Development of Biphenylthiazoles Exhibiting Improved Pharmacokinetics and Potent Activity against Intracellular Staphylococcus aureus

Article abstract of DOI:10.1021/acsinfecdis.0c00137

Exploring the structure-activity relationship (SAR) at the cationic part of arylthiazole antibiotics revealed hydrazine as an active moiety. The main objective of the study is to overcome the inherited toxicity associated with the free hydrazine. A series of hydrocarbon bridges was inserted in between the groups, to separate the two amino groups. Hence, the aminomethylpiperidine-containing analog 16 was identified as a new promising antibacterial agent with efficient antibacterial and pharmacokinetic profiles. Briefly, compound 16 outperformed vancomycin in terms of the antibacterial spectrum against vancomycin-resistant staphylococcal and enterococcal strains with minimum inhibitory concentrations (MICs) ranging from 2 to 4 μg/mL, which is a faster bactericidal mode of action, completely eradicating the high staphylococcal burden within 6-8 h, and it has a unique ability to completely clear intracellular staphylococci. In addition, the initial pharmacokinetic assessment confirmed the high metabolic stability of compound 16 (biological half-life >4 h); it had a good extravascular distribution and maintained a plasma concentration higher than the average MIC value for over 12 h. Moreover, compound 16 significantly reduced MRSA burden in an in vivo MRSA skin infection mouse experiment. These attributes collectively suggest that compound 16 is a good therapeutic candidate for invasive staphylococcal and enterococcal infections. From a mechanistic point of view, compound 16 inhibited undecaprenyl diphosphate phosphatase (UppP) with an IC₅₀value of 29 μM.

Full text of DOI:10.1021/acsinfecdis.0c00137

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Article
Development of biphenylthiazoles exhibiting improved pharmacokinetics
and potent activity against intracellular Staphylococcus aureus
Mohamed Hagras, Nader S. Abutaleb, Noha Elhosseiny, Tamer M. Abdelghany,
Mariam Omara, Mohamed Elsebaei, Marwa Alhashimi, Allison B Norvil, Mark I Gutay,
Humaira Gowher, Ahmed Attia, Mohamed N. Seleem, and Abdelrahman S Mayhoub
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.0c00137 • Publication Date (Web): 08 Sep 2020
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Development of biphenylthiazoles exhibiting improved pharmacokinetics and potent
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activity against intracellular Staphylococcus aureus
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Mohamed Hagras1‖, Nader S. Abutaleb2‖, Noha M. Elhosseiny,3 Tamer M. Abdelghany,4 Mariam
Omara,4 Mohamed M. Elsebaei1, Marwa Alhashimi2, Allison B Norvil5, Mark I Gutay5, Humaira
Gowher5,6, Ahmed S. Attia,3 Mohamed N. Seleem2,7* and Abdelrahman S. Mayhoub1,8*
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1 Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University,
1-Elmokhayem Eldaem Street, Cairo 11884, Egypt.
2 Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University,
725 Harrison Street, West Lafayette, IN 47907, USA.
3 Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo,
Egypt, 11562.
4 Department of Pharmacology and Toxicology, College of Pharmacy, Al-Azhar University, 1-
Elmokhayem Eldaem Street, Cairo 11884, Egypt.
5
Department of Biochemistry, College of Agriculture, Purdue University, West Lafayette, IN
47907, USA.
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Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907,
USA
7 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary
Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA.
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University of Science and Technology, Nanoscience Program, Zewail City of Science and
Technology, Ahmed Zewail Street, October Gardens, 6th of October, Giza 12578, Egypt
Corresponding Authors. *e-mail: mseleem@purdue.edu, amayhoub@azhar.edu.eg
‖These two authors contributed equally
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Exploring the structure-activity-relationship (SAR) at the cationic part of arylthiazole antibiotics
revealed hydrazine as an active moiety. The main objective of the study is to overcome the
inherited-toxicity associated with the free-hydrazine. A series of hydrocarbon bridges was inserted
in between, to separate the two amino groups. Hence, the aminomethylpiperidine-containing
analog 16 was identified as a new promising antibacterial agent with efficient antibacterial and
pharmacokinetic profiles. Briefly, compound 16 outperformed vancomycin in terms of the
antibacterial spectrum against vancomycin-resistant staphylococcal and enterococcal strains with
minimum inhibitory concentrations (MICs) ranging from 2 to 4 µg/mL, faster bactericidal mode
of action, completely eradicating the high staphylococcal burden within 6-8 hours, and a unique
ability to completely clear intracellular staphylococci. In addition, the initial pharmacokinetic
assessment confirmed the high metabolic stability of compound 16 (biological half-life > 4 h),
good extra-vascular distribution and maintaining a plasma concentration higher than the average
MIC value for over 12 h. Moreover, compound 16 significantly reduced MRSA burden in an in
vivo MRSA skin infection mouse experiment. These attributes collectively suggest compound 16
as a good therapeutic candidate for invasive staphylococcal and enterococcal infections. From a
mechanistic point of view, compound 16 inhibited undecaprenyl diphosphate phosphatase (UppP)
with an IC50 value of 29 M.
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Keywords: antibiotic resistance, methicillin-resistant Staphylococcus aureus, vancomycin-
resistant enterococci, intracellular infections, pharmacokinetics.
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According to the World Health Organization (WHO) global priority list of antibiotic-resistant
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bacteria,
Methicillin-resistant,
vancomycin-intermediate
and
vancomycin-resistant
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Staphylococcus aureus (MRSA, VISA and VRSA) are categorized as high priority for which new
antibiotics are urgently needed.1 Although applying strong preventive measures in hospitals over
the last fifteen years alleviated the rapid spread of hospital-acquired MRSA infections, this
progress did not last for a long time. In 2017, reductions of hospital-acquired MRSA infections
stalled and the community-acquired MRSA increased, causing around 20,000 deaths in the USA
alone as per the Centers for Disease Control and Prevention (CDC).2 The majority of the current
antibiotics used to treat MRSA infections are associated with some limitations and toxic side
effects. For instance, vancomycin is not recommended for patients with reduced kidney function
such as the elderly or diabetic patients.3 Additionally, linezolid toxicity is associated with 26%
mortality where some patients develop lactic acidosis, myelosuppression, optic or peripheral
neuropathies, and myopathies.4-5 Furthermore, several incidences of S. aureus strains resistant to
the currently used antibiotics such as vancomycin, linezolid, and daptomycin have been
documented.3, 6, 7 Consequently, the rising MRSA resistance to the commonly used antibiotics,
along with the increased infection cases (both hospital- and community-acquired) highlight the
critical need for new entities to treat MRSA infections.
