Accepted Manuscript Synthesis, Crystal Structure, Structural Characterization and In Vitro Antimicrobial Activities of 1-methyl-4-nitro-1H-imidazole Hafiz Abdullah Shahid, Sajid Jahangir, Sammer Yousuf, Muddasir Hanif, Sikandar Khan Sherwani PII: DOI: Reference:
S1878-5352(14)00249-4 http://dx.doi.org/10.1016/j.arabjc.2014.11.001 ARABJC 1433
To appear in:
Arabian Journal of Chemistry
Received Date: Accepted Date:
23 August 2013 2 November 2014
Please cite this article as: H.A. Shahid, S. Jahangir, S. Yousuf, M. Hanif, S.K. Sherwani, Synthesis, Crystal Structure, Structural Characterization and In Vitro Antimicrobial Activities of 1-methyl-4-nitro-1H-imidazole, Arabian Journal of Chemistry (2014), doi: http://dx.doi.org/10.1016/j.arabjc.2014.11.001
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Synthesis, Crystal Structure, Structural Characterization and In Vitro Antimicrobial Activities of 1-methyl-4-nitro-1Himidazole Hafiz Abdullah Shahid, 1Sajid Jahangir,1* Sammer Yousuf,2 Muddasir Hanif3 and Sikandar Khan Sherwani4 1
Department of Chemistry, Faculty of Science, Federal Urdu University of Arts, Science and Technology Gulshane-Iqbal, Karachi 75300, Pakistan
2
HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
3
Nabiqasim Industries (Pvt) Ltd. Commerce Centre Hasrat Mohani Road, Karachi 74200, Pakistan
4
Department of Microbiology, Faculty of Science, Federal Urdu University of Arts, Science and Technology Gulshan-e-Iqbal, Karachi 75300, Pakistan
*Corresponding Author; E-Mail:
[email protected]
Abstract: We report new reaction conditions (less corrosive reagents) for the regio-specific synthesis, spectroscopic properties, crystal structure, antimicrobial activities and MICs of the 1methyl-4-nitro-1H-imidazole (I). For the structure confirmation, X-ray crystallography was performed using crystals obtained from chloroform and hexane (1:1). The structure of the compound (I) was further characterized by the FTIR, 1H-NMR,
13
C-NMR and EIMS. The
important peaks appearing in the EIMS, at the m/z = 127, 111, 97, 81, and 54 arise due to the loss of single electron, loss of O, NO, NO2, and Loss of NO2 & HCN respectively. The high intensity peak in the EIMS at m/z = 42 arises due to the loss of NO, CO and HCN from the molecular ion. The correct 1H-NMR (400 MHz, CDCl3) of (I) shows peaks at 7.76 (ArH, HC=N), 7.42 (ArH, HC=C), and 3.82 (N-CH3) ppm. Further structural studies showed that the driving force for the crystallization (self-assembly) and the hydrogen bonding (C-H…O-NO, 2.57 Å) originates from the partial negative oxygen of the nitro (NO2) group at one end and the partial positive hydrogen (N-CH3) at another corner of the molecule. Biological assay against many (13) microorganisms and fungi showed that the title compound (I) is moderately active against Gram (+), Gram (-) bacteria and fungi. KEYWORDS: Synthesis, Crystal Structure, Antimicrobial Properties
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1. Introduction Nitration is one of the earliest known, important reactions in organic chemistry. [Schofield et al., 1948] The reaction can be carried out with different nitrating agents, depending on the substrate and reaction conditions to obtain C-NO2, O-NO2 and N-NO2 functionalized compounds. [Katritzky et al., 2005] However, Electrophilic Nitration (C-NO2) of heterocyclic compounds holds higher importance in medicinal organic chemistry. [McCalla et al., 1971, Baumann et al., 2011] Heterocyclic compounds containing nitro group are well recognized for the antibacterial activities and the synthesis of other biologically and industrially valuable active compounds. [Bollo et al., 2004] Nitroimidazole is a class of heterocyclic compounds known as antibacterial agents for long time [Cosar et al., 1959, Anthwal et al., 2013, Anderson et al., 2012] due to its broad spectrum against gram positive and gram negative bacteria and other organisms [Edwards et al., 1993]. Similarly, the 4-nitro derivatives of 1-alkyl-1H-imidazole are well known as active pharmaceutical intermediates (APIs) for example, immunodepressant Azathioprine drug [Yrowell et al., 1973], preparation of 4 aminoimidazoles derivatives as the inhibitors of cyclindependent kinase 5/p25 for the treatment of pain, stroke, Alzheimer’s disease, the treatment of type-II diabetes [Helal et al., 2009], and for the synthesis of various novel APIs as JanusAssociate Kinase (JAK) inhibitors for the treatment of cancer and Myeloproliferative disorders [Chuaqui et al., 2010; Pardanani et al., 2009; Abdel-Magid et al., 2012]. The Nitroimidazole and its derivatives have been widely used by the medical community for various ailments, which is a driving force for further exploration, and more effective pharmaceutical candidate for the treatment of infectious diseases.
