Balancing Physicochemical Properties of Phenylthiazole Compounds with Antibacterial Potency by Modifying the Lipophilic Side Chain

Article abstract of DOI:10.1021/acsinfecdis.9b00211

Bacterial resistance to antibiotics is presently one of the most pressing healthcare challenges and necessitates the discovery of new antibacterials with unique chemical scaffolds. However, the determination of the optimal balance between structural requirements for pharmacological action and pharmacokinetic properties of novel antibacterial compounds is a significant challenge in drug development. The incorporation of lipophilic moieties within a compound's core structure can enhance biological activity but have a deleterious effect on drug-like properties. In this Article, the lipophilicity of alkynylphenylthiazoles, previously identified as novel antibacterial agents, was reduced by introducing cyclic amines to the lipophilic side chain. In this regard, substitution with methylpiperidine (compounds 14-16) and thiomorpholine (compound 19) substituents significantly enhanced the aqueous solubility profile of the new compounds more than 150-fold compared to the first-generation lead compound 1b. Consequently, the pharmacokinetic profile of compound 15 was significantly enhanced with a notable improvement in both half-life and the time the compound's plasma concentration remained above its minimum inhibitory concentration (MIC) against methicillin-resistant Staphylococcus aureus (MRSA). In addition, compounds 14-16 and 19 were found to exert a bactericidal mode of action against MRSA and were not susceptible to resistance formation after 14 serial passages. Moreover, these compounds (at 2× MIC) were superior to the antibiotic vancomycin in the disruption of the mature MRSA biofilm. The modifications to the alkynylphenylthiazoles reported herein successfully improved the pharmacokinetic profile of this new series while maintaining the compounds' biological activity against MRSA.

Full text of DOI:10.1021/acsinfecdis.9b00211

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Article
Balancing Physicochemical Properties of Phenylthiazole Compounds
with Antibacterial Potency by Modifying the Lipophilic Side Chain
Ahmed S. Mancy, Nader S. Abutaleb, Mohamed Elsebaei, Abdallah Yossef saad, Ahmed Kotb, Alsagher
Ali, Jelan A. Abdel-Aleem, Haroon Mohammad, Mohamed N. Seleem, and Abdelrahman S Mayhoub
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00211 • Publication Date (Web): 13 Nov 2019
Downloaded from pubs.acs.org on November 15, 2019
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Balancing Physicochemical Properties of Phenylthiazole Compounds with Antibacterial
Potency by Modifying the Lipophilic Side Chain
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Ahmed Mancya‖, Nader S. Abutalebb‖, Mohamed M. Elsebaeia, Abdullah Y. Saada, Ahmed Kotba,
Alsagher O. Alib,c, Jelan A. Abdel-Aleemb,d, Haroon Mohammadb Mohamed N. Seleemb,e** and
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,
Abdelrahman S. Mayhouba,f*
a.
Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar
University, 1-Elmokhayem Eldaem Streat, Cairo 11884, Egypt.
b.
Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue
University, 725 Harrison Streat, West Lafayette, IN 47907, USA.
c.
Division of Infectious Diseases, Animal Medicine Department, Faculty of Veterinary
Medicine, South Valley University, Qena 83523, Egypt.
d.
Department of Industrial Pharmacy, Faculty of Pharmacy, Assiut University, Assiut
71515, Egypt.
e.
Purdue Institute of Inflammation, Immunology, and Infectious Disease, West
Lafayette, IN 47907, USA.
f.
University of Science and Technology, Nanoscience Program, Zewail City of
Science and Technology, Ahmed Zewail Streat, October Gardens, 6th of October, Giza
12578, Egypt.
Corresponding Authors. *e-mail: mseleem@purdue.edu,
amayhoub@azhar.edu.eg
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Bacterial resistance to antibiotics is presently one of the most pressing healthcare challenges and
necessitates discovering new antibacterials with unique chemical scaffolds. However, determining
the optimal balance between structural requirements for pharmacological action and
pharmacokinetic properties of novel antibacterial compounds is a significant challenge in drug
development. The incorporation of lipophilic moieties within a compound’s core structure can
enhance biological activity but have a deleterious effect on drug-like properties. In this article, the
lipophilicity of alkynylphenylthiazoles, previously identified as novel antibacterial agents, was
reduced by introducing cyclic amines to the lipophilic side chain. In this regard, substitution with
methylpiperidine (compounds 14-16) and thiomorpholine (compound 19) substituents
significantly enhanced the aqueous solubility profile of the new compounds more than 150-fold
compared to the first-generation lead compound 1b. Consequently, the pharmacokinetic profile of
compound 15 was significantly enhanced with a notable improvement both in half-life and the
time the compound’s plasma concentration remained above its minimum inhibitory concentration
(MIC) against methicillin-resistant Staphylococcus aureus (MRSA). In addition, compounds 14-
16 and 19 were found to exert a bactericidal mode of action against MRSA and were not
susceptible to resistance formation after 14 serial passages. Moreover, these compounds (at 2 ×
MIC) were superior to the antibiotic vancomycin in disrupting mature MRSA biofilm. The
modifications to the alkynylphenylthiazoles reported herein successfully improved the
pharmacokinetic profile of this new series while maintaining the compounds’ biological activity
against MRSA.
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Key words: antibiotic resistance; MRSA; VRSA; Suzuki coupling; anti-biofilm.
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Antibiotics are a cornerstone of medical treatment for various bacterial infections and are
prescribed at a rate that exceeds the limit of 800 per 1000 people1. Therefore, the emergence of
bacterial resistance to antibiotics is a notable threat to modern medicine. Though more than 40
antibacterial agents are currently undergoing investigation in clinical trials, many are derivatives
of existing antibiotic classes. The low rate of success (~20%) for infectious disease agents to
receive regulatory approval necessitates the continuous discovery and development of new
antibiotic scaffolds.
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The antimicrobial activity of thiazole-based scaffolds was very well-reported.2-8 In an
intensive effort to identify new antibacterial agents, our group focused on studying the antibacterial
activity of phenylthiazoles as a subclass of thiazoles antimicrobials. These compounds inhibited
growth of multidrug-resistant bacterial species of clinical interest, including methicillin-resistant
S. aureus, including inhibition of staphylococcal biofilm formation, penicillin-resistant
Streptococcus pneumoniae, and vancomycin-resistant enterococci; they exhibited excellent
antivirulent properties9-18. More importantly, bacterial mutants exhibiting resistance to these
phenylthiazoles could not be isolated using multiple approaches. However, a drawback of the first-
generation compounds was their poor physicochemical profile (in particular limited aqueous
solubility and rapid hepatic metabolism) which hindered their investigation in appropriate pre-
clinical animal models of bacterial infection.
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Progress in Development of
Phenylazole antibiotics
Reported strategy to overcome
limited aqueous solubility
N
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S
N
NH
HN
CH3
NH2
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OCH3
HN
1a
N
1st wave of structure optimization:
Rigidifying & increasing the size of
lipophilic side chain
N
OCH3
Lead quinazoline I
F
Acetylene linker
provided
metabolic
stability
N
S
N
NH
HN
Cl
O
O
N
NH2
NH
N
N
N
HN
N
Ionizable group
to enhance
water solubility &
PK profile
NH2
N
OCH3
HN
Guanidine is essential
for antibacterial effect
Gefitinib
1b
1c
R
Adopting the same strategy
Ionizable group
to enhance
water solubility &
PK profile
X
N
acetylene linker
provided metabolic
stability
n
Figure 1. Reported chemical optimization to improve physicochemical properties of the quinoline
epidermal growth factor receptor inhibitor gefitinib19 and schematic representation of the aim of
the current report with the phenylthiazole compounds.
