Antibacterial activity of a series of N2, N4- disubstituted quinazoline-2,4-diamines

Article abstract of DOI:10.1021/jm500039e

A series of N²,N⁴-disubstituted quinazoline-2,4- diamines has been synthesized and tested against multidrug resistant Staphylococcus aureus. A structure-activity and structure-property relationship study was conducted to identify new hit compounds. This study led to the identification of N²,N⁴-disubstituted quinazoline-2,4- diamines with minimum inhibitory concentrations (MICs) in the low micromolar range in addition to favorable physicochemical properties. Testing of biological activity revealed limited potential for resistance to these agents, low toxicity, and highly effective in vivo activity, even with low dosing regimens. Collectively, these characteristics make this compound series a suitable platform for future development of antibacterial agents.

Full text of DOI:10.1021/jm500039e

Article
pubs.acs.org/jmc
Antibacterial Activity of a Series of N2,N4‑Disubstituted Quinazoline-
2,4-diamines
Kurt S. Van Horn,†,§ Whittney N. Burda,‡,§ Renee Fleeman,‡ Lindsey N. Shaw,*,‡
and Roman Manetsch*,†
†Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, CHE 205, Tampa, Florida 33620, United States
‡Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 East Fowler Avenue, ISA 2015,
Tampa, Florida 33620, United States
ABSTRACT: A series of N2,N4-disubstituted quinazoline-2,4-
diamines has been synthesized and tested against multidrug
resistant Staphylococcus aureus. A structure−activity and
structure−property relationship study was conducted to
identify new hit compounds. This study led to the
identification of N2,N4-disubstituted quinazoline-2,4-diamines
with minimum inhibitory concentrations (MICs) in the low
micromolar range in addition to favorable physicochemical
properties. Testing of biological activity revealed limited potential for resistance to these agents, low toxicity, and highly effective
in vivo activity, even with low dosing regimens. Collectively, these characteristics make this compound series a suitable platform
for future development of antibacterial agents.
a return to the preantibiotic era where 80% of staphylococcal
bloodborne infections were fatal.
INTRODUCTION
■
Staphylococcus aureus is a Gram positive bacterium that exists as
a commensal of the human flora, with 30% of the population
harboring this bacterium in their anterior nares.1 S. aureus can
infect many ecological niches within the human body, causing a
wide range of diseases. These can be categorized into skin and
soft tissue infections such as folliculitis and impetigo,
bacteremia, invasive infections such as endocarditis and
pneumonia, and toxinoses such as food poisoning and toxic
shock syndrome. As a result of this complex pathology, S.
aureus is speculated to be the most common cause of infectious
morbidity and mortality in the United States.2 The success of S.
aureus as a pathogen is due, in large part, to its ability to
develop resistance to antibiotics. Indeed, since the introduction
of penicillin, S. aureus has demonstrated an extraordinary
propensity for evading antibiotics, with resistance gained within
the first 12 months following introduction of a new drug.3
Remarkably, resistance to daptomycin, a semisynthetic lip-
opeptide, was seen even before FDA approval.4 The most
severe infections, and public health threats, are posed by
methicillin resistant S. aureus strains (MRSA). These are
isolates that possess a modified penicillin binding protein
(Pbp2a), which effectively renders the entire class of β-lactam
antibiotics inert. This leaves limited choices for therapeutic
intervention, with vancomycin, a glycopeptide antimicrobial
agent, commonly deployed in the direst of cases. In recent
years, however, there has been an increase in vancomycin
intermediate and vancomycin resistant S. aureus, which
threatens to take away our most valuable treatment option
for staphylococcal infections.5 With the rise in antibiotic
resistance and a lack of antimicrobial therapy, we are faced with
To this end, we have explored the potential of quinazoline-
based agents for their anti-MRSA activity. Quinazolines are a
class of compounds that have shown potential as antibacterials.
Recently, Lam et al. showed examples of 7-(benzimidazol-1-yl)-
2,4-diaminoquinazolines such as A that were highly selective
toward S. aureus dihydrofolate reductase (DHFR), displaying a
46700-fold Ki selectivity over human DHFR.6 This active
DHFR inhibitor was found to have an MIC of 0.25 μg/mL
against trimethoprim resistant strains of S. aureus and showed
protection in mice following a lethal challenge of S. aureus,
resulting in a protection of 50% of the mice (PD50) at 2 mg/
mL.
Huband et al. have reported substituted quinazolin-2,4-dione
B as bacterial gyrase and topoisomerase inhibitors (Figure 1).7
They determined a minimum inhibitory concentration against
90% of the tested strains (MIC90) of 0.5 μg/mL versus
staphylococci, 0.06 μg/mL versus streptococci, 2 μg/mL versus
enterococci, and 0.5 μg/mL versus Moraxella catarrhalis,
Haemophilus influenzae, Listeria monocytogenes, Legionella
pneumophila, and Neisseria species. Compound B was used in
a S. aureus in vivo murine acute lethal infection model, where an
oral dose of 2.5 1.8 mg/kg was found to protect 50% of mice
infected with MRSA strain SA-1417.
Indolo[1,2-c]quinazoline C and its analogues have been
shown to have antibacterial and antifungal activity by Rohini et
al. (Figure 1).8 The MIC of these compounds were evaluated
against Gram positive and negative bacteria, including Staph-
Received: January 8, 2014
© XXXX American Chemical Society
A
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Figure 1. Quinazolines with reported antibacterial activity.
Figure 2. Synthesis of N2,N4-disubstituted quinazoline-2,4-diamines.
ylococcus aureus, Bacillus subtilis, Streptococcus pyogenes,
Salmonella typhimurium, Escherichia coli, and Klebsiella pneumo-
nia and also against the fungi Aspergillus niger, Candida albicans,
and Trichoderma viridae. Quinazoline C had MICs between 2
and 5 μg/mL against all tested bacteria and between 2 and 10
μg/mL for fungal isolates.
focusing on the 2-position, the 4-position, and the quinazoline’s
benzenoid ring. Furthermore, we report the selection process
leading to frontrunner compounds displaying low mutation
frequencies, limited cytotoxicity, and promising in vivo efficacy
in murine models of infection.
Mycobacterial growth inhibition and dihydrofolate reductase
inhibition was reported by DeGraw et al. with a series of 2,4-
diaminoquinazolines D (Figure 1).9 An MIC of 1.8 μM was
reported for D and a marked synergistic effect was found with
diaminodiphenyl sulfone. Although activity was found with
Mycobacterium, D was found to be inactive against Staph-
ylococcus aureus, Enterococcus faecalis, Corynebacterium acnes,
Pseudominas aeruginosa, Erysipelothrix insidiosa, Escherichia coli,
Proteus vulgaris, and Salmonella chloraesuis.
In vitro activity against E. coli, S. aureus, and K. pneumonia
was discovered for the quinazoline class E at MICs of 2, 12 μM,
and 12 μM, respectively, as reported by Kung et al. (Figure
1).10 In an in vivo murine model, compound E afforded a 40%
survival rate when injected subcutaneously at 100 mg/kg in
mice infected with K. pneumonia.
RESULTS AND DISCUSSION
■
Synthetic Chemistry. The quinazoline library was
synthesized utilizing known procedures (Figure 2).13 Com-
mercially available anthranilic acids (a) were cyclized with urea,
and the resulting quinazoline-2,4-dione or commercially
available benzoyleneurea (b) was reacted with phosphorus
oxychloride to form the 2,4-dichloroquinazoline (c). Amine
substitution occurred selectively at position 4, yielding 4-amino-
2-chloroquinazoline (d) and subsequently by substitution at
position 2 to give the 2,4-diaminosubstituted quinazoline.
Throughout this synthetic sequence, only 4-amino-2-chlor-
oquinazoline (d) and the final N2,N4-disubstituted quinazoline-
2,4-diamine have been purified and characterized.
Structure−Activity Relationship Studies. Testing of
Quinazolines via Kirby−Bauer Assays. Assessment of the
quinazolin-2,4-diamine structure shows three locales for
substitution patterns: N2-substitution, N4-substitution, and
benzenoid substitution at positions C5−C8. Initial studies
focused on the amine substitutions as shown in Table 1.
Compounds were initially tested using a Kirby−Bauer assay to
determine whether they inhibited bacterial growth. Upon
identification of active compounds, minimum inhibitory
concentrations (MICs) were determined to assess the potency
of the compounds. MICs were not determined for compounds
without zones of inhibition in the Kirby−Bauer assays (Tables
1−3).
́
Gottasova et al. reported the in vitro activity of 4-
aniloquinazoline F against B. subtilis and S. aureus (Figure
1).11 An MIC of 10 μg/mL and an EC50 of 0.8 μg/mL for F
was found against S. aureus. Quinazoline F also had an MIC of
1 μg/mL and an EC50 of 0.7 μg/mL in assays using B. subtilis.
Recently, we tested a small library of quinazolines,
structurally different from previously reported antibacterial
quinazolines and originally designed as potential antileishma-
nials, for antibacterial activity.12 The results were promising as
several compounds had zones of inhibition (ZOI) against
MRSA in Kirby−Bauer tests. A larger library was then prepared,
and minimum inhibitory concentrations (MICs) were
determined for those compounds having ZOIs. Herein, we
report a detailed structure−activity relationship (SAR) study
First, a subset of N2,N4-substituted quinazolines were
synthesized and tested, in which N4 was modified by a furfuryl
moiety, while the N2-group was varied (Table 1). Quinazolines
B
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Table 1. SAR of N-Alkyl-N-furfurylquinazolin-2,4-diamines and N2- and N4-Substitutions Rich in sp3 Hybridized Carbons
1 and 2 with a N2-methyl or N2-ethyl did not have ZOIs in the
Kirby−Bauer assays. Branched alkyl groups on N2 furnished
compounds 3 and 6 displayed inhibition, while unbranched
polar substituents in 4 and 5 lacked any activity. Cycloalkyl
groups on N2 varied in potency based on size: N2-cyclopropyl
and N2-cyclobutyl quinazolines 7 and 8 were inactive while
analogues 9 and 10 with a N2-cyclopentyl and a N2-cyclohexyl
respectively provided ZOIs. Next, compounds were tested with
a furfuryl group at N2. N2-Furfuryl-quinazolines 11 and 12 with
N4-methyl and N4-isopropyl were both active From this small
set it was concluded that when a furfuryl moiety was substituted
at N4, best antibacterial activity would be observed when a
bulky alkyl substituent was at N2. In contrast, small groups were
tolerated at N4 with N2-furfuryl-modified quinazolines.
With the idea to reduce the number of aromatic rings, a
subset of compounds was designed to test whether N2- and N4-
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residues rich in sp3-hybridized carbons are tolerated without
compromising activity. Analogues 13−27 and 30−32 were
synthesized in which one of the two N2,N4-substitutents was a
methyl, an isopropyl, or a 2-hydroxyethyl group, whereas the
other consisted of a branched, an unbranched, or a saturated
cycloalkyl moiety. None of these analogues were particularly
active with the exceptions of the N2-cyclopentyl-N4-isopropyl
derivative 26, the N4-cyclopentyl-N2-isopropyl derivative 31,
and the N4-cyclohexyl-N2-isopropyl derivative 32. Analogues 28
with N2,N4-dibutyl and 29 with N4-2-(methylthio)ethyl and N2-
isopropyl also inhibited growth.
methyl or N4-isopropyl displayed MICs of 390 and 35 μM,
respectively, showing that isopropyl derivation leads to more
active compounds than methyl derivation.
The five active nonaromatic 2,4-diaminoquinazolines from
13−34 displayed quite different MICs. The N2-cyclopentyl-N4-
isopropyl-quinazolin-2,4-diamine 26 had an MIC comparable
to the N4-furfuryl-N2-cyclopentyl derivative 9 or the N4-
furfuryl-N2-isopropyl derivative 3. The N4-cyclopentyl-N2-
isopropyl analogue 31 was more active with an MIC of 37
μM, whereas compound 29, with N4-methylthioethyl-N2-
isopropyl derivation and an MIC >360 μM, was inactive.
N2,N4-Dibutylquinazolin-2,4-diamine 28 and N4-cyclohexyl-N2-
isopropylquinazolin-2,4-diamine 32, with respective MICs of
0.37 and 3.5 μM, were the only analogues of the entire series
displaying submicromolar or single-digit micromolar activity.
These most active dialkyl 2,4-diaminoquinazolines make for
interesting points in the compound subseries.
Following this, a subset of quinazolines was prepared to
probe whether antibacterial activity can be improved via steric
and electronic effects on the quinazoline benzenoid ring (Table
2). N4-Furfuryl-N2-isopropyl-substituted quinazoline 3 was
Table 2. SAR of Benzenoid Substitutions
Study of the benzenoid substitution revealed 6-, 7-, and 8-
chloro-substituted compounds 35−37 to have MICs six times
more active than the quinazoline 3 with an unsubstituted
benzenoid ring (Table 2). 6-Methoxy-quinazoline 45 was more
than twice as active as reference compound 3, while all the
other methyl- or methoxy-substituted quinazolines were less
potent.
ZOI
MIC
R1
−H
R2
−H
R3
−H
R4
−H
−Cl
−H
−H
−H
−CH3
−H
−H
(mm)
(μM)
As the compounds studied previously contained mostly alkyl
or furfuryl derivation, the effect of benzyl and phenyl
substituents was observed (Table 3). N-Benzyl-N-isopropyl
derivatives 47 and 48 with MICs of 34 μM had nearly the same
MIC as the N4-phenyl derivative 49 and the N-butyl N-phenyl
derivatives 51 and 52. N4-Benzyl-N2-butyl 50 had an MIC of
3.3 μM, again showing the favorable effects that butyl groups
have on activity when 50 is compared to 48 while also showing
the preference of benzyl over phenyl when 50 is compared to
51. The N,N-dibenzyl derivative 53 also had excellent activity,
with an MIC of 2.9 μM, again indicating that benzyl derivation
is good for improving the antibacterial activity over phenyl
derivation. The MICs for N4-phenyl derivatives 54 and 55 with
N2- benzyl or phenyl were 31 and 32 μM, respectively. Mixing
N-furfuryl with N-benzyl or p-substituted N-phenyl resulted in
compounds 56−59 with MICs in the range of 30 μM.
Compound Optimization Based on Initial SAR and
Mutation Frequency Studies. The four most active quinazo-
lines found in the initial SAR were 28, 32, 50, and 53. Prior to
optimization efforts, compounds 28, 32, 50, and 53 were
assessed for developability based on mutation frequencies.
Although compounds 28 and 32, without aromatic side chains,
were highly potent quinazolines (Table 1), it was decided that
analogues thereof would not be synthesized due to the less than
favorable mutation frequency (see below). As seen in Table 3,
nearly all tested compounds with aromatic side chains were
active. Thus, analogues were synthesized by using para-
substituted benzyl amines to make analogues of 53 (Table 4).
