1. Introduction

IJOC

International Journal of Organic Chemistry

2161-4687

Scientific Research Publishing

10.4236/ijoc.2017.74031

IJOC-81101

Articles

Biomedical&Life Sciences Chemistry&Materials Science

Green Synthesis of Novel 5-Arylazo-2-[(2S, 3S, 4R, 5R)-3, 4, 5-Trihydroxy-6-(Hydroxymethyl) Tetrahydro-2H-Pyran-2-Yloxy]-4, 6-Dimethyl 3-Nicotinonitrile

Magda

H. Abdellattif

¹^*Mohamed

Mohamed Helmy Arief

²Adel

A. H. Abdel-Rahman

³Abdel-Aleem

H. Abdel Aleem

³Abdel

Moneam Farag Eissa

Chemistry Department, Faculty of Science, Benha University, Benha, Egypt

Deanship of Scientific Research, Taif University, Alhawaya, Taif, KSA

Chemistry Department, Faculty of Science, Menaufia University, Shebin Elkom, Egypt

* E-mail:magdah11uk@hotmail.com(MHA);

25102017

07043894023, October 201712, December 2017 15, December 2017

2014

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Introduction: Pyridone derivatives played important roles in the last decade to approach many and different functionalities, especially as antitumor, antibacterial, anti-fungal, and many of pharmacological activities. Methodology: Novel compounds of 5-Arylazo-2-[(substituted)-3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yloxy]-4, 6-dimethylnicotinonitrile, (3a-e), generally called (fluroarylazopyridine glucosides) were synthesised via green protocol, microwave. Results: The compounds were investigated by (IR, ¹HNMR, ¹³CNMR and mass spectrometry). Where some of pharmacological activities like antibacterial and antifungal studies had been investigated and characterized. It was found that 3a-d had characterized by high activities as antibacterial and antifungal. Where microwave synthetic methods were more efficient, gave higher products quantity, and more saving for time requirement and for using of much more solvents.

Green Chemistry Green Protocol Pyridones Fluroazo Compounds Microwave

1. Introduction

Pyridine nucleus is one of the most interesting nucleus in organic synthesis. Many uses of pyridones derivatives were investigated in the recent decades especially fluorinated derivatives. One of the recent researches discovered that high tuberculostatic activity of pyridone was observed [1] .

Also the amazing character of some pyridone is its high fluorescence activities which were used as molecular sensor of picric acid [2] .

It has been of great importance in the exploring of some novel antimicrobial compounds in veterinary as well as human medicine worldwide.

Genetic mutation and propagation of drug resistance genes of microorganisms are a very great factor that being as a strong barrier in treating the infectious diseases for animal and human patients [3] [4] .

The importance of the synthesis of novel derivatives of pyridone nucleosides is due to their strong affinity to treat many diseases, for example hepatitis, cancer and many of microbial infections [5] [6] [7] [8] [9] .

Fluorinated derivatives of pyridones are of high significance in pharmaceutical and medicinal chemistry [10] [11] . Synthesis of poly substituted fluroarylazopyridone by using green protocol is of great effect in synthetic chemistry and also in pharmaceutical chemistry [12] . Actually in the molecule which contains fluorine atoms became of high change in its lipophilicity, which also affect and change the rate of transportation through lipid membranes [13] .

Achievement of green and sustainable chemistry protocol instead of classical methods synthetic chemistry nowadays is of high interest, especially in synthesis of some novel substituted fluro pyridone derivatives (1a-e) and their nucleosides (3a-e).

2. Results and Discussion

a) Chemistry

General technique for green formation of arylazo pyridone glucosides had been used [14] [15] .

Scheme 1. Represents the general method for synthesis of pyridine derivatives 1(a-e) where, Scheme 2. Represents the general method for synthesis of pyridine derivatives 2(a-e) and 3(a-e).

Where Simple, accurate, green procedure were be used in synthesis of 5-arylazo-2-[(2S, 3S, 4R, 5R)-3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yloxy]-4, 6-dimethylnicotinonitrile [16] [17] [18] [19] .

5-arylazo-2-[(2S, 3S, 4R, 5R)-3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yloxy]-4, 6-dimethylnicotinonitrile (3a-e) had been got in very good yields where microwave irradiation had been used.

