Synthesis, antifungal activity and docking study of 2-amino-4H-benzochromene-3-carbonitrile derivatives

Accepted Manuscript

Synthesis, antifungal activity and docking study of 2-amino-4H-benzochromene-3-

carbonitrile derivatives

B.B.F. Mirjalili, L. Zamani, K. Zomorodian, S. Khabnadideh, Z. Haghighijoo, Z.

Malakotikhah, S.A. Ayatollahi Mousavi, S. Khojasteh

PII:

S0022-2860(16)30197-1

10.1016/j.molstruc.2016.03.002

MOLSTR 22312

DOI:

Reference:

To appear in: Journal of Molecular Structure

Received Date: 6 January 2016

Revised Date: 1 March 2016

Accepted Date: 1 March 2016

Please cite this article as: B.B.F. Mirjalili, L. Zamani, K. Zomorodian, S. Khabnadideh, Z. Haghighijoo,

Z. Malakotikhah, S.A. Ayatollahi Mousavi, S. Khojasteh, Synthesis, antifungal activity and docking study

of 2-amino-4H-benzochromene-3-carbonitrile derivatives, Journal of Molecular Structure (2016), doi:

10.1016/j.molstruc.2016.03.002.

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ACCEPTED MANUSCRIPT

Synthesis, antifungal activity and docking study of 2-amino-4H-benzochromene-3-

carbonitrile derivatives

B. B. F. Mirjalili^a, L. Zamani^b*, K. Zomorodian^c, S. Khabnadideh^b, Z. Haghighijoo^d, Z. Malakotikhah^a, S. A.

Ayatollahi Mousavi^e, S. Khojasteh^e

^aDepartment of Chemistry, College of Science, Yazd University, Yazd, P. O. Box 8915813149, Iran.

^bDepartment of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shiraz

University of Medical Sciences, Shiraz, Iran

^cCenter of Basic Researches in Infectious Diseases and Department of Medical Mycology and Parasitology School

of Medicine, Shiraz University of Medical Sciences, Post code 71348-45794, Shiraz, Iran.

^dMedicinal & Natural Products Chemistry Research Center, Shiraz University of Medical Science, Shiraz-Iran

^eDepartment of Medical Mycology and Parasitology, Kerman University of Medical Sciences, Kerman, Iran.

*Corresponding Author:

Leila Zamani, PhD of Organic Chemistry

Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research

Center, Shiraz University of Medical Sciences, Shiraz, I.R.Iran

.

Tel: (+98) 7132425403; Fax: (+98) 7132425403, E-mails: L.Zamani2008@gmail.com

ABSTRACT:

Pathogenic fungi are associated with diseases ranging from simple dermatosis to life-threatening

infections, particularly in immunocompromised patients. During the past two decades, resistance to

established antifungal drugs has increased dramatically and has made it crucial to identify novel

antimicrobial compounds.

Here, we selected 12 new compounds of 2-amino-4H-benzochromene-3-carbonitrile drivetives (C1-C12)

for synthesis by using nano-TiCl₄.SiO₂as efficient and green catalyst, then nine of synthetic compounds

were evaluated against different species of fungi, positive gram and negative gram of bacteria. Standard

and clinical strains of antibiotics sensitive and resistant fungi and bacteria were cultured in appropriate

1

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media. Biological activity of the 2-amino-4H-benzochromene-3-carbonitrile derivatives against fungi and

bacteries were estimated by the broth micro-dilution method as recommended by clinical and laboratory

standard institute (CLSI). In addition minimal fangicidal and bactericial concenteration of the compounds

were also determined.

Considering our results showed that compound 2-amino-4-(4-methyl benzoate)-4H-benzo[f]chromen-3-

carbonitrile (C9) had the most antifungal activity against Aspergillus clavatus, Candida glabarata,

Candida dubliniensis, Candida albicans and Candida tropicalis at concentrations ranging from 8 to ≤128

µg/mL. Also compounds 2-amino-4-(3,4-dimethoxyphenyl)-4H-benzo[f]chromen-3-carbonitrile (C4) and

2-amino-4-(4-isopropylphenyl)-4H-benzo[f]chromen-3-carbonitrile (C3) had significant inhibitory

activities against Epidermophyton floccosum following 2-amino-4-(4-methylbenzoate)-4H-

benzo[f]chromen-3-carbonitrile (C9), respectively.

Docking simulation was performed to insert compounds C3, C4 and C9 in to CYP51 active site to

determine the probable binding model.

