Second-Generation Phenylthiazole Antibiotics with Enhanced Pharmacokinetic Properties

Article abstract of DOI:10.1021/acs.jmedchem.6b00233

A series of second-generation analogues for 2-(1-(2-(4-butylphenyl)-4-methylthiazol-5-yl)ethylidene)aminoguanidine (1) have been synthesized and tested against methicillin-resistant Staphylococcus aureus (MRSA). The compounds were designed with the object

Full text of DOI:10.1021/acs.jmedchem.6b00233

Article
pubs.acs.org/jmc
Second-Generation Phenylthiazole Antibiotics with Enhanced
Pharmacokinetic Properties
Mohammed A. Seleem,† Ahmed M. Disouky,† Haroon Mohammad,‡ Tamer M. Abdelghany,§
Ahmed S. Mancy,∥ Sammar A. Bayoumi,⊥ Ahmed Elshafeey,#,∇ Ahmed El-Morsy,†
Mohamed N. Seleem,‡,⊗ and Abdelrahman S. Mayhoub*,†
†Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
‡Department of Comparative Pathobiology, Purdue University, College of Veterinary Medicine, West Lafayette, Indiana 47907,
United States
§Department of Pharmacology, College of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
∥Department of Clinical Pharmacy, College of Pharmacy, Heliopolis University, Cairo 11777, Egypt
⊥Department of Pharmaceutics, College of Pharmacy, Heliopolis University, Cairo 11777, Egypt
#Bioequivalence Section, Genuine Research Center, Heliopolis, Cairo 11757, Egypt
∇Department of Pharmaceutics & Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-Ainy, Cairo 11562, Egypt
⊗Purdue Institute for Inflammation, Immunology, and Infectious Diseases, Purdue University, West Lafayette, Indiana 47907, United
States
S
* Supporting Information
ABSTRACT: A series of second-generation analogues for 2-(1-(2-(4-butylphenyl)-4-methylthiazol-5-yl)ethylidene)amino-
guanidine (1) have been synthesized and tested against methicillin-resistant Staphylococcus aureus (MRSA). The compounds
were designed with the objective of improving pharmacokinetic properties. This main aim has been accomplished by replacing
the rapidly hydrolyzable Schiff-base moiety of first-generation members with a cyclic, unhydrolyzable pyrimidine ring. The
hydrazide-containing analogue 17 was identified as the most potent analogue constructed thus far. The corresponding amine 8
was 8 times less active. Finally, incorporating the nitrogenous side chain within an aromatic system completely abolished the
antibacterial character. Replacement of the n-butyl group with cyclic bioisosteres revealed cyclohexenyl analogue 29, which
showed significant improvement in in vitro anti-MRSA potency. Increasing or decreasing the ring size deteriorated the
antibacterial activity. Compound 17 demonstrated a superior in vitro and in vivo pharmacokinetic profile, providing compelling
evidence that this particular analogue is a good drug candidate worthy of further analysis.
methicillin-resistant S. aureus strainknown as MRSAwere
INTRODUCTION
■
published in 1961.3,5 Outbreaks of infections caused by
Infections caused by multidrug-resistant bacteria have become a
global public health crisis. In particular, infections due to
multidrug-resistant staphylococci have been increasing at an
alarming rate. Clinically, Staphylococcus aureus was once
different MRSA strains were reported in hospitals in the
United States in the late 1970s; by the 1980s these strains were
endemic,6,7 leading to the worldwide pandemic of MRSA in
hospitals that continues today. Although global in its
susceptible to most antibiotics. However, the emergence of
distribution and impact, MRSA was still confined mostly to
antibiotic resistance in S. aureus has occurred as a series of
waves. Starting in the mid-1940s,1−3 isolates of S. aureus were
discovered that produced a plasmid-encoded penicillinase
capable of hydrolyzing the β-lactam ring of penicillin, thus
healthcare facilities. In 2013, the Centers for Disease Control
and Prevention (CDC) reported that more than 11 000 people
died from MRSA-related infections in the United States alone.8
rendering the antibiotic ineffective. Penicillin-resistant strains
shortly began to cause community infections, and by the early
Received: February 15, 2016
1950s they had become pandemic.4 The first reports of a
© XXXX American Chemical Society
A
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Egypt is among several Mediterranean countries that are
experiencing a surge in MRSA infections.9 The prevalence of
MRSA in both Egyptian community and hospital-acquired
pyogenic skin and soft tissue infections is currently alarming.10
Over the past 40 years, the ever-increasing burden of MRSA
infections led to the increased use of vancomycin, an agent of
last resort for treatment of recalcitrant MRSA infections. This
intensive, selective pressure resulted in the emergence of
vancomycin-intermediate S. aureus (VISA)11 and vancomcyin-
resistant S. aureus (VRSA) isolates.12 Compounding the
problem further, the effectiveness of vancomycin is limited by
prolonged, persistent, or recurrent bacteraemia during
therapy,13,14 high rates of microbiological and clinical failures,15
nephrotoxicity,16 and the increasing prevalence of non-
susceptible strains.14−17
In this article, a strategy is proposed to overcome the
metabolic instability of the aminoguanidine moiety utilizing
ligand-based drug design approaches, given that the exact
molecular target of these phenylthiazoles has not been
identified yet. To date, all first-generation phenylthiazoles
carry a hydrolyzable CN linkage (Figure 2), which
In addition to exhibiting resistance to vancomycin, MRSA
isolates resistant to a wide variety of antibacterial classes,
including the β-lactam antibiotics,18 macrolides,19 and fluoro-
quinolones,19−22 have been found. Collectively this points to
the pressing need to develop novel antimicrobial agents. These
new agents must have enhanced pharmacokinetic (PK)
properties in order to be useful for clinical applications.
In the search for novel antimicrobials, phenylthiazoles
carrying a nitrogenous moiety at one end and a lipophilic
part at the opposite side (Figure 1) were previously reported as
Figure 2. New design.
contributes to their metabolic instability and short t1/2. All
attempts to replace the hydrolyzable CN with more
metabolically stable C−N or amide bonds resulted in less
active compounds.28 These results, collectively, pointed to the
importance of “linker conformational restriction”. Therefore, in
this study, we propose that incorporating the hydrolyzable
Schiffs base moiety within an aromatic ring system will enhance
the overall metabolic stability of the phenylthiazoles while
keeping the required conformational restriction. Thus, several
phenylthiazoles bearing, at thiazole position-5, a pyrimidine
ring connected with guanidine, or a guanidine-like moiety, have
been proposed (Figure 2).
RESULTS AND DISCUSSION
■
Chemistry. Thioamide 2 was treated with α-chloroacetyl-
acetone to yield phenylthiazole derivative 3 (Scheme 1). The
latter compound was allowed to react with dimethyl-
formamide−dimethylacetal (DMF-DMA) to give the first key
intermediate, 4. Finally, the methylsulfonyl derivative 7 was
easily obtained by allowing enaminone 4 to react with thiourea,
followed by methylation and oxidation of the free thiol group as
detailed in Scheme 1.
Figure 1. MIC values and pharmacokinetic profile of lead structure 1.
Two important structural features are colored: red indicates the
nitrogenous part, and blue indicates the lipophilic tail.
new antimicrobial scaffolds with potent activity against
multidrug-resistant strains of S. aureus, including MRSA and
VRSA.23 These phenylthiazole antibacterials possess a selective
advantage over vancomycin in their ability to rapidly eradicate a
high inoculum of MRSA.23 This is clinically significant for the
treatment of diseases caused by staphylococci, as it will impact
the size and timing of doses administered to MRSA-infected
patients.24,25 The highly promising in vivo antimicrobial activity
of the discovered phenylthiazole class of antibacterials, either as
a single agent or in combination with other therapeutic
agents,26,27 has thus far been hampered by their poor PK
profile. For instance, the lead compound 2-(1-(2-(4-butyl-
phenyl)-4-methylthiazol-5-yl)ethylidene)aminoguanidine (1,
Figure 1) is rapidly cleared by human liver microsomes,
resulting in a relatively short half-life (<30 min).23,26 Designing
compounds with a longer duration is important to ensure the
active compound stays in circulation long enough to reach the
target site of infection and exert its antibacterial effect.
The enaminone moiety of compound 4 revealed two distinct
doublet signals in the H NMR spectrum at δ 7.69 and 5.44,
1
each of one proton, with coupling constant of 12 Hz,
characteristic of the E-configuration. Upon cyclization of
compound 5, the chemical shifts of those two signals were
shifted to a higher field, and their J value decreased to around 5
Hz. The inserted S-methyl group of compound 6 was
1
represented in the H NMR spectrum by one extra singlet
signal in the aliphatic region at δ 2.55. This signal was shifted
downfield to δ 2.93 upon oxidation of the S-mercaptyl group
into the methylsulfonyl analogue 7.
With enaminone key intermediate 4 in hand, nine 5-
pyrimidinylphenylthiazoles were obtained via reaction with
different nucleophiles (Scheme 2).
Finally, methylsulfonyl intermediate 7 was utilized to
complete this series of optimized phenylthiazoles at position-
5. Hence, the methylsulfonyl moiety was replaced by three
nucleophiles, namely, hydrazine hydrate, guanidine hydro-
B
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a
a
Scheme 1. Synthesis of Key Intermediates 4 and 7
Scheme 3. Synthesis of Compounds 17−19
a
Reagents and conditions: (a) NH2NH2·H2O, DMF, heat at 80 °C for
0.5 h; (b) guanidine hydrochloride, DMF, heat at 80 °C for 2 h; (c)
tetramethylguanidine, DMF, heat at 80 °C for 2 h.
have been synthesized in a similar manner as the corresponding
n-butyl analogues 4 and 7, as detailed in Scheme 4.
a
Scheme 4. Synthesis of Compounds 24−35
a
Reagents and conditions: (a) absolute EtOH, 3-chloropentane-2,4-
dione, heat to reflux, 12 h; (b) DMF-DMA, heat at 80 °C, 8 h; (c) S-
methylisothiourea hemisulfate, K2CO3, EtOH, heat to reflux, 6 h; (d)
thiourea, KOH, EtOH, heat to reflux, 8 h; (e) dimethyl sulfate, KOH,
H2O, stirring at 23 °C, 12 h; (f) MCPBA, dry DCM, stirring at 23 °C,
16 h.
a
Scheme 2. Synthesis of Compounds 8−16
a
Reagents and conditions: (a) absolute EtOH, 3-chloropentane-2,4-
dione, heat to reflux, 12 h; (b) DMF-DMA, 100 °C, 8 h; (c) guanidine
hydrochloride, K2CO3, EtOH, heat to reflux, 12 h; (d) boronic acid
derivative, XPhos Pd G2, dry DMF, K2CO3, 85 °C, MW, 1 h; (e)
proper alkene, dry DMF, Pd(OAc)2, Et3N, 80 °C, 2 h.
a
Reagents and conditions: (a) appropriate guanidine, K2CO3, absolute
EtOH, heat to reflux, 8 h; (b) 4H-1,2,4-triazol-3-amine, absolute
EtOH, heat to reflux for 5 h.
So far, three aromatic analogues to the n-butyl moiety have
been obtained via Suzuki cross-coupling using the second-
generation pre-made Xphos Pd catalyst (Scheme 4). The H
NMR spectra of compounds 24−26 showed three extra signals
each, in the aromatic region, corresponding to the furyl and/or
thienyl moieties (see Experimental Section).
1
chloride, and tetramethylguanidine, to afford the final products
17−19, respectively (Scheme 3).
The para-iodo intermediates 21 and 22 necessary for the
subsequent chemical transformations (compounds 23−35)
C
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a
Table 1. Antimicrobial Activity (μg/mL) of Second-Generation Phenylthiazoles vs MRSA (2658 RCMB)
a
Van. = vancomycin.
On the other hand, a series of cycloalkenes have been
tethered to the phenyl group’s para-position via Heck cross-
coupling using a standard protocol, i.e., palladium acetate and
triethylamine in a dry DMF (Scheme 4). Similarly, four
terminal alkenes have been allowed to react with the 4-
iodophenylthiazole 23 using the same protocol (Scheme 4).
Biological Results. A. Anti-MRSA Activity. At the outset of
our study, the hypothesis of replacing the hydrolyzable Schiff’s
moiety with a pyrimidine ring was first tested by synthesizing
the simplest second-generation derivative with an amino-
pyrimidine group at the thiazole position-5. So far, the
aminopyrimidine derivative 8 inhibited MRSA at a value
Table 2. Evaluation of Metabolic Stability of Tested
Compounds, Verapamil, and Warfarin in Human Liver
a
Microsomes
NADPH-dependent
NADPH-free
tested
compd
CLint
(μL/min-mg)
t1/2
(min)
CLint
(μL/min-mg)
t1/2
(min)
123
80.3
3.3
7.5
3
28.8
195
243
308
11
0.5
0.5
0.5
0.5
1
>60
>60
>60
>60
>60
>60
8
11
17
verapamil
warfarin
201
0.3
>60
0.0
(3.12
0.10 μg/mL) slightly better than that of the first-
a
CLint = microsomal intrinsic clearance; t1/2 = half-life.
generation lead compound 1, which had a minimum inhibitory
concentration (MIC) value of 4.8 μg/mL against the same
MRSA strain (Table 1). Fortunately, the intrinsic t1/2 of the first
derivative in the second generation was 7 times (195 min,
Table 2) higher than that of the lead compound 1 (28.8 min,
Table 2).
With this solid evidence in hand, we replaced the free amino
group with different nitrogenous moieties in order to address
the structure−activity relationships (SARs) around what was
identified previously as “the cationic part”. Adding a methyl
D
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group to the terminal nitrogen (compound 9) or nitrile group
(compound 10) dramatically decreased the antibacterial
activity, mostly due to the poor solubility issues under the
testing conditions.
and VRE ATCC 700221). Subsequent aromatization of the
hydrazonyl moiety provided pyrrazolylpyrimindine analogue 14
or its pyridinylpyrimidine derivative 13 that exhibited no
antibacterial activity. This observation further confirms our
previous assumption that the “nitrogenous side chain has to
include, at least, one HBD”.
Next, the guanidine-like moiety of 8 was replaced with a true
guanidinyl group, and compound 18 was prepared. The
antibacterial activity of 18 was on par with that of vancomycin,
the drug of choice for treatment of systemic MRSA infections;
both compounds possessed a MIC value of 1.56 μg/mL (Table
1). This value is 3 times better than that of lead compound 1
and 2-fold better than that of the first lead in the second
generation (compound 8). Altering the lipophilic properties of
compound 18 by replacing the NH with a sulfur atom
(compound 12) led to complete abolishment of the anti-MRSA
activity (Table 1). On the other hand, decreasing the polarity of
the guanidinyl moiety by adding two methyl groups
(compound 11) ameliorated the antibacterial activity and
provided the first derivative in this series with a MIC value
below 1 μg/mL. This observation sheds light on a possible
favorable lipophilic region around the terminal nitrogen. This
hypothesis was explored with the simplest derivative in this set
of compounds (compound 8) by incorporating the free amine
within a pyrrolidine ring (compound 15). Unfortunately, the
pyrrolidinyl derivative 15 lacks any antibacterial activity. This
second observation provided insight into the importance of
having a “hydrogen-bond donor” (HBD) within the cationic
part. Moving back to the 2-guanidinylpyrimidine scaffold, the
tetramethylguanidine derivative 19 was synthesized to test the
latter hypothesis. Unlike the guanidine derivative 18, the
corresponding tetramethyl analogue 19 revealed no antibacte-
rial efficacy (Table 1). This observation confirms our second
hypothesis of the importance of having a HBD within the
nitrogenous side chain is necessary for anti-MRSA activity.
Inspired by the previous results, a second HBD was added to
the amino group of compound 8 and the hydrazonyl analogue
17 was prepared, which inhibited the growth of MRSA at 0.4
μg/mL (Table 1). The hydrazonyl derivative 17 demonstrated
a 12-fold improvement in its MIC value when compared with
the lead compound 1 and was 8 times more active than the first
derivative in the second-generation phenylthiazole antibiotics
(compound 8). The antibacterial activity of 17 was further
evaluated against a panel of MSSA, MSRA, and VRSA clinical
isolates (Table 3), and it was highly comparable with that of
vancomycin and linezolid. In addition, compound 17 showed
drastic superiority with vancomycin-resistant strains (VRSA10
The main drawback of several commercial antimicrobials
used to treat MRSA infections is that they are bacteriostatic
(such as linezolid); i.e., they have the ability of inhibiting
bacterial growth but do not kill the bacteria, or they exhibit a
very slow bactericidal mode of action (such as vancomycin),
resulting in difficulty in clearing an infection.29,30 An
antibacterial agent that demonstrates the ability to rapidly
eradicate MRSA would reduce the likelihood of rapid bacterial
resistance emerging to this agent. To examine the antibacterial
activity of the most promising analogues constructed thus far, a
time−kill assay was performed using the most active compound
17 tested against MRSA US300 strain. This strain is responsible
for most cases of community-acquired MRSA (CA-MRSA)
infections, MRSA skin and soft tissue infections (SSTIs) in the
United States31 and other countries around the world,32 and
pneumonia as well.33 Figure 3 represents the elimination rate of
Figure 3. Time−kill analysis of lead compound 1, compound 17, and
vancomycin at 3.0 × MIC against methicillin-resistant Staphylococcus
aureus (MRSA) strain USA300 over a 12 h incubation period at 37 °C.
DMSO served as the control. Values were obtained from triplicate
samples used for each compound studied.
compound 17 in comparison with vancomycin and lead
compound 1. The lead compound 1 required 6 h to
logarithmically reduce the MRSA inoculum to zero colony
forming units (CFU), while vancomycin required 24 h to
achieve the same effect. Interestingly, the second-generation
phenylthiazole derivative 17 rapidly eliminated the bacterial
cells within a 2 h window. These data confirm that the second-
generation phenylthiazoles maintained the selective advantage
observed with the first-generation compounds over vancomycin
and linezolid in terms of rate of elimination of MRSA cells.
After tuning the proper cationic part, we next directed our
attention toward the lipophilic tail at the phenyl ring’s para-
position, since this part has been previously established to have
a profound impact on the antibacterial activity of the
phenylthiazole compounds.23,28 Thus far, the SAR at phenyl
position-4 has been studied, and the n-butyl moiety of the
second-generation lead compound 8 has been replaced with
different conformationally restricted bioisosteres in order to
rigorously explore the SAR at this position.
Table 3. Minimum Inhibitory Concentration (MIC, μg/mL)
and Minimum Bactericidal Concentration (MBC, μg/mL) of
Compound 17, Vancomycin, and Linezolid Screened against
a Panel of S. aureus and Vancomycin-Resistant Enterococcus
faecium (VRE) Clinical Isolate Strains
17
vancomycin
linezolid
MIC MBC MIC MBC MIC MBC
NRS72 (MSSA)
2
2
2
2
2
2
1
2
8
2
2
4
2
2
2
8
2
1
2
1
1
32
1
2
>64
16
8
NRS119 (MRSA)
NRS123 (MRSA USA400)
NRS382 (MRSA USA100)
NRS383 (MRSA USA200)
NRS384 (MRSA USA300)
VRSA10
1
1
1
1
1
1
1
1
32
4
As a prologue to this new set of structure optimizations, the
intermediate 23, carrying an iodine atom in place of the n-butyl
moiety, was found to lack antibacterial activity (Table 1). This
preliminary observation further highlights the importance of the
1
1
1
>64
>64
>64
>64
1
2
VRE ATCC 700221
1
32
E
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lipophilic tail. Next, three aromatic bioisosteres, 24−26, each
with four carbon units, were prepared and revealed less anti-
MRSA activity. In addition to their reduced potency, the
aromatic derivatives 24−26 also possessed poor aqueous
solubility (Table 4). These results encouraged us to move to
To date, the optimal number of carbon units has been found
to be six. Hence, compound 29, with a cyclohexenyl side chain,
was able to inhibit MRSA growth at a concentration of 0.78 μg/
mL (1-fold less than the reference drug vancomycin and 4
times better than the second-generation lead compound 8).
Shrinking the substituent’s ring size at the phenyl group’s para-
position led to the cyclopentenyl derivative 27 that was on par
with the corresponding n-butyl lead structure 8 in terms of
MIC value (Table 1). Furthermore, adding one extra methyl
group, to structurally resemble the cyclohexenyl moiety
(compound 28), further impacted the antibacterial potency in
a negative manner (Table 1). The same trend of increasing
MIC values was observed by expanding the ring size from six to
seven carbon units, and the cycloheptenyl derivative 30 was 4
times less active than the corresponding cyclohexenyl analogue,
29. Further expanding the ring size generated a less active
derivative: the octenyl 31 had a MIC value of 5.6 μg/mL
(Table 1).
Table 4. Evaluation of Solubility of Tested Compounds,
Reserpine, Tamoxifen, and Verapamil in Phosphate-Buffered
Saline
a
compd tested
solubility limit (μM)
lead compound 1
62.5
42
8
11
53
17
55
24
13
25
14
26
8.5
27
35
We then examined the optimal distance between the cyclic
moiety at the phenyl 4-position and phenylthiazole scaffold. At
this stage of structural optimization, both cycloheptenyl and
cycloctenyl derivatizations were excluded on the basis of their
previously limited antimicrobial potency. Thus, only derivatives
with a smaller ring size (cyclohexenyl and cyclopentenyl) were
considered. Four derivatives were synthesized, two with one
carbon linker (compounds 32 and 33) and two with a two-
carbon units linker (compounds 34 and 35). The two
cycloalkylidenemethyl derivatives 32 and 33 were generally
more active than the corresponding cycloalkylvinyl analogues
34 and 35 (Table 1). However, the compounds 32 and 33 were
as active as vancomycin against MRSA cells; they both were less
29
37
reserpine
tamoxifen
verapamil
31.3
15.6
>500
a
Solubility limit corresponds to the highest concentration of test
compound where no precipitate was detected (OD540).
examine the alicyclic analogues. Therefore, a series of
cycloalkenyl-containing derivatives, 27−31, were prepared,
and their MICs against MRSA (Table 1) were also evaluated
using the broth microdilution method, in accordance with the
recommendations contained in the CLSI guidelines.34
Figure 4. Toxicity analysis of lead compound and second-generation phenylthiazole derivative 17 against human keratinocytes (HaCaT). Percent
viable mammalian cells (measured as average absorbance ratio (test agent relative to DMSO)) for cytotoxicity analysis of thiazole compounds 1 and
17 (tested in triplicate) at 8, 16, 32, 64, 128, and 256 μg/mL against HaCaT cells using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Dimethyl sulfoxide (DMSO) was used as a negative control to determine a
baseline measurement for the cytotoxic impact of each compound. The absorbance values represent an average of a minimum of three samples
analyzed for each compound. Error bars represent standard deviation values for the absorbance values. A two-way ANOVA, with post hoc Dunnet’s
multiple comparisons test, determined statistical difference between the values obtained for each compound and DMSO (denoted by the asterisk, P
< 0.05).
F
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potent than the cyclohexyl analogue 29, which revealed the best
activity in this series (Table 1).
Table 5. Evaluation of Apparent Permeability of Tested
Compounds, Ranitidine, and Warfarin via the Caco-2
Permeability Assay
B. Cytotoxicity. The cytotoxicity impact of the second-
generation compounds, represented by the most potent
derivative 17, was analyzed against human keratinocytes
(HaCaT) and compared with the first-generation compounds,
represented by the lead compound 1. The results are
summarized in Figure 4. Compound 1 was nontoxic up to a
concentration of 16 μg/mL. The toxicity profile of the second-
generation derivative 17 showed significant improvement, as it
was not toxic up to a concentration of 64 μg/mL, which is 160
times more than the MIC value found against MRSA (Figure
4).
mean Papp (×10−6 cm/s)
a
b
c
compd tested
A→B
B→A
efflux ratio
d
lead compound 123
0.0
14.9
1.6
1.2
17.0
2.8
>2
1.1
1.8
8.5
0.4
8
17
ranitidine
warfarin
0.2
1.7
27.6
11.1
a
Mean apparent permeability of test compound from apical to
b
basolateral surface. Mean apparent permeability of test compound
c
d
from basolateral to apical surface. Papp(B→A)/Papp(A→B). Com-
pound not detected in receiver compartment (peak below limit of
detection); permeability may be underestimated.
C. Physiochemical Properties and Pharmacokinetic
Profiling. Thus far, the newly developed second-generation
derivatives displayed improved antibacterial activity and an
enhanced safety profile in relation to the first-generation
compounds. We next moved to examine the “drug-like”
properties of the most promising candidate to determine its
suitability for further investigation. Drug discovery is a complex
process that includes multiple lines of investigation, often with
conflicting goals, that necessitates data integration in order to
achieve a balanced clinical candidate. Those candidates must
possess good PK profiles and physiochemical properties. In this
regard, the solubility and apparent permeability of some
selected members of the second-generation series of com-
pounds were examined in comparison with those of the lead
compound 1 and reference drug molecules. Table 4
demonstrates that tethering the cationic part of the phenyl-
thiazole moiety via a pyrimidine ring resulted in a considerable
decrease in aqueous solubility. The aminopyrimidine derivative
8 was found to be 32% less soluble than the lead compound 1
(Table 4). Expanding the polar surface area of the cationic part
via replacement of the free amino group of 8 with a hydrazide
or guanidine moiety had a positive impact, improving the
compound’s aqueous solubility as measured by the turbido-
metric solubility assay. Hence, the aqueous solubility increased
from 42 μM in the case of compound 8 to 53 and 55 μM in the
case of compounds 11 and 17, respectively (Table 4); both
values are still below that of lead compound 1 but better than
those of the two tested FDA-approved drugs, tamoxifen and
reserpine (Table 4).
Aqueous solubility is not the only factor that determines oral
bioavailability; permeability has also a great impact. In the
present work, the Caco-2 bidirectional permeability assay was
used to examine the permeability profile of the lead compound
in this series, compound 8, relative to that of the first-
generation lead structure, 1. The values obtained were
compared with those from two control drugs, one with limited
permeability properties (ranitidine) and one with a strong
permeability profile (warfarin). Although compound 8 did
possess as strong a permeability profile as warfarin, it exhibited
a notable improvement relative to the previously reported poor
permeability properties of the lead compound 1 (Table 5). The
Caco-2 apparent permeability, Papp(A→B), was significantly
improved from 0.0 in the case of 1 to 14.9 × 10−6 cm/s with
compound 8. In the same vein, the Papp(B→A) value also
improved by a factor of 14. These results collectively point out
the superiority of the second-generation phenylthiazoles over
the older first-generation analogues.
compounds, 8, 11, and 17, one from each subclass, were found
to possess metabolic clearance rates ranging between 3 and 7.5
μL/min-mg (Table 2). These values denote that second-
generation phenylthiazoles exhibit 10- to 27-fold improvements
in how rapidly they are metabolized and cleared from liver cells
compared to the first-generation compounds.
Encouraged by the promising in vitro results, we determined
the PK parameters of the most promising candidate thus far,
compound 17, using Sprague−Dawley (SD) rats. Three
animals were dosed with compound 17 (50 mg/kg) orally,
and plasma samples were collected over a 24 h period. The
preliminary PK curve (Figure 5) suggests a once daily dose.
Figure 5. Oral pharmacokinetic curve in rat after 50 mg/kg oral dose
of compound 17.
Interestingly, compound 17 was detected in plasma in a
microgram range (Cmax = 9.3 μg/mL), well above the MIC
value, which reflects the good permeability and absorption
properties determined from the Caco-2 permeability and
turbidometric solubility analysis described above. The in vivo
mean residence time (MRT) for 17 was approximately 8 h
(Table 6). Finally, these data collectively provide solid evidence
that this new generation of phenylthiazoles, unlike previously
Table 6. Compound 17 Plasma Pharmacokinetic Parameters
Following Single Oral Dose (50 mg/kg) to Rats
Cmax (ng/mL)
tmax (h)
9326.32
4.67
AUCtot (h·(ng/mL)) 100010.96
thalf (h)
8.24
7.74
a
AUClast (h·(ng/mL)) 83173.60
λz (1/h) 0.19
MRT (h)
It has already been reported that the lead compound 1 was
cleared by liver microsomes at a rate of 80.3 μL/min-mg and
had a half-life just below 0.5 h.23 In this study, the three tested
a
Mean residence time.
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Figure 6. Summary of conclusions.
230−400 mesh silica. The progress of reactions was monitored with
Merck silica gel IB2-F plates (0.25 mm thickness). The infrared
spectra were recorded in potassium bromide disks on Pye-Unicam SP
3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers. Mass
spectra were recorded at 70 eV. High-resolution mass spectra were
obtained using a FinniganMAT XL95 instrument. Melting points were
determined using capillary tubes with a Stuart SMP30 apparatus and
are uncorrected. HPLC analyses were performed on an Agiland binary
HPLC system (Model 1260) equipped with a multiple-wavelength UV
absorbance detector set for 254 nM, and using a 5 μM C-18 reverse-
phase column. All cross-coupling reactions were conducted under
nitrogen atmosphere, unless otherwise specified. All yields reported
refer to isolated yields. Compounds 3 and 21 were previously
reported.23
reported derivatives, is no longer P-glycoprotein substrates and
is metabolically stable.
CONCLUSION
■
In spite of the availability of third- and fourth-generation
antibiotics on the market, most of them have fallen prey to the
challenge of microbial resistance. The growing threat from
multidrug-resistant bacterial pathogens highlights a critical need
to expand our currently available arsenal of antibiotics. The
recently discovered phenylthiazoles class of antibacterial agents
exhibited promising antibacterial effect against several highly
resistant strains of S. aureus, including MRSA.
Although the first developed phenylthiazoles had a high
potency, they were plagued by a short half-life that did not
exceed 30 min (due to rapid elimination in human liver
microsomes). The main aim of the work described in this
article was to develop second-generation analogues with
enhanced pharmacokinetic profiles. Thus, the hydrolyzable
CN bond was buried inside a more metabolically stable
pyrimidine nucleus. The SAR at the cationic part was then fully
examined utilizing various nitrogenous moieties at position-2 of
the pyrimidine. Among the tested nitrogenous moieties,
hydrazide and N,N-dimethylguanidine-containing derivatives
17 and 11 were found to be more potent than vancomycin, the
drug of choice for treatment of systemic MRSA infections. By
tuning the lipophilic moiety, we found the cyclohexenyl group
to be the most active conformationally restricted analogue for
the n-butyl moiety. Hence, the MIC value of 29 was 1-fold less
than that of vancomycin and 4 times better than that of the
second-generation lead compound 8 (Figure 6). In addition to
the longer t1/2 and better expected safety margin, the second-
generation phenylthiazoles exhibited a selective advantage over
vancomycin in terms of rapid eradication of MRSA cells.
(E)-1-[2-(4-Butylphenyl)-4-methylthiazol-5-yl]-3-
(dimethylamino)prop-2-en-1-one (4). To compound 3 (3.1 g, 11.3
mmol) was added DMF-DMA (3 mL), and the reaction mixture was
heated at 80 °C for 12 h. After cooling, the formed solid was collected
by filtration, washed with petroleum ether and crystallized from
ethanol to yield the desired product as an orange solid (3.3 g, 89%),
1
mp = 105.4 °C. H NMR (DMSO-d6) δ 7.84 (d, J = 8 Hz, 2H), 7.69
(d, J = 12.3 Hz, 1H), 7.31 (d, J = 8.1 Hz, 2H), 5.44 (d, J = 12 Hz, 1H),
3.15 (s, 3H), 2.89 (s, 3H), 2.65 (s, 3H), 2.60 (t, J = 6.8 Hz, 2H), 1.58
(p, J = 4.5 Hz, 2H), 1.32 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H); EIMS m/z
328 (58%).
Potassium Salt of 4-[2-(4-Butylphenyl)-4-methylthiazol-5-yl]-
pyrimidine-2-thiolate (5). To a solution of potassium hydroxide
(200 mg, 3.5 mmol) and thiourea (500 mg, 6.5 mmol) in ethanol (15
mL), enaminone 4 (1 g, 3 mmol) was added. The reaction mixture was
heated to reflux for 8 h, and then cooled down in a refrigerator at 8 °C.
The formed crystals were filtered and washed with diethyl ether to
yield the final product as yellow crystals (1.1 g, 96%), mp > 300 °C. 1H
NMR (DMSO-d6) δ 8.02 (d, J = 5.1 Hz, 1H), 7.86 (d, J = 7.8 Hz, 2H),
7.32 (d, J = 8.1 Hz, 2H), 6.75 (d, J = 5.4 Hz, 1H), 2.60 (s, 3H), 2.49 (t,
J = 5.1 Hz, 2H), 1.55 (m, 2H), 1.32 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H).
2-(4-Butylphenyl)-4-methyl-5-(2-(methylthio)pyrimidin-4-yl)-
thiazole (6). Method A: To a solution of enaminone 4 (100 mg, 0.3
mmol) in ethanol (10 mL) were added S-methylisothiourea
hemisulfate salt (160 mg, 0.6 mmol) and anhydrous potassium
carbonate (200 mg, 1.4 mmol). The reaction mixture was heated at
reflux for 6 h. After completion of reaction, as monitored by TLC,
ethanol was evaporated under reduced pressure, and the crude product
was purified by flash silica column chromatography using EtOAc−
petroleum ether (1:1) to afford white solid (70 mg, 64%), mp = 112.7
°C. EIMS m/z 355 (31), 308 (100).
EXPERIMENTAL SECTION
■
Chemistry. General. All biologically tested compounds were of a
purity not less than 95%. 1H NMR spectra were run at 300 MHz, and
13C spectra were determined at 75.46 MHz, in deuterated chloroform
(CDCl3) or dimethyl sulfoxide (DMSO-d6) on a Varian Mercury VX-
300 NMR spectrometer. Chemical shifts are given in parts per million
(ppm) on the delta (δ) scale. Chemical shifts were calibrated relative
to those of the solvents.35 Flash chromatography was performed on
H
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Method B: To a solution of compound 5 (750 mg, 2.1 mmol) and
potassium hydroxide (250 mg, 4.2 mmol) in water (15 mL) was added
dimethyl sulfate (0.5 mL, 4 mmol) dropwise with vigorous stirring.
After 2 h, the formed solid was filtered and washed with water to yield
7.34 (d, J = 9.1 Hz, 2H), 6.91 (d, J = 6.8 Hz, 1H), 6.71 (brs, 3H), 2.76
(s, 3H), 2.68 (t, J = 7.6 Hz, 2H), 1.55 (m, 2H), 1.32 (m, 2H), 0.90 (t, J
= 7.2 Hz, 3H); CIMS m/z (rel intensity) 384 (MH+, 100); HRMS
(EI) m/z 383.1228 M+, calcd for C19H21N5S2 383.1238; HPLC purity
95.01% (methanol−H2O, 4:1).
1
a yellowish white powder (63 mg, 89%), mp = 112.7 °C. H NMR
(DMSO-d6) δ 8.63 (d, J = 5.1 Hz, 1H), 7.75 (d, J = 7.8 Hz, 2H), 7.44
(d, J = 5.1 Hz, 1H), 7.31 (d, J = 7.8 Hz, 2H), 2.75 (s, 3H), 2.64 (t, J =
7.2 Hz, 2H), 2.55 (s, 3H), 1.58 (p, J = 7.2 Hz, 2H), 1.32 (m, 2H), 0.90
(t, J = 7.2 Hz, 3H); EIMS m/z 355 (31), 308 (100).
2-(4-Butylphenyl)-4-methyl-5-[2-(pyridin-3-yl)pyrimidin-4-yl]-
1
thiazole (13). White solid (50 mg, 23%), mp = 167 °C. H NMR
(DMSO-d6) δ 9.68 (s, 1H), 9.37 (d, J = 5.1 Hz, 1H), 9.01 (d, J = 6.4
Hz, 1H), 7.91 (d, J = 6.0 Hz, 1H), 7.89 (d, J = 9 Hz, 2H), 7.80 (t, J =
6.4 Hz, 1H), 7.31 (d, J = 9 Hz, 2H), 7.22 (d, J = 5.1 Hz, 1H), 2.83 (s,
3H), 2.60 (t, J = 7.2 Hz, 2H), 1.53 (p, J = 7.2 Hz, 2H), 1.26 (m, 2H),
0.91 (t, J = 7.2 Hz, 3H); EIMS m/z (rel intensity) 386 (M+, 100);
HRMS (EI) m/z 386.1560 M+, calcd for C23H22N4S 386.1565; HPLC
purity 98.46% (methanol−H2O, 4:1).
2-(4-Butylphenyl)-4-methyl-5-(2-(methylsulfonyl)pyrimidin-4-yl)-
thiazole (7). To a solution of compound 6 (500 mg, 1.4 mmol) in dry
DCM (5 mL) was added m-CPBA (514 mg, 1.7 mmol) in DCM (5
mL) portion-wise with continuous stirring. After the reaction mixture
was kept at 23 °C for 16 h, additional DCM (10 mL) was added, and
the reaction mixture was washed with 25 mL of a 5% aqueous solution
of sodium metabisulfite and 25 mL of 5% aqueous sodium carbonate.
The organic layer was separated, dried, and concentrated under
reduced pressure to give the desired product as yellow crystals (520
mg, 95%), mp = 100.5 °C. 1H NMR (DMSO-d6) δ 8.99 (d, J = 5.4 Hz,
1H), 8.66 (d, J = 5.4 Hz, 1H), 7.89 (d, J = 7.8 Hz, 2H), 7.34 (d, J = 7.8
Hz, 2H), 2.93 (s, 3H), 2.63 (t, J = 7.6 Hz, 2 H), 2.61 (s, 3H), 1.57 (p, J
= 7.6 Hz, 2H), 1.33 (m, 2H), 0.90 (t, J = 7.6 Hz, 3H); EIMS m/z 387
(12), 308 (100).
Compounds 8−15. To a solution of enaminone 4 (100 mg, 0.3
mmol) in absolute ethanol (5 mL) was added a suitable guanidine or
carbimidine (1.2 mmol) and anhydrous potassium carbonate (200 mg,
1.4 mmol). The reaction mixture was heated at reflux for 8 h, ethanol
was evaporated under reduced pressure, and the reaction was
quenched with cold water (50 mL). The formed flocculated solid
was filtered, washed with water, and purified with either acid−base
extraction using HCl (1 M, 50 mL) or flash silica column
chromatography using EtOAc−petroleum ether−methanol
(4.5:4.5:1) to yield the desired products. Physical properties and
spectral analysis of isolated products are listed below.
2-(4-Butylphenyl)-5-[2-(1H-pyrazol-1-yl)pyrimidin-4-yl]-4-methyl-
1
thiazole (14). White solid (55 mg, 46%), mp = 163.7 °C. H NMR
(DMSO-d6) δ 8.33 (d, J = 5.8 Hz, 1H), 8.03 (d, J = 4.8 Hz, 1H), 7.85
(m, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.87 (d, J
= 5.1 Hz, 1H), 6.59 (t, J = 4.8 Hz, 1H), 2.71 (s, 3H), 2.60 (t, 2H), 1.6
(m, 2H), 1.3 (m, 2H), 0.9 (t, J = 6.8 Hz, 3H); EIMS m/z (rel
intensity) 375 (M+, 100), calcd for C21H21N5S 375; HPLC purity
95.58% (methanol−H2O, 4:1).
2-(4-Butylphenyl)-4-methyl-5-[2-(pyrrolidin-1-yl)pyrimidin-4-yl]-
thiazole (15). Yellowish white solid (0.032 g, 29%), mp =153.7 °C. 1H
NMR (DMSO-d6) δ 8.40 (d, J = 5.4 Hz, 1H), 7.87 (d, J = 7.8 Hz, 2H),
7.32 (d, J = 7.8 Hz, 2H), 6.88 (d, J = 4.8 Hz, 1H), 3.51 (s, 4H), 2.74
(s, 3H), 2.64 (t, J = 7.2 Hz, 2H), 1.93 (m, 4H), 1.59 (m, 2H), 1.34 (m,
2H), 0.912 (t, J = 7.2 Hz, 3H); CIMS m/z (rel intensity) 379 (MH+,
100); HRMS (EI) m/z 378.1885 M+, calcd for C22H26N4S 378.1878;
HPLC purity 97.91% (methanol−H2O, 4:1).
5-([1,2,4]-Triazolo[1,5-a]pyrimidin-5-yl)-2-(4-butylphenyl)-4-
methylthiazole (16). To a solution of enaminone 4 (200 mg, 6 mmol)
in absolute ethanol (5 mL) were added 4H-1,2,4-triazol-3-amine (100
mg, 0.5 mmol) and anhydrous potassium carbonate (100 mg, 0.7
mmol). The reaction mixture was heated to reflux for 5 h, ethanol was
evaporated under reduced pressure, and the reaction was quenched
with cold water (50 mL). The formed flocculated solid was filtered,
washed with water, and purified with flash silica column chromatog-
raphy using EtOAc−hexane (1:1) to yield the desired product as a
yellowish white solid (55 mg, 52%), mp = 143 °C. 1H NMR (DMSO-
d6) δ 8.95 (d, J = 4.8 Hz, 1H), 8.78 (s, 1H), 7.85 (d, J = 7.8 Hz, 2H),
7.68 (d, J = 4.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 2H), 2.75 (s, 3H), 2.62
(m, 2H), 1.58 (m, 2H), 1.27 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H); CIMS
m/z (rel intensity) 350 (MH+, 100); HRMS (EI) m/z 349.1375 M+,
calcd for C19H19N5S 349.1361; HPLC purity 97.17% (methanol−H2O,
4:1).
2-(4-Butylphenyl)-5-(2-hydrazinylpyrimidin-4-yl)-4-methylthia-
zole (17). To a solution of methylsulfonylpyrimidine 7 (100 mg, 0.26
mmol) in DMF (2 mL) was added hydrazine hydrate (5 mL). The
reaction mixture was heated at 80 °C for 0.5 h. The formed fluffy solid
was filtered and washed with boiled water to remove the residual
hydrazine to finally give the title product as a yellowish white fluffy
powder (70 mg, 80%), mp = 128 °C. 1H NMR (DMSO-d6) δ 8.38 (d,
J = 5.1 Hz, 1H), 8.28 (brs, 1H), 7.87 (d, J = 8.1 Hz, 2H), 7.33 (d, J =
7.8 Hz, 2H), 6.91 (d, J = 6.8 HZ, 1H), 4.22 (brs, 2H), 2.72 (s, 3H),
2.63 (t, J = 7.5 Hz, 2H), 1.58 (m, 2H), 1.32 (m, 2H), 0.90 (t, J = 7.2
Hz, 3H); CIMS m/z (rel intensity) 340 (MH+, 100); HRMS (EI) m/z
339.1526 M+, calcd for C18H21N5S 339.1518; HPLC purity 97.23%
(methanol−H2O, 4:1).
4-[2-(4-Butylphenyl)-4-methylthiazol-5-yl]pyrimidin-2-amine (8).
Yellowish-white solid (70 mg, 71%), mp = 159 °C. 1H NMR (DMSO-
d6) δ 8.32 (d, J = 5.1 Hz, 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.1
Hz, 2H), 6.91 (d, J = 5.1 Hz, 1H), 6.74 (brs, 2H), 2.69 (s, 3H), 2.61 (t,
J = 7.2 Hz, 2H), 1.57 (q, J = 7.2 Hz, 2H), 1.32 (q, J = 7.2 Hz, 2H), 0.9
(t, J = 9.6 Hz, 3H); CIMS m/z (rel intensity) 325 (MH+, 100); HRMS
(EI) m/z 324.1421 M+, calcd for C18H20N4S 324.1409; HPLC purity
95.69% (methanol−H2O, 4:1).
4-[2-(4-Butylphenyl)-4-methylthiazol-5-yl]-N-methylpyrimidin-2-
1
amine (9). White solid (60 mg, 56%), mp = 227 °C (charring). H
NMR (DMSO-d6) δ 8.35 (d, J = 5.4 Hz, 1H), 7.86 (d, J = 7.8 Hz, 2H),
7.50 (brs, 1H), 7.31 (d, J = 8.1 Hz, 2H), 6.93 (d, J = 5.4 Hz, 1H), 2.72
(s, 3H), 2.64 (t, J = 7.5 Hz, 2H), 2.50 (s, 3H), 1.58 (p, J = 7.2 Hz,
2H), 1.3 (m, 2H), 0.89 (t, J = 7.2 HZ, 3H); CIMS m/z (rel intensity)
339 (MH+, 100); HRMS (EI) m/z 338.1569 M+, calcd for C19H22N4S
338.1565; HPLC purity 97.08% (methanol−H2O, 4:1).
N-{4-[2-(4-Butylphenyl)-4-methylthiazol-5-yl]pyrimidin-2-yl}-
cyanamide (10). White solid (60 g, 28%), mp = 118 °C. IR (KBr)
3383 (NH), 3184, 2935, 2160 cm−1; 1H NMR (DMSO-d6) δ 8.17 (d, J
= 5.4 Hz, 1H), 7.91 (d, J = 7.8 Hz, 2H), 7.36 (d, J = 5.4 Hz, 2H), 6.66
(d, J = 4.8 Hz, 1H), 6.55 (brs, 1H), 2.70 (s, 3H), 2.57 (t, J = 7.4 Hz,
2H), 1.57 (p, J = 7.5 Hz, 2H), 1.31 (six, J = 7.5 Hz, 2H), 0.90 (t, J =
7.2 Hz, 3H); CIMS m/z (rel intensity) 350 (MH+, 100); HRMS (EI)
m/z 349.1375 M+, calcd for C19H19N5S 349.1361; HPLC purity 96.1%
(methanol−H2O, 4:1).
2-(4-Butylphenyl)-5-(2-guanidinylpyrimidin-4-yl)-4-methylthia-
zole (18). To a solution of methylsulfonylpyrimidine 7 (100 mg, 0.26
mmol) in dry DMF (5 mL) were added guanidine hydrochloride (50
mg, 0.50 mmol) and potassium carbonate (100 mg, 0.7 mmol). The
reaction mixture was heated at 80 °C for 8 h, ethanol was evaporated
under reduced pressure, and cold water (10 mL) was added. The
formed solid was filtered and crystallized from ethanol (95%) to yield
the desired product as a yellowish brown solid (70 mg, 63%), mp =
227.2 °C (charring). 1H NMR (DMSO-d6) δ 9.98 (brs, 1H), 8.56 (d, J
= 5.1 Hz, 1H), 8.01 (brs, 2H), 7.65 (d, J = 10 Hz, 2H), 7.39 (d, J = 8
Hz, 1H), 7.20 (brs, 2H), 7.05 (d, J = 7.5 Hz, 2H), 2.50 (s, 3H), 2.43
3-{4-[2-(4-Butylphenyl)-4-methylthiazol-5-yl]pyrimidin-2-yl}-1,1-
dimethylguanidine (11). Yellowish white solid (55 mg, 46%), mp =
153.7 °C. 1H NMR (DMSO-d6) δ 8.31 (d, J = 7.2 Hz, 1H), 7.85 (d, J
= 8.4 Hz 2H), 7.32 (d, J = 8.1 HZ, 2H), 7.14 (brs, 1H), 6.84 (d, J = 6.4
Hz, 1H), 6.70 (brs, 1H), 3.33 (s, 6H), 2.69 (s, 3H), 2.61 (t, 2H), 1.56
(m, 2H), 1.3 (m, 2H), 0.90 (t, J = 7.6 Hz, 3H); CIMS m/z (rel
intensity) 395 (MH+, 100); HRMS (EI) m/z 394.1944 M+, calcd for
C21H26N6S 394.1940; HPLC purity 95.9% (methanol−H2O, 4:1).
1-{4-[2-(4-Butylphenyl)-4-methylthiazol-5-yl]pyrimidin-2-yl}-
thiourea (12). White solid (78 mg, 33%), mp = 212 °C (charring); 1H
NMR (DMSO-d6) δ 8.33 (d, J = 6.8 Hz, 1H), 7.86 (d, J = 9.1 Hz, 2H),
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(m, 2H), 1.42 (m, 2H), 1.19 (m, 2H), 0.77 (t, J = 7.5 Hz, 3H); CIMS
m/z (rel intensity) 367 (MH+, 100); HRMS (EI) m/z 366.1620 M+,
calcd for C19H22N6S 366.1627; HPLC purity 99.02% (methanol−H2O,
4:1).
intensity) 351 (MH+, 100); HRMS (EI) m/z 350.0670 M+, calcd for
C18H14N4S2 350.0660; HPLC purity 98.97% (methanol−H2O, 4:1).
4-{2-[4-(Cycloalkenyl)phenyl]-4-methylthiazol-5-yl}pyrimidin-2-
amines (27−35). To a solution of compound 23 (150 mg, 0.43
mmol) in dry DMF (5 mL) were added triethylamine (0.2 mL, 1.9
mmol) and a catalytic amount of palladium acetate (20 mg). The flask
was then charged with an appropriate cycloalkene (2.4 mmol). The
mixture was heated at 80 °C for 1 h, and then was filtered through
Celite 545, extracted with ethyl acetate, and purified with flash silica
column chromatography using eluent EtOAc−hexane (1:9) to yield
the desired product as presented below.
4-{2-[4-(Cyclopent-1-en-1-yl)phenyl]-4-methylthiazol-5-yl}-
pyrimidin-2-amine (27). Off-white solid (50 mg, 39%), mp = 160.6
°C. 1H NMR (DMSO-d6) δ 8.33 (d, J = 5.4 Hz, 1H), 7.89 (d, J = 7.8
Hz, 2H), 7.31(d, J = 7.8 Hz, 2H), 6.91 (d, J = 5.4 Hz, 1H), 6.71 (brs,
2H), 5.81(d, J = 1.5 Hz, 1H), 2.70 (s, 3H), 1.69−1.48 (m, 2H), 1.23−
1.54 (m, 2H), 0.85−0.78 (m, 2H); CIMS m/z (rel intensity) 335
(MH+, 100); HRMS (EI) m/z 334.1250 M+, calcd for C19H18N4S
334.1252; HPLC purity 97.51% (methanol−H2O, 4:1).
4-{4-Methyl-2-[4-(4-methylcyclopent-1-en-1-yl)phenyl]thiazol-5-
yl}pyrimidin-2-amine (28). Off-white solid (60 mg, 45%), mp = 141
°C. 1H NMR (DMSO-d6) δ 8.32 (d, J = 5.1 Hz, 1H), 7.89 (d, J = 7.8
Hz, 2H), 7.52 (d, J = 7.8 Hz, 2H), 6.90 (d, J = 5.1 Hz, 1H), 6.72 (brs,
2H), 5.84 (m, 1H), 2.70 (s, 3H), 1.28−1.69 (m, 4H), 0.92 (d, J = 6.3
Hz, 3H), 0.85 (m, 1H); CIMS m/z (rel intensity) 349 (MH+, 100);
HRMS (EI) m/z 348.1401 M+, calcd for C20H20N4S 348.1409; HPLC
purity 97.29% (methanol−H2O, 4:1).
4-{2-[4-(Cyclohex-1-en-1-yl)phenyl]-4-methylthiazol-5-yl}-
pyrimidin-2-amine (29). Off-white solid (65 mg, 49%), mp = 140.4
°C. 1H NMR (DMSO-d6) δ 8.32 (d, J = 4.8 Hz, 1H), 7.89 (d, J = 7.8
Hz, 2H), 7.39 (d, J = 7.8 Hz, 2H), 6.91 (d, J = 4.8 Hz, 1H), 6.80 (brs,
2H), 5.76 (d, J = 1.5 Hz, 1H), 2.69 (s, 3H), 2.77−1.84 (m, 4H), 1.23−
1.17 (m, 4H); CIMS m/z (rel intensity) 349 (MH+, 100); HRMS (EI)
m/z 348.1414 M+, calcd for C20H20N4S 348.1409; HPLC purity
96.64% (methanol−H2O, 4:1).
4-{2-[4-(Cyclohept-1-en-1-yl)phenyl]-4-methylthiazol-5-yl}-
pyrimidin-2-amine (30). Off-white solid (100 mg, 73%), mp = 164
°C. 1H NMR (DMSO-d6) δ 8.32 (d, J = 4.8 Hz, 1H), 7.89 (d, J = 7.8
Hz, 2H), 7.36(d, J = 7.8 Hz, 2H), 6.90 (d, J = 4.8 Hz, 1H), 6.71 (brs,
2H), 5.86 (m, 1H), 2.69 (s, 3H), 2.69 (m, 2H), 2.263−2.15 (m, 2H),
1.85−1.71 (m, 2H), 1.49−1.38 (m, 2H), 1.22 (m, 2H); CIMS m/z
(rel intensity) 363 (MH+, 100); HRMS (EI) m/z 362.1579 M+, calcd
for C21H22N4S 362.1565; HPLC purity 98.09% (methanol−H2O, 4:1).
4-{2-[4-(Cyclooct-1-en-1-yl)phenyl]-4-methylthiazol-5-yl}-
pyrimidin-2-amine (31). Off-white solid (120 mg, 84%), mp = 188
°C. 1H NMR (DMSO-d6) δ 8.32 (d, J = 5.1 Hz, 1H), 7.89 (d, J = 7.8
Hz, 2H), 7.37 (d, J = 7.8 Hz, 2H), 6.90 (d, J = 5.1 Hz, 1H), 6.72 (brs,
2H), 5.71 (m, 1H), 2.69 (s, 3H), 2.28 (m, 2H), 1.75−1.48 (m, 10H);
CIMS m/z (rel intensity) 377 (MH+, 100); HRMS (EI) m/z 376.1719
M+, calcd for C22H24N4S 376.1722; HPLC purity 95.98% (methanol−
H2O, 4:1).
4-{2-[4-(Cyclopentylidenemethyl)phenyl]-4-methylthiazol-5-yl}-
pyrimidin-2-amine (32). Yellowish white solid (55 mg, 41%), mp =
143 °C. 1H NMR (DMSO-d6) δ 8.33 (d, J = 5.4 Hz, 1H), 7.88 (d, J =
8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 5.4 Hz, 1H), 6.71
(brs, 2H), 5.38 (s, 1H), 2.70 (s, 3H), 2.27 (m, 1H), 2.16 (m, 1H), 1.82
(m, 2H), 1.22 (m, 4H); CIMS m/z (rel intensity) 349 (MH+, 100);
HRMS (EI) m/z 348.1420 M+, calcd for C20H20N4S 348.1409; HPLC
purity 96.18% (methanol−H2O, 4:1).
2-{4-[2-(4-Butylphenyl)-4-methylthiazol-5-yl]pyrimidin-2-yl}-
1,1,3,3-tetramethylguanidine (19). To a solution of methylsulfonyl-
pyrimidine 7 (275 mg, 0.7 mmol) in dry DMF (5 mL) were added
tetramethylguanidine (0.5 mL) and potassium carbonate (100 mg, 0.7
mmol). The reaction mixture was heated at 80 °C for 8 h and then
poured over ice water (50 mL). The formed solid was filtered and
purified by flash silica chromatography using EtOAc−hexane (9:1) to
1
yield the desired product as a yellow solid (21 mg, 7%). H NMR
(DMSO-d6) δ 8.65 (d, J = 5.7 Hz, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.59
(d, J = 5.1 Hz, 1H), 7.34 (d, J = 8.7 Hz, 2H), 2.70 (s, 12H), 2.63 (m,
2H), 2.57 (s, 3H), 1.57 (p, J = 6.0 Hz, 2H), 1.32 (six, J = 6.0 Hz, 2H),
0.90 (t, J = 6.4 Hz, 3H); EIMS m/z (rel intensity) 422 (M+, 31), 407
(100), calcd for C23H30N6S 422; HPLC purity 95.06% (methanol−
H2O, 4:1).
(E)-3-(Dimethylamino)-1-[2-(4-iodophenyl)-4-methylthiazol-5-
yl]prop-2-en-1-one (22). To a solution of compound 21 (1 g, 2.9
mml) in dry DMF (3 mL) was added DMF−DMA (0.5 mL, 4.5
mmol), and the reaction mixture was heated at 80 °C for 8 h. After
cooling down, the reaction mixture was poured over crushed ice with
vigorous stirring. The formed orange solid was collected by filtration
and washed with water to yield the desired product as an orange solid
1
(1.1 g, 95%), mp = 157 °C. H NMR (DMSO-d6) δ 7.86 (d, J = 8.1
Hz, 2H), 7.72 (d, J = 9.8 Hz, 2H), 7.71 (d, J = 12.4 Hz, 1H), 5.42 (d, J
= 12.8 Hz, 1H), 3.10 (s, 3H), 2.89 (s, 3H), 2.66 (s, 3H); EIMS m/z
(rel intensity) 398 (M+, 60), calcd for C15H15IN2OS 398.
4-[2-(4-Iodophenyl)-4-methylthiazol-5-yl]pyrimidin-2-amine
(23). To a solution of enaminone 22 (1.1 g, 2.7 mmol) in absolute
ethanol (5 mL) were added guanidine hydrochloride (1 g, 10.5 mmol)
and anhydrous potassium carbonate (1.5 g, 10.8 mmol). The reaction
mixture was heated at reflux for 12 h. After cooling, the white crystals
that formed were filtered and washed with water to yield the desired
1
product as a white solid (0.6 g, 66%), mp = 114.3 °C. H NMR
(DMSO-d6) δ 8.33 (d, J = 5.1 Hz, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.73
(d, J = 8.4 Hz, 2H), 6.91 (d, J = 5.4 Hz, 1H), 6.91 (brs, 2H), 2.7 (s,
3H); CIMS m/z (rel intensity) 395 (MH+, 100); HRMS (EI) m/z
393.9756 M+, calcd for C14H11IN4S 393.9749; HPLC purity 99.87%
(methanol−H2O, 4:1).
4-[2-(4-Substituted)phenyl)-4-methylthiazol-5-yl]pyrimidin-2-
amines (24−26). To a solution of compound 23 (150 mg, 0.43
mmol) in dry DMF (5 mL) were added anhydrous potassium
carbonate (250 mg, 1.7 mmol) and a catalytic amount of palladium X-
Phos (Xphos Pd G2) (10 mg). The reaction mixture was then charged
with the appropriate boronic acid derivative (1.2 equiv). The mixture
was irradiated in a SINEO microwave (Uwave-1000) for 1 h at 80 °C,
and then the reaction mixture was poured over crushed ice, filtered,
and washed with methanol to yield the desired product. The physical
characteristics and spectral data of separated products are listed below.
4-{2-[4-(Furan-3-yl)phenyl]-4-methylthiazol-5-yl}pyrimidin-2-
1
amine (24). Yellow solid (100 mg, 78%), mp = 161.2 °C. H NMR
(DMSO-d6) δ 8.33 (m, 1H), 8.31 (s, 1H), 7.97 (d, J = 8.1 Hz, 2H),
7.75 (d, J = 8.4 Hz, 2H), 7.04 (m, 1H), 6.92 (m, 2H), 6.73 (brs, 2H),
2.71 (s, 3H); CIMS m/z (rel intensity) 335 (MH+, 100); HRMS (EI)
m/z 334.0892 M+, calcd for C18H14N4OS 334.0888; HPLC purity
99.1% (methanol−H2O, 4:1).
4-{2-[4-(Furan-2-yl)phenyl]-4-methylthiazol-5-yl}pyrimidin-2-
4-{2-[4-(Cyclohexylidenemethyl)phenyl]-4-methylthiazol-5-yl}-
pyrimidin-2-amine (33). Brown solid (60 mg, 44%), mp = 165 °C. 1H
NMR (DMSO-d6) δ 8.33 (d, J = 5.4 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H),
7.33 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 5.4 Hz, 1H), 6.72 (brs, 2H), 6.27
(s, 1H), 2.71 (s, 3H), 2.48 (m, 1H), 2.40 (m, 1H), 1.98−1.23 (m, 6H),
1.05 (m, 2H); CIMS m/z (rel intensity) 363 (MH+, 100); HRMS (EI)
m/z 362.1570 M+, calcd for C21H22N4S 362.1565; HPLC purity
99.72% (methanol−H2O, 4:1).
1
amine (25). Brown solid (105 mg, 82%), mp = 209.3 °C. H NMR
(DMSO-d6) δ 8.33 (d, J = 6.1 Hz, 1H), 8.01 (d, J = 8.6 Hz, 2H), 7.83
(m, 3H), 7.10 (d, J = 3 Hz, 1H), 6.92 (d, J = 4.8 Hz, 1H), 6.73 (brs,
2H), 6.65 (dd, J = 4.5, 4.8 Hz, 1H), 2.70 (s, 3H); CIMS m/z (rel
intensity) 335 (MH+, 100); HRMS (EI) m/z 334.0898 M+, calcd for
C18H14N4OS 334.0888; HPLC purity 98.75% (methanol−H2O, 4:1).
4-{4-Methyl-2-[4-(thiophen-3-yl)phenyl]thiazol-5-yl}pyrimidin-2-
amine (26). Brown solid (120 mg, 90%), mp = 225 °C (charring). 1H
NMR (DMSO-d6) δ 8.33 (d, J = 5.1 HZ, 1H), 7.99 (m, 3H), 7.86 (d, J
= 8.4 Hz, 2H), 7.67 (d, J = 2.7 Hz, 1H), 7.63 (d, J = 3.9 Hz, 1H) 6.91
(d, J = 5.1 Hz, 1H), 6.73 (brs, 2H), 2.71 (s, 3H); CIMS m/z (rel
(E)-4-{2-[4-(2-Cyclopentylvinyl)phenyl]-4-methylthiazol-5-yl}-
pyrimidin-2-amine (34). Brown solid (40 mg, 29%), mp = 180.7 °C.
1H NMR (DMSO-d6) δ 8.32 (d, J = 5.4 Hz, 1H), 7.88 (d, J = 8.1 Hz,
2H), 7.51 (d, J = 8.1 Hz, 2H), 7.31 (dd, J = 12, 6.8 Hz, 1H), 6.90 (d, J
J
DOI: 10.1021/acs.jmedchem.6b00233
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
= 5.4 Hz, 1H), 6.71 (brs, 2H), 6.44 (d, J = 12.0 Hz, 1H), 2.69 (s, 3H),
2.21 (t, J = 6 Hz, 1H), 1.95 (sep, J = 7.5 Hz, 1H), 1.79−1.71 (m, 1H),
1.62−1.45 (m, 4H), 1.22−1.15 (m, 2H); CIMS m/z (rel intensity)
363 (MH+, 100); HRMS (EI) m/z 362.1559 M+, calcd for C21H22N4S
362.1565; HPLC purity 95.33% (methanol−H2O, 4:1).
