Alkynyl-containing phenylthiazoles: Systemically active antibacterial agents effective against methicillin-resistant Staphylococcus aureus (MRSA)

Article abstract of DOI:10.1016/j.ejmech.2018.02.031

The promising activity of phenylthiazoles against multidrug-resistant bacterial pathogens, in particular MRSA, has been hampered by their limited systemic applicability, due to their rapid metabolism by hepatic microsomal enzymes, resulting in short half-lives. Here, we investigated a series of phenylthiazoles with alkynyl side-chains that were synthesized with the objective of improving stability to hepatic metabolism, extending the utility of phenylthiazoles from topical applications to treatment of a more invasive, systemic MRSA infections. The most promising compounds inhibited the growth of clinically-relevant isolates of MRSA in vitro at concentrations as low as 0.5 μg/mL, and exerted their antibacterial effect by interfering with bacterial cell wall synthesis via inhibition of undecaprenyl diphosphate synthase and undecaprenyl diphosphate phosphatase. We also identified two phenylthiazoles that successfully eradicated MRSA inside infected macrophages. In vivo PK analysis of compound 9 revealed promising stability to hepatic metabolism with a biological half-life of ～4.5 h. In mice, compound 9 demonstrated comparable potency to vancomycin, and at a lower dose (20 mg/kg versus 50 mg/kg), in reducing the burden of MRSA in a systemic, deep-tissue infection, using the neutropenic mouse thigh-infection model. Compound 9 thus represents a new phenylthiazole lead for the treatment of MRSA infections that warrants further development.

Full text of DOI:10.1016/j.ejmech.2018.02.031

Accepted Manuscript
Alkynyl-containing phenylthiazoles: Systemically active antibacterial agents effective
against methicillin-resistant Staphylococcus aureus (MRSA)
Mohamed M. Elsebaei, Haroon Mohammad, Mohamed Abouf, Nader S. Abutaleb,
Youssef A. Hegazy, Adel Ghiaty, Lu Chen, Jianan Zhang, Satish R. Malwal, Eric
Oldfield, Mohamed N. Seleem, Abdelrahman S. Mayhoub
PII:
S0223-5234(18)30161-2
10.1016/j.ejmech.2018.02.031
EJMECH 10208
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 January 2018
Revised Date: 9 February 2018
Accepted Date: 10 February 2018
Please cite this article as: M.M. Elsebaei, H. Mohammad, M. Abouf, N.S. Abutaleb, Y.A. Hegazy, A.
Ghiaty, L. Chen, J. Zhang, S.R. Malwal, E. Oldfield, M.N. Seleem, A.S. Mayhoub, Alkynyl-containing
phenylthiazoles: Systemically active antibacterial agents effective against methicillin-resistant
Staphylococcus aureus (MRSA), European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.02.031.
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Alkynyl-Containing Phenylthiazoles: Systemically Active Antibacterial Agents Effective
Against Methicillin-resistant Staphylococcus aureus (MRSA)
a
b
a
b
⊥
⊥
Mohamed M. Elsebaei, Haroon Mohammad, Mohamed Abouf, Nader S. Abutaleb, Youssef
b
a
c
d
f
e,f
A. Hegazy, Adel Ghiaty, Lu Chen, Jianan Zhang, Satish R. Malwal, Eric Oldfield,
b,g**
a,h*
Mohamed N. Seleem,
and Abdelrahman S. Mayhoub
a.
Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar
University, Cairo 11884, Egypt.
b.
Department of Comparative Pathobiology, Purdue University, College of Veterinary
Medicine, West Lafayette, IN 47907, USA.
c.
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana,
IL, 61801, United States
d.
School of Molecular and Cellular Biology, University of Illinois at Urbana-
Champaign, Urbana, IL, 61801, United States
e.
Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-
Champaign, Urbana, IL, 61801, United States
f.
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL,
1801, United States
6
g.
Purdue Institute for Inflammation, Immunology, and Infectious Diseases, West
Lafayette, IN 479067, USA.
h.
University of Science and Technology, Zewail City of Science and Technology,
Giza, Egypt
⊥:
These authors contributed equally to this work
Corresponding Authors
*
* MNS; email, mseleem@purdue.edu
ASM; e-mail, amayhoub@azhar.edu.eg
*
1

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Abbreviations. Caco-2, heterogeneous human epithelial colorectal adenocarcinoma cells; CFUs,
colony forming units; HaCaT, human keratinocytes; P , apparent permeability; VISA,
app
vancomycin-intermediate S. aureus; HBD, hydrogen bond donor; P-gp, P-glycoprotein.
Key words. Antibiotic resistance, MRSA, VRSA, multi-drug resistance, C. elegans
2

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1
. ABSTRACT
The promising activity of phenylthiazoles against multidrug-resistant bacterial pathogens, in
particular MRSA, has been hampered by their limited systemic applicability, due to their rapid
metabolism by hepatic microsomal enzymes, resulting in short half-lives. Here, we investigated a
series of phenylthiazoles with alkynyl side-chains that were synthesized with the objective of
improving stability to hepatic metabolism, extending the utility of phenylthiazoles from topical
applications to treatment of a more invasive, systemic MRSA infections. The most promising
compounds inhibited the growth of clinically-relevant isolates of MRSA in vitro at
concentrations as low as 0.5 µg/mL, and exerted their antibacterial effect by interfering with
bacterial cell wall synthesis via inhibition of undecaprenyl diphosphate synthase and
undecaprenyl diphosphate phosphatase. We also identified two phenylthiazoles that successfully
eradicated MRSA inside infected macrophages. In vivo PK analysis of compound 9 revealed
promising stability to hepatic metabolism with a biological half-life of ~4.5 hours. In mice,
compound 9 demonstrated comparable potency to vancomycin, and at a lower dose (20 mg/kg
versus 50 mg/kg), in reducing the burden of MRSA in a systemic, deep-tissue infection, using
the neutropenic mouse thigh-infection model. Compound 9 thus represents a new phenylthiazole
lead for the treatment of MRSA infections that warrants further development.
3