Keywords: 2-Amino-4H-benzochromenes; Nano-TiCl₄.SiO₂; Docking study; Synthesis; Antifungal;

Antibacterial.

1. Introduction

During the past two decades, resistance to established antimicrobial drugs has increased

dramatically and it is serious public health problem in a wide range of infectious disease [1-3].

These resistant strains cause failure in treatment and enhance mortality risks, and sometimes

contribute to complications. Unlike antibacterial antibiotics, the variety of antifungal drugs is

restricted due to the similarity of structure and metabolism of eukaryotic fungal cells to those of

mammalian cells. Hence, the discovery of antifungal agents that possess selective toxicity against

the eukaryotic fungal cell remains an important scientific challenge. Considering the limited

diversity of antifungal agents and recent resistance of fungi to the known antifungal drugs, the

development of new bioactive compounds effective against resistant strains is highly needed. In

spite of a large number of antibiotics and chemotherapeutics available for medical use, the

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antimicrobial resistance created substantial medical need for new classes of antimicrobial agents.

Design and synthesis of newer antimicrobials will always remain an area of immense significance

[4-5]. Among the important pharmacophores responsible, the chromene moiety is an important

structural shape of many synthetic compounds of biological and pharmaceutical interest such as as

cytotoxicity[6], antioxidant[7], antiplasmodial [8], antimalarial [9], antirhinovirus [10], antifungal [11]

and antibacterial [12].

A particularly interesting group of chromenes are 2-amino-4H-benzo[f]chromenes which are

generally prepared by reaction of malononitrile, aldehydes and naphthols in one-pot

procedure.This multicomponent reaction has been catalyzed with K₂CO₃[13], Mg/Alhydrotalcite

[14], nano-sized magnesium oxide [15], Preyssler heteropolyacid [16], basic alumina [17],

potassium phosphate tribasic trihydrate [18], cetyltrimethylammonium bromide (CTABr) [19],

sodium carbonate [20], DBU [21], 3-butyl-1-methylimidazolium hexafluorophosphate, 1-methyl

imidazoliumiodide [mim]Cl [22], Na₂CO₃[23] and tetrabutylammoniumbromide (TBABr) [24].

However, each method has certain restrictions with regards to scope and reaction conditions; for

example, longer reaction times, purification problems and harsh reaction conditions. To avoid

these limitations, our studies towards the development of more capable methods accompanied

with higher yields for the synthesis of 2-amino-4H-benzochromene-3-carbonitrile in the presence

of nano-TiCl₄.SiO₂. We previously described the design and synthesis of some organic reactions

in princess of solid acid catalysts as a green, cheap and efficient technique [25-30].

Also, some of the synthesized compounds were evaluated for their antifungal and antibacterial

activity.

The antibacterial activities of the above compounds were compared to Ampicillin as positive

control and the antifungal activities of them were evaluated to Fluconazole.

2. Materials and Methods

2.1. Chemistry

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The chemicals were purchased from Merck and used without any additional purification. The products

1

were characterized by FT-IR (ATR), H-NMR, and a comparison of their physical properties with those

reported in the literature. FT-IR (ATR) spectra were acquired on a Bruker, Eqinox 55 spectrometer. A

1

Bruker (DRX-400 Avance) nmr was used to record the H NMR spectra. Spectrophotometer (UV/Vis

biotek model UVIKONXL), Melting points were determined with a Thermo Scientific Electrothermal

digital apparatus (Thermo Fisher Scientific Inc.), Gass Chromatography Mass Mass Spectrometery

(Agilent 7000 Series Triple Quad -MS, Made in USA).

2.2. Docking studies

Molecular vina docking studies were performed using PYRX software [Wolf LK, Chem Eng News. 2009

87: 31], the complex of enzyme Mycobacterium tuberculosis-CYP51 with Fluconazole (PDB ID: 1EA1)

was obtained from Protein Data Bank (http://www.rcsb.org), Water molecules and cognate ligand were

removed from the receptor. The chemical structures were drawn into computer using HyperChem

software (Version 7, Hypercube Inc), then the semi-empirical AM1 method was used for geometry

optimization and saved in pdb file format. Binding mode figures were generated with PYMOL.3.3.

General procedure for synthesis of 2-amino-4H-benzochromene-3-carbonitrile

A mixture of aldehyde (1mmol), malononitrile (1mmole), naphthol (1mmol) and nano-TiCl₄.SiO₂

(0.1 g) was stirred at 90 °C under solvent–free conditions. The progress of the reaction was

monitored by thin layer chromatography (TLC). After completion of the reaction, the reaction

mixture was dissolved to CHCl₃and filtered. The catalyst residue was washed with acetone and

reused. The product was triturated with 2 ml of cooled ethanol to give the solid product. The

spectroscopy of the pure products has been shown in Table 1.