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The 1-methyl-4-nitro-1H-imidazole (Compound (I), CAS: 3034-41-1) is not provided by the standard large chemical suppliers such as TCI, Aldrich, Acros, Alfa Aesar etc. There are 2 important methods [Katritzky et al., 2005; Duddu et al., 2011] to prepare Compound (I). For the nitration, the first method [Katritzky et al., 2005] uses Trifluoroacetic anhydride/conc. HNO3 at 0-5 oC to give Compound (I) in 39% yield as reported in the experimental section (Scheme 1). This manuscript provided the 1H NMR,
13
C NMR, elemental analysis and melting point data
with an error in the 1H NMR data. The second method (Scheme 1) [Duddu et al., 2011] uses Fuming H2SO4/Fum. HNO3 at 0-15 o
C and provided Compound (I) in 46.8% yield (calculated (78:22) from total mixture yield 60%).
This yield is only based on 1H NMR analysis and the 1H NMR data, spectra or any other characterization is absent in the manuscript or the supporting information. Further, the mixture was not isolated, which clearly shows difficulties in separation. The authors also tried higher (110-120 oC) temperatures which resulted in the formation of 5 kinds of nitro products (Scheme 1). Again the products were not isolated and yields are only based on 1H NMR analysis. [Duddu et al., 2011] These two methods [Katritzky et al., 2005; Duddu et al., 2011] used well-known, hazardous materials such as trifluoroacetic anhydride, fuming H2SO4 and fuming HNO3. Some researchers may believe that conc. H2SO4 (as used in our research) and fuming H2SO4 (as used by other researchers) are equally hazardous, but this is not true. This can be confirmed by the standard safety information (Aldrich) which states that fuming H2SO4 containing only 20% SO3 (CAS: 8014-95-7, product code: 435597) has the double hazard codes namely GHS05 (Corrosive to metals, category 1), and GHS06 (Acute toxicity (oral, dermal, inhalation), categories 1,2,3) while 99.9% conc. H2SO4 (CAS: 7664-93-9, product code: 339741) shows single hazard code namely 3
GHS05 (Corrosive to metals, category 1). Similarly, common HNO3 (70%, CAS: 7697-37-2, product code: 438073) shows safety codes GHS03 (Oxidizing gases, category 1), GHS05 (Corrosive to metals, category 1) while the fuming HNO3 (90%, CAS: 7697-37-2, product code: 309079) shows 3 safety codes GHS03 (Oxidizing gases, category 1), GHS05 (Corrosive to metals, category 1), and GHS06 (Acute toxicity (oral, dermal, inhalation), categories 1,2,3). Similarly we can find that trifluoroacetic anhydride (99%, CAS: 407-25-0, product code: 106232) shows 2 safety codes GHS05 (Corrosive to metals, category 1), GHS07 (Acute toxicity (oral, dermal, inhalation), category 4). Beside the toxicity of fuming H2SO4 and fuming HNO3, they contain sulfur and nitrogen oxides, responsible for the global acidification and air pollution. (Kikuchi, R., 2001) All this information is sufficient to prove that the reactions performed in 99.99% conc. H2SO4 or common HNO3 are relatively safer and environmentally friendly compared to those performed in the trifluoroacetic anhydride, fuming H2SO4 and fuming HNO3 due to their acute toxicity and other effects. If the synthesis methods based on more corrosive reagents are applied on an industrial scale, then extraordinary protection measures should be taken for the humans and the environment. The other disadvantage of these methods is that they produce mixture of regio-isomeric products. When isomeric products are obtained, they must be separated through column chromatography which increases production cost. Therefore, more environment friendly reaction conditions, giving single or mainly one regio-isomer are highly desirable. To best of our knowledge, we are the first to report the regio-specific synthesis with less corrosive reagent conditions, unique crystal structure, structural properties, a small typo correction in the 1H NMR data, and Biological assay against 13 microorganisms and fungi. Herein we report our results.
