Vol.:(0123456789)1 3

Journal of the Iranian Chemical Society (2020) 17:1555–1566 https://doi.org/10.1007/s13738-020-01875-5

ORIGINAL PAPER

Robust approach leading to novel densely functionalized four‑cyclic benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5‑b][1,4]diazepines with antibacterial activity toward resistant strains

Seddigheh Sheikhi‑Mohammareh1 · Mansour Mashreghi2,3 · Ali Shiri1

Received: 27 November 2019 / Accepted: 12 February 2020 / Published online: 20 February 2020 © Iranian Chemical Society 2020

Abstract A straightforward approach for the regioselective synthesis of various derivatives of benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5-b] [1, 4] diazepine as a novel heterocyclic system from the annulation of 6-bromo-7-chloro-3-cyano-2-(ethylthio)-5-methylpyrazolo[1,5-a]pyrimidine with several 2-amino-N-substituted benzamides as bident nucleophiles in the presence of K2CO3 and DMF has been disclosed. The right regioisomer was elucidated by 2D-NOESY NMR spectroscopy, as well. Most of the novel synthetic compounds exhibited antimicrobial activity though relatively at high concentrations against multi-drug-resistant Klebsiella pneumoniae and Escherichia coli clinical isolates possessed of strong biofilm formation ability. N-Cyclohexyl-substituted pyrazolopyrimidobenzodiazepine (3 h) as the most potent compound represented 100% growth inhibitory on both types of bacterial strains, which brings promises for further changes to develop potential antimicrobial drugs.

Graphical abstract

Keywords Regioisomeric annulation · Heterocycles · Pyrazolopyrimidobenzodiazepines · Antibacterial activity · Multi-drug resistance

Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1373 8-020-01875 -5) contains supplementary material, which is available to authorized users.

Extended author information available on the last page of the article

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Introduction

The privileged nature of benzodiazepine skeletons has been highlighted as lead compounds in medicinal chem-istry [1] due to the ability of compounds endowed with such a building block to link to multiple biological recep-tors [2]. Benzodiazepines are known to act on the central nervous system, and this vital class of psychotherapeutic compounds is the most widely prescribed minor tranquiliz-ers at present [3]. The 1, 4-diazepine and 1, 4-benzodiaz-epine biomolecules have been described as antibiotics [4], protein farnesyltransferase inhibitors [5] and antimalarial [6], anti-HIV [7] and anticancer agents [8].

Miscellaneous available methods in the literature for the construction of these heterocycles [9, 10] include the condensation reaction of pyrimidine-based 1, 2-diamines with 1, 3-dielectrophilic compounds such as chalcones [11], the intramolecular Michael additions of (o-aminobenzamido)-enones [12], aza-Wittig ring closure of [o-(iminophosphoranyl)benzamido]carbonyls [13], intramolecular cycloadditions of (o-azidobenzamido) alkenes and alkynes under microwave irradiation [14], nucleophilic addition to iminium ethers [15] and one-pot three-component reaction of piperidones, hydroxyalkyl azides and nucleophiles [16]. Although the existence of benzene moiety is an objective criterion for pharmaco-logical activity, a variety of heterocyclic rings fused to the seven-membered diazepine ring system has recently appeared in the literature [17–20]. Pyrimidodiazepines, for instance, have been appeared to be effective in the treat-ment of disorders via inhibition of various enzymes such as phenylalanine hydroxylase [21], Aurora A kinase [22], tyrosine kinase (A) [23], c-Met [18], EGFR [24], ERK5 (MAPK7) [25] and big map kinase 1 (BMK1) [17]. They have been also exhibited a broad spectrum of biological activities as anticonvulsant [26], anti-tumor [18, 27–29], antianxiety [30], antimycobacterial [31], antifungal [27], antimicrobial and antioxidant [32] agents, gastric secretion

inhibitor (B) [33] and second generation of γ-secretase modulators (AZ1136) (C) [34] (Figure 1).

Furthermore, pyrimido[4, 5-b] [1, 4] diazepine is the most common pyrimidodiazepine system reported in the literature which is synthesized via the reaction of 4, 5-diami-nopyrimidines with ethyl pyruvate [35] or chalcones [36, 37], Pictet–Spengler cyclization [38], microwave-induced annulation through the reaction of 4, 5, 6-triaminopyrimi-dine with chalcones in the presence of catalytic amounts of DMF [28] and use of 6-methoxy-5-nitrosopyrimidines in a three-step sequential methodology: aromatic nucleophilic substitution/reduction/cyclocondensation [39].

Urinary tract infections are one of the most common infectious diseases, mainly caused by gram-negative bacteria of multi-drug resistance (MDR). Isolation of various organ-isms from patients with UTI has been reported. Based on the literature, E. coli followed by Klebsiella species are the most prevalent causative pathogens involved in diseases associ-ated with UTI [40–43]. K. pneumoniae has been widely found in the mouth, skin and intestines, as well as in hospi-tal beds and medical devices. K. pneumonia biofilms protect pathogens from the attacks of the host immune system and antibiotics. Also, some of them show multi-drug resistance (MDR) phenotypes. Infections caused by this pathogen include pneumonia, blood infections, urinary tract infec-tions, surgical infections, wound infection and meningitis.

