antibiotics Article

Synthesis of 4,40-(4-Formyl-1H-pyrazole-1,3-diyl) dibenzoic Acid Derivatives as Narrow Spectrum Antibiotics for the Potential Treatment of Acinetobacter Baumannii Infections Evan Delancey 1 , Devin Allison 1 , Hansa Raj KC 1 , David F. Gilmore 2 , Todd Fite 3,4 , Alexei G. Basnakian 3,4 and Mohammad A. Alam 1, * 1

2 3

4

*

Department of Chemistry and Physics, College of Science and Mathematics, Arkansas State University, Jonesboro, AR 72467, USA; [email protected] (E.D.); [email protected] (D.A.); [email protected] (H.R.K.) Department of Biological Sciences, College of Science and Mathematics, Arkansas State University, Jonesboro, AR 72467, USA; [email protected] Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205, USA; [email protected] (T.F.); [email protected] (A.G.B.) Central Arkansas Veterans Healthcare System, W. 7th St., Little Rock, AR 72205, USA Correspondence: [email protected]; Tel.: +1-870-972-3319

Received: 29 July 2020; Accepted: 25 September 2020; Published: 28 September 2020







Abstract: Acinetobacter baumannii has emerged as one of the most lethal drug-resistant bacteria in recent years. We report the synthesis and antimicrobial studies of 25 new pyrazole-derived hydrazones. Some of these molecules are potent and specific inhibitors of A. baumannii strains with a minimum inhibitory concentration (MIC) value as low as 0.78 µg/mL. These compounds are non-toxic to mammalian cell lines in in vitro studies. Furthermore, one of the potent molecules has been studied for possible in vivo toxicity in the mouse model and found to be non-toxic based on the effect on 14 physiological blood markers of organ injury. Keywords: antimicrobial; pyrazole; hydrazone; Acinetobacter baumannii; toxicity; narrow-spectrum antibiotics

1. Introduction The ESKAPE pathogens, a group of six bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) cause the majority of nosocomial infections in the U.S., and these pathogens are effectively escaping the current arsenal of antibiotics [1]. A. baumannii, one of the ESKAPE pathogens, is a commonly found aquatic Gram-negative bacterium, but its drug-resistant strains can cause serious health problems in immunocompromised individuals particularly in clinical settings. In just two decades, this bacterium became well-known to cause serious infections, being notorious opportunist and producing multiresistant strains in hospital wards and in communities [2–5]. A. baumannii infection of U.S. service members has become a major problem since the Iraq War began in 2003 [6]. In recent years, the emergence of A. baumannii, which is extremely drug- and pandrug-resistant, has alarmed the medical community [7–9]. In late February 2017, the WHO released a list of 12 drug-resistant bacteria that pose the greatest threat to human health and for which new antibiotics are desperately needed. Carbapenem-resistant A. baumannii is on the top of the list [10,11]. Therefore, the discovery of new antibacterial agents to treat A. baumannii infections is imperative. Antibiotics 2020, 9, 650; doi:10.3390/antibiotics9100650

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Antibiotics 2020, 9, x FOR PEER REVIEW

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Carbapenem-resistant A. baumannii is on the top of the list [10,11]. Therefore, the discovery of2 new of 14 antibacterial agents to treat A. baumannii infections is imperative. Although broad-spectrum antibiotics play a vital role in treating bacterial infections, there are broad-spectrum antibiotics playfor a vital role in treating bacterial infections, are someAlthough drawbacks to their use such as selection and spread of resistance across multiple there bacterial some drawbacks to their use such as selection for and spread of resistance across multiple bacterial species and the detrimental effect upon the host microbiome. If the causative agent of the infection is species detrimental effect upon the host microbiome. If the causative the infection is known,and thethe use of narrow-spectrum antibiotics can alleviate some of agent theseofproblems. The Antibiotics 2020, 9,of xof FOR PEER REVIEW 2 of 13 known, the use narrow-spectrum antibiotics alleviate of these problems. The development narrow-spectrum antibioticscan that do notsome cause cross-resistance in development non-targeted of narrow-spectrum antibiotics do notupon cause cross-resistance in non-targeted microbes and microbes and elicit less collateralthat damage the host microbiome is an attractive approach to elicit fight Carbapenem-resistant A. baumannii is on the top of the list [10,11]. Therefore, the discovery of new less collateral damage infections upon the host is an attractive approach fight multidrug-resistance [12].microbiome It is also expected that new drugs to will be multidrug-resistance non-toxic in vivo at antibacterial agents to treat A. baumannii infections is imperative. infections [12]. It is also expected that new drugs will be non-toxic in vivo at therapeutic doses. therapeutic doses. Although broad-spectrum antibiotics play a vital role in treating bacterial infections, there are 2. Results and Discussion Discussion some drawbacks to their use such as selection for and spread of resistance across multiple bacterial 2. Results and species andefforts the detrimental effect upon the host microbiome. If the causativereported agent of the thesynthesis infection of is In to find potent agents previously In our our potentantimicrobial antimicrobial agents[13], [13],we wehave have previously the synthesis known, theefforts use to of find narrow-spectrum antibiotics can alleviate some of reported these problems. The phenyl substituted pyrazole derivatives as potent S. aureusaureus and A.and baumannii growthgrowth inhibitors [14,15]. of phenyl substituted pyrazole derivatives as potent A. baumannii inhibitors development of narrow-spectrum antibiotics that doS.not cause cross-resistance in non-targeted Fluoro-substitution on the phenyl ringphenyl increased theincreased potency ofthe molecules significantly [16,17]. Similarly, [14,15]. Fluoro-substitution on the ring potency of molecules significantly microbes and elicit less collateral damage upon the host microbiome is an attractive approach to fight coumarin and naphthalene substituted compounds also showed potent activity against the tested [16,17]. Similarly, coumarin and[12]. naphthalene substituted potent multidrug-resistance infections It is also expected thatcompounds new drugs also will showed be non-toxic in activity vivo at strains, particularly S. aureus [18–20]. Our reported molecules are significantly lipophilic. To get less against the doses. tested strains, particularly S. aureus [18–20]. Our reported molecules are significantly therapeutic lipophilic designed and synthesized a series ofand diylbenzoic acidaderivatives of pyrazole lipophilic.compounds, To get less we lipophilic compounds, we designed synthesized series of diylbenzoic to get compounds with better pharmacological properties (Figure 1). acid derivatives of pyrazole to get compounds with better pharmacological properties (Figure 1). 2. Results and Discussion Antibiotics 2020, 9, 650

In our efforts to find potent antimicrobial agents [13], we have previously reported the synthesis of phenyl substituted pyrazole derivatives as potent S. aureus and A. baumannii growth inhibitors [14,15]. Fluoro-substitution on the phenyl ring increased the potency of molecules significantly [16,17]. Similarly, coumarin and naphthalene substituted compounds also showed potent activity against the tested strains, particularly S. aureus [18–20]. Our reported molecules are significantly lipophilic. To get less lipophilic compounds, we designed and synthesized a series of diylbenzoic acid derivatives of pyrazole to get compounds with better pharmacological properties (Figure 1).

Pyrazole derived derived hydrazones hydrazones as potent anti- Acinetobacter baumannii Figure 1. Pyrazole baumannii agents. agents.

The reaction of hydrazinobenzoic acid (1) with 4-acetylbenzoic acid (2) formed the hydrazone (3), which on reaction with POCl3 /N,N-dimethylformamide (DMF) afforded the diylbenzoic acid-derived pyrazole aldehyde (4) in 90% overall yield. The ease of synthesis of the pure aldehyde derivative (4) in a multigram scale without work-up or column purification helped to synthesize a series of hydrazone derivatives. The starting material was subjected to further reaction with several hydrazine derivatives to get novel hydrazone derivatives. We found the expected products (5–29) in a very good average yield (Scheme 1). 1. Pyrazole derived hydrazones as potent anti- Acinetobacter baumannii agents. Figure

Scheme 1: Synthesis of pyrazole-derived aldehyde (4).