In this method, a homogenous solid mixture of 2(1H)-pyridones (1a-e) and acetyled-α-D-glucopyranose derivatives with silica gel was irradiated in MW for 2-3 minutes. For example, 3-cyano-4, 6-Dimethyl-5-arylazo-2(1H)-pyridinones (1a-e) [14] [15] were allowed to be reacted with acetylaed-α-D-glucopyranosyl bromide derivative for 2 minutes to give (2a-e) in about 91% yield.

Scheme 1. Synthesis of Pyridone derivatives 1a-e.

Scheme 2. Synthesis of Pyridone derivatives 2a-e & 3a-e.

The same nucleosides, (2a-e) were be gained in very high yields by the reaction of the K-salt of pyridnone which was generated in situ, using potassium hydride and an activated sugar moiety. The K-salt of 3-cyano-4, 6-dimethyl-5-arylazo- 2(1H)-pyridinones (1a-e) were allowed to react with α-bromoglucose in DMF for 15 hours to give (2a-e) in average 73% yield.

Deacetylation of 2a-e were almost done by treatment of alkali and although anhydrous media is useful to reduce the amount of alkali but catalytic reaction may be applied. But in fact a mixture of Triethylamine in MeOH and water be used in deacetylation or a mixture of methanol and dry ammonia were be also used.

Table 1 which illustrated the structure of the substituents of Compound 1 (a-e), where One could notice the difference between microwave method and conventional method in time of synthesis and the yield percentage of the product from Table 2. Also it had been noticed the difference between triethyl amine method and methanol and dry ammonia method in the yield percentage of the product as represented in Table 3.

The final structures of the expected compounds had been also represented in Table 4. Where the elemental analysis of the synthetic compounds had been illustrated in Table 5. Also ¹HNMR, ¹³CNMR, Ms-LC, and IR studies had been illustrated at Tables 6-9, respectively.

b) Biology

It has been of great importance in the exploring of some novel antimicrobial compounds in veterinary as well as human medicine worldwide. Genetic mutation and propagation of drug resistance genes of microorganisms are a very

Table 1 Substituted Arylazopyridine glucosides 1a-e

Substituted pyridone	Ar	R²
1a		ـــــCH₃
1b		ـــــCH₃
1c		ـــــCH₃
1d		ـCH₃
1e		ـCH₃

Table 2 Comparison between conventional methods and microwave methods for the Synthesis of Acetylated 5-arylazo-2-[(2S, 3S, 4R, 5R)-3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yloxy]-4, 6-dimethyl 3-nicotinonitrile

Compound number	R	Ar	Microwave method		Conventional
Compound number	R	Ar	time	yield	time	yield
2a	ـــــCH₃	C₇H₃F	2	92	55	60
2b	ـــــCH₃	C₆H₄SF₅	2	94	48	69
2c	ـــــCH₃	C₆H₅NF	2	90	58	60
2d	ـCH₃	C₆H₂Cl₂NO	2	91	56	63
2e	-CH₃	C₁₂H₁₁NF₄	2	93	55	60

Table 3 Yeild % percentage comparison of trimethylamine and dry ammonia methods for novel of nucleosides 3a-e

Compound number	R =	Ar =	Method A	Method B
3a	ـــــCH₃	C₇H₃F	90	82
3b	ـــــCH₃	C₆H₄SF₅	92	80
3c	ـــــCH₃	C₆H₅NF	91	81
3d	ـCH₃	C₆H₂Cl₂NO	92	83
3e	ـCH₃	C₁₂H₁₁NF₄	92	80

