Synthesis and Preliminary Antimicrobial Analysis of Isatin–Ferrocene and Isatin–Ferrocenyl Chalcone Conjugates

Abstract

In this study, we outline the synthesis of isatin–ferrocenyl chalcone and 1H-1,2,3-triazole-tethered isatin–ferrocene conjugates along with their antimicrobial evaluation against the human mucosal pathogen Trichomonas vaginalis. The introduction of a triazole ring among the synthesized conjugates improved the activity profiles with most of the compounds in the library, exhibiting 100% growth inhibition in a preliminary susceptibility screen at 100 μM. IC₅₀ determination of the most potent compounds in the set revealed an inhibitory range between 2 and 13 μM. Normal flora microbiome are unaffected by these compounds, suggesting that these may be new chemical scaffolds for the discovery of new drugs against trichomonad infections.

Introduction

Trichomoniasis, a nonviral sexually transmitted disease caused by the protozoal pathogen Trichomonas vaginalis,¹ mainly infects the urinary tract, vagina, and prostate.² A recent survey indicated that there are about 276 million cases of trichomoniasis occurring globally of which 42.8 million cases occur in Africa (including sub-Saharan Africa)³ and 3.7 million in the United States,⁴ mostly affecting women under the age of 40 years.^5,6 In most of the cases, the infection is asymptomatic,⁷ while the common symptoms might include malodorous vaginal discharge,⁸ dyspareunia, dysuria, lower abdominal pain, or vulvovaginal irritation.⁹ In certain cases, serious complications such as pelvic inflammation, cervical dysplasia, vaginitis, infertility, low birth weight infant, and preterm delivery may occur.¹⁰ Metronidazole and tinidazole are the only US-FDA drugs approved for the treatment of trichomoniasis.¹¹ However, the emergence of drug resistance among the clinical isolates has prompted the need for the development of new and efficient structural entities with potentially low incidence of resistance.

Isatin is a promising class of biologically active scaffolds with tolerance to humans¹² and a good platform for structure modification and derivatization. Isatin derivatives display a myriad of activities including anticancer,¹³ antidepressant,¹⁴ antifungal,¹⁵ anti-HIV,¹⁶ and anti-inflammatory properties.¹⁷ Isatin-β-thiosemicarbazones have been recently investigated for their selectivity to kill gp-over-expressing tumor cells in vitro.¹⁸ SU11248 (Sutent), a 5-fluoro-3-substituted isatin derivative, was approved by FDA in 2006 for the treatment of advanced renal carcinoma and gastrointestinal stromal tumors.¹⁹ Selective inhibitory activity of 5,7-dibromo-N-(p-trifluoromethylbenzyl)isatin against lymphoma and leukemic cancer cell lines over freshly isolated, nontransformed human peripheral blood lymphocytes has been disclosed recently.²⁰ A recent report of Vine et al. has shown the potency of the N-alkyl isatin derivatives against the multidrug-resistant cell lines, viz., U937VbR and MES-SA/Dx5, and observed bioequivalent dose-dependent cytotoxicity to that of the parental control cell lines.²¹ In vitro selective inhibition of aldehyde dehydrogenase has also been reported for the N-substituted isatin–piperazine derivative.²² Many isatin derivatives have also been evaluated for their antimicrobial potential with isatin-β-thiosemicarbazones exhibiting minimum inhibitory concentration (MIC) of 0.78 and 0.39 mg/L against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus.²³ Schiff base of 5-substituted isatin has also been found to be active against Pseudomonas aeruginosa (MIC = 6.25 μg/mL).²⁴

Chalcones, an α,β-unsaturated carbonyl-linked framework,²⁵ belong to the biologically active class of compounds with antileishmanial,²⁶ antibacterial,²⁷ antifungal,²⁸ antitumor,²⁹ antimalarial,³⁰ antiviral,³¹ antitubercular,³² anti-invasive,³³ antioxidant,³⁴ anti-inflammatory,³⁵ and antiplatelet properties.³⁶ Naturally occurring Licochalcone A has shown promising antibacterial activity against Bacillus subtilis, S. aureus, and Micrococcus luteus.³⁷ Chalcone-based conjugates have shown enormous biological potential with chalcone–thiazolidine–dione conjugates, which are proven to be 8 and 64 times more active than the reference drugs, norfloxacin (MIC = 1.0 μg/mL) and oxacillin (MIC = 0.5 μg/mL) against MRSA CCARM 3167 and 3506, respectively.³⁸ Imidazole–chalcone conjugates were found to be active against Aspergillus fumigatus,(39) while ferrocenyl chalcones act as the HepG2 inhibitor and are nontoxic to human fibroblasts.⁴⁰ Recently, metronidazole–chalcone conjugates have proven to be 4 times more potent than the standard drug metronidazole against T. vaginalis.⁴¹

