Naphthylthiazoles: targeting multidrug-resistant and intracellular

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Naphthylthiazoles: targeting multidrug-resistant and intracellular Staphylococcus aureus with biofilm disruption activity Mohamed Hagras, Nader S. Abutaleb, Alsagher Ali, Jelan Abdel-Aleem, Mohamed Elsebaei, Mohamed N. Seleem, and Abdelrahman S Mayhoub ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00172 • Publication Date (Web): 24 Sep 2018 Downloaded from http://pubs.acs.org on September 25, 2018

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ACS Infectious Diseases

Naphthylthiazoles: targeting multidrug-resistant and intracellular Staphylococcus aureus with biofilm disruption activity

Mohamed Hagras1ǁ, Nader S. Abutaleb2,3ǁ, Alsagher O. Ali2,4, Jelan A. Abdel-Aleem2,5 Mohamed M. Elsebaei,1 Mohamed N. Seleem2,6 * and Abdelrahman S. Mayhoub1,7* 1

Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University,

1-Elmokhaiam Eldaem St., Cairo 11884, Egypt 2

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University,

725 Harrison St., West Lafayette, IN, USA 47907 3

Department of Microbiology and Immunology, Faculty of Pharmacy, Zagazig University,

Zagazig, Egypt 44519 4

Division of Infectious Diseases, Animal medicine department, Faculty of Veterinary Medicine,

South Valley University, Qena, Egypt 83523 5

Department of Industrial Pharmacy, Faculty of Pharmacy, Assiut University, Assiut, Egypt

71515 6

Purdue Institute of Inflammation, Immunology, and Infectious Disease, 610 Purdue Mall, West

Lafayette, IN, USA, 47907 7

University of Science and Technology, Zewail City of Science and Technology, Ahmed Zewail

Rd, October Gardens 12578 Giza, Egypt Corresponding Authors. *e-mail: [email protected], [email protected]

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ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Thirty-two new naphthylthiazole derivatives were synthesized with the aim of exploring their antimicrobial effect on multidrug-resistant Gram-positive bacteria. Compounds 25 and 32, with ethylenediamine and methylguanidine side chains, represent the most promising derivatives, as their antibacterial spectrum includes activity against multidrug-resistant staphylococcal and enterococcal strains. Moreover, the new derivatives are highly advantageous over the existing frontline therapeutics for the treatment of multidrug-resistant Gram-positive bacteria. In this vein, compound 25 possesses three attributes: no bacterial resistance was developed against it even after 15 passages, it was very efficient in targeting intracellular pathogens, and it exhibited a concentration-dependent ability to disrupt the preformed bacterial biofilm.

Keywords: antibiotic resistance, vancomycin-resistant Staphylococcus aureus, MRSA biofilm, intracellular bacteria clearance, prolonged half-life.


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The pervasiveness of bacterial resistance to conventional antibiotics over the past decades has become a global epidemic

1-2

. The development of third and fourth generations from the

conventional antibiotic classes did not solve this problem, as most antibiotics have fallen prey to the increased microbial resistance. This problem is rising to dangerously high levels such that modern medicine as we know it today is threatening to collapse due to the scarcity of effective antibiotics that are essential to all major surgeries, organ transplants, and intensive-care units 1, 3. Methicillin-resistant streptococcal infections are with particular concern because of its global spread. Even though MDR-streptococcal infections are resistance to most discovered therapeutics, a handfull of FDA-approved drugs are still clincally effective, which include (lipo)glycopeptides and oxazolidinones. Briefly, vancomycin and linezolid are the recommended first line treatment for systemic MRSA,4 while tedizolid and dalbavancin are approved for skin infections.5 Ceftaroline is the only β-lactame approved for a wide variety of gram-positive infections including MRSA-induced pneumonia and-endocarditis.6-7

Regardless of the

availability of those currently used antibiotics, MDR-streptococcal infections are connected with more than 50% of fatalities-related to antimicrobial resistance.8 Therefore, the World Health Organization (WHO), recommended continued development of new therapeutics for controlling the MDR-streptococcal bacteria.9 In the search for a new chemical scaffold that cannot be identified by current resistant mechanisms, our efforts furnished phenylthiazoles as a new antibacterial scaffold with several advantages over the current frontline antibacterial therapeutics, including antibiofilm activity and fast bactericidal mode of action

10-21

. The structure-activity-relationships (SAR) obtained so far

indicated that cyclizing the terminal butyl group of the lead compound 1a provided the naphthyl analog 1b with enhanced antibacterial activity against several multidrug-resistant (MDR)



pathogens, including methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) 18. On the other hand, connecting the nitrogenous head of the arylthiazole nucleus with a pyrimidine linker had a great positive impact on the pharmacokinetic properties, as the half-life increased from 28 minutes, in the case of the lead compound 1a to more than 3 hours in the case of compound 1c (where R=H) (Figure 1)

20

. In the present work, we are aiming to develop

naphthylthiazoles with a pyrimidine linker to combine both enhanced pharmacokinetic stability criteria and the extended antibacterial effect on multidrug-resistant pathogens.

Figure 1. Combining pre-defined two active moieties in one scaffold.


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RESULTS AND DISCUSSION Chemistry. The naphthylthiazole 2 was prepared as previously reported from the reaction between α-chloroacetylacetone and naphthalene-2-carbothioamide 18, then allowed to react with N,N-dimethylformamide-dimethylacetal (DMF-DMA) to afford the intermediate 3 in high yield (Scheme 1). The 1,3-sequential addition reaction of the carboximidine moiety on the α,βunsaturated carbonyl group of 3, followed by removal of dimethylamine and water molecules, generated the desired pyrimidine linker. So far, the first set of final products 4–9 was obtained directly from the reaction between the enaminone 3 and guanidine or its analogs as outlined in Scheme 1.

The uncommercial availability of large libraries of guanidines and carboximidines limits our ability to diversity the substitutions at pyrimidine position-2. Therefore, a more accessible intermediate was designed (compound 12) in which the presence of a methylsulfone moiety next to two electronegative atoms allows aromatic nucleophilic substitution reations. Accordingly, to complete the rest of the designed final products, the enaminone 3 was charged with thiourea, followed by methylation and oxidation by mCPBA to finally afford the key synthon 12 (Scheme



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2). Lastly, the methylsulfonyl moiety of compound 12 was replaced with a large library of nitrogenous nucleophiles to yield final products 13–39 (Scheme 2). Scheme 2. Synthesis of compounds 13-39 N

N SH

S

N N

S

N

S

Me2SO4, KOH, H2O, 23 oC, 2 h 89%

thiourea, KOH, EtOH, heat at reflux, 8 h 96%

3

N

10

11

MCPBA, dry DCM, 23 oC, 16 h, 95%

N N

R N N

S

N

appropriate amine, hydrazine, guanidine or carboximidate, dry DMF, 80 oC for 0.5 - 8 h 57-91%

N

16, R=

N H

17, R= HN 18,

R= HN

19, R= 20, R=

21, R= 22, R= 23, R=

HN

N N

29, R=

N

30 R=

N

31, R=

34, R=

HN N HN N

N

35, R=

24, R= 25, R=

N H

O

NH

NH2

N

N

32, R=

N

NH

O

N

N

NH

37, R=

NH

N

HN

N OH

N

HN NH

38, R=

HN

27, R=

HN

HN

36, R=

HN NH2

N NH

NH HN

26, R= N

S

12

13-39 13, R= NHEt 14, R= NHPr 15, R= NHBu

O O S

33, R=

N NH

28, R= HNNH2

39, R=

N NH HN NH

N

Biological results and discussion. Antibacterial activity. In this study, 33 congeners were synthesized, and their antibacterial activity was first screened against MRSA USA300, which is considered an increasingly common


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cause of healthcare-associated MRSA infections and one of the most contagious staphylococcal strains

22-23

. Results were summarized in Table 1. Remarkably, the ethylenediaminyl and

methylguanidinyl derivatives 25 and 32 revealed the most promising anti-MRSA activity (MIC = 4 µg/mL). From a structure-activity-relationship (SAR) point of view, removal of the ethylene linker from compound 25 produced the hydrazinyl analog 28 that was void from any MRSA activity (MIC > 128 µg/mL). The role of the terminal amino group of 25 in the antibacterial property was further highlighted by the anti-MRSA activity of related compounds 13 (ethylamino derivative), 14 (propylamino derivative), 26 (piperazinyl derivative), 27 (hydroxyazetidinyl derivative), and 30 (aminopiperazinyl derivative). Briefly, removal of the terminal amino group or replacement with methyl moiety produced the ethylamino and propylamino derivatives 13 and 14, which lack activity against MRSA. Similarly, all cyclic isosteres including the piperazine ring furnished another set of completely inactive analogs 26, 27, and 30 (Table 1). As a general note, all compounds with lipophilic amine side chains demonstrated very weak anti-streptococcal activities with MIC value of 64 µg/mL as in the cases of compounds 8, 17, 18 and 23. A less lipophilic amine such as azetidine (compound 21) was one-fold more potent, while the rest of lipophilic amines, such as compounds 7, 9, 13-16, 20 and 22, produced compounds that lack the antibacterial property. Basicity of the terminal amine seems to be crucial for the antibacterial activity, since the compound 6 with less basic nitrile nitrogen was remarkably less active than the ethylenediamine derivative 25. In the case of the guanidine side chains, mono-methylation (compound 32) produced the most potent derivative in this series, as the guanidinyl analog 31 and the dimethylguanidinyl derivative 33 were four times less active than compound 32 with MIC value of 16 µg/mL.



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Furthermore, incorporating the terminal amino of guanidine moiety within alicyclic structures produces less active compounds (compounds 34-36) with MIC’s values between 16 and 64 µg/mL (Table 1). Also, the anti-MRSA activity was completely abolished by aromatization of terminal cyclic structures connected with the guanidine moiety (compounds 37-39). Next, the two most promising derivatives 25 and 32 were further assessed, and their antibacterial effect was evaluated against additional notorious methicillin-sensitive, methicillinresistant, vancomycin-intermediate, and vancomycin-resistant staphylococcal strains (MSSA, MRSA, VISA, and VRSA). Notably, both compounds 25 and 32 exhibited moderate activity against the tested staphylococcal strains. Advantageously, they kept the superiority of napthylthiazoles over glycopeptides, as they were effective against all VISA and VRSA strains (Table 2). The antibacterial spectrum of naphthylthiazoles 25 and 32 was extended to include important vancomycin-resistant enterococci (VRE) strains that are resistant to glycopeptides (Table 2). VRE are a leading cause of healthcare-associated infections. Thus, there is an unmet need for antibiotic innovation for treatment of VRE infections 24-25. In addition, both compounds exhibited potent activity against a clinical isolate of S. epidermidis. Furthermore, their MBC values were less than three times higher than their corresponding MIC values against the tested strains, indicating that the compounds might have bactericidal activity against the tested strains. Table 1. Initial MIC (µg/mL) screening of Naphthylthiazole compounds against methicillinresistant Staphylococcus aureus NRS 384 (MRSA USA 300).

