which have tremendously captivated attention of medicinal chemists. Figure 3 highlights the molecular manipulation of DZG–thiazole moiety and their resultant antimycobacterial activities.

Figure 3.

Literature reported derivatives containing styryl and thiazole moieties and their antimycobacterial activities along with the designed compounds. Compound 6o exhibited most promising antimycobacterial activity among the synthesized compounds. (A) (E)-3-methoxy-5-styrylcyclohexa-2,4-dien-1-one (MIC against H₃₇Rv = 32 μg/mL);²⁶ (B) (E)-5-bromo-2-(3,4-dimethoxystyryl)-1H-benzo[d] imidazole (MIC against H₃₇Rv = >7.25 μg/mL);¹⁷ (C) 2-amino-5-benzylthiazole-4-carboxylate (MIC against H₃₇Rv = 0.06 μg/mL);²⁷ (D) nitazoxanide (MIC against H₃₇Rv = 16 μg/mL);²⁸ (E) carbazolo-thiazole analogue (MIC against H₃₇Rv = 21 μM).²⁴

In view of the above facts and in continuation of our research program on the design and development of new antimycobacterial agents^19,24,29 it was foreseen to amalgamate two biologically active pharmacophores (styryl portion of DZG and thiazole) in one molecular platform to engender a new scaffold for antimycobacterial evaluation. As shown in Figure 3, the designed hybrid analogues possess both DZG (comprising styryl) and thiazole motifs connected with each other via a hydrazine linker. These unifications were suggested as an effort to explore the possible synergistic influence of such structural hybridizations on the anticipated activity, hoping to discover a new lead structure that would have a promising antimycobacterial activity.

The synthesis of a novel series of styryl hydrazine thiazole hybrids derived from DZG (6a–6o) was achieved through efficient and versatile synthetic routes. The starting material DZG (2) was prepared by using commercially available vanillin (1) by simple aldol condensation with acetone in the presence of base. Methylation of 2 was done with methyl iodide in the presence of potassium carbonate in N,N-dimethylformamide to yield (E)-4-(3,4-dimethoxyphenyl)but-3-en-2-one (3). Further, Schiff base of compound 3 was formed with thiosemicarbazide to yield 4 (Scheme 1). The various appropriately substituted 2-bromo-1-phenylethanones (5c–5o) were synthesized from their respective acetophenones. Compound (4) was then condensed with various freshly synthesized 2-bromo-1-(substituted phenyl)-ethanones (5a–5o) to yield corresponding final compounds, i.e., 2-(2-((2E,3E)-4-(3,4-dimethoxyphenyl)but-3-en-2-ylidene)hydrazinyl)-4-(substituted phenyl)thiazoles (6a–6o; Scheme 2). The anticipated structures of the final compounds were in agreement with the spectral (IR, ¹H NMR, and ¹³C NMR) data obtained and were further substantiated by HRMS data, which is summarized in the Supporting Information.

Scheme 1.

Reaction conditions: (i) acetone, NaOH; (ii) CH₃I, K₂CO₃, DMF, reflux, 1.5 h; (iii) thiosemicarbazide, AcOH, CH₃OH, reflux, 3 h.

Scheme 2.

Reaction conditions: (i) Br₂, ether, 0–5 °C for 5c; Br₂, CHCl₃, reflux, 3 h for 5d and 5g; Br₂, CHCl₃, 0–5 °C for 5e and 5f; CuBr₂, EtOAc, CHCl₃, reflux, 12 h for 5h–5o; (ii) methanol, reflux, 3 h.

The ¹H NMR spectrum of compound 4 exhibited the presence of distinctive singlet signals at around δ 10.22, 8.22–7.76, 7.154–7.150, 3.79–3.76, and 2.11 for the N–H proton, NH₂ proton, second proton of phenyl ring, methoxyl (OCH₃) protons, and methyl (CH₃) protons indicating its formation by a process of simple carbon–nitrogen bond creation with thiosemicarbazide in the presence of acetic acid as catalyst. In addition, the appearance of most informative doublet signals around δ 6.81–6.77 ppm (J = 16.53 Hz) and 7.06–7.01 (J = 16.84 Hz) confirms the presence of olefinic protons.