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Despite the first wave of staphylococcal resistance that was mediated by the secretion of
penicillinase,8 current multidrug-resistant staphylococci developed different mechanisms to
sustain the fight against modern antibacterial agents. One mechanism utilized by the bacteria is its
ability to invade the host phagocytes to avoid the human immune system.9 Staphylococci
developed several mechanisms to suppress the phagocytic lysosomal enzymes, and to survive at
low pH inside macrophages.10 Different multidrug-resistant Staphylococcus aureus strains release
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staphylococcal-borne toxins such as LukAB, which suppress the protective function of
macrophages.11 Moreover, they can compensate for the harsh cytosolic environment by the
slowing down their growth rate to adapt to the intracellular media. Consequently, the ability of S.
aureus to deceive the immune system and pretend to be a cell component resulted in increasing
the resistance burden.12 to further exacerbate the problem, around 40% of patients with MRSA-
induced pneumonia experience a life-threatening slow response to vancomycin, the drug of choice
for systemic staphylococcal infections.13 Therefore, finding a new antibacterial agent with the
ability to resolve the problematic intracellular MRSA is an urgent medical need.
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Figure 1. Progress in phenylthiazole project and aim of the current work.
One of our successfully discovered new antibacterial scaffolds is arylthiazole that demonstrated
confirmed activity against a large panel of multidrug-resistant (MDR)-Gram positive pathogens.14-
24
These compounds outperformed vancomycin in terms of their rapid bactericidal mode of
action.25-26 The structure-activity relationships (SAR) of arylthiazole antibacterial core were
studied from three different perspectives. Briefly, the biphenyl side chain revealed notable
improvement in the antibacterial effect,27 while the short half-life of the first discovered lead
compound was remarkably enhanced by inserting the C=N linker within pyrimidine structure
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(Figure 1).28 Lastly, intensive efforts were reported to optimize the cationic part, in which the
hydrazine moiety potentiates the antibacterial activity (Figure 1).28 Hydrazine and hydrazine-like
structures are considered toxicophores that increase the incidence of oxidative stress in living
tissues.29-30 Hence, hydrazine moiety is an uncommon structural motif in approved therapeutics,
and those containing such toxic moiety, isoniazid as an example, are associated with black box-
warning due to acute hepatotoxicity.31 Hydrazine-containing structures exert their toxic effect via
metabolic activation into diazonium derivative and subsequent generation of aryl free radicals.32
As a strategy to overcome this problem, the two amino groups of hydrazine motif were separated
by a short hydrocarbon chain (Figure 1). Hence, it is chemically or metabolically impossible to
generate a diazonium intermediate. The antibacterial activity of all newly synthesized compounds
were investigated, and the pharmacokinetic behavior of the most promising derivative was studied
as well.
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RESULTS AND DISCUSSION
CHEMISTRY. The synthetic route started with the reaction between bisphenylthioamide 1 and
-chloroacetylacetone, which afforded bisphenylthiazole 3. The later was allowed to react
dimethylformamide-dimethylacetal to yield the intermediate 4, which then subjected to react with
thiourea, and the product was methylated with dimethyl sulfate to give the
methylmercaptopyrimidine derivative 5. Oxidation of 5 with a per-acid provided the key
intermediate 6, which used to obtain the final products 7-25 (Scheme 1).
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S
O
O
NH2
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6
7
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9
Cl
b
Ph
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2
a
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N
S
N
NMe2
O
c, d
S
Ph
Ph
Ph
O
3
4
N
N
O
O
S
N
SMe
N
N
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S
e
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Ph
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f
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R1
S
S
R
N
H
Ph
Ph
7-19
20-25
R
R
R1
NH2
NH
7
(R)-2-methanol
(S)-2-methanol
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NH2
OH
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N
H
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(R)-3-dimethylamine
(S)-3-dimethylamine
OH
OH
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NH2
OH
(R)-2-carboxamide
(S)-2-carboxamide
H2N
OH
OH
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19
OH
OH
12
H2N
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14
OH
OH
Reagents and conditions:
(a) Absolute EtOH, 3-chloropentane-2,4-dione,
heat at reflux, 12 h; (b) DMF-DMA heat at 80 °C, 8h; (c) thiourea, KOH,
EtOH, heat at reflux, 8 h; (d) dimethyl sulfate, KOH, H2O, stirring at 23 oC,
o
2 h; (e) MCPBA, dry DCM, stirring at 23 C, 16 h; (f) appropriate amine,
dry DMF, heat at 80 oC for 0.5 - 8h.
Scheme 1. Synthetic pathway of compounds 7-25
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BIOLOGICAL RESULTS AND DISCUSSIONS. As mentioned earlier, the main purpose of
this study was to find a bioisostere for the toxicophore hydrazine moiety. Since hydrazine is not
an intrinsic toxic group by itself and it needs hepatic activation to generate the corresponding toxic
diazonium intermediate, we decided to first add small alkyl chain between its two amino groups.
Obviously, this chemical modification prevents the formation of diazonium intermediate and
consequently the toxic free radicals. The ethylenediamine and propylenediamine derivatives 7 and
8 were synthesized and tested for their antimicrobial activities. Both compounds were moderately
active against MRSA USA300 with MIC value of 16 g/mL (Table 1). The next set of structural
modifications included conformationally-restricted analogs 15-19. First, by incorporating the
terminal nitrogen within an alicyclic ring system, a divergence in antibacterial activity was
observed. While the pyrrolidinyl derivative 15 was void from any antibacterial activity, the
piperidinyl analog 16 exhibited high potency against MRSA with an MIC value of 2 g/mL (two-
fold higher than the frontline therapeutic vancomycin) (Table 1). Second, restricting the free
rotation of ethylene bridge by loading both amino groups on a cyclic structure gave compounds
18 and 19, in which the geometric relationships of the two amino groups was investigated. In this
vein, both compounds were 2 to 4 times more potent than the ethylenediamine-containing
derivative 7 while the trans-isomer 19 possessed two-fold better anti-MRSA activity than its cis-
form 18. On the other hand, the nullified activity of the trans-1,4-diamine derivative 17 highlights
the crucial importance of the shorter two to three-carbons linker.
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All attempts to functionalize the terminal amine by methylation, replacement with
hydroxyl isostere or incorporating within an amide structure ended up with inactive compounds
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with the exception of the (S)-dimethylaminopyrrolidine 23 that revealed moderate activity against
MRSA with an MIC value of 8 g/mL.
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Table 1. Initial antibacterial assessment, MIC values in g/mL
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N
NRS384
(MRSA
USA300)
R
N
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Ph
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NH2
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16
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H
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NH2
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NH
>64
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>64
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>64
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OH
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N
NRS384
(MRSA
USA300)
R
N
S
Ph
N
7
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9
OH
OH
H
N
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13
>64
>64
OH
OH
H
N
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OH
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20
>64
N
N
OH
>64
1
vancomycin
N/A
For the promising new antibacterial compounds 16 and 19, several attributes need to be
investigated such as studying the activity against a large panel of MDR-bacteria that belong to
different genera, high selectivity to the prokaryotic cells over mammalian cells, mode of killing
and killing kinetics, the likelihood of developing resistance to it, and the ability to target
intracellular pathogens. Therefore, in the next sections, we will present a comprehensive
microbiological profile of the most potent derivatives (compounds 16 and 19).
