J Chem Biol (2014) 7:29–35 DOI 10.1007/s12154-013-0103-8
ORIGINAL ARTICLE
Design, synthesis, and evaluations of antifungal activity of novel phenyl(2H -tetrazol-5-yl)methanamine derivatives Amol B. Salake & Aparna S. Chothe & Shrikant S. Nilewar & Madhavi Khilare & Rutuja S. Meshram & Abhishek A. Pandey & M. K. Kathiravan
Received: 21 January 2013 / Accepted: 22 August 2013 / Published online: 12 September 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl2 in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1.
Keywords Phenyl(2H -tetrazol-5-yl)methanamine . Fluconazole . Antifungal agent . Candida albicans . Aspergillus niger
A. B. Salake : A. S. Chothe Department of Pharmaceutical Chemistry (PG), AISSMS College of Pharmacy, Kennedy road, Near RTO, Pune 411001, Maharashtra, India S. S. Nilewar : M. Khilare : R. S. Meshram : A. A. Pandey : M. K. Kathiravan (*) Department of Pharmaceutical Chemistry (PG), Sinhgad College of Pharmacy, Off. Sinhgad road, Vadgaon (Bk.), Pune 411041, Maharashtra, India e-mail: [email protected]
Introduction Despite advances in preventive, diagnostic, and therapeutic interventions, invasive fungal infections cause significant morbidity and mortality in immune compromised patients. The burden of antifungal resistance in such high-risk patients is becoming a major concern [1]. In the past decade, there is significant increase in incidence of invasive fungal infections specially in hospitalized patients. An increased morbidity and mortality of mycotic infections is related with a growing number of vulnerable patients to opportunistic fungi as a consequence of progress in medicine, improved diagnostics, and awareness of fungi as etiology of these infections [2, 3]. Patients treated with immunosuppressive agents, broadspectrum antibiotics, antineoplastic agents, and anti-HIV agents as well as those undergoing extensive surgery or other invasive procedures are at higher risk of contracting a systemic mycosis [4]. The majority of invasive mycotic diseases are caused by Candida and Aspergillus species [2]. Infections caused by opportunistic pathogens continue to be of public health concern [5]. Candida albicans, Aspergillus niger, Cryptococcus neoformans, and Aspergillus fumigatus are the most common fungal pathogens responsible for causing opportunistic fungal infections in human beings. Patients with AIDS, lymphoma, and those on long-term corticosteroids therapy, organ transplant patients, and even diabetes patients are at risk for opportunistic fungal infections [6]. Heterocyclic tetrazole possesses wide range of activities as antifungal, antibacterial, analgesic, and anti-inflammatory [7–10]. Azole antifungals are strong inhibitors of lanosterol 14-α-demethylase, which is a major component of fungal cell membrane [11]. The triazole antifungal drugs fluconazole, itraconazole, voriconazole, ravuconazole, and posaconazole form an important class of antifungal agents. These drugs act
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by displacing lanosterol from cytochrome P450 14-α-DM and in this manner block the biosynthesis of ergosterol, an essential component of the fungal cell membrane and finally leads to killing of fungi. Cytochrome P450 14-α-DM oxidatively removes the 14-α-methyl group of lanosterol by using oxygen and NADPH [12]. The rising prevalence of multidrug resistant bacteria continues to provide impetus for the search and discovery of novel antifungal agents active against these pathogens. A series of 16 novel phenyl(2H-tetrazol-5-yl)methanamine derivatives are planned to see the effect of tetrazole on replacing triazole of fluconazole (Fig. 1). The designed compounds were also subjected to molecular docking into the active site of cytochrome P450 14α-sterol demethylase. The tetrazole compounds are evaluated in vitro for antifungal activity against A. niger MTCC 281 and C. albicans MTCC227 by disk diffusion method. In the present paper, our aim is to synthesize tetrazole nucleus from α-amino nitrile by modification in fluconazole moiety as chlorine has strong inductive electron-attracting effects, while those of fluorine are very weak. Moreover, these atoms may also influence the stearic characteristics and the hydrophilic– hydrophobic balance of the molecules. So, we have placed substitutions of –Cl, –OCH3, and –CH3 on a designed molecule. The hydrophobic group is required for activity, so for increasing hydrophobicity of the compound, phenyl ring is replaced by triazole moiety [13, 14]. Tetrazole substituent shows antifungal activity so we have replaced one triazole ring of fluconazole by tetrazole ring. An attempt is made for substitution of –CH3 near tetrazole which improves the spectrum of activity [13].
Materials and methods Chemistry One pot condensation of carbonyl compounds, (aldehyde and ketone) amine and cyanide, is known as Strecker reaction as in Scheme 1 [15].
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Scheme 1 General synthetic scheme of Strecker reaction
α-Amino nitriles are significantly important intermediates for the synthesis of a wide variety of amino acids, amides, diamines, and nitrogen-containing heterocycles [15], such as thiadiazoles and imidazole derivatives. Among the methods reported for the synthesis of α-amino nitriles, Strecker reaction, nucleophilic addition of cyanide ion to imines, is of great importance to modern organic chemistry as it offers one of the most direct and viable methods for the synthesis of α-amino nitriles [16, 17]. α-Amino nitriles are synthesized by the reactions of aldehydes/ketones with amines in the presence of a cyanide source such as TMSCN which is the safer and more efficient cyanide anion source. The α-amino nitriles were synthesized as per procedure described in our unpublished work described in TGCL-2011-0076 [18]. The synthetic scheme for (1–16) is shown in Scheme 2. All synthesized compounds were characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and mass spectroscopy. The reagents used were of analytical grade. TLC precoated plates silica gel (G-60 mesh) were used. Melting points were determined in open capillary tube using Labin Melting Point Apparatus and are uncorrected. FTIR spectra of the synthesized compounds were recorded using KBr pellets on a Jasco FTIR V 430 + spectrometer using diffuse reflectance attachment and are reported in per centimeter. Proton nuclear magnetic resonance spectroscopy (1H NMR) spectra were recorded on a Varian Mercury YH300 (300 MHz FT NMR) spectrometer using tetramethylsilane as an internal standard (chemical shift represented in δ part per million). Elemental analysis was performed on Flashea 1112 series equipment by Thermoelectron Corporation. Mass spectra were obtained on a double focusing magnetic sector mass spectrometer using direct insertion probe using electron impact ion source operating at 70 eV (Table 1). General procedure for synthesis of phenyl(2H -tetrazol-5-yl) methanamine derivatives (1–16)
Fig. 1 Structure of fluconazole
To a mixture of α-amino nitrile (1.00 mol), sodium azide (1.05 mol), and zinc chloride (0.50 mol), isopropyl alcohol was refluxed for 16 h. The progress of the reaction was
Scheme 2 General synthetic scheme of phenyl(2H -tetrazol-5yl)methanamine derivatives. Reagents and condition: a TMSCN, IL, RT, and stirring; b NaN3, ZnCl2, and IPA
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Table 1 Different substitutions at 1–16
Compd.
Structures
Compd.
1.
9.
2.
10.
3.
11.
4.
12.
5.
13.
6.
14.
7.
15.
8.
16.