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2
. INTRODUCTION
In recent years, the number of fatal infections caused by antibiotic-resistant bacteria has
increased in many regions around the world. Staphylococcus aureus is a prominent bacterial
pathogen responsible for a diverse array of infections ranging from superficial skin lesions to
invasive diseases including soft-tissue infections, pneumonia, endocarditis, and osteomyelitis.
The emergence in the 1960s of S. aureus isolates exhibiting resistance to β-lactam antibiotics
such as methicillin, methicillin-resistant S. aureus or MRSA, spurred several outbreaks in
hospitals [1]. Treatment of MRSA infections for clinicians has become exacerbated by the
emergence of strains exhibiting resistance to other antibiotic classes, including fluoroquinolones,
tetracyclines, macrolides, lincosamides and aminoglycosides [2]. More recently, strains of drug-
resistant S. aureus have emerged that exhibit intermediate sensitivity or resistance to vancomycin
(VISA and VRSA, respectively), an antibiotic that has been the cornerstone treatment choice for
MRSA infections [3]. Though several antibiotics are currently in the clinical pipeline, the vast
majority of these agents are derivatives of existing antibiotic classes that do not exert their
effects by attacking new molecular targets. Thus, developing new antibacterial agents with
unique chemical scaffolds and molecular target(s), effective against MRSA, still remains a
critical issue for the medicinal chemistry community to address [4].
4

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Figure 1. Chemical structures of two first-generation phenylthiazole lead compounds
with their half-life (t ) and intrinsic clearance (CL ) values identified using human liver
1
/2
int
microsomes.
Recognizing the need for new antibacterial agents, we have been investigating
compounds containing the phenylthiazole scaffold for the development of antibacterial agents
[5]. The first-generation phenylthiazoles we synthesized exhibited antibacterial activity against
key Gram-positive pathogens, including MRSA and vancomycin-resistant enterococci, by
interfering with bacterial cell wall synthesis [6]. However, the compounds were susceptible to
rapid metabolism (t1/2 < 30 minutes), with the exception of the hexynyl derivative 1b (t1/2 > 4
hours), which was not effective in vivo in treating a MRSA infection (Figure 1) [5c].
Incorporating the imine bond of the lead compound 1a within a pyrimidine ring yielded second-
generation phenylthiazoles with enhanced stability to hepatic metabolism [5d], and a complete
metabolite analysis of 1a indicated the presence of an additional metabolic "soft spot" at the
butyl benzylic carbon [7]. Removing this soft spot has the potential to yield phenylthiazole
compounds with better stability to hepatic metabolism. To address this, the n-butyl group of the
lead compound 1a was replaced with an array of alkynyl side-chains having different sizes and
5

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spatial configurations. The antibacterial activity of the new analogues was then examined against
a broad panel of drug-resistant S. aureus clinical isolates, and the metabolic stability and
permeability profiles of the most promising analogues assessed. Additionally, the ability of the
most promising analogue (compound 9) to inhibit the molecular targets of the first-generation
phenylthiazoles (undecaprenyl diphosphate synthase, UPPS, and undecaprenyl diphosphate
phosphatase, UPPP) was investigated. Furthermore, compound 9 was evaluated in three different
animal models of MRSA infection: Caenorhabditis elegans; an uncomplicated skin infection
mouse model, and a neutropenic thigh-infection mouse model.
3
. RESULTS AND DISCUSSION
.1. Chemistry. The key starting compound 3 was prepared as reported [5a], by treatment of
3
p-iodothioamide (2) with α-chloroacetylacetone. The corresponding p-alkynyl analogues 4a-j
were obtained using standard Sonogashira carbon-carbon cross-coupling conditions (Scheme 1).
Condensation of 4a-j with aminoguanidine hydrochloride or 2-hydrazino-2-imidazoline
hydrobromide yielded the final products 5-24 (Scheme 1).
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3
.2. Antibacterial Activity of New Compounds Against MRSA. Initially, the antibacterial
activity of the new analogues was examined against MRSA USA300, a strain responsible for the
majority of S. aureus skin and soft-tissue infections (SSTIs) [8], community-acquired MRSA
(CA-MRSA) infections [8], and MRSA-induced pneumonia [9]. The phenylethynyl derivative 5
inhibited MRSA USA300 at a concentration of 1 µg/mL (Table 1), similar to vancomycin (Table
1
). Replacement of the terminal phenyl ring with alicyclic moieties generated compounds 6, 7
7

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and 9 with cyclohexyl (6), cyclopentyl (7) and cyclopropyl (9) rings. These three alicyclic-
containing derivatives inhibited MRSA growth at 2 µg/mL (Table 1). Separating the terminal
alicyclic ring from the ethynyl moiety via a methylene unit provided compound 8. This
derivative was less active against MRSA USA300 (minimum inhibitory concentration (MIC)
value of 8 = 4 µg/mL, Table 1). The linear analogues, 10-12, inhibited MRSA at concentrations
ranging from 1 to 4 µg/mL (Table 1). Similarly, the antibacterial activity of the branched
analogue 13 was on par with the linear derivatives 11 and 12 (Table 1). Insertion of a polar group
within the lipophilic region (compound 14) abolished the compound’s anti-MRSA activity
(Table 1).
As we have reported before, the guanidinyl head is an essential structural element for
tphenylthiazole anti-MRSA activity [5a]; however, it is also responsible for high affinity to the
P-gp efflux system [5a]. A strategy to decrease P-gp affinity is to decrease the number of HBD’s
[10]. Therefore, hydrazino-2-imidazolinyl analogues 15-24 were synthesized, with only two
HBD’s, and their anti-MRSA activity was initially evaluated against MRSA USA300 (Table 1).
In general, all imidazolinyl analogues were at least one-fold less potent than the corresponding
guanidinyl analogues (Table 1).
Table 1. The minimum inhibitory concentration (MIC in µg/mL) and the minimum bactericidal
concentration (MBC µg/mL) of compounds and vancomycin screened against MRSA USA300.
Compound MIC MBC
5
6
7
8
9
1
2
2
4
2
1
4
4
4
64
1
4
4
4
4
8
4
8
4
1
0
1
2
3
4
1
1
1
1
64
8