Compound

R

MRSA (USA300)

Compound

4

NH2

> 128

24

> 128

5

NHCH3

> 128

25

4


R

MRSA (USA300)

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Compound

R

MRSA (USA300)

Compound

6

NHCN

> 128

26

> 128

7

> 128

27

> 128

8

64

28

> 128

29

> 128

> 128

30

> 128

> 128

31

16

> 128

32

4

> 128

33

16

64

34

32

64

35

64

> 128

36

16

> 128

37

> 128

32

38

> 128

> 128

39

> 128

64

Vancomycin

9 13

NHEt

14

NHPr

15

NHBu

16 17 18 19 20 21 22 23

R

-NHNH2

N/A

MRSA (USA300)

> 128

1-2

Table 2: The minimum inhibitory concentration (MIC in µg/mL) and minimum bactericidal concentration (MBC in µg/mL) of compounds 25 and 32 against a panel of staphylococcal and enterococcal clinically relevant resistant strains Compounds/ Control antibiotics Vancomycin

Compound 32

Compound 25

MIC

MBC

MIC

MBC

MIC

MBC

4

8

8

16

1

2

4

8

8

16

2

2

Bacterial Strains Methicillin-sensitive Staphylococcus aureus ATCC 6538 Methicillin-sensitive Staphylococcus aureus



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NRS 107 MRSA NRS 108

4

8

8

8

1

2

MRSA NRS 194

4

8

8

8

1

1

MRSA NRS 119

4

8

8

8

1

1

MRSA NRS123 (USA400)

4

8

8

8

1

1

MRSA NRS 382 (USA100)

8

16

16

16

2

2


8

16

4

16

1

1


4

32

8

32

0.5

1


8

16

8

16

1

1


4

8

4

8

0.5

0.5


4

16

8

8

1

1


4

16

8

16

2

2

VISA NRS 1

4

8

8

16

4

4

VISA NRS 19

4

4

8

8

4

4

VISA NRS 37

8

8

8

8

4

8

VRSA 2

8

8

8

16

64

64

VRSA 5

8

8

8

16

> 64

> 64

VRSA 6

4

8

8

16

> 64

> 64

VRSA 7

4

8

8

16

> 64

> 64

VRSA 9

8

32

4

8

> 64

> 64

VRSA 10

8

32

8

8

64

> 64

VRSA 11a

8

16

4

8

> 64

> 64

VRSA 12

8

16

8

16

1

64

Enterococcus faecalis ATCC 51299 (VRE)3

8

64

8

16

32

64

Enterococcus faecium ATCC 700221

8

64

8

16

> 64

> 64

2

4

2

8

1

1

1

2

(VRE) Methicillin-resistant Staphylococcus epidermidis NRS101 1 VISA, vancomycin-intermediate Staphylococcus aureus 2 VRSA, vancomycin-resistant Staphylococcus aureus 3 VRE: vancomycin-resistant Enterococci


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Figure 2. Time-kill kinetics assay of compounds 25 and 32 and vancomycin (tested in triplicates at 5 x MIC) against methicillin-resistant Staphylococcus aureus (MRSA USA400) over a 24-hour incubation period at 37 °C. DMSO (solvent for the compounds) served as a negative control. The error bars represent standard deviation values.

In order to confirm the bactericidal activity of compounds 32 and 25, a time-kill assay was performed against MRSA USA400. They exhibited more rapid, potent bactericidal activity than vancomycin; both completely eradicated the high inoculum of MRSA within four and six hours, respectively. Vancomycin (the drug of last resort for treatment of staphylococcal infections) exhibited slower bactericidal activity and required 12 hours to exert its bactericidal activity and completely eradicate the high inoculum of the bacteria.



Figure 3. Analyzing the toxicity of compounds 25 and 32 against human colorectal cells (Caco-2) using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium) assay. Results are presented as percent viable cells relative to DMSO (negative control). The absorbance values represent an average of three samples analyzed for each compound. Error bars represent standard deviation values. Preliminary safety profile. Limiting the toxic effect toward bacterial cells is a crucial factor in developing new antibiotics, as hitting eukaryotic cells will decrease the overall tolerability. To test whether the newly developed naphthylthiazoles 25 and 32 are tolerable or not, their cytotoxicity was tested against human colorectal cell line (Caco-2), and the results are summarized in Figure 3. Briefly, the methylguanidine-containing derivative 25 was non-toxic to Caco-2 cells at a concentration up to 64 µg/mL, at which about 91% of the cells were viable. Thus, its 50% cytotoxic concentration (CC50)—the compound’s concentration (µg/mL) required for the reduction of cell viability by 50%—is greater about 128 µg/mL. On the other hand, compound 32 was less tolerable, with about 54% of the cells viable at a concentration up to 32 µg/mL. Thus, its 50% cytotoxic concentration (CC50) is around 32 µg/mL.


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Antibiofilm activity. New approaches for developing antibacterial drugs must target not only the bacterial viability but also the inhibition of the stages leading to host colonization and virulence itself. Biofilm-related infections are an important cause of healthcare-associated infections, as the bacteria embedded in biofilms are very difficult to treat because they display very high resistance to antibiotics and the host's immune system 26-27. Most traditional antibiotics and drugs of choice for treatment of staphylococcal and enterococcal infections like glycopeptides are highly effective against the planktonic cells, yet they often have limited effects on sessile bacteria that are clumped within the biofilm. Moreover, sessile bacteria tend to form resistance against antibiotics more rapidly

17, 26, 28-29

. Consequently, bacterial adherence and

biofilm formation is one of the most important targets for developing new drugs. We tested the ability of compounds 32 and 25 to disrupt the preformed mature MRSA USA300 biofilm.

Figure 4. Disruption of mature MRSA biofilm by compounds 32 and 25 and vancomycin. Data are presented as percent disruption of MRSA USA300 mature biofilm in relation to DMSO (the solvent for the compounds as a negative control). The values represent an average of four samples analyzed for each compound/drug. Error bars represent standard deviation values. An asterisk (*) denotes statistical significance (P < 0.05) between results for compounds 32 and 25 and vancomycin analyzed via one-way ANOVA with post-hoc Dunnet’s test for multiple comparisons.



Interestingly, both compounds exhibited a concentration-dependent disruption of the preformed MRSA biofilm (Figure 4). Advantageously, they were superior to vancomycin in MRSA biofilm eradication. As reported earlier, many conventional antibiotics are not effective in disrupting biofilms. Vancomycin, the drug of choice for treatment of staphylococcal infections, has a large molecular structure and polar nature. Therefore, it is unable to penetrate biofilms effectively 16. In agreement with what was reported elsewhere 17, vancomycin has little activity in disrupting biofilms. At 1×MIC, vancomycin disrupted about 8% of MRSA biofilm. When its concentration was increased onefold to 2×MIC, it disrupted nearly 17% of the adherent biofilm. Compound 32 exhibited higher biofilm activity than vancomycin. At 1×MIC, compound 32 disrupted about 32% of MRSA biofilm. This disruption increased tremendously, to about 76%, when its concentration was increased onefold to 2×MIC. On the other hand, compound 25 was capable of disrupting the preformed MRSA biofilm by 47% and 62.5% at 1×MIC and 2×MIC, respectively. Multi-step resistant study. Arylthiazoles were previously reported to hit two consecutive proteins involved in bacterial cell wall biosynthesis

19

. This explains the inability of the tested

MDR bacterial cells to develop resistance toward this newly emerging class of antibiotics 11, 14, 16. This attribute guarantees the durability of this novel class of anti-MDR compounds for a long time, in case they reach the clinic. To test whether the new compounds maintained this advantage or not, a multi-step resistance assay was performed (Figure 5).


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Figure 5. Investigation of the ability of MRSA USA400 to develop resistance against naphthylthiazoles 25 and 32 via a multi-step resistance study. Bacteria were serially passaged over a 15-day period and the MIC of each compound was determined after each successive passage. A four-fold increase in MIC indicates bacterial resistance to the test agent. As represented in Figure 5, the MIC values for compounds 25 and 32 increased only onefold after nine passages and did not increase after that. Conversely, the MIC values for rifampicin increased dramatically during the serial passages. After one passage, its MIC increased 14-fold and continued to increase during the following passages (>510,000-fold MIC increase after the fifteenth passage). The result indicates MRSA was unable to develop resistance to any of the tested naphthylthiazoles 25 and 32 and developed resistance rapidly to the antibiotic rifampicin as reported earlier 11, 16. Examination of clearance of intracellular MRSA infection. Apart from the biofilm formation, multidrug-resistant pathogens adopt several other mechanisms to escape from the innate immune system and to minimize the effect of exogenous antibiotics. Hiding and surviving inside the macrophages is one of the most sneaky techniques used by S. aureus to evade the immune system. S. aureus is not considered a typical intracellular pathogen; however, it can invade and survive inside mammalian macrophages and cause chronic and persistent infections.



Furthermore, most antibiotics are unable to access and achieve the optimum therapeutic concentrations within the intracellularly infected niches. Consequently, treatment of the intracellular infections with conventional antibiotics is very challenging 13, 30. Hence, pneumonia induced by MRSA barely responded to vancomycin treatment, with reported clinical failure in nearly half of cases 31. Therefore, we assessed the activity of the naphthylthiazole 25 in clearing the intracellular MRSA USA400 in the infected murine macrophages (J774 cells).

Figure 6. Analyzing the toxicity of compound 25 against murine macrophage (J774) cells using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay. Results are presented as percent viable mammalian cells (measured as average absorbance ratio relative to DMSO) Error bars represent standard deviation values for the absorbance values. A one-way ANOVA, with post hoc Dunnet’s multiple comparisons test, determined statistical difference between the values obtained for each compound and DMSO (denoted by the asterisk) (P < 0.05).


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Figure 7. Examination of the activity of compound 25 on the clearance of intracellular MRSA present in murine macrophage (J774) cells. Data are presented as percent reduction of MRSA USA400 colony forming units inside infected murine macrophage cells after treatment with 2×MIC of either compound 25 or vancomycin (tested in triplicate) for 24 hours. Data were analyzed via a Student's t-test (P < 0.05). Asterisks (*) represent significant difference between treatment of J774 cells with compound 25 in comparison to vancomycin.

