Synthesis and structure-activity relationship of: N 4-benzylamine- N 2-isopropyl-quinazoline-2,4-diamines derivatives as potential antibacterial agents

Article abstract of DOI:10.1039/c7ra10352b

A series of N⁴-benzylamine-N²-isopropyl-quinazoline-2,4-diamine derivatives has been synthesized and tested for antibacterial activity against five bacterial strains. Twelve different substituents on the N⁴-benzylamine group have been investigated along with replacement of the quinazoline core (with either a benzothiophene or regioisomeric pyridopyrimidine ring systems). In order to develop structure activity relationships, all derivatives were tested for their antibacterial activities against Escherichia coli and Staphylococcus aureus via Kirby-Bauer assays and minimum inhibitory concentration assays. Eight of the most potent compounds against S. aureus and E. coli were also screened against one strain of methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis and Salmonella typhimurium to further examine their antibacterial activities. Lead compound A5 showed good activities with MICs of 3.9 μg mL^-1 against E. coli, S. aureus and S. epidermidis and 7.8 μg mL^-1 against MRSA. Selected front runners were also screened for their DMPK properties in vitro to assess their potential for further development.

Full text of DOI:10.1039/c7ra10352b

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Synthesis and structure–activity relationship of N4-
benzylamine-N2-isopropyl-quinazoline-2,4-
diamines derivatives as potential antibacterial
agents
Cite this: RSC Adv., 2017, 7, 52227
Zhengyun Jiang,a W. David Hong,bcde Xiping Cui,a Hongcan Gao,a Panpan Wu,ad
Yingshan Chen,a Ding Shen,a Yang Yang,a Bingjie Zhang,a Mark J. Taylor,b
a
ade
Stephen A. Ward,b Paul M. O'Neill,c Suqing Zhao
*
*
and Kun Zhang
A series of N4-benzylamine-N2-isopropyl-quinazoline-2,4-diamine derivatives has been synthesized and tested
for antibacterial activity against five bacterial strains. Twelve different substituents on the N4-benzylamine group
have been investigated along with replacement of the quinazoline core (with either a benzothiophene or
regioisomeric pyridopyrimidine ring systems). In order to develop structure activity relationships, all derivatives
were tested for their antibacterial activities against Escherichia coli and Staphylococcus aureus via Kirby–
Bauer assays and minimum inhibitory concentration assays. Eight of the most potent compounds against S.
aureus and E. coli were also screened against one strain of methicillin-resistant S. aureus (MRSA),
Staphylococcus epidermidis and Salmonella typhimurium to further examine their antibacterial activities. Lead
compound A5 showed good activities with MICs of 3.9 mg mLꢀ1 against E. coli, S. aureus and S. epidermidis
and 7.8 mg mLꢀ1 against MRSA. Selected front runners were also screened for their DMPK properties in vitro
to assess their potential for further development.
Received 18th September 2017
Accepted 26th October 2017
DOI: 10.1039/c7ra10352b
rsc.li/rsc-advances
cearoline, which are used to treat MRSA infections, have been
reported recently in the literature.15–18 MRSA is on the list of
Introduction
bacteria for which new antibiotics are urgently needed as
recognized by World Health Organization in 2017.19
Novel antibacterial chemotypes are urgently needed because of
the emergence and spread of multidrug resistant microorgan-
isms and pathogenic bacterial infections.1,2 Staphylococcus
aureus is the most common pathogen of surgical site and
wound infections.3–6 Antibiotic-resistant strains of S. aureus
infections oen occur in epidemic waves that are initiated by
one, or a few, successful clones.7–9 Every year in the United
States approximately two million people fall ill and 23 000
people die because of antibiotic-resistant bacterial infections,
according to a report from the Center for Diseases Control and
Prevention.10,11 Antibiotic-resistant bacterial strains, in partic-
ular methicillin-resistant S. aureus (MRSA) infection rates
remain constant within the community and hospital settings in
U.S.12–14 Worryingly, strains of S. aureus showing extended
resistance to vancomycin, daptomycin, linezolid and
Quinazolines and derivatives have shown attractive antibacte-
rial activity.20–22 In previous studies, quinazoline-based compounds
have been investigated for their potential antibacterial activity,
especially anti-MRSA activity. Bedi et al. (2004) reported 2,4-
disubstituted quinazoline, such as compound A (Fig. 1), displayed
antibacterial activity against a wide spectrum of bacteria including
S. aureus, and E. coli.23 Chandrika et al. (2010) reported the in vitro
activity of multiple uoro-substituted triazol-4-yl substituted qui-
nazoline B (Fig. 1) against S. aureus and S. epidermidis with an MIC
of 9.375 mg mL.24 Van Horn et al. (2014) have reported N2,N4-
disubstituted quinazoline-2,4-diamines such as C (Fig. 1) that
aDepartment of Pharmaceutical Engineering, School of Chemical Engineering and Light
Industry, Guangdong University of Technology, Guangzhou, 510006, China. E-mail:
sqzhao@gdut.edu.cn; kzhang@gdut.edu.cn
bResearch Centre for Drugs & Diagnostics, Department of Parasitology, Liverpool
School of Tropical Medicine, Liverpool, L3 5QA, UK
cDepartment of Chemistry, University of Liverpool, Liverpool, L69 7ZD, UK
dFaculty of Chemical & Environmental Engineering, Wuyi University, Jiangmen,
529020, China
eInternational Healthcare Innovation Institute (Jiangmen), Jiangmen, 529000, China
Fig. 1 Quinazolines with reported antibacterial activity.
RSC Adv., 2017, 7, 52227–52237 | 52227
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displayed in vitro and in vivo activities against S. aureus, a low compounds with different heterocyclic aromatic rings fused to
potential for spontaneous resistance and low toxicity.25 Further- the pyrimidine ring in the scaffold were designed and then
more, this proof-of-concept work opened up opportunities for subsequently synthesized via adapted methods from the liter-
further investigation of quinazoline-2,4-diamines in terms of ature (Fig. 2).25–30 Commercially available compound (a) was
structural activity relationships against a broader spectrum of reacted with phosphorus oxychloride and phosphorus penta-
bacteria strains and DMPK related parameters. In this work, we chloride resulting in the corresponding 2,4-dichloro interme-
designed a library of 2,4-diaminoquinazoline analogues and diates (b). Substitution with benzylamines occurred selectively
closely related derivatives, including thirteen 2,4-dia- at the 4-position, yielding 4-benzylamino-2-chloroquinazoline
minoquinazolines, thirteen 2,4-diaminothieno[3,2-d]pyrimidines, (c) under mild conditions. Next, substitution at the 2-position
six 2,4-diaminopyrido[3,2-d]pyrimidine derivatives and six 2,4- with isopropyl amine gave the desired 2,4-diamino products (d).
diaminopyrido[2,3-d]pyrimidine derivatives. These compounds are The synthesis of compounds from series A and B were started
structurally distinct from previously reported quinazoline core from the commercially available as quinazoline-2,4(1H,3H)-
antibacterials. These compounds have been used to further dione and thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione, respec-
explore how the structural variation affects antibacterial activity. tively. However, 2,4-dichloropyrido[3,2-d]-pyrimidine and 2,4-
Herein, a structure–activity relationship (SAR) study is reported dichloropyrido[2,3-d]pyrimidine, exemplied as intermediate
which focuses on the substituent of N4-benzylamine and the (b) which were readily acquired commercially have been used in
variations of the quinazoline scaffold.
the synthesis of series C and D compounds. Throughout the
All derivatives were tested against S. aureus and E. coli in synthetic routes, all intermediates have been puried and the
Kirby–Bauer assays initially. The antibacterial potency of active nal
N4-benzylamine-N2-isopropyl-quinazoline-2,4-diamines
1
compounds was further determined by minimum inhibitory derivatives have been characterized by H NMR, 13C NMR and
concentrations (MICs) assays. Further MICs assays were per- high resolution mass spectrum.
formed against methicillin-resistant S. aureus (MRSA), Staphy-
lococcus epidermidis and Salmonella typhimurium to evaluate the
spectrum of antibacterial activity of the most active compounds.
In addition, DMPK related properties, such as lipophilicity
activities in inhibiting bacterial growth in the Kirby–Bauer assay
(log D7.4), aqueous solubility and in vitro metabolic stability
were also assessed for a selected group of compounds to illus-
trate how the structural modications can potentially inuence
tested for their minimum inhibitory concentrations (MICs) to
the pharmacokinetics of these new lead molecules.
Structure–activity relationship study
Initially all 4 chemical subsets (series A–D) were tested for their
against E. coli and S. aureus. Active compounds with zones of
inhibitions (ZOIs) $ 7.0 mm in the Kirby–Bauer assay were then
quantify their potency against E. coli and S. aureus (Table 1). The
data of ZOIs and MICs presented are the average results from
three independent experiments.
Results and discussion
Chemistry
Kirby–Bauer assay
To systematically explore structure–activity relationships,
a
group of N4-benzylamine-N2-isopropyl-quinazoline-2,4- Kirby–Bauer assay was used to test the compounds at a single
diamine derivatives which include four chemical subsets of concentration of 5 mg mLꢀ1 to determine whether they
Fig. 2 Synthetic route and chemical structures of N4-benzylamino-N2-isopropyl-quinazoline-2,4-diamines. Reagents and conditions: (I) PCl5,
POCl3, 120 ꢁC, 6 h; (II) substituted benzylamine, triethylamine, THF, rt, overnight; (III) isopropylamine, 1,4-dioxane, 120 ꢁC, 48 h.