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2. Result and Discussion 2.1. Synthesis and Spectroscopic Properties Method A shows that 1-methyl-1H-imidazole can undergo nitration in a mixed acid (non fuming 98% H2SO4 (non-fuming) 65% HNO3 at 5-65 oC bath temperature for 8 h. Instead of isomers or mixture of products we obtained only single regio-isomer namely 1-methyl-4-nitro1H-imidazole in 20% yield as shown in the scheme 1. The temperature control and slow addition are important factors to obtain regio-specific nitration. Method B shows that 1-methyl-1H-imidazole can undergo nitration in a mixture of urea nitrate/non-fuming conc. H2SO4 (98%). The method eliminates the use of liquid HNO3 as the source for nitronium ion, and uses urea nitrate as an easy to handle source for the nitration. Urea nitrate is known to convert deactivated aromatic compounds to the corresponding nitro derivatives with high regio-selectivity under mild conditions [Almog et al, 2006] but so far it has not been applied to make 1-methyl-4-nitro-1H-imidazole. The nitration process for the 1-methyl1H-imidazole happens at low temperature (-10 to +10 oC), so there is much less chance for nonregio-specific nitration. In addition the method gives better yield compared to the Method A. Spectroscopy such as, FTIR (Fig SEI-1, Table SEI-1), EIMS (Fig SEI-2, Scheme SEI-1), 1HNMR (Fig. SEI-3),
13
C-NMR (Fig. SEI-4), and X-Ray crystallography (Fig. 1 & 2) confirmed
that the product is 1-methyl-4-nitro-1H-imidazole. The FTIR spectrum data of the compound (I) is compared (Table SEI-1) with the FTIR data (Acros Chemicals) of the starting material, which shows clear nitration of 1-methyl-1H-imidazole. The EIMS spectrum (Fig SEI-2) and clean fragmentation pattern of compound (I) is well explained (Scheme SEI-1). The important peaks appearing in the EIMS, at the m/z = 127, 111, 97, 81, and 54 arise due to the loss of single 5
electron, loss of O, NO, NO2, and Loss of NO2 & HCN respectively. The high intensity peak at m/z = 42 arises due to the loss of NO, CO and HCN from the molecular ion. [Luijten et al., 1981] The 1H-NMR of compound (I) showed 3 peaks at δ: 3.82, 7.42, and 7.76 ppm corresponding to the CH3, and two ArHs. We found that the compound (I) shows 1H-NMR (400 MHz, CDCl3) peaks at 7.76 (ArH, HC=N), 7.42 (ArH, HC=C), and 3.82 (N-CH3) ppm instead of a previously reported [Katritzky et al., 2005] 1H-NMR (300 MHz, CDCl3) 7.78 (ArH), 4.72 (ArH), and 3.83 (N-CH3) ppm. This might be a small correction of the printing error or typo but it is useful for the future research. The
13
C-NMR showed 4 peaks at δ: 34.6 ppm for CH3, 120.1 for ArCH
(HC=C), 136.6 for ArCH (HC=N), and 148.3 ppm for the ArC-NO2 confirming the product compound (I). 2.2. X-Ray Crystallography Single-crystal X-Ray diffraction analysis was carried out to confirm the regio-specific nitration of the title compound (I). The crystal structure in our case is of very high importance to rule out the possibility of any other regio-isomer of the compound (I). The crystal structure shows that compound (I) is composed of nitro and methyl groups attached to a planer imidazole ring. The detailed analysis of the molecule (Fig. 1) shows that all the bond lengths and angles are within expected ranges [Allen et al., 1987]. The crystal structure is stabilized by the intermolecular hydrogen bonds (C-H…O-NO, 2.57 Å) and molecules are linked together to form sheets via C1---H1A...O2 running in zig-zag fashion parallel to the a-axis (Fig. 2). The crystallographic data for the structure determination and refinement of compound (I) are presented in Table 1. CCDC 875050 contains the supplementary crystallographic data for
6
compound (I). The data can be obtained free of charge via http://www.ccdc.cam.ac.uk/cgibin/catreq.cgi, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
