Anticancer and Antimicrobial Activity of Some New 2,3-Dihydro-1,5-benzodiazepine Derivatives

. A series of 2,3-dihydro-1,5-benzodiazepine derivatives have been synthesized and characterized using IR, NMR, GC-MS, single crystal XRD, and microanalysis. Te results of their antibacterial activity against methicillin-resistance Staphylococcus aureus , Escherichia coli , Klebsiella pneumoniae , Bacillus subtilis , Streptococcus mutans , Pseudomonas aeruginosa , Salmonella typhi , and Streptococcus pyrogens indicated that most of the compounds were bacteriostatic (0.125 − 4mg/mL) and also exhibited good bioflm inhibition (0.21 − 72.69%). Te compounds were found to be synergistic when used in combination with other antibiotics. Te antiproliferative and cytotoxic efects were also investigated against PC-3 prostate cancer and RAW 264.7 macrophage cell lines, respectively, using the MTTassay. Apart from compounds 6 and 7 , a good number of the compounds ( 1 , 2 , 3 , 4 , 5, and 8 ) were selectively toxic to the prostate cancer cells at 20 µ M, whilst sparing the normal cells. Compound 3 demonstrated the highest antiprostate cancer efect by reducing the viability of PC-3 cells to (13.75%), which was followed by compounds 1 (47.72%), 2 (48.18%), 4 (62.61%), 5 (66.70%), and 8 (69.55%).


Introduction
Te synthesis of a series of 2,3-dihydro-1,5-benzodiazepines by the condensation reaction of o-phenylenediamine and a group of substituted chalcones in the presence of DMF as a solvent has been reported.When screened for their antibacterial and antifungal activities, the compounds demonstrated signifcant antimicrobial activity, moderate antibacterial and antifungal activity, and high potency against E. coli, S. typhi, and A. oryzae [1].A series of benzodiazepines have also been synthesized and screened for their anti-infammatory, analgesic, and antibacterial activities.Some of the compounds showed potent antiinfammatory, antibacterial, and analgesic activities, the bioactivity of these compounds was attributed to the presence of the oxadiazole ring [2].
Tin layer chromatography was used to follow the reaction using ethyl acetate: petroleum ether (1 : 1).A Bruker Avance AV 400 MHz spectrometer was used to obtain the 1 H NMR and 13 C NMR spectra operating at 400 MHz for 1 H and 100 MHz for 13 C with dimethyl sulfoxide as solvent and tetramethylsilane as internal standard.Te chemical shifts were expressed in ppm.A Bruker Platinum ATR Spectrophotometer Tensor 27 was used to obtain the FT-IR spectral data.Te elemental analysis was performed using a Vario Elementar Microcube ELIII.A Stuart Lasec SMP30 was used to determine the melting points, and were reported uncorrected.To obtain the masses of the compounds Perki-nElmer GC Clarus 580 Gas Chromatograph interfaced to a Mass Spectrometer PerkinElmer (Clarus SQ 8 S) equipped with ZB-5HTMS (5% diphenyl/95% dimethyl poly siloxane) fused capillary column (30 × 0.25 μm ID × 0.25 μm DF) was used polysiloxane.

X-Ray Crystallography.
Te single crystal X-ray diffraction analysis of compound 9 was carried out at 296 K with the aid of a Bruker Kappa Apex II difractometer with monochromated Mo Kα radiation (λ � 0.71073 Å).Data collection was carried out using APEXII and whilst SAINT was used for cell refnement and data reduction [17], the numerical method implemented in SADABS was used for the correction of absorption efects [17].Dual-space methods using SHELXT-2014/7 [18] were used to solve the structure and refned by least-squares procedures using SHELXL-2014/7 [19], with SHELXLE [20] as a graphical interface.Refnement of all nonhydrogen atoms was achieved anisotropically.Carbon-bound H atoms were placed in calculated positions (C-H 0.95 Å for aromatic carbon atoms and C-H 0.99 Å for methylene groups) and were included in the refnement in the riding model approximation, with Uiso (H) set to 1.2Ueq (C).Te H atoms of the methyl groups were allowed to rotate with a fxed angle around the C-C bond to best ft the experimental electron density (HFIX 137 in the SHELXL program [19], with Uiso (H) set to 1.5Ueq (C).Te H atoms of the hydroxyl groups were allowed to rotate with a fxed angle around the C-O bonds to best ft the experimental electron density (HFIX 147 in the SHELXL program [19], with Uiso (H) set to 1.5Ueq(O).Nitrogen-bound H atoms were located on a diferent Fourier map and refned freely.

