Synthesis and Antibacterial Studies of Some Alkynylated Benzo [ a ] phenoxazin-5-one and 1 , 4-Naphthoquinone Derivatives

Organic synthesis has been greatly enhanced by the use of reactions catalyzed by transition metal complexes especially palladium and this has led to the development of new methods of constructing carbon-carbon bonds and carbon heteroatom bonds. One of the most general and widely used palladiumcatalyzed cross-coupling reactions is the alkynylation of aryl halides using terminal alkynes, generally known as the Sonogashira cross-coupling reaction. Other palladium catalyzed coupling reactions that have tremendously changed the face of organic synthesis include Heck-Mizoroki coupling reaction, Buchwald-Hartwig coupling reaction, SuzukiMiyaura coupling reaction and Negishi coupling reaction. The Sonogashira cross-coupling reaction has the advantage of occurring at or slightly above room temperature unlike the harsh conditions required for the alternative Castro-Stephens coupling. It tolerates a wide range of functional groups, making the process a useful tool in total synthesis. However, some drawbacks are experienced when copper co-catalyst is used in the reaction. Researchers have attempted to overcome this by modifying the reaction conditions. Some of the modifications that have proved successful include the use of pyrrolidine as a base with a platinum catalyst in the absence of copper salt, the addition of tetra-n-butylammonium fluoride (TBAF) as a base under a nitrogen atmosphere without using copper or Synthesis and Antibacterial Studies of Some Alkynylated Benzo[a]phenoxazin-5-one and 1,4-Naphthoquinone Derivatives

amine 7 and the use of hydrogen to degas the reaction 5 . The Sonogashira reaction is used in the synthesis of various organic compounds and in the production of pharmaceuticals, agricultural chemicals and natural products. In the present study, the modified Sonogashira cross-coupling reaction is used to synthesize alkynylated derivatives of 6-chloro-5H-benzo[a]phenothiazin-5-one and 2,3-dichloro-1,4-naphthoquinone.
Interest in the synthesis of phenoxazine and its derivatives has continued to grow due to the various biological properties as well as industrial applications. Since the discovery of the parent ring phenoxazine by Bernthsen 8 , researchers have continued to engage in structural modifications to improve the biological properties, reduce side effects and open new areas of applications. Such modifications have led to an array of derivatives of pharmacological and industrial interest. Some of the pharmacological applications of phenoxazine and its derivatives include as anti-epileptic 9 , antitumour 10,11 , anticancer 12 , antituberculosis 13 , antibacterial 14,15 , anthelminthic 16 , spasmolytic, C.N.S. depressants 17,18 , herbicides, tranquillizers, sedatives 19 and parasiticidal agents 20 . Phenoxazine derivatives have also been used as antioxidants 21 , biological stains 22,23 , acid-base indicators 24 and bromometric and stannometric redox indicators [25][26][27][28] .
Naphthoquinone and its derivatives have attracted much interest also due to their biological activities. The naphthoquinone fragments are often encountered in natural biologically active compounds. Natural naphthoquinone derivatives found in plants, such as Juglone Lawsone, Plumbagine and Lapachol, demonstrate antibacterial effect on several species of aerobic and anaerobic organism 29,30 . The natural naphthoquinone products alkannin and shikonin and their derivatives are also active against Gram-positive bacterial such as Staphylococcus aureus, Enterococcus faecium and Bascillus substilis, but they are inactive against Gram-negative bacteria 31 . Reports have also shown that 2,3-disubstituted-1,4-naphthoquinones have potent antibacterial 32 , antifungal 33 , anticoagulant 34,35 , antimalarial 36 , cytotoxic and anticoplastics activities 37,38 . It has been observed that the extensive use of antibiotics has resulted in increased prevalence of antibiotic resistant bacteria. This may render the current antimicrobial agents insufficient to control some bacterial infectious with time 39 . There is therefore the need to synthesize new derivatives of antimicrobial phenoxazines and naphthoquinones to improve their pharmacological properties; peradventure they could form part of the solution to the growing problem of the rising new infectious diseases and ever-increasing multi-drug resistance of microbial pathogen 40 .
Despite the synthesis and various biological activities of phenoxazine derivatives, which abound in the literatures, the antimicrobial study is still under studied. Moreover, synthesis and biological activities of alkynylated phenoxazine and 1,4naphthoquinone derivatives is scarcely and poorly investigated. Herein is reported the successful synthesis of biologically active alkynyl derivatives of 6-chloro-5H-benzo[a]phenoxazin-5-one and 2,3-dichloro-1,4-naphthoquinone via modified Sonogashira cross-coupling reaction.