Structures
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monitored by TLC system as methanol:dichloromethane (1:9). After completion of a reaction, the mixture was cooled to room temperature, the pH was adjusted with concentrated HCl, and the reaction was stirred for 30 min to break up the solid precipitate. The mixture was extracted with ethyl acetate twice. Again, it was washed with brine solution and dried over anhydrous sodium sulphate and evaporate to yield the product [19]. The titled compounds were characterized as follows: N -(phenyl (2H -tetrazol-5-yl) methyl) aniline (1) Yield 72 %, m.p. 193–195 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 4.0 (s, 1H, NH), 5.4 (s, 1H, CH), 9.2–9.3 (s, 1H, tetrazole NH), 7.00– 7.10 (m, 3H, benzene), 7.33–7.55 (m, 7H, benzene), IR (KBr) (per centimeter): 3,328, 3,023, 2,990, 1,515, EIMS (70 eV, m /z ) 251.12 (M+). Anal. calcd for C14H13N5: C, 66.92; H, 5.21; N, 27.87 found C, 66.80; H, 5.15; N, 27.79. 4-chloro-N-(phenyl (2H-tetrazol-5-yl)methyl)aniline (2) Yield: 71 %; m.p. 188–190 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 4.00–4.12 (s, 1H, NH), 5.14–5.19 (s, 1H, CH), 6.67 (d, 2H, benzene), 7.24–7.45 (m, 7H, benzene), 9.1–9.2 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,428, 3,123, 2,979, 1,615, 700. EIMS (70 eV, m/z) 285.1 (M+), Anal. calcd for C14H12ClN5: C, 58.85; H, 4.23; N, 24.51 found C, 58.80; H, 4.18; N, 24.48. 4-methyl-N -(phenyl(2H-tetrazol-5-yl)methyl)aniline (3) Yield 61 %, m.p. 187–190 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 2.34–2.38 (s, 3H, CH3), 3.9–4.1 (s, 1H, NH), 5.1–5.2 (s, 1H, CH), 6.87–6.90 (d, 2H, benzene), 7.12–7.35 (m, 7H, benzene), 10.1–10.3 (s, 1H, tetrazole NH), IR (KBr) (per centimeter): 3,400, 3,003, 2,980, 1,605. EIMS (70 eV, m/z) 265.13 (M+). Anal. calcd for C15H15N5: C, 67.9; H, 5.7; N, 26.4 found C, 67.86; H, 5.6; N, 26.32 4-(phenyl (2H -tetrazol-5-yl)methyl) morpholine (4) Yield 65 %, m.p. 154–156 °C, (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 2.48–2.64 (m, 4H, morpholine), 3.4–3.5 (m, 4H morpholine), 5.2–5.3 (s, 1H, NH), 7.35– 7.8 (m, 5H, benzene), 9.1–9.2 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,428, 3,123, 2,990, 1,615, 1,100. EIMS (70 eV, m /z ) 245.8 (M+). Anal. calcd for C12H15N5O: C, 58.76; H, 6.16; N, 28.55. found C, 58.66; H, 6.11; N, 28.35. N -((4-methoxyphenyl)(2H -tetrazol-5-yl)methyl)aniline (5) Yield: 83 %; m.p. 218–220 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 3.7–3.8 (s, 3H, OCH3), 3.9–4.0 (s, 1H, NH), 5.3–5.4 (s, 1H, CH), 6.96–7.45 (m, 9H, benzene), 9.1–9.3 (s, 1H, tetrazole NH), IR (KBr) (per centimeter): 3,346, 3,125, 2,928, 1,550, 1,123. EIMS (70 eV, m /z ) 281.31 (M+), Anal. calcd
for C15H15N5O: C, 64.04; H, 5.37; N, 24.94. found C, 64.00; H, 5.31; N, 24.84 found C, 63.90; H, 5.29; N, 24.80. 4-chloro-N -((4-methoxyphenyl)(2H -tetrazol-5yl)methyl)aniline (6) Yield: 85 %; 1H NMR (CHCl3, 300 MHz): δ 3.4–3.5 (s, 3H, OCH3), 4.0–4.1 (s, 1H, NH), 5.1–5.2 (s, 1H, CH), 6.66–7.10 (m, 2H, benzene), 7.2–7.56 (m, 6H, benzene), 9.1–9.2 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,326, 2,985, 2,828, 1,670, 1,120, 780. EIMS (70 eV, m/z) 315.76 (M+), Anal. calcd for C15H14ClN5O: C, 57.06; H, 4.47; N, 22.18 found C, 57.00; H, 4.40; N, 22.10. N -((4-methoxyphenyl)(2H -tetrazol-5-yl)methyl)-4methylaniline (7) Yield: 79 %; m.p. 147–150 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 2.48–2.74 (s, 3H, CH3), 3.6–3.7 (s, 3H, OCH3), 4.9–5.1 (s, 1H, NH), 5.4–5.5 (s, 1H, CH), 7.1–7.2 (m, 4H, benzene), 7.4–7.5 (m, 4H, benzene), 9.2–9.4 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,286, 3,125, 2,946, 1,660, 1,140. EIMS (70 eV, m/z) 295.14 (M+), Anal. calcd for C16H17N5O: C, 65.01; H, 5.8; N, 23.71. found C, 65.00; H, 5.78; N, 23.51. 4-((4-methoxyphenyl)(2H-tetrazol-5-yl)methyl)morpholine (8) Yield: 75 %; m.p. 208–210 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 2.35–2.44 (m, 4H, morpholine), 3.3– 3.43 (m, 4H, morpholine), 3.84–3.85 (s, 3H, OCH3), 5.2– 5.3 (s, 1H, CH), 7.35–7.65 (m, 5H, benzene), 9.1–9.3 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,380, 3,025, 2,828, 1,520, 1,120. EIMS (70 eV, m/z) 275.31 (M+), Anal. calcd for C13H17N5O2: C, 56.71; H, 6.22; N, 25.44; O. found C, 56.61; H, 6.12; N, 25.34. N -((2-chlorophenyl)(2H -tetrazol-5-yl)methyl)aniline (9) Yield: 68 %; m.p. 250–251 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 4.0–4.1 (s, 1H, N-H), 5.13–5.24 (s, 1H, CH), 6.90–7.1 (m, 2H, benzene), 7.2–7.6 (m, 6H, benzene), 9.0–9.1 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,389, 3,123, 2,998, 1,600, 723. EIMS (70 eV, m/z) 285.7 (M+). Anal. calcd for C14H12ClN5: C, 58.85; H, 4.23; N, 24.51. found, C, 58.55; H, 4.10; N, 24.41. 4 - c hl or o- N - ( ( 2- ch l o r o ph en y l ) ( 2H - tet ra zo l-5 yl)methyl)aniline (10) Yield: 80 %; m.p. 264–266 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 3.9–4.0 (s, 1H, NH), 5.1–5.2 (s, 1H, CH), 6.7–6.8 (d, 2H, benzene), 7.2–7.44 (m, 6H, benzene), 9.10–9.11 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,376, 2,925, 2,858, 1,550, 765. EIMS (70 eV, m/z) 320.18 (M+), anal. calcd for C14H11Cl2N5: C, 52.52; H, 3.44; N, 21.84. found C, 52.40; H, 3.34; N, 21.64. N -((2-chlorophenyl)(2H -tetrazol-5-yl)methyl)-4methylaniline (11) Yield: 73 %; m.p. 255–257 °C (uncorrected), 1H NMR