52228 | RSC Adv., 2017, 7, 52227–52237
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Table 1 Antibacterial activities of N2-isopropyl-N4-benzylamine-quinazoline-2,4-diamines derivatives
E. colia
S. aureusb
Compound code
R
ZoIs (mm)
MICs (mg mLꢀ1
)
ZoIs (mm)
MICs (mg mLꢀ1
)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
B13
C1
C2
C3
C5
C13
D1
4-CN–phenyl–
2-SO2Me–phenyl–
4-SO2Me–phenyl–
2-CF3–phenyl–
4-CF3–phenyl–
2-F–phenyl–
4-F–phenyl–
2-Cl–phenyl–
4-Cl–phenyl–
2-OCH3–phenyl–
4-OCH3–phenyl–
3,4-Di–OCH3–phenyl–
4-CN–phenyl–
2-SO2Me–phenyl–
4-SO2Me–phenyl–
2-CF3–phenyl–
4-CF3–phenyl–
2-F–phenyl–
20.2 ꢂ 0.6
8.3 ꢂ 0.2
7.5 ꢂ 0.3
20.4 ꢂ 0.7
22.4 ꢂ 0.1
23.8 ꢂ 0.8
22.3 ꢂ 0.5
21.2 ꢂ 0.7
18.6 ꢂ 0.2
15.7 ꢂ 0.6
17.1 ꢂ 0.9
16.2 ꢂ 0.4
21.0 ꢂ 0.4
7.2 ꢂ 0.1
7.9 ꢂ 0.2
Nonec
16.8 ꢂ 0.8
8.6 ꢂ 0.1
8.0 ꢂ 0.3
8.5 ꢂ 0.4
11.4 ꢂ 0.4
8.1 ꢂ 0.5
None
31.2
125.0
$125.0
15.6
3.9
15.6
15.6
15.6
7.8
31.2
31.2
31.2
31.2
$125.0
$125.0
NTd
31.2
125.0
62.5
62.5
62.5
62.5
NT
NT
NT
$125.0
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
#0.12
>31.2
>31.2
11.8 ꢂ 0.5
8.1 ꢂ 0.2
None
31.2
62.5
NT
3.9
3.9
15.6
7.8
3.9
3.9
31.2
7.8
15.6
31.2
62.5
NT
NT
7.8
16.2 ꢂ 0.7
13.2 ꢂ 0.5
12.4 ꢂ 0.3
14.5 ꢂ 0.3
12.0 ꢂ 0.5
11.6 ꢂ 0.2
17.4 ꢂ 0.5
15.4 ꢂ 0.4
14.6 ꢂ 0.5
11.6 ꢂ 0.3
8.9 ꢂ 0.3
None
None
12.1 ꢂ 0.3
12.7 ꢂ 0.3
11.1 ꢂ 0.5
10.9 ꢂ 0.3
13.6 ꢂ 0.6
12.9 ꢂ 0.4
None
7.8
4-F–phenyl–
2-Cl–phenyl–
4-Cl–phenyl–
31.2
31.2
15.6
7.8
NT
NT
62.5
62.5
62.5
NT
NT
15.6
NT
2-OCH3–phenyl–
4-OCH3–phenyl–
3,4-Di–OCH3–phenyl–
4-CN–phenyl–
2-SO2Me–phenyl–
4-SO2Me–phenyl–
4-CF3–phenyl–
4-CN–phenyl–
2-SO2Me–phenyl–
4-SO2Me–phenyl–
4-CF3–phenyl–
4-CF3–pyridyl–
4-CF3–pyridyl–
4-CF3–pyridyl–
4-CF3–pyridyl–
None
None
None
9.2 ꢂ 0.4
8.2 ꢂ 0.1
7.6 ꢂ 0.3
None
7.3 ꢂ 0.1
None
None
None
None
None
None
None
None
None
None
NT
NT
NT
None
8.5 ꢂ 0.6
None
None
NT
31.2
NT
D2
D3
D5
D13
Noroxacin
Vancomycin
Methicillin
8.1 ꢂ 0.2
None
10.2 ꢂ 0.1
8.1 ꢂ 0.8
NT
NT
NT
7.8
62.5
0.24
0.98
0.49
a
c
d
Escherichia coli, CMCC 44102. b Staphylococcus aureus, ATCC 6538. No zone of inhibition was determined. Not tested.
inhibited bacterial growth. The data are exhibited in Table 1. In activity against Gram-negative E. coli. No compound showed
this assay, sterile lter disks with a diameter of 6 mm were used. noticeably stronger activity against Gram-negative E. coli than
The diameter of ZOIs greater than 10.0 mm was dened as Gram-positive S. aureus, represented by the blue coloured area
strongly active, 7.0–10.0 mm was weakly active, and less than in Fig. 3. From this analysis, it also indicated that while
7.0 mm was inactive. As shown in Fig. 3, 13 compounds all from a number of series A quinazoline analogues have activities
series A or B appear in the green area which signies against both Gram-positive and Gram-negative bacteria, series
compounds showing potent activity against both Gram-negative B thieno[3,2-d]pyrimidines were more biased to Gram-positive
bacteria, E. coli and Gram-positive bacteria S. aureus. In the le- bacteria and series C and D compounds had limited activities
hand side yellow area, 5 compounds from series B and D in inhibiting the growth of these bacterial strains.
showed some activity against Gram-positive S. aureus but weak
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same potency against S. aureus but slightly reduced potency
(MIC ¼ 7.8 mg mLꢀ1) against Gram-negative E. coli. The series B
thieno[3,2-d]pyrimidine analogues also showed some activity
against both strains of bacteria, but they were less active than
their corresponding quinazoline core analogues in general. The
SAR observed in series B compounds is very similar to the SAR
of series A. While the replacement of the quinazoline core to
a theino[3,2-d]pyrimidine core had less effect on the potency
against Gram-positive S. aureus (2–4 fold reductions), this
modication resulted in a noticeable reduction of antibacterial
activity against Gram-negative E. coli (2–32 fold reductions).
Compounds in both series C and D with an additional nitrogen
incorporated into the 5- or 8-position of the quinazoline core
showed signicantly reduced potency against both strains of
bacteria. Two compounds with p-CF3 substitutions on the N4-
benzylamine side-chain (C5 and D5) in these two series showed
moderate activities against S. aureus (MIC ¼ 15.6 mg mLꢀ1 and
7.8 mg mLꢀ1, respectively), but no activity against E. coli.
The MIC assay was used to evaluate the eight most active
compounds against S. aureus (as described above) namely, A4,
A5, A7–9, A11, B5 and, B9 against a strain of MRSA. MICs of
these selected compounds against MRSA were only two to four
folds higher than the corresponding MICs against the suscep-
tible strain of S. aureus. The MIC difference seen with this group
of compounds is similar to the MIC differences seen with the
two positive controls, i.e. Noroxacin and vancomycin (2–3
folds), but is signicantly less than that seen with methicillin
(ꢃ16 folds). These results suggest that this new class of
compound has minimal cross resistance with this strain of
MRSA although further studies are required to formally conrm
this using a broader range of MRSA lines with unique and well
characterized resistance mechanisms. The most potent
compound against this strain of MRSA was A5, the p-CF3
substituted analogue from series A.
In order to extend our understanding of the antibacterial
activities of this class of compounds, the same group of eight
active compounds from series A and B were also tested against
additional strains of Gram-positive and Gram-negative bacteria,
namely S. epidermidis and S. typhimurium using the MIC assay.
Overall the antibacterial activities of these eight selected
compounds against these additional bacterial lines matched
very well with those against S. aureus and E. coli (Table 2). Most
of the tested compounds showed good activities against Gram-
positive S. epidermidis at similar levels to those observed with S.
aureus, but their potency against Gram-negative S. typhimurium
was considerably lower than their potency against other tested
strains of bacteria. Both quinazoline analogues with CF3
substituted at either ortho- or para-positions in the N4-benzyl-
amine side-chain (A4 and A5) were the most potent compounds
from the assays against S. epidermidis and S. typhimurium with
MIC ¼ 3.9 mg mLꢀ1 and 15.6 mg mLꢀ1, respectively.
Fig. 3 Zones of inhibition (ZOIs) in Kirby–Bauer assays against E. coli
and S. aureus.
Minimum inhibitory concentrations (MICs) assay
All
N4-benzylamine-N2-isopropyl-quinazoline-2,4-diamines
derivatives with measurable ZOIs in the Kirby–Bauer assays
were tested in minimum inhibitory concentration (MIC) assays
against the same two strains of bacteria to quantitatively
determine their antibacterial potency. Noroxacin, vancomycin
and methicillin were used as positive controls.
In general, the results of the MIC assay mirrored the results
from the single concentration Kirby–Bauer assay, and the
antibacterial potency of the chemical series are in the order of
quinazolines > thieno[3,2-d]pyrimidines > pyrido[3,2-d]pyrimi-
dines z pyrido[2,3-d]pyrimidines, while higher potency was
observed against Gram-positive S. aureus than against Gram-
negative E. coli. (Table 1). In the most active subset, series A,
substitutions on the benzene ring in the N4-benzylamin side-
chain had a signicant effect of the potency of compounds.
Lipophilic electron withdrawing group substitution in this side-
chain, such as CF3 and Cl were benecial for antibacterial
activity. On the other hand, the more polar substitutions i.e.
SO2Me or CN were not tolerated (A1–3). Similar observations
were also seen aer the incorporation of an additional nitrogen
in this side-chain (A13) which reduced both lipophilicity (log D)
and potency compared with the corresponding analogue A5
without the nitrogen (further discussion regarding the effect of
the structural modications to the physiochemical properties
are described in a following session). Series A analogues, with
substitutions at the para-position of the benzene ring, were
more potent against Gram-negative E. coli than the corre-
sponding ortho-substituted analogues, but these substitutions
had little effect on potency against Gram-positive S. aureus (A4
vs. A5 and A8 vs. A9). Quinazoline derivative A5 with a tri-
uoromethyl group at the para-position of the benzene ring was
the most active compound in this subset of compounds and
indeed in this whole set of compounds with MICs
¼
Physicochemical properties and in vitro metabolic stability
studies
3.9 mg mLꢀ1 against both Gram-positive and negative bacteria,
S. aureus and E. coli. It was closely followed by the chloro-
substituted analogue at the same position (A9) that has the
Along with the SAR studies of these compounds, selected
derivatives were also evaluated for their DMPK properties
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Table 2 Antibacterial activities of the most active compounds against
MRSA, S. epidermidis and S. typhimurium
bacterium. The percentage of human plasma protein binding
for those measured compounds also showed positive correla-
tion with lipophilicity. Although the plasma protein bindings
are in the relatively high range of percentage (96.5–99.9%) it is
not uncommon for anti-infective agents. In terms of metabolic
stability, most of the quinazoline analogues showed acceptable
stability in vitro against both human microsome and rat hepa-
tocytes, and are more stable than the corresponding thieno-
pyrimidine analogues (A5 vs. B5 and A9 vs. B9). The
azaquinazoline analogue D5 had the best in vitro metabolic
stability against both human microsome and rat hepatocytes,
but this modication was not well tolerated in the antibacterial
SAR.
MRSAa
S. epidermidisb
S. typhimuriumc
Compound
MICs (mg mLꢀ1
)
MICs (mg mLꢀ1
)
MICs (mg mLꢀ1
)
A4
A5
15.6
7.8
3.9
3.9
15.6
15.6
A7
A8
A9
A11
B5
B9
Noroxacin
Vancomycin
Methicillin
31.2
15.6
15.6
31.2
15.6
15.6
0.97
1.95
7.8
7.8
7.8
7.8
15.6
7.8
7.8
1.95
3.9
15.6
62.5
15.6
31.2
$125.0
31.2
#0.12
$31.2
$31.2
0.24
Conclusions
a
Methicillin-resistant Staphylococcus aureus (MRSA), ATCC 43300.