The ¹H NMR spectrum (400 and 600 MHz, DMSO-d₆) of the final compounds (6a–6o) displayed some distinctive singlet signals at around δ 11.42–10.22 ppm for N–H proton, δ 7.21–7.20 for second and δ 7.12–7.08 for sixth aromatic protons of DZG scaffold, and δ 2.17–2.08 ppm for methyl (N=C–CH₃) protons, respectively. In addition, the most informative singlet signal resonated around δ 7.70–7.31 ppm, which was attributed to the aromatic proton at H-5 of thiazole ring, thus indicating its formation through cyclo-condensation process. Whereas most characteristic doublet signals around δ 6.83–6.64 ppm (J = 16.52–16.24 Hz, Ph–HC=CH−) and δ 7.57–6.91 ppm (J = 16.52–14.76 Hz, Ph–HC=CH−) evidently indicated the presence of olefinic protons. This observation was found in consistence with previously reported similar type of compounds.³⁰ Further, the unique singlet signals resonating around δ 3.82–3.77 ppm indicated the presence of methoxyl protons (OCH₃) on the third and fourth position of the DZG scaffold, while the hydroxyl (OH) protons on aromatic ring resonated as singlet signals around δ 11.24–10.86 ppm. The various signals appearing as either doublets or multiplets around δ 8.29–6.77 ppm accounted for aromatic protons. The E-configuration was ascertained for all final derivatives on the basis of 2D NMR studies. These findings were further corroborated from their respective ¹³C NMR spectra of the title compounds. The characteristic signals resonating at around δ 169.53–156.50 and 108.52–102.10 ppm were assigned to carbons C-2 and C-5 of thiazole ring. The most prominent carbon signals observed around δ 149.27–148.91 and 132.56–126.23 ppm accounted for aromatic carbons having methoxyl groups and olefinic (Ph–HC=CH−) carbons, respectively. Further, the characteristic carbon signals appearing around δ 55.49–55.47 and 12.35–12.15 ppm indicated the presence of methoxyl and methyl groups in the title compounds, while the various aromatic carbons resonated around δ 140.78–108.03 ppm. Further, the fluorine containing compounds 6k and 6m have been discussed, which results in a very characteristic NMR spectra and the J_CF values are represented in Tables S1 and S2 (Supporting Information).

Both level I and II (in vitro) characterizations of antimycobacterial activity of newly synthesized title compounds (4, 6a–6o) were carried out at Infectious Disease Research Institute (IDRI) within the National Institute of Allergy and Infectious Diseases (NIAID) screening program, Bethesda, MD, USA. In the initial studies (level I), minimum inhibitory concentration (MIC) was established against Mtb strain H₃₇Rv grown under aerobic conditions by using a dual read-out (OD₅₉₀ and fluorescence) assay procedure. All the synthesized compounds exhibited interesting and noteworthy activity profiles with MIC ranging from 1.5 to >200 μM against the tested mycobacterial strain (Table S3, Supporting Information).

Interestingly, it was observed that compound 4 (MIC = 2.1 μM) having a thiourea group (without thiazole moiety) displayed encouraging antimycobacterial activity with an IC₅₀ value of 0.98 μM. This evidently indicated that the DZG structural core has greatly contributed for antimycobacterial activity. This finding instigated us to explore brief SAR investigations in order to study the biological effects of various substituents on the aromatic ring at the fourth position of the thiazole moiety, which was in turn attached to DZG scaffold through a hydrazine linkage. Among tested series, compound 6o (MIC = 1.5 μM) with p-amino (NH₂) group on phenyl ring at fourth position of thiazole moiety exhibited excellent antimycobacterial activity with IC₅₀ value of 0.48 μM, whereas compounds 6d (MIC = 15 μM), 6g (MIC = 16 μM), and 6i (MIC = 28 μM) substituted with one or two methoxyl (OCH₃) groups on thiazolylphenyl ring exhibited good inhibitory activity with IC₅₀ value of 8.4, 7.4, and 6.6 μM, respectively. In the case of compounds 6j (MIC = 40 μM) and 6l (MIC = 88 μM) with a hydroxyl (OH) group on the phenyl ring displayed considerable antimycobacterial activity with IC₅₀ value of 24 and 23 μM, respectively. These findings demonstrate that the thiazole core contributed to enhanced activity and played a significant role in the action against Mtb. The activity was also considerably affected by the nature of the substituent on the phenyl ring at the fourth position of the thiazole nucleus. Consistent with our prior report,²⁴ we found that the presence of electron donating (NH₂, OCH₃, and OH) groups on phenyl ring have greatly influenced and conferred good antimycobacterial activity, while the electron withdrawing (CF₃, NO₂, F and Br) substituents have caused a decrease in activity. Thus, compounds 6a, 6c, 6h, and 6m, having either nitro or halogen groups on the phenyl ring, were found to exhibit poor activity with MIC value >200 μM. (Figure S1, Supporting Information). Compounds with promising antimycobacterial activity profile were further subjected for level II screening in order to evaluate their broad spectrum efficiency under assorted conditions against relevant drug resistant isolates of Mtb and other disease causing mycobacterial species.