Although Escherichia coli is a part of typical gut micro-flora, it can cause disease in humans, generally well known as intestinal or extraintestinal pathogenic E. coli strains. Enterotoxinogenic, enteropathogenic and enterohamorrhagic E. coli (ETEC, EPEC and EHEC) are intestinal pathogens that cause more or less severe diarrhea, while extraintestinal pathogenic E. coli (ExPEC) causes some other infections in both humans and animals including urinary tract infections (UTIs), meningitis and septicemia [44]. Both of these dif-ferent types of E. coli strains can harbor various virulence and antibiotic resistance genes.

Regarding the arbitrary use of antibiotics and increasing drug resistance among these species, the use of alternative

Fig. 1 Examples of bioactive pyrimidodiazepines

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drugs to cope with this challenge is essential. New chemi-cal compounds can be a good candidate for the treatment of bacterial infections.

Encouraged by these inspiring facts and due to our insa-tiable desire for the synthesis of new derivatives of heterocy-clic compounds with potential biological activities [45–51], we designed and synthesized a series of highly functional-ized derivatives of a novel heterocyclic scaffold, benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4, 5-b][1, 4]diazepine. The anti-bacterial activity of some of the newly synthesized heterocy-clic compounds against gram-negative bacteria, namely K. pneumoniae and E. coli strains isolated from patients with urinary tract infections, was also investigated.

Results and discussion

Chemistry

Initially, according to the literature procedure [52], ring opening of commercially available isatoic anhydride via reaction of some primary amines was led to the formation of 2-amino-N-substituted benzamide derivatives (2a–i). These compounds as suitable dinucleophiles were reacted with the recently prepared 6-bromo-7-chloro-3-cyano-2-(ethylthio)-5-methylpyrazolo[1,5-a]pyrimidine (1) [45] in the presence of K2CO3 in DMF at 80–90 °C to give the products (3a–i) with structure either (A) or (B) (Scheme 1).

The structural assignment of all the newly synthesized compounds (3a–i) was based upon spectroscopic and

microanalytical data. Although the formation of both iso-mers (A) and (B) was possible, the experimental results did not demonstrate this prediction. The TLC of the products was single spot, and the spectral data such as 1H NMR and 13C NMR revealed the formation of only one product. As an example, the 1H NMR spectrum of (3d) showed a sin-glet peak at δ 2.35 ppm belonging to methyl group of the pyrimidine moiety and two signals at δ 1.55 (doublet) and δ 4.19 ppm (multiple) corresponding to the two equivalent methyl groups and the most deshielded aliphatic NCH pro-ton of the isopropyl moiety, respectively. The thioethyl ether signals were observed at δ 1.24 (triplet) and δ 3.01 ppm (quartet). Also, the signals of the aromatic hydrogen atoms of the benzene ring were assigned at δ 7.60–8.20 ppm. The spectrum did not show the signal of the NH functional group of the diazepine ring, which was probably missed. In 13C NMR, 19 resolved signals were also observed for the carbon atoms of compound (3d). By assigning the C ≡ N location at δ 68.6 ppm, the spectrum was divided into ali-phatic carbons region with five distinct signals at δ 12.0, 15.5, 20.2, 27.4 and 54.5 ppm and aromatic carbons area with 13 dissimilar signals at δ 113.7, 115.4, 122.3, 126.5, 127.7, 128.1, 133.2, 134.9, 141.9, 146.3, 147.2, 151.3 and 162.1 ppm. The IR spectrum of (3d) displayed the stretching vibration band of C ≡ N at 2219 cm−1. The C = O vibration band was blueshifted at 1682 cm−1 in comparison with the amidic carbonyl of precursor (2d) at 1619 cm−1. Including the blueshifted amidic C = O stretching band along with the disappearance of the symmetric and asymmetric stretch-ing bands of NH2 in the IR spectra strongly supports the

(1)

(3a-i)

NNNC

EtS

N

Me

Cl

Br

NNNC

EtS

N

Me

80-90 °C

N

HN

OR

(2a-i)

H2N

O

HN

R

K2CO3/DMF

NNNC

EtS

N

Me

NH

NOR

(A)

(B)

R3a 3b 3c 3d 3e 3f 3g 3h 3iMe Et n-Pr i-Pr n-Bu i-Bu cyclo-Hex p-TolylBn

entry

Scheme 1 Synthesis of benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5-b] [1, 4] diazepine derivatives

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possibility of the heterocyclization leading to compounds (3a–i). The observation of the molecular ion peak of (3d) at m/z 392, together with the elemental analysis, confirmed the occurrence of annulation, as well.

Eventually, the establishment of the correct structure (A) unambiguously came from a 2D-NOESY NMR analysis on the derivative (3d) (Figure 2). The intrinsic significance of the 2D-NOESY NMR is that it excludes the possibility of the cyclization through the path (B) due to the absence of any cross signal between the methyl groups of N-isopropyl moiety and the pyrimidine one. By contrast, the appearance of a somewhat strong cross signal between those aforemen-tioned equivalent methyl groups and the methyl of thioethyl ether moiety is self-evident that they are most probably close enough in space to depict a spatial interaction. Taking these findings into account, the 2D-NOESY NMR clearly identi-fies that the regioisomer (A) is the unequivocal structure.