Staphylococcus epidermidis, ATCC 12228. Salmonella typhimurium, In this article, 36 N2,N4-disubstituted quinazoline-2,4-diamines
b
c
CMCC 50115.
and closely related derivatives with potential antibacterial
activity were designed and synthesized. Our SAR study
demonstrated that lipophilic electron withdrawing groups,
such as triuoromethyl and chloro, in the N4-benzylamine side-
chain were benecial for antibacterial potency, while substitu-
tion at the 4-position of the benzene ring was advantageous
compared with substitutions at the 2-position. Changing the
quinazoline scaffold to a thieno[3,2-d]pyrimidine, series B
compounds, still showed limited antibacterial activity
compared with the corresponding quinazoline analogues.
Scaffold morphing to pyrido[3,2-d]pyrimidine or pyrido[2,3-d]
pyrimidine was not tolerated in the SAR and most of the
compounds bearing these two cores were signicantly less
potent than the parent quinazolines. Screens against one strain
of MRSA suggested this class of compounds had limited cross
resistance to methicillin although the exact protein target(s) for
this class of compounds needs to be further investigated against
a broader selection of MRSA bacteria. The results from the
screens against S. aureus and E. coli in the SAR study and the
additional screens against S. epidermidis and S. typhimurium
demonstrated a broad spectrum of antibacterial activity against
both Gram-positive and Gram-negative bacteria while the
potency against Gram-positive bacteria was generally higher
than the latter. The in vitro DMPK screening results suggested
including physicochemical properties such as log D7.4, aqueous
solubility and plasma protein binding, and in vitro metabolic
stability against human microsome and rat hepatocytes
(Table 3). Overall, the more active compounds, such as A4, A5,
A8 and A9 showed high lipophilicity (log D7.4) and low aqueous
solubility in PBS buffer. Replacing the benzene ring of quina-
zoline scaffold with a thiophene ring (B5 vs. A5 and B9 vs. A9)
did not alter lipophilicity but increased the aqueous solubility
slightly. Incorporation of nitrogens in different parts of the
molecules (A5 vs. A13 or D5) or replacement of CF3 group with
an OMe group (A5 vs. A11) resulted in reductions of lipophilicity
and improvement of solubility, but all manipulations resulted
in decreased antibacterial activities. Based on the available SAR
and the data of log D7.4 in Table 3, it suggested there is
a potential positive correlation between lipophilicity and anti-
bacterial activity. Compounds A4, A5, A8, A9, B5 and B9, which
have log D7.4 values higher than 4, showed good activities
against S. aureus with MICs ¼ 3.9 mg mLꢀ1. On the contrary,
compounds A7, A11 and A13, which have log D7.4 values from
3.3 to 3.6, showed a 2-fold or 8-fold less active against the same
Table 3 Physicochemical properties and in vitro metabolic stability
Aq. Sol.a (mM)
H. Mics CLintb (ml minꢀ1 mg)
R. Heps CLint (ml minꢀ1 per 106 cells)
H. PPBd
%
c
Compound
log D7.4
A4
A5
A7
A8
4.6
4.7
3.6
4.2
4.3
3.3
3.3
4.5
4.8
4.4
0.2
0.5
27
5
9.2
29
32
19
36
37
45
70
156
7.6
25
45
37
98
99.6
99.8
98.0
98.9
99.2
97.4
96.5
99.9
99.8
98.5
A9
4
51
A11
A13
B5
B9
D5
40
17
3.7
11
3
>300
7.8
76
217
6.5
a
b
c
d
Aqueous solubility in pH 7.4 PBS. Human microsomes intrinsic clearance. Rat hepatocytes intrinsic clearance. Human plasma protein
binding.
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that some of the lead series A quinazolines had acceptable equivalent) were mixed in 5 mL 1,4-dioxane in a high pressure
metabolic stability in vitro, their physiochemical properties, reactor and heated to 120 ꢁC for 48 h. The high pressure reactor
aqueous solubility in particular would need to be improved if was cooled down and the solution was concentrated under
they were to be developed further. The most active compound reduced pressure. The remaining residue was puried by ash
A5 showed good potency against E. coli, S. aureus, S. epidermidis, chromatography with dichloromethane and methanol.
S. typhimurium and MRSA. Although the cytotoxicity of these
A1
N4-(4-Cyanobenzyl)-N2-isopropylquinazoline-2,4-diamine.
series of compounds were not determined yet, the cytotoxicity of Yellow solid; yield, 75%; Rf ¼ 0.36 (9 : 1 dichloromethane to
some closed analogues were reported by our group and others methanol); 1H NMR (400 MHz, DMSO-d6) d 10.35 (s, 1H), 8.38 (s,
(Van Horn 2014, Devine 2015 and Johnston 2017), and there is 1H), 7.80 (d, J ¼ 8.3 Hz, 2H), 7.78–7.72 (m, 1H), 7.59 (d, J ¼
no obvious risk of cytotoxicity for these chemotypes in 8.2 Hz, 2H), 7.37 (s, 1H), 4.84 (d, J ¼ 5.5 Hz, 2H), 4.06 (s, 1H),
general.25,31,32 Further investigation of cytotoxicity of selected 1.08 (s, 6H). 13C NMR (100 MHz, DMSO-d6) d 159.90, 158.94,
leads will be carried out in the next stage of optimisation and 144.21, 139.90, 138.89, 135.05, 132.28, 128.26, 128.17, 124.24,
evaluation to mitigate any potential risk in this area. The results 123.70, 118.75, 109.73, 62.76, 42.89, 22.03. HRMS: m/z calcd for
from this work indicate the potential of this chemical series and
directions for further investigation of this chemotype as 257–260 ꢁC.
C
19H20N5 [M + H]+ 318.1713; found 318.1719. Melting point
potential antibacterial agents.
A2 N2-Isopropyl-N4-(2-(methylsulfonyl)benzyl)quinazoline-2,4-
diamine. Brown solid; yield, 49%; Rf ¼ 0.42 (9 : 1 dichloro-
methane to methanol); 1H NMR (400 MHz, CDCl3) d 8.04 (dd, J
¼ 7.9, 1.3 Hz, 1H), 7.71 (dd, J ¼ 7.6, 0.8 Hz, 1H), 7.64–7.56 (m,
2H), 7.55–7.47 (m, 2H), 7.39 (d, J ¼ 8.0 Hz, 1H), 7.14–7.08 (m,
1H), 5.15 (d, J ¼ 5.6 Hz, 2H), 4.29 (m, 1H), 3.18 (s, 3H), 1.27 (d, J
¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) d 160.11, 156.10,
146.37, 137.59, 137.55, 133.57, 133.36, 129.09, 129.02, 127.57,
122.05, 122.00, 120.69, 109.96, 43.48, 42.45, 41.53, 21.67. HRMS:
m/z calcd for C19H23N4O2S [M + H]+ 371.1536; found 371.1540.
Experimental
All chemicals, reagents and solvents were obtained from
commercial sources and used without further purication. H
1
and 13C NMR spectra were recorded on a Bruker Avance III 400
MHz Superconducting Fourier system in the solvent indicated.
Chemical shis were reported in units of ppm on the delta (d)
scale and coupling constants (J) were reported in units of Hz.
Data for 1H NMR were reported as follows: chemical shi (d
ppm), multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼
quartet, m ¼ multiplet), integration and coupling constant (Hz),
while 13C NMR analyses were reported in terms of chemical
shi. Thin-layer chromatography was performed on silica gel 60
F254 aluminium sheets from Merck KGaA. Melting points were
determined by using a microscopic melting point instrument
and were uncorrected. High resolution mass spectra (HRMS)
were performed on a Bruker maXis impact system.
ꢁ
Melting point 171–173 C.
A3 N2-Isopropyl-N4-(4-(methylsulfonyl)benzyl)quinazoline-2,4-
diamine. Brown solid; yield, 53%; Rf ¼ 0.39 (9 : 1 dichloro-
methane to methanol); 1H NMR (400 MHz, CDCl3) d 8.01 (d, J ¼
6.9 Hz, 1H), 7.73 (d, J ¼ 8.3 Hz, 2H), 7.50–7.47 (m, 2H), 7.46 (d, J
¼ 7.2 Hz 1H), 7.32 (d, J ¼ 8.3 Hz, 1H), 7.07 (t, J ¼ 7.6 Hz, 1H),
4.85 (s, 2H), 4.07 (m, 1H), 2.97 (s, 3H), 1.109 (d, J ¼ 6.4 Hz 6H).
13C NMR (100 MHz, CDCl3) d 160.52, 155.15, 151.64, 145.21,
139.02, 134.17, 128.10, 127.42, 122.94, 122.67, 119.93, 110.00,
44.37, 44.11, 43.14, 22.25. HRMS: m/z calcd for C19H23N4O2S [M
+
ꢁ
+ H] 371.1536; found 371.1539. Melting point 175–177 C.
A4 N2-Isopropyl-N4-(2-(triuoromethyl)benzyl)quinazoline-2,4-
General method for the synthesis of compounds A, B, C, D
General synthetic procedures I. One equivalent of diamine. Yellow solid; yield, 26%; Rf ¼ 0.29 (9 : 1 dichloro-
quinazoline-2,4-dione and three equivalents of phosphorus methane to methanol); 1H NMR (400 MHz, DMSO-d6) d 10.01 (s,
pentachloride were mixed in 10 equivalents of phosphorus 1H), 8.35 (s, 1H), 7.76 (d, J ¼ 7.8 Hz, 1H), 7.74 (d, J ¼ 10.0 Hz,
oxychloride and the mixture reuxed for 6 hours under 1H), 7.61 (t, J ¼ 7.5 Hz, 1H), 7.50 (d, J ¼ 8.1 Hz, 1H), 7.47 (d, J ¼
a nitrogen atmosphere. Aer that the mixture was cooled down 7.6 Hz, 1H), 7.33 (s, 1H), 4.95 (d, J ¼ 4.4 Hz, 2H), 4.01–3.83 (m,
to room temperature and added with ice in the amount of 10–15 1H), 1.02 (dd, J ¼ 22.3, 15.7 Hz, 6H). 13C NMR (100 MHz, DMSO-
times the reaction volume. The solution was extracted with ethyl d6) d 163.46, 162.50, 160.05, 150.15, 138.99, 136.68, 136.03,
acetate three times and the combined organic phase was dried 132.66, 131.96, 127.73, 127.34, 126.61, 126.15, 125.85, 125.80,
over anhydrous magnesium sulfate. The organic phase was 123.17, 42.54, 41.04, 21.93. HRMS: m/z calcd for C19H20F3N4 [M
+
ꢁ
ltered and concentrated to give crude product under reduced + H] 361.1635; found 361.1639. Melting point 231–233 C.
pressure. The crude product was puried by ash chromatog-
A5 N2-Isopropyl-N4-(4-(triuoromethyl)benzyl)quinazoline-2,4-
raphy with petroleum ether and ethyl acetate.
diamine. Yellow solid; yield, 19%; Rf ¼ 0.47 (9 : 1 dichloro-
General synthesis procedure II. Benzylamine (1.2 equivalent) methane to methanol); 1H NMR (400 MHz, CDCl3) d 7.65 (d, J ¼
and triethylamine (2 equivalent) were mixed with 1 equivalent 8.0 Hz, 1H), 7.52 (t, J ¼ 5.6 Hz, 2H), 7.48 (dd, J ¼ 6.9, 1.2 Hz, 1H),
of 2,4-dichloroquinazoline in tetrahydrofuran and stirred 7.42 (d, J ¼ 4.5 Hz, 2H), 7.41 (s, 1H), 7.07–7.00 (m, 1H), 6.62 (s,
overnight at room temperature, aer that it was concentrated 1H), 4.82 (d, J ¼ 5.0 Hz, 3H), 4.17 (m, 1H), 1.14 (d, J ¼ 6.5 Hz,
under reduced pressure. The remaining residue was puried by 6H). 13C NMR (100 MHz, CDCl3) d 160.21, 158.84, 151.97,
ash chromatography with petroleum ether and ethyl acetate.