[email protected]. 2.3. Structural Properties Properties of condensed phases are governed by the properties of the individual molecules due to the intermolecular forces between molecules arising from the electrostatic interactions. [Buckingham et al., 1978] To probe into the structural properties of the 1-methyl-4-nitro-1Himidazole or compound (I) we first optimized it’s molecular structure using the Becke’s threeparameter density functional theory (DFT) in combination with Lee-Yang-Parr’s correlation functional (B3LYP) and employed 6-31G(d) basis sets [Lee et al., 1988; Becke et al., 1993] within the Wavefunction Spartan 08. [Yamada et al., 2011] Fig. 3a shows the top view of the optimized structure showing corresponding atomic symbols. The side view (Fig. 3b) of the optimized molecular structure shows that 1-methyl-4nitro-1H-imidazole is a planar molecule except the methyl group (N-CH3) due to the sp3 hybridization. This observation is consistent with its crystal structure (Fig. 1). The Fig. 3c shows the Mulliken charges [Mulliken et al., 1955; Astrand et al., 1998] of different atoms in the optimized molecular structure. The nitro group shows the partial positive nitrogen (0.35) and partial negative oxygen atoms (-0.41 and -0.38) which makes one end of the molecule partial negative (δ-) as shown in Fig. 3b. The nitrogen and carbon (N-CH3) showed the partial negative charges (-0.38 and -0.33 respectively) while the hydrogen (N-CH3) atoms showed partial positive charge (0.19 each), which makes the second corner of the molecule partial positive (δ+) as shown in Fig. 3b. The phenomenon of negative and positive polarization created the driving force (Fig. 4) for the crystallization and the hydrogen bonding between the
7
negatively charged oxygen of the nitro (NO2) group and the positively charged hydrogen atom (Fig. 3b, Fig. 2) of the methyl group (N-CH3). This observation also explains the special orientation of molecules in the crystal structure (Fig. 4). It can be observed from the crystal structure that only one oxygen of the nitro (NO2) group participates in the H-bonding (Fig. 2), likely due to the more partial negative (-0.41 vs. -0.38) charge of one of the oxygen atom bonded to NO2. 2.4. Biological Assay For the nitro-imidazoles two mechanisms of action based on previous studies have been reported. [Van Der Wouden et al., 2000] Nitroimidazole enters the cell by passive diffusion, and metabolized by a reduction step in which the drug behaves as the electron acceptor. For this reduction step several nitroreductases are present in H. pylori (bacteria). It has been suggested that an oxygen-insensitive NADPH nitroreductase encoded by the rdxA gene is the most important. Oxygen-insensitive denotes in this case that the reduction of the nitroimidazole (ArNO2) results in a nitrosoderivate (Ar-NO) by the simultaneous transfer of two electrons. This nitrosoderivate cannot be re-oxidized by molecular oxygen and the nitroreductase facilitating the two-electron transfer step is therefore called oxygen-insensitive. The nitrosoderivate leads to the DNA damage and subsequent cell death of the bacteria (Fig. 5, Mechanism #1).
Fig. 5 Anti-bacterial mechanism of nitroimidazole 8
The second mechanism explains that the other nitroreductases present in H. pylori reduce a nitroimidazole (R-NO2) by a one-electron transfer step to a toxic free radical anion (R-NO2) - that can be metabolized in two ways (Fig. 5, Mechanism #2). First, the free-radical anion can be reoxidized to the original compound (R-NO2) by molecular oxygen with the production of superoxide (O2-). Since molecular oxygen may reverse the reduction step therefore these nitroreductases are called oxygen-sensitive. Theoretically, this process of reduction and reoxidation is repeated endlessly and is called `futile cycling'. The superoxide produced during this `futile cycling' is also toxic but can be eliminated (conversion to water) by superoxide dismutase (SOD) and catalase. The second way to metabolize the toxic free radical anion is another one electron transfer step to the more toxic nitrosoderivate that leads to the DNA damage (inhibiting bacterial nucleic acid synthesis) and resulting in subsequent bacterial cell death of the bacteria. We have