1 s2.0-s1878535213003560-main

Arabian Journal of Chemistry (2013) xxx, xxx–xxx

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sawww.sciencedirect.com

ORIGINAL ARTICLE

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

Received 14 December 2012; accepted 21 October 2013

*

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KEYWORDS

Antifungal;

Brain heart infusion;

Mannich bases;

Thiosemicarbazide

Corresponding author. Te

23751341; fax: +91 231 263

-mail address: sachin_pishaw

er review under responsibilit

Production an

78-5352 Crown Copyright ªtp://dx.doi.org/10.1016/j.arab

lease cite this article in priosemicarbazide for anti-

l.: +91

8833.

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2013 Pu

jc.2013.1

ess as: Pfungal a

Abstract Mannich bases and thiosemicarbazide individually show antimicrobial, antifungal, anti-

convulsant, 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 ali-

phatic, 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 a-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.Crown Copyright ª 2013 Published by Elsevier Ltd. All rights reserved.

231 2637286, mobile: +91

iffmail.com (S.A. Pishawikar).

Saud University.

g by Elsevier

blished by Elsevier Ltd. All rights

0.016

ishawikar, S.A., More, H.N. Sctivity. Arabian Journal of Ch

1. Introduction

Infectious diseases caused by bacteria and fungi affect millionsof people worldwide. Systematic programs to discover and de-velop new antibiotics and antifungals, are the need of the hour

due to considerable extent of development of resistance byorganisms to the existing drugs used against them. Further,the need has arisen largely due to advent of HIV, which has

reserved.

ynthesis, docking and in-vitro screening of mannich bases ofemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.10.016

mailto:[email protected]

http://dx.doi.org/10.1016/j.arabjc.2013.10.016


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CH3R

O

+HR1

O

+ NHR2

R3

HCl

Aldehyde Secondary Amine Hydrochloride

Step 1

R

O

N

R1

R3

R2

HCl

Mannich Base

Step 2NH2

NH

S

NH2

Thiosemicarbazide

R N

NNH

S

NH2R1

R3

R2

Mannich Base of Thiosemicarbazide

2 S.A. Pishawikar, H.N. More

increased the numbers of profoundly and chronically immunesuppressed patients, who are susceptible to both serious inva-sive and superficial fungal infections. The development of new

agents may provide additional options for the treatment offungal infections and may help to overcome the limitationsof current treatments (Denning, 1991).

By definition, mutual prodrugs are carrier-linked prodrugsconsisting of two pharmacologically active agents coupled to-gether so that each acts as a promoiety for the other agent and

vice versa. Individually mannich bases and thiosemicarbazideshow varied pharmacological activities such as, anticancer,antimicrobial, antifungal, anticonvulsant, antimalarial, analge-sic and anti-inflammatory. Present work has used the concept

of mutual prodrug to synthesize mannich bases of thiosemicar-bazide as mutual prodrug and screening them as anti-fungalagents (Bhosle et al., 2006; Pandeya et al., 1999, 2003).

The mannich base is an end product in the mannichreaction, which is nucleophilic addition reaction of a non-enol-izable 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 synthe-sis of a number of mannich bases was done using aldehyde,ketones and amines with aliphatic, aromatic, cyclic and hetero-cyclic nature. In step-2, synthesized mannich bases were con-

densed with thiosemicarbazide to form mannich bases ofthiosemicarbazide (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 identi-

fication to lead optimization. One key methodology is dockingof small molecules to protein binding sites, pioneered duringthe early 1980s. The docking process involves the prediction

of ligand conformation and orientation (or posing) within atargeted binding site. The two aims of docking studies areaccurate structural modeling and correct prediction of activity.Docking studies have become a scientific approach for the

study of macromolecular structures and interactions. Macro-molecular modeling by docking studies provides most detailedpossible view of drug–receptor interaction and has created a

new rational approach to drug design, where the structure ofdrug is designed based on its fit to three dimensional structuresof a receptor site. On the basis of docking scores one can pre-

dict the amount of activity that will be shown by compounds.To conform the mechanism of antifungal activity of synthe-sized compounds, docking of synthesized compounds was per-formed on the structure of Lanosterol 14 a-demethylase

(CYP51A1, P45014DM) an enzyme from cytochrome P450family that catalyzes the oxidative removal of the 14a-methylgroup of lanosterol, an essential step in the production of

sterols. Inhibition of this step leads to inhibition of synthesisof ergosterol, a very essential component of fungal cell mem-brane. 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 dilu-tion method using Candida albicans (ATCC Code 10231),Apergillus niger. (ATCC Code 16404) and using concentrationof Fluconazloe (30 lg/ml) as standard drug (Andrews, 2001;

Please cite this article in press as: Pishawikar, S.A., More, H.N. Sthiosemicarbazide for anti-fungal activity. Arabian Journal of Ch

Report of the Working, 1991; Silverman, 2004; Williamset al., 2007; Committee, 1997; Winstanley et al., 1994; Lennetteet al., 1985).