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Table 1

Spectral data of 2-amino-4H-benzochromene-3-carbonitrile compounds

X

4'

5'

4'

3'

2'

5'

6'

3'

2'

9

1'

4

6'

8

5

1'

12

10

14

3

NC

6

1

2

CN

14

7

11

7

8

13

6

2

11

O

1

H₂N

13

10

12

3

NH₂

O

4

5

9

(C1, C2, C3, C4, C6, C7, C8, C9, C10)

Spectral data

( C5, C11, C12)

Compound

Mp (^ᵒC)

Found Reported^[ref]

273-274 273-275^[20]

FT-IR (neat, ATR) = 3429, 3336 (stretch, NH₂), 2181 (stretch, CN), 1638 (bending, NH₂), 1408, 1588 (stretch,

C1

1

C=C), 1261, 1024 ( stretch, C-O) cm^-1, H NMR (400 MHz, CDCl₃), δ (ppm) = 7.8 (m, 2H_7,10), 7.7 (d, J = 7.2

Hz, 1H₆), 7.4 (m, 2H_8,9), 7.29 (m, 1H_4’), 7.26 (m, 2H_3’,5’), 7.20 (d, J= 7.2 Hz, 3H_{2’, 6’, 5}), 5.26 (s, 1H₁), 4.57 (s,

2H, NH₂), MS: (m/z, %), 297 (M⁺, 10), 221 (100).

217-219

-

FT-IR (neat ATR) = 3463, 3353 (stretch, NH₂), 2189 (stretch, CN), 1656 (bending, NH₂), 1588, 1410 (stretch,

C2

1

C=C),1526, 1348 (stretch, N=O), 1216, 1028 ( stretch, C-O) cm^-1, H NMR (400 MHz, CDCl₃), δ (ppm) = 8.1

(s br, 1H_2’), 8.08 (d, J = 8 Hz, 1H_4’), 7.99 (d, J = 8.8 Hz, 1H₆), 7.9 (m, 2H_7,10), 7.6 (t, J = 8, 1H_5’), 7.4 (d, J= 7.6

Hz, 1H_6’), 7.4 (m, 2H_8,9), 7.38 (d, J = 9.2, 1H₅), 6.42 (s, 2H, NH₂), 5.6 (s, 1H₁), MS: (m/z, %), 343 (M⁺, 15), 221

(100).

217-219

190-192

258-263

-

FT-IR (neat ATR) = 3423, 3335 (stretch, NH₂), 2190 (stretch, CN), 1647 (bending, NH₂), 1589, 1412 (stretch,

C3

C4

C5

C6

C7

C8

C9

1

C=C), 1233, 1038 ( stretch, C-O) cm^-1, H NMR (400 MHz, CDCl₃), δ (ppm) = 7.81(d, J=8.4 Hz, 2H_7,10), 7.72

(m, 1H₆), 7.4 (m, 2H_8,9), 7.26 (m, 1H₅), 7 (s br, 4H_{2’,3’.5’,6’}), 5.23 (s, 1H₁), 4.55 (s, 2H, NH₂), 2.83 (sept. J=7.2

Hz, 1H_isopropyl), 1.18 (d, J=7 Hz, 6H_isopropyl), MS: (m/z, %), 337 (30), 91 (100).

FT-IR (neat ATR) = 3428, 3333 (stretch, NH₂), 2185 (stretch, CN), 1648 (bending, NH₂), 1589, 1403 (stretch,

1

C=C), 1260, 1231, 1134, 1017 (stretch, C-O) cm^-1, H NMR (400 MHz, CDCl₃), δ (ppm) = 7.8 (d, J = 8.4,

2H_7,10), 7.7 (m, 1H₆), 7.4 (m, 2H_8,9), 7.25 (d, J = 9.6 Hz,1H₅), 6.7 (d, J = 6.8, 2H_5’,6’), 6.66 (d, J= 8.4 Hz, 1H_2’),

5.21 (s, 1H₁), 4.6 (s, 2H, NH₂), 3.80 (s, 3H _methoxy), 3.78 (s, 3H_methoxy), MS: (m/z, %), 358 (M⁺, 25), 221 (100).