compared (as reference) the results with the standard drugs. For the antibacterial activity, we have used the gentamicin drug and for the antifungal, we have used the griseofulvin antifungal agent to draw meaningful comparison, the tables in the manuscript were generated considering all these parameters. This is a preliminary screening. We found encouraging results that the compound is active against pathogens. Antimicrobial activities show that the title compound is moderately active towards antibacterial and antifungal activities. Antimicrobial results for the title compound (I) and Minimum inhibitory concentrations (MICs) for the bacterial and fungal isolates reported in the Tables 2 & 3 and range 180-300 µg/mL. Inhibition zones for the gram positive bacteria, gram negative bacterial and fungal isolates were in the range of 16 to 18, 13 to 17 and 15 to 18 mm respectively. The maximum zone of inhibition was observed for the gram positive bacteria on Staphylococcus aureus AB 188 and for fungal isolate Candida tropicalis while 13 mm for the Proteus mirabilis and Pseudomonas aeruginosa, 15 mm
9
for the Morganellamorganii, Candida galbrata and Candida kruzei, 16 mm for the Micrococcus leutus ATCC 9341, Staphylococcus aureus and Candida albicans ATCC 0383 and 17 mm for the Pseudomonas aeruginosa ATCC, Proteus vulgaris and Candida albicans. In summary, Antimicrobial results indicate that the title compound has potential to kill some of the highly pathogenic bacterial and fungal species that has a critical role in a wide range of cutaneous, pyogenic and urinary tract infections. [Gunay et al., 1999; Foroumadia et al., 2009] 3. Conclusions In summary, we report two new reaction conditions for the regio-specific synthesis of 1-methyl4-nitro-1H-imidazole. We have presented a complete characterization by using the EIMS, FTIR, 1
H-NMR,
13
C-NMR, and single crystal X-ray diffraction analysis. The synthesis methods have
the advantages of regio-specificity, easy purification and use of less corrosive reagents. The important peaks appearing in the EIMS, at the m/z = 127, 111, 97, 81, and 54 arise due to the loss of single electron, loss of O, NO, NO2, and Loss of NO2 & HCN respectively. The high intensity peak at m/z = 42 arises due to the loss of NO, CO and HCN from the molecular ion. We found that the title compound shows 1H-NMR (δ, 400 MHz, CDCl3) peaks at 7.76 (ArH), 7.42 (ArH), and 3.82 (N-CH3) ppm. The crystal structure of the compound (I) is stabilized by the intermolecular hydrogen bonds (C-H…O-NO, 2.57 Å) and molecules are linked to form sheets in zig-zag manner, but all the NO2 groups are headed in one direction. This unique mode of crystallization is clearly explained through DFT based structural properties. The compound (I) was found to be moderately active for the antibacterial and antifungal activities and maximum inhibition was examined for the gram positive bacteria on Staphylococcus aureus AB 188 and fungal isolate Candida tropicalis while the MICs of the title compound lies in the range of 180 to 300 µg/mL (observed concentration, at which micro-organisms growth was inhibited). 10
Antimicrobial results indicate that the compound (I) has the potential to kill some of the highly pathogenic bacterial and fungal species playing critical role in a wide range of cutaneous, pyogenic and urinary tract infections. [Gunay et al., 1999; Foroumadia et al., 2009] 4. Experimental Section 4.1. Materials and Methods All chemicals and solvents were of reagent grade and purchased from Aldrich and used without further purification. Thin layer chromatography was performed on Merck silica gel 60F254 plates and observed under UV. Melting point (uncorrected) was recorded in a glass capillary tube on Gallenkamp (sonyo) apparatus. FTIR spectra were recorded directly on Bruker FT-IR spectrophotometer (Vector 22) in the range of 4000-400 cm-1. EIMS spectra were recorded on JeolMS Route. 1H-NMR recorded on BrukerAvance 400 and 13C-NMR recorded on BrukerAvance 300 spectrophotometers at 400 and 75 MHz respectively.