The MIC of test compounds (4, 6d, 6g, 6i, and 6o) was assessed against five drug resistant isolates (INH-R1, INH-R2, RIF-R1, RIF-R2, and FQ-R1) of Mtb strains under aerobic conditions. The antimycobacterial activity results are summarized in Table 1. From perusal of the data, we observed that all tested compounds showed excellent antimycobacterial activity against INH-R1 and INH-R2, while two compounds (4 and 6o) exhibited the most promising antimycobacterial activity against the tested organisms. In particular, both resistant strains (R1 and R2) of INH and RIF were found to be extremely susceptible to compounds 4 and 6o, while these two compounds had an almost comparable activity with that of Levofloxacin against FQ-R1. As compared to reference drug INH (MIC = >200 μM; IC₅₀ = >200 μM), compounds 4 (MIC = 5.3 and 2.5 μM; IC₅₀ = 1.3 and 0.79 μM) and 6o (MIC = 3.2 and 1.5 μM; IC₅₀ = 0.68 and 0.38 μM) displayed highest antimycobacterial activity against INH-R1 and INH-R2, respectively. In the case of RIF-R1 and RIF-R2, compound 6o (MIC = 2.2 and 6.3 μM; IC₅₀ = 0.54 and 0.76 μM) exhibited significant antibacterial activity, whereas compounds 4 (MIC = 4 and 4.8 μM; IC₅₀ = 1.1 and 1.2 μM) showed moderate activity when compared to reference drug RIF (MIC = 2 and >50 μM; IC₅₀ = >50 μM). Nevertheless, the fluoroquinolone-resistant strain (FQ-R1) was found to be less susceptible to these compounds.

Table 1. Antimycobacterial Activity Data of Newly Synthesized Compounds (4, 6d, 6g, 6i, and 6o) against Five Drug-Resistant Isolates of M. tuberculosis H₃₇Rv.

	INH-R1a			INH-R2b			RIF-R1c			RIF-R2d			FQ-R1e
compd	MIC (μM)	IC50 (μM)	IC90 (μM)	MIC (μM)	IC50 (μM)	IC90 (μM)	MIC (μM)	IC50 (μM)	IC90 (μM)	MIC (μM)	IC50 (μM)	IC90 (μM)	MIC (μM)	IC50 (μM)	IC90 (μM)
4	5.3	1.3	6.6	2.5	0.79	2.9	4	1.1	4.6	4.8	1.2	5.9	17	2.8	16
6d	15	12	>50	12	8	>50	25	11	>50	31	8.7	>50	22	23	>50
6g	24	12	>200	13	7.1	12	28	9.2	27	46	19	>200	30	18	>200
6i	32	9.1	>25	19	5.6	>25	17	7	19	41	10	>25	33	13	>25
6o	3.2	0.68	3.8	1.5	0.38	1.7	2.2	0.54	2.6	6.3	0.76	9.2	21	2.3	33
rifampicin	0.018	0.0084	0.022	0.0065	0.0047	0.012	2	1.2	2.3	>50	>50	>50	0.027	0.013	0.039
isoniazid	>200	>200	>200	>200	>200	>200	0.17	0.15	0.21	0.62	0.54	0.6	0.35	0.36	0.47
levofloxacin	1.2	0.64	1.4	1.4	0.84	1.4	0.76	0.59	0.91	1.1	0.6	1.2	20	12	22

^aINH-R1 was derived from H₃₇Rv and is a katG mutant (Y155* = truncation).

