Phenylthiazoles with tert-Butyl side chain: Metabolically stable with anti-biofilm activity

Article abstract of DOI:10.1016/j.ejmech.2018.03.044

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 9 h.

Full text of DOI:10.1016/j.ejmech.2018.03.044

Accepted Manuscript
Phenylthiazoles with tert-Butyl side chain: Metabolically stable with anti-biofilm activity
Ahmed Kotb, Nader S. Abutaleb, Mohamed A. Seleem, Mohamed Hagras, Haroon
Mohammad, Ashraf Bayoumi, Adel Ghiaty, Mohamed N. Seleem, Abdelrahman S.
Mayhoub
PII:
S0223-5234(18)30287-3
10.1016/j.ejmech.2018.03.044
EJMECH 10309
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 February 2018
Revised Date: 28 February 2018
Accepted Date: 15 March 2018
Please cite this article as: A. Kotb, N.S. Abutaleb, M.A. Seleem, M. Hagras, H. Mohammad, A. Bayoumi,
A. Ghiaty, M.N. Seleem, A.S. Mayhoub, Phenylthiazoles with tert-Butyl side chain: Metabolically
stable with anti-biofilm activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.03.044.
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Graphical Abstract

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Phenylthiazoles with tert-Butyl Side Chain: Metabolically Stable
with Anti-Biofilm Activity
Ahmed Kotb,1ǁ Nader S. Abutaleb,2ǁ Mohamed A. Seleem,1,3 Mohamed Hagras,1 Haroon
Mohammad,2 Ashraf Bayoumi,1 Adel Ghiaty,1 Mohamed N. Seleem2,4 * and Abdelrahman S.
Mayhoub1,5*
1Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University,
Cairo 11884, Egypt
2Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University,
West Lafayette, IN, USA 47907
3
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha,
Nebraska 68198, United States
3
Purdue Institute of Inflammation, Immunology, and Infectious Disease, West Lafayette, IN,
USA, 47907
4University of Science and Technology, Zewail City of Science and Technology, Giza, Egypt
Corresponding Authors. *e-mail: mseleem@purdue.edu, amayhoub@azhar.edu.eg
ǁThese two authors contributed equally
Abbreviations. CFU, colony forming unit; Caco-2, human colorectal cells.

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1. 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.
Keywords: antibiotic resistance, methicillin-resistant Staphylococcus aureus, MRSA,
pharmacokinetics.

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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

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incorporated in a heterocyclic linker system; this change led to the potent and metabolically
stable analogue 1c (Figure 1).17
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).
Figure 1. Developmental progress of phenylthiazole antibiotics and the new approach to
improve metabolic stability.

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3. RESULTS AND DISCUSSION
2.1. CHEMISTRY
The key starting phenylthiazole 3 was prepared from 4-t-butylthiobenzamide as reported
previously.12 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).
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).

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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

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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).
MRSA
(2658 RCMB)
MRSA
(2658 RCMB)
Compound
Compound
6.25
25
1.56
1.56
1.56
1.17
> 50
50
5
6
7
20
21
22
23
24
25
26
27
> 50
> 50
> 50
> 50
> 50
> 50
8
12
13
14
15
1.17
3.12

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MRSA
MRSA
(2658 RCMB)
Compound
Compound
(2658 RCMB)
> 50
25
6.25
1.17
0.78
0.78
1.56
16
17
18
19
28
29
Vancomycin
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 cefatroline19-20 (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
Bacterial Strains
19 23 26 28 29
1
16
1
S. aureus
ATCC 6538
MIC
MBC
MIC
MBC
MIC
MBC
4
8
4
8
8
8
8
8
8
8
8
8
4
8
8
8
8
8
4
4
4
4
4
4
2
4
1
1
2
2
1
1
S. aureus
NRS107
64
16
32
>64
>
64
MRSA
NRS119
4
4

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1
MRSA
NRS123 (USA400)
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
4
8
4
8
8
8
8
8
8
8
4
4
4
4
4
4
4
4
4
4
4
4
2
2
4
4
4
4
2
2
2
4
4
4
1
1
1
1
1
1
1
32
1
MRSA
NRS384 (USA300)
32
2
MRSA NRS385
MRSA NRS386
VRS10 (VRSA)
VRS12 (VRSA)
16
2
64
1
1
>64
>64 64
>64
>64 64
1
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
Methicillin-
Methicillin-
resistant
Staphylococcus
epidermidis
NRS101
Cephalosporin-
resistant
Enterococcus
faecium
ATCC
resistant
Streptococcus
pneumoniae
ATCC
Enterococcus
faecalis
ATCC 51299
(VRE)
Listeria
monocytogenes
ATCC 19111
Streptococcus
pneumoniae
ATCC 51916
Compound
/Antibiotic
700221
(VRE)
700677
MIC
MBC MIC MBC MIC MBC MIC
MBC
MIC
MBC MIC MBC
19
23
26
28
8
8
8
8
16
8
16
16
8
8
8
4
8
8
8
8
8
4
8
8
8
16
8
16
16
8
2
16
8
16
16
8
16
8
4
4
4
8
64
> 64
8
16
16
64
4
8
4
8
64
1
> 64
1
64
16
> 64
64
4
32
2
1
32
1
4
2
4
2
8
2
8
2
29
Vancomycin
> 64
> 64

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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.

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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 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.

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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.
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 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

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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).
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).
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.

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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
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

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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 (t1/2 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
(Vd) values. These data collectively would permit a once-daily dosing regimen to be used for
phenylthiazole derivative 19, if administered intravenously.

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Table 4. In vivo PK parameters of compound 19 in rats after a single IV bolus injection.
t1/2
(h)
9.03
CL
(L/hr)
1.26
AUC
mg.hr/L
3.98
Vdss
(L)
3.39
Vβ
(L)
16.36
19
t1/2: half-life; CL: clearance; Vβ: volume of distribution in 2nd 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

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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
1
as an orange solid (3.4 g, 94.4%) mp = 147 °C. H 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:

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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),
1
compound 5 was obtained as yellowish white solid (0.13 g, 71%) mp = 230 °C. H 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
13
(d, J = 5.2 Hz, 1H), 6.72 (brs, 2H), 2.68 (s, 3H), 1.29 (s, 9H); C 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
1
g, 1.2 mmol), compound 6 was obtained as white solid (0.18 g, 89%) mp = 160 °C; H 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,
13
J = 5.2 Hz, 1H), 6.87 (brs,1H), 2.83 (s, 3H), 2.70 (s, 3H), 1.29 (s, 9H); C 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
13
(s, 9H); C 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

ACCEPTED MANUSCRIPT
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
13
(d, J = 8.4 Hz, 2H), 7.37 (d, J = 5.2 Hz, 1H), 2.75 (s, 3H) 1.30 (s, 9H); C 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
1
copious amount of water to yield a yellowish white solid (0.67 g, 89%); mp = 125 °C. H NMR

ACCEPTED MANUSCRIPT
(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
1
reduced pressure to give the desired product as yellow crystals (0.52 g, 95%) mp = 190 °C. H
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-

ACCEPTED MANUSCRIPT
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
1
mmol), compound 13 was obtained as yellow solid (0.07 g, 74%) mp = 168 °C; H 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
13
(m, 4H), 1.29 (s, 9H); C 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

ACCEPTED MANUSCRIPT
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
1
mmol), compound 14 was obtained as brown solid (0.06 g, 57%) mp = 167 °C; H 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
13
(m, 4H), 1.59 (m, 4H), 1.31 (s, 9H); C 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
1
mmol), compound 15 was obtained as yellow solid (0.08 g, 82%) mp = 162 °C; H 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
1
mmol), compound 16 was obtained as brown solid (0.06 g, 60%) mp = 137 °C; H 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

ACCEPTED MANUSCRIPT
(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
1
mmol), compound 17 was obtained as orange solid (0.07 g, 70%) mp = 122.8 °C; H 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.
ol (18).
1-(4-(2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl)pyrimidin-2-yl)azetidin-3-
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 =
13
5.7 Hz, J = 4.8 Hz, 2H), 2.72 (s, 3H), 1.31 (s, 9h); C 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%.

ACCEPTED MANUSCRIPT
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,
1
0.4 mmol), compound 19 was obtained as yellow solid (0.08 g, 85%) mp = 145 °C; H 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
1
mmol), compound 21 was obtained as yellowish solid (0.09 g, 95%) mp = 275 °C; H 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,

ACCEPTED MANUSCRIPT
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

ACCEPTED MANUSCRIPT
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
1
yellow solid (0.1 g, 85%) mp = 220 °C; H 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,

ACCEPTED MANUSCRIPT
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%.

ACCEPTED MANUSCRIPT
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,
1
69%) mp = 170 °C; H 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