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4.1. X-Ray Measurements Single-crystal X-ray diffraction data was collected on Bruker Smart APEX II, CCD 4-K area detector diffractometer [Siemens et al., 1996]. Data reduction was performed by using SAINT program. The structure was solved by direct method [Altomare et al., 1993] and refined by fullmatrix least squares on F2 by using the SHELXTL-PC package [Sheldrick et al., 1997]. The figures were plotted with the ORTEP program [Johnson et al., 1976]. 4.2. Synthesis of Compound (I) Nitrating Mixture: Inside hood, Nitrating mixture was prepared by mixing 4 mL non-fuming H2SO4 (98%) and 3.8 mL of non-fuming HNO3 (65%) in a round bottom flask with continuous stirring. HNO3 was added slowly with the help of dropping funnel into H2SO4 to maintain the temperature at 35-40 oC. 4.3. Synthesis methods for the 1-methyl-4-nitro-1H-imidazole: Method A: Inside hood, to a round bottom flask fitted with a reflux condenser, add nitrating mixture (7.8 mL) and let the mixture cool down to 5-7 oC in an ice-salt cooled water-bath. To the cooled mixture nitrating mixture drop-wise add 1-methyl-1H-imidazole (1.019 g, 12.42 mmol) with continuous stirring. After 0.5 h of addition; bath temperature was allowed to increase temperature (60-65 oC) for 8 h. After this time, the reaction mixture was allowed to cool down to room temperature and the reaction mixture was added to a beaker containing purified water with stirrer. Mixture was transferred to a separatory funnel and methylene chloride (CH2Cl2) was added (50 mL x 3) to separate the organic phase. The organic phase was dried on anhydrous Na2SO4 and filtered. The CH2Cl2 was removed by rotary evaporator to afford the off-white solid, which is recrystallized with small amount of CH2Cl2 to give title compound (I) in 20% yield. 12
Method B: Inside hood, to a 200 mL round bottom flask precooled to -10 oC, freshly prepared dry solid 2.5 g (23.58 mmol) urea nitrate [Shead et al., 1967] was added in small portions to the liquid 1-methyl-1H-imidazole (1.019 g, 12.42 mmol, density = 1.03 g/cm3) and H2SO4 (15 mL). After the complete addition, the temperature was slowly allowed to increase up to 10 oC and maintained for 6 h. The mixture was then poured into a beaker containing crushed ice made from distilled water. After the complete melt down, the acidic solution was neutralized by the aqueous Na2CO3 solution. The organic phase was separated with EtOAc and filtered through a small plug of dry silica and Na2SO4 in a Buchner funnel. EtOAc was used to wash the silica and Na2SO4, and the solvent was removed to obtain pure product. This process afforded the off-white title compound (I) in 45% yield. High quality single crystals suitable for X-ray Crystallography were grown in chloroform and hexane (1:1) by slow evaporation at room temperature; mp: 133-134 oC. FTIR (KBr, cm-1): v 3115 (=C-H arom. str.), 2925 (C-H aliphatic str.), 1424 (C=C ring str.), 1525, 1321 (NO2 Asym./Sym. str.), 982 (C-N). 1H NMR (400 MHz, CDCl3, δ, ppm): 3.82 (s, 3H, CH3), 7.42 (s, 1H, ArH, HC=C), 7.76 (d, J = 1.0 Hz, 1H, ArH, HC=N). 13C NMR (75 MHz, CDCl3, δ, ppm): 34.6 (CH3), 120.1 (ArCH, HC=C), 136.6 (ArCH, HC=N), 148.3 (ArC-NO2). EIMS m/z (%): 127 (M+., 100), 111 (10.6), 97 (5.4), 81 (12.4), 69 (12.7), 54 (11.1), 42 (78.5). Anal. Calcd. for C4H5N3O2: C, 37.80; H, 3.97; N, 33.06. Found C, 38.01; H, 3.93; N, 32.96 7.76 4.4. Antimicrobial Assays Antibacterial Activity: In the present study, synthetic compound (I) was monitored for the antibacterial activities against gram positive and gram negative bacteria. All the bacterial isolates were checked for purity and maintained on nutrient agar at 4 °C in the refrigerator prior to use.