Therefore, it can be rationalized that the reaction has most likely proceeded through two successive SNAr mechanisms via the formation of a non-isolated adduct intermediate that immediately underwent cyclocondensation accompanied by the elimination of HCl and HBr in each nucleophilic attack on pyrazolopyrimidine (1) containing adjacent chlorine and bromine elements (Scheme 2).

Biological evaluation

The in vitro antibacterial activity of the newly synthesized compounds (3b, 3c, 3d, 3f, 3 g, 3 h) was screened for the measurement of minimum inhibitory concentration (MIC)

against multi-drug-resistant pathogenic strains (Escheri-chia coli and Klebsiella pneumoniae) using microbroth dilution technique [53, 54].

Antibiotic susceptibility test

Identification and antibiotic susceptibility test of strains using the Vitek-2 system confirmed that isolated strains belonged to K. pneumoniae species. Also, the Api20E system revealed the identity of the related strains to be E. coli species (100%). The results of the antibiotic sus-ceptibility test showed that K. pneumoniae strains were 100% ampicillin resistant, and the lowest resistances of them were related to nitrofurantoin (14%) and tigecycline (0) antibiotics. However, the highest antibiotic resist-ance and sensitivity among E. coli strains were related to amoxicillin (88.4%) and trimethoprim/sulfamethoxazole (95%), respectively. Furthermore, 60% of the K. pneumo-niae strains had high biofilm-forming potency, 30% had a weak biofilm-forming ability, and less than 10% could not form biofilms, whereas 70% of the E. coli strains had high biofilm-forming potency, 20% had a medium-to-weak biofilm-forming ability, and less than 10% could not form biofilms. Therefore, multi-antibiotic-resistant strains of K. pneumonia (13, 33 and 36) and E. coli (17, 21, 38) that had high biofilm-forming ability along with standard strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923) were considered for further antibacterial assay.

Fig. 2 2D-NOESY NMR of compound (3d)

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Pneumoniae

The highest percentage of growth inhibition of the selected multi-drug-resistant K. pneumoniae strains (13, 33, 36) in the presence of various concentrations of compounds (3b, 3c, 3d, 3 g, 3 h) is shown in Table 1 and Fig. 3.

Based on the antibacterial activity results given in Table 1, except for compound (3 g) that inhibited the growth of 74% of K. pneumoniae strains (13, 33, 36) at 1 mg/ml (1000 ppm) (Fig. 3d), all tested compounds displayed MIC values at 0.5 mg/ml (500 ppm). Nevertheless, compounds (3 h), (3b) and (3c) with 100%, 99% and 96% growth inhibi-tion on strains 13, 13 and 36 were the most effective anti-bacterial agents against multi-drug-resistant K. pneumoniae (Figure 3a, b, e).

E. coli

According to the antibacterial evaluation of compounds (3b, 3c, 3d, 3f, 3 g, 3 h), all derivatives represented good to excellent inhibitory activities in high concentrations

against E. coli strains (17, 21, 38) (Table 2). The percentage of growth inhibition (GI %) of E. coli strains in the presence of various concentrations of the aforementioned compounds is depicted in Fig. 4.

Among the heterocyclic compounds used in this study, derivative (3 h) with 100% growth inhibition at a lower concentration of 0.5 mg/ml (500 ppm) was the most effec-tive antibacterial agent against three multi-drug-resistant E. coli strains (Fig. 4f). Although in 1 mg/ml (1000 ppm), compounds (3c) and (3 g) with also 100% growth inhibition on all tested E. coli strains and compound (3f) with similar effect on only strains 17 and 38 were ranked in the second place of efficacy after derivative (3 h) (Fig. 4b, d, e). The weakest antibacterial activities were obtained for compounds (3b) and (3d), as well (Figure 4a, c).

Standard strains

To compare with clinical isolates, the antimicrobial prop-erties of compounds (3b, 3c, 3d, 3 g, 3f, 3 h) against stand-ard strains (Escherichia coli ATCC 25922, Staphylococcus

NN CN

SEt

N

Me

Cl

Br

NN CN

SEt

N

Me

N

NH

O R

NH2

O

NH

R K2CO3

NH2

O

N RN

N CN

SEt

N

Me

N

OR

ClBrNH2

- HCl

NN CN

SEt

N

Me

N

OR

BrNH2

NN CN

SEt

N

Me

N

NH

O R

Br

H- HBr

(2a-i)

(3a-i)

(1)

Scheme 2 Plausible mechanism for synthesis of compounds (3a–i)

Table 1 The highest percentage of growth inhibition of K. pneumoniae strains in the presence of compounds (3b, 3c, 3d, 3 g, 3 h)

Bacterial strains

The highest percentage of growth inhibition

(3b)(0.5 mg/ml) (%) (3c)(0.5 mg/ml) (%) (3d) (0.5 mg/ml) (%)

(3 g)(1 mg/ml) (%) (3 h) (0.5 mg/ml) (%)

Strain 13 99 90 89 74 100Strain 33 84 57 50 75 87Strain 36 77 96 43 75 81

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aureus ATCC 25923) were evaluated. The results revealed that derivatives (3b), (3 g) and (3 h) 100% inhibited the growth of S. aureus ATCC 25923 in the concentration of 1 mg/ml (1000 ppm) (MIC) (Fig. 5a). The maximum growth inhibitions of E. coli ATCC 25922, however, in

1 mg/ml (1000 ppm) of compounds (3b), (3 g) and (3 h) were 90%, 90% and 93%, respectively (Fig. 5b).