FT-IR (Neat) = 3401(stretch, NH₂), 2194 (stretch, CN), 1710 (stretch, C=O), 1664 (bending, NH₂), 1606,

1

1411(stretch, C=C), 1280, 1106 (stretch, C-O of ester), 1189, 1029 (stretch, C-O) cm^-1, H NMR (400 MHz,

CDCl₃), δ (ppm) = 7.9 (d, J=8.4 Hz, 2H_3’,5’), 7.8 (m, 2H_7,10), 7.6 (m, 1H₆), 7.4 (m, 2H_8,9), 7.27 (t, J=2.8 Hz,

3H_2’,6’,5), 5.3 (s, 1H₄), 4.65 (s, 2H, NH₂), 3.87 (s, 3H_methy), MS: (m/z, %), 357 (M⁺), 314 (60), 221 (100).

238-240 232-234^[21]

271-273 259-261^[20]

FT-IR (neat ATR) = 3461, 3322 (stretch, NH₂), 2200 (stretch, CN), 1658 (bending, NH₂), 1587, 1404 ( stretch,

C=C), 1260, 1045 (stretch, C-O) cm^-1, ¹H NMR (400 MHz, CDCl₃), δ (ppm) = 7.83 (m, 2H_7,10), 7.6 (d, J = 7.2,

1H₆), 7.4 (m, 3H_8,9,5), 7.25 (m, 1H3’), 7.04 (d, J = 8.4, 1H_5’), 6.8 (d, J = 8.4, 1H_6’), 5.84 (s, 1H₁), 4.65 (s, 2H,

NH₂), MS: (m/z, %), 366 (M⁺, 5), 221 (100).

FT-IR (neat ATR) = 3458, 3349 (stretch, NH₂), 2178 (stretch, CN), 1654 (bending, NH₂), 1589, 1409 (stretch,

1

C=C), 1219, 1040 (stretch, C-O), 798 (bending C-H) cm^-1, H NMR (400 MHz, CDCl₃), δ (ppm) = 7.8 (m,

2H_7,10), 7.67 (d, J = 8 Hz, 1H₆), 7.4 (m, 3H_8,9,3’), 7.25 (m, 1H_4’), 7.1 (m, 2H_5’,6’), 6.9 (d, J = 7.2 Hz, 1H₅), 5.9 (s,

1H₁), 4.62 (s, 2H, NH₂), MS: (m/z, %), 332 (M⁺, 15), 221 (100).

256-257

232-235

-

FT-IR (neat ATR) = 3434, 3328 (stretch, NH₂), 2185 (stretch, CN), 1649 (bending, NH₂), 1585, 1408 (stretch,

C=C), 1227, 1024 (stretch, C-O), 798, 724 (bending C-H) cm^-1, ¹H NMR (400 MHz, CDCl₃), δ (ppm) = 7.9 (m,

3H_7,10,6), 7.4 (m, 2H_8,9), 7.3 (d, J = 8.8 Hz, 1H₅), 6.94 (s, 1H_2’), 6.69 (d, J = 8.4 Hz, 1H_6’), 6.6 (d, J = 8 Hz, 1H_5’),

6.14 (s, 2H, NH₂), 5.24 (s, 1H1), MS: (m/z, %), 326 (15), 221 (100).

-

FT-IR (neat ATR) = 3379, 3313 (stretch, NH₂), 2195 (stretch, CN), 1707 (stretch, C=O), 1650 (bending, NH₂),

1590, 1404 (stretch, C=C), 1287, 1187 (stretch, C-O of ester), 1233, 1022 ( stretch, C-O), 726 (bending C-H)

cm^-1,¹H NMR (400 MHz, CDCl₃), δ (ppm) = 7.9 (d, J=8.4 Hz, 2H_3’,5’), 7.8 (m, 2H_7,10), 7.6 (m, 1H₆), 7.4 (m,

2H_8,9), 7.27 (t, J=2.8 Hz, 3H_2’,6’,5), 5.3 (s, 1H₁), 4.65 (s, 2H, NH₂), 3.87 (s, 3H_methy), MS: (m/z, %), 357 (M⁺),

314 (60), 221 (100).

163-164

211-214

-

FT-IR (neat ATR) = 3392, 3346 (stretch, NH₂), 2221(stretch, CN), 1657 bending, NH₂), 1597, 1481 (stretch,

C10

C11

1

C=C), 694 (bending C-H) cm^-1, H NMR (400 MHz, CDCl₃), δ (ppm) = 7.3 (m, 9H_{6,7,8,9,10,3’,4’,5’,6’}), 7.1 (d, J =

6.8 Hz, 1H_2’), 7.0 (d, J = 8 Hz 1H₅), 5.0 (s, 1H₁), 3.8 (s, 2H, NH₂), 3.05 (t, J = 7.6 Hz, 2H_methylene), 2.7 (t, J = 8

Hz, 2H_methylene), MS: (m/z, %), 326 (M⁺, 15), 221 (100).