Mono p-chlorobenzyl 60 and 61 had MICs of 27 and 67 μM,
respectively, while the dichloro derivative 62 had better activity
with an MIC of 2.4 μM. p-Methylbenzyl derivatives 63−65 had
MICs of 28 and 71 μM for the monomethyl derivatives and 27
μM for the di-p-methylbenzyl derivative 65. The N2-p-
methoxybenzyl derivative 66 had an MIC of 0.67 μM, more
than four times more active than the parent 53. The N4-p-
methoxybenzyl and dimethoxy derivatives 67 and 68 were less
active, with MICs of 68 and 25 μM, respectively.
3
10
10
180
32
35
36
37
38
39
40
41
42
43
44
45
46
−H
−H
−H
−Cl
−H
−H
−H
−CH3
−H
−H
−H
−H
−H
−Cl
−H
−H
−H
−CH3
−H
−H
−H
−H
−Cl
−H
−H
−H
-CH3
−H
−H
−H
12
32
11
32
9
>78
340
nd
10
none
none
none
none
none
10
nd
−H
−OCH3
nd
nd
−OCH3 −H
nd
−OCH3 −H
−H
−H
−H
80
−OCH3 −H
9
>80
selected as a reference compound, to which analogues 35−46
were monosubstituted at positions C5−C8 with either a chloro,
a methyl, or a methoxy group. The best compounds were
quinazolines 35−38, with one chloro substitutent suppressing
the growth of bacteria equally or more efficiently than reference
compound 3. The analogues 39−46 substituted with a methyl
or a methoxy group were less potent with inhibition of growth
observed in 25% and 50% of the samples, respectively. The
trends noted for this set are that a chloro- substitution is well
tolerated while methoxy- is less so and methyl- is even less.
A final focus was a selection of compounds with aromatic
residues (Table 3). Analogues 47−59, bearing phenyl and/or
benzyl groups at N2- and/or N4-positions, were successful in
inhibiting the growth of S. aureus in Kirby−Bauer tests.
Minimum Inhibitory Concentration Assays. To obtain a
more precise structure−activity relationship study, quinazolines
displaying ZOIs were tested in a minimum inhibitory
concentration (MIC) assay. The furfuryl substituted quinazo-
lines 1−12 (Table 1) displayed a range of activity against S.
aureus. The N2-isopropyl derivative 3 was twice as active as the
N2-tert-butyl-quinazoline 6, with an MIC of 180 μM. N2-
Cyclopentyl-quinazoline 9 had a MIC comparable to the
isopropyl derivative, while N2-cyclohexyl-quinazoline 10 had an
MIC of 78 μM. N2-Furfuryl-2,4-diaminoquinazoline with N4-
As previous SAR data indicated that a chloro substituent at
the benzenoid ring increases the activity (Table 2), quinazo-
D
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Table 3. N2- and/or N4-Benzyl and/or Phenyl Substitutions
Table 4. Combination with 7-Chloro or p-Substituted Benzyl at N2 and/or N4
lines were synthesized by combining the benzenoid 7-chloro
group with a variety of the most active N2- and N4-benzyl
substitutions (Tables 3 and 4). Addition of a 7-chloro group to
the benzenoid ring of the N2,N4-dibenzylquinazolin-2,4-
diamine 53 reduced the MIC activity from 2.9 to 27 μM in
compound 69 (Table 5). A similar reduction in activity of a
factor of approximately 10 was observed with the addition of a
7-chloro group to the N2-p-methoxybenzyl derivative 66 or to
the di-p-chlorobenzyl derivative 62 in compounds 71 and 72.
Combining the N4-p-chlorobenzyl with N2-p-methoxybenzyl
and 7-chloro resulted in compound 73, with a low antibacterial
activity of 51 μM. The only 7-chloroquinazoline to maintain
E
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Table 5. Combinations with 7-Chloro
activity was the N2-butyl derivative 70 with a MIC of 0.73 μM,
in comparison to its reference 50 with an MIC of 3.3 μM.
Physicochemical Properties. A limited structure−property
relationship study was conducted for the three lead quinazo-
lines 32, 50, and 53. Aqueous solubility, permeability, and the
partition coefficient log D were experimentally determined at
two pHs.14 The properties of all tested compounds were
affected by pH, with better solubilities and poorer perme-
abilities being observed in more acidic environments.
Regardless, because solubility and permeability, as well as log
D, were acceptable for all tested compounds, the quinazoline-
2,4-diamines show promise as a compound series with favorable
physicochemical properties.
In Vitro Biological Evaluation. Testing for Bactericidal
and Bacteriostatic Activity. An important distinction of
antibiotics is whether they are bactericidal or bacteriostatic.
Bactericidal agents kill bacteria, while bacteriostatic agents
prevent their reproduction. The minimum bactericidal
concentrations (MBCs) were tested for the most potent
quinazolines 28, 32, 50, 53, and 62 (Figure 3). Compound 28
was found to have an excellent MBC50 of 0.036 μM compared
to the MIC of 0.37 μM. For compound 32, the MBC50 was
found to be 8.8 μM, which is around nine times the MIC.
Despite this being greater than the MIC, this compound still
appears to possess bactericidal activity. Compound 50 had an
MBC50 of 0.98 μM, compared with the MIC of 3.3 μM,
showing very strong bactericidal capacity. The MBC50 for 62
was 7.2 μM, which is three times the MIC of 2.4 μM, again
showing strong bactericidal capacity. Interestingly, compound
53, with an MIC of 2.9 μM, was not found to be bactericidal at
any of the concentrations tested, thus demonstrating
bacteriostatic activity.
Cytotoxicity and Hemolysis Assays of Selected Com-
pounds. The cytotoxicity of frontrunner compounds was
determined against adenocarcinomic human alveolar basal
epithelial cells (A549 cells, Figure 4). For lead compounds 32,
50, and 53, we observed almost no cytotoxicity, recovering
greater than 60% of cells at all concentrations tested, compared
to untreated controls. Similarly, when compound 28 was tested,
we recovered >50% of cells at all concentrations tested.
Specifically, for compound 32, we recovered greater than 100%
A549 viability at concentrations ranging from 1.8 to 8.8 μM. At
concentrations ranging from 18 to 440 μM, we recovered
98.6% to 70.3% viability of A549 cells, respectively. For
compound 50, we recovered greater than 100% A549 viability
at concentrations ranging from 1.6 to 16 μM. At concentrations
ranging from 41 to 410 μM, we recovered greater than 95%
viable A549 cells. For compound 53, we recovered between
93.2% and 83.6% of the A549 cells at concentrations ranging
from 1.5 to 370 μM, respectively. Finally, for compound 28, we
F
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Figure 3. The minimum bactericidal concentrations of lead quinazolines. The data shown represents percent recovery of inoculum compared to
untreated controls from three independent experiments. Red square = MIC value for each agent.
Figure 4. The effect of lead quinazolines on human alveolar epithelia cells. The effects of lead quinazolines on cell viability were assessed using the
MTT reagent and taken as a percentage of treated cells compared to untreated cells (DMSO). The data presented are the results from three
independent experiments. Error bars are shown as SEM.
observed 100% A549 cell viability at concentrations ranging
from 0 to 9.2 μM, and 58.8% cell viability at concentrations
ranging from 36.7 to 459 μM.
Investigating the Potential for MRSA Resistance to
Quinazolines. An important attribute of potential antimicrobial
agents is that the spontaneous development of resistance is not
easily attained. Thus, we set out to determine the spontaneous
mutation frequency of 28, 32, 50, 53, and 62. We plated 1 ×
109 cells on agar plates containing 2.5× the MIC of either 28,
32, or 62 and observed lawns of bacterial growth for every
replicate. This indicates that resistance to these agents is readily
acquired (Table 6). For compound 53, only seven spontaneous
G
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Table 6. Physicochemical Properties of Quinazolines 32, 50, and 53
a
(*) for solubility ≤5 μM, (**) for 5 μM < solubility ≤10 μM, (***) for 10 μM < solubility ≤20 μM, (****) for 20 μM < solubility ≤30 μM,
b
(*****) for solubility ≥30 μM. For the determination of the Pe values, the following internal controls were utilized: carbazepine pH 4.0
permeability Pe = 108 × 10−6 (cm/s) and pH 7.4 permeability Pe = 130 × 10−6 (cm/s); ranitidine·HCl pH 4.0 permeability Pe = 5.2 × 10−6 (cm/s)
and pH 7.4 permeability Pe = 2.2 × 10−6 (cm/s); verapamil·HCl pH 4.0 permeability Pe = 20.6 × 10−6 (cm/s) and pH 7.4 permeability Pe = 1360 ×
10−6 (cm/s).
mutants were isolated over 10 replicates. The combined
inoculum was 2.31 × 1010, which yielded a mutation frequency
of 3.03 × 10−10. No spontaneous mutants were generated for
compound 50, which yielded a mutation frequency less than 1
DNA sequence analysis of the spontaneous mutants generated
using 53. Accordingly, we sequenced the quinolone resistance-
determining regions (QRDR) of the genes encoding DNA
gyrase (gyrA and gyrB) and topoisomerase IV (grlA and grlB),
as described by us previously.15 We found that each of the
mutants contain the same mutations in the QRDR as that of
the parent: S84L and E88K mutations were observed in gyrA
and S80Y and E84G mutations in grlA. No mutations were
observed in the QRDR of gyrB or grlB of the mutant strains.
This indicates that the mechanism of action likely is not the
inhibition of gyrase or topoisomerase IV. Work by others
suggests that quinazoline-based compounds may interfere with
dihydrofolate reductase (DHFR), an enzyme involved in the
essential process of folic acid synthesis in bacterial cells.16
Therefore, we also employed DNA sequencing analysis of
DHFR with the mutant and parental strains. Upon analysis, we
again observed no mutations in the mutant strains compared to
the wild-type. In spite of this finding, we next performed a
tetrahydrofolic acid assay. DHFR catalyzes the final step in folic
acid production, which is the reduction of dihydrofolic acid to
tetrahydrofolic acid. If quinazolines are DHFR inhibitors,
dihydrofolic acid would not be reduced to tetrahydrofolic acid.
We hypothesized that by adding in exogenous tetrahydrofolic
acid, we would rescue the cells from quinazoline-mediated
killing. Our wild-type strain was thus grown in the presence of
5× MIC of compounds 32, 50, 53, and 62 and varying
concentrations of tetrahydrofolic acid, ranging from 0 to 220
μM. The known DHFR inhibitor trimethoprim was also used as
a positive control, while vancomycin, an antibiotic that inhibits
cell wall biosynthesis, was used as a negative control. After the
18 h incubation period, we observed that even at the highest
concentration of tetrahydrofolic acid used there was no growth
of S. aureus in the presence of vancomycin, as expected. The
reverse was true, again as expected, when cells were incubated
with trimethoprim, with as little as 2.2 μM tetrahydrofolic acid
recovering growth of the MRSA strain. For all compounds we
observed similar results to trimethoprim when exogenous
tetrahydrofolic acid was added, with bacterial growth fully
restored compared to control wells that contain no
tetrahydrofolic acid. These results strongly suggest that our
series of quinazoline agents target DHFR for their antibacterial
mechanism of action, in spite of the fact that no mutations were
observed in the DHFR gene. We suggest that it is perhaps likely
that the mutations occurring in the 53 resistance mutants likely
result in enhanced efflux in these strains.
× 10−11
.
We next explored if cross resistance had developed toward
derivatives of 53 when tested against the spontaneous mutants
obtained from compound 53. First, we tested the MIC of these
strains against the producing compound 53 and found that it
had lost all activity against the mutant strains, with MICs >590
μM compared with 2.9 μM against the parent. The MIC was 27
μM against mutant 1 (M1) and the original strain for 60, while
the compound became more effective against M2−M7, with an
increased MIC of 2.7 μM. Compound 62 was only tested
against M1 and M2 and had a 10-fold drop in activity from 2.4
to 24 μM. Quinazoline 63, with an original activity of 28 μM,
had the same activity toward M1, while it has increased activity
(MIC = 2.8 μM) against M2−M7. The MIC was 27 μM against
the original strain and M1 and M2 for 65, while the activity
against M3−M7 was increased to 2.7 μM. The activity of 66
had a minor decrease in efficacy from 0.67 to 27 μM against
M1−M3 and a remarkably large drop in activity to 270 μM
against M4, while the MIC remained unchanged for M5−M7.
In all mutant strains, 68 had a reduction of activity from 25 μM
against the original strain. Against strains M5 and M6, the MIC
was 125 μM, and for the rest of the strains it was >250 μM.
Compound 69, with an original MIC of 27 μM, had the same
activity for M1 and M2 while activity was increased 10-fold to
2.7 μM against M3−M7. Activity for 70 was found to be 0.73
μM against the mutants and original strain alike. Compounds
61 (original MIC 67 μM), 64 (original MIC 71 μM), and 67
(original MIC 67 μM) were found to be more active against the
mutants than the original strain. Analogue 61 had an MIC of
2.7 μM against all seven mutants. Against M1 and M2, 64 had
MICs of 2.8 μM, whereas the MIC for M3−M7 was 28 μM.
Compound 67 had MICs of 2.7 μM for M4 and M6 and 27 μM
for the rest of the mutants. Collectively, this suggests that the
development of spontaneous resistance to our quinazoline
agents actually leads to a physiological burden, and greater
sensitivity to other compounds from this class, rather than
enhanced resistance. While an immediate observation for this
finding is not entirely clear, it is common for organisms that
incur resistance-facilitating mutations to do so at the expense of
their overall cellular fitness. Thus, it is possible that mutations
gained in these strains to circumvent the activity of 53 renders
them more sensitive to other agents as a result of potentially
deleterious mutations.
Investigating Synergistic and Antagonistic Activities of
Front-Runner Compounds Alongside other Known Folate
Biosynthesis Targeting Antibacterials. To investigate if our
compounds were active against strains that were resistant to
other DHFR targeting antimicrobials, we next assessed activity
Investigating the Mechanism of Action of the Quinazoline
Derivatives. As quinazolines are closely related to quinolones,
which target enzymes that mediate supercoiling, we employed
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Figure 5. Testing the in vivo efficacy of lead quinazolines using a Galleria mellonella model of S. aureus infection. The in vivo efficacy of lead agents
was assessed using a G. mellonella model of infection. Larvae were considered dead if they did not respond to physical stimuli. Mortality was
measured using a log rank and χ-square test with 1degree of freedom. * = p > 0.05, ** = p > 0.01.
Figure 6. Lead quinazoline agents are efficacious in protecting mice during lethal S. aureus peritonitis infection. The significance of mortality was
measured using a log rank and χ-square test with 1 degree of freedom. * = p > 0.05.
of the front-runners using a trimethoprim resistant strain of S.
aureus. After 24 h of incubation, we observed that this strain
was sensitive to all lead quinazolines tested. In addition, we also
performed the reverse test, assessing the MIC of trimethoprim
against our quinazoline resistant mutants. With these experi-
ments, we again observed no changes in MIC, indicating that
cross-resistance between front runner agents and trimethoprim
does not occur despite apparently sharing the same target
within the S. aureus cell. This is very encouraging and suggest
our compounds target a different part of the DHFR protein,
and also that the trimethoprim resistant strains that exist
clinically should still be sensitive to killing by our front runner
compounds.
