Synthesis, docking and in-vitro screening of mannich bases of thiosemicarbazide for anti-fungal activity

Sachin A. Pishawikar; Harinath N. More

doi:10.1016/j.arabjc.2013.10.016

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Original article

10 (

2_suppl

); S2714-S2722

doi:

10.1016/j.arabjc.2013.10.016

Synthesis, docking and in-vitro screening of mannich bases of thiosemicarbazide for anti-fungal activity

Sachin A. Pishawikar^⁎, Harinath N. More

Department of Pharmaceutical Chemistry, Bharti Vidyapeeth College of Pharmacy, Near Chitranagari Kolhapur, Maharashtra 416 013, India

⁎Corresponding author. Tel.: +91 231 2637286, mobile: +91 9823751341; fax: +91 231 2638833. sachin_pishawikar@rediffmail.com (Sachin A. Pishawikar)

Received: 2012-12-14, Accepted: 2013-10-21, Published: 2013-05-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Mannich bases and thiosemicarbazide individually show antimicrobial, antifungal, anticonvulsant, antimalarial, analgesic and anti-inflammatory type of varied pharmacological activities. The novelty of the present work is the synthesis of mannich bases of thiosemicarbazide as mutual prodrugs. In step-1, mannich bases are synthesized using aldehyde, ketones and amines with aliphatic, aromatic, cyclic and heterocyclic nature. In step-2, the synthesized bases were condensed with thiosemicarbazide to form mannich bases of thiosemicarbazide. Structural characterization of synthesized compounds was done using IR, mass and H-NMR spectroscopy. The compounds were screened for anti-fungal activity using BHI (brain heart infusion) broth dilution method against Candida albicans and Apergillus niger. Docking of synthesized compounds was done on CYP51A1, P45014DM (Lanosterol 14 α-demethylase enzyme) using Vlife MDS 3.5 to conform the mechanism of antifungal activity. Docking study showed a strong hydrophobic interaction between amino acid residues Arganine (ARG141), Glutamine (GLU146), Leucine (LEU54), Lycine (LYC227), and Threonine (THR147) with the carbon of ketone, nitrogen of amine and sulfur of thiosemicarbazide. Strong Vander wall’s interactions are also observed with the carbon of ketone, nitrogen of amine and sulfur of thiosemicarbazide.

Analogs with aromatic and substituted aromatic aldehydes showed least activity, while analogs with aliphatic aldehyde, ketones and amines showed greater activity in C. albicans compared to A. niger. Analogs having morpholine as amine showed comparable activity in both. Compounds K₁₇, K₁₈, K₁₉, and K₂₀ have shown comparable highest activities.

Keywords

Antifungal

Brain heart infusion

Mannich bases

Thiosemicarbazide

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1 Introduction

Infectious diseases caused by bacteria and fungi affect millions of people worldwide. Systematic programs to discover and develop new antibiotics and antifungals, are the need of the hour due to considerable extent of development of resistance by organisms to the existing drugs used against them. Further, the need has arisen largely due to advent of HIV, which has increased the numbers of profoundly and chronically immune suppressed patients, who are susceptible to both serious invasive and superficial fungal infections. The development of new agents may provide additional options for the treatment of fungal infections and may help to overcome the limitations of current treatments (Denning, 1991).

By definition, mutual prodrugs are carrier-linked prodrugs consisting of two pharmacologically active agents coupled together so that each acts as a promoiety for the other agent and vice versa. Individually mannich bases and thiosemicarbazide show varied pharmacological activities such as, anticancer, antimicrobial, antifungal, anticonvulsant, antimalarial, analgesic and anti-inflammatory. Present work has used the concept of mutual prodrug to synthesize mannich bases of thiosemicarbazide as mutual prodrug and screening them as anti-fungal agents (Bhosle et al., 2006; Pandeya et al., 1999, 2003 ).

The mannich base is an end product in the mannich reaction, which is nucleophilic addition reaction of a non-enolizable aldehyde and any primary or secondary amine to produce resonance stabilized imine (iminium ion or imine salt).