Identifying the correct balance between excellent physicochemical properties and the
desired pharmacological effect for small molecules is a consistent challenge in drug discovery. In
most cases, the structural motifs required to enhance the biological activity of a compound have a
negative effect on its physicochemical characteristics. In the quest to improve the antibacterial
potency of the first phenylthiazole lead compound 1a, lipophilic moieties were added to the para-
position of the phenyl ring (Figure 1). This tactic successfully enhanced the antibacterial potency
of the second-generation compounds and resulted in derivatives with similar in vitro potency
against MRSA to frontline therapeutics such as vancomycin and linezolid. However, the
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modifications made had a profoundly deleterious effect on the physicochemical properties,
especially aqueous solubility, of these second-generation compounds.11, 20 This problem was
partially solved by replacing the central thiazole nucleus with a more polar pyrazole (structure 1c,
Figure 1).21 In this report, an alternative strategy was adopted in which we kept the essential
structural elements (guanidine head and acetylene linker) tethered to the phenylthiazole core to
maintain the positive attributes previously studied and inserted a cyclic nitrogen group within the
lipophilic side chain (Figure 1). The addition of an ionizable/polar moiety to the active scaffold
has been used by medicinal chemists to enhance the overall aqueous solubility and
pharmacokinetic profile of promising small molecules.19 A successful example of this strategy is
the synthesis of the anticancer drug gefitinib, from the lead quinazoline I,19 highlighted in Figure
1.
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RESULTS AND DISCUSSION
Chemistry. Thioamide starting material 2 was prepared as previously reported.13 A shorter
synthetic pathway to tether the acetylenic part was first tried by allowing compound 3 to react with
propargyl bromide under standard Sonogashira conditions (Scheme 1). However, the desired
compound 5 could not be isolated from the massively produced by-products. Alternatively, N-
propargylcyclic amines 6a-n were prepared first and then allowed to react with iodophenylthiazole
intermediate 3 under the same standard Sonogashira conditions to yield compounds 7a-n.
Subsequent reaction with aminoguanidine in the presence of a catalytic amount of hydrochloric
acid provided the desired final products 8-21 (Scheme 1).
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Scheme 1
O
S
5
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N
S
NH2
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a
b
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c
R
Br
HN
+
R
6a-n
I
I
2
3
4a-n
Br
NH2
b
O
S
HN
N
NH
O
S
N
N
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N
4a-n
d
c
N
N
Br
R
R
5
HN
7a-n
8-21
R
HN
HN
R
HN
R
R
N
N
N
N
N
4a, 6a, 7a, 8
4b, 6b, 7b, 9
4c, 6c, 7c, 10
4d, 6d, 7d, 11
4e, 6e, 7e, 12
4f, 6f, 7f, 13
4g, 6g, 7g, 14
4h, 6h, 7h, 15
4i, 6i, 7i, 16
4j, 6j, 7j, 17
4k, 6k, 7k, 18
4l, 6l, 7l, 19
O
S
N
HO
N
N
N
N
4m, 6m, 7m, 20
4n, 6n, 7n, 21
N
N
N
S
N
N
N
Reagents and conditions
CuI (7.5% mol), Et3N, DME, heat at 50°C for 24 h in sealed flask; (c) anhydrous K2CO3, DMF,
heat at 110°C for 4 h; (d) aminoguanidine HCl, EtOH, conc. HCl, heat to reflux, 3 h.
: (a) Absolute EtOH, heat to reflux, 6 h, (b) PdCl2(PPh3)2 (5% mol),
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Biological Results and Discussion.
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Antibacterial evaluation of new derivatives against MRSA. The newly-synthesized
phenylthiazole derivatives were initially screened against MRSA USA300, which is a significant
source of MRSA skin and soft tissue infections (SSTIs) worldwide.22 The phenylthiazole
derivatives were also screened against an Escherichia coli strain (JW55031) deficient in the
AcrAB-TolC efflux pump, to investigate the compounds’ activity against Gram-negative bacteria.
The initial screening against MRSA USA300 revealed that smaller nitrogenous rings
resulted in compounds with weaker antibacterial activity (Table 1). This activity was slightly
improved upon using pyrrolidine (compound 10, MIC = 32 g/mL) and was significantly enhanced
with 6-membered nitrogenous rings. In this vein, the piperidine-containing derivative 13 (MIC =
8 g/mL) was four-times more potent than the pyrrolidine-containing derivative 10. Methylation
of the piperidine ring in position-2, -3 or -4 had a positive impact on anti-MRSA activity as the
MIC value of compounds 14-16 were one-fold lower than the corresponding unsubstituted
piperidine-containing derivative 13. Interestingly, our attempts to further enhance the polarity of
the side chain by embedding a second polar atom within the terminal side chain ended up with two
inactive compounds; one containing a methylpiperazine side chain (compound 17, MIC = 64
g/mL) and the second consisting of a morpholine substituent (compound 18, MIC > 64 g/mL).
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Unlike the morpholine ring, the thiomorpholine ring resulted in a compound (19, MIC = 4 g/mL)
with similar potency to the first-generation lead 1a. Finally, the side chain consisting of a 6-
membered ring provided the optimum bulkiness for anti-MRSA activity as compounds with a
smaller ring size (compounds 8-12) or larger ring size (compounds 20 and 21) were all either less
active or inactive against MRSA USA300 (Table 1). The MIC for the control antibiotic linezolid
and vancomycin was 1 µg/mL against MRSA USA300. All reported compounds inhibited growth
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of E. coli JW55031 at concentrations identical to or one-fold above/below their MIC against
MRSA USA300 (Table 1). Additionally, all compounds were inactive against E. coli wild-type
strain, which means that this class of antibiotic is efficient only for treatment of Gram-positive
infections.
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Table 1. Initial screening results (MIC in µg/mL) of the new series of phenylthiazoles against
methicillin-resistant S. aureus USA300, E. coli JW55031 and E. coli BW25113.