Determination of Antibacterial Activity of the
Benzodiazepines.Te antibacterial activity of the benzodiazepine derivatives was evaluated using the following assays: Kirby-Bauer agar well difusion, and the broth microdilution [21][22][23].
2.6.Agar Well Difusion.About 20 mL of sterile Muller-Hinton agar was poured out into Petri dishes to set.Tese agar plates were then inoculated with the test bacteria of concentrations 1 × 10 8 colony forming units (CFU)/mL.Eleven (11) wells were immediately made in each plate using a cork borer (No. 3, 5 mm).Next, these wells were flled with 100 µL of 5 mg/mL stock solution of each compound prepared with 20% methanol to give a fnal concentration of 0.5 mg or 500 µg per hole.Methanol was used as a negative control, whereas chloramphenicol (30 µg/disc) was used as a positive control in this study.Te agar plates were fnally subjected to incubation at 37 °C for 24 h, and the zones of inhibitions were recorded.Te procedure was carried out in triplicates.

Determination of Minimum Inhibitory Concentrations (MICs).
Te minimum inhibitory concentrations (MIC) of the test benzodiazepines were obtained by the micro broth dilution method using 96-well microtiter plates according to the protocol previously outlined by the Clinical and Laboratory Standards Institute, 2011 [22,23], with slight modifcations.A stock solution of 2 mg/mL of each compound in methanol as a diluent was used to prepare 10 diferent concentrations using serial dilution by mixing them with double-strength Mueller Hinton Broth (Oxoid Limited, United Kingdom) in the 96-well plates (Citotest Labware Manufacturing Co. Ltd., Jiangsu, China) to arrive at concentrations ranging from 0.0039 to 1.0 mg/mL per well.Wells 11 and 12 on each row of the plates served as positive control (broth + organism only) and negative control (broth with no organism), respectively, for each bacterial strain on each column.Tis procedure was executed for the antibiotics; ciprofoxacin, tetracycline, ampicillin, chloramphenicol, fuconazole, and nystatin at concentrations ranging from 128.0 to 0.125 µg/mL in separate plates against all the bacteria.Te addition of 100 µL of each of the 0.5 McFarland standardized test organisms followed, after which the plates were subjected to incubation at 37 °C for 24 hours for all the bacterial strains.Te MIC values were then noted and recorded by visual analysis after tetrazolium chloride (TTC), (0.1 g/mL) dye for 10 minutes, and the MIC was recorded as the least concentration which did not change colour from colourless/light yellow to red/pink.

Determination of Minimum Bactericidal Concentration (MBC).
In order to confrm if the synthesized benzodiazepine derivatives would be able to kill the diferent bacterial strains, the MBCs against the test strains were determined.

Heteroatom Chemistry
Aliquots from each well from susceptibility testing assays were pipetted into the plates containing nutrient agar and then incubated for 24 hours at 37 °C.Te plates were then checked for the presence or absence of bacterial growth in the nutrient agar [24].
2.9.Evaluation of Synergistic Efects of the Test Benzodiazepines and Test Drugs.Combinatory efects of the benzodiazepines and drugs/antibiotics were assessed using the checkerboard test against the strains of bacteria under study with slight modifcation from the method reported [25][26][27].Briefy, solutions with diferent proportions of each benzodiazepine (fnal volume of 200 μL) were prepared from the MIC solutions of each test benzodiazepine and the individual drugs/antibiotics (1 mg/mL).Te antibacterial activity of each compound and antibiotics combination was determined as described for the MIC determination.Te fractional inhibitory concentration index (FICI) was obtained according to the following equation: Te interaction between the test benzodiazepines and drug/antibiotics was considered synergistic if the FIC index was ≤0.5, partial synergistic if FIC index was >0.5 and <1, additive if FIC index was �1, no diference if the FIC index was >1 and ≤4, and antagonistic if the FIC index was >4.0.