-

FT-IR (Neat ATR) = 3344 (stretch, NH₂), 2194 (stretch, CN), 1667 1647 (bending, NH₂), 1573, 1398 (stretch,

C=C), 1103 ( stretch, C-O), 797 (bending C-H) cm^-1, ¹H NMR (400 MHz, CDCl₃), δ (ppm) = 8.18 (d, J=8 Hz,

1H₇), 7.79 (d, J=7.6 Hz, 1H₁₀), 7.5(m, 3H_6,8,9), 7.15(s br, 4H_{2’,3’.5’,6’}), 7.06 (d, J=8.4, 1H₅), 4.85 (s, 1H₄), 4.72 (s,

2H, NH₂), 2.87(sept. J=6.8 Hz, 1H_isopropyl), 1.2 (d, J=6.8 Hz, 6H_isopropyl), MS: (m/z, %), 337 (30), 91 (100).

ACCEPTED MANUSCRIPT

222-224

-

FT-IR (Neat ATR) = 3343, 3331(stretch, NH₂), 2195(stretch, CN), 1667(bending, NH₂), 1601, 1404 (stretch,

C12

1

C=C), 1272, 1037 (stretch, C-O), 804(bending C-H) cm^-1, H NMR (400 MHz, CDCl₃), δ (ppm) = 7.9 (m,

3H_7,10,6), 7.4 (m, 2H_8,9), 7.3 (d, J = 8.8 Hz, 1H₅), 6.94 (s, 1H_2’), 6.69 (d, J = 8.4 Hz, 1H_6’), 6.6 (d, J = 8 Hz, 1H_5’),

6.14 (s, 2H, NH₂), 5.24 (s, 1H₄), MS: (m/z, %), 326 (15), 221 (100).

2.3. Determination of antifungal activities

2.3.1. Microorganisms

The antifungal activities of the synthetic compounds against some American Type Culture

Collection (ATCC) strains of fungi, including Aspergillus flavus (ATCC 64025), Aspergillus

fumigatus (ATCC 14110), Aspergillus clavatus, Candida albicans (ATCC 1912), Candida

albicans (ATCC 1905), Candida albicans (SUCC 2303), Candida albicans (SUCC 625), Candida

glabarata (ATCC 2192), Candida glabarata (ATCC 863), Candida glabarata (ATCC 2175),

Candida glabarata (ATCC 2175), Candida dubliniensis (ATCC 8501), Candida dubliniensis

(ATCC 7988), Candida tropicalis (SUCC 194), Candida tropicalis (SUCC 611), Candida

tropicalis (ATCC 750), Cryptococcus neoformance (ATCC 9011), as well as two clinical isolates

of yeasts identified by PCR-RFLP were determined. Moreover, the inhibitory activities of the

mentioned compounds against dermatophytes (Trichophyton rubrum, Microsporum canis and

Epidermophyton flocossum) which were identified by morphological and physiological tests were

also examined in this study. The susceptibility of all clinical isolates of fungi against selected

antibiotics was examined by microdilution and disk diffusion methods [31, 32].

Determination of minimum inhibitory concentration

MICs were determined using the broth microdilution method recommended by the CLSI with some

modifications [31, 32]. Briefly, for determination of antimicrobial activities against fungi, serial dilutions

of the synthetic compounds (1–1024 µg/mL) were prepared in 96-well microtiter plates using RPMI-1640

media (Sigma, St. Louis, MO, USA) buffered with MOPS (Sigma). Stock inoculums were prepared by

suspending three colonies of the examined yeast in 5 mL sterile 0.85% NaCl, and adjusting the turbidity

of the inoculums to 0.5 McFarland standards at 530 nm wavelengths (this yields stock suspension of 1-5

× 106 cells/mL). For moulds (Aspergillus spp. and dermatophytes), conidia were recovered from the 7-

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day old cultures grown on potato dextrose agar by a wetting loop with tween-20. The collected conidia

were transferred in sterile saline and their turbidity was adjusted to OD=0.09-0.11 that yields 0.4-5 × 106

conidia/mL. Working suspension was prepared by making a 1/50 and 1/1000 dilution with RPMI of the

stock suspension for moulds and yeasts, respectively. Working inoculums (0.1 mL) were added to the

microtiter plates, which were incubated in a humid atmosphere at 30ºC for 24–48 h. Uninoculated

medium (200 ꢀL) was included as a sterility control (blank). In addition, growth controls (medium with

inoculums but without antibiotics or the synthetic compounds) were also included. The growth in each

well was compared with that of the growth in the control well.