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Antibacterial activity of the title compound (I) against pathogenic bacteria was determined by using agar-well method. Autoclaved Muller Hinton broth (Oxoid, Basingstoke-UK) was used to refresh the bacterial culture, later wells were punched into Muller Hinton Agar and 10 microliters of culture were poured into the wells [Perez et al., 1990]. All plates were incubated at 28+2 °C for 24-48 h and after incubation diameter of inhibition zone measured. Gentamicin antibiotic was used as a control agent in testing. 4.5. Antifungal Activity: The test organisms for this study were members of the 6 saprophytic fungi
Penicilliumsp,
Aspergilusflavus,
Aspergillusniger,
Fusariumsp,
Rhizopus,
Helminthosporum and Neurospora, 5 dermatophytic Microsporumcanis, Microsporumgypseum, Trichophytonrubrum,
Trichophytonmentagrophytes,
Trichophytontonsurans
and
6
yeast
including Candida albicans, Candida albicans ATCC 0383, Saccharomyces cerevisiae, Candida galbrata, Candida tropicalis, Candida kruzei. All the fungal isolates were checked for purity and maintained on Sabourd Dextrose Agar (SDA) (Oxoid, Basingstoke-UK) at 4 °C in the refrigerator until required for use. Antifungal activity of title compound against human, environmental and phyto-pathogenic fungi was determined by using agar-well method. Autoclaved distilled water was used for the preparation of fungal spore suspension and transferred aseptically into each SDA plates [Cappuccino et al., 2007]. All plates were incubated at 28+2 °C for 24-48 h and after incubation, diameter for the zone of inhibition measured. Gresiofulvin antifungal agent was used as a control. 4.6. Minimum Inhibitory Concentration (MIC): Minimum inhibitory Concentration (MIC) of synthetic compound (I) was determined by Micro broth dilution method using 96-well microtitre plate [Samie et al., 2005]. Stock solution of 10 mg/mL was prepared in chloroform, two fold serial dilutions were made in 100 µL broth (Oxoid, Basingstoke-UK) and subsequently 10 µL of 14
2 h old refresh culture matched with 0.5 Mac Farland index was inoculated into each well. One well served as antibiotic and antifungal control while others served as culture control to check MIC. Microtitre plate was incubated for 24 h at 37 ºC. The MIC showed no visible growth. Acknowledgements We highly acknowledge Nabiqasim Pharmaceutical Industries (PVT) LTD for the financial support during the research work.
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Buckingham, A. D., Basic Theory of Intermolecular Forces: Application to Small Molecules. In Intermolecular Interactions: From Diatomics to Biopolymers, Pullman, B., Ed., Wiley: New York, 1978.
Chuaqui, Claudio, Edmundo, Huang, Shan, Ioannidis, Stephanos, Shi, Jie, Su, MeiandSu, Qibin. Heterocyclic JAK kinase inhibitors. WO Patent 2010/038060A1, filed 29 September 2009, and publication 8 April 2010. Cappuccino, J. G., Sherman, N., Microbiology: A Laboratory Manual, 8th edition, London, Benjamin Cummings Publishing Company, 2007. Cosar, C., and Julou. L.,1959. Activity of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (8823 RP) against experimental Trichomonas vaginalis infection. Ann. Inst. Pasteur 96:238-241. Duddu, R., Zhang, M. -X., Damavarapu, R., Gelber, N., 2011. Molten-State Nitration of Substituted Imidazoles: New Synthetic Approaches to the Novel Melt-Cast Energetic Material, 1-Methyl-2, 4, 5-trinitroimidazole. Synthesis. 17, 2859-2864. Edwards, D. I., 1993. Nitroimidazole drugs-action and resistance mechanisms. I. Mechanisms of action. J. Antimicrob. Chemother. 31, 9-20. Foroumadia, A., Sorkhia, M., Moshafib, M. H., Safavia, M., Rineha, A., Siavoshic, F., Shafieea, A., Emamid, S., 2009. 2-Substituted-5-Nitroheterocycles: In Vitro Anti-Helicobacter pylori Activity and Structure-Activity Relationship Study. Medicinal. Chemistry. 5, 529-534. Gunay, N. S., Capan, G., Ulusoy, N., Ergenc, N., Otuk, G., Kaya, D., 1999. 5-Nitroimidazole derivatives as possible antibacterial and antifungal agents. Farmaco. 54, 826-831. Helal, C. J., Kang, Z., Lucas, J. C., Gant, T., Ahlijanian, M. K., Schachter, J. B., Richter, K. E. G., Cook, J. M., Menniti, F. S., Kelly, K., Mente, S., Pandit, J., Hosea, N., 2009. Potent and cellularly active 4-aminoimidazole inhibitors of cyclin-dependent kinase 5/p25 for the treatment of Alzheimer's disease. Bioorg. Med. Chem. Lett. 19, 5703-5707. Johnson, C. K., ‘ORTEPII’ Report ORNL-5138. Oak Ridge National Laboratory, TN, USA, 1976. 17