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(CHCl3, 300 MHz): δ 2.9–3.0 (s, 3H, CH3), 4.13–4.22 (s, 1H, NH), 5.13–5.2 (s, 1H, CH) 6.7–6.8 (d, 2H, benzene), 6.7–6.8 (m, 2H, benzene), 7.2–7.8 (m, 6H, benzene), 9.10–9.11 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,316, 3,095, 2,998, 1,650, 771. EIMS (70 eV, m/z) 299.7 (M+), anal. calcd for C15H14ClN5: C, 60.10; H, 5.04; N, 25.04. found C, 60.00; H, 5.14; N, 25.1. 4-((2-chlorophenyl)(2H -tetrazol-5-yl)methyl)morpholine (12) Yield: 73 %; 1H NMR (CHCl3, 300 MHz): δ 2.45–2.54 (m, 4H, morpholine), 3.43–3.49 (m, 4H morpholine), 5.3–5.44 (s, 1H, CH), 7.25–7.55 (m, 4H, benzene), 9.4–9.6 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,389, 3,123, 2,998, 1,650, 771. EIMS (70 eV, m /z ) 279.7 (M+), anal. calcd for C12H14ClN5O: C, 51.52; H, 5.04; Cl, 12.67; N, 25.04, o, 5.72. N -(1-phenyl-1-(2H-tetrazol-5-yl)ethyl)aniline (13) Yield: 78 %; m.p. 228–230 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 1.8–1.9 (s, 3H, CH3), 3.91–4.00 (s, 1H, NH), 6.95–7.15 (m, 3H, benzene), 7.31–7.55 (m, 6H, benzene), 9.3–9.5 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,320, 3,005, 2,899, 2,600, 1,660. EIMS (70 eV, m/z) 265.31 (M+), anal. calcd for C15H15N5: C, 67.96; H, 5.7; N, 26.4. found C, 67.90; H, 5.61; N, 26.24. 4-chloro-N -(1-phenyl-1-(2H -tetrazol-5-yl)ethyl)aniline (14) Yield: 89 %; m.p. 255–257 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 1.89–2.0 (s, 3H, CH3), 4.00–4.10 (s, 1H, NH), 6.85–6.99 (m, 2H, benzene), 7.11–7.25 (m, 6H, benzene), 9.4–9.5 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,380, 2,928, 2,590, 1,610, 775. EIMS (70 eV, m/z) 299.76 (M+), anal. calcd for C14H15ClN5: C, 60.10; H, 4.71; N, 23.37. 4-methyl-N -(1-phenyl-1-(2H -tetrazol-5-yl)ethyl)aniline (15) Yield: 82 %; m.p. 270–273 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 1.35–1.44 (s, 3H, CH3), 1.6–1.8 (s, 3H, CH3), 4.00–4.12 (s, 1H, NH), 6.8–6.9 (m, 2H, benzene), 7.35–7.65 (m, 6H, benzene), 9.1–9.3 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,480, 3,100, 2,928, 2,590, 1,630. LC-MS: m/z 279.44 (M+1), anal. calcd for C16H17N5: C, 68.79; H, 6.13; N, 25.07. found C, 68.69; H, 6.05; N, 24.97. 4-(1-phenyl-1-(2H -tetrazol-5-yl)ethyl)morpholine (16) Yield: 87 %; m.p. 236–238 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 1.79 (s, 3H, CH3), 3.6 (t, 4H, morpholine), 3.81 (t, 4H, morpholine), 7.3– 7.4 (m, 5H, benzene), 9.1 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,380, 3,008, 2,990, 2,510, 1,610. LC-MS: m /z 259.31 (M+), anal. calcd for C13H17N5O: C, 60.21; H, 6.61; N, 27.01; found C, 60.10; H, 6.55; N, 26.85.
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Molecular docking studies Molecular docking computations were carried out using Glide. Ligand structure preparation: 3D structures of synthesized compounds were constructed based on X-ray crystal structure conformation using the fragment dictionary of Maestro 8.0. Protein structure preparation: The X-ray crystal structure of MTCYP51 in complex with fluconazole (TPF) ligand (PDB ID: LEAL) was obtained from the RCSB Protein Data Bank (PDB) and used to model the protein structure examined in the present study. The protein was optimized for docking using the “Protein Preparation Wizard” and “Prime Refinement Utility” of Maestro 8.0. The heme cofactor and the iron charges and connectivity were carefully inspected. There was a zero-order bond between the heme iron and the fluconazole (TPF) ligand. This bond was broken prior to “picking” the ligand during Glide grid generation. Glide docking: The extra precision (XP) Glide docking method was used to dock compounds into the MTCYP51 azole binding site. Upon completion of each docking calculation, 100 poses at most per ligand were allowed to generate. Identical binding poses with better Glide Score of each of the target compounds were selected for further binding energy/ pose activity relationship study (Fig. 2) [20].
Biological evaluation Antifungal studies The tetrazole compounds were synthesized and evaluated in vitro for antifungal activity against A. niger MTCC 281 and C. albicans MTCC227 by disk diffusion method. The
Fig. 2 XP Glide-predicted binding pose for compound 16 within the MTCYP51 active site. Distances in A0 are shown as dotted lines
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in vitro minimal inhibitory concentrations (MICs) of the target compounds (1–16) were determined by the disk diffusion method according to the methods defined by the National Committee for Clinical Laboratory Standards [21]. The MIC was defined as the lowest concentration that showed no growth. Fungi strains for testing include C. albicans and A. niger. Fluconazole obtained from respective manufacturer served as a positive control [21–23]. The result for antifungal activity against C. albicans and A. niger is summarized in Table 2.
concentration 500 μg/ml against C. albicans and 750 μg/ml against A. niger but less than that of standard fluconazole. Compounds 1, 2, 3, 6, 9, 10, 11, 13, and 14 are active against C. albicans. The result of primary antifungal assay revealed that introduction of morpholine on R1(comp 4 and 12) does not show any antifungal activity against the abovementioned fungal strains; the substitution of toluene on R1(comp 3 and 7) shows antifungal activity against C. albicans. Substitution of chlorobenzene at R1(comp 14 and 15) may exhibit antifungal activity against A. niger. Substitution of methyl on R2 may be responsible for antifungal activity against A. niger.
Result and discussion
Docking results
Synthesis of 16 novel phenyl(2H -tetrazol-5-yl)methanamine derivatives. The synthetic scheme for (1–16) is shown in Scheme 1. Tetrazole compound was synthesized by reaction of α-amino nitrile, ZnCl2, and NaN3 in isopropyl alcohol via known reported procedure. α-Amino nitrile groups is an important intermediate synthesized by reaction of aldehyde/ ketone, amine, and trimethylsilyl cyanide (TMSCN) as a cynating agent in the presence of ionic liquid [(Hbim)+(Cl)−] as modification of Strecker reaction. α-Amino nitriles were synthesized according to the method described in our previous work. The targeted compounds (1–16) were synthesized by converting CN of α-amino nitrile to tetrazole. The results of in vitro antifungal activities showed that the titled compounds did not show much activity against all fungi tested. Compound 3 showed moderate activity active against tested fungi as C. albicans and A. niger. Minimum inhibitory
Sixteen novel phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized. All new synthesized tetrazole compounds and fluconazole were characterized by a docking mode in the active site of the cytochrome P450 14α-sterol demethylase. A tetrazole derivative (compounds 1–16) has more negative docking score in comparison with fluconazole. There is some correlation between antifungal activity and docking score [20, 24]. In vitro antifungal activity of compound 3 against C. albicans (500 μg/ml), A. niger (750 μg/ ml), and Glide Score (G) (−5.96). The further result of docking study is summarized in Table 2 as Glide Score (G).