Eventually, an analogy between antibacterial activities of the newly synthesized compounds against two standard bacteria and hospital bacteria isolated from UTI sufferers

Fig. 3 Antibacterial activity of compounds (3b): A, (3c): B, (3d): C, (3 g): D and (3 h): E against K. pneumoniae strains 13, 33 and 36

Table 2 The highest percentage of growth inhibition of E. coli strains in the presence of various compounds (3b, 3c, 3d, 3f, 3 g, 3 h)

Bacterial strains The highest percentage of growth inhibition

(3b) (1 mg/ml) (3c) (1 mg/ml) (3d) (1 mg/ml) (3f) (1 mg/ml) (3 g) (1 mg/ml) (3 h) (0.5 mg/ml)

Strain 17 90% 100% 64% 100% 100% 100%Strain 21 79% 100% 79% 79% 100% 100%Strain 38 93% 100% 85% 100% 100% 100%

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supports the conclusion that approximately most of the tested compounds demonstrated better antimicrobial activi-ties against standard strains in lower concentrations com-pared with clinical ones.

The application of synthetic chemical compounds as antimicrobial agents has been reported in several research studies. For example, Babalola (1997) synthesized pyridine

and quinoline chlorochromate derivatives and evaluated their antibacterial activity against bacterial strains (P. aeruginosa, E. coli, B. subtilis, S. aureus). Their results indicated that the mixture of the derivatives produces an antagonist effect [55]. The synergistic or antagonistic properties of the compounds that have been synthesized in our study can also be inves-tigated in order to lower the amount of MIC concentration.

Fig. 4 Antibacterial activity of (3b): a, (3c): b, (3d): c, (3f): d, (3 g): e and (3 h): f against E. coli strains 17, 21 and 38

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Also, Bakavoli et al. (2010) evaluated the antimicrobial effects of new pyrazolo[3,4-d]pyrimidine derivatives and concluded that hydroxyl- and halogen-substituted deriva-tives of these compounds had better antimicrobial activities against E. coli and P. aeruginosa pathogens comparable to other substituted pyrazolo[3,4-d]pyrimidines [56]. Our study showed that N-carbocyclic-substituted compound (3 h) with benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5-b] [1, 4] diazepine skeleton had antibacterial effect on clinical pathogenic iso-lates of bacteria beyond compare. In another study, Chanda et al. (2010) showed that most triazole derivatives are capa-ble of inhibiting the growth of K. pneumoniae strains [57]. Inhibition of K. pneumoniae growth also occurred when we used specific concentrations of our heterocyclic compounds but with various percentages for different clinical strains. Shakhatreh et al. (2016) found that benzyl bromides exhibit more antimicrobial activity against gram-positive bacteria than gram-negative ones. In contrast, some ketones and cor-responding chalcone derivatives showed no antimicrobial activity against gram-positive and negative bacterial strains [54]. In our study, the variation on antimicrobial effect was observed from complete inhibition in bacterial growth (100%) to near to half (43%) growth inhibition, too.

Moreover, the structure–activity relationships based on the literature survey revealed that fused diazepines like pyrimidodiazepine heterocyclic compounds have antibac-terial activity [31, 32, 58]. Although higher concentrations were used in our study compared with others, at least one of the compounds, namely (3 h), could hinder the growth of multi-drug-resistant bacteria completely. As it can be con-cluded from the results of our study, the compound bearing N-cyclohexyl substituent (3 h) has shown the highest effect on both Escherichia coli and Klebsiella pneumoniae clinical isolates which may be due to the lipophilicity increase in the moiety, which has enhanced the antibacterial activity. The other compounds were found to exhibit moderate to good activities in higher concentrations against the mentioned

organisms. Therefore, there is a promising scope for further changes in these compounds so as to develop much more effective antimicrobial drugs.

Conclusion

In summary, we disclosed a simple strategy for the synthesis of a robust four-cyclic scaffold benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5-b] [1, 4] diazepine. In this protocol, the treat-ment of precursor (1) with various 2-amino-N-substituted benzamides (2a–i) in the presence of K2CO3 in DMF at 80–90 °C was conveniently led to the construction of the desired promising biologically active compounds (3a–i). The novel compounds were screened for antibacterial activity. Although most of the tested compounds demonstrated bet-ter antimicrobial activities against standard strains in lower concentrations compared with clinical ones, compound (3 h) was the only one synthesized derivative which could hinder the growth of multi-drug-resistant hospital bacteria isolated from UTI sufferers, completely.