N
R
NH
N
S
N
NH2
H
Compounds/
R
MRSA USA300
E. coli JW55031
E. coli BW25113
Control drugs
(TolC mutant)
(wild-type strain)
8
9
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64
64
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> 64
> 64
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HO
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11
N
N
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32
> 64
> 64
> 64
> 64
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N
> 64
64
> 64
N
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14
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> 64
64
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64
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64
> 64
4
64
> 64
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> 64
> 64
> 64
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18
19
N
O
N
N
S
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21
64
64
> 64
> 64
> 64
> 64
N
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1a
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> 64
> 64
NT1
NT1
0.5
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NT
NT
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1
Linezolid
Vancomycin
Gentamicin
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0.5
1NT: Not tested
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Assessment of physicochemical properties and pharmacokinetic profile. After investigating
the antibacterial activity of the compounds against MRSA USA300 and E. coli JW55031, we next
assessed the physicochemical properties of the most promising derivatives. First, the aqueous
solubility of the compounds was analyzed as solubility directly impacts the pharmacokinetic
profile of bioactive compounds and is highly-dependent on a molecule’s polarity/lipophilicity
ratio23-24. Moreover, excessive lipophilic properties can negatively impact the formulation of
compounds into suitable dosage forms.25 It has been shown that compounds with a high logP value
(≥ 4) are challenging to formulate in an oral dosage form.26 To investigate whether our hypothesis
of inserting a nitrogen atom within the lipophilic side chain will positively impact the aqueous
solubility of the phenylthiazole compounds, the four most potent derivatives against MRSA (14-
16 and 19) were evaluated via a turbidometric solubility assay. The methylpiperidine-containing
analogues (14-16) were 150-times more soluble than the lead compound 1b (Table 2). Compound
15 was the most soluble derivative (solubility limit exceeded 500 µM) from this new series and
exhibited a solubility profile similar to the high-solubility control drug verapamil. Compound 19,
containing the thiomorpholine ring, also provided sufficient enhancement in this regard as its
aqueous solubility limit was almost 0.3 µM (Table 2). This value represents a 107-fold
improvement over the solubility limit of the first-generation lead compound 1b.
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Table 2. Evaluation of aqueous solubility limit of phenylthiazole compounds 1a, 1b, 14-16, 19,
and control drugs.
Solubility limit (M)1
Compound/Drug
1a
1b
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15
16
65
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427
> 500
375
292
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Tamoxifen
Verapamil
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> 500
1Solubility limit corresponds to the highest concentration of test compound where no
precipitate was detected (OD540).
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0
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20
25
30
TIME (H)
Figure 2. Pharmacokinetic profile of compound 15 after a single 50 mg/kg oral dose in rats (n =
3).
The initial series of phenylthiazole compounds synthesized could not be administered
orally either due to metabolic instability (resulting in rapid elimination) or poor aqueous solubility
that precluded formulation of the compounds in a proper oral dosage form.17 The current series of
phenylthiazole analogues were designed with an acetylene linker to block the metabolic soft spot
present in compound 1a.20 To test the impact of decreasing the overall lipophilicity and increasing
the hydrophilicity of the new set of alkynylphenylthiazoles on the compounds’ pharmacokinetic
profile, compound 15 was administered orally to rats. Blood samples were collected from each rat
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over a 24 hour period (Figure 2). Compound 15 was selected as it exhibited the best aqueous
solubility profile in vitro. The pharmacokinetic profile of 15 confirmed a notable improvement in
the compound’s stability to metabolism (half-life of three hours). Compound 15’s half-life
exceeded the half-life of the first-generation lead compound 1a by more than six times.13
Furthermore, the maximum plasma concentration (Cmax) of compound 15 was 18.4 µg/mL which
was more than three-fold higher than its MIC against MRSA USA300. Moreover compound 15
reached a plasma concentration equal to its MIC (4 g/mL) against MRSA USA300 within 30
minutes and remained at or above this concentration for nearly eight hours.
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Antibacterial activity of active analogues against a panel of S. aureus isolates. After
confirming the notable improvement in aqueous solubility, metabolic stability, and
pharmacokinetic profile of this new series of phenylthiazole antibacterial agents, we tested their
activity against additional strains of methicillin-sensitive, methicillin-resistant, vancomycin-
intermediate, and vancomycin-resistant staphylococci (MSSA, MRSA, VISA, and VRSA).
Table 3. The minimum inhibitory concentration (MIC in µg/mL) and minimum bactericidal
concentration (MBC in µg/mL) of compounds 14-16 and 19 against 25 strains of methicillin-
sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA),
vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant
Staphylococcus aureus (VRSA).
Bacterial Strain
Compounds/Control antibiotics
14
15
16 19 Linezolid
Vancomycin
MSSA1
ATCC 6538
MSSA
NRS107
MRSA2 NRS108
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16
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8
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1
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2
1
1
1
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1
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1
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MRSA NRS194
MRSA NRS119
1
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64
1
> 64
64
MRSA NRS123
(USA400)
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Bacterial Strain
Compounds/Control antibiotics
16 19 Linezolid
14
15
Vancomycin
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7
8
9
MRSA NRS382
(USA100)
MRSA NRS383
(USA200)
MRSA NRS384
(USA300)
MRSA NRS385
(USA500)
MRSA NRS386
(USA700)
MRSA NRS387
(USA800)
MRSA NRS483
(USA1000)
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>64
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MRSA NRS484
(USA1100)
VISA3 NRS1
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VISA NRS19
VISA NRS37
VRS24
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> 64
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> 64
> 64
> 64
64
> 64
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> 64
> 64
> 64
> 64
> 64
64
VRS5
16
8
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16
16
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1
64
VRS6
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32
VRS7
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16
16
8
16
16
32
16
32
1
> 64
16
VRS9
8
1
VRS10
8
1
> 64
16
VRS11a
VRS12
8
16
16
0.5
1
16
64
1MSSA, Methicillin-sensitive Staphylococcus aureus; 2MRSA, Methicillin-resistant Staphylococcus aureus; 3VISA,
Vancomycin-intermediate Staphylococcus aureus; 4VRS, Vancomycin-resistant Staphylococcus aureus.
Compounds 14-16 and 19 inhibited growth of all methicillin-sensitive, methicillin-
resistant, vancomycin-intermediate and vancomycin-resistant S. aureus (MSSA, MRSA, VISA
and VRSA) strains tested at concentrations ranging from 4 to 16 µg/mL (Table 3). Compounds 15
and 16 were the two most potent compounds with MIC values ranging primarily between 4 to 8
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µg/mL. The compounds also inhibited growth of S. aureus strains exhibiting resistance to the two
frontline treatment options for MRSA infections (linezolid and vancomycin).
7
8
9
To ascertain if the phenylthiazole compounds were bacteriostatic or bactericidal agents
against the tested staphylococcal strains, the MBC was determined. The MBC values were not
more than three-fold higher than the compounds’ MIC values against all S. aureus strains tested
indicating they are bactericidal agents. To confirm the bactericidal activity of the compounds, a
time-kill assay (Figure 3) was performed against MRSA USA400. This is a highly-virulent strain
of MRSA associated with many cases of SSTIs as well as invasive infections such as necrotizing
pneumonia, pulmonary abscesses, and sepsis.27-29
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Time (Hours)
Figure 3. Time-kill assay for compounds 14-16, 19 and vancomycin (tested in triplicates at 4 ×
MIC) against methicillin-resistant Staphylococcus aureus (MRSA) USA400. DMSO (solvent for
the compounds) served as a negative control. The error bars represent standard deviation values.
Compounds 15 and 16 were as effective as vancomycin, the drug of choice for treatment
of MRSA infections, as all three agents reduced the burden of MRSA by 3-log10 within 24 hours.
The 2-methylpiperidine 14 and thiomorpholine-containing derivative 19 achieved the same effect
more rapidly, within 12 hours.
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Selectivity towards prokaryotic cells was evaluated using two cell lines; one carcinogenic
and one non-carcinogenic that are human colorectal adenocarcinoma (Caco-2) and fibroblast-like
monkey kidney cell line (VERO cell) to determine the potential toxic effect in vitro. As
summarized in Figures 1S and 2S, all four tested compounds 14, 15, 16 and 19 were highly
tolerable to both cell lines at concentration as high as 32 µg/mL.
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Multi-step resistance study against MRSA. As reported previously, MRSA was unable to
develop resistance to the initial series of phenylthiazole compounds21,30-31. To test whether the
modifications made to the new compounds affected this feature, a multi-step resistance selection
study was conducted (Figure 4). The MIC values of compounds 15 and 19 remained consistent
and did not change throughout the 14 passages. The MIC values for compounds 14 and 16
increased one-fold after the tenth and ninth passage respectively, but they remained stable
thereafter. The results from the multi-step resistance study indicate that MRSA was unable to
develop rapid resistance to the four phenylthiazole compounds tested.
2
14
15
16
19
1
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Passage Number
Figure 4. Multi-step resistance study of compounds 14-16 and 19 against methicillin-resistant S.
aureus USA400. Bacteria were serially passaged over a 14-day period, and the broth microdilution
assay was used to determine the MIC of each compound after each successive passage. A four-
fold increase in the MIC would be indicative of bacterial resistance forming to the test agent.
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MRSA biofilm eradication assessment. S. aureus is one of the most important bacterial species
capable of forming biofilms. Biofilms can form on medical devices such as indwelling catheters,
implanted artificial bones, knees and hips as well as prosthetic heart valves.32-33 This increases the
chance of developing chronic, recurrent infections.34 Staphylococcal biofilm-related infections are
difficult to eradicate and typically must be treated with antibiotic combinations. Most drugs of
choice for treatment of staphylococcal infections like glycopeptides (vancomycin) have very
limited activity on bacteria embedded within biofilms.18, 35 As a consequence, infected medical
devices have to be surgically extracted and replaced, which can lead to serious risks and
complications.36 Hence, there is a need to develop drugs capable of disrupting staphylococcal
biofilms.
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Compounds 14-16 and 19 were examined for their ability to disrupt pre-formed, mature
MRSA USA300 biofilm. Advantageously, the new phenylthiazoles were superior to vancomycin
in MRSA biofilm eradication. Due to its large molecular structure and polar nature, vancomycin,
the drug of choice for treatment of invasive MRSA infections, does not penetrate biofilms
effectively.37-38 At 2 × MIC, vancomycin only disrupted 12.8% of MRSA biofilm mass (Figure 5).
In contrast, compound 19 (at 2 × MIC) disrupted ~42% of MRSA300 biofilm mass. Compounds
14 and 15 exhibited similar activity as they disrupted ~39% of the pre-formed MRSA300 biofilm.
Compound 16 disrupted ~31% of MRSA300 biofilm mass at the same concentration (2 × MIC).
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*
*
*
*
14
15
16
19
Vancomycin
Compounds
Figure 5. Disruption of mature MRSA USA300 biofilm by compounds 14-16 and 19 and
vancomycin (all at 2 × MIC). Data are presented as percent disruption of MRSA USA300 mature
biofilm in relation to DMSO (the solvent for the compounds that served as a negative control).
The values represent an average of four samples analyzed for each compound/antibiotic. Error bars
represent standard deviation values. An asterisk (*) denotes statistical significance (P < 0.05)
between results for compounds 14-16 and 19 relative to vancomycin as analyzed via an unpaired
t-test.
Conclusion. During the process of developing phenylthiazole antibiotics, the structure-activity-
relationships led us to an “obese molecule” in which increasing the lipophilicity improved the
compounds’ antibacterial activity. However, this had a deleterious effect on the compounds’
physicochemical profile. This article tackled this problem by maintaining the essential structural
elements of the phenylthiazole core and inserting a nitrogen atom within the lipophilic side chain.
A six-membered cyclic amine with methyl substituent or sulfur atom resulted in derivatives with
the most-balanced antibacterial and physicochemical properties. Compound 15 demonstrated a
highly acceptable in vivo pharmacokinetic profile in terms of oral absorbability, metabolic stability
and plasma concentration above the compound’s MIC values against MRSA. The notable
improvement in the pharmacokinetic behavior was accompanied with a slight decrease in
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antibacterial activity as the MIC values reported here were 2-4 times higher than the previously
tested alkynylphenylthiazoles.
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Methods
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General Chemistry: The purity of all biologically-tested compounds
were confirmed to be not less than 95% using elemental analysis.
All starting material chemicals and solvents were obtained from commercial sources. The masses
were weighed on a microbalance with a resolution of 0.0001 g. Visualization on TLC was acquired
using UV light (254 nm) and also staining with iodine. 1H NMR spectra were done at 400 MHz
13
and C spectra were determined at 100 MHz in deuterated dimethyl sulfoxide (DMSO-d6) on a
Varian Mercury VX-400 NMR spectrometer. Chemical shifts are given in parts per million (ppm)
on the delta () 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). 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.
Compounds 7a-n. General procedure: to dry DMF (5 mL) in a round flask, appropriate Sec.
amines 4a-n (200 mg, 1.5-2 mmole), propargyl bromide (476 mg, 300 L, 3-4 mmol, 2 equiv.),
and anhydrous potassium carbonate (830 mg, 4.5-6 mmole, 3 equiv.) were heated at 110 oC and
stirred for 4 h. Next, the reaction mixture was taken without purification and added to a 75-mL
sealed tube charged with compound 3 (300 mg, 0.87 mmol) in dry DME (10 mL) and triethylamine
(2 mL). After the reaction mixture was purged with dry nitrogen gas for 15 min,
dichlorobis(triphenylphosphine)palladium (II) (46 mg, 0.065 mmol) and copper(I) iodide (33 mg,
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o
0.17 mmol) were added. The sealed tube was then maintained at 50 C for 24 h and reaction
progress was monitored by TLC. After completion of the reaction, the reaction mixture was passed
through a pad of silica gel with DCM (50 mL). Organic materials were then collected, concentrated
under vacuum and purified using silica gel chromatography (DCM/ methanol 98:2). Physical
properties, yields, and characterization data of isolated products are listed below:
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1-(2-(4-(3-(Azitidin-1-yl)prop-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one
(7a).
Orange oil (225 mg, 83%); 1H NMR (DMSO-d6) δ: 8.21 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz,
2H), 4.33 (t, J = 8.2 Hz, 4H), 3.77 (s, 2H), 2.62 (s, 3H), 2.59 (s, 3H), 2.52 (m, 2H); 13C NMR
(DMSO-d6) δ: 191.55, 168.38, 158.69, 133.23, 132.94, 127.22, 125.93, 92.81, 83.74, 50.03, 49.66,
30.61, 18.58, 17.88; MS (m/z); 310 (M+, 14.7%).
1-(2-(4-(3-(3-Hydroxyazitidin-1-yl)prop-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one
(7b). Yellowish oil (235 mg, 82.4%); 1H NMR (DMSO-d6) δ: 7.61 (d, J = 8.4 Hz, 2H), 7.58 (d, J
= 8.4 Hz, 2H), 4.40 (brs, 1H), 3.71 (m, 1H), 3.41 (s, 2H), 2.62 (s, 3H), 2.59 (dd, J = 11.2, 3.4 Hz,
2H), 2.57 (dd, J = 11.1, 6.2 Hz, 2H), 2.33 (s, 3H);13C NMR (DMSO-d6) δ: 191.95, 168.14, 158.66,
132.73, 132.44, 129.27, 127.19, 94.51, 84.64, 62.74, 48.23, 44.25, 30.66, 18.54; MS (m/z); 326
(M+, 100%).