Determination of Antibioflm Activity of
Benzodiazepines.Te activity of the benzodiazepines against bacterial bioflms (inhibition of bioflm formation) was evaluated using a 96-well microtiter plate for bacterial bioflm formation and susceptibility testing [28,29].In brief, the media, double-strength Mueller Hinton broth (Oxoid Limited, United Kingdom) was dispensed with multipipette into each well of the fat-bottom 96-well microplate (Citotest Labware Manufacturing Co. Ltd., Jiangsu, China).Each benzodiazepine compound (100 µL) was added to column 1 and diluted until column 10 at concentrations ranging from 0.0039 to 1.0 mg/mL.Afterwards, 100 µL of the microbial suspension of 1 × 10 6 cells/mL was pipetted into the wells of columns 1-11 to arrive at a fnal volume of 200 µL.To each of the solutions in the microplate wells, 2 µL of sterile-fltered TTC, 5% (wt/vol) solution was added to attain fnal TTC concentrations of 0.05% (wt/vol).Te microtiter plates were then subjected to incubation for 24 h at 37 °C.Later, the mixtures were judiciously enunciated, ensuring that there was no interaction with the preformed bioflm, which was then fowed over with PBS (100 µL) two times in order to take away planktonic and nonadherent cells.Te metabolic activity after the antibacterial (benzodiazepines) treatment was assessed by the TTC (CDH) reduction assay [28,29].Finally, plates were taken through spectrophotometry at an OD of 492 nm using a microtiter plate reader, and the percentage of bioflm inhibition was determined using equation (2).Te IC 50 s values were then calculated.%biofilm inhibition � optical density (OD) of control − OD of treatment OD of control   × 100. (2)

Determination of Antiprostate Cancer Efect of
Benzodiazepines.Human (PC-3) prostate cancer cells (2 × 10 5 cells/well) and RAW 264.7 mouse macrophage cells (2 × 10 5 cells/well) were plated in 96-well plates and kept at 37 °C in a humidifed atmosphere of 5% CO 2 and 95% air for 24 h, after which the cells were treated with 20 µM of each compound for 48 h. 100 nM of paclitaxel was used as the positive control.Twenty microliters (20 µL) of MTT solution was added to each well, and incubated for 4 h after which its absorbance was measured at 517 nm (reference wavelength at 670 nm) using a microplate reader (DNM-9602).Cell viability was expressed as a percentage of untreated controls (100%).

Results and Discussion
3.1.Chemistry.Te synthesized compounds were obtained by heating the reagents in methanol for 8 h under refux.Scheme 1 gives a synthetic scheme for the synthesis of the 2,3-dihydro-1,5-benzodiazepine derivatives.Te unsubstituted benzodiazepine derivative which was the precursor for the other compounds was obtained by the reaction of ophenylene diamine with 4-methypent-3-en-2-one.Te reaction was followed by thin-layer chromatography until the disappearance of the spot for the starting material.Most synthesized benzodiazepines have their substituents attached to diferent sites on the diazepine moiety.In this Heteroatom Chemistry work, we have explored the efect of introducing a twocarbon rigid (alkene) gap between diferent substituents and the benzodiazepine ring and studied their activity.Te synthesis of 3-aceyl-bearing-benzodiazepines has been achieved by microwave irradiation.Some of the synthesized compounds have a rigid alkane as part of the substituent.Te compounds were tested for their metal-scavenging activity [30].Palladium (0) has been used to catalyze a series of 1,4benzodiazepinones via a domino sequence by converting Nallenamides and aryl halides in the presence 3-Iodopyridine to give products with an alkene rigid frame as part of the substituents attached to the benzodiazepine ring [31].
Compound 1 reacted with diferent aldehydes in methanol to give the fnal products.Diferent reagents were used to confrm the pathway to the product.In that study, the products were accessed via a one-pot synthesis [32].Te use of electron-withdrawing groups and electron-donating groups were tested for the ability of the diferent compounds to give the alkene-containing benzoxazepines.Te conversion of aldehydes to alkenes is widely reported and the mechanism or pathway is well known [33][34][35][36][37][38][39].In this work, we present a conventional method for making alkenelinked benzodiazepines and their antimicrobial and anticancer activities.Generally, among these compounds the derivatives with electron-donating groups gave lower yields.Te synthesized compounds are listed in Table 1. Figure 1 gives the 1 H NMR spectrum for compound 1, three signals were observed for the 4 protons at 6.95, 6.90, and 6.83 ppm, a signal for a methyl group was observed at 2.23 ppm whilst a signal for the methylene group was observed at 2.16 ppm.Upon addition of 4methoxybenzaldehyde to compound 1 in methanol and heating under refux compound 2 was obtained.Five signals were observed for 10 protons at 7.69, 7.41, 7.06, and 6.93 ppm, confrming the incorporation of six more aromatic protons (Figure 2).A signal for a methoxy group was observed at 3.87 ppm, accompanied by the loss of the signal for the methyl group in Figure 1 confrming the incorporation of the 4-methoxybenzaldehyde into the molecule to form compound 2.
Te method of synthesis was optimized, the condition and time that gave the best yields were used for subsequent derivatives.Table 2 gives the scope and yields of the synthesized compounds.
Te proposed reaction mechanism for the synthesis of 2,3-dihydro-1,5-benzodiazepine derivatives is presented in Scheme 2. Te reaction is proposed to proceed by a proton abstraction from the methyl group connected to the imine on the benzodiazepine ring by the methoxide produced from methanol in 2a, the loss of a proton leads to the formation of a carbanion.Te carbanion formed attacks the carbonyl carbon of the aldehyde in 2b, leading to the formation of a hydroxyl group in 2c.Abstraction of a proton from the methylene group by the methoxide leads to 2d an intermediate, with the partial formation of the double bond.Te subsequent loss of an OH leads to the formation of the fnal product 2e.A similar mechanism has been reported and the path to the product is the same regardless of the heteroatom used [32].
Te infrared spectrum of compound 1 showed the presence of characteristic absorption peaks at 3309 and 2966 cm −1 for the amine (N-H) and aliphatic C-H stretches, respectively.Peaks were observed at 1607, 1555, and 1434 cm −1 for the C�N, C�C, and C-N absorptions, respectively.Compound 1 has been reported to be synthesized by diferent methods [30].For compounds 1-9 (Table S1   13 C NMR, DEPT, and the GC-MS of the synthesized compounds.