In the synthesis of mannich bases, generally the use of thiosemicarbazide is done as amine component, but the novelty of the present work is that, in step-1 successful synthesis of a number of mannich bases was done using aldehyde, ketones and amines with aliphatic, aromatic, cyclic and heterocyclic nature. In step-2, synthesized mannich bases were condensed with thiosemicarbazide to form mannich bases of thiosemicarbazide (Van de Kamp and Mosettig, 1936; March, 1977; Waring, 1979; Mannich and Krosche, 1912; Thompson, 1968 ).

Computational methodologies have become a crucial component of many drug discovery programs, from hit identification to lead optimization. One key methodology is docking of small molecules to protein binding sites, pioneered during the early 1980s. The docking process involves the prediction of ligand conformation and orientation (or posing) within a targeted binding site. The two aims of docking studies are accurate structural modeling and correct prediction of activity. Docking studies have become a scientific approach for the study of macromolecular structures and interactions. Macromolecular modeling by docking studies provides most detailed possible view of drug–receptor interaction and has created a new rational approach to drug design, where the structure of drug is designed based on its fit to three dimensional structures of a receptor site. On the basis of docking scores one can predict the amount of activity that will be shown by compounds. To conform the mechanism of antifungal activity of synthesized compounds, docking of synthesized compounds was performed on the structure of Lanosterol 14 α-demethylase (CYP51A1, P45014DM) an enzyme from cytochrome P450 family that catalyzes the oxidative removal of the 14α-methyl group of lanosterol, an essential step in the production of sterols. Inhibition of this step leads to inhibition of synthesis of ergosterol, a very essential component of fungal cell membrane. For docking study Vlife MDS 3.5 was used (Lengauer and Rarey, 1996; Kitchen et al., 2004).

The characterization of synthesized compounds was done by IR and H NMR. The compounds were screened for antifungal activity using Brain heart infusion (BHI) broth dilution method using Candida albicans (ATCC Code 10231), Apergillus niger. (ATCC Code 16404) and using concentration of Fluconazloe (30 μg/ml) as standard drug (Andrews, 2001; Report of the Working, 1991; Silverman, 2004; Williams et al., 2007; Committee, 1997; Winstanley et al., 1994; Lennette et al., 1985 ).

2 Methods and materials

Scheme of Synthesis:-

2.1

2.1 Step-1 synthesis of mannich base

Proportion of three reactants used for reactions was 1.00 molecular equivalent of carbonyl compound (ketone), 1.05–1.10 molecular equivalent of amine in the form of hydrochloride salt and 1.5–2.0 molecular equivalent of aldehyde.

2.2

2.2 Procedure

Amine was taken in a flat bottom flask and converted into hydrochloride salt using concentrated hydrochloric acid, formation of salt is confirmed by the use of Congo red paper. To this were added ketone and aldehyde. The reaction mixture was exposed to mechanical stirring. For some reactions heating on water bath was done.

Optimization of reaction conditions with respect to time and temperature was done on individual basis for each reaction. Time required varied from 30 min to 12–14 h, with temperature conditions varying from room temperature with mechanical stirring, to heating on water bath at temperature between 80 and 100 °C depending upon the complexity of reactants.

2.3

2.3 Step-I1 synthesis of mannich bases of thiosemicarbazide

Simple condensation reaction was carried out between synthesized mannich bases and one mole quantity of thiosemicarbazide in the presence of alcohol as solvent with refluxation on boiling water bath for around half an hour to form mannich bases of thiosemicarbazide. Synthesized compounds are shown in Table 1.