1-(4-Methyl-2-(4-(3-(pyrrolidin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
(7c).
Dark brown oil (170 mg, 60%); 1H NMR (DMSO-d6) δ: 7.88 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.8
Hz, 2H), 3.63 (s, 2H), 2.61 (s, 3H), 2.49 (m, 4H), 2.31 (s, 3H), 1.73 (m, 4H); 13C NMR (DMSO-
d6) δ: 190.45, 160.34, 148.33, 142.74, 136.33, 132.93, 126.62, 123.91, 89.12, 84.45, 53.66, 43.39,
23.86, 18.55, 16.61; MS (m/z); 324 (M+, 19.51%).
1-(4-Methyl-2-(4-(3-(thiazolidin-3-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one (7d).
Dark yellow oil (163 mg, 54%); 1H NMR (DMSO-d6) δ: 8.02 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.1
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Hz, 2H), 4.69 (s, 2H), 3.86 (s, 2H), 3.14 (t, J = 6.4 Hz, 2H), 2.77 (t, J = 6.4 Hz, 2H), 2.61 (s, 3H),
2.46 (s, 3H); 13C NMR (DMSO-d6) δ: 190.66, 163.16, 158.83, 136.13, 132.64, 131.96, 127.17,
124.31, 93.40, 84.45, 60.06, 53.44, 43.39, 24.11, 18.45, 16.71; MS (m/z); 342 (M+, 37.73%).
1-(2-(4-(3-(1H-Imidazol-1-yl)prop-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one (7e).
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Dark brown oil (215 mg, 76%); H NMR (DMSO-d6) δ: 7.91 (d, J = 8.4 Hz, 2H), 7.78 (s, 1H),
7.57 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 6.8 Hz, 1H), 6.96 (d, J = 6.8 Hz, 1H), 5.22 (s, 2H), 2.68 (s,
13
3H), 2.31 (s, 3H); C NMR (DMSO-d6) δ: 192.15, 166.34, 163.73, 136.93, 133.64, 132.72,
131.91, 129.83, 126.73, 124.96, 121.33, 87.32, 84.55, 43.39, 29.86, 16.45; MS (m/z); 321 (M+,
32.29%).
1-(4-Methyl-2-(4-(piperidin-1-yl)prop-1-yn-1-yl)phenyl)-4-thiazol-5-yl)ethan-1-one
(7f).
Yellow oil (233 mg, 78.9%); 1H NMR (DMSO-d6) δ: 7.99 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz,
2H), 3.51 (s, 2H), 2.71 (s, 3H), 2.57 (s, 3H), 2.49 (m, 4H), 1.51 (m, 4H), 1.39 (m, 2H); 13C NMR
(DMSO-d6) δ: 191.35, 167.92, 158.56, 133.83, 132.54, 132.26, 127.25, 125.61, 89.58, 84.63,
53.10, 48.06, 30.64, 25.94, 24.04, 18.45; MS (m/z); 338 (M+, 29.17%).
1-(4-Methyl-2-(4-(3-(2-methylpiperidin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
(7g). Brown oil ( 214 mg, 69.7%); 1H NMR (DMSO-d6) δ: 7.88 (d, J = 8.0 Hz, 2H), 7.51 (d, J =
7.2 Hz, 2H), 4.51 (s, 2H), 3.73 (m, 1H), 3.51 (m, 2H), 2.69 (s, 3H), 2.52 (s, 3H), 1.60-1.22 (m,
6H), 1.05 (d, J = 6.8 Hz, 3H); 13C NMR (DMSO-d6) δ: 191.15, 168.12, 162.97, 158.83, 132.39,
132.26, 127.34, 126.08, 88.70, 84.88, 54.66, 53.39, 43.87, 34.62, 30.69, 26.34, 24.76, 20.41, 18.31;
MS (m/z); 352 (M+, 49.21%).
1-(4-Methyl-2-(4-(3-(3-methylpiperidin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
(7h). Yellow oil ( 250 mg, 81.4%); 1H NMR (DMSO-d6) δ: 8.00 (d, J = 8.8 Hz, 2H), 7.57 (d, J =
8.4 Hz, 2H), 3.51 (s, 2H), 2.77 (m, 2H), 2.71 (s, 3H), 2.58 (s, 3H), 214-1.46 (m, 7H), 0.87 (d, J =
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6.4 Hz, 3H); 13C NMR (DMSO-d6) δ: 191.14, 168.02, 158.69, 133.53, 133.22, 132.24, 126.91,
125.92, 89.49, 84.43, 60.35, 52.40, 48.01, 32.62, 30.99, 25.64, 18.94, 17.34; MS (m/z); 352 (M+,
49.33%).
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1-(4-Methyl-2-(4-(3-(4-methylpiperidin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
(7i). Light-yellow oil ( 270 mg, 87.9%); 1H NMR (DMSO-d6) δ: 7.88 (d, J = 8.4 Hz, 2H), 7.51 (d,
J = 8.4 Hz, 2H), 3.51 (s, 2H), 2.84 (m, 4H), 2.59 (s, 3H), 2.31 (s, 3H), 2.20 (m, 4H), 1.61-1.11 (m,
1H), 0.90 (d, J = 6.4 Hz, 3H); 13C NMR (DMSO-d6) δ: 191.33, 161.84, 160.13, 136.33, 133.30,
132.44, 126.31, 124.52, 88.59, 84.93, 52.75, 47.60, 34.71, 30.29, 22.24, 18.81, 16.84; MS (m/z);
352 (M+, 49.31%).
1-(4-Methyl-2-(4-(3-(4-methylpiperazin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
(7j). Orange oil ( 234 mg, 75.9%); 1H NMR (DMSO-d6) δ: 8.01 (d, J = 8.4 Hz, 2H), 7.53 (d, J =
8.4 Hz, 2H), 3.55 (s, 2H), 2.72 (s, 3H), 2.59 (s, 3H), 2.52 (s, 8H), 2.16 (s, 3H); 13C NMR (DMSO-
d6) δ: 191.18, 167.73, 158.63, 132.62, 132.43, 127.41, 126.15, 89.25, 84.83, 54.15, 51.15, 47.42,
46.01, 31.04, 18.74; MS (m/z); 353 (M+, 9.14%).
1-(4-Methyl-2-(4-(3-(4-morpholinoprop-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
(7k).
Brown oil ( 263 mg, 88%); 1H NMR (DMSO-d6) δ: 7.99 (d, J = 7.2 Hz, 2H), 7.58 (d, J = 7.2 Hz,
2H), 3.98 (s, 2H), 3.62 (t, J = 6.4 Hz, 4H), 2.71 (s, 3H), 2.57 (s, 3H), 2.53 (t, J = 6.4 Hz, 4H); 13C
NMR (DMSO-d6) δ: 191.23, 161.94, 158.61, 136.43, 133.21, 132.04, 127.56, 125.72, 88.69, 84.53,
62.35, 54.40, 45.41, 18.24, 16.31; MS (m/z); 340 (M+, 100%).