Table 2 In vitro antifungal activity of compounds 1–16 against Candida albicans and Aspergillus niger and Glide Score (G) Compound code
1 2 3 6 7 9 10 11 13 14 15 16 Fluconazole
MIC (μg/ml)
Glide Score (GScore)
Candida albicans
Aspergillus niger
1,000 750 500 500 – 1,000 750 750 750 1,000 – – 0.20
– – 750 – 750 750 – 750 750 1000 750 750 0.20
−6.42 −6.96 −5.96 −7.02 −6.97 −6.54 −6.72 −6.98 −6.64 −6.23 −6.11 −6/97 −5.74
Conclusion A series of 16 novel phenyl(2H -tetrazol-5-yl)methanamine derivatives were synthesized. The compounds were evaluated for their antifungal activity. Compound 3 showed good antifungal activity against C. albicans (500 μg/ml) and A. niger (750 μg/ml). Synthesized tetrazole compounds and fluconazole were characterized and subjected to docking study. Tetrazole derivatives (compounds 1–16) have more negative docking score in comparison with fluconazole; however, many of the compounds did not show good inhibitory activity. Acknowledgments We are grateful to Dr. Mrs. A. R. Madgulkar, Principal, AISSMS College of Pharmacy, Pune for providing us with the necessary financial support and infrastructure for carrying out this work.
References 1. Kanafani ZA, Perfect JR (2008) Antimicrobial resistance: resistance to antifungal agents: mechanisms and clinical impact. Clin Infect Dis 46(1):120–128 2. Senet P, Tichotova L, Votruba I, Buchta V, Spulak M, Kunes J, Nobilis M, Krenk O, Pour M (2010) Antifungal 3,5-disubstituted furanones: from 5-acyloxymethyl to 5-alkylidene derivatives. Bioorg Med Chem 18:1988–2000
J Chem Biol (2014) 7:29–35 3. Rogers TR (2006) Antifungal drug resistance: limited data, dramatic impact? Int J Antimicro Agents 1:7–11 4. Giuseppe LR, Felicia DD, Andrea T, Francesco P (2008) 1-[(3Aryloxy-3-aryl)propyl]-1H-imidazoles, new imidazoles with potent activity against Candida albicans and dermatophytes. Synthesis, structure-activity relationship, and molecular modeling studies. J Med Chem 51:3841–3855 5. Comfort AB, Suresh VKE, Xue YZ, Jagan RE, Barbara AB, Ashfaq MK, Melissa R, Khan S, Larry A, Seth YA (2011) Benzothieno[3,2b]quinolinium and 3-(phenylthio)quinolinium compounds: synthesis and evaluation against opportunistic fungal pathogens. Bioorg Med Chem 19:458–470 6. Boucher HW, Groll AH, Chiou CC, Walsh TJ (2004) Newer systemic antifungal agents pharmacokinetics, safety and efficacy. Drugs 64(18):1997–2020 7. Upadhayaya RS, Jain S, Sinha N, Kishore N, Chandra R, Arora SK (2004) Synthesis of novel substituted tetrazoles having antifungal activity. Eur J Med Chem 39:579 8. Mulwad VV, Pawar Rupesh B, Chaskar Atul C (2008) Synthesis and antibacterial activity of new tetrazole derivatives. J Korean Chem Soc 52:249–256 9. Bachar SC, Lahiri SC (2004) Synthesis of chloro and bromo substituted 5-(indan-1′-yl)tetrazoles and 5-(indan-1′-yl)methyltetrazoles as possible analgesic agents. Pharmazie 59:435–438 10. Mohite PB, Bhaskar VH (2010) Design, synthesis, characterization and biological evaluation of some novel 1, 5 disubstituted tetrazole as potential anti-inflammatory agents. J Optoelectron Biomed Mater 2:231–237 11. Larissa MP, Jure S, Thomas LP, Michael RW et al (2001) Substrate recognition sites in 14a-sterol demethylase from comparative analysis of amino acid sequences and X-ray structure of Mycobacterium tuberculosis CYP51. J Inorg Biochem 87:227–235 12. Cresnar B, Petric S, Trzaskos JM (2011) Cytochrome P450 enzymes in the fungal kingdom. Biochim Biophys Acta 1814:29–35 13. Dhayanithi V, Syed SS, Kumaran K (2011) Synthesis of selected 5thio-substituted tetrazole derivatives and evaluation of their antibacterial and antifungal activities. J Serb Chem Soc 76(2):165–175
35 14. Mesenzani O, Massarotti A, Giustiniano M, Pirali T, Bevilacqua V, Caldarelli A, Canonico P, Sorba G, Novellino E, Genazzani A, Tron GC (2011) Replacement of the double bond of antitubulin chalcones with triazoles and tetrazoles: synthesis and biological evaluation. Bioorg Med Chem Lett 21:764–768 15. Zheng L, Yuanhong M, Jun X, Jinghong S, Hongfang C (2010) Onepot three-component synthesis of α-aminonitriles using potassium hexacyanoferrate(II) as an eco-friendly cyanide source. Tetrahedron Lett 51:3922–3926 16. Ezzat R, Solmaz R, Mohammad J, Hadi C, Kambiz A (2008) Al2O3-supported 12-tungstosilicic acid as an efficient heterogeneous catalyst for the synthesis of α-aminonitrile. Synth Comm 38:2741–2747 17. Duthaler RO (1994) Recent developments in the stereoselective synthesis of α-amino acids. Tetrahedron 50(6):1539–1650 18. Kathiravan MK, Salake AB, Chothe AS, Kale AN, Kulkarni NM, Jankar ST (2012) A rapid and facile synthesis of α-amino nitrile employing ionic liquid. Chem J 2:199–205 19. Zachary PD, Sharpless KB (2001) Preparation of 5-substituted 1Htetrazoles from nitriles in water. J Org Chem 66:7945–7950 20. Patel PD, Patel MR, Talele TT (2010) Design, synthesis and determination of antifungal activity of 5(6)-substituted benzotriazoles. Eur J Med Chem 45:2214–2222 21. Villanova P (2002) Performance standards for antimicrobial susceptibility testing. 8th Informational Supplement, M100S12 22. Gian PT, Luca D, Anna RB, Gian CS, Ornella S, Giacomo F, Stefano A, Nicola C, Vivian T, Massimo A (2004) In vitro fluconazole susceptibility of 1565 clinical isolates of Candida species evaluated by the disk diffusion method performed using NCCLS M44-A guidelines. Diagn Microbiol Infect Dis 50:187–192 23. Izabel CV, Amanda R, Ana LC (2007) Quantitative disk diffusion as a convenient method for determining minimum inhibitory concentrations of oxacillin for staphylococci strains. J Microbiol Methods 71: 186–190 24. Glide, Molecular docking tool of Schrodinger Inc., version 5.0, New York, USA
ORIGINAL ARTICLE
Design, synthesis, and evaluations of antifungal activity of novel phenyl(2H -tetrazol-5-yl)methanamine derivatives Amol B. Salake & Aparna S. Chothe & Shrikant S. Nilewar & Madhavi Khilare & Rutuja S. Meshram & Abhishek A. Pandey & M. K. Kathiravan
Received: 21 January 2013 / Accepted: 22 August 2013 / Published online: 12 September 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl2 in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1.