EXPERIMENTAL
Some of the reactions were carried out under an atmosphere of nitrogen. The intermediate 6-chloro-5H-benzo[a]phenoxazin-5-one (3) was prepared following the literature procedure 41 . Melting points were determined with Fischer John's melting point apparatus and are uncorrected. UV and visible spectra were recorded in ethanol on a unicon UV-2500PC spectrophotometer using matched 1 cm quartz cells; absorptions are measured in nanometer (nm), the figure in parenthesis are the molar absorptivity coefficient (ε) value. IR spectra were recorded on 8400s Fourier transform infrared (FTIR) spectrophotometer using KBr disc and in some cases NaCl disc and are reported in wave numbers (cm -1 ). Nuclear magnetic resonance ( 1 H NMR and 13 C NMR) were determined using Joel 400 MHz at Strathclyde University, Scothland. Chemical shifts are reported in delta (δ) scale. The antimicrobial screening was done at the Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Nigeria. All reagents were of analytical grade and were used as supplied. 2-Aminophenol, bis(triphenylphosphine)palladium(II)chloride [PdCl 2 (PPh 3 ) 2 ], tetrabutylammonium fluoride trihydrate (TBAF·3H 2 O), 2,3-dichloro-1,4-naphthoquinone and all the terminal alkynes were purchased from Sigma Aldrich Chemical Company in sure-seal bottles.

2-Chloro-3-(3-hydroxy-3-methylbut-1-yn-1-yl)-1,4naphthoquinone (6b):
Sensitivity testing: The antibacterial activity of the alkynylated compounds were determined using agar diffusion well method described by Perez et al. 42 . A single colony of each test isolate was suspended in 2 mL sterile Mueller-Hinton agar. The suspension of each isolate was standardized by adjusting to 0.5 McFarland turbidity standards equivalent to approximately 10 8 cfu/mL and used to inoculate the surface of the nutrient agar. The inoculated agar surface was allowed to dry and 6 mm diameter cork borer was used to bore wells (7 mm in diameter and 2.5 mm deep) into the agar. A 20 mg/mL concentration of each compound was prepared by dissolving 0.04 g of each in 2 mL dimethyl sulfoxide (DMSO) and 0.05 mL of each solution was delivered into each agar well. The plates were incubated at 37 °C for 24 h. The diameters of the inhibition zones around the wells were measured with metre rule to the nearest whole millimetre. The test was done in triplicate and the mean inhibition zone calculated.

Minimum inhibitory concentration (MIC):
The minimum inhibitory concentration (MIC) of the alkynylated compounds and the reference standard was determined using agar well diffusion method described by Ojo et al. 43 . Serial dilutions of the synthesized products were prepared from 2 mg/mL solution of the products synthesized to give final concentrations ranging from 10 mg/mL to 0.625 mg/mL. 1 mL of each serial dilution was mixed with 19mL of sterile Mueller-Hinton agar maintained at 45 °C, poured into a sterile plate and allowed to set. The amended culture media was spot-inoculated with 0.025 mL of an overnight broth culture of the test bacterial strains and incubated at 37 °C for 24 h. The plates were examined for presence of visible growth. The minimum concentration that completely inhibited growth of the organisms was taken as the minimum inhibition concentration of the respective alkynylated compounds. The procedure was repeated for the reference standard (gentamycin and ampicillin).