^bINH-R2 is strain ATCC35822.

^cRIF-R1 was derived from H₃₇Rv and is a nrpoB mutant (S522L).

^dRIF-R2 is strain ATCC35828.

^eFQ-R1 is a fluoroquinolone-resistant strain derived from H₃₇Rv and is a gyrB mutant (D94N). INH, isoniazid; RIF, rifampicin; FQ, Fluoroquinolone.

In addition, these five promising compounds (4, 6d, 6g, 6i, and 6o) were systematically assessed against Mtb H₃₇Rv grown under varied conditions. The antimicrobial activity of these compounds under hypoxic conditions was assessed using the low oxygen recovery assay (LORA). Further, the bactericidal (MBC: Minimum Bactericidal Concentration) activity of these compounds was assessed against Mtb H₃₇Rv grown in aerobic conditions in 7H9-Tw-OADC medium. The cytotoxicity and intracellular antimycobacterial activity of compounds was also determined using the THP-1 human monoocytic cell line, and THP1 cells infected with Mtb, respectively. The results of all these investigations are represented in Table S4 (Supporting Information). A systematic analysis of the data revealed that compounds 4 and 6o exhibited an interesting and potent antimycobacterial activity profile as depicted in Figure 4. All the five title compounds displayed an interesting cytotoxicity profile with IC₅₀ values ranging from 11 to >50 μM. Among the series tested, compounds 6o (IC₅₀ = 11 μM) and 6g (IC₅₀ = 38 μM) showed moderate cytotoxicity, while other compounds did not show cytotoxic effect up to concentrations >50 μM. The existence of virulent intracellular Mtb in primary human macrophages compromise its functioning and arrest phagosome maturation, thus coping up with various host threats. The aptitude of the bacteria to assault and survive inside cells may be implicated for the persistence of TB. Therefore, it is of greater corollary for an effective tuberculosis management that these compounds should also be capable of killing intracellular TB in human macrophages, apart from their in vitro activity against TB strains. Accordingly, two compounds (4 and 6o) also displayed effective intracellular antimycobacterial activity with IC₅₀ value of <0.098 μM. However, oxygen restriction also affects adaptive immune responses and triggers antimicrobial effector mechanisms in macrophages and restricts growth of intracellular Mtb.

Figure 4.

Anti-TB activity profile of most active compounds: 6d, R = 4-OCH₃; 6g, R = 3,4-OCH₃; 6i, R = 2,6-OCH₃; 6o, R = 4-NH₂.

The title compounds (4, 6d, 6g, 6i, and 6o) were also evaluated for their in vitro antimycobacterial activity against other disease-relevant Mycobacterial species like Mycobacterium abscessus and Mycobacterium avium by using MABA method (Table S5, Supporting Information). The results reveal that compound 6o (MIC = 100 μM) demonstrated a moderate activity especially against M. avium as compared to the reference drug RIF (MIC = 0.1 μM), while compound 6i displayed a MIC of >100 μM against M. abscessus and M. avium. However, the remaining compounds showed little or poor activity (MIC = >200 μM) against tested organisms.

In summary, in this work we established the synthesis of a series of styryl hydrazine thiazole hybrids derived from dehydrozingerone and their in vitro anti-TB activity. The ease, simply obtainable reactants and reagents, and practically good yields (51–74%) make this synthetic method more attractive and efficient. Moreover, compound 6o emerged as most promising antimycobacterial agent since it has demonstrated most prominent activity under hypoxic condition along with its potential efficiency against drug resistant isolates of Mtb strains and displayed significant bactericidal and intracellular antimycobacterial activity. These findings suggest that the designed compounds highlighted the benefit of incorporating a hydrazine linkage to combine the styryl portion of DZG and the thiazole core, thus providing a good starting point for further lead optimization. The possible enhancement in the antimycobacterial activity can be further accomplished by slender variation in the ring substituents and/or extensive additional functionalization, which warrants further investigation.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.6b00088.

• Synthetic procedures, spectral data, and protocols of bioassay (PDF)

Author Contributions

The manuscript was written through contributions of all authors.

This work was supported by funds from the University of KwaZulu-Natal (UKZN), Westville Campus, Durban, South Africa.

The authors declare no competing financial interest.