Antibacterial Susceptibility Testing of the Test
Benzodiazepines.Te study began with a preliminary test of the diferent benzodiazepines against the test bacteria, the results as shown in Figure 4 indicated that all the compounds were good antibacterial agents when compared to the standard control, chloramphenicol.At concentrations of 500 µg/well for the compounds, diferent zones of inhibition were obtained and compared to the control at 30 µg/mL to determine their activity.Te zones of inhibition ranged from  Heteroatom Chemistry 0.00 to 16.33 mm.Te absence of the zones of inhibition in some instances was attributable to the inability of some of the compounds to dissolve completely in the solution.Tese results are consistent with fndings reported in the literature [9].

Minimum Inhibitory/Bactericidal Concentrations (MICs/ MBCs) and Synergistic Potentials of the Antibiotics and the Test
Benzodiazepine.In exploring the scope of activity of the compounds (benzodiazepines) against the test strains, their MICs, MBCs, and synergistic potentials were determined.S2-S12).Tis has been afrmed by studies that have indicated that similar compounds gave strong microbial inhibition with MIC values equal to or lower than 500 µg/mL  Heteroatom Chemistry [42,43] thus the MIC values recorded for most of these test benzodiazepines in this study, especially with respect to compounds 2 and 7 suggest that they can serve as strong microbial inhibitory agents.Te MICs of the test drugs/ antibiotics determined ranged from 0.98 to 1000 mg/mL (Table S2).In addition to the MIC determination, the MBCs for the benzodiazepines indicated that they were bactericidal against the test strains used except benzodiazepines 4 and 6 which exhibited bacteriostatic activities against S. mutans, S. pyrogens, and P. aeruginosa, respectively, (Tables S2-S12).Tis falls in line with the literature, which indicated that the antimicrobial activity of compounds benzodiazepines against the microbes can be classifed as strong with MIC <2 mg/mL or good with MIC range of (2-10) mg/mL).Te description of a bactericidal agent is one with the ratio of MBC/MIC ≤4, while a bacteriostatic agent has an MBC/MIC ratio of >4 [44,45].Te results of the antimicrobial assay as shown in Tables S5-S12 agree with the fndings in the literature [9].In addition, the synergistic activities of the test compounds with some antibiotics (Ampicillin, Ciprofoxacin, and Tetracycline) were carried out.Te compounds showed indiferent interaction with Ampicillin and Ciprofoxacin.Te compounds were found to be antagonistic to Tetracycline except for a few compounds that had indiferent interactions with Tetracycline.Tese results show that benzodiazepines can be used to enhance the efcacy of standard antibiotics.A similar study has been reported on the use of benzodiazepine as respiratory depressants [46].