Table 1 Synthesized compounds

Sr. No.	Code	R	R1	R2	R3
01	K₁	CH₃	H	C₂H₅	C₂H₅
02	K₂	CH₃	H	CH₃	CH₃
03	K₃	CH₃	CH₃CH₂	CH₃CH₂CH₂	CH₃CH₂CH₂
04	K₄	CH₃CH₂	CH₃CH₂CH₂	CH₂CH₂OH	CH₂CH₂OH
05	K₅	CH₃CH₂CH₂	CH₃–CH–CH₃	CH₃CH₂CH₂CH₂	CH₃CH₂CH₂CH₂
06	K₆		CH₃CH₂	CH₃CH₂CH₂	CH₃CH₂CH₂
07	K₇			C₂H₅	C₂H₅
08	K₈	CH₃		CH₃CH₂CH₂	CH₃CH₂CH₂
09	K₉	CH₃		CH₃CH₂CH₂	CH₃CH₂CH₂
10	K₁₀	CH₃		CH₃CH₂CH₂	CH₃CH₂CH₂
11	K₁₁		CH₃CH₂CH₂	CH₂CH₂OH	CH₂CH₂OH
12	K₁₂		CH₃CH₂	C₂H₅	C₂H₅
13	K₁₃		H	C₂H₅	C₂H₅
14	K₁₄		H	C₂H₅	C₂H₅
15	K₁₅		H	C₂H₅	C₂H₅
16	K₁₆	CH₃		C₂H₅	C₂H₅
17	K₁₇		H	C₂H₅	C₂H₅
18	K₁₈	CH₃	H
19	K₁₉	CH₃CH₂CH₂	H
20	K₂₀		H
21	K₂₁		H
22	K₂₂	CH₃
23	K₂₃	CH₃
24	K₂₄		H
25	K₂₅		H

2.4

2.4 Characterization

Physicochemical characterization of the synthesized compounds was done by the estimation of melting point and R_f values by TLC. Results are mentioned in Table 2.

Table 2 Physicochemical data of synthesized compounds.

Code of compound	Color	Solubility	M.P. (°C)	% Yield	R_f value
K₁	Green	EtOH	120–122C	68	0.5
K₂	Light green	EtOH	84–88	72	0.6
K₃	Mahogany	EtOH	98–104	67	0.5
K₄	Maroon	EtOH	88–92	62	0.6
K₅	Flattery brown	EtOH	116–118	69	0.6
K₆	Drab	EtOH	84–86	78	0.5
K₇	Arsenic	EtOH	134–138	24	0.7
K₈	Orange	EtOH	198–200	38	0.6
K₉	Bistre brown	EtOH	200–202	43	0.6
K₁₀	Orange	EtOH	120–124	58	0.7
K₁₁	Light orange	EtOH	180–182	79	0.8
K₁₂	Maroon	EtOH	186–188	83	0.5
K₁₃	Light red	EtOH	130–134	69	0.7
K₁₄	Light red	EtOH	186–188	74	0.6
K₁₅	Light orange	EtOH	220–226	86	0.9
K₁₆	Earth yellow	EtOH	140–142	54	0.6
K₁₇	Drab	EtOH	138–140	88	0.7
K₁₈	Dark orange	EtOH	112–116	65	0.7
K₁₉	Light orange	EtOH	118–120	60	0.6
K₂₀	Crimson	EtOH	180–184	66	0.8
K₂₁	Dark orange	EtOH	226–228	59	0.7
K₂₂	Light brown	EtOH	123–126	44	0.8
K₂₃	Orange	EtOH	128–130	56	0.7
K₂₄	Brown	EtOH	113–116	71	0.6
K₂₅	Dark brown	EtOH	206–210	71	0.6

Structural characterization was done by using IR, mass and H NMR. Representative results of some proto type compounds in relation to aliphatic, aromatic, cyclic and heterocyclic nature of reactant are as follows:

2.5

2.5 Compound code

2.5.1

2.5.1 K₂: – 4(1-propane 2 one) propane-N-methylamine thiosemicarbazide

IR data for said compound are C–H stretching at 2934.14 cm⁻¹, N–H stretching at 3256.34 cm⁻¹, C⚌S streatching at 1255.19 cm⁻¹, C⚌N stretching at 1587.47 cm⁻¹, and CH₂–CH₂ at 2931.93 cm⁻¹.

NMR Data:-¹H-NMR (DMSO-d₆) δ ppm: 1.2–1.4 (m, 6H, CH₂), 2.2 (3H, CH₃), 2.683 (6H, –N(CH₃)₂), 4.939 (s, 1H, NH). MS (m/z): 165 (M+), [C₈H₁₆N₃S-162].

2.5.2

2.5.2 K₁₄ – 4(1-phenylethanone) propane-N-ethylamine thiosemicarbazide

IR data for said compound are C–H stretching at 2924.44 cm⁻¹, N–H stretching at 3239.85 cm⁻¹, C⚌S stretching at 1239.47 cm⁻¹, C⚌N stretching at 1597.67 cm⁻¹, CH₂–CH₂ at 2938.98 cm⁻¹.