The promising antibacterial activity of compounds 16 and 19 was further confirmed against
a panel of clinically relevant MDR-staphylococcal strains including linezolid-resistant and
vancomycin-resistant S. aureus strains and methicillin-resistant Staphylococcus epidermidis. The
compounds maintained their level of inhibition against all the tested S. aureus isolates (MIC = 2-
4 µg/mL), and compound 16 was superior to 19 MIC value of 2 g/mL against most of the tested
strains (Table 2). They also, exhibited potent activity against S. epidermidis, a common colonizer
of the human skin, and a common source of implanted medical prosthetic devices infections. S.
epidermidis infections are tough to be treated due to their huge ability to form strongly adherent
biofilms that have intrinsic resistance to antibiotics and the host defense systems.33-34 Moreover,
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both compounds exhibited bactericidal activity against the tested strains where their MBC values
were the same as or one-fold higher than their corresponding MICs.
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Table 2. MICs and MBCs (µg/mL) of compounds 16 and 19 against staphylococcal clinical
isolates.
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Compounds/control antibiotics
Bacterial Strains
19
16
Linezolid
Vancomycin
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MSSA ATCC 6538
MSSA NRS 107
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1
0.5
32
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16
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> 64
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2
1
MRSA NRS119
1
MRSA NRS123 (USA400)
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MRSA NRS 385
(USA500)
MRSA NRS 386
(USA700)
0.5
1
32
1
2
64
0.5
64
1
VRSA 10
VRSA 12
0.5
64
> 64
4
2
4
4
2
2
4
2
1
32
16
64
> 64
1
Methicillin-resistant
Staphylococcus
epidermidis NRS101
0.5
0.5
MSSA, methicillin-sensitive Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; VRSA,
vancomycin-resistant Staphylococcus aureus
In coincidence with the activity against S. aureus strains, compounds 16 and 19 exhibited potent
antibacterial activity against other clinically important drug-resistant Gram-positive bacteria,
inhibiting the growth of tested strains at concentrations ranging between 2 and 4 µg/mL (Table 3).
The compounds maintained their activity against vancomycin-resistant enterococci (VRE) (Table
3), a leading cause of nosocomial infections in the USA that cause about 20%-30% of hospital-
acquired infections and the second major cause of such infections across the world.35 Additionally,
compounds 16 and 19 exhibited potent activities against Streptococcus pneumoniae, with MICs
ranging from 2-4 μg/mL. S. pneumoniae is the leading cause of bacterial pneumonia and meningitis
in the USA and a major cause of bloodstream, ear and sinus infections. It is associated with more
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than 2,000,000 infections annually in the USA, resulting in more than 6,000 deaths and an estimate
of $4 billion total costs.36 Moreover, in more than 30% of infections, the bacteria are resistant to
one or more clinically relevant antibiotics.36 Interestingly, the compounds’ MBC values were the
same as or one-fold higher than their corresponding MICs indicating that they exhibited
bactericidal activity against the tested strains.
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Table 3. MICs and MBCs (µg/mL) of compounds 16 and 19 against non-Staphylococcus aureus
clinical isolates.
Bacterial Strains
Compounds/control antibiotics
16 Linezolid
MBC MIC MBC MIC MBC MIC
19
Vancomycin
MBC
MIC
Vancomycin-resistant
Enterococcus faecalis ATCC
51299
Vancomycin-resistant
Enterococcus faecium
ATCC 700221
4
4
8
8
2
2
4
4
0.5
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32
32
> 64
>64
1
>64
Listeria monocytogenes ATCC
19111
Cephalosporin-resistant
Streptococcus pneumoniae
ATCC 51916
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2
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1
16
32
0.5
1
1
1
Methicillin-resistant
Streptococcus pneumoniae
ATCC 700677
4
8
2
8
0.5
16
1
2
Next. in order to test confirm the bactericidal activity of the compounds, a time-killing assay was
performed against MRSA USA400. Figure 2 indicates that compound 19 exhibited a rapid
bactericidal activity by reducing more than 3-log10 CFU within only 2 hours and completely
eradicated MRSA burden after 6 hours. On the other hand, compound 16 reduced the high MRSA
inoculum by more than 3-log10-reduction after 6 hours and completely killed that high inoculum
after 8 hours. In contrast, vancomycin, the drug of choice, eradicated the high MRSA count after
24-hours. This fast-bactericidal mode of action adds an additional clinical value to our newly
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developed compounds as both drugs of choices, in such cases, (vancomycin and linezolid) possess
their own drawbacks that affect the overall clinical efficacy. The former is a very slow-bactericidal
agent,37 while the latter has a bacteriostatic mode of action,38 resulting in difficulty in clearing
bacterial infections in many cases.39-40
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Due to the rapid bacterial killing, it is thought that it is hard (or at least a slow rate) for a bacterium
to develop resistance against an antibacterial agent with a rapid bactericidal mode of action.41 As
reported previously, the main target of phenylthiazoles is the inhibition of two consecutive proteins
involved in bacterial cell wall biosynthesis.26 This consecutive inhibition might explain the
inability of MRSA to develop resistance against phenylthiazoles, as reported earlier.17 To test
whether the new compounds 16 and 19 are susceptible to the development of resistance by MRSA,
a multi-step resistance test was conducted. Unlike rifampicin, to which MRSA developed
resistance rapidly, the MIC values for compounds 19 and 16 increased by only one-fold after the
eighth and fourteenth passage, respectively; but they remained stable thereafter (Figure 3). This
result indicates that MRSA cells could not develop rapid resistance to phenylthiazole antibiotics
which could provide this newly emerging class of antibiotics with an expected long-term clinical
efficacy.
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Figure 2. Killing kinetics of compounds 16 and 19 (tested in triplicates at 5 × MIC) against
methicillin-resistant Staphylococcus aureus (MRSA USA 400) 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.
Figure 3. Multi-step resistance selection of compounds 16, 19 and rifampicin against methicillin-
resistant S. aureus USA400 (NRS123). Bacteria were serially passaged over a 14-day period and
the broth microdilution assay was used to determine the MIC of each compound against MRSA
after each successive passage. A four-fold shift in MIC would be indicative of bacterial resistance
to the test agent.
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Figure 4. Analyzing the toxicity of compounds 16 and 19 (tested in triplicates at 8, 16, 32, 64 and
128 µg/mL) against human colorectal cells (Caco-2) (Left) & against human keratinocyte cells
(HaCaT) (Right) 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 measure 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. Data were analyzed via a two-way
ANOVA with post hoc Dunnett’s test for multiple comparisons. Asterisks (*) denote a statistically
significant difference (P<0.05) between values obtained for the compounds as compared to the
DMSO-treated samples.