143.19, 133.03, 129.75, 127.92, 125.65, 125.29, 122.92, 121.17,
General synthesis procedure III. Isopropylamine (4 equiva- 121.01, 110.72, 44.59, 42.88, 23.26. HRMS: m/z calcd for
lent) and 4-benzylamino-substituted 2-chloroquinazoline (1
52232 | RSC Adv., 2017, 7, 52227–52237
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C19H20F3N4 [M + H]+ 361.1635; found 361.1639. Melting point dichloromethane to methanol); 1H NMR (400 MHz, CDCl3)
ꢁ
69–71 C.
A6
d 8.29 (s, 1H), 7.45 (t, J ¼ 7.7 Hz, 1H), 7.35 (d, J ¼ 8.6 Hz, 2H),
N4-(2-Fluorobenzyl)-N2-isopropylquinazoline-2,4-diamine. 7.27 (d, J ¼ 10.2 Hz, 1H), 7.13 (t, J ¼ 7.6 Hz, 1H), 6.77 (d, J ¼
Yellow oil; yield, 89%; Rf ¼ 0.32 (9 : 1 dichloromethane to 8.4 Hz, 2H), 4.78 (d, J ¼ 4.2 Hz, 2H), 4.24 (m, 1H), 3.73 (s, 3H),
methanol); 1H NMR (400 MHz, CDCl3) d 8.04 (d, J ¼ 5.6 Hz, 1H), 1.24 (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) d 160.18,
7.51 (t, J ¼ 7.7 Hz, 1H), 7.36 (t, J ¼ 7.1 Hz, 2H), 7.19 (d, J ¼ 7.8 Hz, 160.11, 159.21, 134.63, 129.80, 129.56, 129.51, 123.97, 114.26,
1H), 7.17 (dd, J ¼ 4.7, 2.8 Hz, 1H), 7.05–7.00 (m, 1H), 6.98 (d, J ¼ 114.21, 114.08, 110.01, 55.41, 44.98, 43.81, 22.72. HRMS: m/z
10.0 Hz, 1H), 4.86 (d, J ¼ 3.5 Hz, 2H), 4.14 (ddd, J ¼ 18.0, 12.2, calcd for C19H23N4O [M + H]+ 323.1866; found 323.1869. Melting
5.3 Hz, 3H), 1.15 (d, J ¼ 6.7 Hz, 6H). 13C NMR (100 MHz, CDCl3) point 208–210 ꢁC.
d 163.65, 162.04, 160.65, 160.43, 159.59, 152.91, 134.92, 129.74,
A12
N4-(3,4-Dimethoxybenzyl)-N2-isopropylquinazoline-2,4-
129.39, 124.56, 124.32, 124.11, 115.55, 109.62, 43.83, 40.46, diamine. Yellow solid; yield, 15%; Rf ¼ 0.39 (9 : 1 dichloro-
22.46. HRMS: m/z calcd for C18H20N4 [M + H]+ 311.1667; found methane to methanol); 1H NMR (400 MHz, CDCl3) d 8.24 (s, 1H),
311.1669.
A7
7.44 (t, J ¼ 7.7 Hz, 1H), 7.27 (d, J ¼ 10.7 Hz, 1H), 7.11 (t, J ¼
N4-(4-Fluorobenzyl)-N2-isopropylquinazoline-2,4-diamine. 7.6 Hz, 1H), 7.04 (d, J ¼ 1.3 Hz, 1H), 6.95 (dd, J ¼ 8.2, 1.4 Hz,
Yellow solid; yield, 83%; Rf ¼ 0.29 (9 : 1 dichloromethane to 1H), 6.74 (d, J ¼ 8.2 Hz, 1H), 4.77 (s, 2H), 4.25 (m, 1H), 3.81 (s,
methanol); H NMR (400 MHz, CDCl3) d 9.71 (s, NH), 8.49 (s, 3H), 3.80 (s, 3H), 1.24 (d, J ¼ 6.4 Hz, 6H). 13C NMR (100 MHz,
1
NH), 7.47 (t, J ¼ 7.7 Hz, 1H), 7.37 (dd, J ¼ 8.3, 5.5 Hz, 2H), 7.25 CDCl3) d 160.19, 154.16, 149.26, 148.68, 148.65, 134.36, 130.48,
(d, J ¼ 5.7 Hz, 1H), 7.14 (t, J ¼ 7.6 Hz, 1H), 6.87 (t, J ¼ 8.6 Hz, 130.02, 123.87, 123.56, 120.57, 111.85, 111.31, 110.13, 56.08,
2H), 4.80 (s, 2H), 4.17 (m, 1H), 1.18 (d, J ¼ 6.5 Hz, 5H). 13C NMR 56.05, 45.27, 43.66, 22.75. HRMS: m/z calcd for C20H25N4O2 [M +
+
ꢁ
(100 MHz, CDCl3) d 163.51, 161.07, 160.22, 134.50, 133.60, H] 353.1972; found 353.1976. Melting point 192–194 C.
129.77, 129.69, 123.62, 115.66, 115.45, 109.87, 63.88, 44.75,
A13
N2-Isopropyl-N4-((6-(triuoromethyl)pyridin-3-yl)methyl)
43.69, 22.72. HRMS: m/z calcd for C18H20N4 [M + H]+ 311.1667; quinazoline-2,4-diamine. Yellow solid; yield, 23%; Rf ¼ 0.39 (9 : 1
found 311.1669. Melting point 240–243 ꢁC.
dichloromethane to methanol); 1H NMR (400 MHz, CD3OD)
A8 N4-(2-Chlorobenzyl)-N2-isopropylquinazoline-2,4-diamine. d 8.78 (s, 1H), 8.08 (d, J ¼ 6.3 Hz, 1H), 8.07 (s, 1H), 7.79 (d, J ¼
Yellow solid; yield, 39%; Rf ¼ 0.31 (9 : 1 dichloromethane to 8.1 Hz, 1H), 7.70 (t, J ¼ 7.7 Hz, 1H), 7.43 (d, J ¼ 8.1 Hz, 1H), 7.33
methanol); 1H NMR (400 MHz, CDCl3) d 8.19–8.13 (m, 1H), 7.58 (t, J ¼ 7.7 Hz, 1H), 4.95 (s, 2H), 4.12 (dq, J ¼ 12.5, 6.3 Hz, 1H),
(td, J ¼ 7.2, 3.2 Hz, 1H), 7.42 (d, J ¼ 7.0 Hz, 1H), 7.37–7.27 (m, 1.17 (d, J ¼ 6.4 Hz, 6H). 13C NMR (101 MHz, CD3OD) d 162.08,
3H), 7.21–7.15 (m, 2H), 4.90 (d, J ¼ 2.3 Hz, 2H), 4.13 (m, 1H), 155.98, 150.39, 147.70, 144.62, 139.76, 138.25, 135.77, 124.75,
1.14 (d, J ¼ 6.5 Hz 6H). 13C NMR (100 MHz, CDCl3) d 160.37, 124.37, 121.72, 121.70, 120.28, 111.44, 44.59, 43.34, 22.71.
152.21, 138.98, 135.00, 134.60, 133.02, 129.48, 128.69, 128.56, HRMS: m/z calcd for C19H20F3N4 [M + H]+ 362.1587; found
126.88, 124.39, 123.67, 116.89, 109.49, 43.79, 43.03, 21.97. 362.1590. Melting point 202–205 ꢁC.
HRMS: m/z calcd for C18H20ClN4 [M + H]+ 327.1371; found
327.1374. Melting point 209–212 ꢁC.
B1 N4-(4-Cyanobenzyl)-N2-isopropylthieno[3,2-d]pyrimidine-2,4-
diamine. Yellow oil; yield, 61%; Rf ¼ 0.31 (9 : 1 dichloromethane
A9 N4-(4-Chlorobenzyl)-N2-isopropylquinazoline-2,4-diamine. to methanol); H NMR (400 MHz, CDCl3) d 7.63–7.58 (m, 2H),
Yellow oil; yield, 94%; Rf ¼ 0.26 (9 : 1 dichloromethane to 7.56 (d, J ¼ 5.3 Hz, 1H), 7.46 (d, J ¼ 8.4 Hz, 2H), 7.11 (d, J ¼
methanol); 1H NMR (400 MHz, CDCl3) d 8.09 (d, J ¼ 7.7 Hz, 1H), 5.3 Hz, 1H), 5.43 (s, NH), 4.96 (s, NH), 4.84 (d, J ¼ 5.8 Hz, 2H),
7.49 (t, J ¼ 7.7 Hz, 1H), 7.35 (d, J ¼ 8.3 Hz, 1H), 7.29 (d, J ¼ 4.07 (m, 1H), 1.16 (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3)
8.4 Hz, 2H), 7.22 (d, J ¼ 8.4 Hz, 2H), 7.14 (t, J ¼ 7.5 Hz, 1H), 6.21– d 161.09, 160.16, 157.48, 144.78, 132.52, 131.18, 128.13, 123.67,
6.09 (m, 2H), 4.76 (s, 2H), 4.14 (dt, J ¼ 11.4, 5.0 Hz, 1H), 1.19 (d, J 118.88, 111.28, 106.04, 44.43, 43.23, 23.13. HRMS: m/z calcd for
1
¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) d 172.91, 160.45,
C
17H17N5S [M + H]+ 324.1277; found 324.1281.