Experimental

Melting points were recorded on an electrothermal type 9200 melting point apparatus. The IR spectra were obtained on an Avatar 370 FT-IR Thermo Nicolet, and only notewor-thy absorptions are listed. The 1H NMR (300 MHz) and the 13C NMR (75 MHz) spectra were recorded on a Bruker Avance-III 300 NMR Fourier transformer spectrometer. The mass spectra were scanned on a Varian Mat CH-7 at 70 eV. Elemental analyses were performed on a Thermo Finnigan Flash EA 1112 microanalyzer.

6‑Bromo‑7‑chloro‑3‑cyano‑2‑(ethylthio)‑5‑methylpyrazolo[1,5‑a]pyrimidine (1) Compound (1) was prepared

Fig. 5 Antibacterial activity of (3b, 3c, 3d, 3f, 3 g, 3 h) against S. aureus ATCC 25923 (a) and E. coli ATCC 25922 (b)

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through recently reported procedure by our research group [45]. Brown powder; yield 89%; mp 182–184 °C; 1H NMR (CDCl3): δ 1.32 (t, 3H, J = 7.6 Hz, –CH3), 2.11 (s, 3H, –CH3), 2.46 ppm (q, 2H, J = 7.6 Hz, –SCH2) ppm; 13C NMR (CDCl3): δ 14.3, 23.2, 23.5, 89.9, 117.0, 119.6, 148.3, 150.5, 161.0, 164.4 ppm; IR (KBr disk): ν 2962, 2929, 2223 (CN), 1595, 1468, 918 (C–Cl), 718 (C–Br) cm−1; MS: (m/z) 331 [M+], 302 [M+ _ Et], 296 [M+ _ Cl], 270 [M+ _ SEt]. Anal. Calcd. for C10H8BrClN4OS (%): C, 36.22; H, 2.43; N, 16.89; S, 9.67. Found: C, 36.17; H, 2.39; N, 16.85; S, 9.64

General procedure for the synthesis of 3‑cyano‑2‑(ethylthio)‑5‑methyl‑11‑oxo‑12‑substi‑tuted‑11,12‑dihydro‑6H‑benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5‑b][1, 4]diazepine derivatives (3a‑i)

A mixture of compound (1) (1 mmol, 0.331 g), the appro-priate 2-amino-N-substituted benzamide derivatives (2a–i) (1 mmol) and K2CO3 (2 mmol, 0.276 g) in DMF (3 ml) was heated at 80–90 °C for about 20 h. After the completion of the reaction (monitored by TLC, CHCl3/MeOH, 20:1), the mixture was cooled, poured into an ice/water bath and neutralized with aqueous 5% HCl solution. The resulting solid product was collected by filtration, washed with water (2 × 20 ml) and purified by column chromatography.

3‑Cyano‑2‑(ethylthio)‑5,12‑dimethyl‑11‑oxo‑11,12‑di‑hydro‑6H‑benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5‑b][1, 4]diazepine (3a) Cream powder; yield 47%; mp 287 °C–288 °C; 1H NMR (DMSO-d6): 1.20 (t, J = 7.3 Hz, 3H, CH3), 2.35 (s, 3H, CH3), 2.91 (q, J = 7.3  Hz, 2H, SCH2), 3.45 (s, 3H, NCH3), 7.51 (t, J = 7.5 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 8.15 (d, J = 7.9 Hz, 1H) ppm; 13C NMR (CDCl3): δ 15.6, 28.2, 33.0, 63.6, 68.7, 100.0, 112.7, 118.3, 119.6, 126.0, 127.2, 134.6, 144.0, 148.4, 149.5, 162.7 ppm; IR (KBr): ν 3464, 3231, 3141, 2953, 2925, 2847, 2215 (CN), 1656, 1619, 1560, 1475, 1416, 1223, 1045, 770 cm−1. MS: (m/z) 364. Anal. Calcd. for C18H16N6OS (%): C, 59.33; H, 4.43; N, 23.06; S, 8.80. Found: C, 59.30; H, 4.42; N, 23.03; S, 8.78.

3 ‑ C y a n o ‑ 1 2 ‑ e t h y l ‑ 2 ‑ ( e t h y l t h i o ) ‑ 5 ‑ m e ‑thyl‑11‑ oxo ‑11,12‑ dihydro ‑6H‑benzo[e]pyra‑zolo[5 ′ ,1 ′ :2,3]pyrimido[4,5‑b][1, 4]diazepine (3b) Cream powder; yield 53%; mp 264–265  °C; 1H NMR (DMSO-d6): δ 1.04 (t, J = 7.0 Hz, 3H, CH3), 1.23 (t, J = 7.3 Hz, 3H, CH3), 2.37 (s, 3H, CH3), 2.98 (q, J = 7.3 Hz, 2H, SCH2), 4.07 (q, J = 7.0 Hz, 2H, NCH2), 7.58 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.69 (dd, J = 8.4, 1.1 Hz, 1H), 7.86 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 8.21 (dd, J = 8.0, 1.5 Hz, 1H) ppm; 13C NMR (DMSO-d6): δ 12.6, 14.3, 15.4, 27.8, 69.0, 113.2, 115.9, 120.8, 126.7, 127.8, 128.3, 135.0, 135.5, 143.2, 146.2, 147.9, 150.7, 161.7 ppm; IR (KBr): ν 3435,

3235, 3127, 2988, 2926, 2867, 2216 (CN), 1686, 1647, 1618, 1578, 1562, 1473, 1439, 1095, 772 cm−1. MS: (m/z) 378. Anal. Calcd. for C19H18N6OS (%): C, 60.30; H, 4.79; N, 22.21; S, 8.47. Found: C, 60.27; H, 4.74; N, 22.18; S, 8.42.