1-(4-Methyl-2-(4-(3-(4-thiomorpholinoprop-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one (7l).
Yellow oil ( 212 mg, 68%); 1H NMR (DMSO-d6) δ: 8.19 (d, J = 7.2 Hz, 2H), 7.48 (d, J = 7.2 Hz,
2H), 4.61 (s, 2H), 4.02 (t, J = 6.4 Hz, 4H), 2.69 (s, 3H), 2.53 (t, J = 6.4 Hz, 4H), 2.39 (s, 3H); 13C
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NMR (DMSO-d6) δ: 191.63, 163.04, 158.91, 136.43, 133.88, 133.18, 127.16, 124.62, 88.79, 82.43,
54.15, 46.41, 29.45, 18.78, 16.17; MS (m/z); 356 (M+, 9.42%).
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1-(2-(4-(3-(Azepan-1-yl)prop-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one
(7m).
Dark-brown oil (223 mg, 72%); 1H NMR (DMSO-d6) δ: 8.06 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4
Hz, 2H), 4.29 (t, J = 5.4 Hz, 4H), 4.06 (s, 2H), 2.61 (s, 3H), 2.46 (s, 3H), 2.31-1.46 (m, 8H); 13C
NMR (DMSO-d6) δ: 191.35, 167.92, 158.16, 143.83, 133.54, 133.26, 127.25, 125.83, 88.58, 84.63,
54.10, 44.06, 30.64, 25.94, 24.44, 18.45; MS (m/z); 352 (M+, 8.17%).
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1-(4-Methyl-2-(4-(3-(octahydroisoquinolin-2(1H)-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)
ethan-1-one (7n). Dark-brown oil (273 mg, 79.8%); 1H NMR (DMSO-d6) δ: 7.88 (d, J = 8.4 Hz,
2H), 7.51 (d, J = 8.4 Hz, 2H), 3.51 (s, 2H), 2.87 (m, 2H), 2.73 (m, 2H), 2.59 (s, 3H), 2.31 (s, 3H),
2.20-0.89 (m, 12H); 13C NMR (DMSO-d6) δ: 191.15, 160.42, 148.46, 133.53, 132.64, 129.46,
126.22, 124.40, 91.28, 84.83, 59.64, 53.38, 47.01, 42.38, 33.12, 30.74, 27.14, 26.34, 24.04, 19.01,
16.65; MS (m/z); 392 (M+, 20.60%).
2-(1-(2-(4-(3-(Substituted)prop-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)-1-carbox
imidamide (8-21). General procedure: Thiazole derivatives 7a-n (0.57-0.64 mmol) were
dissolved in absolute ethanol (15 mL). Concentrated hydrochloric acid (1 mL) and aminoguanidine
hydrochloride (355 mg, 3.2 mmol, 5 equiv.) were subsequently added. The reaction mixture was
heated at reflux for 3 h. After completion of the reaction, as monitored by TLC, the solvent was
concentrated under reduced pressure, poured onto crushed ice, and neutralized with sodium
carbonate to pH 7-8. The formed precipitated was collected by filtration and washed with copious
amount of water. Crystallization from absolute ethanol afforded the desired products as solids.
Physical properties, yields, and characterization data of isolated final products are listed below:
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hydrazine-1-carboximidamide (8). Yellow solid (160 mg, 68%); mp = 240-241 °C. H NMR
(DMSO -d6) δ: 8.00 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 5.71 (brs, 2H), 4.92 (brs, 2H),
4.35 (t, J = 5.2 Hz, 4H), 3.77 (s, 2H), 2.71 (s, 3H), 2.55 (s, 3H), 2.43 (m, 2H); 13C NMR (DMSO-
d6) δ: 168.38, 163.42, 158.69, 153.21, 132.61, 132.18, 127.31, 125.97, 93.21, 83.64, 51.03, 49.16,
30.51, 18.39, 17.77; HRMS (EI) m/z 366.1621 M+, calc. for C19H22N6S 366.1627; Anal. Calc. for:
C19H22N6S (366): C, 62.27; H, 6.05; N, 22.93%; Found: C, 62.29; H, 6.07; N, 22.96%.
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2-(1-(2-(4-(3-Hydroxyazetidin-1-yl)prop-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl) ethylidene)
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hydrazine-1-carboximidamide (9). Orange solid (167 mg, 71%); mp = 249-251 °C. H NMR
(DMSO -d6) δ: 7.66 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 5.69 (brs, 4H), 3.86 (m, 1H),
3.53 (brs, 1H), 3.47 (s, 2H), 2.61 (s, 3H), 2.49 (s, 3H), 2.47 (dd, J = 10.2, 3.4 Hz, 2H), 2.45 (dd, J
= 10.1, 6.2 Hz, 2H); 13C NMR (DMSO-d6) δ: 168.67, 161.72, 158.53, 152.72, 145.69, 133.05,
132.88, 127.93, 125.17, 94.25, 83.54, 62.35, 53.53, 49.86, 30.81, 18.39; HRMS (EI) m/z 382.1590
M+, calc. for C19H22N6OS 382.1576; Anal. Calc. for: C19H22N6OS (382): C, 59.66; H, 5.80; N,
21.97%; Found: C, 59.61; H, 5.78; N, 21.94%.
2-(1-(4-Methyl-2-(4-(3-(pyrrolidin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)ethylidene)
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hydrazine-1-carboximidamide (10). Brown solid (174 mg, 74%); mp = 245-247 °C. H NMR
(DMSO -d6) δ: 7.87 (d, J = 7.2 Hz, 2H), 7.51 (d, J = 7.2 Hz, 2H), 5.76 (brs, 2H), 5.66 (brs, 2H),
3.76 (s, 2H), 2.59 (m, 4H), 2.57 (s, 3H), 2.31 (s, 3H), 1.73 (m, 4H); 13C NMR (DMSO-d6) δ:
161.57, 160.24, 148.44, 142.99, 136.48, 133.36, 132.27, 126.22, 124.14, 88.99, 84.49, 52.43,
43.89, 23.77, 18.83, 16.55; HRMS (EI) m/z 380.1793 M+, calc. for C20H24N6S 380.1783; Anal.
Calc. for: C20H24N6S (380): C, 63.13; H, 6.36; N, 22.09%; Found: C, 63.16; H, 6.37; N, 22.11%.
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hydrazine-1-carboximidamide (11). Brown solid (174 mg, 74%); mp = 245-247 °C. H NMR
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(DMSO -d6) δ: 7.96 (d, J = 8.4 Hz, 2H), 7.81 (brs, 4H), 7.62 (d, J = 8.4 Hz, 2H), 4.69 (s, 2H),
4.04 (s, 2H), 3.13 (t, J = 6.4 Hz, 2H), 2.87 (t, J = 6.4 Hz, 2H), 2.62 (s, 3H), 2.45 (s, 3H); 13C NMR
(DMSO-d6) δ: 163.54, 158.54, 147.69, 143.56, 136.18, 132.16, 124.52, 121.64, 89.69, 84.69,
61.13, 53.45, 42.68, 29.27, 18.43, 16.65; HRMS (EI) m/z 398.1362 M+, calc. for C19H22N6S2
398.1347; Anal. Calc. for: C19H22N6S2 (398): C, 57.26; H, 5.56; N, 21.09%; Found: C, 57.29; H,
5.58; N, 21.10%.