After confirming the potent bactericidal activity against multiple MDR-bacterial strains, mode of
killing kinetics and a low tendency of resistance development, we moved next to test compounds’
tolerability by examining the cytotoxicity of compounds 16 and 19 against two mammalian cells,
Caco-2 and HaCaT cells (Figure 4). Caco-2 cell line was used to get a preliminary insight about
the tolerability of the compounds to the human colon cells if administered orally, and HaCaT cells
were employed to investigate the toxicity of the compounds to human skin cells if applied
topically. Both compounds exhibited a good toxicity profile on Caco-2 cells, where they exhibited
well tolerability at high concentrations. Briefly, compound 16 was non-toxic to Caco-2 and HaCaT
cells at a concentration as high as 64 µg/mL where about 100% of the cells were viable. This
concentration represents 32-times its corresponding MIC values against MRSA strains. On the
other hand, the diaminocyclohexyl derivative 19 was slightly less tolerable for both tested cell
lines.
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The last studied parameter in the bacteriological profiling is the ability of these compounds to keep
their efficacy against intracellular pathogens. Most antibiotics including the drugs of choice for
MRSA infections such as vancomycin and linezolid, exhibit limited activity against intracellular
bacteria due to several reasons such as low levels of accumulation intracellularly, inactivation by
the acidic pH within macrophages, or binding to lysosomal contents. To assess the intracellular
killing ability of our newly synthesized phenylthiazole compounds, an infected murine
macrophage (J774) model was used. By testing the tolerable concentration to murine macrophage
cells, it was found that both tested compounds 16 and 19 were tolerable to J774 at a concentration
up to 16 µg/mL, where almost 100% of the cells were viable (Figure S1). Therefore, at a non-toxic
concentration (4× MIC), compound 16 had the highest intracellular clearing activity by completely
eradicating the intracellular MRSA burden (below the limit of detection (10 CFU/mL)). On the
other hand, compound 19 reduced the intracellular MRSA burden by 2.08-log10-reduction (about
99.18% reduction) at 4× MIC (16 µg/mL) (Figure 5).
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Figure 5. Examination of the activity of compounds 16 and 19 on the clearance of intracellular
MRSA present in murine macrophage (J774) cells. Data are presented as log10 CFU/mL of
intracellular MRSA USA400 inside infected murine macrophages after treatment with 4× MIC of
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either compounds 16, 19 or vancomycin (tested in quadruplicates) for 24 hours. Data were
analyzed via one-way ANOVA, with post hoc Dunnet’s test for multiple comparisons (P < 0.05),
utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Asterisks (*) denotes
statistically significant difference between treatment with either compounds 16 or 19 compared to
vancomycin.
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So far, the microbiological screening and profiling boil down to suggest compound 16 as a
promising novel antibacterial candidate. Therefore, the next section will focus on exploring the
key pharmacokinetic parameters in order to decide, whether compound 16 can be further
developed into a systemically administered antibiotic or its use will be limited to topical
applications. In this vein, incubation of compound 16 with liver microsomes indicated a high level
of metabolic stability with an intrinsic half-life (intT1/2) value of more than 2.5 hours (Table 4).
This value went down to around 1.5 hours when the same compound was incubated in absence of
NADPH, the co-factor of CYP450, indicating that compound 16 is a substrate for other liver
metabolic enzyme(s) rather than the CYP450. Furthermore, the in vitro metabolic stability of
compound 16 was translated into a biological half-life (t1/2) value of 4.3 h, after being administered
as an intravenous (IV) bolus to rats (Figure 6). The pharmacokinetic curve after single IV dose
revealed, in addition to highly acceptable t1/2 value, a good distribution in biological tissues other
than the vascular system, as indicated by the value of its volume of distribution in the 2nd
compartment (V).
Table 4. In vitro preliminary pharmacokinetic parameters; half-life and intrinsic clearance
(T1/2 and Clint) of compounds 16.
Compound
Incubation
Time (min)
% Compound Remaining Half-Life (min)
Clint
(µL/min/mg)
1st
2nd
Mean
100
106
104
99
1st
2nd
Mean
16 (with NADPH)
0
100.0
111.4
105.2
100.2
76.6
100.0
100.2
102.7
98.0
70.8
162.9
145.8
154.4
44.9
15
30
45
60
0
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Compound
Incubation
Time (min)
% Compound Remaining Half-Life (min)
Clint
(µL/min/mg)
1st
2nd
Mean
1st
2nd
Mean
15
100.0
100.0
100
115.5
70.5
93.0
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30
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{75.0} {44.8}
86.7
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65.8
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*CLint= 0.693/(t1/2*microsomal protein concentration)
PK curve after a bolus IV dose
2.5
2
Key PK
parameters:
T1/2 = 4.28 hr
AUC= 3.40 mg.h/L
CL = 7.35 L/hr
1.5
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TIME (HR)
Figure 6. Pharmacokinetics (PK) curve (average of 3 animals), after a bolus IV dose, of compound
16. T1/2: half-life, V: volume of distribution in the 2nd compartment, Cl: rate of clearance, AUC:
area under the curve.
After testing the in vitro and in vivo pharmacokinetics of compound 16, we moved towards
investigating its mechanism of action. Originally, phenylthiazole scaffold restrains bacterial
growth by perturbing the bacterial cell wall biosynthesis via inhibition of an essential enzyme
called undecaprenyl diphosphate phosphatase (UppP).26 Upon testing the UppP inhibition activity
of compound 16, it showed moderate inhibitory activity against UppP with an IC50 value of around
30 M (Figure 7), which raises the point of having an additional bacterial target.
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B
A
UppP Inhibition
0.20
0.16
0.12
0.08
0.04
0.00
140
120
100
80
log2IC50 = 4.9  0.27 M
IC50 = 29.86  1.2 M
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Inhibitor
-
+
Conc. [Compound 16] in log2(M)
Figure 7. A) The activity of undecaprenyl diphosphate phosphatase (UppP) was measured by the
release of inorganic phosphate in the absence of presence 283µM of the inhibitor, compound 16.
B) Dose-response curve for inhibition of undecaprenyl diphosphate phosphatase (UppP) by
compound 16. The activity of UppP was measured by the release of inorganic phosphate in the
presence of an increasing concentrations of the inhibitor. Untreated sample with the reaction buffer
only, served as a negative control (100% UppP activity). The results are presented as percent UppP
inhibition, in presence of different concentrations of compound 16, relative to the untreated
samples. Error bars represent standard deviation values obtained from six measurements from
three independent experiments.
Next, the promising in vitro activities of compound 16 led us to investigate its activity in an in vivo
murine MRSA skin infection experiment. Mice were infected with MRSA USA300 and treated
for 3 days with either petroleum jelly (PJ), petroleum jelly containing 2% compound 16, or
commercially available topical cream containing 2% fusidic acid (FA). Afterwards, wounds were
harvested, homogenized and plated. Mice treated with petroleum jelly (PJ) only showed a
significant skin lesion development that progressed to extensive skin damage until day 7 of the
infection. Contrary to this, mice treated with PJ containing 2% of compound 16 significantly
showed milder lesions and considerable skin healing by the end of the experiment. Bacterial colony
counts from the drug-treated group were significantly lower than the PJ treated group (**p ≤ 0.01),
and considerably less systemic invasion as measured by colony counts in the spleens of infected
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animals (Figure 8). The fusidic acid-treated control group showed slightly better lesion healing,
less lesion colony counts, and less systemic involvement than the drug-treated group (Figure 8).
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Figure 8. Compound 16 is active in vivo curing S. aureus skin infection in mice. Mice were
infected subcutaneously with approximately 4 x 109 CFUs of S. aureus USA300. Within 72 h post-
infection, an open wound/abscess was formed at the site of injection. Mice were then treated
topically, twice daily for 4 days, with either petroleum jelly (PJ), 2% compound 16 in PJ (16) or a
commercial cream with 2% fusidic acid (FA). A. Photographs of representative mice, on day 7,
showing the infected and treated skin areas in the three groups. B. Box plots of the bacterial burden
recovered from the skin lesions and spleens of the mice of the three groups. The whiskers span the
difference between the minimum and maximum readings, the horizontal bar represents the median,
and the (+) sign represents the mean of the CFU. Statistical analysis was done using One-way
ANOVA followed by Tukey’s multiple comparisons test, the * means p ≤ 0.05, ** means p ≤ 0.01,
*** means p ≤ 0.001, the charts were generated using GraphPad Prism.
Upon testing compound 16 in the S. aureus murine systemic model of infection, it was
noticed that this compound, at a dose of 20 mg/kg, is capable of slightly lowering the mean
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bacterial burdens in the kidneys of the mice as compared to the PBS group (Figure 9). At the same
time, in the group treated with vancomycin (VA), at a dose of 50 mg/kg, the mean bacterial loads
were lowered furthermore (Figure 9). The one-way ANOVA analysis of the three groups results
showed a p-value of 0.0042. However, multiple comparisons (Tukey's test) between the three
groups indicated that there were no significant differences between each two groups individually.
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Figure 9. Box plots of the bacterial burden recovered from the kidneys of the mice of the three
groups. The whiskers span the difference between the minimum and maximum readings, the
horizontal bar represents the median, and the (+) sign represents the mean of the CFU/ml.
Statistical analysis was done using One-way ANOVA followed by Tukey’s multiple comparisons
test, the chart was generated using GraphPad Prism.
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Figure (10). Acute toxicity study of compound 16 on male BALB/c mice; A) serum level of
hepatic transaminases. B) Body weight over 10 days
Finally, the serum level of transaminases, as an indicator of hepatic injury, shows no significant
increase as shown in Figure 10A, which indicates no liver toxicity after oral administration of up
to 250 mg/kg. Furthermore, body weight shows no difference between control and treated animals
after 1,4,7, and 10 days of oral administration of 250 mg/kg (Figure 10B).
Conclusion. The present study aimed at expanding our understanding of the SAR of
phenylthiazole antibacterial scaffold, as a novel class of antibacterial agents. Through studying the
basicity and the stereochemistry of the cationic side chain, it was found that the side chains with
two amino groups possess superior antibacterial properties than their corresponding hydroxy-
containing derivatives. The nullified antibacterial activity of all less basic amide-containing
analogs strongly correlates the antibacterial activity with the basicity of the side chains. From the
spatial arrangement point of view, incorporating the two terminal amino groups with cyclic or
alicyclic structure provided disparity in antibacterial response. In this context, the piperidine-
containing derivative 16 is over 32-times more potent against MRSA than the corresponding
pyrrolidine-containing structure 15. Compound 16 maintained its low MIC value (2 g/mL)
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against most of the additionally tested drug-resistant Gram-positive pathogens including
vancomycin-resistant staphylococcal and enterococcal strains. Moreover, compound 16 possesses
several additional attributes that make it worth considering as an antibiotic candidate. In brief, it
has a rapid bactericidal mode of action, low potential of developing resistance, highly selective to
prokaryotic cells over mammalian cells, and completely cleared the intracellular staphylococci
burden. Initial pharmacokinetic analysis indicated the suitability of compound 16 for systemic
invasive staphylococcal and enterococcal infections with a twice-daily dose-frequency. From a
mechanistic point of view, compound 16 is a moderate inhibitor for undecaprenyl diphosphate
phosphatase (UppP) with an IC50 value of about 30 M. Lastly, compound 16 was found to
significantly reduce MRSA burden in vivo in a MRSA murine skin infection experiment with no
observable acute toxicity.
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Methods
Chemistry
General. Melting points were obtained using a Stuart melting point apparatus and were
uncorrected. Microanalyses for C, H, and N were performed at the Regional Center for Mycology
and Biotechnology, Al-Azhar University. 1H NMR spectra were recorded using a Bruker 400 MHz
(Bruker Corp., Billerica, MA, USA) spectrophotometer at the Faculty of Pharmacy, Cairo
University, Cairo, Egypt, and Varian Mercury-300BB 300 MHz (Varian Corp., Palo Alto, CA,
USA) spectrophotometer at the Cairo University and Faculty of Science, Cairo University, Cairo,
Egypt. Chemical shifts are given in parts per million (ppm) on the delta () scale and coupling
constants (J) were reported in Hz. Chemical shifts were calibrated relative to those of the
solvents.42 13C NMR spectra were recorded using a Bruker 100 MHz spectrophotometer at the
Faculty of Pharmacy, Cairo University, Cairo, Egypt and Varian Mercury-300BB 75 MHz at the
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Faculty of Science, Cairo University, Cairo, Egypt. The progress of the reactions was monitored
with TLC using pre-coated aluminum sheet silica gel MERCK 60F 254. The spots were visualized
using a UV lamp. The solvent system used for this assay was ethyl acetate: hexane [7:3] or [9:1].