We first tested compound 25 for toxicity to macrophage cells at 4×MIC, 8×MIC, and 16×MIC. After a 24-hour incubation of compound 25 with J774 cells, viability was assessed via MTS assay to ensure compound 25 is not toxic for intracellular infection assay (Figure 6). As depicted in Figure 7, after 24 hours, compound 25, at the tested concentration (2×MIC), outperformed vancomycin in reducing the intracellular MRSA inside the infected macrophages. Interestingly, it was capable of reducing the intracellular MRSA by 94% at the tested concentration. On the other hand, vancomycin, the cornerstone for treatment of MDR infections, exhibited very poor activity in reducing the intracellular MRSA USA400: only 30% reduction at



the tested concentration (Figure 7). These results collectively indicated that the naphthylthiazole 25 can gain entry into the infected macrophage cells even at a lower concentration (i.e., 2×MIC) and significantly reduce the burden of MRSA inside them. Conversely, vancomycin, the drug of choice for treatment of MRSA infections, is unable to penetrate the macrophage cells and kill MRSA inside them. Preliminary PK profiling. This class of antibacterials was first reported with metabolic instability, and short half-life as the lead compound 1a was subjected to extensive hepatic metabolism, which leads to a half-life, of less than half an hour 18. To investigate the effect of the new structural modifications on the pharmacokinetic behavior, the key PK parameters of compound 25 were tested in rats. In this regard, the biological t1/2 of compound 25 was around four hours, which allows for a three-times-daily dosing regimen. Other PK parameters such as volume distribution and rate of clearance were also highly acceptable.

PK curve after 5 mg/Kg bolus IV dose Plasma concentration (mg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2.5

T1/2 = 3.9 hr AUC= 2.61 mg.h/L CL = 1.9 L/hr Vβ β = 10.8 L

2 1.5 1 0.5 0 0

2

4

6

8

10

12

14

Time (hr)

Figure 8. PK curve, after bolus IV dose (5 mg/Kg), of compound 25. T1/2: half-life, Vβ β: nd volume of distribution in the 2 compartment. Conclusion. The recent waves of bacterial resistance that involved agents of last resorts such as vancomycin and linezolid are putting us on the cusp of the pre-antibiotic era. Our group has


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introduced arylthiazoles as a promising antibacterial scaffold with several advantages over frontline antibiotics clinically in-use. Paring the conformationally-restricted naphthyl lipophilic tail with pyrimidine linker in one scaffold provided a set of new naphthylthiazole antibacterials that is effective against a wide variety of clinically isolated MDR-pathogens. Studying the SAR of naphthylthiazoles with a pyrimidine linker at thiazole position-5 furnished two antibacterial agents with several advantages over the existing frontline therapeutics (i.e., vancomycin). Apart from

the

extended

spectrum

against

methicillin-

and

vancomycin-resistant

strains,

naphthylthiazole 25 exhibited excellent activity in disrupting the highly resistant MRSA biofilm and had the ability to clear intracellular MRSA, and no rapid resistance was developed against this class of novel antibiotics. Additionally, compound 25 outperformed the first developed arylthiazoles in terms of PK behavior, as it exhibited an eight-times-longer half-life with much lower clearance rate and good distribution throughout biological tissues. Methods General. 1H NMR spectra were run at 400 MHz and

13

C NMR spectra were determined at

100 MHz in deuterated chloroform (CDCl3), or dimethyl sulfoxide (DMSO-d6) on a Varian Mercury VX-400 NMR spectrometer. Chemical shifts are given in parts per million (ppm) on the delta (δ) scale. Chemical shifts were calibrated relative to those of the solvents. Flash chromatography was performed on 230-400 mesh silica. The progress of reactions was monitored with Merck silica gel IB2-F plates (0.25 mm thickness). The infrared spectra were recorded in potassium bromide disks on pye Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometer. Mass spectra were recorded at 70 eV. High-resolution mass spectra for all ionization techniques were obtained from a FinniganMAT XL95. Melting points were



Page 20 of 45

determined using capillary tubes with a Stuart SMP30 apparatus and are uncorrected. All yields reported referring to isolated yields. Compound 2 was prepared as reported 18. (E)-3-(Dimethylamino)-1-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)prop-2-en-1-one

(3).

Dimethylformamide-dimethylacetal (2.7 mL, 2.4 g, 20.4 mmol) was added to a solution of acetylnaphthylthiazole 2 (2.9 g, 11 mmol) in dry DMF (25 mL). The reaction mixture was heated at 80 °C for 8 h. After cooling down to room temperature, the formed solid was collected by filtration, washed with petroleum ether and crystallized from ethanol to yield the titled compound as an orange solid (3.4 g, 95%) mp = 193 °C. 1H NMR (DMSO-d6) δ: 8.54 (s, 1H), 8.08-7.94 (m, 4H), 7.72 (d, J = 12 Hz, 1H), 7.57-7.56 (m, 2H), 5.48 (d, J = 12 Hz, 1H), 3.15 (s, 3H), 2.88 (s, 3H), 2.69 (s, 3H); 13C NMR (DMSO-d6) δ: 184.1, 168.5, 156.9, 150.6, 134.5, 133.1, 129.6, 129.5, 129.2, 128.29, 128.23, 127.6, 126.9, 123.8, 106.2, 92.0, 44.7, 37.11, 18.7; MS (m/z) 322. Anal. Calc. for: (C19H18N2OS): C, 70.78; H, 5.63; N, 8.69 %; Found: C, 70.79; H, 5.64; N, 8.70 %. Compounds 4-9. General procedure A. proper nitrogeneous nucleophiles (guanidine, guanidine derivative or carboximidate (1.25 mmol)) and anhydrous potassium carbonate (200 mg, 1.4 mmol) were added to a solution of compound 3 (200 mg, 0.6 mmol) in absolute ethanol (5-10 mL). The reaction mixture was kept at a reflux temperature for 8 h. After complete conversion of all the reactants, as detected by TLC, the solvent was concentrated under reduced pressure and the reaction was quenched with distilled water (50 mL). The solid was separated by filtration, washed with water and purified by crystallization from absolute ethanol or via acid-base extraction using HCl (1M, 50 mL). Physical properties and spectral analysis of the isolated products are listed below:


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4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-amine (4). Following the general procedure (A), and using guanidine hydrochloride (0.115 g, 1.2 mmol), compound 4 was obtained as pale yellow solid (0.13 g, 71%) mp = 195 °C. 1H NMR (DMSO-d6) δ: 8.54 (s, 1H), 8.34 (d, J = 5.2 Hz, 1H), 8.10-7.95 (m, 4H), 7.58-7.59 (m, 2H), 6.93 (d, J = 5.2 Hz, 2H), 6.74 (brs, 2H), 2.73 (s, 3H); 13C NMR (DMSO-d6) δ: 166.5, 163.8, 159.7, 158.2, 153.4, 134.2, 133.2, 132.5, 130.6, 129.4, 129.2, 128.2, 127.9, 127.5, 126.1, 123.8, 107.0, 18.6; MS (m/z) 318; HRMS (EI) m/z 318.0944 M+, calcd for C18H14N4S 318.0939. N-Methyl-4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-amine (5). Following the general procedure (A), and using N-methylguanidine hydrochloride (0.14 g, 1.2 mmol), compound 5 was obtained as white solid (0.18 g, 89%) mp = 185 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.36 (d, J = 5.2 Hz, 1H), 8.11-7.95 (m, 4H), 7.58-7.56 (m, 2H), 7.19 (brs,1H), 6.92 (d, J = 5.2 Hz, 1H), 2.75 (s, 3H);

13

C NMR (DMSO-d6) δ: 166.5, 162.9, 159.5, 158.2, 153.6,

133.3, 132.5, 130.5, 129.3, 129.2, 128.2, 127.9, 127.5, 126.2, 123.8, 107.0, 38.1, 28.2, 18.6; MS (m/z) 332; HRMS (EI) m/z 332.1106 M+, calcd for C19H16N4S 332.1096. N-(4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)cyanamide (6). Following the general procedure (A), and using cyanoguanidine (0.1 g, 1.2 mmol), compound 6 was obtained as pale yellow solid (0.15 g, 71%) mp = 272 °C. 1H NMR (DMSO-d6) δ: 8.54 (s, 1H), 8.19 (d, J = 5.2 Hz, 1H), 8.10-7.94 (m, 4H), 7.57-7.56 (m, 2H), 7.12 (brs, 1H), 6.70 (d, J = 5.2 Hz, 1H), 2.71 (s, 3H);

13

C NMR (DMSO-d6) δ: 170.1, 165.9, 159.0, 157.8, 152.4, 134.1, 133.8, 133.3,

130.8, 129.3, 129.1, 128.1, 127.7, 127.4, 125.9, 123.8, 123.3, 105.0, 18.5; MS (m/z) 343; HRMS (EI) m/z 343.0890 M+, calcd for C19H13N5S 343.0892.



Page 22 of 45

4-Methyl-2-(naphthalen-2-yl)-5-(2-(pyridin-2-yl)pyrimidin-4-yl)thiazole (7). Following the general procedure (A), and using pyridine-2-carboxamidinehydrochloride (0.25 g, 1.6 mmol), compound 7 was obtained as light brown solid (0.16 g,72 %) mp = 175 °C; 1H NMR (DMSO-d6) δ: 9.02 (d, J = 5.2 Hz, 1H), 8.79-8.76 (m, 2H), 8.64 (s, 1H), 8.44 (d, J = 8 Hz, 1H), 8.14-7.96 (m, 5H), 7.85 (d, J = 5.2 Hz, 1H), 7.58-7.57 (m, 2H), 2.86 (s, 3H);

13

C NMR (DMSO-d6) δ: 167.8,

163.6, 159.2, 158.2, 155.1, 154.7, 150.2, 137.6, 134.4, 133.3, 131.6, 130.4, 129.4, 129.2, 128.2, 128.0, 127.5, 126.5, 125.7, 124.0, 123.8, 117.0, 18.8; MS (m/z) 380; HRMS (EI) m/z 380.1108 M+, calcd for C23H16N4S 380.1096; Anal. Calc. for: (C23H16N4S): C, 72.61; H, 4.24; N, 14.73%; Found: C, 72.63; H, 4.25; N, 14.75%. 4-Methyl-2-(naphthalen-2-yl)-5-(2-(pyridin-3-yl)pyrimidin-4-yl)thiazole (8). Following the general procedure (A), and using pyridine-3-carboxamidine hydrochloride (0.25 g, 1.6 mmol), compound 8 was obtained as brown solid (0.17 g,74 %) mp = 225 °C; 1H NMR (DMSO-d6) δ: 9.54 (s, 1H), 8.96 (d, J = 5.2 Hz, 1H), 8.74 (d, J = 4.4 Hz, 1H), 8.67-8.66 (m, 1H), 8.61 (s, 1H), 8.10-7.94 (m, 4H), 7.75 (d, J = 5.2 Hz, 1H), 7.56-7.55 (m, 3H), 2.83 (s, 3H); 13C NMR (DMSOd6) δ: 169.4, 164.6, 159.7, 159.3, 152.4, 149.3, 133.2, 131.2, 130.3, 129.4, 129.3, 129.2, 129.1, 128.2, 128.1, 127.59, 127.51, 126.5, 126.2, 123.8, 121.9, 117.5, 18.7; MS (m/z) 380; HRMS (EI) m/z 380.1096 M+, calcd for C23H16N4S 380.1096; Anal. Calc. for: (C23H16N4S): C, 72.61; H, 4.24; N, 14.73%; Found: C, 72.62; H, 4.24; N, 14.74% 4-Methyl-2-(naphthalen-2-yl)-5-(2-(pyridin-4-yl)pyrimidin-4-yl)thiazole (9). Following the general procedure (A), and using isonicotinimidamide hydrochloride (0.25 g, 1.6 mmol), compound 9 was obtained as light brown solid (0.17 g,73 %) mp = 150 °C; 1H NMR (DMSO-d6) δ: 9.02 (d, J = 5.6 Hz, 1H), 8.81 (d, J = 4.4 Hz, 2H), 8.63 (s, 1H), 8.30 (d, J = 4.4 Hz, 2H), 8.107.98 (m, 4H), 7.84 (d, J = 5.6 Hz, 1H), 7.56-7.55 (m, 2H), 2.86 (s, 3H); 13C NMR (DMSO-d6) δ:


Page 23 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


168.0, 161.9, 159.4, 158.3, 155.4, 151.0, 144.3, 134.4, 133.2, 131.2, 130.5, 129.4, 129.3, 128.2, 128.1, 127.9, 126.5, 123.8, 121.9, 117.5, 18.9; MS (m/z) 380; HRMS (EI) m/z 380.1091M+, calcd for C23H16N4S 380.1096; Anal. Calc. for: (C23H16N4S): C, 72.61; H, 4.24; N, 14.73%; Found: C, 72.61; H, 4.25; N, 14.74% 4-Methyl-5-(2-(methylthio)pyrimidin-4-yl)-2-(naphthalen-2-yl)thiazole (11). Compound 3 (1 g, 3 mmol) was added to a stirred solution of potassium hydroxide (0.2 g, 3.5 mmol) and thiourea (0.5 g, 6.5 mmol) in absolute ethanol (20 mL). The reaction mixture was kept at reflux temperature for 8 h and then cooled down in an ice-bath to 8 °C. The formed crystals were filtered and washed with diethyl ether to yield the potassium salt intermediate 10 as a yellowish solid (1.1 g, 96%) mp > 300 °C. 1H NMR (DMSO-d6) δ: 8.68 (s, 1H), 8.62 (d, J = 5.2 Hz, 1H), 8.09-7.96 (m, 4H), 7.58-7.57 (m, 2H), 7.48 (d, J = 5.2 Hz, 1H), 2.71 (s, 3H); MS (m/z) 373. The potassium salt 10 (0.8 g, 2.1 mmol) was taken directly without any further purification and added to a solution and potassium hydroxide (0.25 g, 4.2 mmol) in distilled water (20 mL). After stirring for 10 min, dimethyl sulfate (0.5 mL, 4 mmol) was added dropwise, over a period of 30 min, with vigorous stirring. After 2 h, the formed solid materials were separated by filtration, washed with copious amount of water and air-dried to yield a pale yellow solid (0.67 g, 89%); mp = 188 °C. 1H NMR (DMSO-d6) δ: 8.67 (d, J = 5.2 Hz, 1H), 8.60 (s, 1H), 8.10-7.94 (m, 4H), 7.59-7.57 (m, 2H), 7.50 (d, J = 5.2 Hz, 1H), 2.77 (s, 3H), 2.56 (s, 3H); 13C NMR (DMSO-d6) δ: 166.4, 162.9, 159.4, 153.8, 152.7, 134.3, 133.2, 131.6, 130.0, 129.4, 129.2, 128.2, 128.0, 127.5, 126.4, 123.8, 107.6, 22.7, 17.4; MS (m/z) 349. 4-Methyl-5-(2-(methylsulfonyl)pyrimidin-4-yl)-2-(naphthalen-2-yl)thiazole (12). A solution of m-CPBA (0.514 g, 2.9 mmol) in DCM (5 mL) was added cautiously while stirring to a solution of methylmercaptothiazole 11 (0.5 g, 1.4 mmol) in dry DCM (5 mL). The reaction



Page 24 of 45

mixture was stirred at room temperature for additional16 hours. After that, additional amount of DCM (10 mL) was added and the reaction mixture was washed with aqueous solution of sodium metabisulfite (25 mL of 5% w/v) and sodium carbonate solution (25 mL of 5% w/v). The organic layer was separated, dried and concentrated under reduced pressure to give the desired product as yellow crystals (0.52 g, 95%) mp = 201 °C. 1H NMR (DMSO-d6) δ: 9.06 (d, J = 5.2 Hz, 1H), 8.62 (s, 1H), 8.11-7.95 (m, 4H), 7.90 (d, J = 5.2 Hz, 1H), 7.61-7.53 (m, 2H), 3.46 (s, 3H), 2.82 (s, 3H);

13

C NMR (DMSO-d6) δ: 166.3, 163.2, 160.7, 153.8, 152.8, 134.3, 133.2, 131.6, 130.0,

129.4, 129.1, 128.2, 128.0, 127.5, 126.4, 123.8, 108.2, 49.3, 18.3; MS (m/z) 381. Compounds 13-39. General procedure B. To a solution of methylsulfonylpyrimidine derivative 12 (0.1 g, 0.26 mmol) in a dry and degassed DMF (5-10 mL), a proper nitrogenous nucleophiles (0.4 mmol) was added. The reaction mixture was kept at 80-90 °C for 0.5-8 h, and then quenched over iced water (50 mL). The formed solid was separated by filtration and washed with ethanol (50% solution) to get rid of any excess amine and recrystallized from absolute ethanol. In the case of the final product 28, the isolated crude solid was washed with hot water to remove the residual hydrazine and filtered. Physical properties and spectral analysis of isolated products are listed below: N-Ethyl-4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-amine (13). Following the general procedure (B), using ethylamine (18 µL, 0.4 mmol) and heating for 2 h, compound 13 was obtained as yellow solid (0.06 g, 67%) mp = 168 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.36 (d, J = 5.6 Hz, 1H), 8.11-7.95 (m, 4H), 7.59 (m, 2H), 7.26 (brs, 1H), 6.90 (d, J = 5.6 Hz, 1H), 3.36 (q, J = 7.2 Hz, 2H), 2.75 (s, 3H), 1.15 (t, J = 7.2 Hz, 3H);

13

C NMR (DMSO-d6) δ:

166.5, 162.4, 159.4, 158.3, 153.5, 134.2, 133.3, 132.5, 130.6, 129.4, 129.2, 128.2, 127.9, 127.5,


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126.1, 123.8, 106.9, 35.8, 18.6, 15.0; MS (m/z) 346; HRMS (EI) m/z 346.1257 M+, calcd for C20H18N4S 346.1252. 4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)-N-propylpyrimidin-2-amine (14). Following the general procedure (B), using propylamine (23 µL, 0.4 mmol) and heating for 2 h, compound 14 was obtained as yellow solid (0.07 g, 77%) mp = 180 °C; 1H NMR (DMSO-d6) δ: 8.55 (s, 1H), 8.34 (d, J = 5.6 Hz, 1H), 8.11-7.95 (m, 4H), 7.58-7.56 (m, 2H), 7.29 (brs, 1H), 6.89 (d, J = 5.6 Hz, 1H), 3.26 (t, J = 6.8 Hz, 2H), 2.74 (s, 3H), 1.58-1.57 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H); 13

C NMR (DMSO-d6) δ: 166.5, 162.6, 159.3, 158.6, 153.5, 134.2, 133.3, 132.1, 130.6, 129.4,

129.2, 128.2, 127.9, 127.5, 126.1, 123.8, 106.5, 42.9, 22.6, 18.6, 11.9; MS (m/z) 360; HRMS (EI) m/z 360.1425 M+, calcd for C21H20N4S 360.1409. N-Butyl-4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-amine (15). Following the general procedure (B), using butylamine (29 µL, 0.4 mmol) and heating for 2 h, compound 15 was obtained as yellow solid (0.07 g, 74%) mp = 164 °C; 1H NMR (DMSO-d6) δ: 8.55 (s, 1H), 8.34 (d, J = 4.8 Hz, 1H), 8.11-7.95 (m, 4H), 7.58-7.56 (m, 2H), 7.13 (brs, 1H), 6.89 (d, J = 4.8 Hz, 1H), 3.29-3.28 (m, 2H), 2.74 (s, 3H), 1.56-1.55 (m, 2H), 1.36-1.34 (m, 2H), 0.90 (t, J = 7.6 Hz, 3H); 13C NMR (DMSO-d6) δ: 166.5, 162.5, 159.2, 158.3, 153.5, 134.2, 133.3, 132.8, 130.6, 129.4, 129.2, 128.2, 127.9, 127.5, 126.1, 123.8, 110.0, 40.9, 31.5, 20.1, 18.6, 14.2; MS (m/z) 374; HRMS (EI) m/z 374.1574 M+, calcd for C22H22N4S 374.1565. N-Isopropyl-4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-amine (16). Following the general procedure (B), using isopropylamine (23 µL, 0.4 mmol) and heating for 3 h, compound 16 was obtained as yellow solid (0.07 g, 79%) mp = 191 °C; 1H NMR (DMSO-d6) δ: 8.55 (s, 1H), 8.35 (d, J = 5.2 Hz, 1H), 8.11-7.95 (m, 4H), 7.58 (m, 2H), 7.12 (brs, 1H), 6.89 (d, J = 5.2 Hz, 1H), 4.09-4.08 (m, 1H), 2.74 (s, 3H), 1.19 (d, J = 6 Hz, 3H); 13C NMR (DMSO-d6) δ:



Page 26 of 45

166.5, 161.8, 159.4, 158.6, 153.5, 134.2, 133.2, 132.3, 130.5, 129.4, 129.2, 128.2, 127.9, 127.5, 126.1, 123.8, 106.2, 42.5, 22.7, 18.6; MS (m/z) 360; HRMS (EI) m/z 360.1398 M+, calcd for C21H20N4S 360.1409. N-Cyclopentyl-4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-amine

(17).

Following the general procedure (B), using cyclopentylamine (34 µL, 0.4 mmol) and heating for 4 h, compound 17 was obtained as yellow solid (0.08 g, 74%) mp = 207 °C; 1H NMR (DMSOd6) δ: 8.55 (s, 1H), 8.35 (d, J = 4.8 Hz, 1H), 8.11-7.95 (m, 4H), 7.58-7.56 (m, 2H), 7.13 (brs, 1H), 6.89 (d, J = 4.8 Hz, 1H), 3.42 (quint, J = 5.2 Hz, 1H), 2.75 (s, 3H), 1.93-1.92 (m, 3H), 1.691.68 (m, 3H), 1.53-1.52 (m, 4H);

13

C NMR (DMSO-d6) δ: 166.4, 162.5, 159.2, 158.3, 153.5,

134.6, 133.2, 132.3, 130.5, 129.4, 129.2, 128.2, 127.9, 127.5, 126.1, 123.8, 106.1, 32.6, 20.3, 18.5, 14.1; MS (m/z) 386; HRMS (EI) m/z 386.1558 M+, calcd for C23H22N4S 386.1565. N-Cyclohexyl-4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-amine

(18).