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15
16
17
18
19
20
21
22
23
24
8
8
4
8
8
16
8
16
32
16
>64 >64
4
8
2
8
16
4
>64 >64
Vancomycin 1
1
Next, the antibacterial
activity of twelve derivatives
exhibiting the most potent activity against MRSA USA300 was evaluated against six additional
clinical isolates of drug-resistant S. aureus, including three vancomycin-resistant isolates (Table
2
). The compounds inhibited growth of clinical isolates (MICs ranged from 0.5 to 4 µg/mL) of S.
aureus exhibiting high-level resistance to the antibiotics mupirocin (NRS107) and linezolid
NRS119). Additionally, all compounds were significantly more potent (> 100×) than
(
vancomycin against clinical isolates of vancomycin-resistant S. aureus (VRS10, VRS11a, and
VRS12) (Table 2). For example, the pentynyl derivative 10 inhibited growth of VRS11a at a
concentration of 0.5 µg/mL. In contrast, the MIC of vancomycin against VRS11a exceeded 512
µg/mL.
Table 2. Minimum inhibitory concentration (MIC in µg/mL) and minimum bactericidal
concentration (MBC µg/mL) of active compounds screened against additional S. aureus isolates.
S. aureus
NRS107
MRSA
NRS119
MRSA
NRS123
USA400)
VRS10
(VRSA)
VRS11a
(VRSA)
VRS12
(VRSA)
(
Compound
Name
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
5
6
7
8
9
2
16
2
2
16
2
1
4
1
4
2
1
2
4
2
4
2
1
1
4
2
2
2
2
2
1
2
2
2
2
1
2
2
2
4
4
2
1
2
2
2
2
2
2
1
2
2
2
2
2
1
2
2
1
1
2
4
2
4
2
4
4
2
2
2
2
2
1
0
1
1
< 0.5
< 0.5
< 0.5
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S. aureus
NRS107
MRSA
NRS119
MRSA
NRS123
USA400)
VRS10
(VRSA)
VRS11a
(VRSA)
VRS12
(VRSA)
(
Compound
Name
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
1
1
1
1
2
2
1
2
3
7
1
3
2
4
4
8
2
1
2
4
2
4
2
4
2
2
2
4
1
0.5
2
2
2
2
2
2
4
2
4
2
2
4
16
32
32
16
1
4
4
8
4
4
16
16
2
32
32
4
32
16
2
32
32
2
16
16
4
4
8
8
8
16
16
4
16
32
8
8
64
4
8
16
4
4
4
Vancomycin
1
1
1
1
1
512
512
>512
>512
16
256
In agreement with the first- and second-generation phenylthiazoles, the new analogues
possessed minimum bactericidal concentration (MBC) values that were either identical to or up
to three-fold higher than their MIC values against MRSA and VRSA (Tables 1 and 2). This
observation indicates the new phenylthiazoles are bactericidal. To confirm this, six compounds
(5, 6, 7, 9, 10, and 11 all at 4 × MIC) were analyzed against MRSA via a time-kill assay.
As shown in Figure 2A, phenylthiazoles containing linear side chains (10 and 11)
exhibited rapid bactericidal activity against MRSA (complete eradication observed after 2 and 4
hours, respectively). On the other hand, the cyclohexyl-containing compound 6 gradually
reduced MRSA CFU/mL from 1.9-log10 (after 4 hours) to 2.8-log10 (after 12 hours) and
eventually generated a 4.3-log10 reduction in CFU/mL after 24 hours. The two alicyclic-
containing compounds 7 and 9 behaved differently (Figure 2B). Compound 7 generated a near 3-
log10 reduction in MRSA CFU/mL after 8 hours, but bacterial re-growth was observed thereafter.
Compound 9 exhibited slow bactericidal activity against MRSA, similar to vancomycin [10]
.
Both agents gradually reduced MRSA CFU/mL resulting in complete eradication of MRSA
USA400 after 24 hours. The rapid bactericidal characteristic observed with compounds 10 and
1
0

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1
1 in vitro against MRSA may be beneficial in helping to clear an infection in vivo, particularly
for invasive infections such as endocarditis [10]. From a SAR perspective: there is a strong
correlation between antibacterial kinetics and the size and spatial configuration of the lipophilic
side chain. Compound 5, consisting of the phenylethynyl tail, is bacteriostatic. Removal of the
aromaticity of the terminal phenyl ring (compound 6) improved, bactericidal activity—the rate of
bacterial clearance. Furthermore, decreasing the size of the terminal ring by substituting the
cyclohexyl group of compound 6 with the cyclopropyl group in compound 9, yielded a
compound with slow bactericidal activity. Finally, replacement of the terminal phenyl ring of 5
with a linear alkyne chain (compounds 10 and 11) changed the bacteriostatic effect observed, to
rapid bactericidal action against MRSA.
Figure 2: Time-kill analysis of compounds against MRSA A) compounds 6, 10 and 11, and
vancomycin and B) compounds 5, 7 and 9, and vancomycin against MRSA over a 24-hour
1
1

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incubation period at 37 °C. DMSO served as a negative control. The error bars represent
standard deviation values obtained from triplicate samples used for each compound/antibiotic
studied.
3
.3. Investigating the Ability of the Phenylthiazole Analogues to Clear an Intracellular
MRSA Infection. Given the alkynylphenylthiazole bactericidal activity against extracellular
MRSA, we next moved to evaluate activity against intracellular MRSA. MRSA utilizes multiple
approaches to evade the host's immune response to clearing an infection. One mechanism
involves MRSA invading, and surviving, inside host immune cells—including macrophages [13]
.
This tactic leads to recurring infections that are recalcitrant to antibiotic treatment [14], because
the antibiotics that are frequently used to treat MRSA infections, primarily vancomycin, fail to
clear intracellular MRSA. This is due, at least in part, to poor penetration into infected
macrophages [15]. In this regard, pneumonia-induced MRSA has resulted in clinical failure in
more than 40% of cases treated with the standard vancomycin dosing regimen [16]. We thus next
investigated the ability of the alkynylphenylthiazoles to penetrate and target MRSA harboring
inside macrophages (J774) (Figure 3).
We first tested compounds for toxicity to macrophage cells. After a 24-hour incubation of
compounds with J774 cells, viability was assessed via MTS assay. Compounds 6, 10, and 11
were found to be non-toxic to macrophage cells up to a concentration of 16 µg/mL, while
compounds 7 and 9 were non-toxic up to 8 µg/mL (Figure 3A,C). Next, compounds (utilizing the
highest concentration where no toxicity was observed) were evaluated for their ability to reduce
the burden of MRSA inside infected macrophages. Derivatives with linear alkyne side chains, 10
and 11, resulted in complete eradication of intracellular MRSA. In contrast, neither the alicyclic-
containing derivatives 6, 7 or 9 (4 × MIC) nor vancomycin (4 × MIC) were able to produce a
significant reduction in the burden of intracellular MRSA, in comparison to the untreated control
1
2