Further to this, trimethoprim is almost always used clinically
in conjunction with the dihydropteroate synthetase inhibitor
sulfamethoxazole. These two antibiotics work synergistically to
inhibit folic acid production in bacteria, which is an essential
process, at different stages of the folate production pathway.
We next set out to see if similar synergy was obtained when
using our lead quinazolines alongside sulfamethoxazole. To
determine this, we performed a checkerboard microtitration
assay to determine the fractional inhibitor concentration (FIC)
of sulfamethoxazole and compounds 32, 50, 53, and 62. We
determined that the MIC of sulfamethoxazole alone against our
wild-type S. aureus was 2.4 mM, while in combination with our
lead agents, at concentrations ranging from 0.3 up to 300 μM,
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this was lowered to 150 μM, resulting in a FIC of 0.016.
Compounds 32, 50, 53, and 62 have MICs of 3.5, 3.3, 2.9, and
2.4 μM, respectively, while in combination with sulfamethox-
azole (150 μM), these compounds had decreased MICs of 0.3
μM (32, 50, and 53), and 0.24 μM (62). The sum of FIC of
both agents (ΣFIC) for compounds 32, 50, 53, and 62 in
combination with sulfamethoxazole were 0.10 and 0.11 for the
first two agents and 0.12 for the latter two. These results
indicate that these compounds, in combination with
sulfamethoxazole, are synergistic in action as their ΣFIC is
below the ≤0.5 threshold typically used to define such activity.
In Vivo Biological Evaluation. Galleria mellonella Model
of Infection. As a preliminary measure of in vivo efficacy, we
next tested our lead agents in a Galleria mellonella model of S.
aureus infection. For all compounds tested, larvae (N = 10)
were infected with a lethal dose of S. aureus cells (1.0 × 109).
Larvae were then treated 1 h postinfection with either the lead
agents or the vehicle alone (negative control) (Figure 5).
Additionally, we performed mock infection testing, where
larvae were not injected with S. aureus (positive control). For
32 and 50, we observed a 40% increase in survival of treated
larvae when using 5× MIC; however, this was just outside the
range of statistical significance. When dosing was increased to
10× MIC for each of these agents, survival increased to 70%
compared to the vehicle group, which was found to be highly
statistically significant. For compound 62, 5× MIC treatment
resulted in 50% protection (not significant), which rose to 60%
survivability (significant) upon increasing to 10× MIC dosing.
Finally, when using 5× MIC of 53, we observed 50% protection
rates over the infection period that was found to be highly
significant. Increased dosing with this agent did not lead to
enhanced efficacy over the 5× MIC dose (data not shown).
These results indicate the strong potential for in vivo efficacy of
these agents in clearing lethal S. aureus infection.
Murine Model of Lethal Peritonitis. As a final measure of
the suitability of our quinazoline-based compounds to serve as
antibacterial agents, we studied their in vivo efficacy using a
murine model of lethal peritonitis. As such, the ability of 50 and
53 to treat S. aureus infection was determined due to their
limited cytotoxicity, as determined using adenocarinomic
human alveolar basal epithelial cells (A549) and favorable
mutation frequencies. For all compounds tested, mice were
infected with 1 × 108 S. aureus cells via intraperitoneal injection.
At 1 h postinfection, mice were then injected via tail vein with
5× the MIC of either vancomycin (positive control), 50, or 53
(Figure 6). Each group of mice was compared to a negative
control group receiving only vehicle (45% w/v (2-hydrox-
ypropyl)-β-cyclodextrin in water) 1 h postinfection. For
compounds 50 and 53, we only had a single mouse die during
the infection period, which is a 69% increase in survival when
compared to our untreated controls. These data were even
better than that of the positive control, with only 50%
protectivity observed with vancomycin. We were able to
observe protection with vancomycin equivalent to that of 50
and 53 but only at 10× MIC (data not shown). On the basis of
these encouraging results, we also tested our best frontrunner
compound, 53, at 1× the MIC. At this concentration, we
observed complete protection of all mice infected with MRSA,
indicating excellent in vivo activity of these agents, even at very
low concentrations and doses.
CONCLUSION
■
A total of 73 N2,N4-disubstituted quinazoline-2,4-diamines,
many displaying antibacterial activities, have been synthesized
systematically by varying the substitutions in the 2-, 4-, 5-, 6-,
7-, and/or 8-positions. The most potent in vitro activities with
MICs in the submicromolar and single-digit range against
MRSA were obtained with quinazoline-2,4-diamines 28, 32, 50,
53, 62, 66, and 70 bearing N,N-dibutyl substitution, a N4-
cyclohexyl-N2-isopropyl substitution, or a N4-benzyl moiety
combined with either N2-benzyl or N2-n-butyl group. These
substitution patterns are necessary for S. aureus activity, as
supported by DeGraw et al., with quinazoline C (Figure 1), the
quinazoline-2,4-diamine that was unsubstituted on either amine
and inactive against S. aureus.9 Furthermore, the benzenoid ring
of the quinazoline-2,4-diamine scaffold has been identified to
play a secondary role for efficacy, with quinazolines substituted
with one chloro group at the 6-, 7-, or 8-position displaying
lower MICs against S. aureus in comparison to analogues
unsubstituted at the benzenoid ring. Similarly, the data from
Kung et al. in regard to D (Figure 1) shows that benzenoid
substitution can lead to in vitro activity in S. aureus when the
amine at the 4-position is unsubstituted, although excellent
activity was not observed.10 These promising results led to
efficacy testing of the lead compounds, which revealed a limited
capacity for cytotoxicity of the agents as well as highly favorable
spontaneous mutation frequencies for a number of the
frontrunners. Investigation of the mechanism of action suggests
that these agents function as inhibitors of dihydrofolate
reductase within S. aureus cells. Finally, using murine models
of infection we observed excellent in vivo efficacy for two of our
lead agents, giving complete, or almost complete, protection to
mice infected with a lethal dose of S. aureus that was superior to
similar concentrations of the control agent vancomycin. The
murine model data reported earlier shows better protection at
the MIC than the S. aureus in vivo data reported by Huband et
al. who reported a 50% protection at five times the MIC.7 The
observed potencies of frontrunner compounds 32, 50, 53, and
62 in our assays make N2,N4-disubstituted quinazoline-2,4-
diamines an excellent platform for the future development of
antibacterial agents.
EXPERIMENTAL SECTION
■
General. All reagents and solvents were obtained from Aldrich
Chemical Co. and used without further purification. Anthranilic acids
were purchased from Sigma-Aldrich, Oakwood Products, Inc. or TCI
America. NMR spectra were recorded at ambient temperature on a
250 MHz Bruker, 400 MHz Varian, or 500 MHz Varian NMR
1
spectrometer in the solvent indicated. All H NMR experiments are
reported in δ units, parts per million (ppm) downfield of TMS, and
were measured relative to the signals for chloroform (7.26 ppm),
methanol (3.31 ppm), and dimethylsulfoxide (2.50 ppm). All 13C
NMR spectra were reported in ppm relative to the signals for
chloroform (77 ppm), methanol (49 ppm), and dimethylsulfoxide
1
1
(39.5 ppm) with H decoupled observation. Data for H NMR are
reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d
= doublet, t = triplet, q = quartet, p = pentet, sext = sextet, sept =
septet, oct = octet, m = multiplet), integration and coupling constant
(Hz), whereas 13C NMR analyses were reported in terms of chemical
shift. NMR data was analyzed by using MestReNova Software version
5.3.2−4936. The purity of the final compounds was determined to be
≥95% by high-performance liquid chromatography (HPLC) using an
Agilent 1100 LC/MSD-VL with electrospray ionization. Melting
points were determined using a MEL-TEMP 3.0 instrument and are
uncorrected. Low-resolution mass spectra were performed on an
Agilent 1100 LC/MSD-VL with electrospray ionization. High-
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resolution mass spectra (HRMS) were performed on an Agilent LC/
MSD TOF system G3250AA. Analytical thin layer chromatography
(TLC) was performed on silica gel 60 F254 precoated plates (0.25
mm) from EMD Chemical Inc., and components were visualized by
ultraviolet light (254 nm). Reported Rf was determined for TLC.
Silicycle silica gel 230−400 (particle size 40−63 μm) mesh was used
for all flash column chromatography.
General Synthetic Procedure A: Cyclization of Anthranilic
Acids to the Corresponding Quinazoline-2,4-diones. One
equivalent of anthranilic acid and 3.5 equivalents of urea were mortar
and pestled to a powder and heated to 200 °C in a round-bottom flask
open to the atmosphere. After two hours, the mixture was cooled,
triturated with water, and filtered to give the product as crude. No
further purification was performed.
General Synthetic Procedure B: Chlorination of Quinazo-
line-2,4-diones to the Corresponding 2,4-Dichloroquinazo-
lines. One equivalent of quinazoline-2,4-dione and 1 equivalent of
N,N-dimethylaniline were mixed in 12 equivalents of phosphorus
oxychloride and the mixture refluxed under an argon atmosphere until
starting material was no longer present by TLC (3−16 h). The
mixture was then cooled and added to ice in the amount of 10 times
the reaction volume. The solution was filtered to give crude product.
General Synthetic Procedure C: Amine Substitution of 2,4-
Dichloroquinazolines to Yield 4-Amino-substituted 2-Chlor-
oquinazolines. Amine (1.1 equivalent) and sodium acetate were
mixed with 1 equivalent of 2,4-dichloroquinazoline at 0.1 M
concentration in a 3:1 mix of tetrahydrofuran and water and heated
to 65 °C. When the reaction was observed to be finished by TLC, the
solution was diluted with ethyl acetate, the layers were separated, and
the organic phase washed three times with an equal amount of water
and dried over Na2SO4. The crude was then purified by either method
Ca or Cb:
Purification Method Ca. The compound was recrystallized with
ethanol and water, filtered, and rinsed with cold ethanol to yield pure
product.
Purification Method Cb. The crude was purified by flash
chromatography using hexanes and ethyl acetate.
General Synthetic Procedure D: Amine Substitution of 4-
Aminosubstituted-2-chloroquinazolines to Yield 2,4-Diamino-
substituted Quinazolines. Amine (1.5 equivalents) was mixed with
1 equivalent of 4-amino-substituted 2-chloroquinazoline at 0.2 M
concentration in ethanol in a sealed tube and heated to 150 °C. When
the reaction was finished as observed by TLC, the compound was
purified by either method Da or Db:
Purification Method Da. Compound crystallized out of the cool
solution and was filtered and then rinsed with cold ethanol to yield
pure product.
1H), 6.32 (d, J = 1.6 Hz, 1H), 4.84 (d, J = 5.1 Hz, 2H). 13C NMR (63
MHz, CDCl3) δ 160.65, 157.61, 150.90, 150.32, 142.63, 133.70,
127.73, 126.40, 121.16, 113.32, 110.72, 108.73, 38.51. MS (ESI): [M +
H]+ 260; found 260.1. Rf = 0.14 (hexanes to ethyl acetate 4:1).
N4-(Furan-2-ylmethyl)-N2-methylquinazoline-2,4-diamine
(1). 1d (0.12 g, 0.46 mmol) was reacted with methylamine and
purified according to method Db to furnish 0.11 g (0.43 mmol) of the
1
title compound as a light-yellow solid in 93% yield. H NMR (500
MHz, CD3OD) δ 8.00 (d, J = 8.0 Hz, 1H), 7.66−7.56 (m, 1H), 7.44
(d, J = 1.1 Hz, 1H), 7.35 (s, 1H), 7.26 (t, J = 7.7 Hz, 1H), 6.39 (d, J =
3.2 Hz, 1H), 6.36 (dd, J = 3.1, 1.9 Hz, 1H), 4.80 (s, 2H), 3.06 (s, 3H).
13C NMR (126 MHz, CD3OD) δ 159.99, 150.78, 142.14, 142.10,
134.60, 123.99, 123.26, 110.18, 110.12, 107.92, 107.89, 107.87, 37.70,
27.11. HRMS: m/z calcd for C14H15N4O [M + H]+ 255.12404; found
255.12472. Rf = 0.37 (9:1 dichloromethane to methanol). Melting
point decomposition at 205 °C.
N2-Ethyl-N4-(furan-2-ylmethyl)quinazoline-2,4-diamine (2).
1d (0.080 g, 0.31 mmol) was reacted with ethylamine and purified
according to method Db to furnish 0.067 g (0.25 mmol) of the title
compound as a yellow solid in 81% yield. 1H NMR (400 MHz,
CD3OD) δ 8.07−8.04 (m, 1H), 7.66 (dd, J = 11.3, 4.0 Hz, 1H), 7.47−
7.43 (m, 1H), 7.43−7.26 (m, 2H), 6.40−6.35 (m, 2H), 4.83 (s, 2H),
3.56 (dd, J = 14.2, 7.1 Hz, 2H), 1.28 (t, J = 6.9 Hz, 3H). HRMS: m/z
calcd for C15H17N4O [M + H]+ 269.1397; found 269.1406. Rf = 0.43
(9:1 dichloromethane to methanol). Melting point 165−172 °C.
2-(4-(Furan-2-ylmethylamino)quinazolin-2-ylamino)ethanol
(5). 1d (0.12 g, 0.46 mmol) was reacted with 2-aminoethanol and
purified according to method Da to furnish 0.048 g (0.17 mmol) of the
title compound in 37% yield. 1H NMR (500 MHz, CD3OD) δ 8.11 (d,
J = 7.6 Hz, 1H), 7.76 (ddd, J = 8.4, 7.3, 1.3 Hz, 1H), 7.46 (d, J = 0.8
Hz, 1H), 7.40 (t, J = 7.5 Hz, 2H), 6.41 (d, J = 2.9 Hz, 1H), 6.40−6.36
(m, 1H), 3.74 (d, J = 36.6 Hz, 4H). 13C NMR (126 MHz, CD3OD) δ
160.46, 153.34, 150.50, 142.26, 139.06, 135.13, 124.56, 123.47, 116.45,
110.16, 110.12, 107.84, 59.81, 43.38, 37.93. HRMS: m/z calcd for
C15H17N4O2 [M + H]+ 285.13460; found 285.13503. Rf = 0.24 (9:1
dichloromethane to methanol). Melting point 219−221 °C.