B2
N2-Isopropyl-N4-(2-(methylsulfonyl)benzyl)thieno[3,2-d]
152.77, 139.19, 136.22, 134.97, 133.35, 129.25, 128.74, 124.31,
116.95, 109.75, 44.85, 43.89, 22.51. HRMS: m/z calcd for pyrimidine-2,4-diamine. Yellow oil; yield, 38%; Rf ¼ 0.53 (9 : 1
C
18H20ClN4 [M + H]+ 327.1371; found 327.1373.
A10
dichloromethane to methanol); 1H NMR (400 MHz, CDCl3)
N2-Isopropyl-N4-(2-methoxybenzyl)quinazoline-2,4- d 8.03–7.99 (m, 1H), 7.72 (d, J ¼ 7.5 Hz, 1H), 7.56 (t, J ¼ 7.4 Hz,
diamine. White solid; yield, 70%; Rf ¼ 0.31 (9 : 1 dichloro- 1H), 7.50 (d, J ¼ 5.3 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz, 1H), 7.04 (d, J ¼
methane to methanol); 1H NMR (400 MHz, CDCl3) d 7.87 (d, J ¼ 5.3 Hz, 1H), 5.88 (s, 1H), 5.10 (d, J ¼ 6.3 Hz, 2H), 4.70 (d, J ¼
7.4 Hz, 1H), 7.51 (t, J ¼ 7.7 Hz, 1H), 7.38 (d, J ¼ 8.2 Hz, 1H), 7.28 8.0 Hz, 1H), 4.17 (dq, J ¼ 13.0, 6.5 Hz, 1H), 3.14 (s, 3H), 1.20 (d, J
(d, J ¼ 7.2 Hz 1H), 7.23 (d, J ¼ 7.8 Hz, 1H), 7.18 (t, J ¼ 7.6 Hz, 1H), ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) d 161.70, 160.53,
6.87 (d, J ¼ 6.6 Hz, 1H), 6.85 (d, J ¼ 6.6 Hz, 1H), 4.84 (d, J ¼ 157.18, 138.64, 138.40, 134.08, 131.84, 130.89, 129.79, 128.40,
5.0 Hz, 2H), 4.23 (m, 6.3 Hz, 1H), 3.88 (s, 3H), 1.25 (d, J ¼ 6.5 Hz, 123.72, 106.24, 45.10, 42.99, 42.05, 23.21. HRMS: m/z calcd for
6H). 13C NMR (100 MHz, CDCl3) d 160.09, 157.57, 134.60,
129.89, 129.42, 129.18, 125.18, 123.71, 122.98, 120.91, 120.76,
C
17H20N2NaO2S2 [M + Na]+ 399.0920; found 399.0925.
B3
N2-Isopropyl-N4-(4-(methylsulfonyl)benzyl)thieno[3,2-d]
110.75, 110.58, 109.71, 55.59, 43.78, 41.41, 22.62. HRMS: m/z pyrimidine-2,4-diamine. Yellow oil; yield, 31%; Rf ¼ 0.56 (9 : 1
calcd for C19H23N4O [M + H]+ 323.1866; found 323.1870. Melting dichloromethane to methanol); 1H NMR (400 MHz, CDCl3)
point 138–140 ꢁC.
A11
diamine. Yellow solid; yield, 73%; Rf
d 7.80 (d, J ¼ 8.3 Hz, 2H), 7.52 (d, J ¼ 5.3 Hz, 1H), 7.49 (d, J ¼
N2-Isopropyl-N4-(4-methoxybenzyl)quinazoline-2,4- 8.2 Hz, 2H), 7.07 (d, J ¼ 5.3 Hz, 1H), 5.82 (s, 1H), 4.82 (d, J ¼
¼
0.31 (9 : 1 5.9 Hz, 2H), 4.75 (d, J ¼ 7.9 Hz, 1H), 4.05 (dq, J ¼ 13.1, 6.5 Hz,
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1H), 3.00 (s, 3H), 1.12 (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, 5.3 Hz, 1H), 7.32–7.24 (m, 4H), 7.09 (d, J ¼ 5.3 Hz, 1H), 5.65 (b,
CDCl3) d 161.70, 160.46, 157.44, 146.04, 139.18, 130.98, 128.27, 1H), 5.32 (b, 1H), 4.74 (d, J ¼ 5.6 Hz, 2H), 4.14 (dq, J ¼ 13.1,
127.59, 123.78, 106.06, 44.61, 44.08, 43.05, 23.12. HRMS: m/z 6.5 Hz, 1H), 1.20 (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3)
calcd for C17H20N2NaO2S2 [M + Na]+ 399.0920; found 399.0924. d 161.77, 160.61, 157.46, 137.62, 133.07, 130.67, 129.07, 128.72,
B4
N2-Isopropyl-N4-(2-(triuoromethyl)benzyl)thieno[3,2-d] 123.86, 106.00, 44.02, 43.03, 23.18. HRMS: m/z calcd for
pyrimidine-2,4-diamine. Yellow solid; yield, 39%; Rf ¼ 0.33 (9 : 1
C
16H18ClN4S [M + H]+ 333.0935; found 333.0938.
dichloromethane to methanol); 1H NMR (400 MHz, CD3OD)
B10 N2-Isopropyl-N4-(2-methoxybenzyl)thieno[3,2-d]pyrimidine-
d 7.77 (d, J ¼ 5.4 Hz, 1H), 7.68 (d, J ¼ 7.7 Hz, 1H), 7.52 (d, J ¼ 2,4-diamine. Yellow solid; yield, 37%; Rf ¼ 0.31 (9 : 1 dichloro-
4.3 Hz, 2H), 7.38 (dt, J ¼ 8.1, 4.1 Hz, 1H), 7.03 (d, J ¼ 5.4 Hz, 1H), methane to methanol); 1H NMR (400 MHz, CDCl3) d 7.47 (d, J ¼
4.95 (s, 2H), 4.00–3.89 (m, 1H), 1.04 (d, J ¼ 6.5 Hz, 6H). 13C NMR 5.3 Hz, 1H), 7.34 (dd, J ¼ 7.4, 1.3 Hz, 1H), 7.28–7.22 (m, 1H), 7.07
(100 MHz, CD3OD) d 161.85, 161.69, 159.04, 139.67, 133.28, (d, J ¼ 5.3 Hz, 1H), 6.91 (d, J ¼ 7.4 Hz, 1H), 6.88 (d, J ¼ 7.9 Hz,
133.27, 132.88, 128.90, 127.95, 126.81, 123.49, 107.34, 43.87, 1H), 5.42 (s, 1H), 4.82 (d, J ¼ 7.8 Hz, 1H), 4.78 (d, J ¼ 5.8 Hz, 2H),
41.71, 23.02. HRMS: m/z calcd for C17H18F3N4S [M + H]+ 4.22 (qd, J ¼ 13.0, 6.5 Hz, 1H), 3.86 (s, 3H), 1.23 (d, J ¼ 6.5 Hz,
6H). 13C NMR (100 MHz, CDCl3) d 161.31, 160.61, 157.71,
ꢁ
367.1199; found 367.1203. Melting point 135–137 C.
B5
N2-Isopropyl-N4-(4-(triuoromethyl)benzyl)thieno[3,2-d] 157.67, 130.25, 129.75, 128.80, 126.80, 123.81, 120.64, 110.49,
pyrimidine-2,4-diamine. Brown solid; yield, 47%; Rf ¼ 0.31 (9 : 1 106.25, 55.41, 43.02, 40.55, 23.23. HRMS: m/z calcd for
dichloromethane to methanol); 1H NMR (400 MHz, CDCl3)
C
17H21N4OS [M + H]+ 329.1431; found 329.1434. Melting point
ꢁ
d 7.53 (d, J ¼ 8.2 Hz, 2H), 7.50 (d, J ¼ 5.3 Hz, 1H), 7.43 (d, J ¼ 60–62 C.
8.1 Hz, 2H), 7.07 (d, J ¼ 5.3 Hz, 1H), 5.97 (s, 1H), 5.14 (s, 1H),
B11 N2-Isopropyl-N4-(4-methoxybenzyl)thieno[3,2-d]pyrimidine-
4.81 (d, J ¼ 5.6 Hz, 2H), 4.09 (dq, J ¼ 13.2, 6.5 Hz, 1H), 1.15 (d, J 2,4-diamine. Yellow oil; yield, 51%; Rf ¼ 0.22 (9 : 1 dichloro-
¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) d 161.49, 160.45, methane to methanol); 1H NMR (400 MHz, CDCl3) d 7.49 (d, J ¼
157.54, 143.25, 130.91, 129.96, 127.89, 125.69, 123.81, 122.91, 5.3 Hz, 1H), 7.29 (d, J ¼ 8.5 Hz, 2H), 7.09 (d, J ¼ 5.3 Hz, 1H), 6.86
105.96, 44.39, 43.17, 23.16. HRMS: m/z calcd for C17H18F3N4NaS (d, J ¼ 8.6 Hz, 2H), 5.17 (s, 1H), 4.77 (d, J ¼ 7.8 Hz, 1H), 4.70 (d, J
+
ꢁ
[M + Na] 389.1018; found 389.1016. Melting point 62–64 C.
B6
¼ 5.5 Hz, 2H), 4.20 (m, 1H), 3.79 (s, 3H), 1.22 (d, J ¼ 6.5 Hz, 6H).
N4-(2-Fluorobenzyl)-N2-isopropylthieno[3,2-d]pyrimidine- 13C NMR (100 MHz, CDCl3) d 160.71, 159.18, 157.54, 131.00,
2,4-diamine. Yellow oil; yield, 49%; Rf ¼ 0.51 (9 : 1 dichloro- 130.45, 129.29, 123.97, 114.20, 106.10, 55.41, 44.42, 43.08,
methane to methanol); 1H NMR (400 MHz, CDCl3) d 7.50 (d, J ¼ 23.27. HRMS: m/z calcd for C17H21N4OS [M + H]+ 329.1431;
5.3 Hz, 1H), 7.38 (t, J ¼ 7.6 Hz, 1H), 7.23 (m, 1H), 7.10–7.01 (m, found 329.1432.
3H), 5.54 (s, NH), 5.16 (s, NH), 4.83 (d, J ¼ 5.3 Hz, 2H), 4.16 (td, J
B12 N4-(3,4-Dimethoxybenzyl)-N2-isopropylthieno[3,2-d]pyrimi-
¼ 13.0, 6.5 Hz, 1H), 1.20 (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, dine-2,4-diamine. Yellow solid; yield, 51%; Rf ¼ 0.24 (9 : 1
CDCl3) d 162.35, 159.96, 157.55, 130.98, 130.01, 129.21, 125.82, dichloromethane to methanol); 1H NMR (400 MHz, CDCl3)
124.28, 123.25, 115.56, 115.35, 106.21, 43.19, 38.67, 23.14. d 7.52 (d, J ¼ 5.3 Hz, 1H), 7.10 (d, J ¼ 5.3 Hz, 1H), 6.92 (m, 2H),
HRMS: m/z calcd for C16H18FN4S [M + H]+ 317.1231; found 6.86–6.81 (m, 1H), 5.05 (s, 1H), 4.83 (s, 1H), 4.71 (d, J ¼ 5.4 Hz,
317.1234.