Table 4 Structure formulae for compounds 1a-e & 3a-e

Compound number	Compound name	Compound structure
1a	5-[(E)-2-Cyano-4-fluorophenylazo]-4,6-dimethyl-2-oxo-1H-pyridine-3-carbonitrile
1b	5-[(E)-o-(Pentafluorothio)phenylazo]-4,6-dimethyl-2-oxo-1H-pyridine-3-carbonitrile
1c	5-[(E)-2-Amino-5-fluorophenylazo]-4,6-dimethyl-2-oxo-1H-pyridine-3-carbonitrile
1d	5-[(E)-2,4-Dichloro-5-hydroxyphenylazo]-4,6-dimethyl-2-oxo-1H-pyridine-3-carbonitrile
1e	5-[(E)-p-(2,3,5,6-Tetrafluorocyclohexyl)phenylazo]-4,6-dimethyl-2-oxo-1H-pyridine-3-carbonitrile
3a	5-[(E)-2-Cyano-4-fluorophenylazo]-2-[(2S,3S,4R,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy]-4,6-dimethylnicotinonitrile
3c	5-[(E)-o-(Pentafluorothio)phenylazo]-2-[(2S,3S,4R,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy]-4,6-dimethylnicotinonitrile
3d	5-[(E)-2-Amino-4-fluorophenylazo]-2-[(2S,3S,4R,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy]-4,6-dimethylnicotinonitrile
3e	5-[(E)-2,4-Dichloro-5-hydroxyphenylazo]-2-[(2S,3S,4R,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy]-4,6-dimethylnicotinonitrile

Table 5 The Elemental analysis of synthesized compounds

Compd.	Mp. (˚C)	Formula (Mwt)	Solvent of crystallization	Analysis % Calcd. (Found)
Compd.	Mp. (˚C)	Formula (Mwt)	Solvent of crystallization	C	H	N	Cl	F	S
1a	456	C₁₅H₁₀FN₅O	Ethanol/DME	61.01	3.41	23.72		6.43
1b	240.76	C₁₄H₁₁F₅N₄OS	Ethanol/DME	44.45	2.93	14.81		25.11	8.48
1c	399.66	C₁₄H₁₂FN₅O	Ethanol/DME	58.94	4.24	24.55		6.66
1d	487.37	C₁₄H₁₀Cl₂N₄O₂	Ethanol/DME	49.87	2.99	16.62	21.03
1e	363.69	C₂₀H₁₈F₄N₄O	Ethanol/DME	59.11	4.46	13.79		18.7
3a	794	C₂₁H₂₀FN₅O₆	Ethanol/DME	55.14	4.41	15.31		4.15
3b	578	C₂₀H₂₁F₅N₄O₆S	Ethanol/DME	44.45	3.92	10.73		17.58	5.93
3c	737	C₂₀H₂₂FN₅O₆	Ethanol/DME	53.69	4.96	15.72		4.25
3d	825	C₂₀H₂₀Cl₂N₄O₇	Ethanol/DME	48	3.64	16.86			9.65
3e	701	C₂₆H₂₈F₄N₄O₆	Ethanol/DME	54.93	4..04	11.22	14.3

Table 6 1H NMR spectrum data of the novel compounds

Compound number	Spectral1H NMR data
1a	δ 1.77 (s, CH₃, 3H), 1.7 (s, CH₃, 3H), 7.5 (q, 3H, Ar-H)
1b	δ 1.77 (s, CH₃, 3H), 1.7 (s, CH₃, 3H,), 7.2 - 7.5 (m, 4H, Ar-H)
1c	δ 1.9 (s, CH₃, 3H), 1.77 (s, CH₃, 3H), 6.9 (q, 3H, Ar-H),
1d	δ 1.9 (s, CH₃, 3H), 1.77 (s, CH₃, 3H), 7.4 (d, 2H, Ar-H)
1e	Δ 1.77 (s, CH₃, 3H), 1.7 (s, CH₃, 3H), 2.3 (m, CH₂, 2H), 5.4 (m, CH, 4H), 3.98 (m, H, CH), 8.9 (s, H, Ar-H)
3a	δ (s, CH₃, 3H), 1.7 (s, CH₃, 3H), 7.5 (m, 3H, Ar-H), 3.4 (d, 2H, -CH₂’’’’’’), 6.9 (d, 1H, -CH’), 2.4 (m, H, OH), 4.4 (m, 3H, CH’’’, CH’’’’, CH’’’’’)
3b	Δ 1.77 (s, CH₃, 3H), 1.7 (s, CH₃, 3H), 7.5 (m, 4H, Ar-H), 3.4 (d, 2H, -CH₂’’’’’’₎, 6.9 (d, 1H, -CH’), 2.4 (m, H, OH), 4.4 (m, 3H, CH’’’, CH’’’’, CH’’’’’)
3c	Δ 1.77 (s, CH₃, 3H), 1.7 (s, CH₃, 3H), 7.5 (m, 4H, Ar-H), 3.4 (d, 2H, -CH₂’’’’’’₎, 6.1 (d, 1H, -CH’), 3.4 (m, 4H, 4OH, 2H, NH₂), 4.4 (m, 3H, CH’’’, CH’’’’, CH’’’’’)
3d	δ 1.77 (s, CH₃, 3H), 1.7 (s, CH₃, 3H), 6.7-7.5 (d, d, 2H, Ar-OH), 2.9 (m, H, 5OH), 6.1 (d, 1H, -CH’), 3.37 - 4.4 (m, 3H, CH’’’, CH’’’’, CH’’’’’)
3e	δ 1.77 (s, CH₃, 3H), 1.7 (s, CH₃, 3H), 7.5 (q, 3H, Ar-H), 7.2 (s, 2H, -CH₂’’’’’’), 6.9 (d, 1H, -CH’’), 2.4 (m, H, OH), 4.4 (q, 4H, CH’, CH’’’, CH’’’’, CH’’’’’)

