Modifying the lipophilic part of phenylthiazole antibiotics to control their drug-likeness

Article abstract of DOI:10.1016/j.ejmech.2019.111830

Compounds with high lipophilic properties are often associated with bad physicochemical properties, triggering many off-targets, and less likely to pass clinical trials. Two metabolically stable phenylthiazole antibiotic scaffolds having notable high lipo

Full text of DOI:10.1016/j.ejmech.2019.111830

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Modifying the lipophilic part of phenylthiazole antibiotics to control their drug-likeness
Yomna Hosny, Nader S. Abutaleb, Mariam Omara, Marwa Alhashimi, Mohamed M.
Elsebaei, Heba S. Elzahabi, Mohamed N. Seleem, Abdelrahman S. Mayhoub
PII:
S0223-5234(19)30982-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.111830
EJMECH 111830
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 September 2019
Revised Date: 13 October 2019
Accepted Date: 28 October 2019
Please cite this article as: Y. Hosny, N.S. Abutaleb, M. Omara, M. Alhashimi, M.M. Elsebaei, H.S.
Elzahabi, M.N. Seleem, A.S. Mayhoub, Modifying the lipophilic part of phenylthiazole antibiotics to
control their drug-likeness, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111830.
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Modifying the Lipophilic Part of Phenylthiazole Antibiotics to Control Their Drug-
Likeness
aǁ
bǁ
a
b
Yomna Hosny , Nader S. Abutaleb , Mariam Omara , Marwa Alhashimi , Mohamed M.
a
a
b,c**
a,d*
Elsebaei , Heba S. Elzahabi , 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, College of Veterinary Medicine, Purdue
University, West Lafayette, IN 47907, USA.
c.
Purdue Institute of Inflammation, Immunology, and Infectious Disease, West
Lafayette, IN 47907, USA.
d.
University of Science and Technology, Nanoscience Program, Zewail City of
th
Science and Technology, October Gardens, 6 of October, Giza 12578, Egypt.
Corresponding Authors
*
* MNS; email, mseleem@purdue.edu
ASM; email, amayhoub@azhar.edu.eg
*
ǁ
These two authors contributed equally

Abbreviations. MIC, minimum inhibitory concentration; MBC, minimum bactericidal
concentration; MSSA, methicillin-sensitive Staphylococcus aureus; MRSA, methicillin-resistant
Staphylococcus aureus; VRSA, vancomycin-resistant Staphylococcus aureus; PK,
pharmacokinetic; cLogP, partition coefficient value; tPSA, total polar surface area; Cmax
,
maximum plasma concentration; tmax, time required to reach the maximum plasma concentration;
CL, clearance rate; t , half-life; V , volume of distribution;
1
/2
d

Abstract. Compounds with high lipophilic properties are often associated with bad
physicochemical properties, triggering many off-targets, and less likely to pass clinical trials.
Two metabolically stable phenylthiazole antibiotic scaffolds having notable high lipophilic
characters, one with alkoxy side chain and the other one with alkynyl moiety, were derivatized
by inserting a cyclic amine at the lipophilic tail with the objective of improving physicochemical
properties and the overall pharmacokinetic behavior. Only alkynyl derivatives with 4- or 5-
membered rings showed remarkable antibacterial activity. The azetidine-containing compound 8
was the most effective and it revealed a potent antibacterial effect against 15 multi-drug resistant
(
MDR)-Gram positive pathogens including Staphylococcus aureus, Streptococcus pneumoniae,
Staphylococcus epidermidis and enterococci. Compound 8 was also highly effective in clearing
9.7% of the intracellular methicillin- resistant S. aureus (MRSA) harbored inside macrophages.
9
In addition to the remarkable enhancement in aqueous solubility, the in vivo pharmacokinetic
study in rats indicated that compound 8 can penetrate gut cells and reach plasma at a therapeutic
concentration within 15 minutes and maintain effective plasma concentration for around 12
hours. Interestingly, the main potential metabolite (compound 9) was also active as an
antibacterial agent with potent antibiofilm activity.
Key words: MDR-bacteria; Antibiotic resistance; MRSA; Staphylococcal infections;
antibiofilm; intracellular infections

1
. Introduction. The high attrition rate of small molecule drug-candidates from clinical studies
is still the key challenge for the pharmaceutical industry worldwide. For decades, poor
pharmacokinetic behavior was the main reason for the termination of clinical studies. Hence,
many rules were developed to correlate compounds’ physicochemical properties with
pharmacokinetic performance [1-5]. Clearly, it is hard to build a direct correlation between
clinical attrition and a single descriptor, but many reports highlight the influence of excessive
lipophilicity on toxicological outcomes and inappropriate pharmacokinetic profiles [6, 7].
Compounds with high lipophilic property (obese molecules), are promiscuous and commonly
associated with increased off-target side effects [7-9].
For a while, the formulation technology deemed to be a novel alternative to solubilize
fatty molecules allowing their oral absorption [10]. Yet, the advancement in this field led to a
remarkable diminishment in the number of failures related to poor pharmacokinetic profiles [11].
Unfortunately, forcing fatty molecules to enter our systemic circulation and improving the
overall oral bioavailability is only the prominent side of the story. The other dark side of the
story is the excretion step in which the metabolic enzymes have to work assiduously to polarize
these fatty xenobiotics in order to easily excrete them [12]. As a result, the significant decrease in
failures due to pharmacokinetic problems was encountered by a sudden increase of attritions
connected with toxicological aspects and the overall attrition rates from clinical trials are almost
at the same level [11, 13, 14]. Therefore, there is a consensus among big pharmaceutical
developers that “controlling physicochemical properties is beneficial in identifying compounds
of candidate drug qualities” [11].
Phenylthiazole antibiotics, discovered by our group [15-21], originally suffered from the
metabolic instability issue [15, 22]. Identifying the part of the lead compound 1a that is exposed

to rapid metabolism, provided a route to synthesize two sets of metabolically-stable analogs; i.e.,
the alkoxylphenylthiazoles [22, 23] and alkynylphenylthiazoles [17] (Figure 1). The structure-
activity relationships (SAR) of both groups indicated that further addition of methylene units at
the lipophilic tail significantly enhanced the antibacterial activity [17, 23] (Figure 1). The
improvement of the antibacterial activity was combined with a deterioration in drug-likeness.
Hence, the alkynylphenylthiazole, for instance, could not be tested in an animal model in a dose
of more than 40% the therapeutic one due to solubility issue [17].
Figure 1. Progress of phenylthiazole antibiotics development and the general idea of the present
work.
Taking into account the considerations of compounds’ lipophilicity and their impact on
drug-likeness in progressing the development of phenylthiazole antibiotics, this article aimed at
controlling the molecular obesity of phenylthiazole derivatives via inserting a polar atom within
the lipophilic tail connected with the phenyl ring. All designed compounds shown in Figure 1

were then subjected to comprehensive bacteriological profiling versus 15 clinical MDR-
pathogens. In addition, the key physicochemical properties and in vivo pharmacokinetic profile
of the most promising derivative were also investigated.
2
. Results and Discussion
2
.1. Chemistry. The alkynyl derivatives were accessed via two synthetic routes. The first one
included tethering the bromobutyl moiety to the phenylthiazole core and then replacing the
bromine atom with cyclic amines. In the second approach, the -alkynylamines were prepared
N

first and then connected with the phenylthiazole core using traditional Sonogashira C-C cross
coupling reaction conditions. Both routes led us to the desired intermediates 5; however, the
second approach was with better yields, in general (Scheme 1). The final designed derivatives 8-
2
1 were obtained by allowing compounds 5 to react with aminoguanidine in the presence of
catalytical amount of hydrochloric acid.