3. Results and discussion

3.1. Chemistry

Our investigation is based on the development of heterogeneous catalyst for reducing risks to

human and the environment. For finding the best reaction conditions, the reaction of

benzaldehyde, malononitrile and 2-naphthol was examined under various states. According to the

obtained results, the best result was obtained in the presence of nano-TiCl₄.SiO₂(0.1 g) under

solvent free conditions (Table 2, Entry 8).

X

NC

nano-TiCl₄.SiO₂

(E)

OH

O

H₂N

CN

X

O

90 C

CN

H

(E)

O

NH₂

X

Figure 1. Synthesis of 2-amino-4H-benzochromene-3-carbonitrile

Then, scope of synthesis of 2-amino-4

H-benzochromene-3-carbonitrile using a wide range of

aromatic aldehydes was investigated and it can be seen in Table 3 and Figure 1. Clearly, the

reactivity of aldehyde is the key factor for this one-pot protocol. Electron deficient aromatic

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aldehydes produced higher yields of production in a shorter time in comparison to the aldehayes

that are rich in electrons.

Table 2

Synthesis of 2-amino-4-phenyl-4H-benzo[f]chromen-3-carbonitrilein various conditions^a

CN

O

(2)

Catalyst

NC

CN

(E)

O

OH

(E)

O

H₂N

NH₂

H

(1)

(4)

(5)

(3)

Entry

Catalyst

Mol% (g)

ArOH

Condition

Solvent

Product

[Yield(%)][ref]

4 [92%][23]

5 [100%][20]

4 [94%][24]

4[92%][13]

5 [80%][19]

4 [84%][14]

4 [96%][17]

5 [98%]-

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Na₂CO₃

TBABr

K₂CO₃

CTABr

Mg/Al (HT)

γ-alumina.H₂O

Nano-TiCl₄.SiO₂

10

(0.8)

10

(0.01 ml)

50

-

0.1

0.15

0.05

0.1

α-naphthol

β-naphthol

α-naphthol

β-naphthol

α-naphthol

β-naphthol

Grinding

125 ^°C

MW

-

H₂O

-

H₂O

-

H₂O

-

MW

Reflux -110 ^°C

MW

Reflux

90 ^°C

5 [98%]-

5 [85%]-

5 [90%]-

5 [98%]-

5 [70%]-

5 [78%]-

5 [85%]-

5 [80%]-

90 ^°C

80 ^°C

0.1

110 ^°C

Reflux

Sonication

-

Water

EtOH

EtOH: H₂O

EtOAc

n-Hexane

EtOH: H₂O

5 [80%]-

5 [75%]-

^aThe molar ratio of benzaldehyde : naphthol : and malonitrile is 1 : 1 : 1.

Meanwhile, both aldehyde groups of terphthaldialdehyde reacted to malononitrile and 2-naphthol.

The production was 4, 4′-(1, 4-phenylene)bis(2-amino-4H-benzo[f]chromene-3-carbonitrile) with

92% yield after 100 minutes at 90 °C (Scheme 1).

H₂N

CN

NC

NH₂

O

CN

(E)

nano-TiCl₄.SiO₂

O

OH

solvent-free

90 C

H

X

Scheme 1. Synthesis of 4,4′-(1,4-phenylene)bis(2-amino-4H-benzo[f]chromene-3-carbonitrile)

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Table 3

a

Preparation of 2-amino-4H-benzochromene-3-carbonitrile in the presence of nano-TiCl₄.SiO₂

NO₂

CN

(E)

O

(E)

O

(E)

O

NH₂

C1: 45 min, 98%

C2:10 min, 93%

C3: 60 min, 90%

COOCH₃

Cl

OCH₃

Cl

NC

CN

(E)

O

H₂N

O

NH₂

C6: 36 min, 95%

C4: 105 min, 86%

C5: 120 min, 90%

COOCH₃

OH

OCH₃

Cl

CN

(E)

O

(E)

O

(E)

O

NH₂

C7: 30 min, 92%

C9: 160 min, 92%

C8: 140 min, 85%

OH

H₃CO

NC

CN

H₂N

O

H₂N

O

NH₂

C10: 200min, 70%

C12: 150 min, 88%

C11: 70 min, 93%

^aA mixture of aldehyde (1mmol), malononitrile (1mmol), naphthol (1mmol), and nano-TiCl₄.SiO₂(0.1 g) was stirred at 90 °C under solvent–

free conditions

Trioxan as a source of formaldehyde reacted to 2-naphthol and malononitrile. It produced 2-

amino-4H, 4H mixing at 90 °C (Scheme 2).