NMR Data:-¹H-NMR (DMSO-d₆) δ ppm: 1.2–1.6 (m, 6H, CH₂), 2.8–2.977 (10H, N(C₂H₅)₂), 5.173 (s, 1H, NH), 7.498–8.725 (4H, m, aromatic), MS (m/z): 295 (M+), [C₁₅H₂₃N₄S-291].

2.5.3

2.5.3 K₁₇ – 4(1-propane 2 one) propane-N-tetra hydro-1-4 oxazinel thiosemicarbazide

C⚌N stretching at 1577.87 cm⁻¹, C⚌S stretching at 1235.17 cm⁻¹, N–H stretching at 3246.94 cm⁻¹, CH₂–CH₂ at 2936.93 cm⁻¹.

NMR data:-¹H-NMR (DMSO-d₆) δ ppm : 2.5–2.844 (m, 6H, CH₂), 3.631–3.705 (3H, CH₃), 5.171 (s, 1H, NH), 7.1–8.031 (m, 4H, morpholino proton), MS (m/z): 222 (M−), [C₁₀H₁₄N₃SO-224].

2.5.4

2.5.4 Docking study

Lanosterol 14 α-demethylase is a cytochrome P450 family enzyme that catalyzes the oxidative removal of the 14α-methyl group of lanosterol, an essential step in the production of sterols. Inhibition of this step leads to inhibition of the synthesis of ergosterol, a very essential component of fungal cell membrane. Hence it is a target for antifungal drugs.

CYP34A is also an enzyme from the cytochrome P450 family, which plays a role in fast metabolism of thiols and thioamide type of compounds, inhibition of enzyme activity leads to the presence of active compounds at the site of action for a prolonged period of time.

To confirm the mechanism of antifungal activity of synthesized compounds and study the potential interaction, docking of synthesized compounds was performed on the pdb structure of Lanosterol 14 α-demethylase (CYP51A1, P45014DM) using Vlife MDS 3.5 as target. All synthesized molecules were docked into the same binding site.

Docking study showed a strong hydrophobic interaction between amino acid residues, like Arganine (ARG141), Glutamine (GLU146), Leucine (LEU54), Lycine (LYC227), and Threonine (THR147) with the carbon of ketone, nitrogen of amine and sulfur of thiosemicarbazide at distances of 2.998, 4.193 and 4.354, respectively and with hydrogen of aldehyde at 2.279.

Strong Vander wall’s interactions were observed with the carbon of ketone, nitrogen of amine and sulfur of thiosemicarbazide. The amino acid residues involved are Aspartine ASP175A, Glycine GLY176A, Lysine LYS227A, Arganine ARG141B, Threonine THR147B and Phenyl alanine PHE58A, which might be playing an important role in the selective binding of compounds with target. Figs. 1–3 show interactions between K₂₀, the highest activity compound with target.

Representative interactions shown by K20 with amino acid residues of CYP51A1, P45014DM.

Representative interactions shown by K20 with CYP51A1, P45014DM.

Representative docking of K20 in CYP51A1, P45014DM pocket.

2.6

2.6 Estimation of antifungal activity (Andrews, 2001; Report of the Working, 1991; Silverman, 2004; Williams et al., 2007; Committee, 1997; Winstanley et al., 1994; Lennette et al., 1985; Mac Faddin, 1985; N.C.C.L.S., 1990 )

An anti-fungal activity screening is done to determine the MIC (Minimum inhibitory concentrations), using Brain Heart Infusion(BHI) broth dilution method to estimate MIC of compounds using C. albicans (ATCC Code 25923), A. Niger using Fluconazloe as standard drug in concentration 30 μg/ml.

9 dilutions of each drug were done with BHI for MIC.