Katritzky, A. R., Scriven, E. F. V., Majumder, S., Akhmedova, R. G., Vakulenko, A. V., Akhmedov, N. G., Murugan, R., Abboud, K. A., 2005. Preparation of nitropyridines by nitration of pyridines with nitric acid. Org. Biomol. Chem. 3, 538-541. Katritzky, A. R., Scriven, E. F. V., Majumder, S., Akhmedova, R. G., Akhmedov, N. G., Vakulenko, A. V., 2005. Direct nitration of five membered heterocycles. Arkivoc. 3, 179-191. Kikuchi, R., 2001. Environmental Management of Sulfur Trioxide Emission: Impact of SO3 on Human Health, Environmental. Management. 27, 837-844. Luijten, W. C. M. M., Thuijl, J. V., 1981. Mass spectrometry of nitroazoles: 2-the mass spectra of methyl substituted nitroimidazoles. Org. Mass. Spectrom. 16, 199-208. Lee, C., Yang, W., Parr, R. G., 1988. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B. 37, 785-789. McCalla, D. R., Reuvers, A., Kaiser, C., 1971. Breakage of Bacterial DNA by Nitrofuran Derivatives. Cancer research. 31, 2184-2188. Mulliken, R. S., 1955. Electronic Population Analysis on LCAO-MO Molecular Wave Functions. J. Chem. Phys. 23, 1833-1840. Pardanani, A., Lasho, T., Smith, G., Burns, C. J., Fantino E., Tefferi, A., 2009. CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients. Leukemia. 23, 1441-1445. Perez, C., Paul, M., Bazerque, P., 1990. Antibiotic assay by agar-well diffusion method. Acta. Biol. Med. Exp. 15, 113-115. Schofield, K., Swain, T., 1948. Nitration of Simple Heterocycles. Nature. 161, 690-691. Siemens. SMART and SAINT. Siemens Analytical X-ray Instruments Inc. WI, USA: Madison, 1996. Sheldrick G. M., SHELXTL-PC (Version 5.1). In: Siemens Analytical Instruments Inc. WI, USA: Madison, 1997.
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19
From Katritzky et al., 2005 N
F F
O F
O O
O2N
F F Conc. HNO3 F
0-5 oC, 12 h
N
N
N
+
O2N
N 39%
1-methyl-1H-imidazole
N 22%
Experimental
From Duddu et al., 2011 O2N N
0-15 oC, 1 h
N
N
N
Fum. H2SO4/Fum. HNO3
N
+
O2N
N
Isolated mixture 60% 22% (12%) (46.8%) 78% Yields are only Based on NMR Analysis
O2N N Fum. H SO /Fum. HNO 2 4 3 N
N
110-120 oC, 2 h
N
N + O2N
25% N Fum. H SO /Fum. HNO 2 4 3 N
120-150 oC
N
O2N + O2N
N N
O2N +
5%
13%
N N 2%
O2N NO2 O2N
N N
NO2
1%
No nitro product due to decomposition
This Work: Method A: O2N N 98%H SO -65% HNO 2 4 3 60-65 oC, 8 h
N Method B:
N
(I) 20% 1-methyl-4-nitro-1H-imidazole O2N
N N
N
H2SO4-Ureanitrate -10-10 oC, 6 h
N N
(I) 45%
Scheme 1 Synthesis of compound (I).
Figure 1 ORTEP diagram of compound (I) with displacement ellipsoids drawn at 50% probability level. 20
Figure 2 The crystal packing of compound (I); Some Hydrogen atoms were omitted for clarity.
Figure 3 the top view for the optimized structure (a), side view (b) And Mulliken charges (c) for the 1-methyl-4-nitro-1H-imidazole
21
Figure 4 proposed crystallization process for 1-methyl-4-nitro-1H-imidazole
22
Table 1 Crystal Data and Structure Refinement for Compound I Empirical formula
C4H5N3O2
Formula weight
127.11
Temperature (K)
273 (2)
Wavelength (Å)
0.7103
Crystal system
Monoclinic
Space group
P21/c
Unit cell dimensions a (Å)
5.8223(15)
b (Å)
14.882(4)
c (Å)
6.5873(18)
α ( ̊)
90
β ( ̊)
94.705 (7)
γ ( ̊)
90
Volume (Å3)
568.9(3)
Z
4
Calculated density (mg/m3)
1.484
Absorption Coefficient (mm-1)
0.71073
Maximum and minimum transmissions
0.9976 and 0.9417
F (000)
264
Crystal size (mm3)
0.50 x 0.10 x 0.02
Theta ranges for data collection ( ̊)
2.74 – 25.5
Limiting indices
-6