NC

NH₂

O

CN

(E)

H

nano-TiCl₄.SiO₂

H

O

OH

solvent-free

90 C

O

Scheme 2. Synthesis of 2-amino-4H,4H-benzo[f]chromen-3-carbonitrile

ACCEPTED MANUSCRIPT

In reaction of 3-phenyl propionaldehyde as an aliphatic aldehyde with malononitrile and 2-

naphthol, corresponding chromene product was obtained in 80% yield after 100 minutes at 90 °C.

In this protocol, 4-(N,N-dimethylamino) benzaldehyde as an aldehyde with electron releasing

group only reacted to malononitrile and produced benzylidinemalononitrile with 85% yield after

120 minutes mixing at 90 °C. Ketones such as, acetophenone did not produced any chromene

derivatives in condensation with malononitrile and 2-naphthol. It only produced

methylbenzylidinemalononitrile with 88% yield after 150 minutes at 90 °C.

3.2. Antifungal activities of the synthetic compounds

In this study, nine compounds (C1-C9) evaluated against fungi (Table 4). On the other hands, compounds

C1, C2, C5, C6, C7 and C8 showed no antifungal activities against examined Candida, Aspergillus strains

and dermatophytes. Compound C9 exhibited fungicidal activity against A. clavatus, C. glabarata, C.

dubliniensis, C. albicans, and C. tropicalis. In comparing MIC values of the synthetic compounds, C4

exhibited strong inhibitory activities against E. floccosum followed in activity by C3 and C9, respectively.

In this class, replacement of hydrogen with methoxy residue in para and meta-positions of phenyl ring of

C4 increased its antifungal activity against E. floccosum (at concentrations ranging from 1 to 32 µg/mL)

compared to C1 (Table 4).

In addition, isopropyl substitution at para position of the phenyl ring of C3 improved of its antifungal

activity against the tested E. floccosum at concentrations ranging from 2- 8 µg/mL compared to C1 (Table

3). Replacement of methyl ester at para position of phenyl ring provided C9 would result in significant

enhancement of the inhibitory activity against E. floccosum at concentrations ranging from 8-16 µg/mL.

Also this compound showed good fungistatic and fungicidal activity against some species of Candida at

concentrations ranging from 16-128 µg/mL (Table 4). This might be probably due to higher solubility of

C9 than the other compounds in aquatic media.

Also, in this survey, showed that E. floccosum was the most sensitive of the studied dermatophytes, that

good inhibited by C4, C3 and C9 compared to T. rubrum and M. canis.

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None of the synthetic compounds had any effect on negative gram and positive gram of microorganisms

except C1 that it had inhibitory effects on S. aureus microorganism.

In comparison of the antifungal and antibacterial activities of the synthetic compounds based on variation

of substitutions on ortho, meta and para position of phenyl ring, we found that the base compound C1

exhibited a better antibacterial activity against the tested S. aureus (positive garam microorganism) than

the other compounds. We find Compound C9 exhibited fungicidal activity against A. clavatus, C.

glabarata, C. dubliniensis, C. albicans, and C. tropicalis. In comparing MIC values of the synthetic

compounds, C4 exhibited strong inhibitory activities against E. floccosum followed in activity by C3 and

C9, respectively.

3.3. Docking studies

The rasemate compounds were tested but docking studies of binding mode of R and S isomer of

compounds C3, C4 and C9 were performed by PYRX to achieve better understanding on the mechanism,

potency and guide additional structure–activity relationships (SAR). According to the related literature,

there are no any experimental structural information available for the active site of Candida albicans

enzyme so the most close homology X-ray crystallographic structure of the Mycobacterium tuberculosis

enzyme CYP51 (PDB ID:1EA1) were used [33-34]. Interactions of fluconazole in active site of CYP51

for validation and comparison were depicted in Figure 2 (a). The R isomers have no effective interaction

so there are comparisons between isomer S of compounds C3, C4 and C9 and binding model of

fluconazole. Figure 2 (b, c, and d) indicated the polar interaction with Arg 96, His 259 and Thr 260 into

the binding pocket that accommodates well to pharmacophore residues. In summery these H-bond and

hydrophobic interaction might be favor to the anti fungal activity. It seems that suitable substitute on para

and meta site of compounds C3, C4 and C9 orientated toward the front of the pocket of heme iron of

CYP51, These interactions are analogous to interactions typically seen between the triazole ring of

fluconazole and heme iron of CYP51.