6-Chloro-5H-benzo[a]phenoxazin-5-one derivatives:
The intermediate 6-chloro-5H-benzo[a]phenoxazin-5-one (3) was prepared according to a reported procedure by base catalyzed reaction of 2-aminophenol (1) with 2,3-dichloro-1,4-naphthoquinone 2 at 80 °C giving the product of interest 3 as a yellow solid in 97 % yield (Scheme-I). The first step in the reaction is the abstraction of proton from the hydroxyl group of the phenol 1 by the base. The phenoxide ion 1a formed mounts a nucleophilic attack on the 2,3-dichloro-1,4-napthoquinone 2 by displacing one of the halogen atoms to form a diaryl intermediate 7. By doing so, cyclization took place by a second nucleophilic attack from the amino group on the carbon atom of the carbonyl group to form a second intermediate 8, which on elimination of water molecule yields 6-chloro-5Hbenzo[a]phenoxazin-5-one 3 (Scheme 4). Direct nucleophilic alkynylation of compound 3 with various terminal alkynes 4 was done in the absence of copper(I) salt and amine. The reaction was performed in the presence of bis(triphenylphosphine) palladium(II) chloride as a catalyst and tetrabutyl ammonium fluoride trihydrate as a base under nitrogen atmosphere to yield the corresponding 6-alkynyl-5H-benzo[a]phenoxazin-5-ones (5a-e) (Scheme-II). The proposed mechanism follows the modified Sonogashira coupling reaction which begins with the oxidative addition of Pd(0) with 6-chloro-5H-benzo[a]phenoxazin-5-one 3 to form ArPdX complex 9 (Ar = phenoxazine molecule, X = Cl). The application of electron-rich amino phosphine ligand, bis(triphenylphosphine), makes this step easier. This first step is followed by the activation of the terminal alkyne. Because no copper salt was employed and the base is not strong enough to abstract a proton from the alkyne, a transmetallation step could be excluded 10 . The terminal alkyne C-H bond activation is accomplished by the coordination of the alkyne to the ArPdX complex. Upon coordination, the C-H bond is weakened and HX is removed in the presence of the base to form complex 10, which subsequently undergoes reductive elimination to afford the product of interest 5 and regenerates the catalyst (Scheme-V). The electron-rich and bulky aminophosphine ligand is likely to play key roles in facilitating the reductive elimination step. It is also believed that the tetra-n-butylammonium fluoride might have played some beneficial roles in the reaction, which includes the activation of the active Pd(0) species with the formation of anionic Pd species and deprotonation of the acidic hydrogen in the alkyne 7 .
The structures of the 6-alkynyl-5H-benzo[a]phenoxazin-5-ones (5a-e) were established on the bases of FT-IR and NMR spectroscopy. The IR spectra of the compounds 5a-e showed, in each case stretching band of C=O and C=N, C≡C and C=C aromatics in the region of 1667-1635 cm -1 , 2365-2220 cm -1 and 1576-1450 cm -1 respectively. The peaks around 1283-1040 cm -1 that are characteristic of phenoxazine ring C-O-C and C-N stretching frequency were so assigned. The 1 H NMR spectra in each case, showed signals at δ 7.50-8.68 ppm assigned to Ar-H. The 13 C NMR spectra further support the assigned structures. The peaks at 176-177.29 ppm and 123.49-148.06 ppm were attributed to C=O and C=C aromatics respec-tively. Other peaks are in agreement with the rest of the carbons in the structure of the compounds.
Compounds 6a-e were characterized based on their FT-IR, 1 H NMR and 13 C NMR spectra. In each case, the IR of the compounds showed prominent absorption signals due to the stretching vibrations of C≡C, C=O, C=C aromatics and Cl at 2365-2230 cm -1 , 1678-1654 cm -1 , 1486-1455 cm -1 and 755-629 cm -1 respectively. From the 1 H NMR spectra the signals at δ 7.10-8.09 ppm were assigned to aromatic protons while δ 0.92-3.17 ppm were attributed to aliphatic protons. The 13 C NMR spectra showed the absorption bands due to C=O, C=C aromatics, C≡C and Cl at 176-176.49 ppm, 125-143.03 ppm, 89 ppm and 58.09-58.11 ppm respectively. Every other peak is consistent with the assigned structures.
Antimicrobial activities: Due to the considerable biological and pharmaceutical activities of phenoxazines and naphthoquinones, it was necessary to evaluate the antibacterial activity of the alkynylated products (5a-e and 6a-e). The compounds were screened in vitro for antibacterial activities against Gram-positive bacterium (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli I, Escherichia coli 12 and Klebsiella pneumonia) using the disc-agar well diffusion method 42 .
The results of the antimicrobial activity of the compounds are expressed as inhibition zone diameter (IZD, mm) and minimum inhibition concentration (MIC, mg/mL) and shown in Tables 1 and 2 respectively. All the tested compounds showed at least some activity against one or more tested microorganisms. Table-1 shows the IZD produced by each compound against the bacterial organism at 10 mg/mL concentration. The IZD ranged between 9 and 17 mm in diameter; the higher the IZD, the higher the sensitivity. All the synthesized compounds were very active against K. pneumonia. However, compounds 6c and 5d showed activity only to Klebsiella pneumonia with IZD of 15 mm; other bacteria strains were resistant to them. Compound 6d has the highest activity against Klebsiella pneumoniae with IZD of 17 mm. All the tested compounds were active to Staphylococcus aureus except compounds 6c and 5d. Pseudomonas aeruginosa was resistant to compounds Reductive elimination Scheme-VI: Proposed mechanism of the preparation of 2-chloro-3-alkynyl-1,4-naphthoquinones 5a, 6a, 5c, 6c, 5d and 5e but was sensitive to other compounds with IZD ranging between 9 and 11. E. coli 1 was resistant to compounds 5b, 6b, 6c and 5d. E. coli 12 was resistant to compounds 5c, 6c and 5d but sensitive to others. The Grampositive bacterium Staphylococcus aureus was sensitive to all the compounds (IZD 11-13 mm) except 6c and 5d. Minimum inhibitory concentration (MIC) of the compounds was also determined by agar-well diffusion method already described 43 with various concentrations ranging between 10 mg/mL to 0.625 mg/mL. The lowest concentration of each compound that produced no zone was regarded as MIC. Hence, essence of MIC is to determine the least concentration of the compounds that can inhibit the growth of the micro-organism. Table-2 also compares the MICs of the synthesized compounds with those of some standard drugs (ampicillin and gentamycin) that are known to have biological activity. Compared to standard drugs, compounds 5a-e and 6a-e showed significant antibacterial activity against both Gram-positive and negative bacteria. Compound 6d (MIC 0.3 mg/mL) with electron donating group (3-hydroxyprop-1-yne) at position 3 of the quinone ring exhibited highest and excellent activity against Gram-negative bacterium (K. pneumonia). While compound 6b (MIC 1.05 mg/mL) with also an alkynol group at position 3 showed very good and highest activity against the Gram-positive bacterium (S. aureus). All the synthesized products are very active against K. pneumonia. The MIC for gentamycin and ampicillin is 5 mg/mL, which is very high when compared to MIC values of the synthesized compounds, which ranges from 0.30 to 1.26 mg/mL. The MIC value of gentamycin and ampicillin against E. coli I and E. coli 12 is 100 mg/mL, which is still higher than the MIC values for most of the synthesized compounds. The same explanation goes for P. aeruginosa and S. aureus showing that the synthesized phenoxazines and naphthoquinones are highly biologically active, hence they are of pharmaceutical interest.
From structure activity relationship, it was observed that in some cases the antimicrobial activity of compounds 5 and 6 were the same despite the difference in structure. For instance, compounds 5a and 6a (MIC 1.78 mg/mL) and 5e and 6e (MIC 1.99, 1.26 mg/mL) have the same antagonistic effect both on E. coli 1 and E. coli 12 respectively (Table-2). Similarly compounds 5b and 6b (MIC 0.79 mg/mL), have the same antibacterial activity against K. pneumonia. Compounds 5a-e are structurally related with compounds 6a-e, but the only difference is that compounds 5a-e were obtained by first coupling 2,3-dichloro-1,4-naphthoquinone (2) with 2-aminophenol (1) before alkynylating with terminal alkynes whereas compounds 6a-e were obtained by direct alkynylation of 2,3-dichloro-1,4naphthoquinone (2). Considering the fact that highly resistant bacterial strains were used in this study, the potency of the test compounds can be exploited to serve as a chemotherapeutic agent.

Conclusion
The study has shown that palladium catalyzed Sonogashira coupling reaction offers excellent routes to the synthesis of alkynylated angular phenoxazinones and alkynylated napthoquinones. These coupling reactions proceeded excellently under copper-, amine-and solvent free conditions. High yields of products at short time were also recorded. The new compounds were characterized based on FT-IR, 1 H NMR and 13 C NMR spectra. Antimicrobial screening of the synthesized compounds revealed that they have high potency against the test micro-organisms. Therefore, these compounds could be of pharmaceutical interest if properly harnessed.