3‑Cyano‑2‑(ethylthio)‑5‑methyl‑11‑oxo‑12‑pro‑pyl‑11,12‑dihydro‑6H‑benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5‑b][1, 4]diazepine (3c) Cream powder; yield 71%; mp 265 °C; 1H NMR (DMSO-d6): δ 0.62 (t, J = 7.4 Hz, 3H, CH3), 1.24 (t, J = 7.3 Hz, 3H, CH3), 1.49 (q, J = 7.4 Hz, 2H, CH2), 2.38 (s, 3H, CH3), 2.98 (q, J = 7.3 Hz, 2H, SCH2), 3.99 (q, J = 7.2 Hz, 2H, NCH2), 7.61 (t, J = 7.5 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.92–7.84 (m, 1H), 8.22 (dd, J = 8.0, 1.5 Hz, 1H) ppm; 13C NMR (DMSO-d6): δ 11.3, 12.3, 15.4, 21.7, 27.8, 46.5, 69.1, 113.6, 115.5, 120.8, 126.8, 127.8, 128.0, 134.5, 135.1, 142.3, 146.1, 147.5, 151.1, 161.9 ppm; IR (KBr): ν 3227, 3119, 3071, 2964, 2930, 2873, 2216 (CN), 1687, 1648, 1619, 1576, 1560, 1473, 1218, 1095, 771 cm−1. MS: (m/z) 392. Anal. Calcd. for C20H20N6OS (%): C, 61.21; H, 5.14; N, 21.41; S, 8.17. Found: C, 61.18; H, 5.10; N, 21.37; S, 8.14.

3 ‑ C y a n o ‑ 2 ‑ ( e t h y l t h i o ) ‑ 1 2 ‑ i s o p r o p y l ‑ 5 ‑ m e ‑thyl‑11‑ oxo ‑11,12‑ dihydro ‑6H‑benzo[e]pyra‑zolo[5 ′ ,1 ′ :2,3]pyrimido[4,5‑b][1, 4]diazepine (3d) Cream powder; yield 88%; mp 223–225  °C; 1H NMR (DMSO-d6): 1.24 (t, J = 7.3 Hz, 3H, CH3), 1.55 (d, J = 6.7 Hz, 6H, 2CH3), 2.35 (s, 3H, CH3), 3.01 (q, J = 7.3 Hz, 2H, SCH2), 4.19 (m, J = 6.7 Hz, 1H, NCH), 7.60 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.69 (dt, J = 8.1, 1.0 Hz, 1H), 7.86 (ddd, J = 8.4, 7.1, 1.6 Hz, 1H), 8.20 (ddd, J = 8.0, 1.5, 0.6 Hz, 1H) ppm; 13C NMR (DMSO-d6): δ 12.0, 15.5, 20.2, 27.4, 54.5, 68.6, 113.7, 115.4, 122.3, 126.5, 127.7, 128.1, 133.2, 134.9, 141.9, 146.3, 147.2, 151.3, 162.1 ppm; IR (KBr): ν 3131, 3018, 2977, 2929, 2871, 2219 (CN), 1682, 1650, 1606, 1580, 1563, 1221, 1069, 774 cm−1. MS: (m/z) 392. Anal. Calcd. for C20H20N6OS (%): C, 61.21; H, 5.14; N, 21.41; S, 8.17. Found: C, 61.18; H, 5.10; N, 21.37; S, 8.14.

1 2 ‑ B u t y l ‑ 3 ‑ c y a n o ‑ 2 ‑ ( e t h y l t h i o ) ‑ 5 ‑ m e ‑thyl‑11‑ oxo ‑11,12‑ dihydro ‑6H‑benzo[e]pyra‑zolo[5 ′ ,1 ′ :2,3]pyrimido[4,5‑b][1, 4]diazepine (3e) Cream powder; yield 42%; mp 238–240 °C; 1H NMR (DMSO-d6): δ 0.57 (t, J = 7.3  Hz, 3H, CH3), 1.01 (m, J = 7.3 Hz, 2H, CH2), 1.22 (t, J = 7.3 Hz, 3H, CH3), 1.35 (m, J = 6.7, 6.2 Hz, 2H, CH2), 2.34 (s, 3H, CH3), 2.95 (q, J = 7.3 Hz, 2H, SCH2), 4.14 (t, J = 7.3 Hz, 2H, NCH2), 7.53 (ddd, J = 8.1, 7.1, 1.2 Hz, 1H), 7.66 (dd, J = 8.4, 1.1 Hz, 1H), 7.83 (ddd, J = 8.4, 7.1, 1.6 Hz, 1H), 8.18 (dd, J = 8.0, 1.5 Hz, 1H) ppm; 13C NMR (DMSO-d6): δ 13.6, 14.1, 15.5, 19.6, 28.1, 30.3, 44.0, 68.9, 113.0, 117.5, 120.3, 126.8, 127.3, 127.9, 134.9, 139.5, 145.9, 147.7, 148.0, 154.7, 162.16 ppm; IR (KBr): ν 3239, 3137, 3072, 2960, 2928, 2871, 2215 (CN),