The reaction of hydrazinobenzoic acid (1) with 4-acetylbenzoic acid (2) formed the hydrazone (3), which on reaction with POCl3/N,N-dimethylformamide (DMF) afforded the diylbenzoic acidderived pyrazole aldehyde (4) in 90% overall yield. The ease of synthesis of the pure aldehyde derivative (4) in a multigram scale without work-up or column purification helped to synthesize a series of hydrazone derivatives. The starting material was subjected to further reaction with several hydrazine derivatives to get novel hydrazone derivatives. We found the expected products (5-29) in 1: Synthesis of pyrazole-derived aldehyde (4). a very good average yield.Scheme 1. These new compounds were tested against six bacterial strains: Two Gram-positive strains, S. These new compounds were tested against bacterial strains: Twoacid Gram-positive strains, S. aureus The reaction of and hydrazinobenzoic (1)sixwith (2) formed theEnterobacter hydrazone aureus ATCC 25923 Bacillus subtilisacid ATCC 6623,4-acetylbenzoic and four Gram-negative bacteria, ATCC 25923 and Bacillus subtilis ATCC 6623, and four Gram-negative bacteria, Enterobacter aerogenes (3), whichATCC on reaction with POClcoli 3/N,N-dimethylformamide (DMF) afforded the diylbenzoic acidaerogenes 13048, Escherichia ATCC 25922, A. baumannii ATCC 19606, type strain (Ab6), and ATCC 13048, Escherichia coli ATCC baumannii 19606,oftype strain (Ab6), Pseudomonas derived pyrazole aldehyde (4) in25922, 90% A. overall yield.ATCC The ease synthesis of theand pure aldehyde derivative (4) in a multigram scale without work-up or column purification helped to synthesize a series of hydrazone derivatives. The starting material was subjected to further reaction with several hydrazine derivatives to get novel hydrazone derivatives. We found the expected products (5-29) in a very good average yield. These new compounds were tested against six bacterial strains: Two Gram-positive strains, S.

similar potency against thestrains. tested strains of A. baumannii. substitutions, chloro, and fluoro the growth of the tested Extremely electron withdrawing groups such as trifluoromethyl (26), the growth growth of the the tested strains. Extremely electron Mixed-halo withdrawing groups such such as trifluoromethyl trifluoromethyl (26), the of tested strains. electron withdrawing groups as (26), (22 and 23), retained the acid potency ofExtremely the compounds. Tetra-fluoro substitution decreased the potency cyano (27), carboxylic (28), and nitro (29) completely eliminated the potency of the molecules. cyano (27), carboxylic acid (28), and nitro (29) completely eliminated the potency of the molecules. cyano (27), carboxylic (28), and nitro (29) completely eliminated the (25) potency molecules. of the resultant moleculeacid (24) several-fold. Similarly, the pentafluoro compound failedof to the inhibit Based on the antimicrobial data, we could deduce aa clear structure–activity relationship (SAR) Based on the antimicrobial data, we could deduce clear structure–activity relationship (SAR) Based on the antimicrobial data, we could deduce a clear structure–activity relationship (SAR) the growth of the tested strains. Extremely electron withdrawing groups such as trifluoromethyl (26), of narrow spectrum antimicrobial agents. N,N-Disubstituted compounds failed to show any activity of narrow narrow spectrumacid antimicrobial agents. N,N-Disubstituted compounds failed to show show any any activity activity cyano (27), carboxylic (28), and nitro (29) completely eliminatedcompounds the potency of the molecules. of spectrum antimicrobial agents. N,N-Disubstituted failed to against the tested strains. Halogen-substituted N-phenyl hydrazone showed potent activity. Among against the tested strains. Halogen-substituted N-phenyl hydrazone showed potent activity. Among Based on the antimicrobial data, we could deduce a clear structure–activity relationship (SAR)3 of Among against the tested strains. Halogen-substituted N-phenyl hydrazone showed potent activity. Antibiotics 2020, 9, 650 14 these halogen-substituted compounds, mono-substitution showed the best result these halogen-substituted compounds, mono-substitution showed the to best result and the the chlorine chlorine of narrow spectrum antimicrobial agents. N,N-Disubstituted compounds failed show any and activity these halogen-substituted compounds, mono-substitution showed the best result and the chlorine substitution gave the most potent compound (18). Increasing the number of halogen atoms in the against the tested strains. Halogen-substituted N-phenyl potent activity. Among substitution gave the most most potent compound compound (18).hydrazone Increasingshowed the number number of halogen halogen atoms in in the the substitution gave the potent (18). Increasing the of atoms phenyl ring decreased the activity of the molecules as no activity was observed for the pentathese halogen-substituted compounds, mono-substitution showed the best result and the chlorine aeruginosa ATCC 27833. Remarkably, in initial screening tests, none of the compounds showed activity phenyl ring decreased the activity of the molecules as no activity was observed for the pentaphenyl ring decreased the activity of the molecules as no activity was observed for the pentasubstitution gave the most (18). Increasing of halogen substituted compounds. against the bacteria tested otherpotent than compound A. baumannii ATCC 19606the at number concentrations lessatoms than in 50the µg/mL. substituted compounds. substituted compounds. phenyl ring decreased the activity of the molecules as no activity was observed for the pentaBecause of the high specificity of compounds against A. baumannii, these molecules were further tested substituted compounds. Table 1. Synthesis and antimicrobial activity of (pyrazole-1,3-diyl)dibenzoic acid substituted Table 1. Synthesis Synthesis and antimicrobial antimicrobial activity of (pyrazole-1,3-diyl)dibenzoic (pyrazole-1,3-diyl)dibenzoic acid acid substituted substituted against two additional A. baumannii strains (Table 1). of Table 1. and activity hydrazone derivatives. hydrazone derivatives. derivatives. hydrazone Table 1. Synthesis and antimicrobial activity of (pyrazole-1,3-diyl)dibenzoic acid substituted Table 1. Synthesis and antimicrobial activity of (pyrazole-1,3-diyl)dibenzoic acid substituted hydrazone derivatives. hydrazone derivatives.

Comps Comps Comps Comps Comps

MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) Sa Bs Ea Ab6 Ab5 Sa Bs Ea Ab6 Ab5 Sa Sa Ab6Ab6 Ab5Ab5 Ab5 Ab7 Ab7 SaBs BsBsEa EaEa Ab6

Ab7 Ec Ec Ec PaPa Ab7 Ec

Pa Pa Pa

55 55 5

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA NA NANA NANA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA

666 6

NANA NA NA NA NA NA NANA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA NA NA

4 of 14 of 14 14 44 of 4 14 44 of of 14 of 14 NA NA

6

Antibiotics 2020, 9, FOR PEER REVIEW Antibiotics 2020, 2020, 9, 9, xxx FOR FOR PEER PEER REVIEW REVIEW Antibiotics Antibiotics 2020, 9, x FOR PEER REVIEW 7 Antibiotics 2020, 9, x FOR PEER REVIEW Antibiotics 2020, 9, x FOR PEER REVIEW 77 7

7 8 88 8 88 8

Ab7

Ec

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA

NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

9 99 9 99 9

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA NA NA

10 10 10 10 10 10 10

NA NA NA

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA

NA NA NA NA

NA NA NA NA NA NA

11 1111 11 11 11 11

NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA

NA

NA NA NA NA NA NA

12 1212 12 12 12 12

NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA

NA NA NA NA NA NA

13 1313 13 13 13 13

NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA

NA NA

NA NA NA

NA NA

NA NA NA

NA NA

NA NA NA

NA NA

NA NA NA

NA NA

NA NA NA

NA NA

NA NA NA

NA NA NA NA

NA NA NA NA

NA NA NA NA

NA NA NA NA

NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA NA NA

14 14 14 14 14 14

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

15 15 15 15 15 15

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

16 16 16 16 16 16

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

3.125 3.125 3.125 3.125 3.125 3.125

25 25 25 25 25 25

3.125 3.125 3.125 3.125 3.125 3.125

NA NA NA NA NA NA

NA NA NA NA NA NA

against the tested strains. Halogen-substituted N-phenyl hydrazone showed potent activity. Among substitution gave the most potent compound (18). Increasing the number of halogen atoms in the 10 NA NA NA showed NA the NA NAand the NAchlorine NA NA NA these10 halogen-substituted compounds, mono-substitution best result NA NA NA NA NA phenyl ring decreased the activity of the molecules as no activity was observed forNA the penta10 NA NA NA NA NA substitution gave the most potent compoundNA (18). Increasing the number atoms 10 NA NA NA NA NA of halogen NA NA in the NA NA NA 10 NA NA NA NA NA NA NA NA substituted 10 ring compounds. NA NA NA for NA NA NA phenyl decreased the activity of the molecules as no NA activity NA was observed the pentasubstituted compounds.

Table Antibiotics 2020, 9, 6501. Synthesis and antimicrobial activity of (pyrazole-1,3-diyl)dibenzoic acid substituted 4 of 14 11

NA 11 NA hydrazone derivatives. 11 NA Table 1. Synthesis and antimicrobial activity 11 NA of 11 NA hydrazone derivatives. 11 NA

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA (pyrazole-1,3-diyl)dibenzoic acid substituted NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA

12 12 12 12 12 12

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

13 13 13 13 13 13 Comps Comps Comps 14 14 14 14 55 14 14 14

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA MIC (µg/mL) MIC (µg/mL) MIC (µg/mL)

Table 1. Cont.