Keywords Phenyl(2H -tetrazol-5-yl)methanamine . Fluconazole . Antifungal agent . Candida albicans . Aspergillus niger
A. B. Salake : A. S. Chothe Department of Pharmaceutical Chemistry (PG), AISSMS College of Pharmacy, Kennedy road, Near RTO, Pune 411001, Maharashtra, India S. S. Nilewar : M. Khilare : R. S. Meshram : A. A. Pandey : M. K. Kathiravan (*) Department of Pharmaceutical Chemistry (PG), Sinhgad College of Pharmacy, Off. Sinhgad road, Vadgaon (Bk.), Pune 411041, Maharashtra, India e-mail: [email protected]
Introduction Despite advances in preventive, diagnostic, and therapeutic interventions, invasive fungal infections cause significant morbidity and mortality in immune compromised patients. The burden of antifungal resistance in such high-risk patients is becoming a major concern [1]. In the past decade, there is significant increase in incidence of invasive fungal infections specially in hospitalized patients. An increased morbidity and mortality of mycotic infections is related with a growing number of vulnerable patients to opportunistic fungi as a consequence of progress in medicine, improved diagnostics, and awareness of fungi as etiology of these infections [2, 3]. Patients treated with immunosuppressive agents, broadspectrum antibiotics, antineoplastic agents, and anti-HIV agents as well as those undergoing extensive surgery or other invasive procedures are at higher risk of contracting a systemic mycosis [4]. The majority of invasive mycotic diseases are caused by Candida and Aspergillus species [2]. Infections caused by opportunistic pathogens continue to be of public health concern [5]. Candida albicans, Aspergillus niger, Cryptococcus neoformans, and Aspergillus fumigatus are the most common fungal pathogens responsible for causing opportunistic fungal infections in human beings. Patients with AIDS, lymphoma, and those on long-term corticosteroids therapy, organ transplant patients, and even diabetes patients are at risk for opportunistic fungal infections [6]. Heterocyclic tetrazole possesses wide range of activities as antifungal, antibacterial, analgesic, and anti-inflammatory [7–10]. Azole antifungals are strong inhibitors of lanosterol 14-α-demethylase, which is a major component of fungal cell membrane [11]. The triazole antifungal drugs fluconazole, itraconazole, voriconazole, ravuconazole, and posaconazole form an important class of antifungal agents. These drugs act
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by displacing lanosterol from cytochrome P450 14-α-DM and in this manner block the biosynthesis of ergosterol, an essential component of the fungal cell membrane and finally leads to killing of fungi. Cytochrome P450 14-α-DM oxidatively removes the 14-α-methyl group of lanosterol by using oxygen and NADPH [12]. The rising prevalence of multidrug resistant bacteria continues to provide impetus for the search and discovery of novel antifungal agents active against these pathogens. A series of 16 novel phenyl(2H-tetrazol-5-yl)methanamine derivatives are planned to see the effect of tetrazole on replacing triazole of fluconazole (Fig. 1). The designed compounds were also subjected to molecular docking into the active site of cytochrome P450 14α-sterol demethylase. The tetrazole compounds are evaluated in vitro for antifungal activity against A. niger MTCC 281 and C. albicans MTCC227 by disk diffusion method. In the present paper, our aim is to synthesize tetrazole nucleus from α-amino nitrile by modification in fluconazole moiety as chlorine has strong inductive electron-attracting effects, while those of fluorine are very weak. Moreover, these atoms may also influence the stearic characteristics and the hydrophilic– hydrophobic balance of the molecules. So, we have placed substitutions of –Cl, –OCH3, and –CH3 on a designed molecule. The hydrophobic group is required for activity, so for increasing hydrophobicity of the compound, phenyl ring is replaced by triazole moiety [13, 14]. Tetrazole substituent shows antifungal activity so we have replaced one triazole ring of fluconazole by tetrazole ring. An attempt is made for substitution of –CH3 near tetrazole which improves the spectrum of activity [13].
Materials and methods Chemistry One pot condensation of carbonyl compounds, (aldehyde and ketone) amine and cyanide, is known as Strecker reaction as in Scheme 1 [15].
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Scheme 1 General synthetic scheme of Strecker reaction
α-Amino nitriles are significantly important intermediates for the synthesis of a wide variety of amino acids, amides, diamines, and nitrogen-containing heterocycles [15], such as thiadiazoles and imidazole derivatives. Among the methods reported for the synthesis of α-amino nitriles, Strecker reaction, nucleophilic addition of cyanide ion to imines, is of great importance to modern organic chemistry as it offers one of the most direct and viable methods for the synthesis of α-amino nitriles [16, 17]. α-Amino nitriles are synthesized by the reactions of aldehydes/ketones with amines in the presence of a cyanide source such as TMSCN which is the safer and more efficient cyanide anion source. The α-amino nitriles were synthesized as per procedure described in our unpublished work described in TGCL-2011-0076 [18]. The synthetic scheme for (1–16) is shown in Scheme 2. All synthesized compounds were characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and mass spectroscopy. The reagents used were of analytical grade. TLC precoated plates silica gel (G-60 mesh) were used. Melting points were determined in open capillary tube using Labin Melting Point Apparatus and are uncorrected. FTIR spectra of the synthesized compounds were recorded using KBr pellets on a Jasco FTIR V 430 + spectrometer using diffuse reflectance attachment and are reported in per centimeter. Proton nuclear magnetic resonance spectroscopy (1H NMR) spectra were recorded on a Varian Mercury YH300 (300 MHz FT NMR) spectrometer using tetramethylsilane as an internal standard (chemical shift represented in δ part per million). Elemental analysis was performed on Flashea 1112 series equipment by Thermoelectron Corporation. Mass spectra were obtained on a double focusing magnetic sector mass spectrometer using direct insertion probe using electron impact ion source operating at 70 eV (Table 1). General procedure for synthesis of phenyl(2H -tetrazol-5-yl) methanamine derivatives (1–16)
Fig. 1 Structure of fluconazole
To a mixture of α-amino nitrile (1.00 mol), sodium azide (1.05 mol), and zinc chloride (0.50 mol), isopropyl alcohol was refluxed for 16 h. The progress of the reaction was
Scheme 2 General synthetic scheme of phenyl(2H -tetrazol-5yl)methanamine derivatives. Reagents and condition: a TMSCN, IL, RT, and stirring; b NaN3, ZnCl2, and IPA
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Table 1 Different substitutions at 1–16
Compd.
Structures
Compd.
1.
9.
2.
10.
3.
11.
4.
12.
5.
13.
6.
14.
7.
15.
8.
16.