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(
Figure 3B). Interestingly, there appears to be a close association between the results obtained
with the time-kill assay and the ability of the compounds to clear MRSA harboring inside
macrophages. Compounds 10 and 11 exhibited rapid bactericidal activity in the time-kill assay
(completely eradicated a high inoculum of MRSA within four hours), and these two compounds
were able to clear intracellular MRSA harboring inside infected macrophages. In contrast,
compounds 6, 9, and vancomycin exhibited slow bactericidal activity in the time-kill assay (9
and vancomycin completely reduced MRSA CFU to zero only after 24 hours). The clinical
failure rate of standard vancomycin treatment of MRSA-induced pneumonia has been attributed
in part to its time-dependent, slow bactericidal activity [15, 17]. Thus, the presence of the linear
alkyne side chain in compounds 10 and 11 appears to be beneficial in two aspects: contributing
to rapid bactericidal activity against extracellular MRSA, and permitting the compounds to
penetrate and clear MRSA harboring inside infected macrophages.
1
3

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Figure 3. Toxicity analysis and examination of clearance of intracellular MRSA present in
murine macrophage (J774) cells.
A) Percent viable mammalian cells (measured as average absorbance ratio (test agent relative to
DMSO)) for cytotoxicity analysis of compounds 6, 10, and 11 (tested in triplicate) at 8, 16, and
3
2
µg/mL against J774 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. Error bars represent standard deviation values for the absorbance
values. A Student t-test with post-hoc Holm-Sidak test, (P < 0.05), determined statistical
difference between the values obtained for each compound and DMSO (denoted by the asterisk)
(P < 0.05). B) Percent reduction of MRSA USA400 colony forming units inside infected
macrophage (J774) cells after treatment with 16 µg/mL of compounds 6, 10, and 11, and with 4
µg/mL vancomycin (tested in triplicate) for 24 hours. Data was analyzed using a Student’s t-test.
Asterisks (*) indicate statistically significant difference between treatment with compounds 10
and 11 in relation to DMSO-treated wells (P < 0.05). C) Percent viable mammalian cells
(
measured as average absorbance ratio (test agent relative to DMSO)) for cytotoxicity analysis of
compounds 7 and 9 (tested in triplicate) at 8, 16, and 32 µg/mL against J774 cells using the MTS
-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
3
assay. Error bars represent standard deviation values for the absorbance values. A Student’s t-test
with post-hoc Holm-Sidak test, (P < 0.05), determined statistical difference between the values
obtained for each compound and DMSO (denoted by the asterisk) (P < 0.05). D) Percent
reduction of MRSA USA400 colony forming units inside infected macrophage (J774) cells after
treatment with 8 µg/mL of compounds 7, 9, and vancomycin (tested in triplicate) for 24 hours.
3
.4. Toxicity Assessment Using Human Colorectal Cells. After confirming the antibacterial
activity of the new phenylthiazole derivatives against drug-resistant S. aureus, we next sought to
investigate possible cytotoxicity against a human cell line (colorectal cells; HRT-18).
Compounds 5, 6, 7, 9, 10, 11, 12, 13 and 23 were found to be non-toxic up to 32 µg/mL (Figure
4
). This represents a 7- to 31-fold difference between the MIC values obtained against MRSA
and the concentration where the compounds toxic to these mammalian cells.
1
4

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Figure 4: Examination of toxicity of new phenylthiazole analogues against human
colorectal cells (HRT-18) A) Toxicity analysis of tested compounds 5, 7, 12, 13, and 23. B)
Toxicity analysis of compounds 6, 9, 10, and 11.
3
.5. Mechanism of Action of 9. In order to determine whether the new compounds have the
same mechanism of antibacterial action to that of the original lead thiazole compound (1a),
compound 9 was selected for further analysis. In previous work, [6] we found that 1a was an
inhibitor of bacterial cell wall synthesis, inhibiting both undecaprenyl diphosphate phosphatase
(
IC = 6 µM) and undecaprenyl diphosphate synthase (IC = 19 µM), in addition to being a
5
0
50
weak protonophore uncoupler (in inverted membrane vesicles; IC50 ~ 12-25 µM). To determine
to what extent 9 had similar effects, we used the E. coli UPPP expression system described
previously and determined the IC50 for inhibition by 9 [18]. We found an 8.1 ꢀM IC50
(corresponding to 2.7 ꢀg/mL), consistent with a UPPP target, Figure 5A. Under these assay
conditions, the antibiotic bacitracin has a 5.6 µM IC (1.9 µg/mL), as reported previously [6],
5
0
but shown again here for convenience (Figure 5A). We also tested for human FPPS (HsFPPS)
and E. coli UPPS (EcUPPS) inhibition. There was no inhibition of HsFPPS (data not shown), but
UPPS was inhibited with a 38 ꢀM IC (corresponding to 13 ꢀg/mL), Figure 5B, weaker than
5
0
known potent UPPS inhibitors, such as NSC-50460 (Figure 5B). Since UPPS and UPPP are
1
5