15 6 15 15 6 15 15 1515 7

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA

EcEc PaPa Ab7 Ec NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA

Pa NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA

NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA

Sa Sa Ab6Ab6 Ab5 Ab7 Ab5 Ab5 Ab7 SaBs BsBsEa EaEa Ab6

NA NA NA NA NA NA NA NA NA NA

NA

NA

NA

NA

NA

NA

NA

NA

7 16 16 16 16 16 1616

NA NA NA NA NA NA NA NA 3.125 3.125 25 NA NA NA 25 NA NA NA 3.125 25 NA NA NA 3.125 25 NA NA NA 3.125 NA NA NA NA NA NA 3.125 3.125 25 25 253.125

NA 3.125 3.125 3.125 3.125 3.125 NA 3.125

NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

17 17 17 17 1717 17

NA NA NA NA NA 1.56 3.125 NA 1.56 3.125 NA NA NA 1.56 3.125 NA NA NA NA 1.56 3.125 NA 1.56 NA NA NA NA 1.561.563.1253.125 NA NA NA 1.56 3.125

1.56 1.56 1.56 1.56 NA 1.56 1.56

NA NA NA NA NA NA NA

NA NA NA NA NA NA

18 18 18 1818 18 18

NA NA NA 0.78 3.1253.125 NA NA NA 3.125 NA 1.56 NA NA NA NA NA 0.780.78 0.78 3.125

1.56 1.56 1.56 NA 1.56 1.56 1.56

NA NA NA NA

NA

NA NA

NA NA NA NA NA NA

NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA 3.125 3.1256.25 6.25 6.25 3.125 NA 3.125 3.125 NA NA NA NA 3.125

NA NA NA NA NA NA NA

NA NA 5 of 14 NA 5 of 14 NA 5 of 14 NA NA

21 21 2121

NA NA NA NA NA 3.125 3.125 6.25 3.125 NA NA 3.125 3.125 NA NA NA NA NA NA 3.125 3.1256.25 6.25 6.25 3.125

NA NA NA

NA

NA NA NA

22 22 2222

NA NA NA NA NA 6.25 6.25 3.125 NA NA 3.125 3.125 NA NA NA NA NA NA 6.256.25 6.25 6.25 6.25 6.25 3.125

NA NA NA

NA

NA NA NA

2323 23 23

NA NA NA NA NA 3.125 3.1256.25 6.25 6.25 3.125 NA 3.125 3.125 NA NA NA NA 3.125

1919 19 19 19 19 19

2020

20 Antibiotics 2020, 9, x FOR PEER REVIEW 20 Antibiotics 9, x FOR PEER REVIEW 20 2020, Antibiotics 2020, 9, x FOR PEER REVIEW 20 20

24 24 24

NA

NA

NA

0.78

3.125

NA NA

NA NA

NA NA

0.78 0.78

3.125 3.125

NA NA NA NA NA 3.125 3.125 25 NA NA 3.125 NA NA NA 3.125 NA NA NA 3.125 NA NA NA NA NA NA 3.125 3.125

NA NA

NA NA NA NA

NA NA NA NA

NA NA NA NA

3.125 3.125 3.125 3.125

253.125 25 25 25 25 25

6.25 6.25 6.25 6.25

3.125 NA 3.125 3.125 3.125 3.125 3.125

3.125 3.125 3.125 3.125

NA NA

NA

NA

NA

3.125

6.25

3.125

NA NA NA NA

NA NA NA

NA NA NA

NA NA NA

NA NA NA

12.5 12.5 12.5

50 50 50

25 25 25

NA NA NA

NA NA NA

Antibiotics 2020, 9, x FOR PEER REVIEW

5 of 14

against the tested strains. Halogen-substituted N-phenyl hydrazone showed potent activity. Among Antibiotics 2020, 9, x FOR FOR PEER REVIEW of 14 substitution thePEER most potent compound (18). Increasing the number of halogen atoms in555 of the Antibiotics 2020, 9, REVIEW 14 Antibiotics 2020, 2020,gave 9, xx x FOR FOR PEER REVIEW of 14 Antibiotics 9, PEER REVIEW 5 of 14 these halogen-substituted compounds, mono-substitution showed the best result and the chlorine phenyl ring decreased the activity of the molecules as no activity was observed for the pentasubstitution gave the most potent compound (18). Increasing the number of halogen atoms in the substituted compounds. phenyl ring decreased the activity of the molecules as no activity was observed for the penta21 NA NA NA NA 3.125 6.25 3.125 NA NA 21 NA 6.25 3.125 substituted compounds. 21 NA NA NA NA NA 3.125 3.125 6.25 3.125 NA NA NA NA 21 NA NA 3.125 6.25 3.125 NA NA 21 NA NA NA 3.125 3.125 6.25 3.125 NA NA Table activityNA of (pyrazole-1,3-diyl)dibenzoic acid substituted Antibiotics 2020, 9, 6501. Synthesis and antimicrobial NA 5 of 14NA 21 NA 6.25 3.125 NA hydrazone derivatives. Table 1. Synthesis and antimicrobial activity of (pyrazole-1,3-diyl)dibenzoic acid substituted hydrazone derivatives.

22 22 22 22 22 22

Table 1. Cont.

3.125 3.125 3.125 3.125 3.125 3.125

NA NA NA NA NA NA

NA NA NA NA NA NA

3.125 6.25 3.125 6.25 3.125 6.25 3.125 6.25 3.125 6.25 3.125 6.25 MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) Sa Sa Ab6Ab6 Ab5 Ab7 Ab5 Ab5 Ab7 SaBs BsBsEa EaEa Ab6

3.125 3.125 3.125 3.125 3.125 3.125

NA NA NA NA NA NA

NA NA NA NA NA NA

EcEc PaPa Ab7 Ec

Pa

24 55 24 24 24 24 2424

NA NA NA NA NA NA NA NA NA NA

25 NANA NA NA NA 25

NA NA NA NA NA NA

6

NA

7 25 25 25 7 25 25 2525

NA NA NA NA NA NA NA

23 23 23 23 23 23 Comps Comps Comps

6

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

6.25 6.25 6.25 6.25 6.25 6.25

6.25 6.25 6.25 6.25 6.25 6.25

12.5 50 NA NA NA NA 12.5 50 NA NA NA NA NA 12.5 50 NA 12.5 50 NA NA NA 12.5 50 NA NA NA NA NA 12.512.5 50 50 25 NA NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

26 26 2626 26 26 26

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA

27 27 2727 27 27 27

NA NA NA NA NA NA NA NA

25 25 25 25 NA

NA

NA

NA NA NA NA NA

NA

NA

NA

NA NA NANA

NA NA NA NA NA NA

NA NA NA NA

NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA

NA NA NA

NA NA NA NA NA NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA NA NA

NA NA NA

NA NA NANA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA

NA NA NA

NA NA NA NA NA NA

28 28 2828 28 28 28

NA NA NA NA NA NA NA NA

NA NA NANA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA

NA NA NA NA NA NA

2929 29 29 29 29 29

NA NA NA NA NA NA

NA NA NA NA NANA NA

NA NA NA

NA NA NA

NA NA

NA NA NA

NA NA

NA NA NA

NA NA NA

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA DMSO NANA NA NA NA NA NA DMSO NA NA NA DMSO NA NA NA NA DMSO NA NA NA NA NA NA DMSO NA NA NA NA NA Colistin 1.56 3.125 0.78 DMSO NA NA NA NA NA Colistin 1.56 0.78 0.78 Colistin 1.56 DMSO NA 3.1253.125 NA NA NA NA Colistin 1.56 3.125 0.78 Colistin 1.56 3.125 0.78 Colistin 1.56Bacillus 3.125 0.78 Gram-positive bacteria: Staphylococcus Staphylococcus aureus aureus ATCC ATCC 25,923 25,923 (Sa) (Sa) and and Bacillus subtilis 0.78 ATCC 6623 (Bs), (Bs), Colistin 1.56 3.125 Gram-positive bacteria: subtilis ATCC Gram-positive bacteria: Staphylococcus aureus ATCCaureus 25,923 ATCC (Sa) and25,923 Bacillus(Sa) subtilis ATCC 6623 (Bs), Gram-negative Gram-positive bacteria: Staphylococcus and Bacillus subtilis ATCC 6623 6623 (Bs), NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Gram-positive bacteria: Staphylococcus aureus ATCC 25,923 (Sa) and Bacillus subtilis ATCC 6623 (Bs),

Gram-negative bacteria: Enterobacter aerogenes ATCC 13048 (Ea), Escherichia coli ATCC 25922 (Ec), A. Gram-positive bacteria: Staphylococcus aureus ATCC ATCC 25,923 (Sa) and Bacillus subtilis ATCC 6623 (Bs), bacteria: Enterobacter aerogenes ATCC 13048 (Ea),aerogenes Escherichia coli ATCC 25922 (Ec), A.Bacillus baumannii ATCC 19606, type Gram-negative bacteria: Enterobacter ATCC 13048 (Ea), Escherichia coli ATCC 25922 (Ec), A. Gram-positive bacteria: Staphylococcus aureus 25,923 (Sa) and subtilis ATCC 6623 (Bs), Gram-negative bacteria: Enterobacter aerogenes ATCC 13048 (Ea), Escherichia coli ATCC 25922 (Ec), A. Gram-negative bacteria: Enterobacter aerogenes ATCC 13048 (Ea), Escherichia coli ATCC 25922 (Ec), A. baumannii ATCC 19606, type strain (Ab6), A. baumannii ATCC BAA-1605 (Ab5), A. baumannii ATCC strain (Ab6), A. baumannii ATCC BAA-1605 (Ab5), A. baumannii ATCC 747 (Ab7), Pseudomonas aeruginosa 27833 (Pa). Gram-negative bacteria: Enterobacter aerogenes ATCC 13048 13048 (Ea), Escherichia coli ATCC ATCC 25922 (Ec), (Ec), A. baumannii ATCC 19606, strain A. ATCC BAA-1605 (Ab5), A. ATCC Gram-negative bacteria: Enterobacter aerogenes ATCC (Ea), Escherichia coli 25922 A. baumannii ATCC 19606, type type strain (Ab6), (Ab6), A. baumannii baumannii ATCC BAA-1605 (Ab5), A. baumannii baumannii ATCC and NA = no activity. baumannii ATCC 19606, type strain (Ab6), A. baumannii ATCC BAA-1605 (Ab5), A. baumannii ATCC 747 (Ab7), Pseudomonas aeruginosa 27833 (Pa). and NA = no activity. baumannii ATCC 19606,aeruginosa type strain strain27833 (Ab6), A. baumannii baumannii ATCC BAA-1605 BAA-1605 (Ab5), (Ab5), A. A. baumannii baumannii ATCC ATCC 747 Pseudomonas (Pa). and baumannii 19606, type (Ab6), A. ATCC 747 (Ab7), (Ab7), ATCC Pseudomonas aeruginosa 27833 (Pa). and NA NA === no no activity. activity. 747 (Ab7), Pseudomonas aeruginosa 27833 (Pa). and NA no activity. 747 (Ab7), Pseudomonas aeruginosa 27833 (Pa). and NA = no activity. 747 (Ab7), Pseudomonas aeruginosa 27833 (Pa). and NA = no activity.