N2-(tert-Butyl)-N4-(furan-2-ylmethyl)quinazoline-2,4-dia-
mine (6). 1d (0.070 g, 0.27 mmol) was reacted with tert-butylamine
and purified according to method Db to furnish 0.03 g (0.1 mmol) of
1
the title compound as a yellow solid in 37% yield. H NMR (400
MHz, DMSO-d6) δ 8.08 (d, J = 8.0 Hz, 1H), 7.62−7.46 (m, 2H), 7.25
(d, J = 8.3 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 6.42−6.28 (m, 2H), 4.74
(s, 2H), 1.41 (s, 9H). 13C NMR (101 MHz, CD3OD) δ 161.54,
158.37, 153.08, 147.88, 143.22, 134.83, 123.96, 123.43, 121.97, 118.56,
111.37, 107.87, 52.63, 39.25, 29.76. HRMS: m/z calcd for C17H21N4O2
[M + H]+ 297.1710; found 297.1726. Rf = 0.24 (9:1 dichloromethane
to methanol). Melting point 112−115 °C.
N2-Cyclopropyl-N4-(furan-2-ylmethyl)quinazoline-2,4-dia-
mine (7). 1d (0.080 g, 0.31 mmol) was reacted with cyclopropylamine
and purified according to method Db to furnish 0.080 g (0.29 mmol)
Purification Method Db. Solvent was evaporated, and the crude
mixture was purified by flash chromatography using dichloromethane
and methanol.
1
Previously Reported Quinazolines. The following quinazolines
of the title compound as a yellow solid in 92% yield. H NMR (500
have been previously reported: 3, 4, 18, 35−46.12
MHz, CD3OD) δ 7.93 (d, J = 8.2 Hz, 1H), 7.46 (t, J = 6.8 Hz, 2H),
7.37 (dd, J = 1.7, 0.7 Hz, 1H), 7.11 (dd, J = 10.9, 5.4 Hz, 1H), 6.40−
6.32 (m, 1H), 6.29 (dd, J = 3.2, 1.9 Hz, 1H), 4.75 (s, 2H), 2.81 (ddd, J
= 10.7, 7.1, 3.7 Hz, 1H), 0.93−0.80 (m, 2H), 0.67−0.59 (m, 2H). 13C
NMR (126 MHz, CD3OD) δ 159.84, 156.50, 151.26, 142.72, 141.96,
141.93, 133.87, 123.37, 122.98, 119.04, 110.23, 107.79, 37.49, 23.21,
6.63. HRMS: m/z calcd for C16H17N4O [M + H]+ 281.13969; found
281.13955. Rf = 0.36 (9:1 dichloromethane to methanol). Melting
point decomposition at 170 °C.
2,4-Dichloroquinazoline (1c). Commercially available benzoyle-
neurea (0.12 mol) and N,N-dimethylaniline (0.12 mol) were mixed in
60 mL of phosphorus oxychloride and heated to reflux under an
atmosphere of argon. After 5 h, the solution was cooled and slowly
added to 300 mL of ice. Once quenching was finished, the compound
was extracted with chloroform (4 × 125 mL) and purified by flash
chromatography using hexanes and ethyl acetate to yield the title
1
compound in 61% yield (14.5 g, 73 mmol). H NMR (500 MHz,
N2-Cyclobutyl-N4-(furan-2-ylmethyl)quinazoline-2,4-dia-
mine (8). 1d (0.080 g, 0.31 mmol) was reacted with cyclopropylamine
and purified according to method Db to furnish 0.080 g (0.29 mmol)
CDCl3) δ 8.29−8.24 (m, 1H), 8.02−7.99 (m, 2H), 7.74 (ddd, J = 6.3,
5.0, 3.2, 1H). 13C NMR (126 MHz, CDCl3) δ 164.14, 155.25, 152.49,
136.30, 129.49, 128.15, 126.31, 126.09, 122.48. MS (ESI): [M + H]+
199, 201; found 198.9 (100%), 200.9 (64%). Rf = 0.56 (hexanes to
ethyl acetate 4:1).
2-Chloro-N-(furan-2-ylmethyl)quinazolin-4-amine (1d). 1c (1
g,5.0 mmol) was reacted with furfurylamine and purified according to
method Ca to furnish 1.21 g (4.7 mmol) of the title compound in 93%
yield. 1H NMR (250 MHz, CDCl3) δ 7.78−7.67 (m, 3H), 7.41 (ddd, J
= 8.1, 5.4, 2.9 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 6.35 (d, J = 2.8 Hz,
1
of the title compound as a white solid in 92% yield. H NMR (500
MHz, CD3OD) δ 7.89 (d, J = 7.6 Hz, 1H), 7.51−7.48 (m, 1H), 7.43−
7.41 (m, 1H), 7.25 (s, 1H), 7.16−7.11 (m, 1H), 6.37−6.30 (m, 2H),
4.75 (s, 2H), 4.54−4.43 (m, 1H), 2.35 (s, 2H), 2.12−2.06 (m, 2H),
1.81−1.74 (m, 2H). HRMS: m/z calcd for C17H19N4O [M + H]+
295.1553; found 295.1545. Rf = 0.36 (9:1 dichloromethane to
methanol). Melting point decomposition at 170 °C.
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N2-Cyclopentyl-N4-(furan-2-ylmethyl)quinazoline-2,4-dia-
mine (9). 1d (0.12 g, 0.46 mmol) was reacted with cyclopentylamine
and purified according to method Db to furnish 0.083 g (0.27 mmol)
3H), 3.03 (s, 3H). 13C NMR (126 MHz, CD3OD) δ 160.32, 153.64,
139.04, 134.57, 124.19, 123.21, 116.43, 109.50, 27.43, 26.98. HRMS:
m/z calcd for C10H13N4 [M + H]+ 189.11402; found 189.11389. Rf =
0.21 (9:1 dichloromethane to methanol). Melting point decom-
position at 280 °C.
1
of the title compound as a brown solid in 59% yield. H NMR (500
MHz, CD3OD) δ 7.85 (d, J = 8.1 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H),
7.39 (s, 1H), 7.29 (s, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.31 (s, 1H), 6.27
(d, J = 3.1 Hz, 1H), 4.73 (d, J = 14.9 Hz, 2H), 4.38−4.25 (m, 1H),
1.99 (dt, J = 12.1, 5.9 Hz, 2H), 1.76−1.66 (m, 2H), 1.64−1.54 (m,
2H), 1.54−1.45 (m, 2H). 13C NMR (126 MHz, CD3OD) δ 160.46,
153.32, 150.50, 142.26, 139.06, 135.13, 124.56, 123.47, 116.45, 110.16,
110.12, 107.84, 59.81, 43.38, 37.93. HRMS: m/z calcd for C18H21N4O
[M + H]+ 309.17099; found 309.17103. Rf = 0.45 (9:1 dichloro-
methane to methanol). Melting point 113−117 °C.
N2-Isopropyl-N4-methylquinazoline-2,4-diamine (14). 11d
(0.080 g, 0.41 mmol) was reacted with isopropylamine and purified
according to method Db to furnish 0.053 g (0.25 mmol) of the title
compound as a white solid in 60% yield. 1H NMR (500 MHz,
CD3OD) δ 8.00 (dd, J = 8.2, 1.0 Hz, 1H), 7.71 (ddd, J = 8.4, 7.2, 1.3
Hz, 1H), 7.44 (s, 1H), 7.34 (ddd, J = 8.3, 7.3, 1.1 Hz, 1H), 4.33 (s,
1H), 3.16 (s, 3H), 1.33 (d, J = 8.2, 6H). 13C NMR (126 MHz,
CD3OD) δ 160.68, 152.91, 139.59, 134.51, 124.09, 123.18, 116.96,
109.93, 43.52, 27.43, 21.25. HRMS: m/z calcd for C12H17N4 [M + H]+
217.14477; found 217.14485. Rf = 0.33 (9:1 dichloromethane to
methanol). Melting point 259−262 °C.
N2-Cyclohexyl-N4-(furan-2-ylmethyl)quinazoline-2,4-dia-
mine (10). 1d (0.10 g, 0.385 mmol) was reacted with cyclohexylamine
and purified according to method Db to furnish 0.062 g (0.19 mmol)
1
of the title compound as a brown solid in 50% yield. H NMR (400
2-(4-(Methylamino)quinazolin-2-ylamino)ethanol (15). 11d
(0.080 g, 0.41 mmol) of was reacted with ethanol amine and purified
according to method Da to furnish 0.062 g (0.28 mmol) of the title
compound as a cream solid in 69% yield. 1H NMR (500 MHz,
CD3OD) δ 7.92 (d, J = 8.1 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.36 (d, J
= 7.6 Hz, 1H), 7.27 (t, J = 7.7 Hz, 1H), 3.79 (t, J = 5.5 Hz, 2H), 3.67−
3.62 (m, 2H), 3.11 (s, 3H). 13C NMR (126 MHz, CD3OD) δ 160.69,
154.93, 141.31, 134.16, 123.59, 122.95, 118.05, 110.15, 60.30, 43.40,
27.34. HRMS: m/z calcd for C11H15N4O [M + H]+ 219.12459; found
219.12427. Rf = 0.12 (9:1 dichloromethane to methanol). Melting
point 199−204 °C.
MHz, CDCl3) δ 7.81 (s, 1H), 7.44 (t, J = 7.7 Hz, 1H), 7.35 (d, J = 8.1
Hz, 1H), 7.30 (s, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.28 (s, 1H), 6.26 (d, J
= 3.1 Hz, 1H), 4.77 (s, 2H), 3.93 (s, 1H), 2.00 (d, J = 9.6 Hz, 2H),
1.73 (dd, J = 9.3, 3.9 Hz, 2H), 1.60 (dd, J = 8.8, 4.0 Hz, 1H), 1.37 (dd,
J = 24.1, 12.0 Hz, 2H), 1.29−1.16 (m, 3H). HRMS: m/z calcd for
C19H23N4O [M + H]+ 323.1866; found 323.1877. Rf = 0.54 (9:1
dichloromethane to methanol). Melting point decomposition at 165
°C.
N-Methyl-2-chloroquinazolin-4-amine (11d). 1c (1.0 g, 5.0
mmol) was reacted with methylamine and purified according to
method Ca to furnish 0.89 g (4.6 mmol) of the title compound in 92%
yield. 1H NMR (400 MHz, CDCl3) δ 7.74−7.66 (m, 3H), 7.41 (t, J =
6.9 Hz, 1H), 6.28 (s, NH), 3.20 (d, J = 4.7 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 161.82, 158.02, 150.80, 133.64, 127.90, 126.41,
121.02, 113.60, 28.74. MS (ESI): [M + H]+ 194; found 194. Rf = 0.12
(hexanes to ethyl acetate 4:1).
N2-Cyclopropyl-N4-methylquinazoline-2,4-diamine (16). 11d
(0.080 g, 0.41 mmol) was reacted with cyclopropylamine and purified
according to method Db to furnish 0.071 g (0.33 mmol) of the title
compound as a yellow solid in 81% yield. 1H NMR (500 MHz,
CD3OD) δ 7.93 (d, J = 8.0 Hz, 1H), 7.65−7.60 (m, 1H), 7.57 (d, J =
17.6 Hz, 1H), 7.28−7.23 (m, 1H), 3.07 (s, 3H), 2.82 (ddd, J = 10.5,
7.0, 3.6 Hz, 1H), 0.93 (q, J = 6.5 Hz, 2H), 0.68 (td, J = 7.1, 5.0 Hz,
2H). 13C NMR (126 MHz, CD3OD) δ 161.55, 157.10, 141.94, 135.34,
125.25, 124.29, 119.41, 111.66, 28.69, 24.17, 7.81. HRMS: m/z calcd
for C12H15N4 [M + H]+ 215.1291; found 215.1288. Rf = 0.19 (9:1
dichloromethane to methanol). Melting point 204−208 °C.
N2-(Furan-2-ylmethyl)-N4-methylquinazoline-2,4-diamine
(11). 11d (0.080 g, 0.41 mmol) was reacted with furfurylamine and
purified according to method Db to furnish 0.065 g (0.25 mmol) of
1
the title compound as a brown solid in 62% yield. H NMR (500
MHz, CD3OD) δ 7.87 (d, J = 7.6 Hz, 1H), 7.55 (ddd, J = 8.3, 7.4, 1.2
Hz, 1H), 7.47−7.40 (m, 1H), 7.29 (d, J = 7.2 Hz, 1H), 7.18 (t, J = 8.1
Hz, 1H), 6.38−6.33 (m, 2H), 4.65 (s, 2H), 3.10 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ 160.66, 153.79, 150.74, 142.25, 139.03, 134.47,
124.59, 124.25, 117.00, 110.40, 110.25, 107.74, 38.01, 28.65. HRMS:
m/z calcd for C14H15N4O [M + H]+ 255.1240; found 255.1237. Rf =
0.37 (9:1 dichloromethane to methanol). Melting point 145−155 °C.
2-Chloro-N-isopropylquinazolin-4-amine (12d). 1c (1 g, 5.0
mmol) was reacted with isopropylamine and purified according to
method Ca to furnish 0.960 g (4.33 mmol) of the title compound in
N2-Cyclopentyl-N4-methylquinazoline-2,4-diamine (17). 11d
(0.080 g, 0.41 mmol) was reacted with cyclopentylamine and purified
according to method Da to furnish 0.061 g (0.25 mmol) of the title
compound as a white solid in 61% yield. 1H NMR (400 MHz, DMSO-
d6) δ 9.71 (s, 1H), 8.91 (s, 1H), 8.21 (d, J = 29.1 Hz, 2H), 7.63 (s,
1H), 7.23 (d, J = 6.6 Hz, 1H), 4.32 (s, 1H), 3.00 (s, 3H), 1.94 (d, J =
5.6 Hz, 2H), 1.62 (d, J = 25.9 Hz, 2H), 1.51 (s, 4H). 13C NMR (101
MHz, CD3OD) δ 161.21, 159.27, 150.19, 132.56, 123.03, 122.08,
121.02, 111.21, 52.75, 33.07, 26.94, 23.52. HRMS: m/z calcd for
C14H19N4 [M + H]+ 243.16097; found 243.16118. Rf = 0.41 (9:1
dichloromethane to methanol). Melting point 223−228 °C.
N-Ethyl-2-chloroquinazolin-4-amine (19d). 1c (0.20 g, 1.0
mmol) was reacted with ethylamine and purified according to method
Cb to furnish 0.12 g (0.58 mmol) of the title compound in 58% yield.
1H NMR (400 MHz, CD3OD) δ 8.03 (d, J = 8.2 Hz, 1H), 7.75−7.69
(m, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 3.62 (q, J =
7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CD3OD)
δ 161.48, 157.86, 150.07, 133.47, 126.13, 125.81, 122.43, 113.65,
36.10, 13.15. MS (ESI): [M + H]+ 208; found 208.1. Rf = 0.24
(hexanes to ethyl acetate 4:1).
1
87% yield. H NMR (400 MHz, CDCl3) δ 7.76−7.63 (m, 3H), 7.40
(tt, J = 12.8, 6.4 Hz, 1H), 5.75 (d, J = 5.8 Hz, NH), 4.65−4.45 (m,
1H), 1.32 (d, J = 6.5 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.32,
158.08, 151.05, 133.53, 128.04, 126.20, 120.89, 113.41, 43.52, 22.74.