2. Methods and materials

Scheme of Synthesis:-

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 ofhydrochloride salt and 1.5–2.0 molecular equivalent ofaldehyde.

2.2. Procedure

Amine was taken in a flat bottom flask and converted into

hydrochloride salt using concentrated hydrochloric acid, for-mation 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 heat-ing on water bath was done.

Optimization of reaction conditions with respect to time

and temperature was done on individual basis for each reac-tion. Time required varied from 30 min to 12–14 h, with tem-perature conditions varying from room temperature withmechanical stirring, to heating on water bath at temperature



Table 1 Synthesized compounds

R N

NNH

S

NH2R1

R3

R2

Mannich Base of Thiosemicarbazi

.

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

OCH3CH2 CH3CH2CH2 CH3CH2CH2

07 K7

O ClC2H5 C2H5

08 K8 CH3 CH3CH2CH2 CH3CH2CH2

09 K9 CH3

OCH3CH2CH2 CH3CH2CH2

10 K10 CH3

OCH3CH2CH2 CH3CH2CH2

11 K11

OCH3

CH3CH2CH2 CH2CH2OH CH2CH2OH

12 K12

OCH3CH2 C2H5 C2H5

13 K13 O2NH C2H5 C2H5

14 K14 H C2H5 C2H5

15 K15 O H C2H5 C2H5

16 K16 CH3

ClC2H5 C2H5

17 K17

Cl

O

H C2H5 C2H5

Synthesis, docking and in-vitro screening of mannich bases of thiosemicarbazide for anti-fungal activity 3

Please cite this article in press as: Pishawikar, S.A., More, H.N. Synthesis, docking and in-vitro screening of mannich bases ofthiosemicarbazide for anti-fungal activity. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.10.016


Table 1 (continued)

Sr. No. Code R R1 R2 R3

18 K18 CH3 HO N

19 K19 CH3CH2CH2 HO N

20 K20 HO N

21 K21 O HO N

22 K22 CH3

C l O N

23 K23 CH3

O O N

24 K24

OH

O N

25 K25

CH CH

HO N

Table 2 Physicochemical data of synthesized compounds.

Code of compound Color Solubility M.P. (�C) % Yield Rf 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


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


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


Please cite this article in press as: Pishawikar, S.A., More, H.N. Synthesis, docking and in-vitro screening of mannich bases ofthiosemicarbazide for anti-fungal activity. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.10.016



between 80 and 100 �C depending upon the complexity ofreactants.

R CH3

O

+R1

H

O

+ NH.Hcl R

O

N

R1

R3

R2.HCl

SecondaryAmine

Mannich BaseKetone Aldehyde

2.3. Step-I1 synthesis of mannich bases of thiosemicarbazide

Simple condensation reaction was carried out between synthe-sized mannich bases and one mole quantity of thiosemicarba-zide in the presence of alcohol as solvent with refluxation on

boiling water bath for around half an hour to form mannichbases of thiosemicarbazide.

R

O

N

R1R2

R3. Hcl + H2N

NHNH2

S

R N

N

R2

R3

NH

NH2S

Mannich Base Thiosemicarbazide Mannich Base Of Thiosemicarbazide

Synthesized compounds are shown in Table 1.

2.4. Characterization

Physicochemical characterization of the synthesized com-pounds was done by the estimation of melting point and Rf

values by TLC. Results are mentioned in Table 2.Structural characterization was done by using IR, mass

and H NMR. Representative results of some proto type

Figure 1 Representative interactions shown by K20 w


compounds in relation to aliphatic, aromatic, cyclic and het-erocyclic nature of reactant are as follows:

2.5. Compound code

2.5.1. K2: – 4(1-propane 2 one) propane-N-methylaminethiosemicarbazide

IRdata for said compoundareC–Hstretchingat2934.14 cm�1,N–H stretching at 3256.34 cm�1, C‚S streatching at 1255.19 cm�1,

C‚Nstretching at 1587.47 cm�1, andCH2–CH2 at 2931.93 cm�1.NMR Data:-1H-NMR (DMSO-d6) d 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. K14 – 4(1-phenylethanone) propane-N-ethylaminethiosemicarbazide

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 at1239.47 cm�1, C‚N stretching at 1597.67 cm�1, CH2–CH2

at 2938.98 cm�1.