Structures
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monitored by TLC system as methanol:dichloromethane (1:9). After completion of a reaction, the mixture was cooled to room temperature, the pH was adjusted with concentrated HCl, and the reaction was stirred for 30 min to break up the solid precipitate. The mixture was extracted with ethyl acetate twice. Again, it was washed with brine solution and dried over anhydrous sodium sulphate and evaporate to yield the product [19]. The titled compounds were characterized as follows: N -(phenyl (2H -tetrazol-5-yl) methyl) aniline (1) Yield 72 %, m.p. 193–195 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 4.0 (s, 1H, NH), 5.4 (s, 1H, CH), 9.2–9.3 (s, 1H, tetrazole NH), 7.00– 7.10 (m, 3H, benzene), 7.33–7.55 (m, 7H, benzene), IR (KBr) (per centimeter): 3,328, 3,023, 2,990, 1,515, EIMS (70 eV, m /z ) 251.12 (M+). Anal. calcd for C14H13N5: C, 66.92; H, 5.21; N, 27.87 found C, 66.80; H, 5.15; N, 27.79. 4-chloro-N-(phenyl (2H-tetrazol-5-yl)methyl)aniline (2) Yield: 71 %; m.p. 188–190 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 4.00–4.12 (s, 1H, NH), 5.14–5.19 (s, 1H, CH), 6.67 (d, 2H, benzene), 7.24–7.45 (m, 7H, benzene), 9.1–9.2 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,428, 3,123, 2,979, 1,615, 700. EIMS (70 eV, m/z) 285.1 (M+), Anal. calcd for C14H12ClN5: C, 58.85; H, 4.23; N, 24.51 found C, 58.80; H, 4.18; N, 24.48. 4-methyl-N -(phenyl(2H-tetrazol-5-yl)methyl)aniline (3) Yield 61 %, m.p. 187–190 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 2.34–2.38 (s, 3H, CH3), 3.9–4.1 (s, 1H, NH), 5.1–5.2 (s, 1H, CH), 6.87–6.90 (d, 2H, benzene), 7.12–7.35 (m, 7H, benzene), 10.1–10.3 (s, 1H, tetrazole NH), IR (KBr) (per centimeter): 3,400, 3,003, 2,980, 1,605. EIMS (70 eV, m/z) 265.13 (M+). Anal. calcd for C15H15N5: C, 67.9; H, 5.7; N, 26.4 found C, 67.86; H, 5.6; N, 26.32 4-(phenyl (2H -tetrazol-5-yl)methyl) morpholine (4) Yield 65 %, m.p. 154–156 °C, (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 2.48–2.64 (m, 4H, morpholine), 3.4–3.5 (m, 4H morpholine), 5.2–5.3 (s, 1H, NH), 7.35– 7.8 (m, 5H, benzene), 9.1–9.2 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,428, 3,123, 2,990, 1,615, 1,100. EIMS (70 eV, m /z ) 245.8 (M+). Anal. calcd for C12H15N5O: C, 58.76; H, 6.16; N, 28.55. found C, 58.66; H, 6.11; N, 28.35. N -((4-methoxyphenyl)(2H -tetrazol-5-yl)methyl)aniline (5) Yield: 83 %; m.p. 218–220 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 3.7–3.8 (s, 3H, OCH3), 3.9–4.0 (s, 1H, NH), 5.3–5.4 (s, 1H, CH), 6.96–7.45 (m, 9H, benzene), 9.1–9.3 (s, 1H, tetrazole NH), IR (KBr) (per centimeter): 3,346, 3,125, 2,928, 1,550, 1,123. EIMS (70 eV, m /z ) 281.31 (M+), Anal. calcd
for C15H15N5O: C, 64.04; H, 5.37; N, 24.94. found C, 64.00; H, 5.31; N, 24.84 found C, 63.90; H, 5.29; N, 24.80. 4-chloro-N -((4-methoxyphenyl)(2H -tetrazol-5yl)methyl)aniline (6) Yield: 85 %; 1H NMR (CHCl3, 300 MHz): δ 3.4–3.5 (s, 3H, OCH3), 4.0–4.1 (s, 1H, NH), 5.1–5.2 (s, 1H, CH), 6.66–7.10 (m, 2H, benzene), 7.2–7.56 (m, 6H, benzene), 9.1–9.2 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,326, 2,985, 2,828, 1,670, 1,120, 780. EIMS (70 eV, m/z) 315.76 (M+), Anal. calcd for C15H14ClN5O: C, 57.06; H, 4.47; N, 22.18 found C, 57.00; H, 4.40; N, 22.10. N -((4-methoxyphenyl)(2H -tetrazol-5-yl)methyl)-4methylaniline (7) Yield: 79 %; m.p. 147–150 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 2.48–2.74 (s, 3H, CH3), 3.6–3.7 (s, 3H, OCH3), 4.9–5.1 (s, 1H, NH), 5.4–5.5 (s, 1H, CH), 7.1–7.2 (m, 4H, benzene), 7.4–7.5 (m, 4H, benzene), 9.2–9.4 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,286, 3,125, 2,946, 1,660, 1,140. EIMS (70 eV, m/z) 295.14 (M+), Anal. calcd for C16H17N5O: C, 65.01; H, 5.8; N, 23.71. found C, 65.00; H, 5.78; N, 23.51. 4-((4-methoxyphenyl)(2H-tetrazol-5-yl)methyl)morpholine (8) Yield: 75 %; m.p. 208–210 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 2.35–2.44 (m, 4H, morpholine), 3.3– 3.43 (m, 4H, morpholine), 3.84–3.85 (s, 3H, OCH3), 5.2– 5.3 (s, 1H, CH), 7.35–7.65 (m, 5H, benzene), 9.1–9.3 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,380, 3,025, 2,828, 1,520, 1,120. EIMS (70 eV, m/z) 275.31 (M+), Anal. calcd for C13H17N5O2: C, 56.71; H, 6.22; N, 25.44; O. found C, 56.61; H, 6.12; N, 25.34. N -((2-chlorophenyl)(2H -tetrazol-5-yl)methyl)aniline (9) Yield: 68 %; m.p. 250–251 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 4.0–4.1 (s, 1H, N-H), 5.13–5.24 (s, 1H, CH), 6.90–7.1 (m, 2H, benzene), 7.2–7.6 (m, 6H, benzene), 9.0–9.1 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,389, 3,123, 2,998, 1,600, 723. EIMS (70 eV, m/z) 285.7 (M+). Anal. calcd for C14H12ClN5: C, 58.85; H, 4.23; N, 24.51. found, C, 58.55; H, 4.10; N, 24.41. 4 - c hl or o- N - ( ( 2- ch l o r o ph en y l ) ( 2H - tet ra zo l-5 yl)methyl)aniline (10) Yield: 80 %; m.p. 264–266 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 3.9–4.0 (s, 1H, NH), 5.1–5.2 (s, 1H, CH), 6.7–6.8 (d, 2H, benzene), 7.2–7.44 (m, 6H, benzene), 9.10–9.11 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,376, 2,925, 2,858, 1,550, 765. EIMS (70 eV, m/z) 320.18 (M+), anal. calcd for C14H11Cl2N5: C, 52.52; H, 3.44; N, 21.84. found C, 52.40; H, 3.34; N, 21.64. N -((2-chlorophenyl)(2H -tetrazol-5-yl)methyl)-4methylaniline (11) Yield: 73 %; m.p. 255–257 °C (uncorrected), 1H NMR