MS (ESI): [M + H]+ 222; found 222. Rf = 0.27 (hexanes to ethyl
acetate 4:1).
N2-(Furan-2-ylmethyl)-N4-isopropylquinazoline-2,4-diamine
(12). 12d (0.080 g, 0.36 mmol) was reacted with furfurylamine and
purified according to method Db to furnish 0.046 g (0.16 mmol) of
1
the title compound as a brown solid in 45% yield. H NMR (500
MHz, CD3OD) δ 8.06 (d, J = 8.2 Hz, 1H), 7.62−7.54 (m, 1H), 7.41
(d, J = 1.1 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.23−7.16 (m, 1H), 6.33
(dd, J = 3.2, 1.9 Hz, 1H), 6.31−6.27 (m, 1H), 4.66 (s, 2H), 4.63−4.53
(m, 1H), 1.33 (d, J = 6.6 Hz, 6H). 13C NMR (126 MHz, CD3OD) δ
159.49, 155.15, 151.93, 141.98, 141.94, 133.90, 123.18, 123.09, 118.72,
110.25, 110.08, 106.81, 43.52, 37.71, 20.74. HRMS: m/z calcd for
C16H19N4O [M + H]+ 283.15534; found 283.15565. Rf = 0.38 (9:1
dichloromethane to methanol). Melting point 168−178 °C.
N4-Ethyl-N2-isopropylquinazoline-2,4-diamine (19). 19d
(0.10 g 0.50 mmol) was reacted with isopropylamine and purified
according to method Db to furnish 0.045 g (0.020 mmol) of the title
compound as a yellow solid in 40% yield. 1H NMR (500 MHz,
CD3OD) δ 8.05 (d, J = 8.1 Hz, 1H), 7.66 (dd, J = 11.4, 4.1 Hz, 1H),
7.36 (d, J = 36.9 Hz, 1H), 7.31 (dd, J = 14.0, 6.6 Hz, 1H), 4.29 (s, 1H),
3.68 (q, J = 7.2 Hz, 2H), 1.35−1.27 (m, 9H). 13C NMR (126 MHz,
CD3OD) δ 159.93, 152.66, 139.32, 134.56, 124.10, 123.38, 116.73,
109.78, 43.58, 36.43, 21.36, 12.97. HRMS: m/z calcd for C13H19N4 [M
+ H]+ 231.16042; found 231.16093. Rf = 0.47 (9:1 dichloromethane to
methanol). Melting point 205−208 °C.
N2,N4-Dimethylquinazoline-2,4-diamine (13). 11d (0.080 g,
0.41 mmol) was reacted with methylamine and purified according to
method Da to furnish 0.078 g (0.41 mmol) of the title compound as a
white solid in quantitative yield. 1H NMR (500 MHz, CD3OD) δ 7.92
(d, J = 8.1 Hz, 1H), 7.67−7.63 (m, 1H), 7.31−7.25 (m, 2H), 3.09 (s,
L
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N-2,2,2-Trifluoroethyl-2-chloroquinazolin-4-amine (20d). 1c
(0.050 g, 0.25 mmol) was reacted with 2,2,2-trifluoroethylamine and
purified according to method Cb to furnish 0.017 g (0.065 mmol) of
2.84 (m, 1H), 1.29 (d, J = 6.5 Hz, 6H), 0.83 (d, J = 5.4 Hz, 2H), 0.64
(s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 158.47, 155.49, 141.02,
134.19, 124.17, 123.44, 118.51, 110.28, 43.03, 23.45, 21.61, 6.93.
HRMS: m/z calcd for C14H19N4 [M + H]+ 243.1604; found 243.1606.
Rf = 0.34 (9:1 dichloromethane to methanol). Melting point 208−212
°C.
1
the title compound in 26% yield. H NMR (400 MHz, CD3OD) δ
8.16 (d, J = 7.2 Hz, 1H), 7.82 (t, J = 7.4 Hz, 1H), 7.68 (d, J = 7.5 Hz,
1H), 7.58 (t, J = 7.7 Hz, 1H), 4.39 (q, J = 8.9 Hz, 2H). MS (ESI): [M
+ H]+ 235; found 235.0. Rf = 0.11 (hexanes to ethyl acetate 4:1).
N2-Isopropyl-N4-(2,2,2-trifluoroethyl)quinazoline-2,4-dia-
mine (20). 20d (0.017 g, 0.065 mmol) was reacted with isopropyl-
amine and purified according to method Db to furnish 0.005 g (0.018
mmol) of the title compound as a white crystalline solid in 28% yield.
1H NMR (500 MHz, CD3OD) δ 7.91 (dd, J = 8.2, 1.1, 1H), 7.60 (ddd,
J = 8.4, 7.0, 1.4, 1H), 7.38 (d, J = 8.4, 1H), 7.17 (ddd, J = 8.1, 7.0, 1.0,
1H), 4.38 (q, J = 9.3, 2H), 4.24 (hept, J = 6.5, 1H), 1.26 (d, J = 6.5,
6H). 13C NMR (126 MHz, CD3OD) δ 160.87, 157.71, 149.57, 133.28,
124.83 (q, J = 278.55), 122.38, 122.15, 121.53, 110.32, 42.57, 40.98 (q,
J = 34.34), 21.71. HRMS: m/z calcd for C13H16F3N4 [M + H]+
285.13216; found 285.13253. Rf = 0.26 (9:1 dichloromethane to
methanol). Melting point 103−108 °C.
N2-Cyclopentyl-N4-isopropylquinazoline-2,4-diamine (26).
12d (0.080 g, 0.36 mmol) was reacted with cyclopentylamine and
purified according to method Da to furnish 0.080 g (0.30 mmol) of the
title compound as a white crystalline solid in 82% yield. 1H NMR (500
MHz, CD3OD) δ 8.11−8.05 (m, 1H), 7.49 (ddd, J = 7.1, 6.4, 2.3 Hz,
1H), 7.31 (s, 1H), 7.18−7.10 (m, 1H), 4.57−4.43 (m, 1H), 4.35−4.22
(m, 1H), 2.09−1.94 (m, 2H), 1.81−1.66 (m, 2H), 1.64−1.49 (m, 4H),
1.34 (d, J = 6.6 Hz, 6H). 13C NMR (126 MHz, CD3OD) δ 159.16,
154.21, 141.65, 133.92, 123.40, 123.21, 117.97, 109.93, 53.06, 43.69,
32.65, 23.49, 20.94. HRMS: m/z calcd for C16H23N4 [M + H]+
271.1917; found 271.1922. Rf = 0.49 (9:1 dichloromethane to
methanol). Melting point 185−192 °C.
N-tert-Butyl-2-chloroquinazolin-4-amine (27d). 1c (0.17 g,
0.86 mmol) was reacted with tert-butylamine and purified according to
method Cb to furnish 0.064 g (0.27 mmol) of the title compound in
31% yield. 1H NMR (400 MHz, CD3OD) δ 8.16 (d, J = 8.3 Hz, 1H),
7.72 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.46 (t, J = 7.6 Hz,
1H), 1.57 (s, 9H). MS (ESI): [M + H]+ 236; found 236.1. Rf = 0.61
(hexanes to ethyl acetate 2:1).
N4-Isopropyl-N2-methylquinazoline-2,4-diamine (21). 12d
(0.080 g, 0.36 mmol) was reacted with methylamine and purified
according to method Db to furnish 0.075 g (0.35 mmol) of the title
compound as a white solid in 96% yield. 1H NMR (500 MHz,
CD3OD) δ 8.18 (d, J = 8.2 Hz, 1H), 7.70 (ddd, J = 8.4, 7.3, 1.3 Hz,
1H), 7.53−7.24 (m, 2H), 4.68−4.59 (m, 1H), 3.07 (s, 3H), 1.37 (d, J
= 6.6 Hz, 6H). 13C NMR (126 MHz, CD3OD) δ 159.24, 153.79,
139.37, 134.59, 124.18, 123.53, 116.58, 109.88, 43.89, 26.96, 20.52.
HRMS: m/z calcd for C12H17N4 [M + H]+ 217.14477; found
217.14536. Rf = 0.33 (9:1 dichloromethane to methanol). Melting
point 267−270 °C.
N4-tert-Butyl-N2-isopropylquinazoline-2,4-diamine (27). 27d
(0.051 g, 0.22 mmol) was reacted with isopropylamine and purified
according to method Db to furnish 0.047 g (0.018 mmol) of the title
1
compound as a white powder in 83% yield. H NMR (400 MHz,
CD3OD) δ 8.16 (d, J = 8.2 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.40 (d, J
= 6.8 Hz, 1H), 7.31 (t, J = 7.7 Hz, 1H), 4.38−4.21 (m, 1H), 1.60 (s,
9H), 1.31 (d, J = 6.5 Hz, 6H). 13C NMR (101 MHz, CD3OD) δ
160.26, 152.63, 140.27, 134.60, 124.06, 123.83, 117.38, 110.57, 54.04,
43.73, 27.79, 21.70. HRMS: m/z calcd for C15H23N4 [M + H]+
259.19172; found 259.19157. Rf = 0.47 (9:1 dichloromethane to
methanol). Melting point 189−192 °C.
N-Butyl-2-chloroquinazolin-4-amine (28d). 1c (0.30 g, 1.5
mmol) was reacted with n-butylamine and purified according to
method Ca to furnish 0.275 g (1.16 mmol) of the title compound in
77% yield. 1H NMR (250 MHz, CDCl3) δ 7.77−7.53 (m, 3H), 7.44−
7.31 (m, 1H), 5.82 (s, NH), 3.61 (td, J = 7.1, 5.6, 2H), 1.64 (dt, J =
14.7, 7.2, 2H), 1.40 (td, J = 14.5, 7.2, 2H), 0.92 (t, J = 7.3, 3H). 13C
NMR (101 MHz, CDCl3) δ 161.14, 150.99, 133.58, 128.10, 126.29,
121.19, 120.79, 113.42, 41.57, 31.47, 20.35, 14.02. MS (ESI): [M +
H]+ 236, 238; found 236.0 (100%), 238.0 (34%). Rf = 0.21 (hexanes
to ethyl acetate 4:1).
N2-Ethyl-N4-isopropylquinazoline-2,4-diamine (22). 12d
(0.080 g, 0.36 mmol) was reacted with ethylamine and purified
according to method Db to furnish 0.039 g (0.17 mmol) of the title
compound as a yellow solid in 47% yield. 1H NMR (500 MHz,
CD3OD) δ 8.14 (d, J = 7.7 Hz, 1H), 7.63 (ddd, J = 8.3, 7.3, 1.2 Hz,
1H), 7.39 (s, 1H), 7.31−7.25 (m, 1H), 4.58 (dt, J = 13.2, 6.6 Hz, 1H),
3.53 (q, J = 7.2 Hz, 2H), 1.36 (d, J = 6.6 Hz, 6H), 1.28 (t, J = 7.2 Hz,
3H). 13C NMR (126 MHz, CD3OD) δ 159.29, 153.78, 140.32, 134.33,
123.78, 123.46, 117.19, 109.91, 43.79, 36.00, 20.67, 13.67. HRMS: m/z
calcd for C13H19N4 [M + H]+ 231.16042; found 231.16067. Rf = 0.44
(9:1 dichloromethane to methanol). Melting point 209−216 °C.
N2,N4-Diisopropylquinazoline-2,4-diamine (23). 12d (0.080 g,
0.36 mmol) was reacted with isopropylamine and purified according to
method Db to furnish 0.041 g (0.17 mmol) of the title compound as a
white solid in 47% yield. 1H NMR (500 MHz, CD3OD) δ 8.18 (dd, J
= 8.2, 0.9 Hz, 1H), 7.71 (ddd, J = 8.4, 7.2, 1.3 Hz, 1H), 7.45 (s, 1H),
7.35 (ddd, J = 8.3, 7.2, 1.1 Hz, 1H), 4.63 (hept, J = 6.6 Hz, 1H), 4.30
(s, 1H), 1.37 (d, J = 6.6 Hz, 6H), 1.32 (d, J = 6.5 Hz, 6H). 13C NMR
(126 MHz, CD3OD) δ 159.39, 152.89, 139.82, 134.54, 124.02, 123.47,
116.91, 109.90, 43.86, 43.52, 21.32, 20.59. HRMS: m/z calcd for
C14H21N4 [M + H]+ 245.17607; found 245.17613. Rf = 0.38 (9:1
dichloromethane to methanol). Melting point 168−170 °C.
N2,N4-Dibutylquinazoline-2,4-diamine (28). 3d (0.080 g, 0.34
mmol) was reacted with n-butylamine and purified according to
method Da to furnish 0.031 g (0.114 mmol) of the title compound as
a white solid in 34% yield. 1H NMR (500 MHz, DMSO-d6) δ 9.78 (s,
NH), 8.36 (d, J = 8.2 Hz, 1H), 8.14 (s, NH), 7.76 (t, J = 7.7 Hz, 1H),
7.42 (d, J = 7.9 Hz, 1H), 7.37 (t, J = 7.3 Hz, 1H), 3.58 (d, J = 5.6 Hz,
2H), 3.45 (d, J = 5.9 Hz, 2H), 1.72−1.61 (m, 2H), 1.61−1.50 (m,
2H), 1.36 (dd, J = 14.4, 7.1 Hz, 4H), 0.92 (td, J = 7.3, 4.7 Hz, 6H). 13C
NMR (101 MHz, DMSO-d6) δ 160.10, 153.50, 139.26, 135.43, 124.83,
124.36, 116.97, 110.02, 41.37, 40.72, 31.49, 30.69, 20.16, 19.93, 14.12,
14.07. HRMS: m/z calcd for C16H25N4 [M + H]+ 273.20737; found
207.20720. Rf = 0.22 (9:1 dichloromethane to methanol). Melting
point 151−154 °C.
2-(4-(Isopropylamino)quinazolin-2-ylamino)ethanol (24).
12d (0.080 g, 0.36 mmol) was reacted with 2-aminoethanol and
purified according to method Db to furnish 0.082 g (0.33 mmol) of
the title compound as a white solid in 92% yield. 1H NMR (500 MHz,
CD3OD) δ 8.08 (d, J = 8.1 Hz, 1H), 7.64−7.59 (m, 1H), 7.35 (d, J =
8.0 Hz, 1H), 7.24 (dd, J = 11.4, 4.0 Hz, 1H), 4.57 (dt, J = 13.2, 6.6 Hz,
1H), 3.78 (t, J = 5.6 Hz, 2H), 3.63 (t, J = 5.5 Hz, 2H), 1.34 (d, J = 6.6
Hz, 6H). 13C NMR (126 MHz, CD3OD) δ 159.41, 155.11, 141.87,
134.04, 123.34, 123.20, 118.19, 110.17, 60.40, 43.51, 43.42, 20.73.
HRMS: m/z calcd for C13H19N4O [M + H]+ 247.15534; found
247.15531. Rf = 0.23 (9:1 dichloromethane to methanol). Melting
point 185−188 °C.