In the initial tube 20 microliter of drug was added into the 380 microliter of BHI broth.
For dilution 200 microliter of BHI broth was added into the next 9 tubes separately.
Then from the initial tube 200 microliter was transferred to the first tube containing 200 microliter of BHI broth. This was considered as 10⁻¹ dilution.
From 10⁻¹ diluted tube 200 microliter was transferred to the second tube to make 10⁻² dilution.
The serial dilu tion was repeated up to 10⁻⁹ dilution for each drug.
From the maintained stock cultures of required organisms, 5 microliter was taken and added into 2 ml of BHI (brain heart infusion) broth.
In each serially diluted tube 200 microliter of the above culture suspension was added.
The tubes were incubated for 24 h and observed for turbidity

3 Results

3.1

3.1 Results for synthetic scheme

As mannich bases and thiosemicarbazide individually show varied pharmacological activities, the proposed work was carried out with the intention of synthesis of mannich bases of thiosemicarbazide as mutual prodrugs. In step 1 successful synthesis of mannich bases was done using aldehyde, ketones and amines with aliphatic, aromatic, cyclic and heterocyclic nature. Optimization of reaction conditions for the synthesis of mannich bases with respect to time and temperature had to be done on individual basis. Time required for synthesis, varied from 30 min to 12–14 h, with temperature condition varying from room temperature with mechanical stirring to temperature between 80 and 100 °C on the basis of complexity of the structures of reactants. Percentage yield of synthesized compounds varied from 24% to 88 %. The TLC and spectral analysis done for compounds confirmed successful synthesis of expected compounds.

3.2

3.2 Results related to activity

The major components of the fungal cell wall are chitin, glucans, polysaccharides and glycoproteins. The species specific variations in composition of components exist, which were conformed due to variations seen in activities shown by synthesized compounds in C. albicans and A. niger.

Synthesized compounds were screened for antifungal activity using Brain Heart Infusion (BHI) broth dilution method to estimate minimum inhibitory concentration for synthesized compound using C. albicans and A. niger.

Mannich bases with alkyl components have shown comparable activities in both microorganisms to the standard drug used. Mannich bases of thiosemicarbazide having only alkyl components have shown more or less comparable activity in both C. albicans and A. niger. Compounds with morpholine as amine component have shown greater activity on C. albicans than on A. niger.

Highest activity is shown by the compound with ketone having an unsubstituted aromatic ring, formaldehyde/paraformaldehyde and morpholine as amine component.

In compounds with aldehyde other than formaldehyde/paraformaldehyde when antifungal activity is concerned they show lesser activity. Results of activity are mentioned in Table 3.

Table 3 Anti-fungal activity of synthesized compounds.

Sr. No	Product code	Activity on C. albicans MIC (μg/ml)	Activity on Apergillus niger. MIC (μg/ml)
01	K₁	31.25	31.25
02	K₂	31.25	31.25
03	K₃	31.25	31.25
04	K₄	31.25	31.25
05	K₅	31.25	62.5
06	K₆	31.25	62.5
07	K₇	31.25	62.5
08	K₈	31.25	62.5
09	K₉	62.5	62.5
10	K₁₀	31.25	62.5
11	K₁₁	250	31.25
12	K₁₂	62.5	250
13	K₁₃	16.6	62.5
14	K₁₄	16.6	62.5
15	K₁₅	16.6	62.5
16	K₁₆	250	62.5
17	K₁₇	4.0	31.25
18	K₁₈	4.0	16.6
19	K₁₉	16.6	8.3
20	K₂₀	4.0	16.6
21	K₂₁	31.25	62.5
22	K₂₂	16.6	16.6
23	K₂₃	16.6	16.6
24	K₂₄	16.6	16.6
25	K₂₅	16.6	16.6
Std.	Fluconazloe	30	16

3.3

3.3 Results of docking study

For confirmation of antifungal activity, docking study was done on Lanosterol 14 α-demethylase CYP51A1, P45014DM using Vlife MDS 3.5 as target. A low (negative) energy indicates a stable system and thus a likely binding interaction. Negative sign in docking score indicates an association of minimum energy with further emphasis that the orientation of synthesized compounds in the pocket of the said target was proper. Compound K20 showed a docking score of −78.008461 while Original Ligand Score was −71.342323. Docking study showed strong hydrophobic interaction between amino acid residues, like Arganine (ARG141), Glutamine (GLU146), Leucine (LEU54), Lycine (LYC227), and Threonine (THR147) with the carbon of ketone, nitrogen of amine and sulfur of thiosemicarbazide at distances of 2.998, 4.193 and 4.354, respectively and with hydrogen of aldehyde at 2.279. Also strong Vander wall’s interactions were observed with carbon of ketone, nitrogen of amine and sulfur of thiosemicarbazide. The amino acid residues involved are Aspartine ASP175A, Glycine GLY176A, Lysine LYS227A, Arganine ARG141B, Threonine THR147B and Phenylalanine PHE58A, which might be playing an important role in selective binding of compounds with target.