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(CHCl3, 300 MHz): δ 2.9–3.0 (s, 3H, CH3), 4.13–4.22 (s, 1H, NH), 5.13–5.2 (s, 1H, CH) 6.7–6.8 (d, 2H, benzene), 6.7–6.8 (m, 2H, benzene), 7.2–7.8 (m, 6H, benzene), 9.10–9.11 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,316, 3,095, 2,998, 1,650, 771. EIMS (70 eV, m/z) 299.7 (M+), anal. calcd for C15H14ClN5: C, 60.10; H, 5.04; N, 25.04. found C, 60.00; H, 5.14; N, 25.1. 4-((2-chlorophenyl)(2H -tetrazol-5-yl)methyl)morpholine (12) Yield: 73 %; 1H NMR (CHCl3, 300 MHz): δ 2.45–2.54 (m, 4H, morpholine), 3.43–3.49 (m, 4H morpholine), 5.3–5.44 (s, 1H, CH), 7.25–7.55 (m, 4H, benzene), 9.4–9.6 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,389, 3,123, 2,998, 1,650, 771. EIMS (70 eV, m /z ) 279.7 (M+), anal. calcd for C12H14ClN5O: C, 51.52; H, 5.04; Cl, 12.67; N, 25.04, o, 5.72. N -(1-phenyl-1-(2H-tetrazol-5-yl)ethyl)aniline (13) Yield: 78 %; m.p. 228–230 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 1.8–1.9 (s, 3H, CH3), 3.91–4.00 (s, 1H, NH), 6.95–7.15 (m, 3H, benzene), 7.31–7.55 (m, 6H, benzene), 9.3–9.5 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,320, 3,005, 2,899, 2,600, 1,660. EIMS (70 eV, m/z) 265.31 (M+), anal. calcd for C15H15N5: C, 67.96; H, 5.7; N, 26.4. found C, 67.90; H, 5.61; N, 26.24. 4-chloro-N -(1-phenyl-1-(2H -tetrazol-5-yl)ethyl)aniline (14) Yield: 89 %; m.p. 255–257 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 1.89–2.0 (s, 3H, CH3), 4.00–4.10 (s, 1H, NH), 6.85–6.99 (m, 2H, benzene), 7.11–7.25 (m, 6H, benzene), 9.4–9.5 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,380, 2,928, 2,590, 1,610, 775. EIMS (70 eV, m/z) 299.76 (M+), anal. calcd for C14H15ClN5: C, 60.10; H, 4.71; N, 23.37. 4-methyl-N -(1-phenyl-1-(2H -tetrazol-5-yl)ethyl)aniline (15) Yield: 82 %; m.p. 270–273 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 1.35–1.44 (s, 3H, CH3), 1.6–1.8 (s, 3H, CH3), 4.00–4.12 (s, 1H, NH), 6.8–6.9 (m, 2H, benzene), 7.35–7.65 (m, 6H, benzene), 9.1–9.3 (s, 1H, NH tetrazole). IR (KBr) (per centimeter): 3,480, 3,100, 2,928, 2,590, 1,630. LC-MS: m/z 279.44 (M+1), anal. calcd for C16H17N5: C, 68.79; H, 6.13; N, 25.07. found C, 68.69; H, 6.05; N, 24.97. 4-(1-phenyl-1-(2H -tetrazol-5-yl)ethyl)morpholine (16) Yield: 87 %; m.p. 236–238 °C (uncorrected), 1H NMR (CHCl3, 300 MHz): δ 1.79 (s, 3H, CH3), 3.6 (t, 4H, morpholine), 3.81 (t, 4H, morpholine), 7.3– 7.4 (m, 5H, benzene), 9.1 (s, 1H, tetrazole NH). IR (KBr) (per centimeter): 3,380, 3,008, 2,990, 2,510, 1,610. LC-MS: m /z 259.31 (M+), anal. calcd for C13H17N5O: C, 60.21; H, 6.61; N, 27.01; found C, 60.10; H, 6.55; N, 26.85.
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Molecular docking studies Molecular docking computations were carried out using Glide. Ligand structure preparation: 3D structures of synthesized compounds were constructed based on X-ray crystal structure conformation using the fragment dictionary of Maestro 8.0. Protein structure preparation: The X-ray crystal structure of MTCYP51 in complex with fluconazole (TPF) ligand (PDB ID: LEAL) was obtained from the RCSB Protein Data Bank (PDB) and used to model the protein structure examined in the present study. The protein was optimized for docking using the “Protein Preparation Wizard” and “Prime Refinement Utility” of Maestro 8.0. The heme cofactor and the iron charges and connectivity were carefully inspected. There was a zero-order bond between the heme iron and the fluconazole (TPF) ligand. This bond was broken prior to “picking” the ligand during Glide grid generation. Glide docking: The extra precision (XP) Glide docking method was used to dock compounds into the MTCYP51 azole binding site. Upon completion of each docking calculation, 100 poses at most per ligand were allowed to generate. Identical binding poses with better Glide Score of each of the target compounds were selected for further binding energy/ pose activity relationship study (Fig. 2) [20].
Biological evaluation Antifungal studies The tetrazole compounds were synthesized and evaluated in vitro for antifungal activity against A. niger MTCC 281 and C. albicans MTCC227 by disk diffusion method. The
Fig. 2 XP Glide-predicted binding pose for compound 16 within the MTCYP51 active site. Distances in A0 are shown as dotted lines
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in vitro minimal inhibitory concentrations (MICs) of the target compounds (1–16) were determined by the disk diffusion method according to the methods defined by the National Committee for Clinical Laboratory Standards [21]. The MIC was defined as the lowest concentration that showed no growth. Fungi strains for testing include C. albicans and A. niger. Fluconazole obtained from respective manufacturer served as a positive control [21–23]. The result for antifungal activity against C. albicans and A. niger is summarized in Table 2.
concentration 500 μg/ml against C. albicans and 750 μg/ml against A. niger but less than that of standard fluconazole. Compounds 1, 2, 3, 6, 9, 10, 11, 13, and 14 are active against C. albicans. The result of primary antifungal assay revealed that introduction of morpholine on R1(comp 4 and 12) does not show any antifungal activity against the abovementioned fungal strains; the substitution of toluene on R1(comp 3 and 7) shows antifungal activity against C. albicans. Substitution of chlorobenzene at R1(comp 14 and 15) may exhibit antifungal activity against A. niger. Substitution of methyl on R2 may be responsible for antifungal activity against A. niger.
Result and discussion
Docking results
Synthesis of 16 novel phenyl(2H -tetrazol-5-yl)methanamine derivatives. The synthetic scheme for (1–16) is shown in Scheme 1. Tetrazole compound was synthesized by reaction of α-amino nitrile, ZnCl2, and NaN3 in isopropyl alcohol via known reported procedure. α-Amino nitrile groups is an important intermediate synthesized by reaction of aldehyde/ ketone, amine, and trimethylsilyl cyanide (TMSCN) as a cynating agent in the presence of ionic liquid [(Hbim)+(Cl)−] as modification of Strecker reaction. α-Amino nitriles were synthesized according to the method described in our previous work. The targeted compounds (1–16) were synthesized by converting CN of α-amino nitrile to tetrazole. The results of in vitro antifungal activities showed that the titled compounds did not show much activity against all fungi tested. Compound 3 showed moderate activity active against tested fungi as C. albicans and A. niger. Minimum inhibitory
Sixteen novel phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized. All new synthesized tetrazole compounds and fluconazole were characterized by a docking mode in the active site of the cytochrome P450 14α-sterol demethylase. A tetrazole derivative (compounds 1–16) has more negative docking score in comparison with fluconazole. There is some correlation between antifungal activity and docking score [20, 24]. In vitro antifungal activity of compound 3 against C. albicans (500 μg/ml), A. niger (750 μg/ ml), and Glide Score (G) (−5.96). The further result of docking study is summarized in Table 2 as Glide Score (G).