ACCEPTED MANUSCRIPT

We have demonstrated a simple method for the synthesis of 2-amino-4H-benzochromene-3-carbonitrile

by nano-TiCl₄.SiO₂as efficient catalyst under solvent-free condition. The antibacterial and antifungal

activities of nine synthetic compounds were investigated.

It is evident that synthesized compounds, C3, C4 and C9 exhibited strong inhibitory activities against

E. floccosum and thereby, these compounds can constituting promising antifungal both individually and

in combined therapy.

A computer model of the interaction of compound C3, C4 and C9 with the Mycobacterium tuberculosis

enzyme CYP51 binding pocket was proposed. The model involved a favorable polar interaction that

accommodated in complete pharmacophoric equivalency to the known anti-fungal activity binding mode.

A

B

D

C

Figure 2 (a). Molecular docking modeling perpendicular orientation of fluconazole to the heme iron of CYP51, (b) Orientation

compound C3, (c) Orientation compound C4 and (d) Orientation compound C9 in active site of CYP51. Note: For clarity, only

interacting residues in 12 Å were displayed.

ACCEPTED MANUSCRIPT

Table 4

Result of Minimum inhibitory concentrations of the 2-amino-4H-benzochromenes drivatives (µg/mL) against fungi

Compounds

C1

C2

C3

C4

C5

C6

C7

C8

C9

Fluconazolel

Fungi(number of strains)

A. flavus (ATCC 64025) )

A. fumigatus(ATCC 14110) )

A. clavatus (ATCC )

C. glabarata (ATCC 863)

C. dubliniensis (ATCC 8501)

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

1>

MFC

1>

128

1>

>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128

>128 >128 >128 >128 >128 >128

>128 >128 >128 >128

>128 >128 >128 >128 >128 >128

32

128

64

32

16

128

16

>128

C. dubliniensis (ATCC 7988) >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128

C. glabarata (ATCC 863)

C. albicans (ATCC 1912)

C. glabarata (ATCC 2192)

C. glabarata (ATCC 2175)

C. albicans (ATCC 1905)

C. albicans (SUCC 2303)

C. albicans (SUCC 625)

C. tropicalis (SUCC 194)

C. tropicalis (SUCC 611)

C. tropicalis (ATCC 750)

M. canis

>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128

>128 >128 >128 >128 >128

>128 >128 >128 >128 >128 >128

32

T. rubrum

E. floccosum

>128 >128 >128 >128

2

8

1

32

>128 >128 >128 >128

8

16

MIC: Minimum inhibitory concentration, MFC: Minimum fungicidal concentration

13

ACCEPTED MANUSCRIPT

4. Conclusion

We have demonstrated a simple method for the synthesis of 2-amino-4H-benzochromene-3-carbonitrile

by nano-TiCl₄.SiO₂as efficient catalyst under solvent-free condition. The antibacterial and antifungal

activities of nine synthetic compounds were investigated.

It is evident that synthesized compounds, C3, C4 and C9 exhibited strong inhibitory activities against E.

floccosum, these compounds can constituting promising antifungal both individually and in combined

therapy.

A computer model of the interaction of compound C3, C4 and C9 with the Mycobacterium tuberculosis

enzyme CYP51 binding pocket was proposed. The model involved a favorable polar interaction that

accommodated in complete pharmacophoric equivalency to the known anti-fungal activity binding mode.

Acknowledgements

The Research Council of Yazd University is gratefully acknowledged for the financial support of

design and synthesis of compounds. The antifungal activities of the synthetic compounds were

evaluated in Shiraz University of Medical Sciences. The authors would like to thank the Research

Consulting Center (RCC) at Shiraz University of Medical Sciences for their assistant in editing

this article.

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ACCEPTED MANUSCRIPT

Highlights

►A series of novel 2-amino-4H-benzochromene-3-carbonitrile derivatives in the presence of

nano-TiCl₄.SiO₂were synthesized. ► Some of the compounds showed good fungi static and

fungicidal activity against some species of Candida ►Docking simulation was performed to

insert compounds in to CYP51 active site to determine the probable binding model.

.

Article Doi

Synthesis, antifungal activity and docking study of 2-amino-4H-benzochromene-3-carbonitrile derivatives

DOI: 10.1016/j.molstruc.2016.03.002

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.molstruc.2016.03.002

Full text of DOI:10.1016/j.molstruc.2016.03.002

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