2.1. Experimental Datahydrazine derivatives (5, 6, 7, and 8) did not show any activity against Products of aliphatic 2.1. 2.1. Experimental Experimental Data Data 2.1. Experimental Data the tested bacterial strains. N,N-Disubstituted hydrazone derivatives (9, 10, 11, 12, 1 13, 14, 2.1. Experimental Data 2.1. Experimental Data 4,4′-(4-formyl-1H-pyrazole-1,3-diyl)dibenzoic acid (4). (4). Yellow solid solid (3.02g (3.02g 90%). 90%). 1H H NMR, 300 300 4,4′-(4-formyl-1H-pyrazole-1,3-diyl)dibenzoic 1H NMR, and 15) also failed to show any noticeable antimicrobial acid property for thesolid tested strains. N-Phenyl 4,4′-(4-formyl-1H-pyrazole-1,3-diyl)dibenzoic acid (4). Yellow Yellow solid (3.02g 90%). NMR, 300 300 1H 13C NMR 4,4′-(4-formyl-1H-pyrazole-1,3-diyl)dibenzoic acid (4). Yellow (3.02g 90%). NMR, 1H6NMR, MHz (DMSO-d 6): δ 9.99 (s, 1H), 9.41 (s, 1H), 8.09-8.05acid (m, 8H); (75MHz, DMSO-d ) δ = 189.6, 4,4′-(4-formyl-1H-pyrazole-1,3-diyl)dibenzoic (4). Yellow solid (3.02g 90%). 300 13 1H6) MHz (DMSO-d 6): δ 9.99 (s, 1H), 9.41 (s, 1H), 8.09-8.05acid (m, (75MHz, DMSO-d δ = 189.6, (4).potent Yellow solid (3.02g 90%). NMR, 300 13C derivatives with halogen in the ring exhibited activity against A. baumannii. MHz4,4′-(4-formyl-1H-pyrazole-1,3-diyl)dibenzoic (DMSO-d 6): δ substitution 9.99 (s, (s, 1H), 1H), 9.41 9.41 (s,phenyl 1H), 8.09-8.05 8.09-8.05 (m, 8H); 8H); C NMR NMR (75MHz, DMSO-d 6) δ = 189.6, 13C MHz (DMSO-d 6): δ146.5, 9.99 (s, 1H), (m, 8H); NMR (75MHz, DMSO-d 6) δ = (m/z): 189.6, 13C 172.1, 171.6, 156.9, 141.1, 140.2, 136.1, 134.6, 133.9, 127.9, 124.0. HRMS (ESI-FTMS Mass MHz (DMSO-d 6): δ 9.99 (s, 1H), 9.41 (s, 1H), 8.09-8.05 (m, 8H); NMR (75MHz, DMSO-d 6) δ = 189.6, 13 172.1, 171.6, 156.9, 146.5, 141.1, 140.2, 136.1, 134.6, 133.9, 127.9, 124.0. HRMS (ESI-FTMS Mass (m/z): MHz 6): δ 9.99 (s, 1H), 9.41 (s, 1H), 8.09-8.05 (m, 8H); C NMR (75MHz, DMSO-d6) δ = 189.6, 2-Fluorphenyl-susbstituted hydrazone (16)136.1, showed potent activity the A. (ESI-FTMS baumannii strains 172.1,(DMSO-d 171.6, 156.9, 156.9, 146.5, 141.1, 140.2, 136.1, 134.6, 133.9, 127.9, against 124.0. HRMS HRMS (ESI-FTMS Mass (m/z): (m/z): + = 337.0819, 172.1, 171.6, 146.5, 141.1, 140.2, 134.6, 133.9, 127.9, 124.0. Mass calcd for for C18 18H 12N2O 5 [M+H] found 337.0821. 172.1, 171.6, 156.9, 146.5, 141.1, 140.2, 136.1, 136.1, 134.6, 133.9, 127.9, 127.9, 124.0. 124.0. HRMS HRMS (ESI-FTMS (ESI-FTMS Mass Mass (m/z): (m/z): + = calcd C H 12N2O 5 [M+H] 337.0819, found 337.0821. 172.1, 171.6, 156.9, 146.5, 141.1, 140.2, 134.6, 133.9, + with calcd MIC values low as 3.125= µg/mL. calcd for C C18 18H 12N 2O5 [M+H] 337.0819,3-Fluorophenyl found 337.0821.derivative (17) showed better activity for Has 12N2O5 [M+H]+ = 337.0819, found 337.0821. 4-[1-(4-carboxyphenyl)-4-[(E)-(dimethylhydrazono)methyl]pyrazol-3-yl]benzoic acid (5). (5). calcd4-[1-(4-carboxyphenyl)-4-[(E)-(dimethylhydrazono)methyl]pyrazol-3-yl]benzoic for C C18 18H12N2O5 [M+H]++ = 337.0819, found 337.0821. calcd for H12N2O5strains [M+H] with = 337.0819, foundas 337.0821. 4-[1-(4-carboxyphenyl)-4-[(E)-(dimethylhydrazono)methyl]pyrazol-3-yl]benzoic acid (5). against the4-[1-(4-carboxyphenyl)-4-[(E)-(dimethylhydrazono)methyl]pyrazol-3-yl]benzoic A. baumannii MIC values low as 1.56 µg/mL. Chloro-substitution acid gave (5). 1 acid Yellow solid (347 (347 mg, 92%). 92%). 1H H NMR (300 (300 MHz MHz DMSO-d DMSO-d66): ): 8.77 8.77 (s, 1H), 1H), 8.11-8.04 8.11-8.04 (m, (m, 6H), 6H), acid 7.90 (d,(5). J= 4-[1-(4-carboxyphenyl)-4-[(E)-(dimethylhydrazono)methyl]pyrazol-3-yl]benzoic (5). Yellow solid 7.90 4-[1-(4-carboxyphenyl)-4-[(E)-(dimethylhydrazono)methyl]pyrazol-3-yl]benzoic 1H NMR the most potent molecule in the series.(300 ThisMHz molecule (18)66): inhibited the growth of(m, A. baumannii Yellow solid (347 mg, mg,(18) 92%). NMR (300 MHz DMSO-d ): 8.77 (s, (s, 1H), 1H), 8.11-8.04 (m, 6H), acid 7.90 (d, (d, JJJ === 1H 13C NMR Yellow solid (347 mg, 92%). NMR DMSO-d 8.77 (s, 8.11-8.04 6H), 7.90 (d, 1H(s, 8.2 Hz, 2H), 7.29 (s, 1H), 2.88 6H); (75 MHz DMSO-d 6 ): 167.5, 167.1, 149.9, 142.5, 137.2, Yellow solid (347 mg, 92%). NMR (300 MHz DMSO-d 6 ): 8.77 (s, 1H), 8.11-8.04 (m, 6H), 7.90 (d, J 13 1 (s, 8.2 Hz, 2H), (s, 1H), 2.88 6H); (75 ): 167.1, 149.9, 142.5, 137.2, (347 mg, 92%). (300 MHz DMSO-d ): 8.77 (s, 661H), 8.11-8.04 6H), 7.90 (d, J == 13C 8.2 Hz, solid 2H), 7.29 (s, 1H), 2.88 (s,NMR 6H); C NMR NMR (75 MHz MHz 6DMSO-d DMSO-d ): 167.5, 167.5, 167.1, 149.9, 142.5, 137.2, ATCCYellow 19606, type7.29 strain with anHMIC value at submicrogram concentration. Two (m, other strains, 13C 8.2 Hz, 2H), 7.29 (s, 1H), 2.88 (s, 6H); NMR (75 MHz DMSO-d 6): 167.5, 167.1, 149.9, 142.5, 137.2, 13 8.2 Hz, Hz, 2H), 2H), 7.29 (s, (s, 1H), 1H), 2.88 2.88 (s, 6H); 6H); 13C C NMR NMR (75 (75 MHz DMSO-d DMSO-d66): ): 167.5, 167.5, 167.1, 167.1, 149.9, 142.5, 142.5, 137.2, 8.2 A. baumannii ATCC7.29 BAA-1605 and A.(s,baumannii ATCC 747,MHz were also inhibited efficiently 149.9, by this novel137.2, molecule with minimum inhibitory concentration (MIC) values 3.125 and 1.56 µg/mL, respectively. The bromo-substituted compound (19) also showed potent activity against the tested A. baumannii