2-Chloro-N-(2-(methylthio)ethyl)quinazolin-4-amine (29d).
1c (0.20 g, 1.0 mmol) was reacted with 2-(methylthio)ethylamine
and purified according to method Cb to furnish 0.15 g (0.59 mmol) of
1
the title compound in 59% yield. H NMR (250 MHz, CD3OD) δ
7.93 (dd, J = 8.3, 0.8 Hz, 1H), 7.65 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H),
7.50−7.44 (m, 1H), 7.38 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 3.70 (dd, J =
7.5, 6.6 Hz, 2H), 2.75−2.65 (m, 2H), 2.08 (s, 3H). MS (ESI): [M +
H]+ 254; found 254.0. Rf = 0.17 (hexanes to ethyl acetate 4:1).
N2-Isopropyl-N4-(2-(methylthio)ethyl)quinazoline-2,4-dia-
mine (29). 29d (0.12 g, 0.47 mmol) was reacted with isopropylamine
and purified according to method Db to furnish 0.056 g (0.20 mmol)
N2-Cyclopropyl-N4-isopropylquinazoline-2,4-diamine (25).
12d (0.080 g, 0.36 mmol) was reacted with cyclopropylamine and
purified according to method Db to furnish 0.035 g (0.14 mmol) of
the title compound as a cream solid in 40% yield. 1H NMR (400 MHz,
DMSO-d6) δ 9.00 (s, 1H), 8.43 (d, J = 8.2 Hz, 1H), 7.72 (t, J = 7.5 Hz,
1H), 7.33 (t, J = 7.9 Hz, 1H), 4.55 (dq, J = 13.2, 6.5 Hz, 1H), 2.92−
M
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1
of the title compound as a white solid in 43% yield. H NMR (500
MHz, CD3OD) δ 7.81 (d, J = 8.1 Hz, 1H), 7.47 (dd, J = 8.2, 7.2 Hz,
1H), 7.28 (d, J = 8.2 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 4.20 (dt, J =
13.0, 6.5 Hz, 1H), 3.76−3.71 (m, 2H), 2.79−2.74 (m, 2H), 2.11 (s,
3H), 1.22 (d, J = 6.5 Hz, 6H). 13C NMR (126 MHz, CD3OD) δ
160.39, 157.49, 148.19, 132.93, 122.28, 121.64, 121.48, 110.56, 42.62,
40.15, 32.36, 22.03, 14.11. HRMS: m/z calcd for C14H21N4S [M + H]+
277.14814; found 277.14880. Rf = 0.52 (9:1 dichloromethane to
methanol). Melting point 87−90 °C.
(dichloromethane to methanol 9:1). Melting point decomposition at
345 °C.
2-Chloro-N-((tetrahydrofuran-2-yl)methyl)quinazolin-4-
amine (33d). 1c (0.12 g, 0.60 mmol) was reacted with
tetrahydrofurfurylamine and purified according to method Cb to
1
furnish 0.13 g (0.49 mmol) of the title compound in 82% yield. H
NMR (400 MHz, CD3OD) δ 7.98 (d, J = 7.8 Hz, 1H), 7.70−7.65 (m,
1H), 7.50 (d, J = 8.2 Hz, 1H), 7.43−7.37 (m, 1H), 4.22 (qd, J = 6.9,
4.7 Hz, 1H), 3.88 (dd, J = 14.7, 6.9 Hz, 1H), 3.79−3.65 (m, 2H), 3.58
(dd, J = 13.7, 7.6 Hz, 1H), 2.09−1.98 (m, 1H), 1.98−1.81 (m, 2H),
1.70−1.59 (m, 1H). 13C NMR (101 MHz, CD3OD) δ 161.74, 157.50,
149.97, 133.60, 126.21, 125.72, 122.43, 113.46, 77.23, 67.83, 45.17,
28.86, 25.35. MS (ESI): [M + H]+ 264; found 264.1. Rf = 0.15
(hexanes to ethyl acetate 2:1).
2-Chloro-N-cyclopropylquinazolin-4-amine (30d). 1c (0.16 g,
0.78 mmol) was reacted with cyclopropylamine and purified according
to method Cb to furnish 0.14 g (0.62 mmol) of the title compound in
80% yield. 1H NMR (250 MHz, CD3OD) δ 7.92 (d, J = 7.9 Hz, 1H),
7.62 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.37−
7.28 (m, 1H), 2.95 (ddd, J = 11.1, 7.4, 3.9 Hz, 1H), 0.82−0.74 (m,
2H), 0.62 (ddd, J = 7.8, 6.1, 4.7 Hz, 2H). 13C NMR (63 MHz,
CD3OD) δ 164.23, 158.86, 151.04, 134.80, 127.46, 126.98, 123.74,
114.77, 25.53, 7.05. MS (ESI): [M + H]+ 220; found 220.0. Rf = 0.12
(hexanes to ethyl acetate 2:1).
N2-Isopropyl-N4-((tetrahydrofuran-2-yl)methyl)quinazoline-
2,4-diamine (33). 33d (0.10 g, 0.38 mmol) was reacted with
isopropylamine and purified according to method Db to furnish 0.068
g (0.24 mmol) of the title compound as a clear amorphous solid in
63% yield. 1H NMR (400 MHz, CD3OD) δ 7.78 (d, J = 7.4 Hz, 1H),
7.46 (t, J = 7.1 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.01 (t, J = 7.1 Hz,
1H), 4.27−4.16 (m, 2H), 3.87 (dt, J = 13.5, 6.8 Hz, 1H), 3.77−3.55
(m, 3H), 2.04−1.78 (m, 3H), 1.66 (dq, J = 11.8, 6.8 Hz, 1H), 1.22 (d,
J = 6.5 Hz, 6H). 13C NMR (101 MHz, CD3OD) δ 160.84, 159.31,
151.40, 132.53, 123.52, 122.07, 120.75, 111.04, 77.69, 67.81, 44.66,
42.45, 28.85, 25.39, 22.29. HRMS: m/z calcd for C16H23N4O [M +
H]+ 287.18664; found 287.18737. Rf = 0.13 (2:1 hexanes to ethyl
acetate).
Methyl-2-(2-chloroquinazolin-4-ylamino)acetate (34d). 1c
(0.198 g, 0.99 mmol) was reacted with glycine methyl ester and
purified according to method Ca to furnish 0.18 g (0.72 mmol) of the
title compound in 73% yield. 1H NMR (400 MHz, CD3OD) δ 7.85 (d,
J = 8.1 Hz, 1H), 7.62 (dd, J = 11.3, 4.1 Hz, 1H), 7.45 (d, J = 8.3 Hz,
1H), 7.33 (t, J = 7.6 Hz, 1H), 4.26 (s, 2H), 3.71 (s, 3H). 13C NMR
(101 MHz, CD3OD) δ 170.77, 161.67, 157.12, 150.04, 133.76, 126.38,
125.93, 122.25, 113.24, 51.72, 42.35. MS (ESI): [M + H]+ 252; found
252.1. Rf = 0.17 (hexanes to ethyl acetate 2:1).
N4-Cyclopropyl-N2-isopropylquinazoline-2,4-diamine (30).
30d (0.10 g, 0.48 mmol) was reacted with isopropylamine and
purified according to method Db to furnish 0.094 g (0.39 mmol) of
1
the title compound as a yellow solid in 81% yield. H NMR (500
MHz, CD3OD) δ 7.80 (d, J = 8.1 Hz, 1H), 7.44 (dd, J = 11.2, 4.1 Hz,
1H), 7.30 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 4.24 (dt, J =
12.8, 6.4 Hz, 1H), 2.92 (dt, J = 10.8, 3.6 Hz, 1H), 1.25−1.21 (m, 6H),
0.82−0.77 (m, 2H), 0.67−0.63 (m, 2H). 13C NMR (126 MHz,
CD3OD) δ 161.84, 158.25, 149.46, 132.59, 122.51, 122.20, 121.04,
110.79, 42.46, 23.75, 22.04, 5.87. HRMS: m/z calcd for C14H19N4 [M
+ H]+ 243.16042; found 243.16004. Rf = 0.47 (9:1 dichloromethane to
methanol). Melting point 81−85 °C.
2-Chloro-N-cyclopentylquinazolin-4-amine (31d). 1c (0.20 g,
1.0 mmol) was reacted with cyclopentylamine and purified according
to method Cb to furnish 0.15 g (0.61 mmol) of the title compound in
61% yield. 13C NMR (101 MHz, DMSO-d6) δ 135.10, 133.42, 127.29,
126.35, 126.26, 126.04, 125.73, 122.64, 53.15, 31.91, 23.76. MS (ESI):
[M + H]+ 248; found 248.1. Rf = 0.44 (hexanes to ethyl acetate 4:1).
N4-Cyclopentyl-N2-isopropylquinazoline-2,4-diamine (31).
31d (0.12 g, 0.49 mmol) was reacted with isopropylamine and
purified according to method Db to furnish 0.047 g (0.17 mmol) of
the title compound as a cream crystalline solid in 35% yield. 1H NMR
(500 MHz, CD3OD) δ 8.16 (d, J = 7.9 Hz, 1H), 7.66−7.61 (m, 1H),
7.40 (s, 1H), 7.28 (dd, J = 11.6, 4.3 Hz, 1H), 4.61 (dd, J = 14.5, 7.1
Hz, 1H), 4.34−4.24 (m, 1H), 2.18−2.07 (m, 2H), 1.88−1.79 (m, 2H),
1.78−1.69 (m, 2H), 1.69−1.62 (m, 2H), 1.31 (d, J = 6.5 Hz, 6H). 13C
NMR (126 MHz, CD3OD) δ 159.89, 153.38, 140.82, 134.25, 123.62,
123.47, 117.46, 109.97, 53.59, 43.43, 31.82, 23.81, 21.45. HRMS: m/z
calcd for C16H22N4 [M + H]+ 271.19172; found 271.19178. Rf = 0.50
(9:1 dichloromethane to methanol). Melting point 145−150 °C.
2-Chloro-N-cyclohexylquinazolin-4-amine (32d). 1c (0.16 g,
0.80 mmol) was reacted with cyclohexylamine and purified according
to method Cb to furnish 0.15 g (0.55 mmol) of the title compound in
69% yield. 1H NMR (250 MHz, CD3OD) δ 8.05−7.99 (m, 1H), 7.60
(ddd, J = 8.4, 7.0, 1.4 Hz, 1H), 7.46−7.41 (m, 1H), 7.33 (ddd, J = 8.3,
7.0, 1.3 Hz, 1H), 4.17−4.01 (m, 1H), 1.97−1.84 (m, 2H), 1.70 (d, J =
2.6 Hz, 2H), 1.65−1.52 (m, 1H), 1.38−1.25 (m, 4H), 1.20−1.00 (m,
1H). 13C NMR (63 MHz, CD3OD) δ 161.94, 159.05, 151.35, 134.67,
127.25, 126.90, 123.83, 114.81, 51.68, 33.32, 26.71, 26.48. MS (ESI):
[M + H]+ 262; found 262.1. Rf = 0.43 (hexanes to ethyl acetate 2:1).
N4-Cyclohexyl-N2-isopropylquinazoline-2,4-diamine (32).
32d (0.10 g, 0.40 mmol) was reacted with isopropylamine and
purified according to method Db to furnish 0.060 g (0.21 mmol) of
Ethyl-2-(2-(isopropylamino)quinazolin-4-ylamino)acetate
(34). 34d (0.12 g, 0.47 mmol) was reacted with isopropylamine and
purified according to method Db to furnish 0.060 g (0.21 mmol) of
the title compound as an amorphous solid in 44% yield. 1H NMR (400
MHz, CD3OD) δ 7.84 (d, J = 8.2 Hz, 1H), 7.57−7.51 (t, J = 7.8 Hz,
1H), 7.32 (d, J = 8.2 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H), 4.26−4.14 (m,
5H), 1.25 (t, J = 7.1 Hz, 3H), 1.21 (d, J = 6.5 Hz, 6H). 13C NMR (101
MHz, CD3OD) δ 172.23, 162.13, 159.29, 150.86, 134.31, 123.71,
123.57, 122.61, 111.84, 62.22, 43.87, 43.66, 23.25, 14.52. HRMS: m/z
calcd for C15H21N4O2 [M + H]+ 289.1659; found 289.1668. Rf = 0.34
(dichloromethane to methanol 9:1).
N2-Benzyl-N4-isopropylquinazoline-2,4-diamine (47). 12d
(0.080 g, 0.36 mmol) was reacted with benzylamine and purified
according to method Db to furnish 0.019 g (0.065 mmol) of the title
1
compound as a yellow crystalline solid in 18% yield. H NMR (400
MHz, DMSO-d6) δ 8.12 (d, J = 6.7 Hz, NH), 7.49 (t, J = 8.2 Hz, 2H),
7.44−7.32 (m, 3H), 7.28 (dd, J = 12.3, 4.7 Hz, 3H), 7.19 (t, J = 7.2 Hz,
1H), 7.08 (s, 1H), 4.57 (s, 2H), 4.44 (d, J = 6.1 Hz, 1H), 1.24 (dd, J =
25.1, 6.5 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 159.75, 141.48,
134.84, 133.28, 129.56, 129.21, 129.04, 128.75, 127.85, 127.08, 123.92,
121.31, 44.69, 42.85, 22.63. HRMS: m/z calcd for C18H21N4 [M + H]+
293.17607; found 293.17641. Rf = 0.40 (9:1 dichloromethane to
methanol). Melting point 200−202 °C.
N-Benzyl-2-chloroquinazolin-4-amine (48d). 1c (0.30 g, 1.5
mmol) was reacted with benzylamine and purified according to
method Ca to furnish 0.345 g (1.28 mmol) of the title compound in
85% yield. 1H NMR (400 MHz, CDCl3) δ 7.70 (ddd, J = 11.3, 8.4, 4.9
Hz, 3H), 7.48−7.26 (m, 5H), 6.27 (s, 1H), 4.83 (d, J = 5.3 Hz, 2H).
13C NMR (101 MHz, CDCl3) δ 160.93, 157.93, 151.09, 137.48,
133.76, 129.11, 128.51, 128.21, 128.00, 126.46, 121.07, 113.38, 45.94.
MS (ESI): [M + H]+ 270; found 270.0. Rf = 0.21 (hexanes to ethyl
acetate 4:1).