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adjacent to each other in the cell wall biosynthetic pathway, such multi-target inhibition may
nevetheless contribute to the activity of 9, similar to the dual UPPS/UPPP inhibition we have
reported with other compounds [25].
To determine whether 9 is a protonophore uncoupler, we used the E. coli inverted
membrane vesicle (IMV) system, again as previously reported [6]. Results with compound 9 and
the potent, known uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone) are shown in
2
Figure 5 (panels C and D) with both ATP-powered PMF generation as well as succinate/O -
powered PMF generation. The IMVs have their ATPase on the outside of the vesicle so ATP
+
hydrolysis through the ATPase, or succinate/O
2
, drives H into the vesicles, the fluorophore
ACMA (9-amino-6-chloro-2-methoxyacridine) accumulates, and its fluorescence is self-
quenched (the signal goes down). Addition of CCCP or 9 collapses the PMF and fluorescence
increases (back to normal). For CCCP, the EC is 0.47 ꢀM in ATP and 0.21 ꢀM in succinate;
5
0
for 9, the EC50 for PMF collapse is 24 ꢀM in ATP and 28 ꢀM in succinate, Figure 5C,D
corresponding to 8 and 9.5 µg/mL, respectively). This is relatively weak uncoupling, but could
lead to inhibition of some transporters, including drug efflux pumps.
(
1
6

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Figure 5. Dose response curves for enzyme inhibition by 9 and its effects on the PMF. A) UPPP
(YubB) inhibition; bacitracin control. B) UPPS inhibition, bisamidine NSC-50460 control. (C)
PMF collapse in E. coli IMVs, ATP driven PMF, CCCP control. (D) As C but succinate/O2-
driven PMF generation.
3
.6. Resensitization of VRSA to the Effect of Vancomycin. Vancomycin is a valuable
therapeutic agent for treatment of drug-resistant staphylococcal infections. However, the
emergence of clinical isolates exhibiting resistance to vancomycin is of great concern. One
approach we have investigated in order to try and counter vancomycin resistance is to use small
molecules to resensitize VRSA to the effect of vancomycin. This approach has shown promise in
vitro, with small molecules such as the phenylthiazole analogues [7, 19] that target the early
steps in cell wall biosynthesis, formation of undecaprenyl phosphate. We thus next investigated
1
7

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whether the new phenylthiazoles would be capable of re-sensitizing VRSA to the effect of
vancomycin (which acts downstream of UPPS and UPPP). We tested compounds 5 (with a
phenyl side- chain), 7 (with an alicyclic side-chain) and 9 (with linear side-chain), at sub-
inhibitory concentration (½ × MIC) in combination with vancomycin against a clinical isolate of
S. aureus (VRS10) that exhibits high-level resistance to vancomycin (MIC of 512 µg/mL). All
three compounds were effective at sensitizing VRS10 to the effect of vancomycin, producing a
5
12-fold improvement in vancomycin’s MIC value (Table 3), opening up the possibility that
these or related alkynylphenylthiazoles might serve as partners to vancomycin in combating
extremely drug-resistant staphylococcal infections.
Table 3. The minimum inhibitory concentration (MIC, in µg/mL) of vancomycin (VAN) in the
absence and presence of a sub-inhibitory concentration (½ × MIC) of tested compounds against
vancomycin-resistant S. aureus (VRS10).
VRSA
strain
MIC of VAN
MIC of
Fold re-
MIC of
Fold re-
MIC of
Fold re-
VAN + 5 sensitization VAN + 7 sensitization VAN + 9 sensitization
of VRSA to
vancomycin
512-fold
of VRSA to
vancomycin
512-fold
of VRSA to
vancomycin
512-fold
VRS10
512
1
1
1
3
.7. In vitro Pharmacokinetic Profiling. Nearly 90% of drugs currently in the drug
discovery/development pipeline exhibit poor physicochemical properties such as limited aqueous
solubility, poor permeability, or both [20]. The first-generation phenylthiazoles indeed exhibited
a poor physicochemical profile, including limited ability to cross the gastrointestinal tract (as
evaluated via the Caco-2 bidirectional permeability assay), due in large part to being a substrate
for the P-gp efflux system [5a]. Additionally, first-generation phenylthiazoles containing the n-
alkyl lipophilic tail were found to be substrates for CYP450. This resulted in very short half-lives
and high clearance rates (t1/2 of 1a is below 30 minutes, Figure 1) [5a]. The only exception
among all first-generation phenylthiazoles was the hexynyl analogue (1b) that possessed a longer
1
8

ACCEPTED MANUSCRIPT
t1/2 and lower clearance rate (Figure 1), but 1b was not effective in treating a systemic MRSA
infection in vivo [5c, 21].
To examine if the modifications made in the new series of phenylthiazoles improved the
permeability and metabolic stability profiles, compound 9 and its imidazolinyl analogue 19 were
selected for further analysis. Initially, the permeability profile of the compounds was evaluated
via the Caco-2 bidirectional permeability assay. Both compounds 9 and 19 showed low
permeability through Caco-2 cells, comparable to the well-known H2 antagonist ranitidine
(Table 4). The guanidinyl derivative 9 was a good substrate for P-gp, with an efflux ratio of 69.
Table 4. Caco-2 bidirectional permeability analysis for compounds 9 and 19.
Test
Assay
Mean
B P
(10
Mean
A P
(10
Efflux ratio
(Papp(B→A)
/Papp(A→B)
P-gp
substrate
Notes
compound Duration A
→
B
→
app
app
-
6
-6
(
minutes)
cm/sec)
0.13
0.27
cm/sec)
5.54
1.71
Colchicine
Ranitidine
60
60
44.30
6.30
Yes
Yes
Poor-
permeability
control
Labetolol
Propranolol
9
60
60
8.81
35.05
42.44
Yes
No
Moderate-
permeability
control
High-
permeability
control
Low
permeability
Low
24.99
1.70
60
60
0.05
0.13
0.35
0.13
69
Yes
No
19
0.96
permeability
(Table 4). Interestingly, the imidazolinyl analogue 19, which contains of fewer hydrogen bond
donor (HBD) groups, was found to not be a P-gp substrate (efflux ratio < 1, similar to the control
drug propranolol). Compound 19 demonstrated similar rates of permeation in both directions:
from apical (A) to basolateral (B), and vice-versa (Table 4). This result suggests that neither
1
9