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2-(1-(2-(4-(3-(1H-Imidazol-1-yl)prop-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)
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hydrazine-1-carboximidamide (12). Dark-brown solid (148 mg, 63%); mp = 205-207 °C. H
NMR (DMSO -d6) δ: 7.91 (d, J = 8.4 Hz, 2H), 7.78 (s, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.32 (d, J =
7.2 Hz, 1H), 6.96 (d, J = 7.2 Hz, 1H), 5.76 (brs, 2H), 5.66 (brs, 2H), 5.22 (s, 2H), 2.59 (s, 3H),
2.31 (s, 3H); 13C NMR (DMSO-d6) δ: 165.25, 163.63, 155.63, 147.65, 136.43, 134.74, 133.52,
132.94, 131.91, 127.53, 124.43, 122.65, 94.52, 84.55, 43.39, 29.96, 18.45; HRMS (EI) m/z
377.1411 M+, calc. for C19H19N7S 377.1423; Anal. Calc. for: C19H19N7S (377): C, 60.46; H, 5.07;
N, 25.98%; Found: C, 60.42; H, 5.08; N, 26.00%.
2-(1-(4-Methyl-2-(4-(3-(piperidin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)ethylidene)
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hydrazine-1-carboximidamide (13). Yellow solid (196 mg, 84%); mp = 255-257 °C. H NMR
(DMSO -d6) δ: 11.53 (brs, 1H), 11.06 (brs, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.80 (brs, 2H), 7.67 (d,
J = 8.4 Hz, 2H), 4.32 (s, 2H), 3.51 (m, 4H), 3.02 (m, 4H), 2.63 (s, 3H), 2.45 (s, 3H), 1.84 (m, 2H);
13C NMR (DMSO-d6) δ: 167.64, 161.53, 158.62, 147.29, 136.46, 132.90, 132.07, 127.33, 125.94,
89.57, 84.63, 53.17, 48.14, 30.90, 25.93, 24.91, 18.55; HRMS (EI) m/z 394.1938 M+, calc. for
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C21H26N6S 394.1940; Anal. Calc. for: C21H26N6S (394): C, 63.93; H, 6.64; N, 21.30%; Found: C,
63.96; H, 6.65; N, 21.34%.
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2-(1-(4-Methyl-2-(4-(3-(2-methylpiperidin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)
ethylidene)hydrazine-1-carboximidamide (14). Yellow solid (196 mg, 84%); mp = 255-257 °C.
1H NMR (DMSO -d6) δ: 7.88 (d, J = 7.2 Hz, 2H), 7.51 (d, J = 7.2 Hz, 2H), 5.77 (brs, 4H), 3.78 (s,
2H), 3.55 (m, 2H), 3.51 (m, 1H), 2.59 (s, 3H), 2.31 (s, 3H), 1.61-1.19 (m, 6H), 1.06 (d, J = 4.8 Hz,
3H); 13C NMR (DMSO-d6) δ: 161.69, 160.15, 148.55, 136.43, 133.09, 132.58, 126.33, 124.31,
87.68, 85.23, 54.72, 53.26, 43.91, 34.73, 26.32, 24.71, 20.33, 18.70, 16.49; HRMS (EI) m/z
408.2118 M+, calc. for C22H28N6S 408.2096; Anal. Calc. for: C22H28N6S (408): C, 64.68; H, 6.91;
N, 20.57%; Found: C, 64.70; H, 6.95; N, 20.54%.
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2-(1-(4-Methyl-2-(4-(3-(3-methylpiperidin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)
ethylidene)hydrazine-1-carboximidamide (15). Yellow-white solid (203 mg, 87%); mp = 259-
261 °C. 1H NMR (DMSO -d6) δ: 11.37 (brs, 1H), 10.92 (brs, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.72
(brs, 2H), 7.70 (d, J = 8.4 Hz, 2H), 4.34 (s, 2H), 3.57 (d, J = 9.6 Hz, 2H), 3.47 (m, 2H), 2.63 (s,
3H), 2.45 (s, 3H), 1.86 (m, 1H), 1.12-103 (m, 4H), 0.95 (d, J = 6.4 Hz, 3H); 13C NMR (DMSO-d6)
δ: 163.51, 161.25, 158.12, 147.83, 136.74, 132.78, 132.11, 127.53, 124.97, 94.38, 86.43, 54.72,
53.26, 42.81, 33.73, 28.32, 24.71, 23.12, 18.53, 16.69; HRMS (EI) m/z 408.2105 M+, calc. for
C22H28N6S 408.2096; Anal. Calc. for: C22H28N6S (408): C, 64.68; H, 6.91; N, 20.57%; Found: C,
64.71; H, 6.94; N, 20.55%.
2-(1-(4-Methyl-2-(4-(3-(4-methylpiperidin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)
ethylidene)hydrazine-1-carboximidamide (16). Yellow solid (212 mg, 90.9%); mp = 263-265
°C. 1H NMR (DMSO -d6) δ: 7.88 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H), 5.74 (brs, 4H), 3.51
(s, 2H), 2.84 (t, J = 12.2 Hz, 4H), 2.59 (s, 3H), 2.31 (s, 3H), 2.17 (t, J = 10.8 Hz, 4H), 1.17 ( m,
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1H), 0.91 (d, J = 6.4 Hz, 3H); 13C NMR (DMSO-d6) δ: 161.61, 160.19, 148.52, 143.11, 136.38,
133.30, 132.52, 126.30, 124.27, 88.60, 84.82, 52.52, 47.43, 34.35, 30.36, 22.24, 18.65, 16.49;
HRMS (EI) m/z 408.2096 M+, calc. for C22H28N6S 408.2122; Anal. Calc. for: C22H28N6S (408): C,
64.68; H, 6.91; N, 20.57%; Found: C, 64.71; H, 6.94; N, 20.55%.
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2-(1-(4-Methyl-2-(4-(3-(4-methylpiperazin-1-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)
ethylidene)hydrazine-1-carboximidamide (17). Orange solid (166 mg, 71.5%); mp = 253-255
°C. 1H NMR (DMSO -d6) δ: 8.01 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 5.75 (brs, 4H), 3.55
(s, 2H), 2.84 (m, 4H), 2.72 (s, 3H), 2.58 (s, 3H), 2.52 (m, 4H), 2.16 ( s, 3H); 13C NMR (DMSO-
d6) δ: 168.18, 165.19, 158.55, 142.31, 132.98, 131.95, 127.21, 124.93, 89.17, 84.12, 55.01, 52.03,
47.33, 46.34, 30.08, 18.55; HRMS (EI) m/z 409.2051 M+, calc. for C21H27N7S 409.2049; Anal.
Calc. for: C21H27N7S (409): C, 61.59; H, 6.65; N, 23.94%; Found: C, 61.61; H, 6.64; N, 23.97%.