1
the title compound as a white crystalline solid in 53% yield. H NMR
(400 MHz, CD3OD) δ 7.95 (d, J = 8.0 Hz, 1H), 7.55 (t, J = 7.5 Hz,
1H), 7.31 (d, J = 8.3 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 4.20 (sept, J =
6.5 Hz, 2H), 2.04 (d, J = 2.2 Hz, 2H), 1.83 (d, J = 4.5 Hz, 2H), 1.70
(d, J = 12.4 Hz, 1H), 1.41 (m, 5H), 1.25 (d, J = 6.5 Hz, 6H). 13C
NMR (101 MHz, CD3OD) δ 159.82, 157.34, 147.57, 133.16, 122.65,
121.83, 121.25, 110.84, 50.46, 42.93, 32.26, 25.37, 21.93. HRMS: m/z
calcd for C17H25N4 [M + H]+ 285.2074; found 285.2077. Rf = 0.38
N4-Benzyl-N2-isopropylquinazoline-2,4-diamine (48). 48d
(0.029 g, 0.11 mmol) was reacted with isopropylamine and purified
N
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according to method Db to furnish 0.016 g (0.055 mmol) of the title
compound as a white crystalline solid in 50% yield. H NMR (400
δ 10.46 (s, NH), 8.64 (s, NH), 8.45 (d, J = 8.0 Hz, 1H), 7.74 (dt, J =
7.8, 1.5 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.36 (dt, J = 7.8, 1.3 Hz,
1H), 7.36−7.12 (m, 10H), 4.75 (d, J = 5.5 Hz, 2H), 4.63 (d, J = 4.8
Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ 160.26, 153.49, 139.44,
138.85, 138.29, 135.67, 129.93, 128.85, 128.80, 128.24, 128.12, 127.72,
127.57, 124.93, 124.64, 117.19, 110.22, 110.12, 44.86, 44.35. HRMS:
m/z calcd for C22H21N4 [M + H]+ 341.1761; found 341.1751. Rf =
0.45 (dichloromethane to methanol 9:1). Melting point 237−240 °C.
N2-Benzyl-N4-phenylquinazoline-2,4-diamine (54). 49d (0.10
g, 0.39 mmol) was reacted with benzylamine and purified according to
method Da to furnish 0.010 g (0.031 mmol) of the title compound as
a white crystalline solid in 10% yield. 1H NMR (500 MHz, DMSO-d6)
δ 9.56 (s, 1H), 9.13 (s, 1H), 8.37 (d, J = 7.5 Hz, 1H), 7.92 (d, J = 7.5
Hz, 2H), 7.86 (d, J = 8.1 Hz, 2H), 7.66 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H),
7.48 (d, J = 8.4 Hz, 1H), 7.42−7.35 (m, 2H), 7.27 (ddd, J = 8.1, 7.0,
1.2 Hz, 1H), 7.25−7.20 (m, 2H), 7.13 (t, J = 7.4 Hz, 1H), 6.89 (t, J =
7.3 Hz, 1H), 1.89 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.01,
157.09, 152.33, 141.80, 140.10, 133.64, 129.12, 128.92, 126.26, 124.14,
123.77, 123.10, 122.52, 121.45, 119.58, 112.43, 21.76. HRMS: m/z
calcd for C21H19N4 [M + H]+ 327.16097; found 327.16140. Rf = 0.53
(9:1 dichloromethane to methanol). Melting point decomposition at
310 °C.
1
MHz, DMSO-d6) δ 9.55 (s, NH), 8.18 (s, NH), 7.59 (s, 1H), 7.34 (dt,
J = 22.9, 7.5 Hz, 6H), 7.23 (t, J = 7.2 Hz, 1H), 7.17 (s, 1H), 4.75 (d, J
= 5.6 Hz, 2H), 4.14 (dd, J = 13.1, 6.5 Hz, 1H), 1.13 (d, J = 6.3 Hz,
6H). 13C NMR (101 MHz, DMSO-d6) δ 160.45, 156.58, 145.74,
139.91, 134.13, 128.93, 128.00, 127.46, 123.99, 123.05, 122.11, 111.11,
44.42, 42.88, 23.37. HRMS: m/z calcd for C18H20N4O [M]+
308.16316; found 308.15874. Rf = 0.47 (9:1 dichloromethane to
methanol). Melting point 161−164 °C.
2-Chloro-N-phenylquinazolin-4-amine (49d). 1c (0.30 g, 1.5
mmol) was reacted with aniline and purified according to method Ca
to furnish 0.36 g (1.4 mmol) of the title compound as a white solid in
94% yield. 1H NMR (400 MHz, CDCl3) δ 7.90−7.73 (m, 2H), 7.70−
7.48 (m, 3H), 7.47−7.32 (m, 1H), 7.24−7.11 (m, 2H), 7.01 (dd, J =
15.4, 8.1 Hz, 1H). MS (ESI): [M + H]+ 256; found 256.0. Rf = 0.19
(hexanes to ethyl acetate 4:1).
N2-Isopropyl-N4-phenylquinazoline-2,4-diamine (49). 49d
(0.039 g, 0.15 mmol) was reacted with isopropylamine and purified
according to method Db to furnish 0.010 g (0.036 mmol) of the title
compound as a white solid in 24% yield. 1H NMR (400 MHz, DMSO-
d6) δ 9.49 (s, NH), 8.31 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 7.2 Hz, 2H),
7.57 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 7.7 Hz, 3H), 7.15 (t, J = 7.4 Hz,
1H), 7.07 (t, J = 7.2 Hz, 1H), 6.85 (s, NH), 4.11 (s, 1H), 1.15 (d, J =
6.5 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 158.10, 157.06,
151.00, 139.37, 132.99, 128.35, 127.63, 125.56, 123.28, 121.97, 120.90,
110.77, 42.03, 22.33. HRMS: m/z calcd for C17H19N4 [M + H]+
279.16042; found 279.16094. Rf = 0.50 (9:1 dichloromethane to
methanol). Melting point decomposition at 240 °C.
N2,N4-Diphenylquinazoline-2,4-diamine (55). 49d (0.10 g, 0.39
mmol) was reacted with aniline and purified according to method Da
to furnish 0.011 g (0.035 mmol) of the title compound in 9% yield. 1H
NMR (500 MHz, DMSO-d6) δ 11.27 (s, 1H), 10.63 (s, 1H), 8.75 (d, J
= 8.2 Hz, 1H), 7.95−7.85 (m, 1H), 7.68 (d, J = 7.7 Hz, 2H), 7.62−
7.56 (m, 1H), 7.56−7.49 (m, 1H), 7.49−7.38 (m, 4H), 7.34−7.26 (m,
3H), 7.16 (t, J = 6.4 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ
159.50, 151.56, 139.50, 136.94, 136.64, 135.74, 128.80, 128.59, 126.29,
124.95, 124.92, 124.86, 124.82, 122.09, 117.53, 110.49, 56.03, 18.57.
HRMS: m/z calcd for C20H17N4 [M + H]+ 313.14477; found
313.14503. Rf = 0.55 (9:1 dichloromethane to methanol). Melting
point decomposition at 305 °C.
N4-Benzyl-N2-butylquinazoline-2,4-diamine (50). 48d (0.10 g,
0.37 mmol) was reacted with n-butylamine and purified according to
method Da to furnish 0.047 g (0.15 mmol) of the title compound as a
white solid in 41% yield. 1H NMR (400 MHz, CDCl3) δ 9.54 (s, 1H),
8.43 (d, J = 8.1 Hz, 1H), 7.82 (s, 1H), 7.44−7.37 (m, 3H), 7.25−7.11
(m, 5H), 4.83 (d, J = 5.7 Hz, 2H), 3.39 (q, J = 6.8 Hz, 2H), 1.49 (p, J
= 7.3 Hz, 2H), 1.31 (sext, J = 7.3 Hz, 2H), 0.85 (t, J = 7.3 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 160.08, 153.46, 137.43, 134.65,
128.41, 127.83, 127.40, 124.41, 124.12, 116.62, 109.99, 109.68, 45.31,
41.02, 31.30, 19.95, 13.66. HRMS: m/z calcd for C19H23N4 [M + H]+
307.1917; found 307.1926. Rf = 0.33 (dichloromethane to methanol
9:1). Melting point 210−212 °C.
N2-Benzyl-N4-(furan-2-ylmethyl)quinazoline-2,4-diamine
(56). 1d (0.10 g, 0.39 mmol) was reacted with benzylamine and
purified according to method Da to furnish 0.080 g (0.24 mmol) of the
title compound as a white crystalline solid in 62% yield. 1H NMR (400
MHz, CD3OD) δ 7.86 (t, J = 10.0 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H),
7.36−7.08 (m, 8H), 6.26 (dd, J = 2.9, 1.9 Hz, 1H), 6.15 (s, 1H), 4.75−
4.55 (m, 4H). 13C NMR (101 MHz, CD3OD) δ 160.60, 151.87,
141.95, 139.64, 133.80, 133.64, 128.30, 127.41, 127.16, 126.85, 122.76,
122.44, 120.86, 110.71, 110.21, 107.28, 44.73, 37.61. HRMS: m/z
calcd for C20H19N4O [M + H]+ 331.15589; found 331.15673. Rf =
0.49 (9:1 dichloromethane to methanol). Melting point decom-
position at 150 °C.
N2-Butyl-N4-phenylquinazoline-2,4-diamine (51). 49d (0.10 g,
0.39 mmol) was reacted with n-butylamine and purified according to
method Da to furnish 0.011 g (0.038 mmol) of the title compound as
a white crystalline solid in 10% yield. 1H NMR (400 MHz, DMSO-d6)
δ 10.96 (s, 1H), 8.62 (d, J = 8.1 Hz, 1H), 8.25 (s, 1H), 7.85−7.70 (m,
3H), 7.51−7.37 (m, 4H), 7.25 (t, J = 6.7 Hz, 1H), 3.31−3.22 (m, 2H),
1.49 (dd, J = 24.5, 17.8 Hz, 2H), 1.39−1.14 (m, 2H), 0.92−0.70 (m,
3H). 13C NMR (126 MHz, DMSO-d6) δ 159.34, 153.33, 139.99,
137.54, 136.02, 128.96, 126.44, 125.16, 124.68, 123.20, 117.25, 110.23,
41.01, 31.40, 19.89, 14.01. HRMS: m/z calcd for C18H21N4 [M + H]+
293.17662; found 293.17630. Rf = 0.33 (9:1 dichloromethane to
methanol). Melting point 189−191 °C.
2-Chloro-N-(4-chlorobenzyl)quinazolin-4-amine (57d). 1c
(0.22 g, 1.1 mmol) was reacted with 4-chlorobenzylamine and purified
according to method Ca to furnish 0.27 g (0.89 mmol) of the title
1
compound in 81% yield. H NMR (400 MHz, CDCl3) δ 7.80−7.65
(m, 3H), 7.45 (t, J = 6.4 Hz, 1H), 7.32 (s, 4H), 6.31 (s, 1H), 4.83 (d, J
= 5.3 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 160.68, 157.57,
150.87, 135.80, 133.79, 133.63, 129.60, 128.96, 127.82, 126.32, 120.76,
113.08, 44.89. MS (ESI): [M + H]+ 290; found 290.0. Rf = 0.31
(hexanes to ethyl acetate 2:1).
N4-Butyl-N2-phenylquinazoline-2,4-diamine (52). 28d (0.080
g, 0.34 mmol) was reacted with aniline and purified according to
method Da to furnish 0.021 g (0.071 mmol) of the title compound as
a white crystalline solid in 21% yield. 1H NMR (400 MHz, DMSO-d6)
δ 10.51 (s, 1H), 9.97 (s, 1H), 8.41 (d, J = 8.1 Hz, 1H), 7.82 (t, J = 7.6
Hz, 1H), 7.62 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 8.2 Hz, 1H), 7.48−7.39
(m, 3H), 7.20 (t, J = 7.4 Hz, 1H), 3.56 (q, J = 6.8 Hz, 2H), 1.65 (p, J =
7.5 Hz, 2H), 1.36 (h, J = 7.4 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 159.95, 151.41, 138.60, 136.95, 135.24,
128.88, 124.74, 124.63, 124.31, 121.76, 117.29, 110.21, 41.43, 30.08,
19.72, 13.61. HRMS: m/z calcd for C18H21N4 [M + H]+ 293.17662;
found 293.17709. Rf = 0.49 (9:1 dichloromethane to methanol).
Melting point 234−236 °C.
N4-(4-Chlorobenzyl)-N2-(furan-2-ylmethyl)quinazoline-2,4-
diamine (57). 57d (0.032 g, 0.11 mmol) was reacted with
furfurylamine and purified according to method Db to furnish 0.015
1
g (0.0 mmol) of the title compound in 39% yield. H NMR (500
MHz, CD3OD) δ 8.06 (dd, J = 8.3, 0.9 Hz, 1H), 7.72 (t, J = 10.9 Hz,
2H), 7.55 (ddd, J = 8.4, 5.1, 1.3 Hz, 1H), 7.41−7.35 (m, 2H), 7.29−
7.25 (m, 2H), 7.15 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 6.30 (dd, J = 3.2, 1.8
Hz, 1H), 6.16 (s, 1H), 4.55 (s, 2H). 13C NMR (126 MHz, CD3OD) δ
160.05, 159.84, 154.60, 142.88, 142.86, 139.13, 134.41, 129.81, 129.39,
125.19, 124.83, 123.55, 123.06, 112.34, 111.32, 107.31, 39.44. HRMS:
m/z calcd for C19H15ClN4O [M + H]+ 351.1007; found 351.1000. Rf =
0.60 (9:1 dichloromethane to methanol). Melting point decom-
position at 180 °C.
N2,N4-Dibenzylquinazoline-2,4-diamine (53). 48d (0.10 g, 0.37
mmol) was reacted with benzylamine and purified according to
method Da to furnish 0.101 g (0.297 mmol) of the title compound as
a white crystalline solid in 80% yield. 1H NMR (500 MHz, DMSO-d6)
N2-(4-Chlorophenyl)-N4-(furan-2-ylmethyl)quinazoline-2,4-
diamine (58). 1d (0.10 g, 0.39 mmol) was reacted with benzylamine
O
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and purified according to method Da to furnish 0.089 g (0.25 mmol)
of the title compound as a white crystalline solid in 64% yield. H
2-Chloro-N-(4-methylbenzyl)quinazolin-4-amine (64d). 1c
(0.22 g, 1.1 mmol) was reacted with 4-methylbenzylamine and
purified according to method Ca to furnish 0.29 g (1.0 mmol) of the
1
NMR (400 MHz, CD3OD) δ 7.92−7.87 (m, 1H), 7.65 (d, J = 8.9 Hz,
2H), 7.57−7.52 (m, 1H), 7.41 (d, J = 8.6 Hz, 2H), 7.19 (t, J = 5.8 Hz,
2H), 7.18−7.12 (m, 1H), 6.33 (dd, J = 3.1, 1.9 Hz, 1H), 6.25 (d, J =
2.9 Hz, 1H), 4.76 (s, 2H). 13C NMR (101 MHz, CD3OD) δ 161.92,
158.22, 153.69, 151.81, 142.98, 140.74, 133.99, 129.32, 127.39, 125.75,
123.38, 122.08, 113.04, 111.37, 107.93, 99.72, 38.86. HRMS: m/z
calcd for C19H16ClN4O [M + H]+ 351.1007; found 351.1027. Rf = 0.52
(9:1 dichloromethane to methanol). Melting point 143−145 °C.