The overall results of synthesis, docking study and activity leads to conformation that the protocol of plane of work to synthesis of mannich bases of thiosemicarbazide as novel mutual prodrug was successful.

4 Discussion

Complexity of aldehyde, ketones and amines played a significant role in the optimization of time, temperature and % yield of synthesized compounds on individual basis. It was observed that in the presence of formaldehyde or paraformaldehyde as aldehyde, reaction goes to completion at a faster rate with better yield of end product.

Screening of compounds for antifungal activity led to the following observations,

Mannich bases of thiosemicarbazide having only alkyl components have shown more or less comparable activity in both C. albicans and A. niger.
Acyl derivatives are found to be partly active.
Mannich bases of Thiosemicarbazides formed from aliphatic carbonyl compounds showed good antifungal activity against C. albicans compared to A. niger.
Use of unsubstituted aromatic components in the synthesis of mannich bases of thiosemicarbazide gave active derivatives, but compounds with 3- and 4-substituted aromatic ring showed to be less active.
Highest activity was shown by thiosemcarbazides with morpholine as amine and ketone component having aromatic nature.
Mannich bases of thiosemicarbazide with aromatic aldehyde showed somewhat lesser activity than the thiosemicarbazone with aromatic ketones
Mannich bases of thiosemicarbazide with heterocyclic aldehydes showed comparable activity to alkyl derivatives.

In docking study specific structural features like the carbon of ketone, nitrogen of amine and sulfur of thiosemicarbazide present in compounds were found to be responsible for strong interactions like strong hydrophobic, Vander wall’s interactions and hydrogen bonding with amino acid residues from chosen target. The result obtained conform the mechanism of antifungal activity shown by the synthesized compounds.

Acknowledgements

Authors wish to acknowledge Dr. Kishore G. Bhat, HOD. Of Microbiology from the Maratha Mandal’s Nathajirao G. Halgekar Institute of Dental Science and Research Centre Belgaum for their kind cooperation in allowing us to carry out desired pharmacological activity. Author is also thankful to the Principal, Dr. H.N. More of the Bharati Vidyapeeth College of Pharmacy, Kolhapur for providing excellent facilities to carry out synthetic work.

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Introduction

Methods and materials

Step-1 synthesis of mannich base
Procedure
Step-I1 synthesis of mannich bases of thiosemicarbazide
Characterization
Compound code
Estimation of antifungal activity (Andrews, 2001; Report of the Working, 1991; Silverman, 2004; Williams et al., 2007; Committee, 1997; Winstanley et al., 1994; Lennette et al., 1985; Mac Faddin, 1985; N.C.C.L.S., 1990)

Results

Results for synthetic scheme
Results related to activity
Results of docking study

Discussion

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[1] Andrews Jennifer M., . Determination of minimum inhibitory concentrations. J. Antimicrob. Chemother.. 2001;48 (5–16 © 2001, UK)
[Google Scholar]

[2] Bhosle D., Bharambe S., Gairola N., Dhaneshwar S.S., . Mutual prodrug concept: fundamentals and applications. Indian J. Pharm. Sci.. 2006;68:286-294.
[Google Scholar]

[3] National Committee for Clinical Laboratory Standards, . Specialty Collection: Susceptibility Testing. Wayne, PA: NCCLS; 1997. (SC21-L, M7-A4)

[4] Denning D.W., . Epidemiology and pathogenesis of systemic fungal infections in the immunocompromised host. J. Antimicrob. Chemother.. 1991;28(Suppl. B):1-6.
[Google Scholar]

[5] Kitchen D.B., Decornez H., Furr J.R., Bajorath J., . Docking and scoring in virtual screening for drug discovery: methods and applications. Nat. Rev. Drug Discovery. 2004;3(11):935-949.
[CrossRef] [PubMed] [Google Scholar]

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Synthesis, docking and in-vitro screening of mannich bases of thiosemicarbazide for anti-fungal activity