B7
2H), 4.21 (td, J ¼ 13.1, 6.5 Hz, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 1.24
N4-(4-Fluorobenzyl)-N2-isopropylthieno[3,2-d]pyrimidine- (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) d 161.33, 160.52,
2,4-diamine. Yellow oil; yield, 58%; Rf ¼ 0.56 (9 : 1 dichloro- 157.58, 149.40, 148.77, 131.41, 130.66, 123.88, 120.37, 111.54,
methane to methanol); 1H NMR (400 MHz, CDCl3) d 7.48 (dd, J 111.52, 106.15, 56.14, 56.07, 44.95, 43.18, 23.28. HRMS: m/z
¼ 5.3, 0.6 Hz, 1H), 7.29 (dd, J ¼ 8.2, 5.5 Hz, 2H), 7.04 (d, J ¼ calcd for C18H23N4O2S [M + H]+ 359.1536; found 359.1542.
ꢁ
5.3 Hz, 1H), 6.96 (t, J ¼ 8.3 Hz, 2H), 5.94 (s, 1H), 5.06 (s, 1H), 4.71 Melting point 134–136 C.
(d, J ¼ 4.6 Hz, 2H), 4.13 (td, J ¼ 13.4, 6.5 Hz, 1H), 1.17 (d, J ¼
B13
N2-Isopropyl-N4-((6-(triuoromethyl)pyridin-3-yl)methyl)
6.4 Hz, 6H). 13C NMR (100 MHz, CDCl3) d 163.24, 160.80, 159.34, thieno[3,2-d]pyrimidine-2,4-diamine. Yellow oil; yield, 46%; Rf ¼
1
157.42, 134.63, 131.31, 129.40, 129.32, 122.55, 115.30, 106.33, 0.29 (9 : 1 dichloromethane to methanol); H NMR (400 MHz,
43.95, 43.12, 22.97. HRMS: m/z calcd for C16H18FN4S [M + H]+ CDCl3) d 8.71 (s, 1H), 7.85 (d, J ¼ 7.1 Hz, 1H), 7.60 (d, J ¼ 8.1 Hz,
317.1231; found 317.1234.
1H), 7.53 (d, J ¼ 5.3 Hz, 1H), 7.08 (d, J ¼ 5.3 Hz, 1H), 5.69 (s, 1H),
B8
N4-(2-Chlorobenzyl)-N2-isopropylthieno[3,2-d]pyrimidine- 4.84 (d, J ¼ 5.7 Hz, 2H), 4.77 (d, J ¼ 7.6 Hz, 1H), 4.05 (m, 1H),
2,4-diamine. Yellow oil; yield, 28%; Rf ¼ 0.33 (9 : 1 dichloro- 1.14 (d, J ¼ 6.5 Hz, 6H). 13C NMR (101 MHz, CDCl3) d 161.85,
methane to methanol); 1H NMR (400 MHz, CDCl3) d 7.48 (d, J ¼ 160.44, 157.36, 149.41, 147.19, 138.42, 136.49, 131.14, 123.86,
5.3 Hz, 1H), 7.39 (dd, J ¼ 5.0, 4.2 Hz, 1H), 7.34 (dd, J ¼ 5.8, 123.03, 120.39, 106.02, 43.16, 41.96, 23.13. HRMS: m/z calcd for
3.5 Hz, 1H), 7.19–7.17 (m, 1H), 7.16 (d, J ¼ 5.1 Hz, 1H), 7.06 (d, J
¼ 5.3 Hz, 1H), 4.84 (d, J ¼ 5.9 Hz, 2H), 4.13 (dq, J ¼ 19.5, 6.5 Hz,
C
16H16F3N5NaS [M + Na]+ 390.0971; found 390.0973.
C1
N4-(4-Cyanobenzyl)-N2-isopropylpyrido[3,2-d]pyrimidine-
1H), 1.16 (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) 2,4-diamine. Yellow oil; yield, 62%; Rf ¼ 0.56 (9 : 1 dichloro-
d 160.61, 160.09, 157.43, 136.19, 133.37, 130.89, 129.56, 129.45, methane to methanol); 1H NMR (400 MHz, CDCl3) d 8.28 (d, J ¼
128.60, 126.89, 123.30, 106.14, 43.05, 42.46, 23.06. HRMS: m/z 3.4 Hz, 1H), 7.68 (d, J ¼ 7.5 Hz, 1H), 7.57 (d, J ¼ 7.9 Hz, 2H), 7.43
calcd for C16H18ClN4S [M + H]+ 333.0935; found 333.0937.
(d, J ¼ 7.0 Hz, 2H), 7.40–7.30 (m, 1H), 5.19 (s, 1H), 4.80 (d, J ¼
B9
N4-(4-Chlorobenzyl)-N2-isopropylthieno[3,2-d]pyrimidine- 5.9 Hz, 2H), 4.17 (m, 1H), 1.18 (d, J ¼ 5.8 Hz, 6H). 13C NMR (100
2,4-diamine. Yellow oil; yield, 50%; Rf ¼ 0.38 (9 : 1 dichloro- MHz, CDCl3) d 159.91, 158.77, 146.89, 144.35, 142.89, 132.24,
methane to methanol); 1H NMR (400 MHz, CDCl3) d 7.52 (d, J ¼ 128.75, 127.98, 127.71, 118.75, 110.90, 72.69, 43.85, 42.83,
52234 | RSC Adv., 2017, 7, 52227–52237
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23.01. HRMS: m/z calcd for C18H19N6 [M + H]+ 319.1666; found dichloromethane to methanol); 1H NMR (400 MHz, CD3OD)
319.1669.
C2
d 8.64–8.60 (m, 1H), 8.34 (d, J ¼ 7.5 Hz, 1H), 8.05 (d, J ¼ 7.8 Hz,
N2-Isopropyl-N4-(2-(methylsulfonyl)benzyl)pyrido[3,2-d] 1H), 7.63 (d, J ¼ 5.5 Hz, 2H), 7.51 (dd, J ¼ 10.9, 5.5 Hz, 1H), 7.10
pyrimidine-2,4-diamine. White solid; yield, 71%; Rf ¼ 0.49 (9 : 1 (dd, J ¼ 7.9, 4.6 Hz, 1H), 5.22 (s, 2H), 4.18 (s, 1H), 3.31 (s, 3H),
dichloromethane to methanol); 1H NMR (400 MHz, CDCl3) 1.12 (s, 6H). 13C NMR (101 MHz, CD3OD) d 162.64, 155.76,
d 8.30 (d, J ¼ 4.2 Hz, 1H), 8.05 (d, J ¼ 7.9 Hz, 1H), 7.75 (s, NH), 151.44, 139.95, 139.49, 135.12, 133.48, 130.85, 130.57, 130.28,
7.72 (d, J ¼ 7.7 Hz, 1H), 7.65 (d, J ¼ 8.4 Hz, 1H), 7.59 (t, J ¼ 130.08, 128.96, 117.56, 44.66, 30.82, 30.31, 22.99. HRMS: m/z
7.5 Hz, 1H), 7.48 (t, J ¼ 7.6 Hz, 1H), 7.41 (dd, J ¼ 8.5, 4.2 Hz, 1H), calcd for C18H21NaN5O2S [M + Na]+ 394.1308; found 394.1313.
5.17 (d, J ¼ 6.5 Hz, 2H), 4.91 (s, NH), 4.28–4.18 (m, 1H), 3.17 (s,
D3
N2-Isopropyl-N4-(4-(methylsulfonyl)benzyl)pyrido[2,3-d]
3H), 1.24 (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) pyrimidine-2,4-diamine. Yellow solid; yield, 20%; Rf ¼ 0.36 (9 : 1
d 160.03, 158.90, 146.86, 143.35, 138.75, 138.43, 134.14, 132.35, dichloromethane to methanol); 1H NMR (400 MHz, CD3OD)
131.63, 130.08, 128.57, 127.86, 72.93, 45.27, 43.08, 42.06, 23.32. d 8.56 (dd, J ¼ 4.6, 1.6 Hz, 1H), 8.30 (d, J ¼ 7.7 Hz, 1H), 7.84 (d, J
HRMS: m/z calcd for C18H22N5O2S [M + H]+ 372.1489; found ¼ 8.3 Hz, 2H), 7.57 (d, J ¼ 8.3 Hz, 2H), 7.02 (dd, J ¼ 8.0, 4.6 Hz,
372.1495. Melting point 161–162 ꢁC.
C3
1H), 4.82 (s, 2H), 3.30–3.27 (m, 1H), 3.04 (s, 3H), 1.14 (d, J ¼
N2-Isopropyl-N4-(4-(methylsulfonyl)benzyl)pyrido[3,2-d] 34.1 Hz, 6H). 13C NMR (101 MHz, CD3OD) d 162.53, 162.33,
pyrimidine-2,4-diamine. Yellow oil; yield, 62%; Rf ¼ 0.50 (9 : 1 161.90, 155.62, 147.41, 140.48, 133.56, 129.21, 128.51, 117.47,
dichloromethane to methanol); 1H NMR (400 MHz, CDCl3) 107.15, 45.16, 44.44, 43.64, 23.07. HRMS: m/z calcd for C18H21-
d 8.32–8.28 (m, 1H), 7.90 (d, J ¼ 8.3 Hz, 2H), 7.70 (d, J ¼ 8.4 Hz, NaN5O2S [M + Na]+ 394.1308; found 394.1311. Melting point
1H), 7.58 (d, J ¼ 8.1 Hz, 2H), 7.44 (dd, J ¼ 8.5, 4.2 Hz, 1H), 4.87 162–164 ꢁC.