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1681, 1648, 1618, 1562, 1472, 1219, 1096, 772 cm−1. MS: (m/z) 407. Anal. Calcd. for C21H22N6OS (%): C, 62.05; H, 5.46; N, 20.67; S, 7.89. Found: C, 62.00; H, 5.42; N, 20.63; S, 7.83.

3 ‑ C y a n o ‑ 2 ‑ ( e t h y l t h i o ) ‑ 1 2 ‑ i s o b u t y l ‑ 5 ‑ m e ‑thyl‑11‑ oxo ‑11,12‑ dihydro ‑6H‑benzo[e]pyra‑zolo[5 ′ ,1 ′ :2,3]pyrimido[4,5‑b][1, 4]diazepine (3f) Cream powder; yield 75%; mp 219  °C; 1H NMR (DMSO-d6): 0.59 (d, J = 6.7  Hz, 6H, 2CH3), 1.23 (t, J = 7.3 Hz, 3H, CH3), 1.70 (m, J = 6.8 Hz, 1H, CH), 2.37 (s, 3H, CH3), 2.95 (q, J = 7.3  Hz, 2H, SCH2), 4.02 (d, J = 7.3 Hz, 2H, NCH2), 7.54 (t, J = 7.5 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.84 (t, J = 7.3 Hz, 1H), 8.18 (dd, J = 8.0, 1.5 Hz, 1H) ppm; 13C NMR (DMSO-d6): δ 14.4, 15.4, 20.0, 27.3, 28.2, 51.2, 69.1, 113.3, 117.3, 120.1, 126.9, 127.5, 134.9, 140.2, 146.2, 148.0, 148.8, 154.5, 162.5, 172.6 ppm; IR (KBr): ν 3231, 3122, 3071, 3043, 2960, 2929, 2870, 2215 (CN), 1682, 1647, 1619, 1562, 1472, 1438, 1219, 1096, 771 cm−1. MS: (m/z) 407. Anal. Calcd. for C21H22N6OS (%): C, 62.05; H, 5.46; N, 20.67; S, 7.89. Found: C, 62.00; H, 5.42; N, 20.63; S, 7.83.

1 2 ‑ B e n z y l ‑ 3 ‑ c y a n o ‑ 2 ‑ ( e t h y l t h i o ) ‑ 5 ‑ m e ‑thyl‑11‑ oxo ‑11,12‑ dihydro ‑6H‑benzo[e]pyra‑zolo[5 ′ ,1 ′ :2,3]pyrimido[4,5‑b][1, 4]diazepine (3 g) Cream powder; yield 73%; mp 211–212 °C; 1H NMR (DMSO-d6): 1.28 (t, J = 7.3 Hz, 3H, CH3), 1.99 (s, 3H, CH3), 3.03 (q, J = 7.3 Hz, 2H, SCH2), 5.38 (s, 2H, NCH2), 7.17 (m, 3H), 7.28 (m, 2H), 7.64 (t, J = 7.6 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.91 (t, J = 7.6 Hz, 1H), 8.26 (d, J = 7.9 Hz, 1H) ppm; 13C NMR (DMSO-d6): δ 11.8, 15.5, 27.9, 46.6, 69.2, 115.5, 120.6, 126.5, 127.1, 127.4, 127.7, 127.9, 128.2, 128.7, 128.9, 135.4, 137.1, 141.4, 146.0, 147.5, 151.3, 162.1 ppm; IR (KBr): ν 3391, 3150, 3068, 3027, 2966, 2925, 2872, 2213 (CN), 1677, 1667, 1608, 1584, 1564, 1472, 1452, 1337, 1240, 1150, 775 cm−1. MS: (m/z) 440. Anal. Calcd. for C24H20N6OS (%): C, 65.44; H, 4.58; N, 19.08; S, 7.28. Found: C, 65.41; H, 4.55; N, 19.06; S, 7.25.