2-(1-(4-Methyl-2-(4-(3-morpholinoprop-1-yn-1-yl)phenyl)thiazol-5-yl)ethylidene)hydrazine
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-1-carboximidamide (18). Dark-brown solid (192 mg, 82.4%); mp = 253-255 °C. H NMR
(DMSO -d6) δ: 7.99 (d, J = 7.2 Hz, 2H), 7.58 (d, J = 7.2 Hz, 2H), 5.65 (brs, 2H), 5.48 (brs, 2H),
3.62 (s, 2H), 3.56 (m, 4H), 2.71 (s, 3H), 2.57 (s, 3H), 2.53 (m, 4H); 13C NMR (DMSO-d6) δ:
167.28, 165.49, 157.75, 141.37, 133.98, 132.45, 132.05, 126.21, 124.95, 89.17, 83.72, 58.01,
50.23, 44.13, 21.28, 18.55; HRMS (EI) m/z 396.1721 M+, calc. for C20H24N6OS 396.1732; Anal.
Calc. for: C20H24N6OS (396.5): C, 60.58; H, 6.10; N, 21.20%; Found: C, 60.61; H, 6.14; N,
21.17%.
2-(1-(4-Methyl-2-(4-(3-thiomorpholinoprop-1-yn-1-yl)phenyl)thiazol-5-yl)ethyl-idene)
hydrazine-1-carboximidamide (19). Yellow solid (172 mg, 74.4%); mp = 263-264 °C. 1H NMR
(DMSO -d6) δ: 7.96 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 5.65 (brs, 4H), 4.53 (s, 2H), 4.26
(m, 4H), 2.83 (m, 4H), 2.59 (s, 3H), 2.33 (s, 3H); 13C NMR (DMSO-d6) δ: 167.11, 165.39, 158.75,
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142.37, 133.78, 132.35, 126.11, 124.85, 89.17, 85.72, 58.34, 48.53, 43.13, 30.51, 18.98, 16.55;
HRMS (EI) m/z 412.1488 M+, calc. for C20H24N6S2 412.1504; Anal. Calc. for: C20H24N6S2 (412):
C, 58.22; H, 5.86; N, 20.37%; Found: C, 58.21; H, 5.84; N, 20.35%.
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hydrazine-1-carboximidamide (20). Brown solid (196 mg, 84%); mp = 275-277 °C. H NMR
(DMSO -d6) δ: 8.02 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 5.49 (brs, 2H), 5.48 (brs, 2H),
4.41 (t, J = 6.8 Hz, 4H), 4.02 (s, 2H), 2.57 (s, 3H), 2.34 (s, 3H), 1.95-1.05 (m, 8H); 13C NMR
(DMSO-d6) δ: 165.49, 161.15, 157.86, 147.45, 136.43, 133.19, 132.48, 126.23, 124.41, 89.68,
84.93, 59.76, 44.41, 34.71, 27.33, 23.70, 18.49; HRMS (EI) m/z 408.2081 M+, calc. for C22H28N6S
408.2096; Anal. Calc. for: C22H28N6S (408): C, 64.68; H, 6.91; N, 20.57%; Found: C, 64.70; H,
6.95; N, 20.54%.
2-(1-(4-Methyl-2-(4-(3-(octahydroisoquinolin-2(1H)-yl)prop-1-yn-1-yl)phenyl)thiazol-5-yl)
ethylidene)hydrazine-1-carboximidamide (21). Yellow solid (183 mg, 80%); mp = 253-255 °C.
1H NMR (DMSO -d6) δ: 7.88 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 5.76 (brs, 2H), 5.66
(brs, 2H), 3.51 (s, 2H), 2.87 (t, J = 10.8 Hz, 2H), 2.73 (d, J = 8.8 Hz, 2H), 2.59 (s, 3H), 2.31 (s,
3H), 1.86-0.92 (m, 12H);13C NMR (DMSO-d6) δ: 160.51, 158.90, 148.96, 143.81, 136.58, 132.50,
128.63, 126.39, 124.17, 88.58, 84.92, 59.12, 52.89, 47.69, 41.95, 41.44, 32.35, 29.91, 26.60, 26.17,
18.55, 16.47; HRMS (EI) m/z 448.2417 M+, calc. for C25H32N6S 448.2409; Anal. Calc. for:
C25H32N6S (448.6): C, 66.93; H, 7.19; N, 18.73%; Found: C, 66.91; H, 7.16; N, 18.75%.
Microbiological assays:
Bacterial strains, mammalian cell lines and antibiotics. Bacterial strains used in this study were
obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI
Resources) and the American Type Culture Collection (ATCC). Human colorectal
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adenocarcinoma (Caco-2) cell line, human keratinocyte cell line (HaCaT) and murine macrophage
(J774) cells were purchased from American Type Culture Collection (ATCC). Linezolid (Chem-
Impex International, Wood Dale, IL, USA) and vancomycin hydrochloride (Gold Biotechnology,
St. Louis, MO, USA), were purchased from commercial vendors. Phenylthiazole compounds were
prepared as a stock concentration of 10 mg/mL in DMSO.
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Determination of MICs and MBCs of the new phenylthiazole compound series against
Staphylococcus aureus clinical isolates. The broth microdilution method was utilized to test the
antibacterial activity of the new set of phenylthiazole compounds against a panel of clinically-
important S. aureus strains according to the guidelines outlined by the Clinical and Laboratory
Standards Institute (CLSI).39
Time-kill assay against MRSA. The test was performed against MRSA USA400, as described
previously10. Briefly, MRSA USA400 cells in logarithmic growth phase were diluted and
compounds were added at 4 × MIC (using triplicate samples for each test agent). At the
corresponding time intervals, samples were collected, diluted and plated onto Tryptic soy agar
plates. Plates were incubated aerobically at 37 ℃ for at least 18 hours before determining the
number of viable colony forming units (CFU)/mL.
Multi-step resistance study against MRSA. The ability of MRSA USA400 to develop resistance
to the new set of phenylthiazoles was investigated via a multi-step resistance study, as described
previously11. Resistance was defined as a greater than four-fold increase from the initial MIC40.
MRSA biofilm eradication assessment. Compounds 14-16 and 19 were examined for their ability
to disrupt a well-established, mature MRSA USA300 biofilm using the microtiter plate biofilm
formation assay, following a procedure described previously.18
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In vivo pharmacokinetic study. Compound 15’s pharmacokinetic profile was determined
following Institutional Animal Care and Use Committee guidelines, as described in a previous
report.17
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ASSOCIATED CONTENTS
Supporting information. The supporting information is available free of charge on the ACS
publication website. The strains used in the manuscript and their corresponding sites of isolation,
1H and 13C NMR spectra of all new described compounds.
Abbreviations. PK, pharmacokinetic; t1/2, half-life; CL, clearance; Cmax, maximum plasma
concentration; MIC, minimum inhibitory concentration; MBC, minimum bactericidal
concentration; MSSA, methicillin-sensitive Staphylococcus aureus; MRSA, methicillin-resistant
Staphylococcus aureus; VISA, vancomycin-intermediate Staphylococcus aureus; VRSA,
vancomycin-resistant Staphylococcus aureus.
Author information.
‖These two authors contributed equally
ORCID
Abdelrahman S. Mayhoub: 0000-0002-3987-3680
Acknowledgement. This work was funded by Science & Technology Development Funds (STDF-
Egypt) and the Egyptian Ministry of Higher Education and Scientific Research (MHESR). Article
is derived from the Subject Data funded in whole or part by NAS and USAID, and that any
opinions, findings, conclusions, or recommendations expressed in such article are those of the
authors alone, and do not necessarily reflect the views of USAID or NAS.
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