Following the general procedure (B), using cyclohexylamine (39 µL, 0.4 mmol) and heating for 4 h, compound 18 was obtained as yellow solid (0.06 g, 57%) mp = 205 °C; 1H NMR (DMSOd6) δ: 8.54 (s, 1H), 8.34 (d, J = 5.6 Hz, 1H), 8.10-7.95 (m, 4H), 7.58-7.56 (m, 2H), 7.12 (brs, 1H), 6.87 (d, J = 5.6 Hz, 1H), 3.73-3.72 (m, 1H), 2.74 (s, 3H), 1.90-1.89 (m, 2H), 1.72-1.71 (m, 2H), 1.61-1.59 (m, 2H), 1.28-1.27 (m, 4H); 13C NMR (DMSO-d6) δ: 166.4, 161.8, 159.6, 158.3, 153.5, 134.2, 133.2, 132.4, 130.5, 129.4, 129.2, 128.2, 127.9, 127.5, 126.1, 123.8, 106.6, 49.9, 32.7, 25.8, 25.3, 18.6; MS (m/z) 400; HRMS (EI) m/z 400.1726 M+, calcd for C24H24N4S 400.1722. N,N-Dimethyl-4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-amin

(19).

Following the general procedure (B), using dimethylamine (18 µL, 0.4 mmol) and heating for 2 h, compound 19 was obtained as yellow solid (0.07 g, 82%) mp = 193 °C; 1H NMR (DMSO-d6)


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δ: 8.57 (s, 1H), 8.43 (d, J = 5.2 Hz, 1H), 8.11-7.94 (m, 4H), 7.57-7.56 (m, 2H), 6.92 (d, J = 5.2 Hz, 1H), 3.16 (s, 6H), 2.75 (s, 3H);

13

C NMR (DMSO-d6) δ: 166.6, 161.8, 159.3, 157.9, 153.8,

134.2, 133.3, 130.5, 129.3, 129.2, 128.2, 127.9, 127.5, 126.2, 123.8, 121.5, 105.9, 36.9, 18.7; MS (m/z) 346; HRMS (EI) m/z 346.1241 M+, calcd for C20H18N4S 346.1252. N,N-Diethyl-4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-amine (20). Following the general procedure (B), using dimethylamine (28 µL, 0.4 mmol) and heating for 2 h, compound 20 was obtained as yellow solid (0.06 g, 62%) mp = 210 °C; 1H NMR (DMSO-d6) δ: 8.53 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 8.09-7.92 (m, 4H), 7.57-7.55 (m, 2H), 6.85 (d, J = 5.2 Hz, 1H), 3.59 (q, J = 6.8 Hz, 4H), 2.77 (s, 3H), 1.13(t, J = 5.2 Hz, 6H);

13

C NMR (DMSO-d6) δ:

166.5, 160.6, 159.2, 157.9, 153.5, 134.2, 133.2, 132.6, 130.5, 129.3, 129.1, 128.1, 127.8, 127.4, 126.1, 123.8, 105.9, 40.8, 18.7, 13.4; MS (m/z) 374; HRMS (EI) m/z 374.1577 M+, calcd for C22H22N4S 374.1565. 5-(2-(Azetidin-1-yl)pyrimidin-4-yl)-4-methyl-2-(naphthalen-2-yl)thiazole (21). Following the general procedure (B), using azetidine hydrochloride (0.04 g, 0.4 mmol) and heating for 2 h, compound 21 was obtained as yellow solid (0.06 g, 60%) mp = 188 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.40 (d, J = 5.2 Hz, 1H), 8.10-7.94 (m, 4H), 7.57-7.55 (m, 2H), 6.96 (d, J = 5.2 Hz, 1H), 4.07 (t, J = 7.6 Hz, 4H), 2.74 (s, 3H), 2.31-2.29 (m, 2H);

13


162.8, 159.3, 158.0, 154.0, 134.2, 133.3, 132.0, 130.5, 129.3, 129.2, 128.2, 127.9, 127.5, 126.2, 123.8, 107.1, 50.3, 18.7, 16.3; MS (m/z) 358; HRMS (EI) m/z 358.1259 M+, calcd for C21H18N4S 358.1252. 4-Methyl-2-(naphthalen-2-yl)-5-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)thiazole (22). Following the general procedure (B), using pyrrolidine (27 µL, 0.4 mmol) and heating for 2 h, compound 22 was obtained as yellow solid (0.09 g, 91%) mp = 260 °C; 1H NMR (DMSO-d6) δ: 8.56 (s,



Page 28 of 45

1H), 8.41 (d, J = 5.2 Hz, 1H), 8.11-7.94 (m, 4H), 7.58-7.55 (m, 2H), 6.91 (d, J = 5.2 Hz, 1H), 3.52-3.51 (m, 4H), 2.76 (s, 3H), 1.94-1.93 (m, 4H); 13C NMR (DMSO-d6) δ: 166.5, 160.1, 159.3, 157.9, 153.7, 134.2, 133.3, 132.4, 130.5, 129.3, 129.2, 128.2, 127.9, 127.5, 126.2, 123.8, 106.0, 46.7, 25.4, 18.7; MS (m/z) 372; HRMS (EI) m/z 372.1399 M+, calcd for C22H20N4S 372.1409. 4-Methyl-2-(naphthalen-2-yl)-5-(2-(piperidin-1-yl)pyrimidin-4-yl)thiazole (23). Following the general procedure (B), using piperidine (34 µL, 0.4 mmol) and heating for 2 h, compound 23 was obtained as yellow solid (0.08 g, 79%) mp = 262 °C; 1H NMR (DMSO-d6) δ: 8.58 (s, 1H), 8.43 (d, J = 5.2 Hz, 1H), 8.12-7.95 (m, 4H), 7.58-7.56 (m, 2H), 6.92 (d, J = 5.2 Hz, 1H), 3.783.77 (m, 4H), 2.74 (s, 3H), 1.64-1.63 (m, 2H), 1.55-1.54 (m, 4H);

13

C NMR (DMSO-d6) δ:

166.3, 161.3, 159.5, 158.0, 153.6, 134.2, 133.3, 132.6, 130.5, 129.3, 129.2, 128.2, 127.9, 127.5, 126.3, 123.8, 106.2, 44.6, 25.7, 24.7, 18.7; MS (m/z) 386; HRMS (EI) m/z 386.1569 M+, calcd for C23H22N4S 386.1565. 4-(4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)morpholine (24). Following the general procedure (B), using morpholine (34 µL, 0.4 mmol) and heating for 2 h, compound 24 was obtained as orange solid (0.07 g, 70%) mp = 155 °C; 1H NMR (DMSO-d6) δ: 8.58 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.10-7.98 (m, 4H), 7.58-7.57 (m, 2H), 7.02 (d, J = 5.2 Hz, 1H), 3.75 (t, J = 4.4 Hz, 4H), 3.69 (t, J = 4.4 Hz, 4H), 2.75 (s, 3H); 13C NMR (DMSO-d6) δ: 167.0, 161.5, 159.5, 158.0, 153.6, 134.2, 133.3, 132.6, 130.5, 129.3, 129.2, 128.2, 127.7, 127.5, 126.3, 123.8, 107.1, 66.4, 56.4, 18.7; MS (m/z) 388; HRMS (EI) m/z 388.1366 M+, calcd for C22H20N4OS 388.1358. N-(4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)ethane-1,2-diamine

(25).

Following the general procedure (B), using ethylenediamine (24 µL, 0.4 mmol) and heating for 2 h, compound 25 was obtained as yellow solid (0.08 g, 81%) mp = 186 °C; 1H NMR (DMSO-d6)


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δ: 8.54 (s, 1H), 8.35 (d, J = 5.2 Hz, 1H), 8.08-7.96 (m, 4H), 7.58-7.56 (m, 2H), 7.18 (brs, 1H), 6.90 (d, J = 5.2 Hz, 1H), 3.63 (t, J = 4.4 Hz, 2H), 2.75 (s, 3H) 2.28 (t, J = 4.4 Hz, 2H), 1,0 (brs, 1H);

13

C NMR (DMSO-d6) δ: 166.5, 162.4, 159.3, 158.0, 153.9, 153.2, 134.3, 133.2, 130.0,

129.4, 129.2, 128.2, 128.0, 127.5, 126.5, 123.7, 105.9, 48.0, 25.5, 18.7; MS (m/z) 361; HRMS (EI) m/z 361.1361 M+, calcd for C20H19N5S 361.1361. 4-Methyl-5-(2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)-2-(naphthalen-2-yl)thiazole

(26).

Following the general procedure (B), using methylpiperazine (40 µL, 0.4 mmol) and heating for 2 h, compound 26 was obtained as yellow solid (0.08 g, 72%) mp = 222 °C; 1H NMR (DMSOd6) δ: 8.60 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.12-7.95 (m, 4H), 7.60-7.58 (m, 2H), 7.00 (d, J = 5.2 Hz, 1H), 3.79-3.78 (m, 4H), 2.73 (s, 3H), 2.30-2.29 (m, 4H), 2.14 (s,3H); 13C NMR (DMSOd6) δ: 166.6, 162.7, 161.1, 159.4, 158.3, 134.1, 133.1, 132.3, 130.9, 129.4, 129.1, 128.4, 127.4, 127.1, 126.4, 123.6, 106.5, 54.8, 46.1, 39.7, 18.7; MS (m/z) 401; HRMS (EI) m/z 401.1682 M+, calcd for C23H23N5S 401.1674. 1-(4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)azetidin-3-ol (27). Following the general procedure (B), using azetidin-3-ol hydrochloride (40 g, 0.4 mmol) and heating for 5 h, compound 27 was obtained as yellow solid (0.08 g, 62%) mp = 295 °C; 1H NMR (DMSO-d6) δ: 8.57 (s, 1H), 8.41 (d, J = 5.6 Hz, 1H), 8.10-7.94 (m, 4H), 7.59-7.58 (m, 2H), 6.99 (d, J = 5.6 Hz, 1H), 5.79 (brs, 1H), 4.58-4.57 (m, 1H), 4.26 (dd, J = 7.2 Hz, J = 9.2 Hz, 2H), 3.83 (dd, J = 4.4 Hz, J = 10 Hz, 2H), 2.74 (s, 3H); 13C NMR (DMSO-d6) δ: 166.8, 162.9, 159.4, 158.1, 154.1, 134.2, 133.3, 132.3, 130.5, 129.3, 129.2, 128.2, 127.9, 127.5, 126.3, 123.8, 107.2, 61.2, 60.3, 18.7; MS (m/z) 374; HRMS (EI) m/z 374.1207 M+, calcd for C21H18N4OS 374.1201. 5-(2-Hydrazinylpyrimidin-4-yl)-4-methyl-2-(naphthalen-2-yl)thiazole (28). Following the general procedure (B), using hydrazine hydrate (5 mL) and heating for 0.5 h, compound 28 was



Page 30 of 45

obtained as pale yellow fluffy powder (0.07 g, 80%) mp = 172 °C; 1H NMR (DMSO-d6) δ: 8.55 (s, 1H), 8.40 (d, J = 5.2 Hz, 1H), 8.32 (brs, 1H), 8.10-7.96 (m, 4H), 7.59-7.58 (m, 2H), 6.95 (d, J = 5.2 Hz, 1H), 4.23 (brs, 2H), 2.72 (s, 3H); 13C NMR (DMSO-d6) δ: 166.7, 162.8, 159.3, 158.1, 154.0, 134.2, 133.3, 132.0, 130.5, 129.3, 129.2, 128.2, 127.9, 127.5, 126.2, 123.8, 107.1, 18.7; MS (m/z) 333; HRMS (EI) m/z 333.1030 M+, calcd for C18H15N5S 333.1048. 4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)-N-(piperidin-1-yl)pyrimidin-2-amine

(29).

Following the general procedure (B), using piperidin-1-amine (40 µL, 0.4 mmol) and heating for 3 h, compound 29 was obtained as yellow solid (0.07 g, 67%) mp = 150 °C; 1H NMR (DMSOd6) δ: 8.58 (s, 1H), 8.42 (d, J = 5.6 Hz, 1H), 8.11-7.94 (m, 4H), 7.57-7.56 (m, 2H), 7.14 (brs, 1H), 6.91 (d, J = 5.6 Hz, 1H), 3.78 (t, J = 5.6 Hz, 4H), 2.74 (s, 3H), 1.64-1.63 (m, 2H), 1.55-1.54 (m, 4H); 13C NMR (DMSO-d6) δ: 166.7, 161.3, 159.5, 158.1, 153.6, 134.2, 133.3, 132.6, 130.5, 129.3, 129.2, 128.2, 127.9, 127.5, 126.2, 123.8, 106.1, 44.6, 25.7, 24.7, 18.7; MS (m/z) 401; HRMS (EI) m/z 401.1671 M+, calcd for C23H23N5S 401.1674. 4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)-N-(4-methylpiperazin-1-yl)pyrimidin-2-amine (30). Following the general procedure (B), using 1-amino-4-methylpiperazine (46 µL, 0.4 mmol) and heating for 3 h, compound 30 was obtained as yellowish brown solid (0.07 g, 67%) mp = 176 °C; 1H NMR (DMSO-d6) δ: 8.57 (s, 1H), 8.44 (d, J = 4.8 Hz, 1H), 8.11-8.00 (m, 4H), 7.577.56 (m, 2H), 7.22 (brs, 1H), 6.97 (d, J = 4.8 Hz, 1H), 3.77 (t, J = 4.8 Hz, 4H), 2.74 (s, 3H), 2.37 (t, J = 4.8 Hz, 1H), 2.21 (s, 3H);

13

C NMR (DMSO-d6) δ: 166.8, 161.4, 159.5, 158.1, 153.8,

134.3, 133.3, 132.5, 130.5, 129.3, 129.2, 128.2, 127.9, 127.5, 126.3, 123.8, 106.8, 54.8, 46.2, 43.7, 18.7; MS (m/z) 416; HRMS (EI) m/z 416.1788 M+, calcd for C23H24N6S 416.1783. 1-(4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)guanidine (31). Following the general procedure (B), using guanidine hydrochloride (0.05 g, 0.4 mmol) and heating for 8 h,


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compound 31 was obtained as yellowish brown solid (0.09 g, 93%) mp = 230 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.10-7.95 (m, 4H), 7.58-7.56 (m, 2H), 7.04 (d, J = 5.2 Hz, 1H), 6.94 (brs, 4H), 2.72 (s, 3H);

13

C NMR (DMSO-d6) δ: 166.7, 166.5, 159.7,

156.4, 157.6, 153.1, 134.2, 133.28, 133.22, 130.6, 129.3, 129.2, 128.2, 127.9, 127.5, 126.1, 123.9, 107.7, 18.6; MS (m/z) 360; HRMS (EI) m/z 360.1173 M+, calcd for C19H16N6S 360.1157. 1-Methyl-3-(4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)guanidine

(32).

Following the general procedure (B), using methylguanidine hydrochloride (0.06 g, 0.4 mmol) and heating for 8 h, compound 32 was obtained as pale brown solid (0.06 g, 65%) mp = 189 °C; 1

H NMR (DMSO-d6) δ: 8.58 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.10-7.93 (m, 4H), 7.57-7.56 (m,

2H), 7.30 (brs, 1H), 7.03 (d, J = 5.2 Hz, 1H), 6.89 (brs, 2H), 3.48 (s, 3H), 2.72 (s, 3H); 13C NMR (DMSO-d6) δ: 167.3, 166.3, 163.8, 159.0, 158.0, 152.7, 134.1, 133.3, 130.1, 129.8, 129.1, 128.4, 127.7, 127.4, 126.7, 126.1, 123.6, 107.2, 28.1, 18.3; MS (m/z) 374; HRMS (EI) m/z 374.1304 M+, calcd for C20H18N6S 374.1314. 1,1-Dimethyl-3-(4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin2-yl)guanidine (33). Following the general procedure (B), using 1,1-dimethylguanidine hydrochloride ( 0.06 g, 0.4 mmol) and heating for 8 h, compound 33 was obtained as yellow solid (0.06 g, 65%) mp = 183 °C; 1H NMR (DMSO-d6) δ: 8.57 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 8.17 (brs, 2H), 8.09-7.95 (m, 4H), 7.58-7.56 (m, 2H), 7.06 (d, J = 5.2 Hz, 1H), 3.02 (s, 6H), 2.73 (s, 3H); 13C NMR (DMSOd6) δ: 166.3, 165.3, 163.1, 158.5, 156.9, 152.7, 134.2, 133.33, 133.30, 130.6, 129.3, 129.1, 128.2, 127.4, 126.4, 126.1, 123.8, 107.9, 37.2, 18.5; MS (m/z) 388; HRMS (EI) m/z 388.1475 M+, calcd for C21H20N6S 388.1470. N-(4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)pyrrolidine-1carboximidamide (34). Following the

general procedure (B),


using pyrrolidine-1-


Page 32 of 45

carboximidamide hydroiodide ( 0.1 g, 0.4 mmol) and heating for 8 h, compound 34 was obtained as yellowish brown solid (0.1 g, 91%) mp = 220 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.43 (brs, 2H), 8.08-7.95 (m, 4H), 7.58-7.57 (m, 2H), 7.04 (d, J = 5.2 Hz, 1H), 3.36-3.35 (m, 4H), 2.72 (s, 3H), 1.87-1.86 (m, 4H); 13C NMR (DMSO-d6) δ: 166.4, 166.1, 158.6, 157.3, 156.5, 152.9, 134.2, 133.2, 133.1, 130.6, 129.3, 129.1, 128.2, 127.9, 127.5, 126.1, 123.8, 107.5, 46.5, 25.3, 18.5; MS (m/z) 414; HRMS (EI) m/z 414.1625 M+, calcd for C23H22N6S 414.1627. N-(4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)morpholine-4carboximidamide (35). Following the general procedure (B), using morpholine-4carboximidamide hydroiodide ( 0.1 g, 0.4 mmol) and heating for 8 h, compound 35 was obtained as yellow solid (0.1 g, 85%) mp = 298 °C; 1H NMR (DMSO-d6) δ: 8.57 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.43 (brs, 2H), 8.09-7.98 (m, 4H), 7.59-7.57 (m, 2H), 7.11 (d, J = 5.2 Hz, 1H), 3.633.62 (m, 4H), 3.58-3.57 (m, 4H), 2.73 (s, 3H);

13

C NMR (DMSO-d6) δ: 166.6, 166.1, 158.6,

157.7, 157.5, 153.2, 134.2, 133.2, 133.1, 130.5, 129.4, 129.1, 128.2, 127.9, 127.5, 126.2, 123.8, 108.2, 66.3, 44.7, 18.5; MS (m/z) 430; HRMS (EI) m/z 430.1590 M+, calcd for C23H22N6OS 430.1576. 4-Methyl-N-(4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)piperazine-1carboximidamide (36). Following the general procedure (B), using 4-methylpiperazine-1carboximidamide hydroiodide ( 0.1 g, 0.4 mmol) and heating for 8 h, compound 36 was obtained as yellow solid (0.07 g, 60%) mp = 250 °C; 1H NMR (DMSO-d6) δ: 8.57 (s, 1H), 8.50 (d, J = 5.6 Hz, 1H), 8.33 (brs, 2H), 8.08-7.95 (m, 4H), 7.59-7.57 (m, 2H), 7.09 (d, J = 5.6 Hz, 1H), 3.593.58 (m, 4H), 2.73 (s, 3H), 2.34-2.32 (m, 4H), 2.19 (s, 3H);

13


165.9, 158.6, 157.6, 157.5, 153.1, 134.2, 133.2, 133.0, 130.5, 129.4, 129.3, 129.1, 128.2, 127.9,


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127.5, 126.2, 123.8, 107.6, 54.8, 46.1, 44.0, 18.5; MS (m/z) 443; HRMS (EI) m/z 443.1905 M+, calcd for C24H25N7S 443.1892. N-(4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)nicotinimidamide

(37).

Following the general procedure (B), using nicotinimidamide hydrochloride ( 0.06 g, 0.4 mmol) and heating for 8 h, compound 37 was obtained as orange solid (0.08 g, 69%) mp = 155 °C; 1H NMR (DMSO-d6) δ: 9.21 (s, 1H), 8.77 (d, J = 5.2 Hz, 1H), 8.72 (d, J = 5.2 Hz, 1H), 8.61 (s, 1H), 8.41 (d, J = 5.2 Hz, 1H), 8.26 (brs, 2H), 8.11-7.96 (m, 4H), 7.54-7.52 (m, 3H), 7.43 (d, J = 5.2 Hz, 1H), 2.74 (s, 3H); 13C NMR (DMSO-d6) δ: 167.1, 166.6, 159.2, 159.1, 158.3, 154.1, 152.1, 149.1, 135.6, 134.3, 133.2, 132.3, 131.9, 130.4, 129.4, 129.2, 128.2, 128.1, 127.5, 126.3, 126.0, 123.8, 111.3, 18.6; MS (m/z) 422; HRMS (EI) m/z 422.1310 M+, calcd for C24H18N6S 422.1314. N-(4-(4-methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)isonicotinimidamide

(38).

Following the general procedure (B), using isonicotinimidamide hydrochloride ( 0.06 g, 0.4 mmol) and heating for 8 h, compound 38 was obtained as orange solid (0.07 g, 60%) mp = 221 °C; 1H NMR (DMSO-d6) δ: 8.75-8.73 (m, 3H), 8.60 (s, 1H), 8.33 (brs, 2H), 8.10-7.95 (m, 6H), 7.59-7.57 (m, 2H), 7.44 (d, J = 5.2 Hz, 1H), 2.78 (s, 3H); 13C NMR (DMSO-d6) δ: 167.2, 166.6, 159.1, 158.9, 158.4, 154.1, 150.6, 143.5, 134.3, 133.2, 132.3, 130.4, 129.4, 129.2, 128.2, 128.1, 127.5, 126.9, 123.8, 121.9, 111.6, 18.7; MS (m/z) 422; HRMS (EI) m/z 422.1319 M+, calcd for C24H18N6S 422.1314. N-(4-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)pyrimidin-2-yl)picolinimidamide

(39).

Following the general procedure (B), using picolinmidamide hydrochloride ( 0.06 g, 0.4 mmol) and heating for 8 h, compound 39 was obtained as orange solid (0.1 g, 89%) mp = 239 °C; 1H NMR (DMSO-d6) δ: 8.79 (d, J = 5.6 Hz, 1H), 8.72 (d, J = 4.8 Hz, 1H), 8.62 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.33 (brs, 2H), 8.10-7.95 (m, 5H), 7.60-7.58 (m, 3H), 7.45 (d, J = 5.2 Hz, 1H), 2.80



(s, 3H);

13

Page 34 of 45

C NMR (DMSO-d6) δ: 167.1, 166.6, 159.0, 158.6, 157.9, 154.1, 151.3, 148.9, 138.2,

135.7, 134.0, 133.3, 132.3, 129.8, 129.1, 128.8, 128.4, 127.7, 127.4, 126.7, 126.4, 122.5, 111.4, 18.6; MS (m/z) 422; HRMS (EI) m/z 422.1332 M+, calcd for C24H18N6S 422.1314. Microbiological Assays Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Staphylococcus aureus and important Gram-positive bacterial pathogens. Broth microdilution method was utilized

32-33

against a panel of clinically

important Gram positive bacterial pathogens. Time-kill assay against MRSA. It was performed against MRSA USA400 as described previously 34-35. In brief, bacterial cells in logarithmic phase were diluted and drugs were added at 5 × MIC (in triplicates). After time points, cells were collected, diluted and plated on tryptic soy agar plates. Plates were incubated for 18-20 h and the viable CFU/mL was determined. MRSA biofilm eradication assessment. Phenylthiazole compounds 25, 32 were tested for their ability to eradicate established and mature staphylococcal biofilm using the microtiter plate biofilm formation assay following the procedure reported elsewhere

17, 36

. Briefly, an overnight

culture of MRSA USA300 (NRS384) was diluted 1:100 in culture medium (Tryptic soy broth + 1% glucose) and incubated at 37 °C for 24 hours to form a adherent biofilm. The bacterial suspension was removed and compounds were added. Compounds were incubated with the biofilm at 37 °C for 24 hours. In order to quantify the biofilm mass, the bacterial suspension was removed and wells were washed with phosphate-buffered saline to remove planktonic bacteria. An aliquot of 0.1% crystal violet was added to each well to stain biofilm mass. After 30 minutes, wells were washed with sterile water and dried. Wells were de-stained using 100% ethanol prior to quantifying biofilm mass using a spectrophotometer (OD595). Data are presented as percent


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eradication of MRSA USA300 biofilm for each test agent relative to the negative (DMSO) control wells. Data were analyzed using two-way ANOVA with post-hoc Dunnet’s test for multiple comparisons (P < 0.05). Multi- step resistance study for naphthylthiazoles 25 and 32 against MRSA. The capability of MRSA to develop resistance against the naphthylthiazoles was further investigated via a multistep resistance study as described previously 34. Resistance was classified as a greater than four-fold increase in the initial MIC, as reported elsewhere 37. In vitro cytotoxicity analysis of naphthylthiazoles 25 and 32 against human colorectal adenocarcinoma (Caco-2) cells and murine macrophages (J774) cells. Naphthylthiazoles were assayed for their toxicity profile against a human colorectal adenocarcinoma (Caco-2) cell line and murine macrophage (J774) cells as reported earlier 16, 38. Examination of clearance of intracellular MRSA present in murine macrophage (J774) cells. An intracellular bacterial clearance experiment was utilized as described elsewhere 13, 39 to investigate the ability of naphthylthiazoles to pass inside the macrophages and clear the intracellular MRSA USA400. Murine macrophage (J774) cells were seeded at a density of approximately 1 × 105 cells per well in 96-well plates (Corning Incorporated) for 24 hours before being infected with the bacteria. The cells were routinely grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum. Following incubation, the cells were washed once with DMEM. Then, the cells were infected with MRSA USA 400 (at multiplicity of infection 10:1) in DMEM supplemented with 10% FBS for 60 minutes. At the end of the infection, the cells were washed three times with DMEM medium containing 100µg/ mL gentamicin (Sigma) and was further incubated for 30 minutes to kill and wash off non-phagocytized bacteria. After that, DMEM medium supplemented with 10% fetal



bovine serum containing the drugs at the indicated concentrations was added to the cells. Control cells received DMSO at a concentration equal to that in drug-treated cell samples. The plate was then incubated for 24 hours. Afterwards, the infected cells were washed three times and lysed with 100 µL of 0.01% triton X in PBS to collect the intracellular bacteria. The colony forming units (CFUs) of the bacteria in the lysates were determined by plating a series of 10-fold serial dilutions onto tryptic soy agar (TSA) and incubating the plates at 37 °C for 24-hour. Experiments were performed in triplicate in two independent experiments. Statistical significance was assessed with one-way ANOVA, with post hoc Dunnet’s multiple comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). In vivo Pharmacokinetics. Pharmacokinetic studies were performed in male naïve Sprague− Dawley (SD) rats (three animals) following Institutional Animal Care and Use Committee guidelines. An IV bolus of a 5 mg/Kg was directly administered. Blood samples were collected over a 12-hour period post dose into Vacutainer tubes containing EDTA-K2. Plasma was isolated, and the concentration of tested compounds in plasma was determined with LC/MS/MS after protein precipitation with acetonitrile. Two-compartmental pharmacokinetic analysis was performed on plasma concentration data in order to calculate pharmacokinetic parameters as previously reported.14 ASSOCIATED CONTENTS Supporting information. The supporting information is available free of charge on the ACS publication website. The strains used in the manuscript and their corresponding sites of isolation, 1

H and 13C NMR spectra of all new described compounds.

Author information


Page 36 of 45

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Abbreviations.. MIC, minimum inhibitory concentration; CFU, colony forming unit; Caco-2, human colorectal cells; DMF-DMA, dimethylformamide-dimethylacetal; MDR, Multi-drug resistance; PK, pharmacokinetic, T1/2, half-life; Vβ: volume of distribution in the 2nd compartment Author information. ǁ

These two authors contributed equally

ORCID Abdelrahman S. Mayhoub: 0000-0002-3987-3680 Acknowledgments. This work was funded by Science & Technology Development Funds (STDF), Grant#5334. The authors would like to thank BEI Resources for providing clinical isolates of S. aureus used in this study.



References

1.

Laxminarayan, R.; Duse, A.; Wattal, C.; Zaidi, A. K.; Wertheim, H. F.; Sumpradit, N.;

Vlieghe, E.; Hara, G. L.; Gould, I. M.; Goossens, H.; Greko, C.; So, A. D.; Bigdeli, M.; Tomson, G.; Woodhouse, W.; Ombaka, E.; Peralta, A. Q.; Qamar, F. N.; Mir, F.; Kariuki, S.; Bhutta, Z. A.; Coates, A.; Bergstrom, R.; Wright, G. D.; Brown, E. D.; Cars, O., Antibiotic resistance-the need for global solutions. Lancet Infect Dis 2013, 13 (12), 1057-98. DOI: 10.1016/S14733099(13)70318-9. 2.

Mohammad, H.; Thangamani, S.; Seleem, M. N., Antimicrobial peptides and

peptidomimetics - potent therapeutic allies for staphylococcal infections. Curr Pharm Des 2015, 21 (16), 2073-88. 3.

Laxminarayan R; Malani A; Howard D; DL., S. Extending the cure: policy responses to

the growing threat of antibiotic resistance; Washington: Resources for the Future, 2007. 4.

Liu, C.; Bayer, A.; Cosgrove, S. E.; Daum, R. S.; Fridkin, S. K.; Gorwitz, R. J.; Kaplan,

S. L.; Karchmer, A. W.; Levine, D. P.; Murray, B. E., Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children. Clin. Infect. Dis. 2011, 52 (3), e18-e55. DOI: 10.1093/cid/ciq146. 5.

Boucher, H. W.; Wilcox, M.; Talbot, G. H.; Puttagunta, S.; Das, A. F.; Dunne, M. W.,

Once-weekly dalbavancin versus daily conventional therapy for skin infection. N. Engl. J. Med. 2014, 370 (23), 2169-2179. DOI: 10.1056/NEJMoa1310480. 6.

Lin, J. C.; Fierer, J.; Aung, G.; Johns, S.; Thomas, A.; Jahng, M., The use of ceftaroline

fosamil in methicillin-resistant Staphylococcus aureus endocarditis and deep-seated MRSA infections: a retrospective case series of 10 patients. J.Inf.Chemother. 2013, 19 (1), 42-49. DOI: 10.1007/s10156-012-0449-9.


Page 38 of 45

Page 39 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7.

Pasquale, T. R.; Tan, M. J.; Trienski, T. L.; File, T. M., Methicillin-resistant

Staphylococcus aureus nosocomial pneumonia patients treated with ceftaroline: retrospective case

series

of

10

patients.

J.

Chemother.

2015,

27

(1),

29-34.

DOI:

10.1179/1973947813Y.0000000156. 8.

de Lencastre, H.; Oliveira, D.; Tomasz, A., Antibiotic resistant Staphylococcus aureus: a

paradigm of adaptive power. Curr. Opin. Microbiol. 2007, 10 (Copyright (C) 2014 American Chemical Society (ACS). All Rights Reserved.), 428-435. DOI: 10.1016/j.mib.2007.08.003. 9.

Antibacterial agents in clinical development: an analysis of the antibacterial clinical

development pipeline, including tuberculosis. Geneva: World Health Organization; 2017 (WHO/EMP/IAU/2017.11). 10.

Mohammad, H.; Reddy, P. V.; Monteleone, D.; Mayhoub, A. S.; Cushman, M.; Seleem,

M. N., Synthesis and antibacterial evaluation of a novel series of synthetic phenylthiazole compounds against methicillin-resistant Staphylococcus aureus (MRSA). Eur J Med Chem 2015, 94, 306-16. DOI: 10.1016/j.ejmech.2015.03.015. 11.

Hagras, M.; Hegazy, Y. A.; Elkabbany, A. H.; Mohammad, H.; Ghiaty, A.; Abdelghany,

T. M.; Seleem, M. N.; Mayhoub, A. S., Biphenylthiazole antibiotics with an oxadiazole linker: An approach to improve physicochemical properties and oral bioavailability. Eur J Med Chem 2018, 143, 1448-1456. DOI: 10.1016/j.ejmech.2017.10.048. 12.

Mohammad, H.; Reddy, P. V.; Monteleone, D.; Mayhoub, A. S.; Cushman, M.; Hammac,

G. K.; Seleem, M. N., Antibacterial Characterization of Novel Synthetic Thiazole Compounds against Methicillin-Resistant Staphylococcus pseudintermedius. PLoS One 2015, 10 (6), e0130385. DOI: 10.1371/journal.pone.0130385.



13.

Eid, I.; Elsebaei, M. M.; Mohammad, H.; Hagras, M.; Peters, C. E.; Hegazy, Y. A.;

Cooper, B.; Pogliano, J.; Pogliano, K.; Abulkhair, H. S.; Seleem, M. N.; Mayhoub, A. S., Arylthiazole antibiotics targeting intracellular methicillin-resistant Staphylococcus aureus (MRSA) that interfere with bacterial cell wall synthesis. Eur J Med Chem 2017, 139, 665-673. DOI: 10.1016/j.ejmech.2017.08.039. 14.

Elsebaei, M. M.; Mohammad, H.; Abouf, M.; Abutaleb, N. S.; Hegazy, Y. A.; Ghiaty, A.;

Chen, L.; Zhang, J.; Malwal, S. R.; Oldfield, E.; Seleem, M. N.; Mayhoub, A. S., Alkynylcontaining phenylthiazoles: Systemically active antibacterial agents effective against methicillinresistant Staphylococcus aureus (MRSA). Eur J Med Chem 2018, 148, 195-209. DOI: 10.1016/j.ejmech.2018.02.031. 15.

Hagras, M.; Mohammad, H.; Mandour, M. S.; Hegazy, Y. A.; Ghiaty, A.; Seleem, M. N.;

Mayhoub, A. S., Investigating the Antibacterial Activity of Biphenylthiazoles against Methicillin- and Vancomycin-Resistant Staphylococcus aureus (MRSA and VRSA). J Med Chem 2017, 60 (9), 4074-4085. DOI: 10.1021/acs.jmedchem.7b00392. 16.

Kotb, A.; Abutaleb, N. S.; Seleem, M. A.; Hagras, M.; Mohammad, H.; Bayoumi, A.;

Ghiaty, A.; Seleem, M. N.; Mayhoub, A. S., Phenylthiazoles with tert-Butyl side chain: Metabolically stable with anti-biofilm activity. Eur J Med Chem 2018, 151, 110-120. DOI: 10.1016/j.ejmech.2018.03.044. 17.

Mohammad, H.; Mayhoub, A. S.; Cushman, M.; Seleem, M. N., Anti-biofilm activity and

synergism of novel thiazole compounds with glycopeptide antibiotics against multidrug-resistant staphylococci. J Antibiot (Tokyo) 2015, 68 (4), 259-66. DOI: 10.1038/ja.2014.142. 18.

Mohammad, H.; Mayhoub, A. S.; Ghafoor, A.; Soofi, M.; Alajlouni, R. A.; Cushman,

M.; Seleem, M. N., Discovery and characterization of potent thiazoles versus methicillin- and


Page 40 of 45

Page 41 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


vancomycin-resistant Staphylococcus aureus. J Med Chem 2014, 57 (4), 1609-15. DOI: 10.1021/jm401905m. 19.

Mohammad, H.; Younis, W.; Chen, L.; Peters, C. E.; Pogliano, J.; Pogliano, K.; Cooper,

B.; Zhang, J.; Mayhoub, A.; Oldfield, E.; Cushman, M.; Seleem, M. N., Phenylthiazole Antibacterial Agents Targeting Cell Wall Synthesis Exhibit Potent Activity in Vitro and in Vivo against Vancomycin-Resistant Enterococci. J Med Chem 2017, 60 (6), 2425-2438. DOI: 10.1021/acs.jmedchem.6b01780. 20.

Seleem, M. A.; Disouky, A. M.; Mohammad, H.; Abdelghany, T. M.; Mancy, A. S.;

Bayoumi, S. A.; Elshafeey, A.; El-Morsy, A.; Seleem, M. N.; Mayhoub, A. S., SecondGeneration Phenylthiazole Antibiotics with Enhanced Pharmacokinetic Properties. J Med Chem 2016, 59 (10), 4900-12. DOI: 10.1021/acs.jmedchem.6b00233. 21.

Yahia, E.; Mohammad, H.; Abdelghany, T. M.; Fayed, E.; Seleem, M. N.; Mayhoub, A.

S., Phenylthiazole antibiotics: A metabolism-guided approach to overcome short duration of action. Eur J Med Chem 2017, 126, 604-613. DOI: 10.1016/j.ejmech.2016.11.042. 22.

Freitas, E. A.; Harris, R. M.; Blake, R. K.; Salgado, C. D., Prevalence of USA300 strain

type of methicillin-resistant Staphylococcus aureus among patients with nasal colonization identified with active surveillance. Infect Control Hosp Epidemiol 2010, 31 (5), 469-75. DOI: 10.1086/651672. 23.

Carrel, M.; Perencevich, E. N.; David, M. Z., USA300 Methicillin-Resistant

Staphylococcus aureus, United States, 2000-2013. Emerg Infect Dis 2015, 21 (11), 1973-1980. DOI: 10.3201/eid2111.150452.



24.

Mohammad, H.; AbdelKhalek, A.; Abutaleb, N. S.; Seleem, M. N., Repurposing

niclosamide for intestinal decolonization of vancomycin-resistant enterococci. Int J Antimicrob Ag 2018, 51 (6), 897-904. DOI: 10.1016/j.ijantimicag.2018.02.003. 25.

AbdelKhalek, A.; Abutaleb, N. S.; Elmagarmid, K. A.; Seleem, M. N., Repurposing

auranofin as an intestinal decolonizing agent for vancomycin-resistant enterococci. Sci Rep-Uk 2018, 8. DOI: Artn 835310.1038/S41598-018-26674-0. 26.

Lebeaux, D.; Ghigo, J. M.; Beloin, C., Biofilm-Related Infections: Bridging the Gap

between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics. Microbiol Mol Biol R 2014, 78 (3), 510-543. DOI: 10.1128/Mmbr.00013-14. 27.

del Pozo, J. L.; Patel, R., The challenge of treating biofilm-associated bacterial infections.

Clin Pharmacol. Ther. 2007, 82 (2), 204-9. DOI: 10.1038/sj.clpt.6100247. 28.

Antunes, A. L. S.; Bonfanti, J. W.; Perez, L. R. R.; Pinto, C. C. F.; de Freitas, A. L. P.;

Macedo, A. J.; Barth, A. L., High vancomycin resistance among biofilms produced by Staphylococcus species isolated from central venous catheters. Mem I Oswaldo Cruz 2011, 106 (1), 51-55. DOI: Doi 10.1590/S0074-02762011000100008. 29.

Mohamed, M. F.; Brezden, A.; Mohammad, H.; Chmielewski, J.; Seleem, M. N.,

Targeting biofilms and persisters of ESKAPE pathogens with P14KanS, a kanamycin peptide conjugate. Bba-Gen Subjects 2017, 1861 (4), 848-859. DOI: 10.1016/j.bbagen.2017.01.029. 30.

Mohamed, M. F.; Abdelkhalek, A.; Seleem, M. N., Evaluation of short synthetic

antimicrobial peptides for treatment of drug-resistant and intracellular Staphylococcus aureus. Sci Rep-Uk 2016, 6. DOI: Artn 2970710.1038/Srep29707. 31.

Moise-Broder, P. A.; Forrest, A.; Birmingham, M. C.; Schentag, J. J., Pharmacodynamics

of vancomycin and other antimicrobials in patients with Staphylococcus aureus lower respiratory


Page 42 of 45

Page 43 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tract infections. Clin Pharmacokinet 2004, 43 (13), 925-942. DOI: Doi 10.2165/00003088200443130-00005. 32.

Thangamani, S.; Mohammad, H.; Abushahba, M. F.; Sobreira, T. J.; Hedrick, V. E.; Paul,

L. N.; Seleem, M. N., Antibacterial activity and mechanism of action of auranofin against multidrug resistant bacterial pathogens. Sci. Rep. 2016, 6, 22571. DOI: 10.1038/srep22571. 33.

Mohammad, H.; Cushman, M.; Seleem, M. N., Antibacterial Evaluation of Synthetic

Thiazole Compounds In Vitro and In Vivo in a Methicillin-Resistant Staphylococcus aureus (MRSA) Skin Infection Mouse Model. PloS One 2015, 10 (11). DOI: ARTN e0142321 10.1371/journal.pone.0142321. 34.

Mohammad, H.; Younis, W.; Ezzat, H. G.; Peters, C. E.; AbdelKhalek, A.; Cooper, B.;

Pogliano, K.; Pogliano, J.; Mayhoub, A. S.; Seleem, M. N., Bacteriological profiling of diphenylureas as a novel class of antibiotics against methicillin-resistant Staphylococcus aureus. PLoS One 2017, 12 (8), e0182821. DOI: 10.1371/journal.pone.0182821. 35.

Thangamani, S.; Younis, W.; Seleem, M. N., Repurposing celecoxib as a topical

antimicrobial agent. Front Microbiol 2015, 6. DOI: 10.3389/fmicb.2015.00750. 36.

Mohamed, M. F.; Hamed, M. I.; Panitch, A.; Seleem, M. N., Targeting Methicillin-

Resistant Staphylococcus aureus with Short Salt-Resistant Synthetic Peptides. Antimicrob Agents Chemther 2014, 58 (7), 4113-4122. DOI: 10.1128/Aac.02578-14. 37.

Farrell, D. J.; Robbins, M.; Rhys-Williams, W.; Love, W. G., Investigation of the

potential for mutational resistance to XF-73, retapamulin, mupirocin, fusidic acid, daptomycin, and vancomycin in methicillin-resistant Staphylococcus aureus isolates during a 55-passage study. Antimicrob Agents Chemother 2011, 55 (3), 1177-81. DOI: 10.1128/AAC.01285-10.



38.

AbdelKhalek, A.; Ashby, C. R.; Patel, B. A.; Talele, T. T.; Seleem, M. N., In Vitro

Antibacterial Activity of Rhodanine Derivatives against Pathogenic Clinical Isolates. PloS One 2016, 11 (10). DOI: ARTN e0164227 10.1371/journal.pone.0164227. 39.

Abushahba, M. F. N.; Mohammad, H.; Thangamani, S.; Hussein, A. A. A.; Seleem, M.

N., Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens. Sci Rep-Uk 2016, 6. DOI: Artn 2083210.1038/Srep20832.


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Table of graphical contents


Naphthylthiazoles: targeting multidrug-resistant and intracellular

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