(d, J ¼ 6.2 Hz, 2H), 4.18 (dt, J ¼ 13.4, 6.7 Hz, 1H), 3.03 (s, 3H),
D5
N2-Isopropyl-N4-(4-(triuoromethyl)benzyl)pyrido[2,3-d]
1.21 (d, J ¼ 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) d 160.00, pyrimidine-2,4-diamine. Yellow solid; yield, 74%; Rf ¼ 0.47 (9 : 1
1
158.51, 145.20, 143.20, 139.61, 132.14, 128.36, 127.92, 127.78, dichloromethane to methanol); H NMR (400 MHz, DMSO-d6)
127.71, 72.80, 44.57, 43.91, 43.02, 23.06. HRMS: m/z calcd for d 8.72–8.44 (m, 1H), 8.44 (d, J ¼ 42.8 Hz, 1H), 7.62 (d, J ¼ 8.1 Hz,
C
18H22N5O2S [M + H]+ 372.1489; found 372.1491.
C5
2H), 7.52 (d, J ¼ 7.9 Hz, 2H), 7.05 (d, J ¼ 35.0 Hz, 1H), 4.74 (d, J ¼
N2-Isopropyl-N4-(4-(triuoromethyl)benzyl)pyrido[3,2-d] 4.3 Hz, 2H), 4.14–3.88 (m, 1H), 1.01 (dd, J ¼ 23.4, 16.7 Hz, 6H).
pyrimidine-2,4-diamine. Yellow solid; yield, 91%; Rf ¼ 0.55 (9 : 1 13C NMR (101 MHz, DMSO-d6) d 166.89, 160.53, 144.22, 143.15,
dichloromethane to methanol); 1H NMR (400 MHz, CDCl3) 131.44, 127.87, 127.60, 127.29, 125.65, 125.10, 122.95, 64.96,
d 8.31 (d, J ¼ 4.2 Hz, 1H), 7.72 (d, J ¼ 8.5 Hz, 1H), 7.60 (d, J ¼ 62.77, 22.37. HRMS: m/z calcd for C18H18F3N5Na [M + Na]+
ꢁ
8.1 Hz, 2H), 7.50 (d, J ¼ 8.0 Hz, 2H), 7.45 (dd, J ¼ 8.5, 4.2 Hz, 384.1407; found 384.1410. Melting point 107–110 C.
1H), 4.85 (d, J ¼ 6.1 Hz, 2H), 4.21 (dq, J ¼ 13.4, 6.7 Hz, 1H), 1.23
D13 N2-Isopropyl-N4-((6-(triuoromethyl)pyridin-3-yl)methyl)
(d, J ¼ 6.5 Hz, 6H). 13C NMR (101 MHz, CDCl3) d 160.00, 158.03, pyrido[2,3-d]pyrimidine-2,4-diamine. Yellow solid; yield, 7%; Rf ¼
1
143.44, 142.43, 130.35, 128.06, 127.98, 127.88, 125.57, 122.78, 0.26 (9 : 1 dichloromethane to methanol); H NMR (400 MHz,
72.81, 44.08, 43.19, 22.99. HRMS: m/z calcd for C18H19F3N5 [M + CD3OD) d 8.76 (s, 1H), 8.64 (s, 1H), 8.39 (s, 1H), 8.06 (d, J ¼
+
ꢁ
H] 362.1587; found 362.1595. Melting point 92–94 C.
8.0 Hz, 1H), 7.78 (d, J ¼ 8.1 Hz, 1H), 7.17 (s, 1H), 4.90 (d, J ¼
C13
N2-Isopropyl-N4-((6-(triuoromethyl)pyridin-3-yl)methyl) 2.4 Hz, 2H), 4.18 (d, J ¼ 8.2 Hz, 1H), 1.15 (s, 6H). 13C NMR (101
pyrido[3,2-d]pyrimidine-2,4-diamine. Yellow solid; yield, 45%; Rf MHz, CD3OD) d 162.36, 155.77, 150.41, 147.57, 140.24, 138.24,
¼ 0.54 (9 : 1 dichloromethane to methanol); 1H NMR (400 MHz, 133.92, 130.83, 127.14, 124.43, 121.67, 121.64, 119.00, 107.13,
CDCl3) d 8.77 (s, 1H), 8.28 (dd, J ¼ 4.2, 1.1 Hz, 1H), 7.88 (d, J ¼ 64.33, 43.99, 43.20, 22.95. HRMS: m/z calcd for C17H17F3N6Na
7.9 Hz, 1H), 7.68 (d, J ¼ 8.4 Hz, 1H), 7.63 (d, J ¼ 8.1 Hz, 1H), 7.43 [M + Na]+ 385.1359; found 385.1366. Melting point 108–110 ꢁC.
(dd, J ¼ 8.5, 4.2 Hz, 1H), 4.94 (s, 1H), 4.85 (d, J ¼ 6.2 Hz, 2H),
4.17 (d, J ¼ 6.3 Hz, 1H), 1.20 (d, J ¼ 6.4 Hz, 6H). 13C NMR (101
Antibacterial activities assay
MHz, CDCl3) d 160.09, 158.83, 149.61, 147.54, 147.20, 147.10,
143.21, 137.97, 136.52, 132.68, 127.99, 123.05, 120.47, 43.08,
Bacterial strains and growth conditions. E. coli CMCC 44102,
41.71, 23.19. HRMS: m/z calcd for C17H18F3N6 [M + H]+ S. aureus ATCC 6538, S. epidermidis ATCC 12228, S. typhimurium
ꢁ
363.1540; found 363.1546. Melting point 111–113 C.
CMCC 50115 and MRSA ATCC 43300 used in this study were
D1
N4-(4-Cyanobenzyl)-N2-isopropylpyrido[2,3-d]pyrimidine- acquired from Guangdong Microbiology Culture Center. E. coli,
2,4-diamine. Yellow oil; yield, 24%; Rf ¼ 0.32 (9 : 1 dichloro- S. aureus, S. epidermidis and MRSA were grown in Mueller–
methane to methanol); 1H NMR (400 MHz, CD3OD) d 8.53 (dd, J Hinton Broth (MHB) and S. typhimurium was grown in tryptic
¼ 4.5, 1.6 Hz, 1H), 8.27 (d, J ¼ 7.7 Hz, 1H), 7.59 (d, J ¼ 8.1 Hz, soy broth (TSB) at 37 ꢁC, and agar was added to a nal
2H), 7.47 (d, J ¼ 8.2 Hz, 2H), 7.00 (dd, J ¼ 8.0, 4.6 Hz, 1H), 4.77 concentration of 1.5% (w/v) for growth plates.25
(s, 2H), 4.11 (s, 1H), 3.28–3.26 (m, 1H), 1.12 (d, J ¼ 23.6 Hz, 6H).
Kirby–Bauer assay. These assays for antibacterial activity
13C NMR (101 MHz, CD3OD) d 162.49, 162.34, 161.84, 155.60, were performed as reported previously.25,33 Briey, streaked
146.66, 133.54, 133.28, 129.18, 119.76, 117.46, 111.56, 107.13, bacterial strains were put onto agar plates and incubated over-
45.26, 43.64, 23.06. HRMS: m/z calcd for C18H18N6Na [M + Na]+ night. A single colony was picked to inoculate into broth media
341.1485; found 341.1489.
D2
N2-Isopropyl-N4-(2-(methylsulfonyl)benzyl)pyrido[2,3-d] diluted 1 : 1000 into 5 mL of broth media. 400 mL of diluted
pyrimidine-2,4-diamine. Yellow solid; yield, 6%; Rf ¼ 0.40 (9 : 1 culture was added to an agar plate with a diameter of 120 mm
and incubated overnight. The activated broth cultures were
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and was coated uniformly, allowed to dry for 15 min before use. extracted ion chromatograms and the in vitro half-life (t1/2) of
Aer that, nine sterile lter disks with a diameter of 6 mm were parent compound determined by regression analysis of the ln
added and 5 mL of 5 mg mLꢀ1 test compound dissolved in percent parent disappearance vs. time curve. The in vitro
DMSO were dropwise added to each lter disk. Bacterial plates intrinsic clearance (in vitro CLint, in mL minꢀ1 per 106 cells or mg
were incubated overnight. The assays were performed in trip- protein) was determined from the slope value using the
licate and zones of inhibition were measured in millimeters.
following equation: in vitro CLint ¼ kV/N where V ¼ incubation
Microtiter MIC determination assay. Two assays were carried volume; N ¼ number of hepatocytes or protein concentration
out based on previous methods.25,33 The minimum inhibitory per well.
concentrations (MIC) were determined for compounds which
showed antibacterial activity against corresponding strains.
Broth cultures were prepared as above and 1000 fold diluted
Conflicts of interest
cultures were added to a sterile 96-well plate with a dosage of Compounds described in this manuscript are included in a UK
200 mL per well. 5 mL compounds, with two-times gradient patent application led by the Liverpool School of Tropical
dilution, were added to wells and mixed by pipetting, then, the Medicine and the University of Liverpool (application No.
plates were incubated at 37 ꢁC overnight. MICs were determined 1700814.5, led 17 January 2017). The authors declare that they
by visual inspection of minimum concentration of compound have no other competing interests.
inhibiting bacterial growth. Each concentration was performed
parallelly in triplicate to verify the MIC determinations. Pure
DMSO was used as blank control which didn't show inhibition
Acknowledgements
about bacterial growth.
The authors want to thank the DMPK group (led by Peter
Webborn) in AstraZeneca U.K. for providing the in vitro
measurement of DMPK properties, including log D7.4, aqueous
solubility, human plasma protein binding, mouse microsome
DMPK properties assays
The DMPK properties data described above were measured clearance and rat hepatocytes clearance of hit and lead mole-
through a high through-put platform kindly provided by cules. Majority of the synthetic and biological research work was
AstraZeneca U.K. The methods of the ve assays, including carried out in the Liverpool-GDUT Joint Laboratory for Drug
log D7.4, aqueous solubility, plasma protein binding, micro- Discovery located in Guangdong University of Technolgy
some and hepatocyte clearance measurements has been re- (GDUT). Financial support was provided by the Guangzhou
ported previously (Basarab et al., 2014 and Doyle et al., 2016) Municipal Science and technology project for major project of
and are described briey as below34,35
.
industry-university-research cooperation and collaborative
log D7.4 Determination assay. The partition coefficient innovation (project No. 2016201604030025), Liverpool-GDUT
(log D) was measured by shake ask method, using 10 mM Drug Discovery Initiative (project No. CA131122SWGDUT), the
phosphate buffer at pH 7.4 and n-octanol. The samples were Department of Science and Technology of Guangdong province
allowed to reach equilibrium by shaking for 1 hour at 1200 rpm, (project No. 2017A050501034), the Department of Education of
and sample analysis was done by LC/UV, with MS for mass Guangdong Province (project No. 2013JDXM27) and Guangzhou
conrmation.