All yields reported refer to isolated yields. Compounds 1-6 were prepared and characterized as
reported elsewhere.27
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Compounds 7-25. General procedure. To a solution of 6 (0.1 g, 0.25 mmol) in dry DMF (5 mL),
a proper amine (0.4 mmol) was added. The reaction mixtures were heated at 80 °C for 4-8 h, and
then poured over ice water (50 mL), the formed solid was filtered and washed with 50% ethanol
and recrystallized from absolute ethanol to give the desired products. Physical properties and
spectral analysis of all isolated products are listed below:
N-{4-[2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl]pyrimidin-2-yl}ethane-1,2-diamine (7)
Following the general procedure, and using ethylenediamine (21 µL, 0.4 mmol), compound 7 was
obtained as yellow solid (0.09 g, 94%) mp = 204 °C; 1H NMR (DMSO-d6) δ: 8.33 (d, J = 4.8 Hz,
1H), 8.03 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 7.2 Hz, 2H), 7.47 (t, J = 7.6
Hz, 2H), 7.39 (t, J = 6.8 Hz, 1H), 7.33 (brs, 1H), 6.86 (d, J = 4.8 Hz, 1H), 3.35- 3.31 (m, 2H), 2.71
13
(s, 3H), 2.65-2.60 (m, 2H), 1.67 (brs, 2H); C NMR (DMSO-d6) δ: 166.1, 162.5, 161.5, 153.5,
142.5, 139.4, 132.1, 129.5, 129.4, 128.5, 127.8, 127.19, 127.11, 127.0, 106.2, 38.7, 31.5, 18.6;
HRMS (EI) m/z 387.5060 M+, calcd for C22H21N5S 387.5050; Anal. Calc. for: (C22H21N5S): C,
68.19; H, 5.46; N, 18.07%; Found: C, 68.28; H, 5.55; N, 18.15%.
N-{4-[2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl]pyrimidin-2-yl}propane-1,3-diamine (8)
Following the general procedure, and using propane-1,3-diamine (27 µL, 0.4 mmol), compound 8
was obtained as yellow solid (0.09 g, 91%) mp = 165 °C; 1H NMR (DMSO-d6) δ: 8.34 (d, J = 4.8
Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.2 Hz, 2H), 7.48 (t, J =
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7.6 Hz, 2H), 7.39 (t, J = 6.8 Hz, 1H), 7.24 (brs, 1H), 6.89 (d, J = 4.8 Hz, 1H), 3.20- 3.17 (m, 2H),
2.86- 2.85 (m, 2H), 2.71 (s, 3H), 2.30-2.28 (m, 2H), 1.17 (brs, 2H); 13C NMR (DMSO-d6) δ: 166.1,
162.6, 161.4, 159.3, 153.5, 142.5, 139.4, 132.1, 131.9, 129.5, 128.5, 127.8, 127.19, 127.12, 106.9,
44.4, 41.4, 35.0, 18.6; HRMS (EI) m/z 401.5318 M+, calcd for C23H23N5S 401.5320; Anal. Calc.
for: (C23H23N5S): C, 68.80; H, 5.77; N, 17.44%; Found: C, 68.87; H, 5.85; N, 17.21%.
2-{[4-(2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl]amino}ethan-1-ol (9)
Following the general procedure, and using 2-aminoethan-1-ol (21 µL, 0.4 mmol), compound 9
was obtained as yellow solid (0.09 g, 94%) mp = 173 °C; 1H NMR (DMSO-d6) δ: 8.35 (d, J = 4.8
Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.2 Hz, 2H), 7.48 (t, J =
7.6 Hz, 2H), 7.39 (t, J = 6.8 Hz, 1H), 7.13 (brs, 1H), 6.90 (d, J = 4.8 Hz, 1H), 4.68 (brs, 1H), 3.55-
3.54 (m, 2H), 3.40- 3.35 (m, 2H), 2.72 (s, 3H); 13C NMR (DMSO-d6) δ: 166.1, 162.5, 159.4, 157.2,
153.6, 142.5, 139.4, 132.1, 131.9, 129.5, 128.5, 127.8, 127.2, 127.1, 106.5, 60.0, 43.9, 18.6; HRMS
(EI) m/z 388.4909 M+, calcd for C22H20N4OS 388.4890; Anal. Calc. for: (C22H20N4OS): C, 68.02;
H, 5.19; N, 14.42%; Found: C, 68.09; H, 5.26; N, 14.49%.
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3-{[4-(2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl]amino}propan-1-ol (10)
Following the general procedure , and using 3-aminopropan-1-ol (27 µL, 0.4 mmol), compound
10 was obtained as yellow solid (0.09 g, 86%) mp = 113°C; 1H NMR (DMSO-d6) δ: 8.34 (d, J =
4.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.2 Hz, 2H), 7.48 (t, J
= 7.6 Hz, 2H), 7.39 (t, J = 6.8 Hz, 1H), 7.23 (brs, 1H), 6.88 (d, J = 4.8 Hz, 1H), 4.51 (brs, 1H),
3.50- 3.45 (m, 2H), 3.37- 3.34 (m, 2H), 2.72 (s, 3H), 1.70 (t, J = 6 Hz, 2H); 13C NMR (DMSO-d6)
δ: 166.1, 162.5, 159.4, 157.2, 153.5, 142.5, 139.4, 132.1, 131.9, 129.5, 128.5, 127.8, 127.2, 127.12,
106.5, 59.1, 38.5, 32.5, 18.6; HRMS (EI) m/z 402.5164 M+, calcd for C23H22N4OS 402.5160; Anal.
Calc. for: (C23H22N4OS): C, 68.63; H, 5.51; N, 13.92%; Found: C, 68.73; H, 5.59; N, 13.99%.
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1-{[4-(2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl]amino}ethane-1,2-diol
(11). Following the general procedure, and using 3-aminopropane-1,2-diol (33 µL, 0.4 mmol),
compound 11 was obtained as yellow solid (0.07 g, 63%) mp = 176 °C; 1H NMR (DMSO-d6) δ:
8.34 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.2 Hz,
2H), 7.46 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 6.8 Hz, 1H), 7.01 (brs, 1H), 6.90 (d, J = 4.8 Hz, 1H), 4.76
(brs, 1H), 4.54 (brs, 1H), 3.88- 3.67 (m, 1H), 3.44- 3.37 (m, 2H), 3.15- 3.13 (m, 2H), 2.71 (s, 3H);
13C NMR (DMSO-d6) δ: 166.3, 162.5, 159.4, 157.6, 153.6, 142.5, 139.4, 132.1, 131.9, 129.5,
128.5, 127.8, 127.2, 127.1, 106.5, 70.6, 64.5, 44.8, 18.6; HRMS (EI) m/z 418.5153 M+, calcd for
C23H22N4O2S 418.5150; Anal. Calc. for: (C23H22N4O2S): C, 66.01; H, 5.30; N, 13.39%; Found: C,
66.09; H, 5.38; N, 13.47%.
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(R)-1-{[4-(2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl]amino}ethane-1,2-
diol (12). Following the general procedure, and using (R)-3-aminopropane-1,2-diol (33 mg, 0.4
mmol), compound 12 was obtained as yellow solid (0.06 g, 57%) mp = 166 °C; 1H NMR (DMSO-
d6) δ: 8.34 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.2
Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 6.8 Hz, 1H), 7.01 (brs, 1H), 6.90 (d, J = 4.8 Hz, 1H),
4.77 (brs, 1H), 4.55 (brs, 1H), 3.69- 3.68 (m, 1H), 3.41- 3.38 (m, 2H), 3.23- 3.20 (m, 2H), 2.71 (s,
3H); 13C NMR (DMSO-d6) δ: 166.2, 162.5, 159.4, 157.2, 153.6, 142.5, 139.4, 132.1, 131.6, 129.5,
128.5, 127.8, 127.2, 127.1, 106.2, 70.6, 64.5, 44.8, 18.7; HRMS (EI) m/z 418.5142 M+, calcd for
C23H22N4O2S 418.5150; Anal. Calc. for: (C23H22N4O2S): C, 66.01; H, 5.30; N, 13.39%; Found: C,
66.12; H, 5.39; N, 13.44%.
(S)-3-{[4-(2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl]amino}propane-1,2-
diol (13). Following the general procedure, and using (S)-3-aminopropane-1,2-diol (33 mg, 0.4
mmol), compound 13 was obtained as yellow solid (0.07 g, 75%) mp = 164 °C; 1H NMR (DMSO-
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d6) δ: 8.35 (d, J = 4.8 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.2
Hz, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 6.8 Hz, 1H), 7.01 (brs, 1H), 6.91 (d, J = 4.8 Hz, 1H),
4.77 (brs, 1H), 4.53 (brs, 1H), 3.73- 3.68 (m, 1H), 3.38- 3.30 (m, 2H), 3.28- 3.20 (m, 2H), 2.73 (s,
3H); 13C NMR (DMSO-d6) δ: 166.2, 162.5, 159.3, 157.6, 153.6, 142.5, 139.4, 132.1, 131.9, 129.5,
128.5, 127.9, 127.2, 127.1, 106.2, 70.6, 64.5, 44.8, 18.6; HRMS (EI) m/z 418.5166 M+, calcd for
C23H22N4O2S 418.5150; Anal. Calc. for: (C23H22N4O2S): C, 66.01; H, 5.30; N, 13.39%; Found: C,
66.07; H, 5.36; N, 13.47%.
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2-{[4-(2-((1,1'-biphenyl)-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl]amino}propane-1,3-diol
(14). Following the general procedure, and using 2-aminopropane-1,3-diol (33 mg, 0.4 mmol),
compound 14 was obtained as yellow solid (0.08 g, 81%) mp = 154 °C; 1H NMR (DMSO-d6) δ:
8.36 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.2 Hz,
2H), 7.48 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 6.8 Hz, 1H), 6.90 (d, J = 4.8 Hz, 1H), 6.67 (brs, 1H), 4.62
(brs, 2H), 3.95 (p, J = 6 Hz, 1H), 3.15- 3.13 (m, 4H), 2.72 (s, 3H); 13C NMR (DMSO-d6) δ: 166.1,
162.3, 159.3, 157.6, 153.6, 142.5, 139.4, 132.1, 129.5, 128.5, 127.8, 127.2, 127.19, 127.13, 106.5,
60.6, 55.0, 18.3; HRMS (EI) m/z 418.5137 M+, calcd for C23H22N4O2S 418.5150; Anal. Calc. for:
(C23H22N4O2S): C, 66.01; H, 5.30; N, 13.39%; Found: C, 66.09; H, 5.38; N, 13.43%.
4-{2-[(1,1'-biphenyl)-4-yl]-4-methylthiazol-5-yl}-N-(pyrrolidin-3-yl)pyrimidin-2-amine (15)
Following the general procedure, using 3-amino-pyrrolidine dihydrochloride (60 mg, 0.4 mmol)
and potassium carbonate anhydrous (0.1 g, 0.7 mmol), compound 15 was obtained as orange solid
(0.1 g, 98%) mp = 90 °C; 1H NMR (DMSO-d6) δ: 8.37 (d, J = 4.8 Hz, 1H), 8.02 (d, J = 8.4 Hz,
2H), 7.93 (brs, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 7.2 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H),
7.38 (t, J = 6.8 Hz, 1H), 6.85 (d, J = 4.8 Hz, 1H), 3.62- 3.28 (m, 3H), 2.73 (s, 3H), 2.03- 1.98 (m,
2H), 1.72- 1.70 (m, 2H), 1.18 (brs, 1H); 13C NMR (DMSO-d6) δ: 166.1, 160.1, 159.2, 157.8, 153.7,
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142.5, 139.4, 132.1, 131.9, 129.5, 128.5, 127.8, 127.18, 127.10, 106.0, 54.8, 51.0, 45.2, 34.0, 18.7;
HRMS (EI) m/z 413.5436 M+, calcd for C24H23N5S 413.5430; Anal. Calc. for: (C24H23N5S): C,
69.71; H, 5.61; N, 16.94%; Found: C, 69.79; H, 5.71; N, 17.04%.
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4-{2-[(1,1'-biphenyl)-4-yl]-4-methylthiazol-5-yl}-N-(piperidin-2-ylmethyl)pyrimidin-2-
amine (16). Following the general procedure, and using 2-(aminomethyl)piperidine (42 µL, 0.4
mmol), compound 16 was obtained as yellow solid (0.1 g, 94%) mp = 146 °C; 1H NMR (DMSO-
d6) δ: 8.38 (d, J = 4.8 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.2
Hz, 2H), 7.51 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 6.8 Hz, 1H), 7.20 (brs, 1H), 6.89 (d, J = 4.8 Hz, 1H),
3.31- 3.15 (m, 2H), 2.93- 2.91 (m, 2H), 2.72 (s, 3H), 2.63- 2.60 (m, 1H), 1.75- 1.23 (m, 6H), 1.02
(brs, 1H); 13C NMR (DMSO-d6) δ: 166.1, 162.6, 159.4, 157.2, 153.6, 142.5, 139.4, 132.1, 131.9,
129.5, 128.5, 127.8, 127.19, 127.13, 106.2, 56.2, 47.2, 46.6, 30.7, 26.5, 24.7, 18.6; HRMS (EI)
m/z 441.5971 M+, calcd for C26H27N5S 441.5970; Anal. Calc. for: (C26H27N5S): C, 70.72; H, 6.16;
N, 15.86%; Found: C, 70.83; H, 6.25; N, 15.93%.
N-{4-[2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl]pyrimidin-2-yl}cyclohexane-trans-1,4-
diamine (17). Following the general procedure, and using trans-1,4-diaminocyclohexane (42 mg,
1
0.4 mmol), compound 17 was obtained as yellow solid (0.1 g, 97%) mp = 260 °C; H NMR
(DMSO-d6) δ: 8.34 (d, J = 4.8 Hz, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.74 (d,
J = 7.2 Hz, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 6.8 Hz, 1H), 7.13 (brs, 1H), 6.91 (d, J = 4.8
Hz, 1H), 3.72- 3.55 (m, 2H), 2.74 (s, 3H), 1.97-1.87 (m, 4H), 1.33-1.28 (m, 4H), 1.20 (brs, 2H);
13C NMR (DMSO-d6) δ: 166.3, 161.8, 159.7, 157.6, 153.1, 142.5, 139.4, 132.1, 131.9, 129.5,
128.5, 127.9, 127.16, 127.13, 106.5, 49.6, 37.1, 33.3, 31.2, 30.5, 18.6; HRMS (EI) m/z 441.5951
M+, calcd for C26H27N5S 441.5970; Anal. Calc. for: (C26H27N5S): C, 70.72; H, 6.16; N, 15.86%;
Found: C, 70.83; H, 6.25; N, 15.94%.
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N-{4-[2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl]pyrimidin-2-yl}cyclohexane-cis-1,2-
diamine (18). Following the general procedure, and using (±)-cis-1,2-diaminocyclohexane (42
µL, 0.4 mmol), compound 18 was obtained as brown solid (0.06 g, 51%) mp = 104 °C; 1H NMR
(DMSO-d6) δ: 8.36 (d, J = 4.8 Hz, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.72 (d,
J = 7.2 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.42 (t, J = 6.8 Hz, 1H), 6.91 (d, J = 4.8 Hz, 1H), 4.65
(brs, 1H), 4.03- 3.86 (m, 2H), 3.11- 3.08 (m, 2H), 2.71 (s, 3H), 1.90-1.28 (m, 6H), 1.20 (brs, 2H);
13C NMR (DMSO-d6) δ: 166.3, 161.8, 159.0, 157.6, 153.8, 142.5, 139.4, 132.1, 129.5, 128.5,
127.8, 127.2, 127.19, 127.13, 105.5, 54.5, 49.3, 39.3, 26.4, 21.21, 19.7, 18.6; HRMS (EI) m/z
441.5955 M+, calcd for C26H27N5S 441.5970; Anal. Calc. for: (C26H27N5S): C, 70.72; H, 6.16; N,
15.86%; Found: C, 70.81; H, 6.25; N, 15.93%.
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N-{4-[2-((1,1'-biphenyl)-4-yl)-4-methylthiazol-5-yl]pyrimidin-2-yl}cyclohexane-trans-1,2-
diamine (19). Following the general procedure, and using (±)-trans-1,2-Diaminocyclohexane (42
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µL, 0.4 mmol), compound 19 was obtained as brown solid (0.1 g, 94%) mp = 93 °C; H NMR
(DMSO-d6) δ: 8.33 (d, J = 4.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.73 (d,
J = 7.2 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 6.8 Hz, 1H), 7.14 (brs, 1H), 6.86 (d, J = 4.8
Hz, 1H), 3.48- 3.46 (m, 2H), 3.30- 3.15 (m, 2H), 2.72 (s, 3H), 2.05-1.08 (m, 2H), 1.64 (brs, 2H),
1.23-1.10 (m, 4H); 13C NMR (DMSO-d6) δ: 166.0, 162.7, 159.4, 157.6, 153.5, 142.5, 139.4, 132.3,
132.1, 129.5, 128.5, 127.9, 127.18, 127.12, 106.9, 53.7, 37.1, 34.9, 31.2, 25.3, 25.2, 18.6; HRMS
(EI) m/z 441.5981 M+, calcd for C26H27N5S 441.5970; Anal. Calc. for: (C26H27N5S): C, 70.72; H,
6.16; N, 15.86%; Found: C, 70.81; H, 6.23; N, 15.95%.
(R)-{1-[4-(2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl]pyrrolidin-2-
yl}methanol (20). Following the general procedure, using (R)-(+)-2-pyrrolidinemethanol (37 µL,
1
0.4 mmol), compound 20 was obtained as yellow solid (0.1 g, 96%) mp = 105 °C; H NMR
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(DMSO-d6) δ: 8.40 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.73 (d,
J = 7.2 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 6.8 Hz, 1H), 6.91 (d, J = 4.8 Hz, 1H), 4.76
(brs, 1H), 4.18- 4.12 (m, 1H), 3.68- 3.66 (m, 2H), 3.53- 3.47 (m, 2H), 2.71 (s, 3H), 2.03- 1.89 (m,
4H); 13C NMR (DMSO-d6) δ: 166.2, 160.2, 158.6, 153.4, 142.5, 139.4, 132.3, 131.6, 129.5, 128.5,
128.1, 127.8, 127.2, 127.1, 106.3, 60.7, 59.4, 47.7, 28.0, 22.9, 18.7; HRMS (EI) m/z 428.5549 M+,
calcd for C25H24N4OS 428.5540; Anal. Calc. for: (C25H24N4OS): C, 70.07; H, 5.65; N, 13.07%;
Found: C, 70.18; H, 5.73; N, 13.16%.
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(S)-{1-[4-(2-((1,1'-Biphenyl)-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl]pyrrolidin-2-
yl}methanol (21). Following the general procedure, using (S)-(-)-2-pyrrolidinemethanol (37 µL,
0.4 mmol), compound 21 was obtained as grey solid (0.09 g, 87%) mp = 256 °C; 1H NMR (DMSO-
d6) δ: 8.39 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.2
Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 6.8 Hz, 1H), 6.90 (d, J = 4.8 Hz, 1H), 4.80 (brs, 1H),
4.12- 4.11 (m, 1H), 3.65- 3.53 (m, 2H), 3.48- 3.43 (m, 2H), 2.73 (s, 3H), 2.02- 1.88 (m, 4H); 13C
NMR (DMSO-d6) δ: 166.2, 160.2, 159.0, 157.6, 153.1, 142.5, 139.4, 132.3, 132.1, 129.5, 128.5,
127.8, 127.2, 127.1, 106.3, 60.7, 59.7, 47.7, 28.0, 22.9, 18.7; HRMS (EI) m/z 428.5542 M+, calcd
for C25H24N4OS 428.5540; Anal. Calc. for: (C25H24N4OS): C, 70.07; H, 5.65; N, 13.07%; Found:
C, 70.13; H, 5.69; N, 13.11%.
(R)-1-{4-[2-((1,1'-biphenyl)-4-yl)-4-methylthiazol-5-yl]pyrimidin-2-yl}-N,N-
dimethylpyrrolidin-3-amine (22). Following the general procedure, using (R)-(+)-3-
(dimethylamino)pyrrolidine dihydrochloride (71 mg, 0.4 mmol) and potassium carbonate
anhydrous (0.1 g, 0.7 mmol), compound 22 was obtained as yellow solid (0.1 g, 91%) mp = 127
°C; 1H NMR (DMSO-d6) δ: 8.38 (d, J = 4.8 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.4 Hz,
2H), 7.72 (d, J = 7.2 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 6.8 Hz, 1H), 6.87 (d, J = 4.8 Hz,
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