One of the auspicious branches of bioorganometallic chemistry includes the synthesis of sandwich and half-sandwich complexes with important biological activity.⁴² Ferrocene represents a momentous scaffold in present day drug discovery paradigm because of its robustness, reactivity, redox properties, lipophilicity, and low cytotoxicity.⁴³ Replacement of a purely organic component with ferrocene has shown to improve the biological potential of the target compounds as evidenced by ferrocenyl conjugates of commercial antiestrogen tamoxifen and antiandrogen nilutamide.⁴⁴ Ferroquine is the most exemplary contribution of ferrocene to the improvement of antiplasmodial potential of chloroquine and is under clinical trials.⁴⁵

Previous reports from our group have shown the synthesis of an isatin-based scaffold, viz., mono and bis-uracil–isatin, isatin–4-aminoquinoline, and isatin−β-lactam conjugates tethered via 1H-1,2,3-triazole and β-amino alcohol along with their preliminary in vitro evaluation against T. vaginalis.⁴⁶ In continuation with our research efforts,⁴⁷ the present study describes the synthesis of isatin–ferrocenyl chalcone and 1H-1,2,3-triazole-tethered isatin–ferrocene conjugates along with their antitrichomonad activities.

Results and Discussion

The methodology for the synthesis of isatin–ferrocenyl chalcone conjugates involved base-promoted N-alkylation of C-5-functionalized isatins with O-alkyl-bromo-ferrocenyl chalcones. The precursor, viz., O-alkyl-bromo-ferrocenyl chalcones 3, was prepared via an initial base-promoted O-alkylation of 4-hydroxy-acetophenone 1 with various dibromoalkanes at 60 °C for 8 h to afford the corresponding O-alkyl bromo-acetophenones 2. Subsequent aldol condensation of O-alkyl-bromoacetophenones with ferrocene carboxyaldehyde delivered the precursor O-alkyl-bromo-ferrocenyl chalcones in good to excellent yield (Scheme 1).

Scheme 1. Synthesis of Compounds 5a–x.

Sodium hydride-promoted reaction of C-5-substituted isatin with O-alkyl-bromo-ferrocenyl chalcone at room temperature (rt) for 3 h resulted in the isolation of desired isatin–ferrocenyl chalcone conjugates which were purified via flash chromatography using a mixture of ethyl acetate/hexane (20:80) as the eluent. The structure to the conjugates 5 was assigned on the basis of spectral data and analytical lines of evidence. Compound 5g, for example, exhibited a molecular ion peak at 519.3688 (m/z) in its high-resolution mass spectrum (HRMS). The salient features of its ¹H NMR spectrum included the appearance of a multiplet at δ 4.16–4.19 (7H) corresponding to cyclopentadiene ring protons (5H) along with a methylene and singlets at δ 4.49 (2H) and 4.60 (2H) corresponding to ferrocene ring protons. The appearance of doublets at δ 7.12 (1H, J = 15.3 Hz) and 7.74 (1H, J = 15.3 Hz) confirmed the presence of trans-olefinic protons of ferrocenyl chalcones. The appearance of characteristics peaks at δ 161.40 and 188.03 ppm corresponding to isatin ring carbonyls along with the requisite number of carbons in ¹³C NMR spectra further corroborated the assigned structure.

Synthetic methodology for the synthesis of 1H-1,2,3-triazole-tethered isatin–ferrocene conjugates 12 involved an initial base-promoted N-alkylation of isatin with dibromoalkanes at 80 °C with subsequent treatment with sodium azide to afford the corresponding N-alkylazidoisatins 8 (Scheme 2). The second precursor, viz., ((prop-2-yn-1-yloxy)methyl)ferrocene 11, was prepared via an initial reduction of ferrocene carboxyaldehyde 9 with NaBH₄ in dry tetrahydrofuran to afford ferrocenyl-methanol 10, which was subsequently O-propargylated. Cu-promoted azide–alkyne cycloaddition of 8 with O-propargylated ferrocene methanol 11 led to the isolation of crude product, which was purified on silica gel chromatography (60:120) using methanol/chloroform (10:90) mixture as the eluent to yield 12 in good to excellent yields (Scheme 3). The structure to the compound was assigned on the basis of spectral studies and analytical evidence. For example, compound 12m exhibited a molecular ion peak at 504.0652 in its HRMS. Its ¹H NMR spectrum showed the presence of a singlet at δ 4.14 corresponding to 5H (cyclopentadiene ring of ferrocene) along with singlets at δ 4.16 (2H) and δ 4.22 (2H) because of the presence of −OCH₂ protons along with the characteristic singlet at δ 7.49 (1H) corresponding to the triazole ring proton. The appearance of absorption peaks at δ 68.5, 68.6, 68.8, and 82.7 in its ¹³C NMR spectrum corresponding to ferrocene ring carbons along with the presence of isatin ring carbonyl at δ 181.2 corroborated the assigned structure.