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strains with MIC value as low as 3.125 µg/mL. Dihalosubstitution, fluoro (20), and chloro (21), in the phenyl ring resulted in similar potency against the tested strains of A. baumannii. Mixed-halo substitutions, chloro, and fluoro (22 and 23), retained the potency of the compounds. Tetra-fluoro substitution decreased the potency of the resultant molecule (24) several-fold. Similarly, the pentafluoro compound (25) failed to inhibit the growth of the tested strains. Extremely electron withdrawing groups such as trifluoromethyl (26), cyano (27), carboxylic acid (28), and nitro (29) completely eliminated the potency of the molecules. Based on the antimicrobial data, we could deduce a clear structure–activity relationship (SAR) of narrow spectrum antimicrobial agents. N,N-Disubstituted compounds failed to show any activity against the tested strains. Halogen-substituted N-phenyl hydrazone showed potent activity. Among these halogen-substituted compounds, mono-substitution showed the best result and the chlorine substitution gave the most potent compound (18). Increasing the number of halogen atoms in the phenyl ring decreased the activity of the molecules as no activity was observed for the penta-substituted compounds. 2.1. Experimental Data 4,40 -(4-formyl-1H-pyrazole-1,3-diyl)dibenzoic acid (4). Yellow solid (3.02 g 90%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 9.99 (s, 1H), 9.41 (s, 1H), 8.09–8.05 (m, 8H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 189.6, 172.1, 171.6, 156.9, 146.5, 141.1, 140.2, 136.1, 134.6, 133.9, 127.9, 124.0. HRMS (ESI-FTMS Mass (m/z): calcd for C18 H12 N2 O5 [M + H]+ = 337.0819, found 337.0821. 4-[1-(4-carboxyphenyl)-4-[(E)-(dimethylhydrazono)methyl]pyrazol-3-yl]benzoic acid (5). Yellow solid (347 mg, 92%). 1 H NMR (300 MHz DMSO-d6 ): 8.77 (s, 1H), 8.11–8.04 (m, 6H), 7.90 (d, J = 8.2 Hz, 2H), 7.29 (s, 1H), 2.88 (s, 6H); 13 C NMR (75 MHz DMSO-d6 ): 167.5, 167.1, 149.9, 142.5, 137.2, 131.3, 130.7, 130.1, 128.8, 128.5, 126.4, 124.2, 120.9, 118.4, 42.8. HRMS (ESI-FTMS Mass (m/z): calcd for C20 H18 N4 O4 [M + H]+ = 379.1401, found 379.1398. 4-[1-(4-carboxyphenyl)-4-[(E)-1-piperidyliminomethyl]pyrazol-3-yl]benzoic acid (6). Yellow solid, (376 mg, 91%). 1 H NMR 300 MHz (DMSO-d6 ): 8.77 (s, 1H), 8.06–8.03 (m, 6H), 7.88 (d, J = 8.0 Hz, 2H), 7.59 (s, 1H), 3.05 (s, 4H), 1.63 (s, 4H), 1.46 (s, 2H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.6, 167.2, 150.2, 142.4, 137.0, 131.3, 131.0, 130.0, 129.2, 128.6, 126.8, 126.1, 120.7, 118.4, 51.8, 25.0, 24.0. HRMS (ESI-FTMS Mass (m/z): calcd for C23 H23 N5 O4 [M + H]+ = 434.1823, found 434.1819. 4,40 -{4-[(E)-(4-methylpiperazin-1-yl)imino)methyl]-1H-pyrazole-1,3-diyl}dibenzoic acid (7). Yellow solid (402 mg, 93%). 1 H NMR 300 MHz (DMSO-d6 ): δ 8.80 (s, 1H), 8.06 (s, 4H), 8.04 (d, J = 8.7 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.64 (s, 1H), 3.13 (br s, 4H), 2.60 (br s, 4H), 2.29 (s, 3H); 13 C NMR (75 MHz, DMSO-d + CDCl ) δ = 167.8, 167.4, 150.3, 142.3, 136.7, 131.4, 131.3, 130.0, 129.7, 6 3 128.6, 128.0, 127.1, 120.3, 118.5, 53.9, 50.4, 45.4. HRMS (ESI-FTMS Mass (m/z): calcd for C23 H23 N5 O4 [M + H]+ = 434.1823, found 434.1819. 4-[1-(4-carboxyphenyl)-4-[(E)-(4-cyclopentylpiperazin-1-yl)iminomethyl]pyrazol-3-yl]benzoic acid (8). Yellow solid (457 mg, 94%). 1 H NMR, 300 MHz (DMSO-d6 ): 8.83 (s, 1H), 8.11–8.04 (m, 6H), 7.89 (d, J = 8. 1 Hz, 2H), 7.62 (s, 1H), 3.08 (s, 4H), 2.60 (s, 4H), 2.50 (s, 1H), 1.79 (s, 2H), 1.59–1.34 (m, 6H), 13 C NMR (75 MHz, DMSO-d ) δ = 167.6, 167.1, 150.3, 142.4, 136.9, 131.3, 131.0, 130.1, 129.3, 128.6, 127.5, 6 127.0, 120.4, 118.5, 66.8, 51.2, 51.0, 30.3, 24.1. HRMS (ESI-FTMS Mass (m/z): calcd for C27 H29 N5 O4 [M + H]+ = 488.2292, found 488.2301. 4-[1-(4-carboxyphenyl)-4-[(E)-(diphenylhydrazono)methyl] pyrazol-3-yl] benzoic acid (9). Yellow solid (446 mg, 89%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 9.12 (s, 1H), 8.14–8.06 (m, 4H), 7.97 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.1 Hz, 2H), 7.44 (t, J = 7.8 Hz, 4H), 7.28–7.14 (m, 7H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.4, 167.0, 150.5, 143.2, 142.4, 136.9, 131.3, 130.9, 130.4, 129.8, 129.1, 128.6, 128.0, 127.8, 125.0, 122.5, 119.5, 118.5. HRMS (ESI-FTMS Mass (m/z): calcd for C30 H22 N4 O4 [M + H]+ = 503.1714, found 503.1713. 4-[4-[(E)-[benzyl(phenyl)hydrazono]methyl]-1-(4-carboxyphenyl)pyrazol-3-yl] benzoic acid (10). Yellow solid (464 mg, 90%). (1 H NMR, 300 MHz (DMSO-d6 ): δ 9.04 (s, 1H), 8.10–8.07 (m,