(E)-4-{2-[4-(2-Cyclohexylvinyl)phenyl]-4-methylthiazol-5-yl}-
pyrimidin-2-amine (35). Brown solid (65 mg, 45%), mp = 189 °C. 1H
NMR (DMSO-d6) δ 8.32 (d, J = 5.0 Hz, 1H), 7.95 (m, 1H), 7.88 (d, J
= 7.8 Hz, 2H), 7.51 (d, J = 7.8 Hz, 2H), 6.90 (d, J = 4.8 Hz, 1H), 6.80
(brs, 2H), 6.41 (m, 1H), 2.69 (s, 3H), 2.17 (m, 1H), 1.74 (m, 2H),
1.54−1.49 (m, 4H), 1.22−1.14 (m, 4H); CIMS m/z (rel intensity)
377 (MH+, 100); HRMS (EI) m/z 376.1731 M+, calcd for C22H24N4S
376.1722; HPLC purity 95.03% (methanol−H2O, 4:1).
Antimicrobial Testing. Determination of Minimum Inhibitory
Concentration (MIC). MRSA clinical isolates and vancomycin-resistant
Staphylococcus aureus (VRSA) strain were obtained through the
Network of Antimicrobial Resistance in Staphylococcus aureus
(NARSA) program. In addition, vancomycin-resistant Enterococcus
faecium (VRE) strain was obtained from the ATCC.
The MICs of the newly synthesized compounds, tested against
isolates of S. aureus and VRE, were determined using the broth
microdilution method in accordance with the recommendations
contained in the Clinical and Laboratory Standards Institute
guidelines.34 Bacteria were cultured in cation-adjusted Mueller Hinton
broth in a 96-well plate. Compounds, using triplicate samples, were
added to the plate and serially diluted. Plates were incubated at 37 °C
for 20 h prior to determining the MIC. Plates were visually inspected,
and the MIC was categorized as the concentration at which no visible
growth of bacteria was observed. The average of triplicate MIC
determinations is reported.
Time−Kill Assay. MRSA (USA300) cells, in the logarithmic growth
phase, were diluted to 1.0 × 106 CFU/mL and exposed to
concentrations equivalent to 3.0 × MIC (in triplicate) of tested
compounds, and vancomycin in tryptic soy broth (Becton, Dickinson
and Company, Sparks, MD, USA). Aliquots (100 μL) were taken from
each sample after 2, 4, 6, 8, 10, 12, and 24 h, serially diluted in PBS,
and transferred to trypticase soy agar (Becton, Dickinson and
Company) plates. Plates were incubated at 37 °C for at least 16 h
before counting viable CFU/mL to determine the time required to
reduce the bacterial cell count by 3 log10.
In Vitro Cytotoxicity Analysis. Compound 17 was assayed at
concentrations of 8, 16, 32, 64, 128, and 256 μg/mL against an
immortal keratinocyte cell line (HaCaT) in order to determine the
potential toxic effect against mammalian cells in vitro. Cells were
cultured in Dulbeco’s modified Eagle’s medium (Sigma-Aldrich, St.
Louis, MO, USA) with 10% fetal bovine serum (USA Scientific, Inc.)
at 37 °C with 5% CO2. Controls received DMSO alone at a
concentration equal to that in drug-treated cell samples. The cells were
incubated with the compounds in a 96-well plate at 37 °C and 5.0%
CO2 for 2 h prior to addition of the assay reagent MTS (Promega,
Madison, WI, USA). Corrected absorbance readings (actual
absorbance readings for each treatment subtracted from background
absorbance) were taken using a kinetic ELISA microplate reader
(Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells
after treatment with each compound is expressed as a percentage of
the control, DMSO.
Pharmacokinetic Assays. Caco-2 Permeability Assay. Caco-2
cells grown in tissue culture flasks were trypsinized and suspended in
medium, and the suspensions were applied to wells of a Millipore 96-
well Caco-2 plate. The cells were allowed to grow and differentiate for
3 weeks, feeding at 2-day intervals. For apical-to-basolateral (A→B)
permeability, the tested compound was added to the apical (A) side,
and the amount of permeation was determined on the basolateral (B)
side; for basolateral-to-apical (B→A) permeability, the tested
compound was added to the B side, and the amount of permeation
was determined on the A side. The A-side buffer contained 100 μM
Lucifer yellow dye, in transport buffer (1.98 g/L glucose in 10 mM
HEPES, 1.0× Hank’s Balanced Salt Solution), pH 6.5, and the B-side
buffer contained transport buffer at pH 7.4. Caco-2 cells were
incubated with these buffers for 2 h, and the receiver-side buffer was
removed for analysis by LC/MS/MS. To verify that the Caco-2 cell
monolayers were properly formed, aliquots of the cell buffers were
analyzed by fluorescence to determine the transport of the
impermeable Lucifer yellow dye. Any deviations from control values
are reported. Data are expressed as permeability Papp = (dQ/dt)/C0A,
where dQ/dt is the rate of permeation, C0 is the initial concentration
of test agent, and A is the area of the monolayer. In bidirectional
permeability studies, the efflux ratio is also calculated: RE = Papp(B→
A)/Papp(A→B). An RE > 2 indicates a potential substrate for P-
glycoprotein or other active efflux transporters.
PBS Solubility Screen. Serial dilutions of the tested compounds,
reserpine, tamoxifen, and verapamil were prepared in phosphate-
buffered saline (PBS) at 100× the final concentration. The solutions
were diluted 100-fold into PBS in a 96-well plate and mixed. The
absorbance of the PBS-containing plate was measured prior to
addition of the test agents to determine the background absorbance.
After 2 h, the presence of precipitate was detected by turbidity
(absorbance at 540 nm). An absorbance value of greater than (mean +
3× standard deviation of the blank), after subtracting the pre-
experiment background, is indicative of turbidity. The solubility limit is
reported as the highest experimental concentration with no evidence
of turbidity.
Human Microsomal Stability Analysis. The tested compounds
were incubated in duplicate with human liver microsomes at 37 °C.
The reaction contained microsomal protein in 100 mM potassium
phosphate, 2 mM NADPH, 3 mM MgCl2, pH 7.4. A control was run
for each test agent omitting NADPH to detect NADPH-free
degradation. At 0, 10, 20, 40, and 60 min, an aliquot was removed
from each experimental and control reaction and mixed with an equal
volume of ice-cold stop solution (methanol containing haloperidol,
diclofenac, or other internal standard). Stopped reactions were
incubated at least 10 min at −20 °C, and an additional volume of
water was added. The samples were centrifuged to remove precipitated
protein, and the supernatants were analyzed by LC/MS/MS to
quantitate the remaining parent. Data are converted to % remaining by
dividing by the time zero concentration value. Data are fit to a first-
order decay model to determine half-life. Intrinsic clearance is
calculated from the half-life and the protein concentrations: CLint
=
ln(2)/T1/2[microsomal protein].
Statistical Analysis. All statistical analysis was conducted using
Kaleida Graph, version 4.03 (Synergy Software, Reading, PA).
Statistical significance was determined using ANOVA and the Fisher’s
least significant difference test with α = 0.05.
In Vivo Pharmacokinetics. This assay was conducted at an
accredited bioequivalence center (http://www.grc-me.com/pk_pd.
̈
html). Pharmacokinetic studies were performed in male naive
Sprague−Dawley (SD) rats (three animals) following Institutional
Animal Care and Use Committee guidelines. Oral dosing was
administered by gavage in a vehicle containing 5% ethanol, 45%
PEG 400, and 50% water. Blood samples were collected over a 24 h
period post dose into Vacutainer tubes containing EDTA-K2. Plasma
was isolated, and the concentration of compound 17 in plasma was
determined by LC/MS/MS after protein precipitation with acetoni-
trile.
Non-compartmental PK analysis was performed on plasma
concentration data to calculate PK parameters using Kinetica 2000
(release 4.4.1).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00233.
Molecular formulas for 2−35 (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: 0020-100-771-5002. E-mail: amayhoub@azhar.edu.eg.
■
K
DOI: 10.1021/acs.jmedchem.6b00233
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Journal of Medicinal Chemistry
Article
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Academy of Scientific
Research & Technology, Egypt, JESOUR-D program (Grant
ID No. 42). The authors also acknowledge The World
Academy of Sciences, COMSTECH-TWAS program (grant
no. 14-389 RG/PHA/AF/AC_C) for their generous support.
ABBREVIATIONS USED
■
Caco-2, heterogeneous human epithelial colorectal adenocarci-
noma cells; CA-MRSA, community-acquired MRSA; CFUs,
colony-forming units; Cmax, maximum serum concentration;
DMF-DMA, dimethylformamide−dimethylacetal; HaCaT,
human keratinocytes; MRSA, methicillin-resistant Staphylococ-
cus aureus; MRT, mean residence time; Papp, apparent
permeability; PK, pharmacokinetic; SSTIs, skin and soft tissue
infections; VISA, vancomycin-intermediate S. aureus
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