Scheme 2. Synthesis of N-Alkyl Azido Isatin.

Scheme 3. Synthesis of Compounds 12a–x.

The synthesized compounds, viz., 5a–x and 12a–x, were evaluated for their inhibitory activity against T. vaginalis, and the results are summarized in Table 1. As evident, the activity profiles of synthesized scaffolds, viz., 5a–x, showed an interesting structure–activity relationship (SAR) with activity being dependent upon the nature of substituent at the C-5 position of the isatin ring as well as the length of the alkyl chain, introduced as a linker. Apparently, among conjugates with unsubstituted (R = H) isatin, the activities showed improvement with increase in the chain length from n = 2 to n = 4 as evidenced by 5a–c, while a further increase in the chain length reduced the activity. The introduction of a methyl substituent at the C-5 position of the isatin ring decreased the activities except in the case of 5g (n = 2) and 5i (n = 4). Introduction of electron-withdrawing substituents, viz., fluoro and chloro, in general improved the growth inhibition activity against T. vaginalis irrespective of the length of the alkyl chain linker. The conjugates, viz., 5o (R = F, n = 4) and 5s (R = Cl, n = 2), with an optimal combination of electron-withdrawing substituents at the C-5 position of the isatin ring and short alkyl chain length proved to be the most potent among the test compounds with 100% growth inhibition. Interestingly, the introduction of a 1H-1,2,3-triazole ring among isatin–ferrocene conjugates, 12a–x, substantially improved the activities with most of the synthesized conjugates exhibiting 100% growth inhibition. Analysis of SAR among 1H-1,2,3-triazole-tethered conjugates revealed the activity to be independent on the nature of the substituent present at the C-5 position of the isatin ring as well as the length of the alkyl chain used as the spacer except in cases where the octyl (n = 8) chain was used. The presence of the octyl chain length reduced the antitrichomonad activities as evident by conjugates 12f, 12l, 12r, and 12x exhibiting 64.70, 49.16, 52.70, and 35.38% growth inhibition, respectively.

Table 1. Antitrichomonad Activity of 5a–x and 12a–x at 100 μM against T. vaginalis.

entry	% inhibition	entry	% inhibition	entry	% inhibition
5a	75.47 ± 12.23	5q	86.37 ± 03.20	12i	100 ± 0.0
5b	89.43 ± 04.66	5r	93.79 ± 02.45	12j	100 ± 0.0
5c	99.15 ± 01.46	5s	100 ± 0	12k	100 ± 0.0
5d	63.67 ± 11.32	5t	99.51 ± 00.83	12l	49.16 ± 16.91
5e	83.07 ± 11.99	5u	97.02 ± 01.21	12m	100 ± 0.0
5f	81.43 ± 11.20	5v	93.33 ± 03.86	12n	100 ± 0.0
5g	95.83 ± 04.35	5w	93.12 ± 07.15	12o	100 ± 0.0
5h	82.83 ± 14.59	5x	96.04 ± 02.44	12p	100 ± 0.0
5i	90.88 ± 08.21	12a	100 ± 0.0	12q	100 ± 0.0
5j	78.77 ± 07.45	12b	100 ± 0.0	12r	52.70 ± 5.92
5k	23.02 ± 13.36	12c	100 ± 0.0	12s	28.32 ± 20.55
5l	N.D	12d	100 ± 0.0	12t	100 ± 0.0
5m	98.83 ± 01.05	12e	100 ± 0.0	12u	100 ± 0.0
5n	90.17 ± 04.26	12f	64.70 ± 17.76	12v	100 ± 0.0
5o	100 ± 00	12g	100 ± 0.0	12w	52.20 ± 13.12
5p	90.46 ± 01.81	12h	100 ± 0.0	12x	35.38 ± 13.97

Most potent conjugates of the two series were further evaluated for their IC₅₀ values and compared with metronidazole, the only FDA-approved drug used for the treatment of trichomoniasis (Table 2). As evident, although the conjugates were not as active as metronidazole, most of the compounds displayed low IC₅₀ values. The conjugates, viz., 5w, 12c, 12d, 12j, and 12p, emerged as the most active conjugates with IC₅₀ values of 7.13, 5.53, 5.82, 2.26, and 2.96 μM, respectively.