The p-hydroxyphenylthiazole 22 was prepared as reported [22] and then allowed to react
with bromochloroalkane under a mild nucleophilic substitution reaction conditions to yield
compounds 23 (Scheme 2). The chlorine atom was them replaced with a proper cyclic amine and
the final products were obtained as described previously in Scheme 1.
2
2
.2. Biological Results and Discussion.
.2.1. Antibacterial activity of new analogs against MRSA. Iinitial screening of the newly
prepared compounds 8-21 and 63-101 against MRSA USA300, one of the most clinically
important and highly-pathogenic MDR-pathogen, [24-26] resulted in only four derivatives
(compounds 8, 9, 11 and 12) with promising antibacterial activity (Table 1S). The active
derivatives (MIC values g/mL) are all belonging to the alkynylphenylthiazole series. None
≤4 µ
of the alkoxyphenylthiazole derivatives 63-101 showed any promising antibacterial activity;
however, the ones with shorter linker and smaller cyclic amines demonstrated moderate potency
with MIC value of 8-16 µg/mL (Table 1S).
The SAR of this new set of alkynylphenylthiazole analogs seemed to be very
straightforward as the antibacterial potency was directly correlated to the ring size. In this vein,
the smaller ring (azetidine, compound 8) provided the most potent compound in this series with
only one-fold higher MIC value than vancomycin (MIC value of 8 is 2
µg/mL & MIC of
vancomycin is 1 g/mL; Table 1S). The promising antibacterial activity of compound 8 was
µ
further confirmed against a panel of clinically relevant MDR-staphylococcal strains including
linezolid-resistant S. aureus (MRSA NRS119) and two vancomycin-resistant strains (VRSA 10
and VRSA 12), in addition to a couple of methicillin-sensitive strains (MSSA) (Table 1). Briefly,
compound 8 was equipotent to vancomycin against MSSA NRS107 and one-fold less active than
vancomycin against most other tested MRSA strains with MIC value of 4 µg/mL. Moreover, the

azetidine-containing compound 8 maintained its potency when tested against linezolid-resistant
and vancomycin-resistant S. aureus strains (MRSA NRS119, VRSA 10 and VRSA 12) (Table 1).
Table 1. Antibacterial activity (MICs and MBCs in µg/mL) of alkynylphenylthiazoles 8, 9, 11
and 12 against a panel of clinical staphylococcal strains.
Compounds/ Control antibiotics
Bacterial Strains
8
9
11
12
Linezolid
Vancomycin
MSSA ATCC 6538
MSSA NRS 107
MRSA NRS119
MRSA NRS123
2
2
2
2
2
4
2
2
4
8
8
4
4
4
4
4
4
4
4
4
4
4
8
8
4
4
8
8
8
4
8
4
4
4
4
4
4
4
32
16
16
32
32
16
16
32
32
4
4
8
4
4
4
4
4
4
16
8
1
1
16
64
1
2
2
2
8
32
8
64
1
> 64
32
1
2
4
1
1
(
USA400)
MRSA NRS384
USA300)
MRSA NRS 385
4
16
16
16
8
1
32
1
1
(
4
1
32
1
1
(
USA500)
MRSA NRS 386
8
2
64
1
1
(
USA700)
VRSA 10
8
1
> 64
64
64
64
> 64
64
VRSA 12
16
16
1
MSSA, methicillin-sensitive Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; VRSA,
vancomycin-resistant Staphylococcus aureus
Next, increasing ring size of the terminal cyclic amine to five units provided two active
derivatives 11 (with thiazolidine ring) and 12 (with imidazole ring). Both are one- to two-fold
less active than the azetidine-containing compound 8, against most of the tested strains, with
MIC values ranging from 4 to 8
µg/mL (Table 1). Compounds with larger nitrogenous rings; 6 or
larger units are all with very mild antibacterial activity (Table 1S).

Table 2. Key physicochemical properties of compound 8 and compound 1d
Compound 8
2.68
89.89
Previously prepared compound 1d [17]
5.23
86.62
1
cLogP
tPSA
1
2
Aqueous Solubility 0.5 mM
< 5 µM
1
2
calculated values using ChemBioDraw Ultra 14.0; measured in phosphate buffer pH 6.8
Table 3. Compound 8 plasma pharmacokinetic parameters following a single oral
dose (50 mg/Kg) to rats
Cmax
tmax
AUC
CL
thalf
Vd
(µg/mL)
(h)
((h)*(ng/mL))
(L/h)
(h)
(L)
1
5.6
1.0
161840
0.31
2.9
1.29
Taking the antibacterial data together, and before going deeper in comprehensive
biological studies, compound 8 was selected to test our hypothesis, whether the new chemical
modification presented in this study would improve the overall PK profile of phenylthiazole
compounds or not. It is worth mentioning that both alkoxythiazole 1b and alkynyl derivative 1c
were orally unavailable, and suffered from severe aqueous solubility issue, in which they even
could not be formulated in an injectable form [17, 23]. Measurements of physicochemical
properties indicate that compound 8 is with optimum values for orally administrated
therapeutics, in which its partition coefficient value (cLog P) is 2.68 and its total polar surface
2
area (tPSA) value is of 90 Å (Table 2). Notably, the tPSA value of compound 8 is very close to
that of 1d, which has the same number of carbons; however, inserting a single nitrogen atom to
the lipophilic side chain of 1d magnificently reduced its cLogP value to the half (Table 2). As a
result, the aqueous solubility of compound 8 was enhanced by a factor of more than one hundred
(Table 2).

With lower lipophilic property and descent aqueous solubility, compound 8 was easily
formulated, in a relatively high dose (50 mg/kg) and administrated orally to rats. The
experimental in vivo PK data of a single oral dose showed that compound 8 is well absorbed
from the gut and reached the maximum plasma concentration (Cmax) within only one hour.
Interestingly, the plasma concentration maintained over its maximum value of MIC (4
for more than 12-hours (Figure 1S). The other key preliminary PK data indicated that compound
is metabolically stable (t1/2 = 2.9 h) with low clearance rate (less than 1 L/h) and it is very well
µg/mL)
8
distributed as indicated by its volume of distribution value (Table 3).
Our initial analysis of the metabolites using LC/MS/MS indicated that compound 8 is
mainly metabolized by oxidation to M+18 metabolite, and the oxidation step occurs most
probably at the azetidine ring (data is not shown). Therefore, compound 9, as a potential main
metabolite for 8, was prepared, as the only hydroxylated derivative; however, it was not in the
first set of synthesized alkynylphenylthiazoles. Intriguing, the main potential metabolite 9
maintained most of the antimicrobial potency of the parent compound 8, in which it showed an
MIC value of 4 µg/mL against most of the tested clinical isolates (Table 1).
Bacteriological Profiling. Next, the spectrum of antibacterial activity of the active new
compounds, including the metabolite 9, was further examined against a panel of clinically
relevant Gram-positive bacterial pathogens. The four tested compounds exhibited a potent
activity against S. epidermidis, a common colonizer of the human skin, which represents the
most common source of infections on implanted medical prosthetic devices. S. epidermidis
infections are difficult to treat due to their ability to form strong adherent biofilms that have
intrinsic resistance to antibiotics and the host defense [27, 28]. Most importantly, tested
compounds 8, 9, 11 and 12 kept their superiority over vancomycin against vancomycin-resistant

enterococci (VRE). VRE is one of the major leading cause of nosocomial infections in USA and
worldwide causing about 20-30% of hospital-acquired [29]. Moreover, according to the World of
Health Organization (WHO), vancomycin-resistant E. faecium is categorized as one of twelve
bacterial pathogens that urgently need the development of new therapeutics and alternative
strategies to combat their infections [30].
Table 4. Antibacterial activity (MICs and MBCs in µg/mL) of alkylphenylthiazoles 8, 9, 11 and
1
2 against clinically important Gram-positive bacterial pathogens including Staphylococcus
epidermidis Enterococcus faecalis Enterococcus faecium Listeria monocytogenes and
,
,
,
Streptococcus pneumoniae
.
Bacterial Strains
Compounds/control antibiotics
11 12
Linezolid
8
9
Vancomycin
Methicillin-resistant
Staphylococcus
1
4
2
16
2
8
4
16
1
16
1
2
epidermidis
NRS101
Enterococcus faecalis
ATCC 51299 (VRE)
Enterococcus faecium
4
2
16
8
8
4
16
16
8
4
32
16
8
4
32
16
1
1
16
16
32
64
1
>64
>64
ATCC 700221
(
VRE)
Listeria
4
2
8
8
4
4
32
16
4
8
32
32
8
8
32
16
≤0.5
16
32
1
1
1
2
monocytogenes
ATCC 19111
Cephalosporin-
resistant
Streptococcus
pneumoniae ATCC
1
5
1916
Methicillin-resistant
Streptococcus
2
8
4
16
8
32
4
16
1
16
2
2
pneumoniae ATCC
7
00677
1
VRE: vancomycin-resistant Enterococci
’
From Tables 2 and 4, it was noted that compounds MBC values were one- to three-folds
higher than their corresponding MICs against the tested strains indicating the compounds are

bactericidal against the tested strains. This has been further confirmed by a time kill Assay
Figure 2), which indicated that the most important compounds in this study 8 and 9. Both
(
compounds surpassed vancomycin; the drug of choice for treatment of staphylococcal infections,
in terms of the time required to exert their bactericidal activity against MRSA USA400. Briefly,
vancomycin required 12-hours to exert its bactericidal activity by causing more than 3-log
reduction in the high inoculum of the bacteria and completely eradicated the inoculum after 24-
hours. Advantageously, our newly synthesized active compounds 8, 9, 11 and 12 exhibited a
more rapid killing time than vancomycin, as they required 10 hours to exert their bactericidal
activity and reduce the high initial MRSA USA400 count by 3-log reduction. Surprisingly, both
azetidine-containing derivative 8 and its potential main metabolite 9 behave similarly in their
mode of antibacterial action, in which compound 8 required 12 hours to completely eradicate the
high MRSA count while its metabolite 9 required 10 hours to completely eradicate it. On the
other hand, five-membered containing derivatives 11 and 12 reduced the initial count by 3-log
reduction (99.9% reduction) after 10 hours but they were not able to completely eradicate it after
2
4 hours. This suggests that these two compounds will need to be frequently dosed when used
clinically especially because the bacterial count tended to remain as it is or decreased slightly
Figure 2).
(

1
0
9
8
7
6
5
4
3
2
1
0
DMSO
8
9
1
1
1
2
Vancomycin
0
2 4 6 8 10 12 14 16 18 20 22 24
Time (hours)
Figure 2. Killing kinetics of selected phenylthiazole compounds (tested in triplicates at 5 × MIC)
against methicillin-resistant Staphylococcus aureus (MRSA USA400) over a 24-hour incubation
period at 37
℃. DMSO (solvent for the compounds) served as a negative control and
vancomycin served as a control drug. The error bars represent standard deviation values obtained
from triplicate samples used for each compound/antibiotic studied.
Assessment of cytotoxicity. Selectivity towards prokaryotic cells is an essential attribute
for any antibiotic candidates. In this regard, compound 8 and its potential hydroxylated
metabolite 9 were highly tolerable to caco-2 cells at a concentration higher than 128 µg/mL as
represented by their 50% cytotoxic concentration (CC ) (Figure 3). On the other hand, the
50
imidazolyl derivative 12 was less tolerable as it was non-toxic to caco-2 cells at a concentration
as high as 64 µg/mL where about 70% of the cells were viable at this concentration (Figure 3).

Figure 3. Analyzing the cytotoxicity of tested alkynylphenylthiazoles 8, 9, 11 and 12 (tested in
triplicates at 32, 64 and 128 µg/mL) against human colorectal cells (Caco-2) using the MTS 3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
assay. Results are presented as percent viable cells relative to DMSO (negative control to
determine a baseline measurement for the cytotoxic impact of each compound). The absorbance
values represent an average of three samples analyzed for each compound. Error bars represent
standard deviation values. CC (the compound’s concentration that causes 50% cells viability)
5
0
was determined for each compound.
Figure 4. Disruption of mature MRSA USA300 biofilm by tested phenythiazoles and
vancomycin (at 2× MIC and 4× MIC). The 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 8, 9 and 12 and vancomycin
analyzed via two-way ANOVA test with post-hoc Dunnett’s test for multiple comparisons.
A second important attribute required in antibiotic drug discovery is inhibiting the
bacterial virulence factors like biofilm. Biofilm represents a shell that protects pathogens from
the effect of antibiotics. Therefore, many antibiotics currently used in the clinics are unable to
reach hidden bacteria within the biofilms [31]. Moreover, bacterial biofilm is the key source of
infections on indwelling medical devices [32, 33]. Thus, development novel antibacterial agent
with antibiofilm activity is highly advantageous. Upon testing three of our active compounds (8,
9
and 12) for their ability to eradicate pre-formed, mature staphylococcal biofilm, it was found
that they were superior to vancomycin in MRSA biofilm eradication, which disrupted about 9%
and 16% of MRSA biofilm mass at 2× MIC and 4× MIC, respectively (Figure 4). The tested
phenylthiazoles exhibited a concentration-dependent biofilm disruption activity. The most
important compound in this series (compound 8) showed the least biofilm eradication activity as
it disrupted only 31% and 41% of MRSA USA300 biofilm (around 3-times better than
vancomycin) at 2× MIC and 4× MIC, respectively. Interestingly, the potential metabolite of
compound 8 (i.e., compound 9) showed double efficacy in terms of anti-biofilm activity as it
disrupted about 72% of the pre-formed MRSA USA300 biofilm, at 4× MIC, (Figure 4) and it
was the same as 8 in terms of biofilm disruption at 2× MIC. Compound 12 exhibited the most
effective biofilm disruption activity among the tested compounds where it disrupted about 65%
and 81% of the pre-formed biofilm 2× MIC and 4× MIC, respectively.
A third important attribute for a future antibiotic is the ability to reach intracellular
pathogens as this is one of the most deceitful mechanisms of microbial virulence. Most
antibiotics are unable to target intracellular bacteria due to low levels of accumulation

intracellularly and inactivation or loss of activity due to the acidic pH within macrophages or
binding to lysosomal contents [33]. For instance, vancomycin, one of our last resorts for fighting
MDR-Gram positive infections, is unable to sufficiently accumulate inside macrophage cells and
clear the intracellular MRSA infection even after 24-hours of treatment at a high concentration as
depicted in Figure 5. On the other hand, azetidine-containing compound 8 (at 2
× MIC),
generated a 1.82-log10 reduction (equivalent to 98% reduction) of intracellular MRSA. This
increased to reach about 2.56 log reduction (equivalent to 99.7% reduction) of intracellular
1
0
MRSA when its concentration was increased to 4 × MIC. The hydroxyazetidine analog 9 was
less active as it generated a 0.31-log reduction (equivalent to 55% reduction) of intracellular
1
0
MRSA. This increased to reach about 2.14 log10 reduction (equivalent to 99% reduction) of
intracellular MRSA when the concentration was increased to 4 × MIC (Figure 5). Collectively,
these data indicated that the most promising compound 8, and its potential metabolite 9, had the
ability to gain entry into the infected macrophage cells at non-toxic concentrations, at non-toxic
concentration as indicated in Figure 3S - (4 and 8 µg/mL), and this concentration is capable of
significantly reducing the burden of MRSA inside them.
Figure 5. Examination of the activity of compounds 8 and 9 on the clearance of intracellular
MRSA present in murine macrophage (J774) cells. Data are presented as log colony forming
1
0

units of MRSA USA400 per mL inside infected murine macrophages after treatment with
2
2
×MIC and 4×MIC of either tested two compounds or vancomycin (tested in quadruplicates) for
4-hours. Data were analyzed via two-way ANOVA, with post hoc Dunnet’s multiple
comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).
Asterisks (*) represent a significant difference between the treatment of J774 cells with
compound 8 and 9 in comparison to vancomycin.
3
. Conclusion. Statistically, there is a strong connection between the high rate of drug-
candidates attrition from clinical trials and their physicochemical properties. To advance the
development of phenylthiazole antibiotics, we turned our attention to limit their lipophilic
property. In this regard, two phenylthiazole cores, one carries alkoxy moiety and the other one
with alkynyl side chain, were selected for the next round of structural optimization. Both selected
cores have the advantage of metabolic stability over the lead compound 1a. By using nitrogen-
containing cycles as part of the lipophilic part of phenylthiazole molecules, the physicochemical
properties of alkynyl-containing phenythiazoles were remarkably enhanced. Briefly, the cLogP
value of azetidine-containing derivative 8 was reduced to the half value of the corresponding
derivative 1d with the same number of carbons, resulting in more than 100-times improvement
in the aqueous solubility. Consequently, compound 8 was found to be well-orally absorbed and
reach a plasma concentration that exceeds its maximum MIC value within only few minutes.
Other advantageous PK attributes include descent metabolic stability, low rate of clearance and
good body distribution. Remarkably, the hydroxyazetidine derivative 9 was identified as the
main potential metabolite and it turned to be biologically potent with a notable antibiofilm
activity that exceeds that of the parent molecule 8. Therefore, this work suggests compound 8 as
a good therapeutic candidate for S. epidermidis and S. aureus that are significant sources of
biofilm-related infections [34]. Finally, both compounds 8 and 9 possess high ability to penetrate
inside the infected macrophages at lower concentrations capable of killing the intracellular
MRSA. Consequently, due to their efficient intracellular clearance activity, compounds 8 and 9

could be nominated as perfect therapeutic options for the treatment of pneumonia-induced
MRSA infection, which is difficult to treat using current antibiotics including vancomycin [35].
4
4
4
. Experimental
.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 dimethyl sulfoxide (DMSO- 6
d
) on a Varian Mercury VX-400 NMR
) scale.
spectrometer. Chemical shifts are given in parts per million (ppm) on the delta (
δ
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.
4
.1.2. 1-(2-(4-(4-Bromobut-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one (4). To dry
DME (5 mL) in a sealed tube, compound 3 (300 mg, 0.87 mmol), triethylamine (2 mL) were
added. After the reaction mixture was purged with dry nitrogen gas for 15 min,
dichlorobis(triphenylphosphine)palladium (II) (46 mg, 0.065 mmol), copper (I) iodide (33 mg,
0
.17 mmol) and 4-bromobut-1-yn (174 mg, 0.123 mL, 1.3 mmol) were added. The sealed tube
o
was then heated and stirred at 50 C for 24 h. and monitored by thin-layer chromatography
(TLC). After completion of the reaction, the reaction mixture was passed through a pad of silica
gel with ethylacetate to remove some insoluble salts. The desired product was obtained by silica
gel chromatography using eluent (EtOAc/ Hexane 9:1). Yellow solid (275 mg, 90.7%); mp =
1
1
45-147 °C; H NMR (DMSO-d6
)
δ: 8.03 (d,
J
= 8.4 Hz, 2H), 7.62 (d,
J
= 8.4 Hz, 2H), 6.22 (dd,

1
3
J
= 16.0, 7.0 Hz, 2H), 5.86 (dd,
J = 16.3, 6.8 Hz, 2H), 2.72 (s, 3H), 2.59 (s, 3H); C NMR
(
DMSO-d6 : 190.5, 165.1, 152.6, 133.2, 132.9, 130.3, 127.9, 122.3, 88.6, 81.7, 33.5, 30.6, 24.9,
) δ
+
+2
1
5
4
6.1; MS (m/z); 346 (M , 29.7%), 348 (M , 30.8%); Anal. Calc. for: C H BrNOS (348): C,
16 14
5.18; H, 4.05; N, 4.02%; Found: C, 55.26; H, 4.11; N, 4.09%.
.1.3. 1-(2-(4-(4-(Sec. amine derivatives-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)
ethan-1-one (5a-n). General procedure: to dry DMF (5 mL) in a round flask, compound 4 (200
mg, 0.57 mmol), anhydrous potassium carbonate (237 mg, 1.7 mmol, 3 equiv.), and appropriate
Sec. amines (2.8 mmol, 5 equiv.), with a catalytic amount of potassium iodide was heated at
o
1
10 C with stirring overnight. The reaction mixture was cooled, poured on crushed ice after
then, the organic material was extract 3 times with ethyl acetate and dried over anhydrous
sodium sulphate and concentrated over reduced pressure to get yellowish-brown oil. Yields,
physical properties, and spectral data of isolated purified products are listed below:
4
.1.3.1.
1-(2-(4-(4-(Azitidin-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one
1
(
5a). Orange oil (140 mg, 75%); H NMR (DMSO-d6
)
δ
: 8.21 (d,
= 6.8 Hz, 2H), 3.51 (t,
H), 2.58 (s, 3H), 2.11-1.98 (m, 2H); C NMR (DMSO- : 191.5, 164.8, 150.0, 133.7, 129.4,
27.2, 122.9, 120.1, 87.8, 79.9, 57.2, 49.6, 30.1, 18.8, 17.7, 16.5; MS ( ).
.1.3.2. 1-(2-(4-(4-(3-Hydroxyazitidin-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)
J
= 8.4 Hz, 2H), 7.62 (d,
J =
8
3
1
4
.4 Hz, 2H), 3.83 (t,
J
= 7.2 Hz, 4H), 3.71 (t,
J
J
= 6.4 Hz, 2H), 2.69 (s,
1
3
6
d ) δ
m\z
1
ethan-1-one (5b). Yellowish oil (163 mg, 83.5%); H NMR (DMSO-d6
)
δ
: 7.98 (d,
= 13.1, 3.4 Hz, 2H),
J = 5.2 Hz, 2H), 2.61 (s, 3H),
J = 8.4 Hz,
2
4
2
8
H), 7.56 (d,
.39 (dd, = 12.3, 3.1 Hz, 2H), 4.06 (t,
.33 (s, 3H); C NMR (DMSO-
J
= 8.4 Hz, 2H), 5.76 (brs, 1H), 4.71-4.68 (m, 1H), 4.41 (dd,
= 5.4 Hz, 2H), 2.98 (t,
J
J
J
1
3
d
6
)
δ
: 191.9, 164.7, 150.1, 134.2, 129.4, 127.4, 122.3, 121.1,
+
3.5, 79.2, 62.7, 57.2, 49.8, 30.6, 20.5, 18.3, 16.8; MS (
m
\z
); 340 (M , 26.2%).

4
.1.3.3. 1-(4-Methyl-2-(4-(4-(pyrrolidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
1
(
5c). Brown oil (138 mg, 71%); H NMR (DMSO-d6
)
δ
: 8.21 (d,
J
= 8.8 Hz, 2H), 7.61 (d,
J = 8.8
Hz, 2H), 3.63 (dd,
J
= 11.3, 5.4 Hz, 2H), 3.55-3.48 (m, 4H), 3.22 (dd,
J
= 10.3, 3.4 Hz, 2H), 2.71
1
3
(
s, 3H), 2.56 (s, 3H), 1.93-1.89 (m, 4H); C NMR (DMSO-d6
)
δ: 191.4, 167.3, 154.8, 132.7,
+
1
1
4
31.6, 127.4, 122.6, 121.8, 94.7, 83.5, 57.6, 50.9, 24.8, 22.5, 18.1, 16.7; MS (
9.9%).
m
\z
); 338 (M ,
.1.3.4. 1-(4-Methyl-2-(4-(4-(thiazolidin-3-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
1
(
5d). Yellow oil (167 mg, 81.8%); H NMR (DMSO-
d
6
)
δ
: 8.12 (d,
J
J
= 8.1 Hz, 2H), 7.66 (d,
= 6.4 Hz, 2H), 3.23 (t,
J
J
=
=
8
6
1
1
4
.1 Hz, 2H), 4.42 (s, 2H), 3.76 (dd,
.4 Hz, 2H), 2.76 (s, 3H), 2.61 (s, 3H), 2.06 (t,
J = 11.3, 5.4 Hz, 2H), 3.59 (t,
1
3
J
= 5.4 Hz, 2H); C NMR (DMSO-d6) δ: 191.6,
65.1, 150.0, 134.8, 129.6, 127.7, 122.7, 120.1, 88.4, 79.8, 66.1, 61.2, 55.3, 30.1, 25.5, 18.5,
+
6.7; MS (
m
\z
); 356 (M , 39.73%).
.1.3.5.
1-(2-(4-(4-(1
H
-Imidazol-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-
1
one (5e). Dark brown oil (143 mg, 74%); H NMR (DMSO-d6
)
δ
: 7.81 (d,
= 6.8 Hz, 1H), 6.96 (d, = 6.8 Hz, 1H), 4.55 (t,
= 9.6 Hz, 2H), 2.47 (s, 3H); C NMR (DMSO- : 191.1,
J = 8.4 Hz, 2H), 7.78
(s, 1H), 7.52 (d,
J
= 8.4 Hz, 2H), 7.32 (d,
J
J
J =
1
3
9
1
1
4
.8 Hz, 2H), 2.68 (s, 3H), 2.61 (t,
J
6
d ) δ
63.4, 154.7, 142.3, 140.4, 138.2, 129.8, 127.8, 127.3, 121.9, 119.2, 90.2, 79.7, 46.1, 32.2, 24.4,
+
7.7; MS (
m
\z
); 335 (M , 43.29%).
.1.3.6. 1-(4-Methyl-2-(4-(4-(piperidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
1
(
5f). Yellow oil (170 mg, 84%); H NMR (DMSO-d6
)
δ
: 7.85 (d,
= 6.4 Hz, 4H), 2.75 (s, 3H), 2.68 (t,
H), 2.52 (s, 3H), 1.67-1.49 (m, 6H); C NMR (DMSO- : 193.8, 163.2, 153.6, 145.8, 132.4,
J
= 8.2 Hz, 2H), 7.51 (d,
J =
8
2
.2 Hz, 2H), 3.81 (t,
J
= 6.8 Hz, 2H), 3.56 (t,
J
J
= 5.7 Hz,
1
3
6
d ) δ

1
30.6, 127.5, 122.1, 118.6, 89.8, 82.3, 60.1, 49.2, 26.4, 25.1, 24.4, 18.3, 17.8; MS (m\z); 352
+
(
M , 39.17%).
4
.1.3.7. 1-(4-Methyl-2-(4-(4-(2-methylpiperidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)ethan
1
-
1-one (5g). Brown oil (183 mg, 87%); H NMR (DMSO-d6
)
δ
: 7.88 (d,
J = 8.4 Hz, 2H), 7.33 (d,
J
2
d
2
4
= 8.2 Hz, 2H), 3.53-3.50 (m, 2H), 2.71 (t,
.52 (s, 3H), 1.98-1.89 (m, 2H), 1.58-1.27 (m, 6H), 0.85 (d,
: 192.1, 168.4, 158.7, 146.8, 143.3, 131.6, 129.8, 126.8, 87.7, 82.3, 62.6, 55.3, 53.7, 33.6,
J = 5.4 Hz, 2H), 2.65 (s, 3H), 2.62-2.59 (m, 1H),
1
3
J
= 6.8 Hz, 3H); C NMR (DMSO-
6
) δ
+
7.4, 24.6, 22.6, 18.3, 17.6, 16.1; MS (m\z); 366 (M , 49.21%).
.1.3.8. 1-(4-Methyl-2-(4-(4-(3-methylpiperidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)ethan
1
-
1-one (5h). Yellow oil ( 190 mg, 90%); H NMR (DMSO-d6
)
δ
: 7.97 (d,
J
= 8.8 Hz, 2H), 7.52
(d,
J = 8.4 Hz, 2H), 4.08-3.98 (m, 2H), 3.63-3.57 (m, 2H), 3.25-3.22 (m, 2H), 2.88-2.81 (m, 2H),
1
3
2
1
2
4
.76 (s, 3H), 2.62 (s, 3H), 1.75-1.46 (m, 5H), 1.26 (d,
J = 6.4 Hz, 3H); C NMR (DMSO-d6) δ:
91.4, 168.9, 158.1, 146.5, 142.2, 131.7, 129.9, 125.8, 87.4, 81.7, 61.5, 55.1, 48.3, 32.6, 26.2,
+
4.7, 21.6, 18.8, 18.1, 16.8; MS (
m
\z
); 366 (M , 48.61%).
.1.3.9. 1-(4-Methyl-2-(4-(4-(4-methylpiperidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)ethan
1
-
1-one (5i). Light-yellow oil ( 194 mg, 92%); H NMR (DMSO-
d
6
)
δ
: 7.88 (d,
J
= 8.4 Hz, 2H),
7
2
1
1
4
.51 (d,
J = 8.4 Hz, 2H), 3.53-3.51 (m, 2H), 3.02-2.94 (m, 2H), 2.81-2.76 (m, 4H), 2.61 (s, 3H),
1
3
.31 (s, 3H), 2.24 (m, 4H), 1.61-1.11 (m, 1H), 0.90 (d,
J
= 6.4 Hz, 3H); C NMR (DMSO-d6) δ:
91.3, 161.4, 158.4, 144.3, 136.6, 132.8, 126.7, 124.5, 88.6, 84.9, 58.2, 47.6, 34.8, 30.2, 22.4,
+
8.1, 17.3, 16.4; MS (
m
\z
); 366 (M , 55.61%).
.1.3.10.
1-(4-Methyl-2-(4-(4-(4-methylpiperazin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)
1
ethan-1-one (5j). Orange oil (153 mg, 72.8%); H NMR (DMSO-
d
6
)
δ
: 7.76 (d,
J
= 8.4 Hz, 2H),
7
.48 (d, J = 8.4 Hz, 2H), 3.65 (s, 4H), 3.48-3.29 (m, 4H), 3.24 (s, 4H), 2.71 (s, 3H), 2.41 (s, 3H),

1
3
2
8
4
.24 (s, 3H); C NMR (DMSO-
d
6
)
δ
: 191.8, 167.3, 158.6, 148.3, 132.6, 132.3, 127.4, 122.5,
+
8.5, 84.3, 57.5, 54.7, 53.5, 44.1, 26.3, 21.4, 18.7; MS (
m
\z
); 367 (M , 19.14%).
.1.3.11. 1-(4-Methyl-2-(4-(4-(4-morpholinobut-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-one
1
(
5k). Brown oil (148 mg, 73%); H NMR (DMSO-d6
)
δ
: 7.76 (d,
J
= 8.2 Hz, 2H), 7.48 (d,
J
=
8
3
5
4
.2 Hz, 2H), 3.83 (t, J = 6.4 Hz, 4H), 3.63-3.51 (m, 6H), 2.76-2.67 (m, 2H), 2.51 (s, 3H), 2.33 (s,
1
3
H); C NMR (DMSO-d6
)
δ
: 191.6, 168.4, 158.6, 148.3, 133.2, 132.9, 127.5, 122.7, 96.6, 84.8,
+
7.3, 55.4, 48.4, 26.2, 21.5, 18.3; MS (
m
\z
); 354 (M , 9.2%).
.1.3.12. 1-(4-Methyl-2-(4-(4-(4-thiomorpholinobut-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-1-
1
one (5 l). Yellow oil ( 152 mg, 71%); H NMR (DMSO-
d
6
)
δ: 7.76 (d,
J = 8.2 Hz, 2H), 7.48 (d, J
=
8.2 Hz, 2H), 4.23 (t,
J
= 6.4 Hz, 4H), 3.79-3.75 (m, 2H), 2.53 (t,
J
= 6.4 Hz, 4H), 2.76-2.71 (m,
1
3
2
1
4
H), 2.51 (s, 3H), 2.39 (s, 3H); C NMR (DMSO-d6
)
δ
: 191.6, 168.4, 158.9, 148.3, 133.8, 133.1,
+
27.6, 122.2, 96.4, 84.6, 58.5, 55.7, 34.8, 26.5, 21.1, 18.3; MS (
.1.3.13.
m
\z
); 370 (M , 14.4%).
1-(2-(4-(4-(Azepan-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one
1
(
5m). Dark-brown oil (143 mg, 68%); H NMR (DMSO-d6
)
δ: 7.88 (d,
J = 8.4 Hz, 2H), 7.52 (d,
J
= 8.4 Hz, 2H), 3.73 (t,
.49 (s, 3H), 1.61-0.76 (m, 8H); C NMR (DMSO-
J = 5.4 Hz, 4H), 3.61-3.52 (m, 2H), 2.71 (s, 3H), 2.61-2.58 (m, 2H),
1
3
2
1
4
5
2
3
1
1
d
6
)
δ
: 191.5, 167.9, 158.6, 148.8, 133.7,
+
33.2, 127.2, 122.8, 96.5, 84.6, 56.1, 47.1, 34.4, 25.9, 16.8, 14.7; MS (
m
\z
); 366 (M , 8.17%).
.1.3.14. 1-(4-Methyl-2-(4-(4-(octahydroisoquinolin-2(1
H)-yl)but-1-yn-1-yl)phenyl)thiazol-
1
-yl)ethan-1-one (5n). Brown oil (175 mg, 75%); H NMR (DMSO-d6
)
δ
: 7.88 (d, = 8.4 Hz,
J
H), 7.51 (d,
J = 8.4 Hz, 2H), 3.51-3.48 (m, 2H), 3.23-3.18 (m, 2H), 2.77-2.64 (m, 4H), 2.56 (s,
1
3
H), 2.26 (s, 3H), 2.20-0.89 (m, 12H); C NMR (DMSO-d6
) δ: 191.1, 167.4, 162.6, 148.5,
46.4,131.7, 128.3, 120.2, 92.2, 80.8, 61.4, 59.8, 47.1, 36.3, 34.9, 33.7, 32.4, 26.5, 25.1, 23.7
+
9.1, 16.5; MS (
m
\z
); 406 (M , 20.6%).

4
.1.4. 2-(1-(2-(4-(3-(Substituted-sec-amine-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)
ethylidene)-1-carboximidamide (8-21). General procedure: Thiazole derivatives 5a-n (0.57-
.64 mmol) were dissolved in absolute ethanol (20 mL), concentrated hydrochloric acid (1 mL),
0
aminoguanidine hydrochloride (355 mg, 3.2 mmol, 5 equiv.), were added. The reaction mixture
was heated at reflux for 4 h. The solvent was concentrated under reduced pressure, then poured
in crushed ice and neutralized with sodium carbonate to pH 7-8, and the formed precipitated
solid was collected by filtration, washed with copious amount of water. Crystallization from
ethylacetate afforded the desired products as solids.
4
.1.4.1. 2-(1-(2-(4-(4-(Azetidin-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (8). Orange solid (176 mg, 75%); mp = 246-247 °C. H NMR
(
DMSO -d6
)
δ
: 8.05 (d,
J
= 8.4 Hz, 2H), 7.61 (d,
J
= 8.4 Hz, 2H), 5.77 (brs, 2H), 5.75 (brs, 2H),
= 6.4 Hz, 2H), 2.74 (s, 3H), 2.55 (s,
3
3
1
.98 (t,
J
= 5.2 Hz, 4H), 3.87 (t,
J
= 6.4 Hz, 2H), 3.51 (t, J
1
3
H), 2.13-1.99 (m, 2H); C NMR (DMSO-d6
)
δ: 163.3, 158.4, 151.6, 145.2, 133.7, 133.1, 126.3,
+
24.7, 117.5, 93.2, 82.4, 61.1, 48.6, 24.8, 23.9, 18.3, 17.8; HRMS (EI) m/z 380.1785 M , calc.
for C20
H
24
N
6
S 380.1783; Anal. Calc. for: C20
Found: C, 63.23; H, 6.44; N, 22.19%.
.1.4.2. 2-(1-(2-(4-(4-(3-Hydroxyazetidin-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)
24 6
H N S (380): C, 63.13; H, 6.36; N, 22.09%;
4
ethylidene)hydrazine-1-carboximidamide (9). Orange solid (175 mg, 75%); mp = 255-256 °C.
1
H NMR (DMSO - 6
brs, 3H), 5.61 (brs, 1H), 4.65-4.58 (m, 1H), 4.15 (dd,
1.2, = 3.2 Hz, 2H), 2.88-2.83 (m, 2H), 2.53 (s, 3H), 2.34 (s, 3H), 1.77-1.72 (m, 2H); C NMR
DMSO-
d
)
δ
: 11.12 (brs, 1H), 7.86 (d,
J
= 8.4 Hz, 2H), 7.11 (d,
J = 8.4 Hz, 2H), 5.91
(
J
= 12.2, = 5.2 Hz, 2H), 3.98 (dd,
J
J =
1
3
1
J
(
d
6
)
δ
: 163.6, 158.2, 151.3, 145.1, 133.7, 133.2, 127.3, 124.5, 118.1, 89.2, 81.4, 64.6,
+
5
9.7, 48.8, 23.7, 23.1, 18.3, 16.9; HRMS (EI) m/z 396.1745 M , calc. for C20
H
24
N
6
OS 396.1732;

24 6
Anal. Calc. for: C20H N OS (396): C, 60.58; H, 6.10; N, 21.20%; Found: C, 61.04; H, 6.17; N,
2
1.28%.
4
.1.4.3.
2-(1-(4-Methyl-2-(4-(4-(pyrrolidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-
yl)ethylidene) hydrazine-1-carboximidamide (10). Brown solid (171 mg, 73%); mp = 241-240
1
°
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),
1
3
5
.66 (brs, 2H), 3.76 (s, 2H), 2.59 (m, 4H), 2.57 (s, 3H), 2.31 (s, 3H), 1.73 (m, 4H); C NMR
(DMSO-
d
6
)
δ: 161.57, 160.24, 148.44, 142.99, 136.48, 133.36, 132.27, 126.22, 124.14, 88.99,
+
8
3
6
4
4.49, 52.43, 43.89, 23.77, 18.83, 16.55; HRMS (EI) m/z 394.1933 M , calc. for C21
H
26
N
6
S
94.1940; Anal. Calc. for: C21
.37; N, 22.11%.
26 6
H N S (394): C, 63.13; H, 6.36; N, 22.09%; Found: C, 63.16; H,
.1.4.4.
2-(1-(4-Methyl-2-(4-(4-(thiazolidin-3-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)ethylid-
1
ene)hydrazine-1-carboximidamide (11). Brown solid (186 mg, 80%); mp = 235-237 °C. H
NMR (DMSO - 6
d
)
δ
: 7.96 (d,
J
= 8.4 Hz, 2H), 7.49 (d,
= 6.4 Hz, 2H), 3.11-3.08 (m, 2H), 2.72 (s, 3H),
= 6.4 Hz, 2H); C NMR (DMSO- : 163.5, 158.6, 151.6, 145.3, 133.1,
J = 8.4 Hz, 2H), 5.67 (brs, 2H), 5.53 (brs,
2
2
1
4
5
4
H), 3.97 (s, 2H), 3.68-3.61 (m, 2H), 3.53 (t,
J
1
3
.53 (s, 3H), 1.97 (t,
J
6
d ) δ
32.8, 126.2, 124.4, 117.7, 91.4, 79.5, 66.2, 65.5, 52.3, 33.7, 21.4, 18.4, 16.5; HRMS (EI) m/z
+
12.1491 M , calc. for C H N S 412.1504; Anal. Calc. for: C H N S (412): C, 58.22; H,
20
24
6
2
20 24
6 2
.86; N, 20.37%; Found: C, 58.29; H, 5.95; N, 20.42%.
.1.4.5. 2-(1-(2-(4-(4-(1
H-Imidazol-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethylid-
1
ene)hydrazine-1-carboximidamide (12). Brown solid (177 mg, 76%); mp = 201-202 °C. H
NMR (DMSO - 6
d
)
δ
: 11.23 (brs, 1H), 8.01 (d,
= 7.2 Hz, 1H), 7.02 (d, = 7.2 Hz, 1H), 5.68 (brs, 3H), 3.82 (t,
.52 (s, 3H), 2.48 (t, = 5.6 Hz, 2H), 2.31 (s, 3H); C NMR (DMSO-
J
= 8.4 Hz, 2H), 7.83 (s, 1H), 7.61 (d,
= 6.2 Hz, 2H),
: 163.5, 158.6, 151.3,
J = 8.8 Hz,
2
2
H), 7.38 (d,
J
J
J
13
J
6
d ) δ

1
45.1, 138.3, 134.7, 133.7, 132.8, 126.4, 124.5, 118.6, 116.5, 91.2, 79.7, 45.3, 21.6, 18.5, 16.8;
+
HRMS (EI) m/z 391.1595 M , calc. for C H N S 391.1579; Anal. Calc. for: C H N S (391):
2
0
21
7
20 21
7
C, 61.36; H, 5.41; N, 25.04%; Found: C, 61.42; H, 5.48; N, 25.10%.
4
.1.4.6. 2-(1-(4-Methyl-2-(4-(4-(piperidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (13). Yellow solid (196 mg, 85%); mp = 245-246 °C. H NMR
(DMSO -d6
) δ: 11.03 (brs, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.80 (brs, 3H), 7.67 (d, J = 8.4 Hz, 2H),
3
.72-3.67 (m, 4H), 3.56-3.48 (m, 2H), 2.73 (s, 3H), 2.68-2.62 (m, 2H), 2.52 (s, 3H), 1.75-1.56
1
3
(
m, 6H); C NMR (DMSO-
d
6
)
δ
: 163.4, 158.3, 151.6, 145.6, 136.7, 133.9, 133.2, 126.8, 118.1,
+
9
1.3, 79.6, 63.5, 59.6, 27.8, 27.2, 20.9, 18.5, 16.8; HRMS (EI) m/z 408.2099 M , calc. for
C H N S 408.2096; Anal. Calc. for: C H N S (408.5): C, 64.68; H, 6.91; N, 20.57%; Found:
22
28
6
22 28
6
C, 64.75; H, 6.98; N, 20.66%.
.1.4.7. 2-(1-(4-Methyl-2-(4-(4-(2-methylpiperidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)
4
ethylidene)hydrazine-1-carboximidamide (14). Yellow solid (196 mg, 84%); mp = 235-236
1
°
C. H NMR (DMSO -d6
)
δ
: 11.12 (brs, 1H), 7.88 (d,
J
= 8.4 Hz, 2H), 7.31 (d,
J
= 8.4 Hz, 2H),
5
.61 (brs, 3H), 3.55-3.49 (m, 2H), 2.82-2.76 (m, 1H), 2.71-2.69 (m, 2H), 2.68 (s, 3H), 2.59-2.53
13
(m. 2H), 2.48 (s, 3H), 1.96-1.48 (m, 6H), 0.85 (d,
J
= 4.8 Hz, 3H); C NMR (DMSO-
6
d ) δ:
1
2
63.6, 158.5, 151.3, 145.4, 136.7, 134.2, 133.8, 126.7, 117.2, 91.6, 79.7, 63.4, 54.8, 53.9, 27.5,
+
6.8, 23.7, 20.3, 18.7, 16.4; HRMS (EI) m/z 422.2260 M , calc. for C H N S 422.2253; Anal.
2
3
30
6
Calc. for: C H N S (422.5): C, 65.37; H, 7.16; N, 19.89%; Found: C, 65.45; H, 7.22; N,
2
3
30
6
1
9.95%.
4
.1.4.8.
2-(1-(4-Methyl-2-(4-(4-(3-methylpiperidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)
ethylidene)hydrazine-1-carboximidamide (15). Yellow solid (203 mg, 88%); mp = 257-259
1
°
C. H NMR (DMSO -
d
6
)
δ: 10.87 (brs, 1H), 7.92 (d,
J
= 8.4 Hz, 2H), 7.82 (brs, 2H), 7.76 (d,
J
=

8
.4 Hz, 2H), 6.84 (brs, 1H), 4.05-3.89 (m, 2H), 3.62-3.51 (m, 2H), 3.25-3.20 (m, 2H), 2.85 (d,
J
=
9.6 Hz, 2H), 2.79 (s, 3H), 2.75 (s, 3H), 1.86 (m, 1H), 1.58-1.41 (m, 4H), 1.31 (d, = 6.4 Hz,
J
1
3
3
7
H); C NMR (DMSO-d6
)
δ
: 163.5, 158.6, 152.3, 145.1, 136.4, 133.8, 133.1, 127.3, 117.7, 91.4,
+
9.9, 63.2, 54.2, 53.8, 30.1, 27.6, 24.1, 22.6, 18.5, 16.9; HRMS (EI) m/z 422.2257 M , calc. for
C H N S 422.2253; Anal. Calc. for: C H N S (422.5): C, 65.37; H, 7.16; N, 19.89%; Found:
23
30
6
23 30
6
C, 65.48; H, 7.25; N, 19.99%.
.1.4.9. 2-(1-(4-Methyl-2-(4-(4-(4-methylpiperidin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)
4
ethylidene)hydrazine-1-carboximidamide (16). Yellow solid (212 mg, 92%); mp = 258-260
1
°
C. H NMR (DMSO -
d
6
)
δ
: 7.88 (d,
J
= 8.2 Hz, 2H), 7.53 (d,
J
= 8.2 Hz, 2H), 5.71 (brs, 2H),
5
3
1
2
.53 (brs, 2H), 3.62-3.51 (m, 2H), 3.03-2.92 (m, 2H), 2.63-2.51 (m, 4H), 2.59 (s, 3H), 2.31 (s,
13
H), 2.22-2.16 (m, 4H), 1.61-1.18 ( m, 1H), 0.91 (d,
J = 6.4 Hz, 3H); C NMR (DMSO-d6) δ:
63.6, 158.9, 152.2, 145.1, 133.2, 132.8, 127.4, 124.9, 117.7, 92.3, 90.8, 57.5, 43.4, 34.9, 31.6,
+
3.4, 21.8, 18.6, 16.4; HRMS (EI) m/z 422.2263 M , calc. for C H N S 422.2253; Anal. Calc.
2
3
30
6
for: C H N S (422.5): C, 65.37; H, 7.16; N, 19.89%; Found: C, 65.47; H, 7.22; N, 19.99%.
2
3
30
6
4
.1.4.10. 2-(1-(4-Methyl-2-(4-(4-(4-methylpiperazin-1-yl)but-1-yn-1-yl)phenyl)thiazol-5-yl)
ethylidene)hydrazine-1-carboximidamide (17). Orange solid (166 mg, 72%); mp = 233-235
1
°
C. H NMR (DMSO -d6
)
δ
: 7.79 (d,
J
= 8.8 Hz, 2H), 7.48 (d,
J
= 8.8 Hz, 2H), 5.77 (brs, 2H),
5
3
1
.65 (brs, 2H), 3.81-3.75 (m, 4H), 3.48-3.38 (m, 2H), 3.21-3.18 (m, 4H), 2.72 (s, 3H), 2.52 (s,
13
H), 2.41-2.38 (m, 2H), 2.25 ( s, 3H); C NMR (DMSO-d6) δ: 163.8, 158.6, 152.5, 145.3, 133.8,
33.1, 127.2, 124.3, 117.7, 87.7, 81.1, 59.9, 58.7, 56.3, 46.7, 22.2, 19.7, 18.5, 16.6; HRMS (EI)
+
m/z 423.2218 M , calc. for C H N S 423.2205; Anal. Calc. for: C H N S (423.5): C, 62.38;
22
29
7
22 29
7
H, 6.90; N, 23.15%; Found: C, 62.55; H, 6.99; N, 23.25%.

4
.1.4.11.
2-(1-(4-Methyl-2-(4-(4-morpholinobut-1-yn-1-yl)phenyl)thiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (18). Brown solid (192 mg, 83%); mp = 250-253 °C. H NMR
(DMSO -d6
)
δ
: 7.77 (d,
J = 7.2 Hz, 2H), 7.45 (d, J = 7.2 Hz, 2H), 5.65 (brs, 2H), 5.48 (brs, 2H),
3
3
1
.85-3.76 (m, 2H), 3.65-3.60 (m, 4H), 3.58-3.52 (m, 4H), 2.79-2.7 (m, 2H), 2.53 (s, 3H), 2.32 (s,
1
3
H); C NMR (DMSO-d6
17.6, 88.4, 80.6, 66.7, 62.3, 52.8, 22.2, 19.5, 18.5; HRMS (EI) m/z 410.1882 M , calc. for
OS 410.1889; Anal. Calc. for: C21 OS (410.5): C, 61.44; H, 6.38; N, 20.47%;
Found: C, 61.61; H, 6.46; N, 20.57%.
.1.4.12. 2-(1-(4-Methyl-2-(4-(4-thiomorpholinobut-1-yn-1-yl)phenyl)thiazol-5-yl)
) δ: 163.8, 158.9, 152.3, 145.2, 133.7, 133.5, 132.9, 126.5, 124.9,
+
C
21
H
26
N
6
26 6
H N
4
ethylidene)hydrazine-1-carboximidamide (19). Yellow solid (178 mg, 77%); mp = 265-266
1
°
C. H NMR (DMSO -d6
)
δ
: 7.78 (d,
J
= 8.4 Hz, 2H), 7.48 (d,
J
= 8.4 Hz, 2H), 5.71 (brs, 2H),
5
.68 (brs, 2H), 4.21-4.11 (m, 4H), 3.83-3.72 (m, 2H), 3.62-3.52 (m, 4H), 2.79-2.69 (m, 2H), 2.53
1
3
(
s, 3H), 2.31 (s, 3H); C NMR (DMSO-d6
)
δ
: 163.7, 158.3, 152.5, 145.2, 133.7, 133.1, 126.8,
+
1
24.5, 117.9, 88.4, 80.2, 62.3, 58.9, 33.1, 22.2, 18.9, 16.7; HRMS (EI) m/z 426.1658 M , calc.
for C21
H
26
N
6
S
2
426.1660; Anal. Calc. for: C21
26 6 2
H N S (426.6): C, 59.13; H, 6.14; N, 19.70%;
Found: C, 59.21; H, 6.23; N, 19.85%.
4
.1.4.13. 2-(1-(2-(4-(4-(Azepan-1-yl)but-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (20). Brown solid (166 mg, 72%); mp = 265-267 °C. H NMR
(
DMSO -d6
.80 (brs, 1H), 3.92 (t,
s, 3H), 1.76-1.25 (m, 8H); C NMR (DMSO-d6
27.4, 124.3, 117.5, 91.6, 90.0, 58.6, 47.6, 32.1, 30.1, 23.2, 18.4, 16.7; HRMS (EI) m/z 422.2238
)
δ
: 11.12 (brs, 1H), 7.77 (d,
J = 8.4 Hz, 2H), 7.74 (brs, 2H), 7.32 (d, J = 8.4 Hz, 2H),
6
J
= 6.8 Hz, 4H), 3.61-3.51 (m, 2H), 2.56 (s, 3H), 2.49-2.33 (m, 2H), 2.29
1
3
(
) δ: 164.2, 158.9, 151.8, 145.1, 133.7, 132.9,
1