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4H), 7.87 (d, J = 8.0 Hz, 2H), 7.47–7.35 (m, 9H), 7.30 (t, J = 7.9 Hz, 1H), 7.21 (d, J = 7.1 Hz, 2H), 6.91 (t, J = 7.2 Hz, 1H), 5.30 (s, 2H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 168.0, 167.7, 150.3, 147.7, 142.0, 136.5, 136.1, 132.5, 131.2, 130.0, 129.5, 129.3, 127.9, 127.5, 126.8, 126.6, 125.8, 120.5, 120.0, 118.4, 114.7, 48.9. HRMS (ESI-FTMS Mass (m/z): calcd for C31 H24 N4 O4 [M + H]+ = 517.1870, found 517.1878. 4-[1-(4-carboxyphenyl)-4-[(E)-(dibenzylhydrazono)methyl]pyrazol-3-yl]benzoic acid (11). Yellow solid (487 mg, 92%). 1 H NMR, 300 MHz (DMSO-d6 ): 8.79 (s, 1H), 8.11–8.03 (m, 4H), 7.82 (d, J = 8.1 Hz, 2H), 7.39–7.21 (m, 12H), 7.08 (s, 1H), 4.56 (s, 4H); 13 C NMR (75 MHz DMSO-d6 ): 167.4, 167.1, 149.6, 142.5, 138.3, 136.8, 131.3, 130.4, 129.9, 129.0, 128.6, 127.9, 127.8, 127.5, 125.9, 123.9, 120.6, 118.5, 58.1. HRMS (ESI-FTMS Mass (m/z): calcd for C32 H26 N4 O4 [M + H]+ = 531.2027, found 531.2031. 4-[1-(4-carboxyphenyl)-4-[(E)-[ethyl(phenyl)hydrazono]methyl]pyrazol-3-yl]benzoic acid (12). Yellow solid (399 mg, 88%). 1 H NMR, 300 MHz (DMSO-d6 ): 9.04 (s, 1H), 8.17–8.07 (m, 6H), 7.91 (m, 2H), 7.69 (s, 1H), 7.32–7.21 (m, 4H), 6.85 (t, 1H), 4.02 (q, J = 6.5 Hz, 2H), 1.13 (t, J = 6.6 Hz, 3H); 13 C NMR (75 MHz DMSO-d6 ): 167.5, 167.1, 150.4, 146.5, 142.5, 137.3, 131.3, 130.8, 130.1, 129.4, 128.9, 128.8, 127.3, 123.7, 120.8, 120.1, 118.5, 114.4, 38.7, 9.9. HRMS (ESI-FTMS Mass (m/z): calcd for C26 H22 N4 O4 [M + H]+ = 455.1714, found 455.1720. 4-[4-[(E)-[butyl(phenyl)hydrazono]methyl]-1-(4-carboxyphenyl)pyrazol-3-yl]benzoic acid (13). Yellow solid (428 mg, 89%). 1 H NMR, 300 MHz (DMSO-d6 ): 9.06 (s, 1H), 8.17–8.05 (m, 6H), 7.89 (d, J = 8.2 Hz, 2H), 7.65 (s, 1H), 7.33–7.21 (m, 4H), 6.84 (t, J = 6.9 Hz, 1H), 3.92 (s, 2H), 1.55–1.53 (m, 2H), 1.42–1.34 (m, 2H), 0.93 (t, J = 7.1 Hz, 3H); 13 C NMR (75 MHz DMSO-d6 ): 167.5, 167.1, 150.4, 146.9, 142.5, 137.2, 131.3, 130.8, 130.0, 129.3, 128.9, 128.7, 127.1, 123.7, 120.7, 120.1, 118.5, 114.4, 43.9, 26.4, 20.0, 14.2. HRMS (ESI-FTMS Mass (m/z): calcd for C28 H26 N4 O4 [M + H]+ = 483.2027, found 483.2027. 4-[1-(4-carboxyphenyl)-4-[(E)-[methyl(m-tolyl)hydrazono]methyl]pyrazol-3-yl]benzoic acid (14). Yellow solid (408 mg, 90%). 1 H NMR (300 MHz DMSO-d6 ): δ 9.00 (s, 1H), 8.15–8.06 (m, 6H), 7.94 (d, J = 8.1 Hz, 2H), 7.64 (s, 1H), 7.13–7.09 (m, 3H), 6.66 (s, 1H), 3.37 (s, 3H), 2.25 (s, 3H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.5, 167.1, 150.3, 147.6, 142.5, 138.4, 137.4, 131.4, 130.7, 130.1, 129.1, 128.9, 128.8, 127.6, 124.3, 121.2, 120.7, 118.5, 115.5, 112.2, 32.9, 21.9. HRMS (ESI-FTMS Mass (m/z): calcd for C26 H22 N4 O4 [M + H]+ = 455.1714, found 455.1720. 4-[1-(4-carboxyphenyl)-4-[(E)-[ethyl(p-tolyl)hydrazono]methyl]pyrazol-3-yl]benzoic acid (15). Yellow solid (411 mg, 88%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 9.01 (s, 1H), 8.18–8.06 (m, 6H), 7.90 (d, J = 8.1 Hz, 2H), 7.63 (s, 1H), 7.20 (d, J = 8.3 Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H), 3.98 (q, J = 6.4 Hz, 2H), 2.23 (s, 3H), 1.11 (t, J = 6.3 Hz, 3H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.5, 167.1, 150.3, 144.4, 142.6, 137.3, 131.3, 130.7, 130.1, 129.8, 128.9, 128.8, 128.7, 127.2, 123.0, 120.9, 118.5, 114.6, 20.5, 9.9. HRMS (ESI-FTMS Mass (m/z): calcd for C27 H24 N4 O4 [M + H]+ = 469.1870, found 469.1872. 4,40 -(4-{(E)-[2-(2-fluorophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl)dibenzoic acid (16). Brownish solid (346 mg, 78%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.52 (s, 1H), 9.12 (s, 1H), 8.16–8.07 (m, 6H), 7.99 (s, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.22–7.15 (m, 1H), 6.86 (d, J = 11.7 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.49 (t, J = 6.7 Hz, 1H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.5, 167.1, 163.8 (1 JC-F = 238.8 Hz), 150.4, 147.7 (3 JC-F = 11.1 Hz), 142.4, 136.9, 131.3, 131.03 (d, 3 JC-F = 10.0 Hz), 131.0, 130.1, 130.0, 129.1, 128.8, 127.7, 119.6, 118.6, 108.4, 104.5 (3 JC-F = 21.2 Hz), 98.9 (3 JC-F = 26.0 Hz). HRMS (ESI-FTMS Mass (m/z): calcd for C24 H17 FN4 O4 [M + H]+ = 445.1307, found 445.1309. 4,40 -(4-{(E)-[2-(3-fluorophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl)dibenzoic acid (17). Brownish solid (346 mg, 78%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.47 (s, 1H), 9.08 (s, 1H), 8.15–7.90 (m, 4H), 7.97 (s, 1H), 7.75 (d, J = 7.4 Hz, 2H), 7.55–7.48 (m, 2H), 7.19 (q, J = 6.8 Hz, 1H), 6.88 (d, J = 11.7 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.49 (t, J = 8.3 Hz, 1H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.1, 163.9 (d, 1 J =238.6 Hz), 151.6, 147.8 (d, 3 J = 11.1 Hz), 142.6, 132.7, 131.3, 131.0 (d, 3 J = 10.1 Hz), 130.4, 129.0, 128.8, 127.2, 119.2, 118.9, 118.4, 108.4, 105.0 (2 J = 20.5), 104.7, 98.8 (d, 2J = 26.1 Hz). HRMS (ESI-FTMS Mass (m/z): calcd for C24 H17 FN4 O4 [M + H]+ = 445.1307, found 445.1310. 4,40 -(4-{(E)-[2-(4-chlorophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl)dibenzoic acid (18). Brownish solid (349 mg, 76%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.42 (s, 1H), 9.07 (s, 1H), 8.16–8.07 (m, 6H), 7.98 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 8.7 Hz, 2H), 7.02 (d, J = 8.7 Hz, 2H);

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NMR (75 MHz, DMSO-d6 ) δ = 167.5, 167.1, 150.4, 144.6, 142.4, 136.9, 131.3, 131.0, 130.0, 129.8, 129.7, 128.8, 127.5, 122.1, 119.7, 118.6, 113.6. HRMS (ESI-FTMS Mass (m/z): calcd for C24 H17 ClN4 O4 [M + H]+ = 461.1011, found 461.1008. 4,40 -(4-{(E)-[2-(3-bromophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl)dibenzoic acid (19). Brownish solid (388 mg, 77%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.47 (s, 1H), 9.22 (s, 1H), 8.15–8.08 (m, 2H), 7.99 (s, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.21–7.20 (m, 1H), 7.11 (t, J = 7.9 Hz, 1H), 6.91–6.84 (m, 2H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.5, 167.1, 150.5, 147.2, 142.4, 136.9, 131.3, 131.1, 130.9, 130.5, 130.0, 129.3, 128.9, 127.9, 123.0, 121.2, 119.5, 118.6, 114.3, 111.3. HRMS (ESI-FTMS Mass (m/z): calcd for C24 H17 BrN4 O4 [M + H]+ = 505.0506, found 505.0512. 4,40 -(4-{(E)-[2-(2,5-difluorophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl)dibenzoic acid (20). Brownish solid (337 mg, 73%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.42 (s, 1H), 9.18 (s, 1H), 8.33 (s, 1H), 8.17–8.07 (m, 6H), 7.89 (d, J = 8.2 Hz, 2H), 7.25–7.10 (m, 2H), 6.52–6.47 (m, 1H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.5, 167.1, 160.1 (1 JC-F = 236.4 Hz), 150.6, 145.5 (d, J = 234.9 Hz), 142.4, 136.8, 135.3–135.1 (m), 133.2, 131.3, 131.1, 130.0, 129.3, 128.8, 127.9, 119.4, 118.6, 118.6–116.1 (m), 104.0 (2,3 JC-F = 25.0, 7.5 Hz), 100.8 (2 JC-F = 30.4 Hz). HRMS (ESI-FTMS Mass (m/z): calcd for C24 H16 F2 N4 O4 [M + H]+ = 463.1212, found 463.1214. 4,40 -(4-{(E)-[2-(2,4-dichlorophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl)dibenzoic acid (21). Brownish solid (376 mg, 76%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.04 (s, 1H), 9.11 (s, 1H), 8.47 (s, 1H), 8.16–8.08 (m, 6H), 7.88 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.5, 167.1, 150.7, 142.4, 141.0, 136.7, 133.7, 131.3, 131.1, 130.0, 129.3, 128.9, 128.7, 128.2, 127.6, 122.3, 119.5, 118.6, 116.7, 115.3. HRMS (ESI-FTMS Mass (m/z): calcd for C24 H16 Cl2 N4 O4 [M + H]+ = 495.0621, found 495.0618. 4-[1-(4-carboxyphenyl)-4-[(E)-[(3-chloro-2-fluoro-phenyl)hydrazono]methyl]pyrazol-3-yl] benzoic acid (22). Brownish solid (358 mg, 75%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.40 (s, 1H), 9.09 (s, 1H), 8.32 (s, 1H), 8.11–8.07 (m, 6H), 7.88 (s, J = 7.8 Hz, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.05 (t, J = 7.7 Hz, 1H), 6.85 (t, J = 6.4 Hz, 1H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.5, 167.1, 150.6, 144.7 (d, 1 J = 235.8 Hz), 142.4, 136.8, 135.3 (d, 3 J = 9.2), 133.2, 131.3, 131.1, 130.0, 129.3, 128.8, 127.7, 125.8 (d, J = 3.5 Hz), 119.9 (d, 2 J = 14.2 Hz), 119.4, 118.8, 118.6, 113.0. HRMS (ESI-FTMS Mass (m/z): calcd for C24 H16 ClFN4 O4 [M + H]+ = 479.0917, found 479.0920. 4,40 -(4-{(E)-[2-(3-chloro-4-fluorophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl) dibenzoic acid (23). Brownish solid (368 mg, 77%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.83 (br s, 1H), 9.05 (s, 1H), 8.10–8.98 (m, 7H), 7.81 (d, J = 8.1 Hz, 2H), 7.25–7.19 (m, 2H), 6.98–6.94 (m, 1H); 13 C NMR (75 MHz, DMSO-d ) δ = 168.8, 168.4, 150.7, 151.1 (1 J 6 C-F = 234.6 Hz), 150.7, 143.4, 141.2, 135.8, 135.1, 134.0, 131.0, 129.9, 128.2, 127.1, 120.3 (2 JC-F = 18.2 Hz), 119.2, 118.1, 117.6 (2 JC-F = 22.1 Hz), 112.6, 111.9. HRMS (ESI-FTMS Mass (m/z): calcd for C24 H16 ClFN4 O4 [M + H]+ = 479.0917, found 479.0919. 4,40 -(4-{(E)-[2-(2,3,5,6-tetrafluorophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl) dibenzoic acid (24). Brownish solid (398 mg, 80%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.38 (s, 1H), 8.81 (s, 1H), 8.36 (s, 1H), 8.05–7.98 (m, 6H), 7.81 (d, J = 6.9 Hz, 2H), 7.25–7.13 (m, 1H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 168.4, 168.1, 157.5, 151.0, 140.9, 138.5, 136.1, 135.8, 134.7, 131.0, 129.8, 128.2, 127.2, 118.3, 95.7 (2 JC-F = 25.0 Hz). HRMS (ESI-FTMS Mass (m/z): calcd for C24 H14 F4 N4 O4 [M + H]+ = 499.1024, found 499.1021. 4,40 -(4-{(E)-[2-(2,3,4,5,6-pentafluorophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl) dibenzoic acid (25). Brownish solid (423 mg, 82%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.13 (br s, 1H), 8.72 (s, 1H), 8.21 (s, 1H), 8.01 (s, 7H), 7.84–7.81 (m, 2H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.6, 167.2, 150.5, 142.1, 139.9–139.7 (m), 138.9, 136.4, 135.5, 135.3, 131.2, 129.8, 129.6–129.2 (m), 129.4, 128.6, 127.4, 121.6, 118.7, 118.5. HRMS (ESI-FTMS Mass (m/z): calcd for C24 H13 F5 N4 O4 [M + H]+ = 517.0930, found 517.0932. 4,40 -(4-{(E)-[2-(4-trifluoromethylphenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl) dibenzoic acid (26). Brownish solid (370 mg, 75%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.78 (s, 1H), 9.07 (s, 1H), 8.14–8.07 (m, 8H), 7.90 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.5 Hz, 13 C