Table 2. IC₅₀ Values of Compounds 5a–x and 12a–x.

entry	IC50 (μM)	entry	IC50 (μM)	entry	IC50 (μM)
5b	11.90	12c	5.53	12m	12.16
5o	9.01	12d	5.82	12n	9.54
5t	12.83	12e	13.16	12p	2.96
5v	9.55	12g	11.40	12t	8.70
5w	7.13	12h	9.83	12u	9.25
5x	11.96	12j	2.26	12v	13.09
12a	7.85	12k	8.46	MTZ	0.76

To ascertain if the observed inhibitory effect of these compounds is due to their activity and not cytotoxicity, the conjugates were evaluated on normal human flora consisting of the nonpathogenic strains, Lactobacillus reuteri (ATCC 23272), Lactobacillus acidophilus (ATCC 43560), and Lactobacillus rhamnosus (ATCC 53103). No cytotoxic effects from any of these synthesized compounds were observed for any of the normal flora, which is suggestive of the fact that these compounds are antiparasitic.

Conclusions

In conclusion, a series of isatin–ferrocenyl chalcone and isatin–ferrocene conjugates were synthesized either via aliphatic nucleophilic substitution reaction or via azide–alkyne cycloaddition reaction, respectively, and were evaluated for their inhibitory activities against T. vaginalis. The lack of a chalcone moiety and the introduction of a 1H-1,2,3 triazole ring substantially improved the inhibitory activities with most of the conjugates exhibiting 100% growth inhibition at the preliminary screening concentration of 100 μM. The lack of dependence of activity upon the nature of substituent at the C-5 position of the isatin ring or the length of the alkyl chain introduced as the linker among 1H-1,2,3-triazole-tethered isatin–ferrocenes is suggestive of the fact that such conjugates may act as therapeutic templates for the design of new antitrichomonads.

Experimental Section

General Information

Melting points are uncorrected and determined via an open capillary method using a Veego Precision digital melting point (MP-D) apparatus. ¹H and ¹³C NMR spectra were recorded in CDCl₃ with Bruker AVANCE II (500 and 125 MHz) using tetramethylsilane as the internal standard. Chemical shift and coupling constant values are expressed in parts per million and hertz, respectively, while splitting patterns are designated as s: singlet, d: doublet, t: triplet, m: multiplet, dd: double doublet, ddd: doublet of a doublet of a doublet, and br: broad peak. Elemental analysis was performed on a Heraeus CHN-O rapid elemental analyzer, while mass spectra were recorded on a Bruker high-resolution mass spectrometer (microOTOF-QII). Column chromatography was carried out on a silica gel (60–120 mesh) with ethylacetate/hexane as the eluent.^47e

Materials and Methods for Antimicrobial Susceptibility Assays

T. vaginalis strain G3 was cultured for 24 h at 37 °C. To perform the preliminary antimicrobial screen, the compounds were dissolved in dimethyl sulfoxide (DMSO) to obtain stock concentrations of 100 μM; 5 μL aliquots of these suspensions were diluted in 5 mL of trypticase–yeast extract–maltose diamond’s media to obtain a final concentration of the compound of 100 μM. After 24 h of exposure to the compounds, any remaining motile cells were visualized and manually counted using a hemacytometer. Cell counts were normalized to the DMSO controls. These data sets were then transformed using GraphPad Prism software, and the sample size consists of four independent trials carried out on four different days (to account for possible variation in the parasite population). The IC₅₀ values were determined by titration assays of increasing concentrations of each compound, usually within a range of 5–40 μM, by a regression analysis using Prism software from GraphPad. The calculated IC₅₀ values of the compounds were then confirmed by using the same assay described previously.

Cytotoxicity Assay for Normal Flora Microbiota

Cultures of nonpathogenic bacteria such as L. reuteri (ATCC 23272), L. acidophilus (ATCC 43560), and L. rhamnosus (ATCC 53103), known to comprise the human mucosal microbiome, were grown in Lactobacilli MRS media at 37 °C under anaerobic conditions. Escherichia coli K12 MG1655 (nonpathogenic) was cultured in Luria broth at 37 °C aerobically. Stock solutions (100 μM) of synthesized compounds and vehicle control DMSO were diluted to 100 μM in media to afford working dilutions. Empty BDL-sensi disks (6 mm) were incubated in the working dilutions at rt for 20 min. Streaked agar plates of chosen bacteria were incubated with disks containing the vehicle control, compounds, or various antibiotic disks [levofloxacin (5 μg), gentamicin (10 μg), and gentamicin (120 μg)] and incubated overnight at 37 °C. Sensitivity to the vehicle control, compounds, and antibiotics was determined via the measurement of zones of inhibition around each disk in millimeter. All organisms were purchased from the American Type Culture Collection (ATCC).^47c

Glossary

Abbreviations

MIC

minimum inhibitory concentration

SAR

structure–activity relationship

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00553.

• Scanned (¹H, ¹³C) NMR spectra for the compounds, viz., 5c, 5d, 5g, 5n, 5p, 5s, 5t, 5u, 5v, 12a, 12c, 12e, 12f, 12j, 12k, 12o, 12p, 12r, 12s, 12t, and 12u (PDF)

The authors declare no competing financial interest.