NMR Data:-1H-NMR (DMSO-d6) d 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. K17 – 4(1-propane 2 one) propane-N-tetra hydro-1-4oxazinel 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 at2936.93 cm�1.

NMR data:-1H-NMR (DMSO-d6) d 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].

ith amino acid residues of CYP51A1, P45014DM.




2.5.4. Docking study

Lanosterol 14 a-demethylase is a cytochrome P450 family en-

zyme that catalyzes the oxidative removal of the 14a-methylgroup of lanosterol, an essential step in the production of ster-ols. Inhibition of this step leads to inhibition of the synthesis of

Figure 3 Representative docking of K

Figure 2 Representative interactions show


ergosterol, a very essential component of fungal cell mem-brane. 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 andthioamide type of compounds, inhibition of enzyme activity

20 in CYP51A1, P45014DM pocket.

n by K20 with CYP51A1, P45014DM.




leads to the presence of active compounds at the site of actionfor a prolonged period of time.

To confirm the mechanism of antifungal activity of synthe-

sized compounds and study the potential interaction, dockingof synthesized compounds was performed on the pdb structureof Lanosterol 14 a-demethylase (CYP51A1, P45014DM) using

Vlife MDS 3.5 as target. All synthesized molecules weredocked into the same binding site.

Docking study showed a strong hydrophobic interaction be-

tween amino acid residues, likeArganine (ARG141),Glutamine(GLU146), Leucine (LEU54), Lycine (LYC227), andThreonine(THR147) with the carbon of ketone, nitrogen of amine and sul-fur 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 thiosemicar-

bazide. The amino acid residues involved are AspartineASP175A, Glycine GLY176A, Lysine LYS227A, ArganineARG141B, Threonine THR147B and Phenyl alanine PHE58A,

which might be playing an important role in the selective bind-ing of compounds with target. Figs. 1–3 show interactions be-tween K20, the highest activity compound with target.

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 theMIC (Minimum inhibitory concentrations), using Brain

Heart Infusion(BHI) broth dilution method to estimateMIC of compounds using C. albicans (ATCC Code

Table 3 Anti-fungal activity of synthesized compounds.

Sr. No Product code Activity on C. albicans M

01 K1 31.25

02 K2 31.25

03 K3 31.25

04 K4 31.25

05 K5 31.25

06 K6 31.25

07 K7 31.25

08 K8 31.25

09 K9 62.5

10 K10 31.25

11 K11 250

12 K12 62.5

13 K13 16.6

14 K14 16.6

15 K15 16.6

16 K16 250

17 K17 4.0

18 K18 4.0

19 K19 16.6

20 K20 4.0

21 K21 31.25

22 K22 16.6

23 K23 16.6

24 K24 16.6

25 K25 16.6

Std. Fluconazloe 30


25923), A. Niger using Fluconazloe as standard drug in con-centration 30 lg/ml.

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

1. In the initial tube 20 microliter of drug was added into the380 microliter of BHI broth.

2. For dilution 200 microliter of BHI broth was added intothe next 9 tubes separately.

3. Then from the initial tube 200 microliter was transferred to

the first tube containing 200 microliter of BHI broth. Thiswas considered as 10�1 dilution.

4. From 10�1 diluted tube 200 microliter was transferred tothe second tube to make 10�2 dilution.

5. The serial dilution was repeated up to 10�9 dilution foreach drug.

6. From the maintained stock cultures of required organisms,

5 microliter was taken and added into 2 ml of BHI (brainheart infusion) broth.

7. In each serially diluted tube 200 microliter of the above cul-

ture suspension was added.8. The tubes were incubated for 24 h and observed for

turbidity

3. Results

3.1. Results for synthetic scheme

As mannich bases and thiosemicarbazide individually showvaried pharmacological activities, the proposed work was car-ried out with the intention of synthesis of mannich bases of thi-

osemicarbazide as mutual prodrugs. In step 1 successfulsynthesis of mannich bases was done using aldehyde, ketones

IC (lg/ml) Activity on Apergillus niger. MIC (lg/ml)

31.25

31.25

31.25

31.25

62.5

62.5

62.5

62.5

62.5

62.5

31.25

250

62.5

62.5

62.5

62.5

31.25

16.6

8.3

16.6

62.5

16.6

16.6

16.6

16.6

16




and amines with aliphatic, aromatic, cyclic and heterocyclicnature. Optimization of reaction conditions for the synthesisof 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 conditionvarying from room temperature with mechanical stirring to

temperature between 80 and 100 �C on the basis of complexityof the structures of reactants. Percentage yield of synthesizedcompounds varied from 24% to 88 %. The TLC and spectral

analysis done for compounds confirmed successful synthesis ofexpected compounds.