Science and Technology Plan (project No. 201604030020 and
Aqueous (thermodynamic) solubility assay. It is a shake-ask No. 2017A050501034). Compounds descripted in this manu-
approach that uses compounds dried from 10 mM DMSO script are included in an UK Patent Application No. 1700814.5.
solutions. The dried compounds are equilibrated in an aqueous
phosphate buffer (pH 7.4) for 24 hours at 25 ꢁC, the portion with
the dissolved compound is then separated from the remains.
References
The solutions are analysed and quantied using UPLC/MS/MS.
Plasma protein binding determination assay. Human
plasma protein binding was determined from a 10 mM
compound solution in a Dianorm plasma well incubating at
37 ꢁC for 16 hours. Free fractions were calculated from ratios of
drug concentration in buffer and plasma wells determined by
LC-MS/MS.
In vitro hepatocyte and microsomal clearance assays. Cry-
opreserved or fresh hepatocytes were incubated with test
compounds at a nal concentration of 1 mM in incubation
medium containing 1.0 ꢄ 106 viable cells per mL (hepatocyte
incubations) or with a microsomal protein concentration of
1 mg mLꢀ1 + 1 mM NADPH. Incubations were performed at
37 ꢁC and samples taken at 5, 15, 30, 45, 60, 80, 100 and 120 min
(hepatocytes) and 0.5, 5, 10, 15, 20 and 30 min (microsomes) for
analysis using LC-MS/MS. Peak areas were determined from
1 C. Walsh, Nat. Rev. Microbiol., 2003, 1, 65–70.
2 L. Feng, M. M. Maddox, M. Z. Alam, L. S. Tsutsumi,
G. Narula, D. F. Bruhn, X. Wu, S. Sandhaus, R. B. Lee,
C. J. Simmons, Y. C. Tse-Dinh, J. G. Hurdle, R. E. Lee and
D. Sun, J. Med. Chem., 2014, 57, 8398–8420.
3 L. G. M. Bode, J. A. J. W. Kluytmans, H. F. L. Wertheim,
D. Bogaers, C. M. J. E. Vandenbroucke-Grauls,
R. Roosendaal, A. Troelstra, A. T. A. Box, A. Voss, I. van der
Tweel, A. van Belkum, H. A. Verbrugh and M. C. Vos, N.
Engl. J. Med., 2010, 362, 9–17.
4 H. Mohammad, P. V. Reddy, D. Monteleone, A. S. Mayhoub,
M. Cushman and M. N. Seleem, Eur. J. Med. Chem., 2015, 94,
306–316.
5 S. M. Lehar, T. Pillow, M. Xu, L. Staben, K. K. Kajihara,
R. Vandlen, L. DePalatis, H. Raab, W. L. Hazenbos,
J. H. Morisaki, J. Kim, S. Park, M. Darwish, B. C. Lee,
52236 | RSC Adv., 2017, 7, 52227–52237
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
H. Hernandez, K. M. Loyet, P. Lupardus, R. Fong, D. Yan, 20 D. Wang and F. Gao, Chem. Cent. J., 2013, 7, 95–109.
C. Chalouni, E. Luis, Y. Khaln, E. Plise, J. Cheong, 21 E. Jafari, M. R. Khajouei, F. Hassanzadeh, G. H. Hakimelahi
J. P. Lyssikatos, M. Strandh, K. Koefoed, P. S. Andersen, and G. A. Khodarahmi, Res. Pharm. Sci., 2016, 11, 1–14.
J. A. Flygare, M. Wah Tan, E. J. Brown and S. Mariathasan, 22 Y. L. Gao, Q. Z. Xiong, R. An and X. P. Bao, Chin. J. Org.
Nature, 2015, 527, 323–328. Chem., 2011, 31, 1529–1537.
6 S. R. Martinez, D. M. Rocca, V. Aiassa and M. C. Becerra, RSC 23 P. M. Bedi, V. Kumar and M. P. Mahajan, Bioorg. Med. Chem.
Adv., 2016, 6, 101023–101028. Lett., 2004, 14, 5211–5213.
7 H. F. Chambers and F. R. Deleo, Nat. Rev. Microbiol., 2009, 7, 24 P. M. Chandrika, T. Yakaiah, G. Gayatri, K. P. Kumar,
629–641.
B. Narsaiah, U. S. N. Murthy and A. R. R. Rao, Eur. J. Med.
Chem., 2010, 45, 78–84.
8 B. A. Diep, S. R. Gill, R. F. Chang, T. H. Phan, J. H. Chen,
M. G. Davidson, F. Lin, J. Lin, H. A. Carleton, 25 K. S. Van Horn, W. N. Burda, R. Fleeman, L. N. Shaw and
E. F. Mongodin, G. F. Sensabaugh and F. Perdreau-
Remington, Lancet, 2006, 367, 731–739.
9 L. C. Chan, A. Gilbert, L. Basuino, T. M. da Costa,
S. M. Hamilton, K. R. Dos Santos, H. F. Chambers and
S. S. Chatterjee, Antimicrob. Agents Chemother., 2016, 60,
3934–3941.
R. Manetsch, J. Med. Chem., 2014, 57, 3075–3093.
26 D. Gonzalez Cabrera, C. Le Manach, F. Douelle, Y. Younis,
T. S. Feng, T. Paquet, A. T. Nchinda, L. J. Street, D. Taylor,
C. de Kock, L. Wiesner, S. Duffy, K. L. White,
K. M. Zabiulla, Y. Sambandan, S. Bashyam, D. Waterson,
M. J. Witty, S. A. Charman, V. M. Avery, S. Wittlin and
K. Chibale, J. Med. Chem., 2014, 57, 1014–1022.
10 R. J. Guidos, B. Spellberg, M. Blaser, H. W. Boucher,
J. S. Bradley, B. I. Eisenstein, D. Gerding, R. Lyneld, 27 M. T. Kenji Yoshida, J. Chem. Soc. Pak., 1992, 1, 919–923.
L. B. Reller, J. Rex, D. Schwartz, E. Septimus, F. C. Tenover, 28 K. Kanuma, K. Omodera, M. Nishiguchi, T. Funakoshi,
D. N. Gilbert and IDSA, Clin. Infect. Dis., 2011, 52, S397–S428.
11 J. P. Surivet, C. Zumbrunn, G. Rueedi, D. Bur, T. Bruyere,
H. Locher, D. Ritz, P. Seiler, C. Kohl, E. A. Ertel, P. Hess,
S. Chaki, Y. Nagase, I. Iida, J. Yamaguchi, G. Semple,
T. A. Tran and Y. Sekiguchi, Bioorg. Med. Chem., 2006, 14,
3307–3319.
J. C. Gauvin, A. Mirre, V. Kaegi, M. Dos Santos, S. Kraemer, 29 P. R. Gilson, C. Tan, K. E. Jarman, K. N. Lowes, J. M. Curtis,
M. Gaertner, J. Delers, M. Enderlin-Paput, M. Weiss,
R. Sube, H. Hadana, W. Keck and C. Hubschwerlen, J.
Med. Chem., 2015, 58, 927–942.
W. Nguyen, A. E. Di Rago, H. E. Bullen, B. Prinz, S. Duffy,
J. B. Baell, C. A. Hutton, H. Jousset Subroux, B. S. Crabb,
V. M. Avery, A. F. Cowman and B. E. Sleebs, J. Med. Chem.,
2017, 60, 1171–1188.
12 R. J. Gordon and F. D. Lowy, Clin. Infect. Dis., 2008, 46(suppl.
5), S350–S359.
13 C. Vuong, A. J. Yeh, G. Y. Cheung and M. Otto, Expert Opin.
Invest. Drugs, 2016, 25, 73–93.
30 X. Zhu, K. S. Van Horn, M. M. Barber, S. Yang, M. Z. Wang,
R. Manetsch and K. A. Werbovetz, Bioorg. Med. Chem., 2015,
23, 5182–5189.
14 C. M. Macal, M. J. North, N. Collier, V. M. Dukic, 31 W. Devine, J. L. Woodring, U. Swaminathan, E. Amata,
D. T. Wegener, M. Z. David, R. S. Daum, P. Schumm,
J. A. Evans, J. R. Wilder, L. G. Miller, S. J. Eells and
D. S. Lauderdale, J. Transl. Med., 2014, 12, 124–135.
G. Patel, J. Erath, N. E. Roncal, P. J. Lee, S. E. Leed,
A. Rodriguez, K. Mensa-Wilmot, R. J. Sciotti and
M. P. Pollastri, J. Med. Chem., 2015, 58, 5522–5537.
15 L. M. Weigel, D. B. Clewell, S. R. Gill, N. C. Clark, 32 K. L. Johnston, D. A. N. Cook, N. G. Berry, W. David Hong,
L. K. McDougal, S. E. Flannagan, J. F. Kolonay, J. Shetty,
G. E. Killgore and F. C. Tenover, Science, 2003, 302, 1569–
1571.
R. H. Clare, M. Goddard, L. Ford, G. L. Nixon,
P. M. O'Neill, S. A. Ward and M. J. Taylor, Sci. Adv., 2017,
3, 1551–1560.
16 M. K. Hayden, K. Rezai, R. A. Hayes, K. Lolans, J. P. Quinn 33 W. N. Burda, K. B. Fields, J. B. Gill, R. Burt, M. Shepherd,
and R. A. Weinstein, J. Clin. Microbiol., 2005, 43, 5285–5287.
17 G. Morales, J. J. Picazo, E. Baos, F. J. Candel, A. Arribi,
X. P. Zhang and L. N. Shaw, Eur. J. Clin. Microbiol. Infect.
Dis., 2012, 31, 327–335.
B. Pelaez, R. Andrade, M. A. de la Torre, J. Fereres and 34 G. S. Basarab, P. J. Hill, C. E. Garner, K. Hull, O. Green,
M. Sanchez-Garcia, Clin. Infect. Dis., 2010, 50, 821–825.
18 L. C. Chan, L. Basuino, B. Diep, S. Hamilton, S. S. Chatterjee
and H. F. Chambers, Antimicrob. Agents Chemother., 2015, 59,
2960–2963.
B. A. Sherer, P. B. Dangel, J. I. Manchester, S. Bist,
S. Hauck, F. Zhou, M. Uria-Nickelsen, R. Illingworth,
R. Alm, M. Rooney and A. E. Eakin, J. Med. Chem., 2014,
57, 6060–6082.
19 W. H. Organization, WHO publishes list of bacteria for 35 K. Doyle, H. Lonn, H. Kack, A. Van de Poel, S. Swallow,
which new antibiotics are urgently needed, http://www.
who.int/mediacentre/news/releases/2017/bacteria-antibiotics-
needed/en/.
P. Gardiner, S. Connolly, J. Root, C. Wikell, G. Dahl,
K. Stenvall and P. Johannesson, J. Med. Chem., 2016, 59,
9457–9472.
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