Table 7 13C NMR spectrum data of the novel compounds

Compound number	Spectral 13C NMR data
1a	δ, 19 (s, C, CH₃), 81 (s, C3, Ar), 118 (s, C, CN), 184 (s, C2, -C-O), 163 (s, C4,), 108 (s, C5, Ar-), 165 (s, C6, Ar-), 111 (s, C1’, Ar’), 119 (s, C3’, Ar’), 114 (s, C2’, Ar’), 118 (C, CN’) 164 (s, C4’, Ar’), 119 (s, C5’, Ar’), 130 (s, C6’, Ar’),
1b	δ, 19 (s, C, CH₃), 81 (s, C3, Ar), 118 (s, C, CN), 180 (s, C2, -C-O), 157 (s, C4,), 105 (s, C5, Ar-), 164 (s, C6, Ar-), 128 (s, C1’, Ar’), 130 (s, C3’, Ar’), 128 (s, C2’, Ar’) 124 (s, C4’, Ar’), 154 (s, C5’, Ar’), 128 (s, C6’, Ar’),
1c	δ, 19 (s, C, CH₃), 81 (s, C3, Ar), 118 (s, C, CN), 184 (s, C2, -C-O), 157 (s, C4,), 108 (s, C5, Ar-), 165 (s, C6, Ar-), 111 (s, C1’, Ar’), 103 (s, C3’, Ar’), 148 (s, C2’, Ar’) 164 (s, C4’, Ar’), 106 (s, C5’, Ar’), 131 (s, C6’, Ar’),

1d	δ, 19 (s, C, CH₃), 81 (s, C3, Ar), 118 (s, C, CN), 184 (s, C2, -C-O), 157 (s, C4,), 105 (s, C5, Ar-), 164 (s, C6, Ar-), 128 (s, C1’, Ar’), 130 (s, C3’, Ar’), 128 (s, C2’, Ar’) 124 (s, C4’, Ar’), 154 (s, C5’, Ar’), 118 (s, C6’, Ar’).
1e	δ, 19 (s, C, CH₃), 118 (s, C, CN), 153 (s, C, -C=O), 117 (s, C3, Ar-), 157 (s, C4, Ar-), 115 (s, C5, Ar-), 163 (s, C6, Ar-), 128 (s, C1’, Ar’), 130 (s, C3’, C5’, Ar’), 128 (s, C2’, C6’, Ar’) 124 (dd, C4’, Ar’), 31 (m, C1’’’), 92 (m, C2’’’, C6’’’ (, 85 (m, C3’’’, C5’’’) 27 (C4’’’)
3a	δ, 19 (s, C, CH₃), 81 (s, C3, Ar), 118 (s, C, CN), 184 (s, C2, -C-O), 163 (s, C4,), 108 (s, C5, Ar-), 165 (s, C6, Ar-), 111 (s, C1’, Ar’), 119 (s, C3’, Ar’), 114 (s, C2’, Ar’), 118 (C, CN’) 164 (s, C4’, Ar’), 119 (s, C5’, Ar’), 130 (s, C6’, Ar’), 131 (s, C1’’), 67 (s, C2’’), 67 (s, C3’’), 67 (s, C4’’), 81 (s, C5’’), 60 (s, C6’’),
3b	δ, 19 (s, C, CH₃), 81 (s, C3, Ar), 118 (s, C, CN), 180 (s, C2, -C-O), 157 (s, C4,), 105 (s, C5, Ar-), 164 (s, C6, Ar-), 128 (s, C1’, Ar’), 130 (s, C3’, Ar’), 128 (s, C2’, Ar’) 124 (s, C4’, Ar’), 154 (s, C5’, Ar’), 128 (s, C6’, Ar’), 131 (s, C1’’), 67 (s, C2’’), 67 (s, C3’’), 67 (s, C4’’), 81 (s, C5’’), 60 (s, C6’’),
3c	δ, 19 (s, C, CH₃), 81 (s, C3, Ar), 118 (s, C, CN), 184 (s, C2, -C-O), 157 (s, C4,), 108 (s, C5, Ar-), 165 (s, C6, Ar-), 111 (s, C1’, Ar’), 103 (s, C3’, Ar’), 148 (s, C2’, Ar’) 164 (s, C4’, Ar’), 106 (s, C5’, Ar’), 131 (s, C6’, Ar’), 131 (s, C1’’), 67 (s, C2’’), 67 (s, C3’’), 67 (s, C4’’), 81 (s, C5’’), 60 (s, C6’’),
3d	δ, 19 (s, C, CH₃), 81 (s, C3, Ar), 118 (s, C, CN), 184 (s, C2, -C-O), 157 (s, C4,), 105 (s, C5, Ar-), 164 (s, C6, Ar-), 128 (s, C1’, Ar’), 130 (s, C3’, Ar’), 128 (s, C2’, Ar’) 124 (s, C4’, Ar’), 154 (s, C5’, Ar’), 118 (s, C6’, Ar’), 131 (s, C1’’), 67 (s, C2’’), 67 (s, C3’’), 67 (s, C4’’), 81 (s, C5’’), 60 (s, C6’’),
3e	δ, 19 (s, C, CH₃), 81 (s, C3, Ar), 118 (s, C, CN), 184 (s, C2, -C-O), 157 (s, C4,), 105 (s, C5, Ar-), 164 (s, C6, Ar-), 128 (s, C1’, Ar’), 130 (s, C3’, C5’, Ar’), 128 (s, C2’, C6’, Ar’) 124 (dd, C4’, Ar’), 131 (s, C1’’), 67 (s, C2’’), 67 (s, C3’’), 67 (s, C4’’), 81 (s, C5’’), 60 (s, C6’’), 31 (m, C1’’’), 92 (m, C2’’’, C6’’’ (, 85 (m, C3’’’, C5’’’) 27 (C4’’’)

Table 8 LC/Ms Fragmentation spectrum data of synthesized compounds of Scheme 13

Compound	m/z	Abundance%
1a	295.27	98
1b	378.32	99
1c	285.28	97
1d	337.16	99
1e	406	99
3a	457.41	96
3b	540.5	98
3c	472	97
3d	498	98
3e	568	99

Table 9 IR spectrum data for the synthesized compounds Scheme 12

Compound	IR υ cm⁻¹
1a	3123 (NH), 2220 (CN), 1646 (CO)
1b	3226 (NH), 2215 (CN), 1717 (CO).
1c	3300, 3400 (NH₂); 1645 (C=O) 3195 (NH), 2223 (CN)
1d	3300 (OH), 2230 (CN), 1690 (CO), 3500 (NH)
1e	3200 (OH), 1640 (CO), 3300 (NH), 2250 (CN)
3a	3300, 3400 (NH₂); 1645 (C=O) 3195 (NH), 2223 (CN)
3b	3100 (NH), 1650 (CO), 2228 (CN)
3c	3195 (NH), 2223 (CN), 1645 (C=O)
3d	3300 (OH); 1680 (C=O); 2224 (CN); 3455 (NH)
3e	3300 (NH), 2225 (CN), 1650 (C=O)

great factor that being as a strong barrier in treating the infectious diseases for animal and human patients.

All investigated compounds show different antibacterial and antifungal activities, these results were be due to the newly derivatives formed from fluroazo pyridone and their glucosides.

The most active compounds were 1a, 3a, 1c, 3c although most of them showed good activity.

One could notice that Table 10, represented the antibacterial and the anti-fungal effects of the new synthetic compounds. Where Table 11 represented the strain of organism.

3. Methodolgy3.1. Chemistry3.1.1. General Coupling Procedures of Synthesis of Acetylated-Arylazo- 2-[(2S, 3S, 4R, 5R)-3, 4, 5-Trihydroxy-6-(Hydroxymethyl) Tetrahydro-2H-Pyran-2-Yloxy]-4,6 Dimethylnicotinonitrile

Microwave synthesis will be performed using CEM Microwave system. Melting

Table 10 Virtual screening for in vitro Antibacterial and antifungal of the novel fluroarylazopyridin-2-one derivatives

Samples		Inhibition zone diameter
		Bacterial species					Fungi
		Gram positive			Gram negative		Fungi
		Bacillus subtilis	Staphylococcus aureus		Escherichia coli	Pseudomonas aeruginosa	Aspergillus flavas	Candida albicans
control DMSO		0.0		0.0	0.0	0.0	0.0	0.0
Standard	Ampicillin Antibacterial agent	26		21	25	26	---	---
Standard	Amphotericin Antifungal agent	-----		----	----	----	16	19
0		21		17	17	15	12	12
1a		14		13	12	13	11	11
3a		16		16	14	14	11	11
1b		10		12	12	13	0.0	0.0
3b		15		13	14	13	0.0	0.0
1c		11		11	11	11	0.0	0.0
3c		19		29	20	22	0	0
1d		12		13	12	14	0.0	0.0
3d		17		27	17	23	0.0	0.0
1e		0.0		0.0	0.0	9	0.0	0.0

Table 11 The type strain of microorganisms

Microorganism	Gram Reaction	ATCC
Escherichia coli	G⁻	11,775
Staphylococcus aureus	G⁺	12,600
Candida albicans	Fungus	7102
Aspergillus flavus	Link

points will be determined on (Pyrex capillary) Gallenkamp apparatus. Infrared spectra will be recorded with a Thermo Nicolet Nexus 470 FT-IR spectrometer in the range 4000 - 400 cm⁻¹ using potassium bromide disks. ¹H-NMR spectra, ¹³C-NMR spectra will be obtained on Varian Gemini 400 and 200 MHz FT NMR spectrometer in CDCl₃and DMSO-d₆; chemical shifts will be recorded in d (ppm) units, relative to Me₄Si as an internal standard. The mass spectra will be recorded on Shimadzu LCMS-QP 800 LC-MS and AB-4000 Q-trap LC-MS/MS. Thin-layer chromatography (TLC) will be carried out on pre-coated Merck silica gel F₂₅₄ plates. Column chromatography will be performed on a Merck silica gel. The reagents will be purchased from Aldrich and used without further purification.

a) Green Microwave method

A solution of 2(1H)-pyridones (1a-e) (10 mmol) and acetylated-α-D-glucopyranose derivatives (11 mmol, 4.29 g) had been prepared by dissolving in methylene chloride/methanol (80/20) then silica gel (200 - 400 mesh) mixture. Then the excess solvent had been subjected to vaporization to be removed. The dried residue had been transferred into a vial and subjected to microwave irradiation for 2 - 3 minutes using CEM Microwave system. The product will be purified using column to gain (2a-e).

b) Conventional method

A solution of 2(1H)-pyridone (1a-e) (10 mmol) in DMF (15 ml) had been mixed with potassium hydride (4.76 mmol) under nitrogen they had been stirred at 60˚C. After 2 h, the acetylated glucopyranosyl bromide (5) (15 mmol,), had been added and the solution had been stirred at normal temperature for 15 h. The solvent had been evaporated and reminder prepared material had been partitioned between CHCl₃ (30 mL) and water (30 mL). Mixed organic extracts had been dried on (Na₂SO₄), filtered and vaporized until complete dryness. The crude synthetic compounds had been dried and purified using column chromatography to gain the compounds (2a-e).

3.1.2. General Procedure for Nucleoside Deacetylation

a) Triethyl amine method

Triethylamine (1.0 ml) had been mixed with glucosides solution of (2a-e). (1 m mol) in (10 ml methanol and few drops of water). The mixture had been stirred for 15 hours at normal. Reduced pressure had been applied for the vaporization and the residue was evaporated with methanol until triethylamine had removed. The synthetic compounds had been crystallized using suitable solvents to get compounds (3a-e).

b) Methanol and dry ammonia

Dry methanol (20 ml) solution of protected glucosides (0.5 g) of at 0˚C (2a-e) had been treated by dry ammonia for 25 minutes. The reaction mixture had been stirred till it had done and finally had been investigated by TLC. Reduced pressure had been applied to get the resultant products concentrated till crude solid had been reached. A solution (chloroform: methanol, 20:1), and silica gel chromatography had been applied to the products for purification. Methanol had been applied for crystallization to gain (3a-e).

3.2. Biology

A method of Kirby-Bauer had been used for the determination of antimicrobial activity for novel fluroazopyridone compounds [20] .

This had been carried out using special concentration of the test bacteria/fungi and had been grown up in a certain concentration of fresh media and then left it to grow up to 10⁸ cells/mL for bacteria 10⁵ cells/mL for fungi [21] .

Onto agar plates a concentration of 100 µl of bacterial of fungal suspension had been spread according to the broth for their maintenance. Each isolated colony for any organism used had played a pathological effect on primary and tested. Utility of disc diffusion method that many media were available, NCCLS [22] gave a recommendation of using Mueller-Hinton agar because of [23] [24] their reproducibility in good batch-to-batch.

An approved standard method (M38-A) disc diffusion method which had been applied to filamentous fungi tested [24] [25] [26] to evaluate the pathological activity of filamentous fungi to antifungal agents. Using an approved standard method (M44-P) had been used for disc diffusion method for yeasts developed by [25] . Aspergillus flavus used as an example of fungi had been incubated at 25˚C for 48 hours. Staphylococcus aureus, Bacillus subtilis used as an example of Gram positive bacteria, where Escherichia coli, Pseudomonas aeuroginosa used as an example of Gram negative bacteria, both had been incubated at 35˚C - 37˚C for 24 - 48 hours.

Where; Candida albicans used as yeast had been incubated at 30˚C for 24 - 48 hours.

The inhibition zones diameter had been measured in millimetres [20] .

A positive control for microbial activity had been served by standard discs of Ampicillin (Antibacterial agent), Amphotericin B (Antifungal agent). A negative control had been achieved by immersing of filter discs in 10 µl of solvent (distilled water, chloroform, DMSO). Meuller-Hinton agar was used for tested the composition and pH.

In addition a factor had been considered in the disc diffusion method was the depth of the agar in the plate. This method was a very known one and well documented and standard zones of inhibition had been determined for susceptible and resistant values. Blank paper disks (Schleicher & Schuell, Spain) with a diameter of 8.0 mm were impregnated 10 µl of tested concentration of the stock solutions. An impregnated filter paper disc with a tested chemical had been put on agar and the novel chemical compound had been diffused from the disc into the agar. By this diffusion the chemical compound had been transferred in the agar only around the disc. The size of the area of chemical infiltration around the disc had been determined by the chemical compound solubility and its molecular size. Bacteria organism had not been grown up when placed in the agar which contains the chemical compounds under investigation. This area of no growth around the disc had been named as a “Zone of inhibition” or “Clear zone”. The diffusion, the zone diameters were measured with the National Committee for Clinical Laboratory Standards. Good alternatives methods had been characterized such as E-test and disk diffusion and they were simpler and faster techniques [26] .

Cite this paper

Abdellattif, M.H., Arief, M.M.H., Abdel-Rahman, A.A.H., Aleem, A.-A.H.A. and Eissa, A.M.F. (2017) Green Synthesis of Novel 5-Arylazo-2-[(2S, 3S, 4R, 5R)-3, 4, 5-Trihydroxy-6-(Hydrox- ymethyl) Tetrahydro-2H-Pyran-2-Yloxy]-4, 6-Dimethyl 3-Nicotinonitrile. International Journal of Organic Chemistry, 7, 389-402. https://doi.org/10.4236/ijoc.2017.74031

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