ACCEPTED MANUSCRIPT
compound would be suitable to be administered orally, given their slow rate of permeation
across the GI tract (simulated by the Caco-2 monolayer). Intravenous infusion of these
compounds may be an alternative option, however, depending on the metabolic stability, which
we thus investigated using pooled human liver microsomes. Both compounds 9 and 19 were
found to have a markedly improved stability to hepatic metabolism (with a clearance rate that
was < 115.5 µL/min-mg) compared to the first-generation phenylthiazoles. This resulted in
noticeably longer half-lives for compounds 9 and 19 of 4.8 and 8 hours, respectively (Table 5).
Table 5. Metabolic stability analysis for compounds 9 and 19 in HLM.
Test
Article
Test
Concentration (µL/min-mg)
M)
Mean CLint
Mean t1/2
(minutes)
Notes
(µ
Terfenadine
Verapamil
Propranolol
Imipramine
9
0.1
0.1
0.1
0.1
0.1
0.1
774.0
8.95
High clearance
control
High clearance
control
Low clearance
control
Low clearance
control
286.6
24.20
< 115.5
<115.5
< 115.5
<115.5
143.80
87.20
292.00
476.30
Stable to
metabolism
Stable to
1
9
metabolism
3
.8. In vivo Pharmacokinetic Profiling. To further confirm that the chemical modifications
made to the new generation of phenylthiazoles positively impacted their pharmacokinetic profile,
naïve Sprague−Dawley rats were intravenously dosed with compound 9. The results obtained
when fit within a two-compartment model indicate a biological half-life of > 4 hours (Table 6),
in agreement with the hepatic microsomal stability result described above. Taken together, these
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results would permit a twice-daily dosing regimen to be used for compound 9, if administered
intravenously.
Table 6. In vivo PK parameters of compound 9 in rat after single IV bolus injection.
t1/2
h)
4.43
CL
(L/hr)
5.36
AUC
mg.hr/L
V
(L)
18.46

Vdss
(L)
5.51
(
9
0.69
nd
t : half-life; CL: clearance; Vb: volume of distribution in 2 compartment (peripheral tissues); Vdss: volume of
1
/2
distribution at the steady state
3
.9. Investigating the Anti-MRSA Activity of 9 and 11 in a C. elegans Model of MRSA
Infection. The improvement in stability to hepatic metabolism observed with compound 9 led us
to investigate its effect in treating in vivo MRSA infections. Compound 9, and the rapidly-
bactericidal analogue 11, were initially evaluated in a C. elegans model of MRSA infection. The
C. elegans animal model is an established system for investigating the efficacy of antibacterial
agents in vivo in the early stages of drug discovery [22]. C. elegans is a useful model as it
expresses cytochrome P450 enzymes that are homologous to mammalian enzymes [22].
Therefore, it permits the investigation of the pharmacodynamic effect of rapidly metabolized
xenobiotics, prior to evaluating molecules in higher-order animal models. To examine the
efficacy of the alkynyl-containing phenylthiazoles to reduce the burden of MRSA in infected C.
elegans, worms were treated with compounds 9 (alicyclic-containing derivative) or 11 (linear
side chain analogue). As shown in Figure 6, both compounds were as potent as vancomycin, and
they reduced the burden of MRSA USA300 in infected C. elegans by ~90% (Figure 6). This
value is almost nine times higher than the initial lead compound 1b [24], indicating that the
modifications made in this series of phenylthiazoles resulted in significantly improved efficacy.
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1

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Figure 6. Antibacterial activity of 9, 11, and vancomycin in vivo against MRSA-infected C.
elegans. Examination of the antibacterial activity of test agents (at 20 µg/mL) in C. elegans
AU37 infected with methicillin-resistant Staphylococcus aureus USA300. Vancomycin served as
a positive control. Worms (in L4 stage of growth) were infected with bacteria for six hours
before transferring 25-30 worms to wells of a 96-well plate. Test agents were added and
incubated with worms for 18 hours. Worms were sacrificed and the number of viable colony-
forming units of MRSA USA300 in infected worms was determined for each treatment regimen.
The figure presents the average percent reduction of MRSA USA300 (relative to worms
receiving the negative control, PBS) for each treatment. A one-way ANOVA with post-hoc
Dunnet’s multiple comparisons found statistical difference between worms treated with
compounds 9, 11, or vancomycin relative to the negative control (vehicle alone) (*, P < 0.05).
3
.10. MRSA Murine Skin Infection. MRSA is a leading cause of uncomplicated (simple
abscess) and complicated (infected burns and foot ulcers) skin infections in humans [25].
Treatment of uncomplicated skin infections often involves multiple doses of a topical antibiotic
agent, such as fusidic acid or mupirocin [26]. Given the promising result obtained from the C.
elegans study, we thus next moved to evaluate the effectiveness of compounds 9 and 11 in mice
using an established MRSA skin infection model. Mice were injected intradermally with MRSA
USA300, and an abscess formed near the injection site three days after infection. The wounds
were subsequently treated twice daily (for five consecutive days) with a topical suspension of
2
2

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either compounds 9, 11, or fusidic acid. The wounds present in one group of mice were treated
with the vehicle (petroleum jelly) alone, for comparison. Compounds 9 (0.61-log , 73 %
1
0
reduction) and 11 (0.58-log , 71% reduction) were found to be about as effective as the control
1
0
antibiotic fusidic acid (0.71-log , 78% reduction) in reducing the burden of MRSA in the
1
0
wounds of infected mice (compared to mice treated with the vehicle alone) (Figure 7). There was
no excess inflammation (redness or swelling around the wound site) or toxicity observed in
wounds after exposure to the compounds, or fusidic acid.
Figure 7. Reduction of MRSA USA300 in infected skin wounds of mice. Mice (n = 5 per
group) received an intradermal injection of MRSA USA300. After formation of an abscess at the
site of injection, wounds were treated twice daily (for five days) with a 2% suspension of
compounds 9, 11, or fusidic acid (using petroleum jelly as the vehicle). Twelve hours after
administering the last dose, mice were euthanized, wounds extracted and homogenized to
determine the presence of MRSA in wounds. The figure presents the average percent reduction
of MRSA CFU/mL in murine skin lesions. A one-way ANOVA with post-hoc Dunnet’s multiple
comparisons found statistical difference between mice treated with compounds 9, 11, or fusidic
acid compared to mice receiving the vehicle (petroleum jelly) alone (*, P < 0.05).
2
3