Antibioflm Formation Potential of Test Benzodiazepines.
With several studies reporting that bioflm formation by most microbes has been contributing to the occurrence of most infectious diseases that have been difcult to treat lately, many clinically vital microbes including Grampositive methicillin-resistant S. aureus bioflms have been singled out for many nosocomial infections [47].Moreover, recent studies have shown that the pathogenicity of these many organisms mainly depends on their virulence factors, such as adherence and invasion, hyphal and bioflm formation, cell wall integrity, and hydrolase secretion [48].
In efect, the quest to fnd a lasting solution to this resistance factor such as bioflm formation of most organisms implicated in most infections has become imperative.In this regard, the antibioflm formation activity of our compounds determined has been found to be appreciable with concentrations ranging between 0.02 and 1 mg/mL.Te percentage inhibition recorded against the bioflms ranged from 0.21 to 72.69% against the test organisms, respectively, (Figures 5(a) and 5(b)).Te IC 50 determined for each of the compounds when compared to the standard antibiotic against the bacterial bioflms gave IC 50 values between 0.03 and 0.94 mg/mL.However, the IC 50 s of some of the compounds could not be determined due to their low activity at Bond lengths ( Å) Bond angles ( 10 Heteroatom Chemistry determined concentrations (Table S10).Tis variation is expected because studies have shown that bioflm formation depends on the structure, nature, and composition of the organism in question [49].
In a related report recorded, similar benzodiazepines tested have been found to be efective against similar microbial bioflms [50,51].Tese abilities of the benzodiazepines have been alluded to by the variation in the sensitivity of the bioflm to these benzodiazepine derivatives.More importantly, the bioflm inhibition mechanisms of these benzodiazepines may also be attributed to the release of ions in the internal structures of the test bacteria to disrupt cells walls and essential structure, thereby enhancing cell-to-cell adhesion [52].Again, it must be noted that bioflm formation is largely afected by cell surface hydrophobicity, extracellular appendages such as fagella, and extracellular polymeric substances, and these properties may diverge from cell to cell hence the transformation in strength of bioflm formation as a result [53].Terefore, the fact established about the inhibition of microbial bioflms by benzodiazepines as reported in many studies has been their possession of abilities to go into the bioflm matrix to subvert bacterial cell walls [54].
Compound 1 was the most active against E. coli with an IC 50 of 0.1 mg/mL, whilst compound 2 was most active against methicillin-resistance S. aureus and K. pneumoniae with IC 50 s of 0.07 and 0.2 mg/mL, respectively.Against S. mutans compound 4 was the most active with an IC 50 of 0.14 mg/mL.Te most active compounds against B. subtilis were compounds 4 and 9 were with an IC 50 of 0.08 mg/mL whilst compounds 7 and 9 were the most active against S. pyrogens with an IC 50 of 0.11 mg/mL.For S. typhi the most active compounds were compound 4 and 7 with an IC 50 of 0.07 mg/mL.Compound 8 was most active against P. aeruginosa with an IC 50 of 0.11 mg/mL.Figures S42−S45 give pictures of the zones of inhibition for all the compounds and organisms.