3 ‑ Cy a n o ‑ 1 2 ‑ c y c l o h e x y l ‑ 2 ‑ ( e t h y l t h i o ) ‑ 5 ‑ m e ‑thyl‑11‑ oxo ‑11,12‑ dihydro ‑6H‑benzo[e]pyra‑zolo[5 ′ ,1 ′ :2,3]pyrimido[4,5‑b][1, 4]diazepine (3 h) Cream powder; yield 62%; mp 208 °C; 1H NMR (CDCl3): 1.45 (t, J = 7.4  Hz, 3H, CH3), 1.75 (m, 4H, 2CH2), 1.84–1.96 (m, 4H, 2CH2), 2.56 (s, 3H, CH3), 3.28 (q, J = 7.4 Hz, 2H, SCH2), 3.53 (m, 1H, NCH), 7.07 (d, J = 7.8 Hz, 1H), 7.24 (ddd, J = 8.2, 7.3, 1.0 Hz, 1H), 7.62 (ddd, J = 8.1, 7.3, 1.5 Hz, 1H), 8.15 (dd, J = 8.0, 1.4 Hz, 1H), 9.48 (s, 1H, NH) ppm; 13C NMR (CDCl3): δ 14.9, 24.7, 25.3, 26.4, 28.9, 32.7, 51.3, 88.2, 113.4, 114.4, 115.1, 123.2, 128.5, 134.8, 138.6, 145.0, 151.6, 151.8, 156.2, 162.8, 163.1 ppm; IR (KBr): ν 3282, 3194, 3125, 3063, 2929, 2855,

2220 (CN), 1716, 1660, 1619, 1583, 1445, 1291, 758 cm−1. MS: (m/z) 432. Anal. Calcd. for C23H24N6OS (%): C, 63.87; H, 5.59; N, 19.43; S, 7.41. Found: C, 63.83; H, 5.55; N, 19.40; S, 7.39.

3 ‑ C y a n o ‑ 2 ‑ ( e t h y l t h i o ) ‑ 5 ‑ m e ‑thyl‑11‑oxo‑12‑(p‑tolyl)‑11,12‑dihydro‑6H‑benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5‑b][1, 4]diazepine (3i) Cream powder; yield 38%; mp 272–276 °C; 1H NMR (CDCl3): 1.25 (t, J = 7.3 Hz, 3H, CH3), 2.20 (s, 3H, CH3), 2.22 (s, 3H, CH3), 2.96 (q, J = 7.3 Hz, 2H, SCH2), 6.96 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 7.53 (ddd, J = 8.2, 6.6, 1.7 Hz, 1H), 7.69–7.82 (m, 2H), 8.30 (d, J = 6.6 Hz, 1H) ppm; 13C NMR (CDCl3): δ 10.9, 15.2, 21.2, 28.3, 69.2, 114.8, 121.6, 127.3, 127.6, 128.4, 128.6, 129.4, 133.5, 134.9, 139.2, 139.8, 144.1, 147.3, 152.5, 162.2 ppm; IR (KBr): ν 3252, 3170, 3056, 2925, 2872, 2215 (CN), 1669, 1611, 1559, 1471, 1438, 1340, 1219, 776 cm−1. MS: (m/z) 440. Anal. Calcd. for C24H20N6OS (%): C, 65.44; H, 4.58; N, 19.08; S, 7.28. Found: C, 65.41; H, 4.55; N, 19.06; S, 7.25.

Bacterial culture and purification of the samples

In order to purify and identify bacterial isolates, aliquots of the bacterial strains suspension from each patient were separately inoculated and cultivated on the plates containing Mueller–Hinton agar medium. Then, all plates were incu-bated at 37 °C for 24 h in the incubator. After the incuba-tion period, the culture medium was investigated for colony formation. In the following, the strains that could grow on the plate were selected, cultured and, after recultivation in nutrient broth (NB) medium, cultivated on the plates con-taining MacConkey agar medium. All plates were incubated at 37 °C for 24 h in the incubator. The presence of bile salts and crystal violet in MacConkey media results in lactose-positive gram-negative bacteria and lactose-negative gram-negative bacteria producing purple and colorless colonies, respectively. Finally, the strains that could grow on the Mac-Conkey agar medium were purified and further examined using the biochemical tests. These tests confirmed the 100% purity and identity of Escherichia coli and Klebsiella pneu-moniae isolates.

Antibacterial test

For evaluation of antibacterial activity of the synthetic com-pounds against selected K. pneumoniae strains (13, 33 and 36) and E. coli strains (17, 21 and 38), certain concentrations of (3b, 3c, 3d, 3f, 3 g, 3 h) (15–1000 ppm) were prepared by a twofold serial dilution method. Then, 100 μl of the bacterial suspension (1 McFarland) and 100 μl of the above-mentioned compounds were separately inoculated into wells of a 96-well plate and transferred to the incubator (37 ºC,

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150  rpm) along with samples of the positive and nega-tive controls. Finally, the bacterial growth in the different concentrations of compounds (3b, 3c, 3d, 3f, 3 g, 3 h) was determined by reading the absorbance at 630 nm by ELISA (0 and 24 h), and the percentage of growth inhibition (GI %) for each strain was calculated in 24 h. It is worth to mention that the quantities of compounds (3a), (3e) and (3i) were too low to test for in vitro antibacterial potency.

Acknowledgements The authors gratefully acknowledge the Research Council of Ferdowsi University of Mashhad for financial support of this project (3/44510).

Compliance with ethical standards

Conflict of interest The authors declare no conflict of interest.

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Affiliations

Seddigheh Sheikhi‑Mohammareh1 · Mansour Mashreghi2,3 · Ali Shiri1

* Ali Shiri alishiri@um.ac.ir

1 Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

2 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

3 Industrial Biotechnology Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran