Phenylthiazoles with tert-Butyl Side Chain: Metabolically Stable with Anti-Biofilm Activity

Abstract

A new series of phenylthiazoles with t-butyl lipophilic component was synthesized and their antibacterial activity against a panel of multidrug-resistant bacterial pathogens was evaluated. Five compounds demonstrated promising antibacterial activity against methicillin-resistant staphylococcal strains and several vancomycin-resistant staphylococcal and enterococcal species. Additionally, three derivatives 19, 23 and 26 exhibited rapid bactericidal activity, and remarkable ability to disrupt mature biofilm produced by MRSA USA300. More importantly, a resistant mutant to 19 couldn’t be isolated after subjecting MRSA to sub-lethal doses for 14 days. Lastly, this new series of phenylthiazoles possesses an advantageous attribute over the first-generation compounds in their stability to hepatic metabolism, with a biological half-life of more than six hours.

Graphical Abstract

2. INTRODUCTION

The European Centre for Disease Prevention and Control (ECDC) has reported that healthcare-associated infections (HAIs), together with antimicrobial resistance (AMR), represent one of the most serious health threats not only in Europe but also globally.¹ Nearly 8% of hospitalized European patients experienced adverse events mainly as a result of nosocomial infections.² Additionally, HAIs account for 37,000 annual fatalities.³ More importantly, around half of the healthcare-associated fatalities are connected with superbugs and antibiotic resistance.

Of the bacterial pathogens that are a source of HAIs, methicillin-resistant Staphylococcus aureus (MRSA) remains a persistent problem. MRSA is resistant to not only methicillin but to many antibiotics in our therapeutic arsenal including other β-lactams,⁴ quinolones,^5–8 and macrolides,⁸ in addition to agents of last resorts such as vancomycin⁹ and linezolid.¹⁰ Though new derivatives of oxazolidinones and lipoglycopeptides have recently been developed and have increased the therapeutic options available for treatment of MRSA and other Gram-positive multidrug-resistant pathogens, the World Health Organization (WHO), in a recent report,¹¹ recommended continued development of new therapeutics for these bacterial pathogens to keep up with the anticipated evolution of resistance.¹¹

Phenylthiazoles represent a new antibiotic class of compounds developed by our research team that are potent inhibitors of multidrug-resistant bacterial species including MRSA, vancomycin-resistant Staphylococcus aureus (VRSA)^12–15 and vancomycin-resistant Enterococci (VRE).¹⁵ The initial series of phenylthiazole compounds (represented by compound 1a in Figure 1) suffered from short half-lives¹² which limited their pharmacological application. A detailed metabolic study identified a metabolic soft spot at the butyl benzylic carbon,¹⁶ which upon replacement with an oxygen atom provided derivatives 1b with longer duration of action.¹⁶ A second set of structural modifications focused on the cationic head, where the C=N was incorporated in a heterocyclic linker system; this change led to the potent and metabolically stable analogue 1c (Figure 1).¹⁷

Figure 1.

Developmental progress of phenylthiazole antibiotics and the new approach to improve metabolic stability.

This article explores a different tactic to block the metabolic soft spot via replacement of the n-butyl tail of the lead compound 1a with a tertiary isostere (where no benzylic C-H functionality is available for microsomal oxidation).¹⁸ Then, the Schiff bond was replaced with a pyrimidine linker in order to maximize both pharmacodynamic and pharmacokinetic profiles, and the structure-activity-relationship (SAR) at the nitrogenous head were rigorously studied (Figure 1).

3. RESULTS AND DISCUSSION

2.1. CHEMISTRY

The key starting phenylthiazole 3 was prepared from 4-t-butylthiobenzamide as reported previously. Treatment of 3 with DMF-DMA under solvent-free conditions afforded enaminone 4 in almost quantitative yield, which was allowed to react with different carboximidamides to provide the final products 5–8 (Scheme 1).

Scheme 1. Synthesis of compounds 5–8.

Reagents and conditions: (a) Absolute EtOH, 3-chloropentane-2,4-dione, heat at reflux, 12 h; (b) DMF-DMA heat at 80 °C, 8h; (c) proper imidamidate, K₂CO₃, absolute EtOH, heat at reflux, 3–8 h.

Next, the 2-(methylsulfonyl)pyrimidine 11 was obtained from the corresponding enaminone 4 via three consecutive steps; reaction with thiourea followed by methylation and oxidation with mCPBA (Scheme 2). Nucleophilic substitution on the methylsulfonyl moiety with different nitrogenous nucleophiles afforded the final products 12–29 (Scheme 2).

Scheme 2. Synthesis of compounds 12–29.

Reagents and conditions: (a) thiourea, KOH, EtOH, heat at reflux, 8 h; (b) dimethyl sulfate, KOH, H₂O, stirring at 23 °C, 2 h; (c) MCPBA, dry DCM, stirring at 23 °C, 16 h; (d) appropriate amine, hydrazine, guanidine or carboximidate, dry DMF, heat at 80 °C for 0.5 – 8 h

2.2. BIOLOGICAL RESULTS AND DISCUSSION

Initial antibacterial screening was conducted against one MRSA strain and the result is summarized in Table 1. First, the aminopyrimidine derivative 5 showed very moderate anti-MRSA potency, inhibiting growth of MRSA at 6.25 µg/mL. Adding a small alkyl group provided the methyaminopyrimidine analogue 6 with four-fold weaker antibacterial activity. Further extension of the alkyl side chain abolished the anti-MRSA activity (minimum inhibitory concentration (MIC) values of compounds 12–14 were above 50 µg/mL). Second, alkylation of the pendent amine of aminopyrimidine derivative 5 provided three N,N-dialkylated derivatives 7, 15 and 16 with MIC values above 50 µg/mL (Table 1). The antibacterial activity was partially retained upon adding a polar group to the carbon chain connected with the 2-aminopyrimidine moiety; hence, the hydroxyazetidine-containing compound 18 was moderately active when compared with its congener without the terminal hydroxyl group (compound 16). Similarly, adding a second amino group to the ethylamine moiety of 12 furnished compound 19 with a nearly 40-fold improvement in the antibacterial activity found (MIC = 1.17 µg/mL, Table 1). On the same vein, the hydrazine- and guanidine-containing analogues 20–23 exhibited notable anti-MRSA potency with MIC values equipotent to vancomycin (MIC = 1.56 µg/mL). It is important to note that dialkylation of the guanidine moiety (compound 23) had a slight positive impact on anti-MRSA activity. On the other hand, further methylation (compound 24) nullified the phenylthiazole compound from exhibiting antibacterial activity against MRSA. Finally, further extension of the guanidinyl moiety by replacement with substituted carboximidines provided compounds 25–29, in which only pyrrolidinyl derivative 25 was found to be inactive. Analogues with more polar side chains, 26–29, possessed potent anti-MRSA activity (MIC values range from 0.78 to 3.12 µg/mL). The lowest MIC values were obtained from picolinimidamide- and nicotinimidamide-containing derivatives 28 and 29 in which both demonstrated sub-microgram/mL inhibitory activity (Table 1).

Table 1.

The minimum inhibitory concentration (MIC in µg/mL) of compounds initially screened against methicillin-resistant Staphylococcus aureus (2658 RCMB).

Compound	MRSA (2658 RCMB)	Compound	MRSA (2658 RCMB)
5	6.25	20	1.56
6	25	21	1.56
7	> 50	22	1.56
8	> 50	23	1.17
12	> 50	24	> 50
13	> 50	25	50
14	> 50	26	1.17
15	> 50	27	3.12
16	> 50	28	0.78
17	25	29	0.78
18	6.25	Vancomycin	1.56
19	1.17

The five compounds identified from the initial screening with MIC values less than or equal to 1.2 µg/mL were selected for further evaluation (Table 2). In general, the tested compounds maintained their promising antibacterial activity against all tested methicillin-sensitive S. aureus (MSSA), MRSA and VRSA strains inhibiting growth at concentrations ranging from 2 to 8 µg/mL (except 29 which was inactive against S. aureus NRS107). This result correlates with the activity found for vancomycin (MIC ranges from 1 – 2 µg/mL against MRSA) and activity reported for cefatroline^19–20 (MIC ranges from 0.5 – 4 µg/mL against MRSA and VRSA), a recently-approved antibacterial agent for treatment of MRSA infections. The MBC values for the compounds were equal to or one-fold higher than the compounds’ MIC values against the tested strains which suggests that this series of phenylthiazoles are bactericidal agents.

Table 2.

The minimum inhibitory concentration (MIC in µg/mL) and minimum bactericidal concentration (MBC in µg/mL) of tested compounds against methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA) strains.

		Compound					Vancomycin	Linezolid
Bacterial Strains		19	23	26	28	29
S. aureus ATCC 6538	MIC	4	8	4	4	2	1	1
	MBC	8	8	8	4	4	1	16
S. aureus NRS107	MIC	4	8	8	4	64	2	1
	MBC	8	8	8	4	>64	2	16
MRSA NRS119	MIC	8	8	8	4	4	1	32
	MBC	8	8	8	4	4	1	>64
MRSA NRS123 (USA400)	MIC	8	8	8	4	2	1	1
	MBC	8	8	8	4	2	1	32
MRSA NRS384 (USA300)	MIC	8	8	4	4	4	1	1
	MBC	8	8	8	4	4	1	32
MRSA NRS385	MIC	8	8	4	4	4	1	2
	MBC	8	8	8	4	4	1	16
MRSA NRS386	MIC	8	8	8	4	2	1	2
	MBC	8	8	8	4	2	1	64
VRS10 (VRSA)	MIC	8	8	8	4	2	>64	1
	MBC	8	8	8	4	4	>64	64
VRS12 (VRSA)	MIC	8	8	8	4	4	>64	1
	MBC	8	8	8	4	4	>64	64

We next moved to examine the antibacterial spectrum of this new series of phenylthiazoles against other clinically-relevant Gram-positive bacterial pathogens. Therefore, compounds 19, 23, 26, 28 and 29 were tested against S. epidermidis, Enterococcus faecalis, E. faecium, Listeria monocytogenes, and Streptococcus pneumoniae isolates (Table 3).

Table 3.

The minimum inhibitory concentration (MIC in µg/mL) and minimum bactericidal concentration (MBC in µg/mL) of phenylthiazoles against Gram-positive bacterial pathogens including S. epidermidis, E. faecalis, E. faecium, L. monocytogenes, and S. pneumoniae.

	Bacterial Strains
Compound /Antibiotic	Methicillin- resistant Staphylococcus epidermidis NRS101		Enterococcus faecalis ATCC 51299 (VRE)		Enterococcus faecium ATCC 700221 (VRE)		Listeria monocytogenes ATCC 19111		Cephalosporin- resistant Streptococcus pneumoniae ATCC 51916		Methicillin- resistant Streptococcus pneumoniae ATCC 700677
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
19	8	8	16	16	8	8	8	8	8	16	2	16
23	8	8	8	16	8	16	8	8	16	16	16	16
26	4	4	4	8	4	8	8	8	8	8	8	8
28	64	> 64	8	16	8	16	4	64	4	8	4	8
29	64	> 64	64	> 64	4	32	2	32	4	4	8	8
Vancomycin	1	1	16	64	> 64	> 64	1	1	2	2	2	2

Out of all pathogenic strains listed in Table 3, vancomycin-resistant enterococcal strains are a significant concern as they are leading sources of nosocomial infections.²¹ Interestingly, the nitrogenous side chains connected to the pyrimidine position-2 seem to have high impact on the anti-enterococcal activity of the compounds. The ethylenediamine, dimethylguanidine, morpholine carboxamidine and picolinimidamide (compounds 19, 23, 26 and 28) derivatives were found to be effective against both vancomycin-resistant E. faecalis and E. faecium. On the other hand, repositioning the nitrogen atom in the pyridine ring from position-3 to position-2 abolished the antibacterial activity observed against E. faecalis (Table 3).

The MBC values for most of the compounds were found to be equal to or one-fold higher than the compounds’ MIC values against the tested bacterial strains indicating the compounds are bactericidal. The MBC values for 28 and 29 against L. monocytogenes were found to be more than three-fold higher than the compounds’ MIC values indicating these compounds might be bacteriostatic against this particular strain or species.

To confirm the rapid bactericidal kinetics of this series of phenylthiazoles against MRSA, the five most promising derivatives were further evaluated via a time-kill assay (Figure 2). Compounds 19, 23 and 26 exhibited rapid bactericidal activity in vitro, completely eradicating the high inoculum of MRSA within two to four hours. Vancomycin required 24 hours to exert its bactericidal activity by causing a three-log₁₀ reduction in the initial inoculum of MRSA. Compound 29 exhibited rapid bactericidal activity in vitro, decreasing the bacterial count by three-log₁₀ within six hours and completely eradicated the bacterial CFU within 24 hours. Interestingly, compound 28 exhibited bacteriostatic activity by decreasing the MRSACFU by only 2.3-log over 24 hours (Figure 2). This result confirms that phenylthiazoles with t-butyl lipophilic tail maintained the previously reported¹⁶ unique advantage (i.e. rapid bactericidal activity in vitro) over the existing drug of choice; i.e. vancomycin, used in the treatment of invasive Gram-positive infections.

Figure 2. Time-kill analysis of tested compounds and vancomycin (both at 4 × 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 obtained from triplicate samples used for each compound/antibiotic evaluated via two independent experiments.

To detect the ability of MRSA to develop resistance, MRSA USA400 was exposed to sub-lethal doses of the ethylenediamine-containing derivative 19, to try to generate drug-resistant mutants via a multi-step resistance selection experiment (over 14 daily passages). The MIC for compound 19 increased only one-fold after the ninth passage but remained stable thereafter. In contrast, MRSA developed resistance rapidly to the antibiotic rifampicin as the MIC of the antibiotic increased 29-fold after only one passage and continued to increase rapidly (>500,000-fold increase in MIC by the ninth passage) (Figure 3). The result indicates MRSA was unable to develop rapid resistance to 19, similar to the first-generation phenylthiazole compounds.

Figure 3. Multi-step resistance selection of compound 19 and rifampicin against methicillin-resistant S. aureus USA400 (NRS123).

Bacteria were serially passaged over a 14-day period and the broth microdilution assay was used to determine the minimum inhibitory concentration of each compound against MRSA after each successive passage. A four-fold shift in MIC would be indicative of bacterial resistance having formed to the test agent.

Next, we moved to investigate the selectivity of our compounds towards bacterial cells by measuring their toxicity to human colorectal cells. With the exception of compounds 23 and 29 that were intolerable at 32 µg/mL, the other three derivatives 19, 28 and 29 were highly tolerable at this concentration, which represents an eight to 30-fold difference when compared with the MIC value required to inhibit MRSA growth (reported in Tables 1 and 2).

Surface bound biofilm formation and prosthetic joints are inextricable. With the consistent upward increase in prosthetic joint replacements and other medical devices, the problem of recalcitrance to antibiotic treatment increased mainly due to biofilm-related infections.²² Bacterial biofilms are estimated to be a major source of infection (i.e. around 65% of human bacterial infections),²³ particularly on indwelling medical devices.^24–25 Staphylococci (namely S. aureus and S. epidermidis) are significant sources of biofilm-related infections.²⁶ The biofilm is a complex structure that provides a natural shield to bacterial cells to most conventional antibiotics as these drugs cannot effectively penetrate the biofilm mass at an effective concentration.²⁷ Thus finding antibacterial agents capable of disrupting these bacterial biofilms is important.

We evaluated the ability of the new series of phenylthiazole analogues to effectively disrupt adherent biofilm formed by MRSA via the microtiter dish biofilm formation assay. As noted earlier, many conventional antibiotics are ineffective at disrupting bacterial biofilms. This was observed with vancomycin, a cornerstone therapeutic for treatment of invasive MRSA infections, which exhibited very limited success in reducing MRSA biofilm mass, even at a high concentration (Figure 5), as has been previously reported.²⁸ Briefly, at 1 × MIC, vancomycin only disrupted 3% of MRSA biofilm mass. Even at a concentration of 32 × MIC, vancomycin only was capable of reducing the biofilm mass by 34% (data not published). The findings are congruent with the reported reduced susceptibility of staphylococcal infections encased in biofilm to vancomycin.^29–30 Other front-line alternative antibiotics such as linezolid and daptomycin have also shown limited success in disrupting/eradicating MRSA biofilm.^31–32

Figure 5. Eradication of mature MRSA biofilm by the five tested compounds and vancomycin.

Percent eradication of MRSA USA300 mature biofilm assessed via the microtiter dish biofilm formation assay. Wells treated with DMSO (the solvent for tested compounds) served as a negative control while vancomycin served as the control antibiotic. The values represent an average of a minimum of three samples analyzed for each compound/drug (from two independent experiments). Error bars represent standard deviation values. Asterisk (*) denotes statistical significance (P < 0.05) between results for tested compounds and the control antibiotic (vancomycin) analyzed via two-way ANOVA with post-hoc Sidak’s test for multiple comparisons.

Remarkably, all tested compounds were significantly more effective than vancomycin in disrupting mature MRSA biofilm, as shown in Figure 5. All five compounds evaluated exhibited a concentration-dependent disruption of MRSA biofilm. Briefly, compound 28 exhibited the highest biofilm eradication activity of the tested compounds; at 1 × MIC, this compound disrupted 45.8% of MRSA300 biofilm mass. This increased to 65.2% eradication of adherent biofilm mass when the concentration of 28 was increased one-fold (2 × MIC). Compound 29 similarly exhibited very good biofilm eradication activity at 1 × MIC, as it disrupted almost 44% of MRSA300 biofilm mass. This increased to nearly 49% eradication of adherent biofilm mass when compound 29’s concentration was increased one-fold (2×MIC). Compound 23 disrupted 35.5 % and 48.2% of MRSA300 biofilm mass at 1×MIC and 2×MIC, respectively. In brief, at 1×MIC, compound 19 disrupted 28% of MRSA biofilm mass. This increased to 64% eradication of adherent biofilm mass when the concentration of 19 was increased to two-fold (2 × MIC). Compound 26 was the least effective compound as it only disrupted 18.1 % and 28.3% of MRSA300 biofilm mass at 1 × MIC and 2 × MIC, respectively.

As indicated previously, first-generation phenylthiazoles, represented by the lead compound 1a (Figure 1), exhibited an ultra-short duration of action due to rapid hepatic metabolism. Therefore, a major objective of this study was to elongate the half-life of this new series in order to accelerate these compounds’ progress toward further pre-clinical evaluation. To investigate the biological half-life of this new series of phenylthiazoles, the pharmacokinetic profile of compound 19 was evaluated in vivo in male naïve Sprague–Dawley (SD) rats. The pharmacokinetic data obtained for the ethylenediamine derivative 19, as a representative example of t-butylphenylthiazoles, showed a remarkable increase in the biological half-life (t_1/2 of compound 19 = 9.03 h, Table 4) compared to 1a (more than 18-fold increase). Additionally, 19 exhibited a high ability to distribute throughout biological tissues as indicated by the volume of distribution (V_d) values. These data collectively would permit a once-daily dosing regimen to be used for phenylthiazole derivative 19, if administered intravenously.

Table 4.

In vivo PK parameters of compound 19 in rats after a single IV bolus injection.

	t1/2 (h)	CL (L/hr)	AUC mg.hr/L	Vβ (L)	Vdss (L)
19	9.03	1.26	3.98	16.36	3.39

t_1/2: half-life; CL: clearance; Vβ: volume of distribution in 2^nd compartment (peripheral tissues); Vdss: volume of distribution at the steady state

4. SUMMARY

Replacement of the n-butyl moiety of the lead compound 1a with a t-butyl moiety provided a new series of phenylthiazoles characterized by a longer biological half-life. In addition to their promising antibacterial effect against different staphylococcal and enterococcal bacterial isolates, five derivatives were superior to vancomycin in their ability to disrupt MRSA biofilm mass in a concentration-dependent manner. More importantly, the multi-step resistance selection study indicated MRSA is unlikely to form rapid resistance to the new series of phenylthiazoles.

5. EXPERIMENTAL PART

5.1. Chemistry

5.1.1. General

¹H NMR spectra were run at 400 MHz and ¹³C 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 determined using capillary tubes with a Stuart SMP30 apparatus and are uncorrected. All yields reported refer to isolated yields.

5.1.2. (E)-1-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)-3-(dimethylamino)prop-2-en-1-one (4)

To compound 2 (3 g, 11 mmol), DMF-DMA (2.7 mL, 2.4 g, 20.4 mmol) was added and the reaction mixture was heated at 80°C for 8 h. After cooling, the formed solid was collected by filtration, washed with petroleum ether and crystallized from ethanol to yield the desired product as an orange solid (3.4 g, 94.4%) mp = 147 °C. ¹H NMR (DMSO-d₆) δ: 7.86 (d, J = 8.4 Hz, 2H),7.71 (d, J = 12.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 5.44 (d, J = 12.4 Hz, 1H), 3.14 (s, 3H), 2.87 (s, 3H), 2.64 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 179.5, 166.1, 154.6, 154.5, 154.1, 134.2, 130.6, 126.5, 126.4, 94.2, 45.1, 37.7, 35.1, 31.3, 18.3; MS (m/z) 328. Anal. Calc. for: (C₁₉H₂₄N₂OS): C, 69.48; H, 7.36; N, 8.53 %; Found: C, 69.46; H, 7.37; N, 8.55%.

5.1.3. Compounds 5–8. General procedure

To a solution of enaminone 3 (0.2 g, 0.6 mmol) in absolute ethanol (5mL), proper guanidine or carboximidate (1.25 mmol); namely: guanidine hydrochloride, N-methylguanidine hydrochloride, pyrrolidine-1-carboximidamide hydroiodide, nicotinimidamide hydrochloride, and anhydrous potassium carbonate (0.2 g, 1.4 mmol) were added. The reaction mixture was heated at reflux for 8h, ethanol was evaporated under reduced pressure and the reaction was quenched with cold water (50 mL). The formed flocculated solid was filtered, washed with water and purified by crystallization from absolute ethanol or via acid-base extraction using HCl (1M, 50 mL). Upon neutralization with sodium carbonate to pH 7–8, the desired products were precipitated. The obtained solid was filtered, washed with a copious amount of distilled water and dried. Physical properties and spectral analysis of isolated products are listed below:

5.1.3.1. 4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-amine (5)

Following the general procedure (5.1.3), and using guanidine hydrochloride (0.115 g, 1.2 mmol), compound 5 was obtained as yellowish white solid (0.13 g, 71%) mp = 230 °C. ¹H NMR (DMSO-d₆) δ: 8.31 (d, J = 5.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 5.2 Hz, 1H), 6.72 (brs, 2H), 2.68 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.6, 163.8, 159.6, 158.3, 154.0, 153.2, 131.9, 130.6, 126.5, 126.4, 106.9, 35.1, 31.3, 18.6; MS (m/z) 324; HRMS (EI) m/z 324.1421 M⁺, calcd for C₁₈H₂₀N₄S 324.1409; Anal. Calc. for: (C₁₈H₂₀N₄S): C, 66.64; H, 6.21; N, 17.27 %; Found: C, 66.65; H, 6.22; N, 17.29%.

5.1.3.2. 4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)-N-methylpyrimidin-2-amine (6)

Following the general procedure (5.1.3), and using N-methylguanidine hydrochloride (0.14 g, 1.2 mmol), compound 6 was obtained as white solid (0.18 g, 89%) mp = 160 °C; ¹H NMR (DMSO-d₆) δ: 8.33 (d, J = 5.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H),7.52 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 5.2 Hz, 1H), 6.87 (brs,1H), 2.83 (s, 3H), 2.70 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.6, 162.9, 159.4, 158.3, 154.0, 153.4, 131.8, 130.6, 126.5, 126.4, 106.6, 38.1, 31.3, 28.2, 18.6; MS (m/z) 338; HRMS (EI) m/z 338.1569 M⁺, calcd for C₁₉H₂₁N₄S 338.1565; Anal. Calc. for: (C₁₉H₂₁N₄S): C, 67.42; H, 6.55; N, 16.55%; Found: C, 67.41; H, 6.56; N, 16.56%.

5.1.3.3. 2-(4-((tert-Butyl)phenyl)-4-methyl-5-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)thiazole (7)

Following the general procedure (5.1.3), and using pyrrolidine-1-carboximidamide hydroiodide (0.3 g, 1.2 mmol), compound 7 was obtained as brown solid (0.21 g, 93%) mp =203 °C; ¹H NMR (DMSO-d₆) δ: 8.38 (d, J = 5.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 5.2 Hz, 1H), 3.50 (m, 4H), 2.72 (s, 3H), 1.92 (m, 4H),1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.5, 160.1, 159.2, 158.0, 154.0, 153.6, 131.6, 130.5, 126.5, 126.4, 106.1, 46.7, 35.1, 31.3, 25.3, 18.7; MS (m/z) 378; HRMS (EI) m/z 378.1883 M⁺, calcd for C₂₂H₂₆N₄S 378.1878; Anal. Calc. for: (C₂₂H₂₆N₄S): C, 69.81; H, 6.92; N, 14.80%; Found: C, 69.80; H, 6.93; N, 14.82%.

5.1.3.4. 2-(4-(tert-Butyl)phenyl)-4-methyl-5-(pyridin-3-yl)thiazole (8)

Following the general procedure (5.1.3), and using nicotinimidamide hydrochloride (0.25 g, 1.6 mmol), compound 8 was obtained as light brown solid (0.14 g, 73.5 %) mp = 150 °C; ¹H NMR (DMSO-d₆) δ: 9.53 (s, 1H), 8.95 (d, J = 5.2 Hz, 1H), 8.74 (d, J = 6.4 Hz, 1H), 8.66 (d, J = 6.8 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 5.6 Hz, 1H), 7.60 (t, J = 6.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 2H), 2.81 (s, 3H), 1.30 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 167.9, 162.1, 159.2, 158.2, 155.2, 154.3, 152.1, 149.4, 135.5, 132.7, 130.6, 130.3, 126.6, 126.5, 124.3, 116.7, 36.1, 31.3, 18.9; MS (m/z) 308; HRMS (EI) m/z 308.1340 M⁺, calcd for C₁₉H₂₀N₂S 308.1347; Anal. Calc. for: (C₁₉H₂₀N₂S): C, 73.99; H, 6.54; N, 9.08%; Found: C, 73.98; H, 6.52; N, 9.09%.

5.1.4. 2-(4-(tert-Butyl)phenyl)-4-methyl-5-(2-(methylthio)pyrimidin-4-yl)thiazole (10)

To a solution of potassium hydroxide (0.2 g, 3.5 mmol) and thiourea (0.5 g, 6.5 mmol) in ethanol (15 mL), enaminone 4 (1 g, 3 mmol) was added. The reaction mixture was heated to reflux for 8h 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 9 as yellow solid (1.1 g, 96%) mp > 300 °C. ¹H NMR (DMSO-d₆) δ:11.52 (brs, 1H), 8.64 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 5.2 Hz, 1H), 2.75 (s, 3H) 1.30 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 167.6, 159.4, 159.4, 158.0, 155.1, 154.3, 151.8, 130.3, 126.6, 126.5, 111.7, 35.1, 31.3, 18.7; MS (m/z) 341. To a solution of the obtained intermediate 9 (0.8 g, 2.1 mmol) and potassium hydroxide (0.25 g, 4.2 mmol) in water (15 mL), dimethyl sulfate (0.5 mL, 4 mmol) was added dropwise with vigorous stirring. After 2h, the formed solid was filtered and washed with a copious amount of water to yield a yellowish white solid (0.67 g, 89%); mp = 125 °C. ¹H NMR (DMSO-d₆) δ: 8.35 (d, J = 4.8 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 4.8 Hz, 1H), 2.77 (s, 3H), 2.68 (s, 3H) 1.31 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.6, 162.9, 159.4, 158.1, 154.0, 153.4, 131.8, 130.5, 126.5, 126.4, 106.5, 35.1, 31.3, 28.2, 18.6; MS (m/z) 355. Anal. Calc. for: (C₁₉H₂₁N₃S₂): C, 64.19; H, 5.95; N, 11.82%; Found: C, 64.17; H, 5.97; N, 11.84%.

5.1.5. 2-(4-(tert-Butyl)phenyl)-4-methyl-5-(2-(methylsulfonyl)pyrimidin-4-yl)thiazole (11)

To a solution of compound 10 (0.5 g, 1.4 mmol) in dry DCM (5 mL), m-CPBA (0.514 g, 2.9 mmol) in DCM (5 mL) was added portion-wise with continuous stirring. After the reaction mixture was kept at 23 °C for 16 h, additional DCM (10mL) was added and the reaction mixture was washed with 25 mL of 5% aqueous solution of sodium metabisulfite and 25 mL of 5% aqueous sodium carbonate. The organic layer was separated, dried and concentrated under reduced pressure to give the desired product as yellow crystals (0.52 g, 95%) mp = 190 °C. ¹H NMR (DMSO-d₆) δ: 8.93 (d, J = 5.2 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H),7.82 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 5.2 Hz, 1H), 3.47 (s, 3H), 2.76 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 168.6, 165.3, 159.0, 158.3, 156.3, 154.7, 130.1, 130.0, 126.7, 126.6, 106.9, 50.3, 35.1, 31.2, 18.9; MS (m/z) 387; Anal. Calc. for: (C₁₉H₂₁N₃O₂S₂): C, 58.89; H, 5.46; N, 10.84%; Found: C, 58.87; H, 5.47; N, 10.86%.

5.1.6. Compounds 12–29. General procedure

To a solution of 11 (0.1 g, 0.26 mmol) in dry DMF (5mL), a proper amine, hydrazine, guanidine or caboximidate (0.4 mmol); namely: ethylamine, cyclopentylamine, cyclohexylamine, dimethylamine, azetidine hydrochloride, morpholine, azetidin-3-ol hydrochloride, ethylenediamine, hydrazine hydrate, guanidine hydrochloride, methylguanidine hydrochloride, 1,1-dimethylguanidine hydrochloride, N,N-tetramethyl guanidine, pyrrolidine-1-carboximidamide hydroiodide, morpholine-4- carboximidamide hydroiodide, 4-methylpiperazine-1-carboximidamide hydroiodide, picolinmidamide hydrochloride, nicotinimidamide hydrochloride, was added. The reaction mixture was heated at 80 °C for 0.5–8 h, and then poured over ice water (50 mL). The formed solid was filtered and washed with 50% ethanol and recrystallized from absolute ethanol. For 18, the crude solid was washed with boiling water to remove the residual hydrazine. Physical properties and spectral analysis of isolated products are listed below:

5.1.6.1. 4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)-N-ethylpyrimidin-2-amine (12)

Following the general procedure (5.1.6), and using ethylamine (18 µL, 0.4 mmol), compound 12 was obtained as yellow solid (0.06 g, 67%) mp = 145.5 °C; ¹H NMR (DMSO-d₆) δ: 8.33 (d, J = 4.8 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 4.8 Hz, 1H), 6.86 (brs, 1H), 3.43 (q, J = 4.8 Hz, 2H), 2.70 (s, 3H), 1.29 (s, 9H), 1.13 (t, J = 4.8 Hz, 3H); ¹³C NMR (DMSO-d₆) δ: 166.6, 162.3, 159.4, 158.3, 154.0, 153.4, 131.8, 130.6, 126.5, 126.4, 106.6, 35.8, 35.1, 31.3, 18.6, 15.0; MS (m/z) 352; HRMS (EI) m/z 352.1721 M⁺, calcd for C₂₀H₂₄N₄S 352.1722; Anal. Calc. for: (C₂₀H₂₄N₄S): C, 68.15; H, 6.86; N, 15.89%; Found: C, 68.14; H, 6.87; N, 15.88%.

5.1.6.2. 4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)-N-cyclopentylpyrimidin-2-amine (13)

Following the general procedure (5.1.6), and using cyclopentylamine (34 µL, 0.4 mmol), compound 13 was obtained as yellow solid (0.07 g, 74%) mp = 168 °C; ¹H NMR (DMSO-d₆) δ: 8.32 (d, J = 4.8 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.25 (brs, 1H), 6.84 (d, J = 4.8 Hz, 1H), 4.21 (m, 1H), 2.70 (s, 3H), 1.93 (m, 2H), 1.69 (m, 2H), 1.53 (m, 4H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.6, 162.2, 159.3, 158.3, 153.9, 153.3, 131.9, 130.6, 126.4, 126.4, 106.5, 52.7, 35.0, 32.6, 31.3, 23.9, 18.6; MS (m/z) 392; HRMS (EI) m/z 392.2028 M⁺, calcd for C₂₃H₂₈N₄S 392.2035; Anal. Calc. for: (C₂₃H₂₈N₄S): C, 70.37; H, 7.19; N, 14.27%; Found: C, 70.35; H, 7.18; N, 14.26%.

5.1.6.3. 4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)-N-cyclohexylpyrimidin-2-amine (14)

Following the general procedure (5.1.6), and using cyclohexylamine (39 µL, 0.4 mmol), compound 14 was obtained as brown solid (0.06 g, 57%) mp = 167 °C; ¹H NMR (DMSO-d₆) δ: 8.33 (d, J = 4.8 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 4.8 Hz, 2H), 7.11 (brs, 1H), 6.85 (d, J = 4.8 Hz, 1H), 3.71 (m, 1H), 3.16 (m, 1H), 2.71 (s, 3H), 1.91 (m, 1H), 1.73 (m, 4H), 1.59 (m, 4H), 1.31 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.6, 161.7, 159.6, 158.3, 154.0, 153.2, 131.8, 130.6, 126.5, 126.4, 106.5, 44.3, 35.1, 32.7, 31.3, 25.8, 25.3, 18.5; MS (m/z) 406; HRMS (EI) m/z 406.2202 M⁺, calcd for C₂₄H₃₀N₄S 406.2191; Anal. Calc. for: (C₂₄H₃₀N₄S): C, 70.90; H, 7.44; N, 13.78%; Found: C, 70.91; H, 7.45; N, 13.77%.

5.1.6.4. 4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)-N,N-dimethylpyrimidin-2-amine (15)

Following the general procedure (5.1.6), and using dimethylamine (18 µL, 0.4 mmol), compound 15 was obtained as yellow solid (0.08 g, 82%) mp = 162 °C; ¹H NMR (DMSO-d₆) δ: 8.40 (d, J = 4.8 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 4.8 Hz, 1H), 3.15 (s, 6H), 2.71 (s, 3H), 1.30 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.7, 161.9, 159.2, 157.9, 154.0, 153.5, 131.8, 130.5, 126.4, 126.4, 105.9, 36.9, 35.1, 31.3, 18.7; MS (m/z) 352; HRMS (EI) m/z 352.1745 M⁺, calcd for C₂₀H₂₄N₄S 352.1722; Anal. Calc. for: (C₂₀H₂₄N₄S): C, 68.15; H, 6.86; N, 15.89%; Found: C, 68.16; H, 6.88; N, 15.90%.

5.1.6.5. 5-(2-(Azetidin-1-yl)pyrimidin-4-yl)-2-(4-(tert-butyl)phenyl)-4-methylthiazole (16)

Following the general procedure (5.1.6), and using azetidine hydrochloride (0.04 g, 0.4 mmol), compound 16 was obtained as brown solid (0.06 g, 60%) mp = 137 °C; ¹H NMR (DMSO-d₆) δ: 8.37 (d, J = 4.8 Hz, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 4.8 Hz, 1H), 4.07 (t, J = 7.6 Hz, 4H), 2.7 (s, 3H), 2.28 (p, J = 7.6 Hz, 2H),1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.7, 162.8, 159.2, 158.1, 154.0, 153.8, 131.2, 130.5, 126.5, 126.4, 107.1, 50.3, 35.1, 31.3, 18.7, 16.2; MS (m/z) 364; HRMS (EI) m/z 364.1718 M⁺, calcd for C₂₁H₂₄N₄S 364.1722; Anal. Calc. for: (C₂₁H₂₄N₄S): C, 69.20; H, 6.64; N, 15.37%; Found: C, 69.22; H, 6.65; N, 15.38%.

5.1.6.6. 4-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl) morpholine (17)

Following the general procedure (5.1.6), and using morpholine (35 µL, 0.4 mmol), compound 17 was obtained as orange solid (0.07 g, 70%) mp = 122.8 °C; ¹H NMR (DMSO-d₆) δ: 8.42 (d, J = 5.2 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 5.2 Hz, 1H), 3.70 (t, J = 4.4 Hz, 4H), 3.67 (t, J = 4.4 Hz, 4H), 2.69 (s, 3H), 1.27 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.7, 161.9, 159.4, 158.1, 154.0, 153.6, 131.6, 130.5, 126.5, 126.4, 107.2, 66.4, 44.3, 35.1, 31.3, 18.7; MS (m/z) 394; HRMS (EI) m/z 394.1812 M⁺, calcd for C₂₂H₂₆N₄OS 394.1827; Anal. Calc. for: (C₂₂H₂₆N₄OS): C, 66.98; H, 6.64; N, 14.20%; Found: C, 66.99; H, 6.65; N, 14.21%.

5.1.6.7. 1-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl)azetidin-3-ol (18)

Following the general procedure (5.1.6), and using azetidin-3-ol hydrochloride (0.04 g, 0.4 mmol), compound 18 was obtained as orange solid (0.06 g, 61%) mp = 214 °C; ¹H NMR (DMSO-d₆) δ: 8.40 (d, J = 4.8 Hz, 1H),7.90 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 4.8 Hz, 1H), 5.74 (brs, 1H),4.59 (m, 1H), 4.29 (dd, J = 4.4 Hz, J = 4.8 Hz, 2H), 3.84 (dd, J = 5.7 Hz, J = 4.8 Hz, 2H), 2.72 (s, 3H), 1.31 (s, 9h); ¹³C NMR (DMSO-d₆) δ: 166.8, 162.9, 159.3, 158.2, 154.1, 153.9, 131.2, 130.5, 126.5, 126.4, 107.2, 61.2, 60.3, 35.1, 31,3, 18,7; MS (m/z) 380; HRMS (EI) m/z 380.1675 M⁺, calcd for C₂₁H₂₄N₄OS 380.1671; Anal. Calc. for: (C₂₁H₂₄N₄OS): C, 66.29; H, 6.36; N, 14.72%; Found: C, 66.28; H, 6.34; N, 14.70%.

5.1.6.8. N-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl)ethane-1,2-diamine (19)

Following the general procedure (5.1.6), and using ethylenediamine (24 µL, 0.4 mmol), compound 19 was obtained as yellow solid (0.08 g, 85%) mp = 145 °C; ¹H NMR (DMSO-d₆) δ: 8.35 (d, J = 5.2 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.27 (brs, 1H), 6.89 (d, J = 5.2, 1H), 3.17 (t, J = 4.8, 2H), 2.85 (t, J = 4.8, 2H), 2.71 (s, 3H), 1.82 (brs, 2H), 1.31 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.7, 162.6, 159.4, 158.4, 154.0, 153.4, 131.8, 130.6, 126.5, 126.4, 106.8, 43.3, 40.5, 35.1, 31.3, 18.6; MS (m/z) 367; HRMS (EI) m/z 367.1830 M⁺, calcd for C₂₀H₂₅N₅S 367.1831; Anal. Calc. for: (C₂₀H₂₅N₅S): C, 65.36; H, 6.86; N, 19.06%; Found: C, 65.35; H, 6.87; N, 19.07%.

5.1.6.9. 2-(4-(tert-Butyl)phenyl)-5-(2-hydrazinylpyrimidin-4-yl)-4-methylthiazole (20)

Following the general procedure (5.1.6), and using hydrazine hydrate (5mL), compound 20 was obtained as yellowish white fluffy powder (0.07g, 80%) mp = 151 °C; ¹H NMR (DMSO-d₆) δ: 8.38 (d, J = 5.2 Hz, 1H), 8.29 (brs, 1H), 7.89 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 5.2 Hz, 1H), 4.21 (brs, 2H), 2.73 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.7, 164.6, 159.3, 158.1, 154.0, 153.7, 131.6, 130.5, 126.5, 126.4, 107.2, 35.1, 31,3, 18.7; MS (m/z) 339; HRMS (EI) m/z 339.1527 M⁺, calcd for C₁₈H₂₁N₅S 339.1518; Anal. Calc. for: (C₁₈H₂₁N₅S): C, 63.69; H, 6.24; N, 20.63%; Found: C, 63.67; H, 6.25; N, 20.64%.

5.1.6.10. 1-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl)guanidine (21)

Following the general procedure (5.1.6), and using guanidine hydrochloride (0.05 g, 0.5 mmol), compound 21 was obtained as yellowish solid (0.09 g, 95%) mp = 275 °C; ¹H NMR (DMSO-d₆) δ: 8.87 (brs, 1H), 8.44 (d, J = 5.2 Hz, 1H), 8.41 (brs, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 8.2 Hz, 2H), 7.01 (d, J = 5.2 Hz, 1H), 6.89 (brs, 2H), 2.75 (s, 3H), 1.30 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.7, 159.8, 159.2, 158.3, 157.8, 154.0, 153.3, 131.9, 130.6, 126.5, 126.4, 106.0, 35.1, 31.3, 18.7; MS (m/z) 366; HRMS (EI) m/z 366.1640 M⁺, calcd for C₁₉H₂₂N₆S 366.1627; Anal. Calc. for: (C₁₉H₂₂N₆S): C, 62.27; H, 6.05; N, 22.93%; Found: C, 62.28; H, 6.06; N, 22.92%.

5.1.6.11. 1-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl)-3-methylguanidine (22)

Following the general procedure (5.1.6), and using methylguanidine hydrochloride (0.06 g, 0.5 mmol), compound 22 was obtained as yellowish solid (0.07 g, 65%) mp = 236 °C; ¹H NMR (DMSO-d₆) δ: 8.78 (brs, 1H), 8.53 (d, J = 5.2, 1H), 8.33 (brs, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.15 (brs, 1H), 6.87 (d, J = 5.2 Hz, 1H), 2.85 (s, 3H), 2.77 (s, 3H), 1.31(s, 9H); ¹³C NMR (DMSO-d₆) δ: 168.1, 166.7, 162.4, 159.2, 158.1, 154.0, 153.5, 140.8, 130.5, 126.5, 126.4, 107.0, 36.9, 35.1, 31.3, 18.6; MS (m/z) 380; HRMS (EI) m/z 380.1790 M⁺, calcd for C₂₀H₂₄N₆S 380.1783; Anal. Calc. for: (C₂₀H₂₄N₆S): C, 63.13; H, 6.36; N, 22.09%; Found: C, 63.12; H, 6.35; N, 22.11%.

5.1.6.12. 3-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl)-1,1-dimethylguanidine (23)

Following the general procedure (5.1.6), and using 1,1-dimethylguanidine hydrochloride (0.06 g, 0.5 mmol), compound 23 was obtained as yellowish solid (0.1 g, 85%) mp = 215 °C; ¹H NMR (DMSO-d₆) δ: 8.46 (d, J = 5.2 Hz, 1H), 8.22 (brs,2H), 7.89 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 5.2 Hz, 1H), 3.01 (s, 6H), 2.68 (s, 3H), 1.30 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.5, 165.9, 158.6, 158.5, 157.4, 154.0, 152.8, 132.4, 130.6, 126.5, 126.4, 107.7, 37., 35.1, 31.3, 18.6; MS (m/z) 394; HRMS (EI) m/z 394.1944 M⁺, calcd for C₂₁H₂₆N₆S 394.1940; Anal. Calc. for: (C₂₁H₂₆N₆S): C, 64.37; H, 6.21; N, 17.27%; Found: C, 64.35; H, 6.22; N, 17.28%.

5.1.6.13. N-2-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl)-1,1,3,3-tetramethylguanidine (24)

Following the general procedure (5.1.6), and using N,N-tetramethyl guanidine (50 µL, 0.4 mmol), compound 24 was obtained as yellowish solid (0.09 g, 85%) mp = 115 °C; ¹H NMR (DMSO-d₆) δ: 8.46 (d, J = 5.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 5.2 Hz, 1H), 2.73 (s, 12H), 2.68 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 167.1, 166.7, 162.8, 159.5, 158.1, 153.9, 152.9, 131.6, 130.6, 126.5, 126.4, 107.8, 50.1, 35.1, 31.3, 18.64; MS (m/z) 422; HRMS (EI) m/z 422.2255 M⁺, calcd for C₂₃H₃₀N₆S 422.2253; Anal. Calc. for: (C₂₃H₃₀N₆S): C, 65.37; H, 7.16; N, 19.89%; Found: C, 65.35; H, 7.15; N, 19.87%.

5.1.6.14. N-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl) pyrrolidine-1-carboximidamide (25)

Following the general procedure (5.1.6), and using pyrrolidine-1-carboximidamide hydroiodide (0.1 g, 0.4 mmol), compound 25 was obtained as yellowish solid (0.1 g, 92%) mp = 190 °C; ¹H NMR (DMSO-d₆) δ: 8.47 (d, J = 4.8 Hz, 1H), 8.02 (brs, 2H),7.90 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 4.8 Hz, 2H), 7.02 (d, J = 4.8 Hz, 1H), 3.42 (m, 4H), 2.69 (s, 3H), 1.88 (m, 4H), 1.31 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.5, 166.0, 158.6, 157.4, 156.6, 154.0, 152.8, 132.5, 130.6, 126.5, 126.4, 107.5, 46.6, 35.1, 31.3, 25.3, 18.5; MS (m/z) 420; HRMS (EI) m/z 420.2110 M⁺, calcd for C₂₃H₂₈N₆S 420.2096; Anal. Calc. for: (C₂₃H₂₈N₆S): C, 65.68; H, 6.71; N, 19.98%; Found: C, 65.67; H, 6.73; N, 19.97%.

5.1.6.15. N-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl) morpholine-4-carboximidamide (26)

Following the general procedure (5.1.6), and using morpholine-4-carboximidamide hydroiodide (0.1 g, 0.4 mmol), compound 26 was obtained as yellow solid (0.1 g, 85%) mp = 220 °C; ¹H NMR (DMSO-d₆) δ: 8.50 (d, J = 4.8 Hz, 1H), 8.39 (brs, 1H), 7.91 (brs, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 4.6 Hz, 1H), 3.63 (m, 4H), 3.59 (m, 4H), 2.69 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 166.6, 166.0, 158.5, 157.8, 157.6, 154.0, 153.0, 132.3, 130.5, 126.5, 126.4, 108.2, 66.4, 44.7, 35.1, 31.3, 18.6; MS (m/z) 436; HRMS (EI) m/z 436.2053 M⁺, calcd for C₂₅H₂₄N₆OS 436.2045; Anal. Calc. for: (C₂₃H₂₈N₆OS): C, 63.28; H, 6.45; N, 19.25%; Found: C, 63.26; H, 6.46; N, 19.27%.

5.1.6.16. N-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl)-4-methylpiperazine-1-carboximidamide (27)

Following the general procedure (5.1.6), and using 4-methylpiperazine-1-carboximidamide hydroiodide (0.11 g, 0.4 mmol), compound 27 was obtained as yellow solid (0.07 g, 60%) mp = 165 °C; ¹H NMR (DMSO-d₆) δ: 8.48 (d, J = 5.2 Hz, 1H), 8.37 (brs, 1H), 7.94 (brs, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 5.2 Hz, 1H), 3.58 (m, 4H), 2.72 (s, 3H), 2.33 (m, 4H), 2.19 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 168.7, 166.7, 162.9, 159.4, 158.3, 154.0, 153.1, 131.4, 130.6, 126.5, 126.5, 107.0, 54.8, 46.1, 36.3, 35.1, 31.3, 18.9; MS (m/z) 449; HRMS (EI) m/z 449.2368 M⁺, calcd for C₂₄H₃₁N₇S 449.2362; Anal. Calc. for: (C₂₄H₃₁N₇S): C, 64.11; H, 6.95; N, 21.81%; Found: C, 64.13; H, 6.97; N, 21.83.

5.1.6.17. N-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl) nicotinimidamide (28)

Following the general procedure (5.1.6), and using picolinmidamide hydrochloride (0.06 g, 0.4 mmol), compound 28 was obtained as orange-yellowish solid (0.1 g, 92%) mp = 150 °C; ¹H NMR (DMSO-d₆) δ: 9.53 (brs, 1H), 8.77 (d, J = 4.4 Hz, 1H), 8.71 (d, J = 5.2 Hz, 1H), 8.47 (d, J = 4.8 Hz, 1H), 8.22 (brs, 1H), 7.93 (t, J = 7.2 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.61 (t, J = 6.8 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 4.8 Hz, 1H), 2.68 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 167.2, 166.9, 162.8, 159.0, 158.4, 158.0, 154.2, 151.9, 151.6, 149.0, 138.0, 131.7, 130.5, 126.5, 126.4, 122.6, 111.4, 35.1, 31.3, 18.7; MS (m/z) 428; HRMS (EI) m/z 428.1788 M⁺, calcd for C₂₄H₂₄N₆S 428.1783; Anal. Calc. for: (C₂₄H₂₄N₆S): C, 67.26; H, 5.64; N, 19.61%; Found: C, 67.25; H, 5.65; N, 19.63%.

5.1.6.18. N-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl) picolinimidamide (29)

Following the general procedure (5.1.6), and using nicotinimidamide hydrochloride (0.06 g, 0.4 mmol), compound 29 was obtained as orange-yellowish solid (0.08 g, 69%) mp = 170 °C; ¹H NMR (DMSO-d₆) δ: 9.25 (brs, 1H), 8.73 (m, 2H), 8.44 (d, J = 6.8 Hz, 1H), 8.22 (brs, 1H), 7.89 (m, 2H), 7.52 (m, 4H), 7.37 (d, J = 4.8 Hz, 1H), 2.73 (s, 3H), 1.29 (s, 9H); ¹³C NMR (DMSO-d₆) δ: 167.2, 166.6, 159.4, 158.9, 158.3, 154.1, 153.8, 152.0, 149.1, 135.7, 131.9, 131.6, 130.4, 126.5, 126.5, 123.7, 111.3, 35.1, 31.3, 18.7; MS (m/z) 428; HRMS (EI) m/z 428.1790 M⁺, calcd for C₂₄H₂₄N₆S 428.1783; Anal. Calc. for: (C₂₄H₂₄N₆S): C, 67.26; H, 5.64; N, 19.61%; Found: C, 67.28; H, 5.66; N, 19.62%.

5.2. Microbiological Assays

5.2.1. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Staphylococcus aureus and important Gram-positive bacterial pathogens

The minimum inhibitory concentration (MIC) of tested compounds and control antibiotics was determined using the broth microdilution method³³ against methicillin-sensitive (ATCC 6538 and NRS107), methicillin-resistant (MRSA), and vancomycin-resistant (VRSA) Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, E. faecium, Listeria monocytogenes, and Streptococcus pneumoniae clinical isolates. A bacterial solution equivalent to 0.5 McFarland standard was prepared and diluted in cation-adjusted Mueller-Hinton broth (CAMHB) to achieve a bacterial concentration of about 5 × 10⁵ CFU/mL and seeded in 96-well plates. Enterococcus faecium was diluted in brain heart infusion broth. Enterococcus faecalis, Streptococcus pneumonia and Listeria monocytogenes were diluted in tryptone soya broth (TSB). Compounds and control drugs were added in the first row of 96-well plates and serially diluted (to achieve a concentration gradient ranging from 128 to 1 µg/mL). Plates were then incubated aerobically at 37° C for at least 18 hours. The minimum bactericidal concentration (MBC) of the active compounds was tested by plating 5 µL from wells with no growth onto Tryptic soy agar (TSA) plates. Plates were incubated at 37 °C for at least 18 hours before recording the MBC.

5.2.2. Time-kill assay against MRSA

MRSA USA400 cells in logarithmic growth phase (OD₆₀₀ ∼1.00) were diluted to 3.42 × 10⁶ colony-forming units (CFU/mL) and exposed to concentrations equivalent to 4 × MIC (in triplicate) of the compounds and vancomycin in TSB. Aliquots (100 µL) were collected from each treatment after 0, 2, 4, 6, 8, 12, and 24 hours of incubation at 37 °C and subsequently serially diluted in PBS. Bacteria were then transferred to Tryptic soy agar (TSA) plates and incubated at 37 °C for at least 18 hours before viable CFU/mL was determined.

5.2.3. Resistance study against MRSA

To determine if MRSA would be capable of forming resistance to the compounds quickly, a multi-step resistance selection experiment was conducted, as described previously.³⁴ The broth microdilution assay was utilized to determine the MIC of the tested compounds and rifampicin exposed to MRSA USA400 (NRS123) for 14 passages over a period of two weeks. Resistance was classified as a greater than four-fold increase in the initial MIC, as reported elsewhere.³⁵

5.2.4. In vitro cytotoxicity analysis of tested compounds against Caco-2 cells

Tested compounds were assayed (at concentrations of 16, 32, and 64 µg/mL) against a human colorectal (Caco-2) cell to determine the potential toxic effect to mammalian cells in vitro. Briefly, cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), non-essential amino acids (1X), and penicillin-streptomycin at 37 °C with CO₂ (5%). Control cells received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells were incubated with the compounds (in triplicate) in a 96-well plate at 37 °C with CO₂ (5%) for two hours. The assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was subsequently added and the plate was incubated for four hours. Absorbance readings (at OD₄₉₀) were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data was analyzed via a two-way ANOVA, with post hoc Sidak’s multiple comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).

5.2.5. MRSA biofilm eradication assessment

The compounds and vancomycin were examined for their ability to eradicate pre-formed, mature staphylococcal biofilm using the microtiter dish biofilm formation assay,³⁶ following the procedure described in a previous report.²⁸ 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 strong adherent biofilm. The bacterial suspension was removed and compounds were added at concentrations ranging from 128 to 1 µg/mL in TSB. 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 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 Sidak’s test for multiple comparisons (P < 0.05).

5.2.6. 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 µM solution of compound 19 was directly administered via tail-vein injection. 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 (Supporting information).

Figure 4. Toxicity analysis of compounds 19, 23, 26, 28, and 29 against human colorectal cells (Caco-2).

Percent viable mammalian cells (measured as average absorbance ratio (test agent relative to DMSO) for cytotoxicity analysis of tested compounds (tested in triplicate) at 16, 32, and 64 µg/mL against Caco-2 cells using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Dimethyl sulfoxide (DMSO) was used as a negative control to determine a baseline measurement for the cytotoxic impact of each compound. The absorbance values represent an average of a minimum of three samples analyzed for each compound from two independent experiments. Error bars represent standard deviation values for the absorbance values. A two-way ANOVA, with post hoc Sidak’s multiple comparisons test, determined statistical difference (denoted by the asterisk) (P < 0.05) between the values obtained for each compound and DMSO (negative control, used as solvent for the compounds).

Highlights.

• Phenylthiazoles with t-buty tail are with the ability to penetrate the bacterial biofilm mass

• Three derivatives 19, 23 and 26 exhibited an advantage over vancomycin being rapid bactericidal agent

• Unlike the lead compound, the new analogues possess high metabolic stability

• The most potent analogue 19 exhibited biological t1/2 of almost 9 h

Abbreviations

CFU

colony forming unit

Caco-2

human colorectal cells