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2H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.5, 167.1, 150.6, 148.6, 142.4, 136.8, 131.5, 131.3, 131.0, 130.0, 129.1, 128.8, 127.8, 126.7, 123.6, 119.4, 118.6, 111.8. HRMS (ESI-FTMS Mass (m/z): calcd for C25 H17 F3 N4 O4 [M + H]+ = 495.1275, found 495.1272. 4-[1-(4-carboxyphenyl)-4-[(E)-[(4-cyanophenyl)hydrazono]methyl]pyrazol-3-yl]benzoic acid (27). Brownish solid (342 mg, 76%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 10.92 (s, 1H), 9.12 (s, 1H), 8.16–8.07 (m, 8H), 7.90 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 8.7 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.4, 167.0, 150.7, 148.9, 142.4, 136.8, 134.0, 132.6, 131.4, 131.1, 130.0, 129.2, 128.8, 128.0, 120.5, 119.2, 118.7, 112.4, 99.5. HRMS (ESI-FTMS Mass (m/z): calcd for C25 H17 N5 O4 [M + H]+ = 452.1353, found 452.1360. 4,40 -(4-{(E)-[2-(4-corboxyphenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl)dibenzoic acid (28). Brownish solid (347 mg, 74%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 12.8 (br s, 3H), 10.77 (s, 1H), 9.12 (s, 1H), 8.17–8.07 (m, 8H), 7.91 (s, 1H), 7.79–7.77 (d, J = 8.58 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.7, 167.5, 167.1, 150.6, 149.1, 142.4, 136.9, 131.5, 131.4, 131.0, 130.0, 129.9, 129.1, 129.0, 128.9, 120.5, 119.4, 118.6, 111.4. HRMS (ESI-FTMS Mass (m/z): calcd for C25 H18 N4 O6 [M + H]+ = 471.1299, found 471.1299. 4,40 -(4-{(E)-[2-(4-nitrophenyl)hydrazinylidene]methyl}-1H-pyrazole-1,3-diyl)dibenzoic acid (29). Reddish solid (372 mg, 79%). 1 H NMR, 300 MHz (DMSO-d6 ): δ 11.31 (s, 1H), 9.17 (s, 1H), 8.15–8.08 (m, 9H), 7.89 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H); 13 C NMR (75 MHz, DMSO-d6 ) δ = 167.7, 167.2, 151.0, 150.9, 142.2, 138.5, 136.5, 134.6, 131.6, 131.3, 130.0, 129.8, 128.8, 128.3, 126.5, 118.9, 118.7, 111.5. HRMS (ESI-FTMS Mass (m/z): calcd for C24 H17 N5 O6 [M + H]+ = 472.1252, found 472.1250. All the Spectra can be seen in the Supplementary File. 2.2. In Vitro and In Vivo Toxicity Synthesized molecules were tested for their possible toxicity to human cell lines. These compounds did not show any noticeable growth inhibition for the NCI-60 cancer cell lines at 10 µM concentration. Furthermore, compounds showing activity against A. baumannii were tested for their possible toxicity against human embryonic kidney (HEK293) cell line and they did not show any significant toxicity up to 50 µg/mL concentration. After finding the benign nature of potent compounds against human cell lines, we tested the most potent compound 18 for any toxic in vivo effect on mice (Figure 2). We chose the doses of 20 and 50 mg/kg, which were used in our previous studies [20]. In vivo effects of a single IP injection of the compound were assessed by 14 different parameters for organs’ functions as described in Methods section and shown in Figure 2. This measurement clearly showed that none of the organ function markers indicated a toxicity by the used criteria. The majority of blood tests after the compound 18 administration showed no significant difference from control samples, and all except one of them were within the normal ranges. In particular, a normal concentration of albumin indicated no harm to the liver and kidneys. Unaffected blood urea nitrogen, creatinine, sodium, potassium levels indicated no harm to the kidneys of the treated animals. A normal concentration of amylase indicated that this potent anti-A baumannii agent did not adversely affect the pancreas. A normal alkaline phosphatase (ALP) level in blood is a key indicator of a healthy liver and bones. This compound treatment showed a slightly lower level of ALP but within the normal range. Normal alanine transaminase (ALT), and glucose levels at 20 mg/kg treatment further confirm the tolerance of this compound by the liver. Although a slight elevation of glucose was observed at 50 mg/kg dose, liver injury was not confirmed by other liver injury markers, such as total bilirubin, ALP, and ALT. Glucose level is very labile and it changes constantly during the day depending, for example, on food consumption. Calcium and phosphorus levels indicated the normal function of several organs such as liver, kidney, and bones. Unaffected total protein and globulin levels indicate healthy liver and kidney, and immune system respectively. Thus, this narrow-spectrum antibiotic is non-toxic in therapeutic doses, and seems to be very safe for further drug development. Future studies may need to focus on the development of a final pharmaceutical product. For this, additional tests would be needed to detect pharmacological

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needed to detect pharmacological responses through absorption, distribution, metabolism and excretion studies. All those will need to be done prior to Phase I testing in humans. Antibiotics 2020, 9, 650

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responses through absorption, distribution, metabolism and excretion studies. All those will need to be done prior to Phase I testing in humans.

Figure 2. Cont.

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Figure 2. In vivo toxicity assessment of the compound 18. Two doses (20 and 50 mg/kg) were administered intraperitoneally (IP) in CD-1 mice (n = 5 for each dose). Blood samples were collected 24 h later and tested by 14 parameters for organ functions. The red and green lines indicate the normal ranges for the assays. U, untreated animals; S, animals injected with saline (vehicle control).

