β-Lactamase inhibition profile of new amidine substituted diazabicyclooctanes

Diazabicyclooctane (DBO) scaffold is the backbone of non -β-lactam based second generation β-lactamase inhibitors. As part of our efforts we have synthesized a series of DBO derivatives A1-A23 containing amidine substituents at C2 position of the bicyclic ring. These compounds, alone and in combination with meropenem, were tested against ten bacterial strains for their antibacterial activity in vitro. All compounds didn’t show antibacterial activity when alone ( MIC, >64 mg/L), however exhibited moderate inhibition activity in the presence of meropenem by lowering its MIC values. Compound A12 proved most potent among the other counterparts against all bacterial species with MIC from <0.125 mg/L – 2 mg/L, and is comparable to avibactam against both E. coli strains with MIC value of <0.125 mg/L.

Amidine, β-lactamases inhibitors, diazabicyclooctane, synthesis, antibacterial activity.  Corresponding authors. Tel.: +86-951-861-7686. E-mail addresses: [email protected] (H. Yang) , [email protected] (Z. Yang). 2

Survival stress posed by the antimicrobial agents triggers multiple mechanisms1in microorganisms ultimately leading to the initiation of antibiotic resistance and survival of the microorganisms2. In case of Gram-negative pathogenic bacteria, production of β-lactamases3 is the main arsenal of these microorganisms against antibiotics. The number of β-lactamases is increasing day by day thereby indicating the strength of these pathogens in compromising the efficacy of new antibiotics after certain period of time. Recently WHO warned about the seriousness of carbapenemase resistant Gram-negativebacteria as a global threat and urged for the development of new remedies4. β-Lactams (BL) have served as the first line antibiotics since the introduction of penicillin. However, due to existence and continuous increase in β-lactamases5, multidrug therapy is becoming the new modality of bacterial treatment against multiple-drug resistant (MDR) bacteria. Multidrug therapy employs the combination of an existing antibiotic with a β-lactamase inhibitor (BLI). A few BLI/BL combinations have been approved6so far for clinical applications by different countries, clavulanic acid7/amoxicillin (Augmentin)8 being the first one , while others are in clinical trials6. Although Augmentin9 was successfully applied to treat the infection s caused by bacterial strains producing Ambler class A and extended spectrum β-lactamases (ESBLs)10, however the emergence of new and mutant class A β-lactamases compromised its effectiveness overtime9, 11. Subsequently sulbactam and tazobactam12 evolved as the BLI of class A, B and few of class D β-lactamases13. These inhibitors were advantageous to clavulanic acid due to their lack of chromosomal induction of AmpC but found susceptible to a few of class A enzymes such as TEM type9 and CTX-M (ESBL), identified in Escherichia coli clinical isolate13-14. 3 Diazabicyclooctane (DBO) 15ring suggested as an alternative to β-lactam ring16 by the Hoe chst researchers15 could not prove its antibacterial strength in early experiments rather showed β- lactamase inhibition activity. This discovery led the researchers to develop second generation β- lactamase inhibitors, finally succeeded with the approval of avibactam and relebactam as non-β- lactam based BLIs. Avibactam proved potent inhibitor of KPCs, AmpCs and some of class D β- lactamases17 is now in clinical practice in combination with ceftazidime6. Followed by avibactam, relebactam/imipenem/cilastatin6 combinationhas been approved by FDA for the treatment of clinical indications ag ainst carbapenemases, ESBLs, and MDR Enterobacteriaceae as well as Pseudomonas aeruginosa18. Of note these combinations are not effective against class B metallo- lactamases and most of class D (OXA) β-lactamases. Therefore, several other DBO based BLIs16, such as durlobactam, nacubactam 19, zidebactam, ETX0282 and ARX -1796 (prodrug of Avibactam)20, WCK 4234 17, 21, are passing through phase I and phase III clinical trials 6, 22 in combination with different types of β -lactams. Of these, WCK 4234 has shown promise against class A, class C and class D carbapenemases17, 21. These multidrug combinations have shown promise for future antibiotic regime n and drug development based on non -β-lactam inhibitors. Nonetheless, partial loss of activity has been reported in case of ceftazidime-avibactam combination due to overproduction of AmpC cephalosporinases23. In another report it has been concluded that ESBLs of the GES, PER and BEL types in E. coli and P . aeruginosa conferred resistance against sulbactam and avibactam combinations24. Therefore, it is utmost necessary to continue the struggle with exploring new inhibitors capable of improved resistance and activity against all classes of β-lactamases. Based on 4 our ongoing efforts towards the synthesis of new DBO based BLIs, we have synthesized a number of amidine conjugated derivatives of avibactam. We report the synthesis and antibacterial as well as inhibitory activities o f these compounds in combination with avibactam in comparison to avibactam and meropenem (MER), an existing antibiotic in clinics.

and discussion Synthesis of intermediates 1-5 Synthesis of intermdiate 1 is the key step for the synthesis of final compounds (scheme 1). Compound 1 was synthesized by the dehydration of amide 256 which is commercially available. Dehydration was acheived by reacting 6 with trifluoroacetic anhydride in CH2Cl2 at room temperature (RT) and is described elsewhere17. Conversion of the cyano compound 7 into corresponding amidine compound 1, the key intermediate, proved cumbersome. Several experiments and reagents were tried before finding the trimethylaluminum (Al(Me)3) and NH4Cl as the reagents of choice for this conversion. As a result compound 7 was reacted with Al(Me)3 and NH4Cl to furnish amidine in CH2Cl2 starting the reaction at low temperature followed by at ambient te mperature for 16 h. Amidine 1 was obtained in 44% yield after purification by colummn chromatography using MeOH and CH2Cl2. Lower yield of this reaction was due to the formation of two isomeric products revealed by TLC and subsequent analysis by analytical LCMS. The NMR spectra of both isomers, after chromatographic separation, showed different chemical shifts for the protons at C2 position of DBO ring, indicating the racemization during the reaction process. Less polar isomer with R-configuration at C2 showed complete loss of β-lactamase inhibition activity as compared to the more polar isomer. Therefore less polar isomer was discarded while saving the more polar S-isomer, (relative ratio of S:R isomers = 6:1). Racemization at C2 of DBO 5 suggests the amidation reaction proceeds through carbocation formation at C2 as well. Scheme 1. Synthesis of intermediate 1. Reagents and condition: (i) trifluoroacetic anhydride, CH2Cl2, 0 oC-RT, 3h; (ii) Al(Me)3, NH4Cl, CH2Cl2, 0 oC-RT, 16h. Scheme 2. Synthesis of intermediate 2. Reagents and conditions: (i) Pd/C (wet), EtOAc/ CH2Cl2, H2, 45 psi, RT, 2h; (ii) TBSCl, Imidazole, CH2Cl2, RT, 16h; (iii) Al(Me)3, NH4Cl, CH2Cl2, 0 oC-RT, 40h. Scheme 3. Synthesis of intermediates 3-5. Reagents and conditions: (i) (Ac)2O, CH2Cl2, RT, 24h; (ii) Aqueous NaOH, 0 oC, 2h; (iii) (Ac)2O, H2O, RT, 3h. Synthesis of intermediate 2 started from the hydrogenation of 7 by following previously described

using N,N-dimethylformamide (DMF)/CH2Cl217 as solvent led to low yield in our hands. 6 Therefore, we planned to switch the solvent from DMF to EtOAc whereupon the yield improved however, still amino derivative as side product was observed. Addition of CH2Cl2 with ethylacetate proved helpful in increasing the yield and NMR of crude product 8 was acceptable to use it for further reaction without purification. Hydroxyl group in 8 was then protected by TBS (tert- butyldimethylsilane) using tert-butyldimethylsilyl chloride (TBSCl) and imidazole in CH2Cl2. Thus obtained derivative 9 was subjected to amidination by Al(Me) 3 and NH 4Cl to afford amidine 2 (scheme 2). Compounds 3,4 were prepareed from commercially available compounds 11 and 12 respectively in two steps. In first step ester derivatives were acetylated by acetic anhydride in CH2Cl2, followed by the hydrolysis by aqueous NaOH in tertrahydrofuran (THF) to afford the required intermediates 3 and 4 in overall good yields. Compound 5 was obtained by direct acetylation of commercially available acid 13 using acetic anhydride and stoicheometric amount of water, at room temeprature (scheme 3). Synthesis of compounds A1-A23 Synthesis of compounds A1-A21 starting from intermediate 1 was accomplished as depicted in scheme 4. Coupling of the organic acids with amidine 1 to form the corresponding derivatives B1-B21 was achieved by coupling reagents such as or N,N'-dicyclohexylcarbodiimide (DCC) or (O-(7-Aza-1H- benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate ) (HATU)26 in DMF or CH2Cl2 whereas N,N-diisopropylethylamine (DIPEA) or 4-dimethylaminopyridine (DMAP) were used as base. Palladium catalyzed hydrogenation of compounds B1-B21 in THF or EtOAc led to afford hydroxy derivatives C1-C21. It has been observed that catalytic amount of triethylamine (TEA) in EtOAc enhances the rate of hydrogenolysis of benzyl ethers. Compounds C1-C21 are then reacted with 7 SO3-pyridine to form sulfonic acid derivatives A1-A21 after purification by preparative HPLC. Sodium salts of these compounds are obtained by ion exchange using column filled with Dowex-50wx Na + resin. Water is used as eluant which is lyophilized to get the sodium salts of desired compounds. In case of A18, Boc deprotection was applie d using trifluoroacetic acid (TFA) before preparative HPLC. Synthesis of compound s A22 and A23 was accomplished by an alternative route elaborated in scheme 5. Coupling of compound 5 with intermediate 2 was done by using HATU and DIPEA in DMF/ CH2Cl2 mixture to form the derivative B22 which was treated with tetrabutylammonium fluoride (TBAF) in THF to obtain the hydroxy derivative C22. The compound C22 was converted to the sodium salt of A22 by using the procedure described for A1. Compound A23 was prepared following aforementioned scheme 5 methods starting from 4-aminothiazole-2-carboxylic acid and amidine derivative 2 according to the procedures described for A22. Scheme 4. Synthesis of compounds A1-21. Reagents and conditions : (i) Acetylchloride, TEA, CH2Cl2, RT, 16h (for B1); HA TU, DIPEA or DCC, DMAP, DMF or THF, RT, 16-24h; (ii) Pd/C (wet), THF or EtOAc/TEA, H2, RT, 16h; (iii) SO3-pyridine, pyridine, or SO3-pyridine, TEA, THF/Water, RT, 16h, then Dowex-50wx Na+. 8 Scheme 5: Synthesis of compounds A22-23. Reagents and conditions: (ii) HA TU, DIPEA or DCC, DMAP, DMF or THF, RT, 16h. (iii) TBAF, THF. (iv) SO3-pyridine, pyridine, or SO3-pyridine, TEA, THF/Water, RT, 16h, then Dowex-50wx Na+. In vitro antibacterial efficacy We synthesized a series of amidine derivative s of avibactam containing a variety of substituents , forming amide linkage with NH 2 of amidine of the parent intermediate 1 or 2. Different kinds of substituents (R) introduced in final compounds A1-A23 are depicted in table 1. In vitro antibacterial activities of compounds A1-A23 were determined without combining it with an antibacterial drug and minimum inhibitor concentration (MIC) of each compound was determined for each of the ten bacterial strains i.e. E. coli clinical isolate; E. coli 8739; K. pneumoniae clinical isolate; K. pneumoniae 700603; E. cloacae clinical isolate; E. cloacae 700323; A. baumannii clinical isolate; A. baumannii 19606; P . aeruginosa clinical isolate and P . aeruginosa 9027 (table 1). All the synthesized compounds showed MIC value of >64 mg/L against all tested bacterial species. For comparison, MIC values of avibactam against all of these bacteria were also determined and were found comparable to our synthesized compounds (MIC, >64 mg/L). This indicates that both avibactam and compounds A1-A23 are not 9 antibacterial in action when used alone. Next, we deter mined the antibacterial activity of meropenem (MER) alone and its combination with avibactam as well as in combination with newly synthesized compounds A1-A23. From the table 1, it can be deduced that the antibacterial activity of MER increases after addition (4 mg/L) of avibactam against all bacterial strains under observation. The MIC values of MER without avibactam were observed to be in the range of 2 mg/L to 4 mg/L, whereas after the addition of avibactam this range modified to <0.125 mg/L – 1 mg/L indicating the enzyme inhibition effect of the avibactam. In order to establish the lactamase inhibition effect of our synthesized avibactam derivatives A1-A23, we determined the antibacterial activity of MER in combination with compounds A1-A23 individually. The results are summarised in table 1 as MIC values of each compound against each bactrial strain. From the table it is evident that all of the compounds enhanced the antibacterial activity of MER (MIC, <0.125 mg/L – 2 mg/L) as compared to meropenem alone (MIC, 2 mg/L to 4 mg/L). Compound A12 proves most potent among the other counterparts against all bacterial species with MIC from <0.125 mg/L – 2 mg/L, and is comparable to avibactam against both E. coli strains and K. pneumoniae strains with MIC value of <0.125 mg/L. From the data in table 1 it is clear that A. baumannii clinical isolate is the most resistant strain against all newly synthesized compounds as well as avibactam showing MIC value of 2 mg/L and 1 mg/L respectively. However, E. coli 8739 is the most susceptible strain to most of the synthesized compounds for example, A1, A2, A8, A12, A13 and A16 with MIC value of <0.125 mg/L. 10 Table 1. In vitro antibacterial activity of avibactam and compounds A1-A23 alone as well as in combination with meropenem (MER). R Substituents in A1-A23 Sample Minimum Inhibitory Concentration (MIC, mg/L) E. colia E. colib K. pc K. pd E.ce E.cf A.bg A.bh P.ai P.aj A1-A23& avibactam alone >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 MER alone 4 4 4 2 4 4 4 2 4 4 MER+Avibactam <0.125 <0.125 <0.125 <0.125 <0.125 <0.125 1 0.5 0.5 0.25 Me A1 0.5 <0.125 2 0.5 2 1 2 0.5 0.5 1 Et A2 0.5 <0.125 2 1 2 2 2 0.5 0.5 0.5 AcNHEt A3 <0.125 0.25 1 0.25 1 1 2 0.5 0.5 1 Ph A4 2 0.25 1 0.25 2 0.25 2 0.5 2 1 4-FPh A5 1 0.25 2 0.25 2 1 2 1 1 1 4-CF3Ph A6 0.5 0.25 2 0.5 2 2 2 0.5 1 0.5 A7 1 0.25 2 0.5 2 1 2 0.5 0.5 1 A8 0.5 <0.125 2 0.5 2 2 2 0.5 0.5 1 A9 0.5 0.5 2 1 2 2 2 1 1 2 A10 1 0.5 2 0.5 2 1 2 0.5 0.5 1 A11 0.25 0.25 0.25 0.25 0.5 1 2 0.5 0.25 1 A12 <0.125 <0.125 <0.125 <0.125 0.5 0.5 2 1 0.25 0.5 A13 0.5 <0.125 2 0.25 2 1 2 0.5 0.5 1 11 A14 0.25 0.25 0.25 0.25 2 0.25 2 0.5 0.25 1 A15 0.25 0.25 0.25 0.25 2 1 2 1 0.25 1 A16 1 <0.125 2 0.25 2 0.5 2 0.5 0.5 0.5 A17 0.25 0.25 0.25 0.25 1 1 2 0.5 0.25 0.5 A18 0.25 0.5 0.25 0.25 2 1 2 2 1 0.25 A19 0.25 0.25 2 0.25 1 0.5 2 1 1 0.5 A20 0.25 0.25 1 0.25 2 0.5 2 0.5 1 0.5 A21 0.5 0.5 2 0.5 2 0.5 2 0.5 0.25 1 A22 1 0.5 2 0.25 1 0.5 2 0.5 0.25 0.5 A23 0.25 0.25 1 0.25 0.5 1 2 0.5 0.25 0.5 aE. coli clinical isolate; bE. coli 8739; cK. pneumoniae clinical isolate; dK. pneumoniae 700603; eE. cloacae clinical isolate; fE. cloacae 700323; gA. baumannii clinical isolate; hA. baumannii 19606; iP . aeruginosa clinical isolate; jP . aeruginosa 902.

We have successfully synthesized a series of amidine substituted avibactam derivatives in moderate to good overall yields. In vitro antibacterial testing for these compounds showed lack of antibacterial efficacy, however all compounds showed moderate lactamase inhibition activity depicted by minimized the MIC values of meropenem in the presence of test compounds. Compound A12 was most potent inhibitor in case of all bacterial strains under observation and may be a lead compound for further development. Acknowledgments 12 This work was supported by the grant from Science and Technology Department of Ningxia, P.R. China (No. 2018BCG01001). Ministry of Science and Technology, P.R. China is gratefully acknowledged for the award of foreign expert program to Dr. Haikang Yang and Dr. Zafar Iqbal.

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Eur. J. 2019, 25, 14912-14920. 14 Graphical Abstract β-Lactamase inhibition profile of new amidine substituted diazabicyclooctanes Zafar Iqbal,†a Yuanyu Gao,†a Dong Tang,a Xueqin Ma,b Jinbo Ji,a Jian Sun,a Jingwen Ji,a Yuanbai Liu,a Lijuan Zhai,a Rui Jiang,a Yangxiu Mu,a Lili He,a Haikang Yang*a and Zhixiang Yang*a