Antiprostate Cancer Efect of Compounds.
Tere are various methods of treating cancers, which include chemotherapy, radiotherapy, surgery, and immunotherapy.Although there are several limitations associated with these methods, drugs ofer the only approach in treating cases where the disease has spread through the body.A good number of chemotherapeutic drugs are available for the treatment of cancers, but for most of them the applications are limited due to adverse efects including anaemia, fatigue, hair loss, nephrotoxicity, hepatotoxicity, and skin infections [55].
Tere is therefore the need to search for novel agents with little or no side efects for the treatment of cancers.Some benzodiazepine derivatives containing the thiochromeno moiety have been synthesized and tested for their anticancer, antimicrobial, and antitubercular activities.Te incorporation of the thiochromeno moiety provides further rigidity to the compound and allows it to ft into a wider space than is possible in most benzodiazepine compounds.It was also observed that the presence of the thiochromeno and the benzothiepino derivatives had higher activity against the test organisms [56].Te selective anticancer activity of some benzodiazepine and benzothiazepine derivatives has been reported.2-Methoxy-4-(4-phenyl-1H-1,5-benzodiazepin-2yl)phenol which is a benzodiazepine gave the best anticancer activity.Te higher activity was attributable to the benzodiazepine scafold [57].Te synthesis of some chloro-and fuoro-substituted 5-aryl-1,4-benzodiazepines has been achieved.Te compounds were tested for their anti-infammatory, myeloperoxidase, and anticancer properties.7-Chloro-5-(2-chlorophenyl)-1H-benzo[e] [1,4]diazepin-2(3H)-one gave the best activity amongst the compounds tested, the activity was attributable to the presence of the chloro group on the benzodiazepine ring [58].
Apart from compounds 6 and 7, the compounds did not demonstrate signifcant toxicity to normal cells and had no negative efect on their viability at 20 µM compared to the untreated controls (100%) (Figure 6).were selectively toxic to the prostate cancer (PC-3) cells, whereas sparing the normal (RAW 264.7) cells.Compound 3 demonstrated the highest antiprostate cancer efect by reducing the viability of PC-3 cells to a minimum (13.75%), indicating that the presence of the hydroxyl group at position 4 on the aromatic ring leads to a higher anticancer activity amongst these compounds.Compound 1 was moderately active (47.72%),confrming that the benzodiazepine frame was active on its own as an anticancer scafold.Te moderate activity of compound 2 (48.18%) also confrmed the anticancer efect of the compound when a methoxy group is at position 4 on the aromatic ring.Te activity of compounds 4 (62.61%), 5 (66.70%), and 8 (69.55%) (Figure 6) confrmed that substitution at position 3 with a nitro group, position 2 with a chloro group or the aromatic ring without any substituent leads to mild activity amongst these compounds.Compounds 6 and 7, however, did not reduce the viability of PC-3 cells signifcantly, indicating that the presence of the N,N-diethylamino group at position 4 on the aromatic ring makes the compounds less active than the benzodiazepine frame.Although compound 9 did not demonstrate a significant antiprostate cancer efect, it still appears to be selectively toxic to the PC-3 cell line and hence may exhibit mild to moderate antiprostate cancer efects at a higher dose.Prostate cancer is the second-deadliest malignancy in males after skin cancer and also the most diagnosed cancer type in men [59].More than 1,400,000 new cases of prostate cancer are diagnosed annually with 375,000 deaths worldwide [60].Te search for novel therapies for the treatment of prostate cancers is therefore warranted.Benzodiazepines are noted to have a variety of therapeutic efects including antimicrobial, antiviral, and antioxidant efects [61].However, research into the anticancer efect of benzodiazepines is scanty.Despite a handful of studies on the anticancer activities of benzodiazepines, research into the antiprostate cancer efect of these compounds remains limited.Te result of this study is therefore imperative as it gives insight into the possibility of exploring the antiprostate cancer efects of benzodiazepines.Figure S46 gives a pictorial presentation of the anticancer results.

Conclusion
Tese synthesized compounds were evaluated for their antibacterial activity using agar well difusion, microdilution, and bioflm inhibition assays.Subsequently, the determination of the combined antimicrobial activity of these benzodiazepines with antibiotics (ampicillin, tetracycline, and ciprofoxacin) against microbial strains was evaluated by checkerboard microdilution assay.Results from the study indicated that the antimicrobial activity of most of these compounds was bacteriostatic with their MICs ranging from 0.125 to 4 mg/mL.Interestingly, all the compounds were proven as good bioflm inhibitors with percentage inhibition ranging from 0.21 to 72.69%.Te combination interaction of the benzodiazepine derivatives with antibiotics gave results ranging from synergy to antagonism according to the parameters used.Te results showed that these benzodiazepines have signifcant antibacterial properties.Furthermore, benzodiazepines alone or in combination with the tested antibiotics could provide a promising approach to the management of microbial infections caused by drug-resistant strains.Te interaction of the compounds with other antibacterial agents would be helpful in combating common infections caused by methicillin-resistance Staphylococcus aureus (NCTC 29212), Escherichia coli (ATCC25922), Klebsiella pneumoniae (NCTC 13440), Bacillus subtilis (ATCC 10004), Streptococcus mutans (ATCC 700610), Pseudomonas aeruginosa (ATCC 4853), Salmonella typhi (ATCC14028), and Streptococcus pyrogens (Clinical).
Tis study has demonstrated the efect of some 2,3dihydro-1H-benzo[b] [1,4]diazepine derivatives against PC-3 prostate cancer cells, a good number of which were found to be selectively toxic to the PC-3 cells, whilst sparing the normal macrophage cells.Tese compounds are therefore promising candidates for further research into their mechanism of action against the proliferation of PC-3 prostate cancer cells.

Figure 5 :
Figure 5: (a) Antibioflm inhibition of some benzodiazepine against test strains of bacteria.(b) Antibioflm inhibition of various benzodiazepine against test strains of bacteria.

Table 1 :
List of synthesized compounds.

Table 2 :
Scope and yields of synthesized benzodiazepines.

Table 3 :
Crystallographic data and structure refnement summary for compounds 9.