2.3. Calculated Physicochemical Properties Figure 2. In vivo assessmentproperties of the compound 18. Two doses (20 and 50 were We calculated the toxicity physicochemical of the most potent compound 18mg/kg) by using a free administered intraperitoneally (IP) in CD-1 mice (n = 5 for each dose). Blood samples were collected online software, SwissADME (http://www.swissadme.ch/index.php). The n-octanol/water partition 24 h later and tested 14this parameters organ functions. red and green lines the range normal[21]. coefficient (ilogP) valueby for potentfor molecule is 2.67, The which is within the indicate optimum ranges for the assays. U, untreated animals; S, animals injected with saline (vehicle control). ◦ The Topological Total Surface Area (TPSA) is 116.81 A , which should allow good passive transport across the cell membrane. There is no violation of Lipinski’s rule of five and zero alert for PAINS. All 2.3. Calculated Physicochemical Properties these favorable parameters and very good biological activities indicate the suitability of the molecule calculated thedevelopment. physicochemical properties of the most potent compound 18 by using a free 18 for We further antibiotic online software, SwissADME (http://www.swissadme.ch/index.php). The n-octanol/water partition 3.coefficient Materials(ilogP) and Methods value for this potent molecule is 2.67, which is within the optimum range [21]. The Topological Total Surface Area (TPSA) is 116.81 A°, which should allow good passive transport General Consideration across the cell membrane. There is no violation of Lipinski’s rule of five and zero alert for PAINS. All All the products were obtained reactions carried out in round-bottom flasksofunder an air these favorable parameters and very by good biological activities indicate the suitability the molecule atmosphere. Reagents, and substrates were purchased from Oakwood Chemical (Estill, SC, 18 for further antibioticsolvents, development. USA) and Fisher Scientific (Hanover Park, IL, USA.). 1 H and 13 C spectra were recorded with a Varian 3. Materials Methods Mercury −300and MHz and 75 MHz respectively in DMSO-d6 solvent with TMS as internal standard. ESI-FTMS mass spectra were recorded in Brucker Apex II-FTMS system. Growth media and bacterial General Consideration broth were purchased from Fisher Scientific, ATCC, and ATCCHardy Diagnostics (Santa Maria, CA, USA). All the products were obtained by reactions carried out in round-bottom flasks under an air SynthesisReagents, of pyrazole aldehyde A mixture 4-actylbenzoic acid (2, 1.64 g, 10 mmol) atmosphere. solvents, and(4): substrates wereofpurchased from Oakwood Chemical (Estill,and SC, 4-hydrazinobenzoic acid (1, 1.980 g, 10.5 mmol) in ethanol was refluxed for 8 h and ethanol was 1 13 USA) and Fisher Scientific (Hanover Park, IL, USA.). H and C spectra were recorded with a Varian removed evaporation following drying in vacuo to get 6the hydrazone intermediate which was Mercuryby -300 MHz and 75 MHz respectively in DMSO-d solvent with TMS as internal(3), standard. ESIsubjected to further reaction without purification. The hydrazone derivative (3) was dissolved in FTMS mass spectra were recorded in Brucker Apex II-FTMS system. Growth media and bacterial N,N-dimethylformamide (30 mL), and cooled under iceand for ATCCHardy ~15 min followed by the dropwise addition broth were purchased from Fisher Scientific, ATCC, Diagnostics (Santa Maria, CA, ofUSA). phosphorous oxychloride (POCl3 , 4.67 mL, 50 mmol). The reaction mixture was stirred under ice ◦ C for 12 h. The reaction mixture was poured onto ice and the aqueous for 30 Synthesis min, and heated to 80aldehyde of pyrazole (4): A mixture of 4-actylbenzoic acid (2, 1.64 g, 10 mmol) and 4mixture was stirred for 12 h. The solid product was in filtered by gravity filtration washed repeatedly hydrazinobenzoic acid (1, 1.980 g, 10.5 mmol) ethanol was refluxed forand 8 h and ethanol was with water followed by drying under vacuum to get the pure product (4). removed by evaporation following drying in vacuo to get the hydrazone intermediate (3), which was Synthesis of hydrazones (4–29): A mixture ofThe aldehyde (4, 336 mg, 1 mmol) and the hydrazine subjected to further reaction without purification. hydrazone derivative (3) was dissolved in N,Nderivative (1.05 mmol) and sodium acetate (86 mg, 1.05 mmol) in case of the hydrochloride salt of theof dimethylformamide (30 mL), and cooled under ice for ~15 min followed by the dropwise addition hydrazine derivatives in anhydrous ethanol refluxedThe forreaction 8 h. Water was added in the mixture and phosphorous oxychloride (POCl3, 4.67 mL,was 50 mmol). mixture was stirred under ice for the solid product was filtered followed by washing with water and ethanol to get the pure products for 30 min, and heated to 80 °C for 12 h. The reaction mixture was poured onto ice and the aqueous biological studies. mixture was stirred for 12 h. The solid product was filtered by gravity filtration and washed Culturing ofwater Bacteria: Tryptic agarunder (TSA)vacuum slants were to maintain cultures. repeatedly with followed by soy drying to getused the pure product bacterial (4). Cation-Adjusted Mueller Hinton Broth (CAMHB) was used to grow bacteria in liquid culture including

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96-well plates for minimum inhibition concentration (MIC) testing and phosphate-buffered saline (PBS) was used to make bacterial dilutions. MIC studies: The MICs of potent compounds were determined by using the broth microdilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. The starting concentration of compounds was 50 µg/mL with two-fold serial dilutions below for MIC determination. The MIC values were measured in three independent experiments on different days with fresh bacterial culture and confirmed with at least duplicate occurrence. The concentration of compound in diluent dimethyl sulfoxide (DMSO) was maintained at 2.5%, which is below the cytotoxicity level for bacteria. In vitro Cytotoxicity Studies: Cytotoxicity of compounds against human embryonic (HEK293) kidney cell line and NCI-60 cancer cell lines were performed in 96-well black plate using resazurin cell viability assay. Cells (4000 per well) were plated in Eagle’s Minimum Essential Medium (EMEM) with 10% Fetal Bovine Serum (FBS) and incubated at 37 ◦ C in the presence of 5% carbon dioxide for 24 h. After incubation, test compounds at different concentrations were added to each well and placed in 96-well plate shaker for 5 min for proper mixing of compounds. The plate was re-incubated for 24 h. After the second incubation, 40 µL resazurin (0.15 mg/mL, w/v) was added in each well and mixed gently by pipetting. The plate was incubated for 4 additional hours and plate reading was conducted using Bio TekTM CytationTM 5 plate reader with excitation at 560 nm and emission at 590 nm. Data were processed in Microsoft Excel® for Office 365 MSO. In Vivo Toxicity Studies: All animal experiments were performed at the Central Arkansas Veterans Healthcare System (John L. McClellan Memorial Veterans Hospital in Little Rock, AR) and have been approved by the Institutional Animal Care and Use Committee. CD-1 male mice (8 weeks old, 33–37 g) were purchased from Charles River Laboratories (Wilmington, MA). The test compound 18 was freshly dissolved in 0.9% saline, sterilized by ultrafiltration, and injected intraperitoneally (IP) in mice at two doses of 20 or 50 mg/kg (n = 5 per dose). The two additional control groups (n = 5/group) were untreated or administered with the vehicle (saline). The mice were euthanized 24 h after the injection, and blood was collected by cardiac puncture. Toxicity was assessed by measuring 14 blood markers of various organ function available in the Comprehensive Diagnosis Kit (Abaxis, Union City, CA, USA) and using VetScan VS2 instrument (Abaxis). The markers included: alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), amylase (AMY), calcium (CA), creatinine (CRE), globulin (GLOB), glucose (GLU), phosphorus (PHOS), potassium (K+ ), sodium (NA+ ), total bilirubin (TBIL), total protein (TP), and blood urea nitrogen (BUN). Our criterium for toxicity was the combination of: (a) measurements being beyond the normal ranges, (b) statistically significantly difference from the untreated and vehicle (saline) controls, and (c) consistency between several functional markers of the same organ. 4. Conclusions We reported the synthesis of 25 novel pyrazole compounds. They were synthesized efficiently by using readily available starting materials and benign conditions. Column purification was not required to get pure products. The molecules were tested against both Gram-positive and Gram-negative bacteria and found to be potent and specific inhibitors of A. baumannii at low concentrations. These molecules are non-toxic in in vitro and in vivo studies. Thus, these potent compounds with very good selectivity factor could be pursued for further drug development as narrow-spectrum antibiotics. Mode of action and further antimicrobial studies are going on and will be reported elsewhere. 5. Patents Alam, M. A. Antimicrobial agents and the method of synthesizing the antimicrobial agents. US Patent. 10,596,153, 2020. Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/10/650/s1, Supplement File:1 H and 13 C NMR spectra.

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Author Contributions: Conceptualization, M.A.A. and D.F.G.; methodology, E.D., D.A., H.R.K. and T.F.; resources, M.A.A., D.G. and A.G.B.; writing—original draft preparation, M.A.A.; writing—review and editing, D.F.G., supervision, M.A.A., D.F.G. and A.G.B.; funding acquisition, M.A.A., D.F.G. and A.G.B. All authors have read and agreed to the published version of the manuscript. Funding: “This research was funded by National Institute of General Medical Sciences (NIGMS), grant number P20 GM103429” and “The APC was funded by P20 GM103429, 2I01BX002425, and P20 GM109005”. Acknowledgments: This manuscript was made possible by the Research Technology Core of the Arkansas INBRE program, supported by a grant from the National Institute of General Medical Sciences, (NIGMS), P20 GM103429 from the National Institutes of Health to record the Mass Spectrometry data and grants 2I01BX002425 and P20 GM109005 to perform in vivo toxicity study. This publication was made possible by the Arkansas INBRE Summer Research Grant, supported by a grant from the National Institute of General Medical Sciences, (NIGMS), P20 GM103429 from the National Institutes of Health. Conflicts of Interest: The authors declare no conflict of interest.

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