3.2. Results related to activity

The major components of the fungal cell wall are chitin, glu-cans, polysaccharides and glycoproteins. The species specificvariations in composition of components exist, which were

conformed due to variations seen in activities shown by syn-thesized compounds in C. albicans and A. niger.

Synthesized compounds were screened for antifungal activ-

ity using Brain Heart Infusion (BHI) broth dilution method toestimate minimum inhibitory concentration for synthesizedcompound using C. albicans and A. niger.

Mannich bases with alkyl components have shown compa-rable activities in both microorganisms to the standard drugused. Mannich bases of thiosemicarbazide having only alkylcomponents have shown more or less comparable activity in

both C. albicans and A. niger. Compounds with morpholineas amine component have shown greater activity on C. albi-cans than on A. niger.

Highest activity is shown by the compound with ketonehaving an unsubstituted aromatic ring, formaldehyde/parafor-maldehyde and morpholine as amine component.

In compoundswith aldehyde other than formaldehyde/para-formaldehyde when antifungal activity is concerned they showlesser activity. Results of activity are mentioned in Table 3.

3.3. Results of docking study

For confirmation of antifungal activity, docking study wasdone on Lanosterol 14 a-demethylase CYP51A1, P45014DM

using Vlife MDS 3.5 as target. A low (negative) energy indi-cates 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 orientationof synthesized compounds in the pocket of the said targetwas proper. Compound K20 showed a docking score of

�78.008461 while Original Ligand Score was �71.342323.Docking study showed strong hydrophobic interaction be-tween amino acid residues, like Arganine (ARG141), Gluta-

mine (GLU146), Leucine (LEU54), Lycine (LYC227), andThreonine (THR147) with the carbon of ketone, nitrogen ofamine 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 observedwith carbon of ketone, nitrogen of amine and sulfur of thio-semicarbazide. The amino acid residues involved are Aspartine

ASP175A, Glycine GLY176A, Lysine LYS227A, ArganineARG141B, 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 activityleads to conformation that the protocol of plane of work tosynthesis of mannich bases of thiosemicarbazide as novel mu-

tual prodrug was successful.

4. Discussion

Complexity of aldehyde, ketones and amines played a signifi-cant role in the optimization of time, temperature and % yieldof synthesized compounds on individual basis. It was observed

that in the presence of formaldehyde or paraformaldehyde asaldehyde, reaction goes to completion at a faster rate with bet-ter yield of end product.

Screening of compounds for antifungal activity led to thefollowing observations,

(1) Mannich bases of thiosemicarbazide having only alkylcomponents have shown more or less comparable activ-ity in both C. albicans and A. niger.

(2) Acyl derivatives are found to be partly active.

(3) Mannich bases of Thiosemicarbazides formed from ali-phatic carbonyl compounds showed good antifungalactivity against C. albicans compared to A. niger.

(4) Use of unsubstituted aromatic components in the syn-thesis of mannich bases of thiosemicarbazide gave activederivatives, but compounds with 3- and 4-substituted

aromatic ring showed to be less active.(5) Highest activity was shown by thiosemcarbazides with

morpholine as amine and ketone component having aro-matic nature.

(6) Mannich bases of thiosemicarbazide with aromatic alde-hyde showed somewhat lesser activity than the thiosem-icarbazone with aromatic ketones

(7) Mannich bases of thiosemicarbazide with heterocyclicaldehydes showed comparable activity to alkyl derivatives.

In docking study specific structural features like the carbonof ketone, nitrogen of amine and sulfur of thiosemicarbazidepresent in compounds were found to be responsible for strong

interactions like strong hydrophobic, Vander wall’s interac-tions and hydrogen bonding with amino acid residues fromchosen target. The result obtained conform the mechanismof antifungal activity shown by the synthesized compounds.

Acknowledgements

Authors wish to acknowledge Dr. Kishore G. Bhat, HOD. OfMicrobiology from the Maratha Mandal’s Nathajirao G. Hal-

gekar Institute of Dental Science and Research Centre Bel-gaum for their kind cooperation in allowing us to carry outdesired pharmacological activity. Author is also thankful tothe Principal, Dr. H.N. More of the Bharati Vidyapeeth Col-

lege of Pharmacy, Kolhapur for providing excellent facilitiesto carry out synthetic work.

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http://dx.doi.org/10.1038/nrd1549.PMID15520816

http://dx.doi.org/10.1038/nrd1549.PMID15520816

http://dx.doi.org/10.1016/S0959-440X(96)80061-3.PMID8804827

http://dx.doi.org/10.1016/S0959-440X(96)80061-3.PMID8804827




































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