Table 2 In vitro antifungal activity of compounds 1–16 against Candida albicans and Aspergillus niger and Glide Score (G) Compound code
1 2 3 6 7 9 10 11 13 14 15 16 Fluconazole
MIC (μg/ml)
Glide Score (GScore)
Candida albicans
Aspergillus niger
1,000 750 500 500 – 1,000 750 750 750 1,000 – – 0.20
– – 750 – 750 750 – 750 750 1000 750 750 0.20
−6.42 −6.96 −5.96 −7.02 −6.97 −6.54 −6.72 −6.98 −6.64 −6.23 −6.11 −6/97 −5.74
Conclusion A series of 16 novel phenyl(2H -tetrazol-5-yl)methanamine derivatives were synthesized. The compounds were evaluated for their antifungal activity. Compound 3 showed good antifungal activity against C. albicans (500 μg/ml) and A. niger (750 μg/ml). Synthesized tetrazole compounds and fluconazole were characterized and subjected to docking study. Tetrazole derivatives (compounds 1–16) have more negative docking score in comparison with fluconazole; however, many of the compounds did not show good inhibitory activity. Acknowledgments We are grateful to Dr. Mrs. A. R. Madgulkar, Principal, AISSMS College of Pharmacy, Pune for providing us with the necessary financial support and infrastructure for carrying out this work.
References 1. Kanafani ZA, Perfect JR (2008) Antimicrobial resistance: resistance to antifungal agents: mechanisms and clinical impact. Clin Infect Dis 46(1):120–128 2. Senet P, Tichotova L, Votruba I, Buchta V, Spulak M, Kunes J, Nobilis M, Krenk O, Pour M (2010) Antifungal 3,5-disubstituted furanones: from 5-acyloxymethyl to 5-alkylidene derivatives. Bioorg Med Chem 18:1988–2000
J Chem Biol (2014) 7:29–35 3. Rogers TR (2006) Antifungal drug resistance: limited data, dramatic impact? Int J Antimicro Agents 1:7–11 4. Giuseppe LR, Felicia DD, Andrea T, Francesco P (2008) 1-[(3Aryloxy-3-aryl)propyl]-1H-imidazoles, new imidazoles with potent activity against Candida albicans and dermatophytes. Synthesis, structure-activity relationship, and molecular modeling studies. J Med Chem 51:3841–3855 5. Comfort AB, Suresh VKE, Xue YZ, Jagan RE, Barbara AB, Ashfaq MK, Melissa R, Khan S, Larry A, Seth YA (2011) Benzothieno[3,2b]quinolinium and 3-(phenylthio)quinolinium compounds: synthesis and evaluation against opportunistic fungal pathogens. Bioorg Med Chem 19:458–470 6. Boucher HW, Groll AH, Chiou CC, Walsh TJ (2004) Newer systemic antifungal agents pharmacokinetics, safety and efficacy. Drugs 64(18):1997–2020 7. Upadhayaya RS, Jain S, Sinha N, Kishore N, Chandra R, Arora SK (2004) Synthesis of novel substituted tetrazoles having antifungal activity. Eur J Med Chem 39:579 8. Mulwad VV, Pawar Rupesh B, Chaskar Atul C (2008) Synthesis and antibacterial activity of new tetrazole derivatives. J Korean Chem Soc 52:249–256 9. Bachar SC, Lahiri SC (2004) Synthesis of chloro and bromo substituted 5-(indan-1′-yl)tetrazoles and 5-(indan-1′-yl)methyltetrazoles as possible analgesic agents. Pharmazie 59:435–438 10. Mohite PB, Bhaskar VH (2010) Design, synthesis, characterization and biological evaluation of some novel 1, 5 disubstituted tetrazole as potential anti-inflammatory agents. J Optoelectron Biomed Mater 2:231–237 11. Larissa MP, Jure S, Thomas LP, Michael RW et al (2001) Substrate recognition sites in 14a-sterol demethylase from comparative analysis of amino acid sequences and X-ray structure of Mycobacterium tuberculosis CYP51. J Inorg Biochem 87:227–235 12. Cresnar B, Petric S, Trzaskos JM (2011) Cytochrome P450 enzymes in the fungal kingdom. Biochim Biophys Acta 1814:29–35 13. Dhayanithi V, Syed SS, Kumaran K (2011) Synthesis of selected 5thio-substituted tetrazole derivatives and evaluation of their antibacterial and antifungal activities. J Serb Chem Soc 76(2):165–175
35 14. Mesenzani O, Massarotti A, Giustiniano M, Pirali T, Bevilacqua V, Caldarelli A, Canonico P, Sorba G, Novellino E, Genazzani A, Tron GC (2011) Replacement of the double bond of antitubulin chalcones with triazoles and tetrazoles: synthesis and biological evaluation. Bioorg Med Chem Lett 21:764–768 15. Zheng L, Yuanhong M, Jun X, Jinghong S, Hongfang C (2010) Onepot three-component synthesis of α-aminonitriles using potassium hexacyanoferrate(II) as an eco-friendly cyanide source. Tetrahedron Lett 51:3922–3926 16. Ezzat R, Solmaz R, Mohammad J, Hadi C, Kambiz A (2008) Al2O3-supported 12-tungstosilicic acid as an efficient heterogeneous catalyst for the synthesis of α-aminonitrile. Synth Comm 38:2741–2747 17. Duthaler RO (1994) Recent developments in the stereoselective synthesis of α-amino acids. Tetrahedron 50(6):1539–1650 18. Kathiravan MK, Salake AB, Chothe AS, Kale AN, Kulkarni NM, Jankar ST (2012) A rapid and facile synthesis of α-amino nitrile employing ionic liquid. Chem J 2:199–205 19. Zachary PD, Sharpless KB (2001) Preparation of 5-substituted 1Htetrazoles from nitriles in water. J Org Chem 66:7945–7950 20. Patel PD, Patel MR, Talele TT (2010) Design, synthesis and determination of antifungal activity of 5(6)-substituted benzotriazoles. Eur J Med Chem 45:2214–2222 21. Villanova P (2002) Performance standards for antimicrobial susceptibility testing. 8th Informational Supplement, M100S12 22. Gian PT, Luca D, Anna RB, Gian CS, Ornella S, Giacomo F, Stefano A, Nicola C, Vivian T, Massimo A (2004) In vitro fluconazole susceptibility of 1565 clinical isolates of Candida species evaluated by the disk diffusion method performed using NCCLS M44-A guidelines. Diagn Microbiol Infect Dis 50:187–192 23. Izabel CV, Amanda R, Ana LC (2007) Quantitative disk diffusion as a convenient method for determining minimum inhibitory concentrations of oxacillin for staphylococci strains. J Microbiol Methods 71: 186–190 24. Glide, Molecular docking tool of Schrodinger Inc., version 5.0, New York, USA