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3
.11. Neutropenic Murine Thigh Infection Experiment. Encouraged by the previous results in
C. elegans and the uncomplicated murine skin wound model, in addition to the in vivo PK study,
compound 9 was tested in a systemic murine model of MRSA soft-tissue infection. Mice
6
received an intramuscular injection of MRSA USA300 (5.35 × 10 CFU) in the right thigh. Each
group of mice were treated twice (2 and 12 hours post-infection) with compound 9, or
vancomycin. Mice receiving the vehicle alone served as the negative control. Mice were
humanely euthanized (24 hours post-infection) and the infected thighs were aseptically removed
in order to count MRSA CFU post-treatment. Compound 9 (0.71-log10, 77% reduction) was
slightly more effective than the control antibiotic vancomycin (0.46-log10, 66% reduction), the
drug of choice for treatment of invasive MRSA infections, in reducing the burden of MRSA in
the thigh of the infected mice (Figure 8). Importantly, compound 9 (20 mg/kg) achieved its effect
at 40% the dose of vancomycin (50 mg/kg), further confirming the potent anti-MRSA activity of
compound 9, and its stability.
Figure 8: Reduction of MRSA USA300 in infected thigh of mice after treatment with
compound 9 or vancomycin. Average percent reduction of MRSA CFU/g tissue in murine right
thigh after treatment (two doses per day). A one-way ANOVA with post-hoc Dunnet’s multiple
2
4

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comparisons found statistical significance (*, P < 0.05) between mice treated with compound 9
and vancomycin compared to mice receiving the vehicle (10% DMSO, 10% Tween 80, 80%
PBS) alone.
4
. CONCLUSIONS
The results we have described here are of general interest since we have developed new
phenylthiazole compounds that have good activity both in vitro and in vivo against methicillin-
resistant Staphylococcus aureus. The most promising compounds killed MRSA in vitro at
concentrations as low as 0.5
µg/mL, and were active against extracellular as well as intracellular
(macrophage resident) bacteria. They act by targeting the enzymes undecaprenyl diphosphate
synthase and undecaprenyl diphosphate phosphatase, involved in bacterial cell wall biosynthesis.
Compound 9 was of particular interest since it had good resistance to microsomal P450 enzymes,
and was active against MRSA in three animal models: C. elegans, a murine skin infection model,
and a neutropenic murine thigh-infection model. In mice, compound 9 demonstrated comparable
potency to vancomycin and at lower doses, and thus represents a new lead for the treatment of
MRSA infections.
5
5
5
. EXPERIMENTAL METHODS
.1. Chemistry
1
13
.1.1. General. H NMR spectra were run at 400 MHz and C spectra were determined at 100
MHz in deuterated chloroform (CDCl ), or dimethyl sulfoxide (DMSO-d ) on a Varian Mercury
3
6
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
2
5

ACCEPTED MANUSCRIPT
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. 1-(4-Methyl-2-(4-substituted alkynyl) phenyl) thiazol-5-yl) ethanone 4a-j.
General procedure: To dry DMF (5 mL) in a 25-mL sealed tube, compound 3 (500 mg, 1.45
mmol), appropriate alkynes (2.91 mmol), triethylamine (1 mL), and cesium carbonate (947 mg,
2
.91 mmol) were added. After the reaction mixture was purged with dry nitrogen gas for 20
minutes, dichloro-bis(triphenylphosphine)palladium (II) (52 mg, 0.072 mmol) and copper(I)
iodide (28 mg, 0.145 mmol) were added. The sealed tube was then placed in an oil bath and
o
stirred at 65 C for 16 hours. After cooling to room temperature, the reaction mixture was passed
through celite, followed by chloroform (2×50 mL). The organic materials were then concentrated
under reduced pressure. The crude materials were purified and washed via silica gel flash
column chromatography using hexane-ethyl acetate (9:1). Yields, physical properties, and
spectral data of isolated purified products are listed below:
5
.1.2.1. 1-(4-Methyl-2-(4-phenylethynyl)phenyl)thiazol-5-yl)ethanone (4a). Yellow solid
1
(
200 mg, 43%) mp = 125 °C. H NMR (CDCl )
δ: 7.97 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8 Hz,
3
2
H), 7.55 (d, J = 6.4 Hz, 2H), 7.37 (d, J = 6.8 Hz, 2H), 7.36 (t, J = 8 Hz, 1H), 2.79 (s, 3H), 2.57
13
(
s, 3H); C NMR (CDCl )
δ
: 190.40, 168.45, 159.62, 132.22, 132.19, 131.68, 131.49, 128.68,
3
1
28.10, 127.64, 126.19, 122.78, 92.20, 88.79, 29.65, 18.64; Anal. Calc. for: C H NOS (317):
2
0
15
C, 75.68; H, 4.76; N, 4.41%; Found: C, 75.69; H, 4.75; N, 4.40%.
2
6

ACCEPTED MANUSCRIPT
5
.1.2.2.
1-(2-(4-(Cyclohexylethynyl)phenyl)-4-methylthiazol-5-yl)ethanone
(4b).
1
Yellowish oil (420 mg, 89%). H NMR (CDCl )
δ: 7.90 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz,
3
1
3
2
H), 2.77 (s, 3H), 2.64 (p, J = 8.8 Hz, 1H), 2.56 (s, 3H), 1.90-1.25 (m, 10H); C NMR (CDCl )
3
δ
: 190.40, 186.75, 159.56, 132.18, 131.46, 131.27, 127.23, 126.60, 97.73, 80.13, 32.55, 30.77,
2
9.68, 25.86, 24.88, 18.43; Anal. Calc. for: C H NOS (323): C, 74.27; H, 6.54; N, 4.33%;
20 21
Found: C, 74.25; H, 6.53; N, 4.31%.
5
.1.2.3. 1-(2-(4-(Cyclopentylethynyl)phenyl)-4-methylthiazol-5-yl)ethanone (4c). Orange oil
1
(
400 mg, 89%). H NMR (CDCl )
δ: 7.89 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8 Hz, 2H), 2.84 (m, 1H), 2.76
3
(
s, 3H), 2.55 (s, 3H). 1.77-1.24 (m, 8H); Anal. Calc. for: C H NOS (309): C, 73.75; H, 6.19; N, 4.53%;
19
19
Found: C, 73.73; H, 6.20; N, 4.52%.
5
.1.2.3. 1-(2-(4-(Cyclopentylprop-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethanone (4d).
1
Orange oil (350 mg, 74%). H NMR (CDCl
3
)
δ
: 7.97 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 8.7 Hz,
2
8
H), 2.70 (s, 3H), 2.57 (s, 3H), 2.49 (dd, J = 1.8 Hz, J = 6.6 Hz, 2H), 1.79 (m, 1H), 1.56-1.30 (m,
1
3
H); C NMR (CDCl3)
δ: 190.40, 168.96, 159.55, 132.14, 131.42, 131.35, 127.28, 126.59, 97.95, 79.69,
3
4
3.80, 30.27, 25.07, 20.92, 18.41, 13.64; Anal. Calc. for: C H NOS (323): C, 74.27; H, 6.54; N,
20
21
.33%; Found: C, 74.28; H, 6.52; N, 4.32%.
5
.1.2.4. 1-(2-(4-(Cyclopropylethynyl)phenyl)-4-methylthiazol-5-yl)ethanone (4e). Pale
1
yellow oil (360 mg, 88%). H NMR (CDCl )
δ
: 7.89 (d, J = 8 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H),
3
2
2
.77 (s, 3H), 2.56 (s, 3H), 1.64 (p, J = 6.8 Hz, 1H), 1.49 (q, J = 8.4 Hz, 2H), 1.39 (q, J = 5.2 Hz,
1
3
H); C NMR (CDCl
9.04, 13.64, 8.72, 0.28; Anal. Calc. for: C17
C, 72.56; H, 5.38; N, 4.99%.
3
)
δ
: 190.42, 168.71, 159.57, 132.15, 131.48, 131.35, 127.02, 126.63, 96.80, 78.15,
1
H15NOS (281): C, 72.57; H, 5.37; N, 4.98%; Found:
5
.1.2.5. 1-(4-Methyl-2-(4-(pent-1-yn-1-yl)phenyl)thiazol-5-yl)ethanone (4f). Yellow oil
1
(
370 mg, 90%). H NMR (CDCl )
δ
: 7.91 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 2.77 (s,
3
2
7

ACCEPTED MANUSCRIPT
1
3
3
H), 2.56 (s, 3H), 2.43 (t, J = 6.9 Hz, 2H), 1.69 (m, 2H), 1.07 (t, J = 7.8 Hz, 3H); C NMR
CDCl : 190.42, 168.72, 159.56, 132.16, 131.54, 131.31, 127.15, 126.64, 93.56, 80.30, 30.76, 22.07,
1.51, 18.42, 13.64; Anal. Calc. for: C H NOS (283): C, 72.05; H, 6.05; N, 4.94%; Found: C,
(
3
) δ
2
7
1
7
17
2.06; H, 6.07; N, 4.95%.
5
.1.2.6. 1-(2-(4-(Hept-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethanone (4g). Yellow oil
1
(
350 mg, 78%). H NMR (CDCl )
δ: 7.91 (d, J = 8 Hz, 2H), 7.47 (d, J = 8 Hz, 2H), 2.77 (s, 3H),
3
2
3
.56 (s, 3H), 2.40 (t, J = 7.2 Hz, 2H), 1.64 (p, J = 6.8 Hz, 2H), 1.47 (m, 4H), 0.90 (t, J = 5.6 Hz
1
3
H); C NMR (CDCl3)
δ: 190.40, 168.72, 159.56, 132.14, 131.52, 131.30, 127.18, 126.64, 93.81, 80.15,
3
1.33, 29.68, 28.61, 28.57, 22.53, 19.55, 18.42, 14.04; Anal. Calc. for: C H NOS (311): C, 73.27;
1
9
21
H, 6.80; N, 4.50%; Found: C, 73.26; H, 6.81; N, 4.52%.
5
.1.2.7. 1-(4-Methyl-2-(4-(oct-1-yn-1-yl)phenyl)thiazol-5-yl)ethanone (4h). Yellow oil (385
1
mg, 81%). H NMR (CDCl
3
)
δ
: 7.90 (d, J = 8 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 2.77 (s, 3H),
2
3
.56 (s, 3H), 2.44 (t, J = 7.2 Hz, 2H), 1.65 (p, J = 7.2 Hz, 2H), 1.47 (m, 6H), 0.94 (t, J = 9.6 Hz
1
3
H); C NMR (CDCl3)
δ: 190.40, 168.72, 159.56, 132.14, 131.52, 131.30, 127.18, 126.64, 93.81, 80.15,
3
7
1.33, 30.53, 29.68, 28.61, 28.57, 22.53, 19.55, 18.42, 14.04; Anal. Calc. for: C H NOS (325): C,
20
23
3.81; H, 7.12; N, 4.30%; Found: C, 73.82; H, 7.13; N, 4.29%.
5
.1.2.8. 1-(-2-[4-(3,3-Dimethylbut-1-yn-1-yl)phenyl]-4-methylthiazol-5-yl)ethanone (4i).
1
Faint yellow oil (351 mg, 80%). H NMR (CDCl )
δ
: 7.90 (d, J = 8 Hz, 2H), 7.45 (d, J = 7.6 Hz,
3
1
3
2
1
H), 2.77 (s, 3H), 2.56 (s, 3H), 1.33 (s, 9H); C NMR (CDCl
3
) δ: 190.41, 168.97, 159.53,
32.17, 131.42, 131.26, 127.20, 126.59, 101.72, 78.67, 30.88, 19.04, 18.43, 13.65; Anal. Calc.
for: C H NOS (297): C, 72.69; H, 6.44; N, 4.71%; Found: C, 72.68; H, 6.46; N, 4.70%.
1
8
19
5
.1.2.9. 1-(2-(4-(3-Amino-3-methylbut-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethanone
1
(
4j). Faint yellow oil (300 mg, 69%). H NMR (CDCl )
δ: 8.01 (brs, 2H), 7.91 (d, J = 8 Hz, 2H),
3
1
3
7
.46 (d, J = 8.4 Hz, 2H), 2.94 (s, 3H), 2.87 (s, 3H), 2.77 (s, 3H), 2.56 (s, 3H); C NMR (CDCl )
3
2
8