N4-(Furan-2-ylmethyl)-N2-(p-tolyl)quinazoline-2,4-diamine
(59). 1d (0.34 g, 1.3 mmol) was reacted with p-toluidine and purified
according to method Da to furnish 0.42 g (0.1.27 mmol) of the title
compound as a yellow solid in 98% yield. 1H NMR (400 MHz,
CD3OD) δ 8.15 (d, J = 8.1 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.54 (d, J
= 8.3 Hz, 1H), 7.48−7.38 (m, 4H), 7.26 (d, J = 7.9 Hz, 2H), 6.34 (d, J
= 17.1 Hz, 1H), 6.26 (s, 1H), 4.82 (s, 2H), 2.37 (s, 3H). 13C NMR
(101 MHz, CD3OD) δ 160.92, 152.41, 150.44, 142.49, 139.19, 136.57,
135.44, 133.39, 129.74, 125.24, 124.05, 123.62, 117.26, 110.53, 110.30,
108.06, 38.20, 19.84. HRMS: m/z calcd for C20H19N4O [M + H]+
331.1553; found 331.1544. Rf = 0.60 (9:1 dichloromethane to
methanol). Melting point decomposition at 225 °C.
1
title compound in 90% yield. H NMR (400 MHz, CDCl3) δ 7.76−
7.63 (m, 3H), 7.40 (t, J = 8.0 Hz, 1H), 7.26 (d, J = 7.9 Hz, 2H), 7.14
(d, J = 7.8 Hz, 2H), 6.23 (s, 1H), 4.78 (d, J = 5.2 Hz, 2H), 2.32 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 160.63, 157.70, 150.82, 137.79,
134.15, 133.46, 129.52, 128.30, 127.74, 126.16, 120.83, 113.16, 45.52,
21.11. MS (ESI): [M + H]+ 284; found 283.9. Rf = 0.39 (hexanes to
ethyl acetate 2:1).
N2-Benzyl-N4-(4-methylbenzyl)quinazoline-2,4-diamine (64).
64d (0.086 g, 0.30 mmol) was reacted with benzylamine and purified
according to method Da to furnish 0.058 g (0.16 mmol) of the title
1
compound as a white powder in 55% yield. H NMR (400 MHz,
DMSO-d6) δ 10.44 (s, 1H), 8.63 (s, 1H), 8.46 (d, J = 8.0 Hz, 1H),
7.76 (t, J = 7.7 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.37 (t, J = 7.7 Hz,
1H), 7.26 (s, 5H), 7.19 (d, J = 6.9 Hz, 2H), 7.03 (d, J = 6.8 Hz, 2H),
4.70 (d, J = 5.2 Hz, 2H), 4.66 (s, 2H), 2.24 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 160.13, 153.50, 139.40, 138.83, 136.60, 135.58,
135.21, 129.29, 128.79, 128.05, 127.67, 127.50, 124.88, 124.56, 117.17,
110.12, 44.61, 44.31, 21.11. HRMS: m/z calcd for C23H23N4 [M + H]+
355.1917; found 355.1907. Rf = 0.38 (9:1 dichloromethane to
methanol). Melting point 221−223 °C.
N4-Benzyl-N2-(4-chlorobenzyl)quinazoline-2,4-diamine (60).
48d (0.075 g, 0.28 mmol) was reacted with 4-chlorobenzylamine and
purified according to method Da to furnish 0.098 g (0.26 mmol) of the
title compound as a white crystalline solid in 93% yield. 1H NMR (400
MHz, DMSO-d6) δ 13.09 (s, 1H), 10.45 (s, 1H), 8.65 (s, 1H), 8.46 (d,
J = 8.0 Hz, 1H), 7.77 (t, J = 7.7 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.39
(t, J = 7.7 Hz, 1H), 7.26 (d, J = 13.1 Hz, 8H), 4.74 (d, J = 5.2 Hz, 2H),
4.63 (d, J = 4.3 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.25,
153.49, 139.45, 138.22, 137.99, 135.64, 131.99, 129.43, 128.73, 128.70,
127.97, 127.51, 124.86, 124.65, 117.25, 110.14, 44.79, 43.71. HRMS:
m/z calcd for C22H20ClN4 [M + H]+ 375.1371; found 375.1384. Rf =
0.34 (9:1 dichloromethane to methanol). Melting point 268−270 °C.
N2-Benzyl-N4-(4-chlorobenzyl)quinazoline-2,4-diamine (61).
57d (0.084 g, 0.28 mmol) was reacted with benzylamine and purified
according to method Da to furnish 0.084 g (0.22 mmol) of the title
N2,N4-Bis(4-methylbenzyl)quinazoline-2,4-diamine (65). 64d
(0.086 g, 0.30 mmol) was reacted with 4-methylbenzylamine and
purified according to method Da to furnish 0.080 g (0.22 mmol) of the
title compound as a fluffy white crystalline solid in 72% yield. 1H NMR
(400 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.59 (s, 1H), 8.45 (d, J = 8.1
Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.37 (t, J =
7.7 Hz, 1H), 7.16 (dd, J = 19.9, 7.2 Hz, 4H), 7.08−7.00 (m, 4H), 4.71
(d, J = 5.5 Hz, 2H), 4.59 (d, J = 4.3 Hz, 2H), 2.25 (s, 3H), 2.24 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 160.10, 153.41, 139.32,
136.57, 135.74, 135.60, 135.21, 129.33, 129.28, 128.01, 127.62, 124.88,
124.57, 117.10, 110.07, 44.59, 44.08, 21.11. Rf = 0.34 (9:1
dichloromethane to methanol). HRMS: m/z calcd for C24H25N4 [M
+ H]+ 369.2074; found 369.2091. Rf = 0.34 (9:1 dichloromethane to
methanol). Melting point 249−251 °C.
N4-Benzyl-N2-(4-methoxybenzyl)quinazoline-2,4-diamine
(66). 48d (0.089 g, 0.33 mmol) was reacted with 4-methoxybenzyl-
amine and purified according to method Da to furnish 0.096 g (0.26
mmol) of the title compound as a white crystalline solid in 79% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.57 (s, 1H), 8.48
1
compound as a white crystalline solid in 80% yield. H NMR (400
MHz, DMSO-d6) δ 13.06 (s, 1H), 10.54 (s, 1H), 8.64 (s, 1H), 8.47 (d,
J = 8.1 Hz, 1H), 7.77 (t, J = 7.7 Hz, 1H), 7.48 (d, J = 7.9 Hz, 1H),
7.41−7.36 (m, 1H), 7.28 (dd, J = 23.2, 6.5 Hz, 8H), 4.73 (d, J = 5.5
Hz, 2H), 4.63 (d, J = 4.9 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ
160.30, 153.44, 139.37, 138.79, 137.30, 135.68, 132.06, 129.81, 128.77,
128.67, 127.58, 127.47, 124.93, 124.64, 117.15, 110.08, 44.29, 44.15.
HRMS: m/z calcd for C22H20ClN4 [M + H]+ 375.1371; found
375.1377. Rf = 0.29 (9:1 dichloromethane to methanol). Melting point
246−248 °C.
(d, J = 8.1 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H),
7.40−7.23 (m, 6H), 7.17 (d, J = 7.6 Hz, 2H), 6.79 (d, J = 7.7 Hz, 2H),
4.79 (d, J = 5.8 Hz, 2H), 4.56 (d, J = 4.2 Hz, 2H), 3.70 (s, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 160.23, 158.82, 153.39, 139.38, 138.32,
135.59, 130.63, 129.15, 128.77, 127.98, 127.51, 124.91, 124.54, 117.10,
114.18, 110.07, 55.46, 44.81, 43.79. HRMS: m/z calcd for C23H23N4O
[M + H]+ 371.1866; found 371.1872. Rf = 0.38 (9:1 dichloromethane
to methanol). Melting point 246−248 °C.
N2,N4-Bis(4-chlorobenzyl)quinazoline-2,4-diamine (62). 57d
(0.081 g, 0.27 mmol) was reacted with 4-chorobenzylamine and
purified according to method Db to furnish 0.028 g (0.068 mmol) of
the title compound as a white solid in 25% yield. 1H NMR (400 MHz,
DMSO-d6) δ 8.59 (s, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.50 (t, J = 7.5 Hz,
1H), 7.25 (d, J = 8.4 Hz, 9H), 7.06 (t, J = 7.5 Hz, 1H), 4.67 (s, 2H),
4.48 (s, 2H), 3.70 (s, 1H). HRMS: m/z calcd for C22H19Cl2N4 [M +
H]+ 409.0981; found 409.0983. Rf = 0.27 (9:1 dichloromethane to
methanol). Melting point 174−177 °C.
2-Chloro-N-(4-methoxybenzyl)quinazolin-4-amine (67d). 1c
(0.22 g, 1.1 mmol) was reacted with 4-methoxybenzylamine and
purified according to method Ca to furnish 0.24 g (0.82 mmol) of the
1
title compound in 74% yield. H NMR (400 MHz, CDCl3) δ 7.77−
7.65 (m, 3H), 7.41 (ddd, J = 8.2, 6.5, 1.7 Hz, 1H), 7.31 (d, J = 8.6 Hz,
2H), 6.87 (dd, J = 9.1, 2.5 Hz, 2H), 6.24 (s, 1H), 4.76 (d, J = 5.2 Hz,
2H), 3.78 (d, J = 5.7 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 160.57,
159.37, 157.69, 150.81, 133.46, 129.72, 129.22, 127.72, 126.15, 120.85,
114.22, 113.16, 55.29, 45.25. MS (ESI): [M + H]+ 300; found 300.0.
Rf = 0.27 (hexanes to ethyl acetate 2:1).
N4-Benzyl-N2-(4-methylbenzyl)quinazoline-2,4-diamine (63).
48d (0.065 g, 0.24 mmol) was reacted with 4-methylbenzylamine and
purified according to method Da to furnish 0.066 g (0.19 mmol) of the
title compound as a white crystalline solid in 78% yield. 1H NMR (400
MHz, DMSO-d6) δ 10.47 (s, 1H), 8.60 (s, 1H), 8.47 (d, J = 8.0 Hz,
1H), 7.76 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.40−7.35 (m,
1H), 7.31 (s, 2H), 7.25 (d, J = 3.4 Hz, 3), 7.10 (dd, J = 36.0, 6.9 Hz,
4H), 4.77 (d, J = 5.6 Hz, 2H), 4.60 (s, 2H), 2.25 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 160.21, 153.46, 139.39, 138.28, 136.59,
135.73, 135.62, 129.35, 128.75, 128.07, 127.67, 127.52, 124.90, 124.57,
117.12, 110.09, 44.81, 44.11, 21.12. HRMS: m/z calcd for C23H23N4
[M + H]+ 355.1917; found 355.1915. Rf = 0.35 (9:1 dichloromethane
to methanol). Melting point 258−260 °C.
N2-Benzyl-N4-(4-methoxybenzyl)quinazoline-2,4-diamine
(67). 67d (0.089 g, 0.30 mmol) was reacted with benzylamine and
purified according to method Da to furnish 0.092 g (0.25 mmol) of the
title compound as a white crystalline solid in 83% yield. 1H NMR (400
MHz, DMSO-d6) δ 10.46 (s, 1H), 8.65 (s, 1H), 8.47 (d, J = 8.1 Hz,
1H), 7.75 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.40−7.15 (m,
8H), 6.76 (d, J = 7.7 Hz, 2H), 4.68 (d, J = 5.5 Hz, 4H), 3.69 (s, 3H).
13C NMR (101 MHz, DMSO-d6) δ 159.99, 158.81, 153.48, 139.28,
138.85, 135.57, 130.13, 129.57, 128.83, 127.57, 127.49, 124.90, 124.56,
117.07, 114.10, 110.12, 55.45, 55.44, 44.29. HRMS: m/z calcd for
P
dx.doi.org/10.1021/jm500039e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
C23H23N4O [M + H]+ 371.1866; found 371.1877. Rf = 0.38 (9:1
dichloromethane to methanol). Melting point 222−224 °C.
of the title compound as a white crystalline solid in 36% yield. H
NMR (400 MHz, CDCl3) δ 7.42 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H),
7.28−7.17 (m, 10H), 7.00 (dd, J = 8.7, 2.1 Hz, 1H), 4.68 (d, J = 5.5
Hz, 2H), 4.61 (d, J = 5.7 Hz, 2H). 13C NMR (101 MHz, CD3OD) δ
160.25, 160.15, 152.34, 139.29, 138.37, 138.05, 132.16, 131.88, 128.45,
128.13, 127.97, 127.89, 127.17, 123.78, 122.33, 121.02, 43.63, 43.18. .
HRMS: m/z calcd for C22H18Cl3N4 [M + H]+ 443.0592; found
443.0582. Rf = 0.77 (9:1 dichloromethane to methanol). Melting point
190−191 °C.
N2,N4-Bis(4-methoxybenzyl)quinazoline-2,4-diamine (68).
67d (0.090 g, 0.30 mmol) was reacted with 4-methoxybenzylamine
and purified according to method Da to furnish 0.098 g (0.24 mmol)
1
of the title compound as a white crystalline solid in 82% yield. H
NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.57 (s, 1H), 8.45 (d, J
= 8.1 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.45 (d, J = 7.5 Hz, 1H), 7.36
(t, J = 7.7 Hz, 1H), 7.21 (dd, J = 19.9, 7.6 Hz, 4H), 6.80 (t, J = 8.5 Hz,
4H), 4.70 (d, J = 5.7 Hz, 2H), 4.60 (s, 2H), 3.71 (s, 3H), 3.70 (s, 3H).
13C NMR (101 MHz, DMSO-d6) δ 160.05, 158.81, 153.38, 139.33,
135.57, 130.72, 130.18, 129.42, 129.02, 124.89, 124.54, 117.10, 114.18,
114.11, 110.07, 55.43, 44.27, 43.78. HRMS: m/z calcd for C24H25N4O2
[M + H]+ 401.1972; found 401.1985. Rf = 0.46 (9:1 dichloromethane
to methanol). Melting point 257−259 °C.
N4-Benzyl-7-chloro-N2-(4-methoxybenzyl)quinazoline-2,4-
diamine (72). 69d (0.10 g, 0.35 mmol) was reacted with 4-
methoxybenzylamine and purified according to method Da to furnish
0.094 g (0.23 mmol) of the title compound as a white crystalline solid
in 67% yield. 1H NMR (400 MHz, CDCl3) δ 7.44 (s, 1H), 7.36 (d, J =
8.7 Hz, 1H), 7.34−7.23 (m, 7H), 6.97 (dd, J = 8.7, 1.2 Hz, 1H), 6.82
(d, J = 8.4 Hz, 2H), 5.70 (bs, 1H), 5.26 (bs, 1H), 4.73 (d, J = 5.4 Hz,
2H), 4.60 (d, J = 5.7 Hz, 2H), 3.77 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 172.49, 159.86, 159.70, 158.70, 153.19, 138.61, 138.32,
131.76, 128.91, 128.75, 127.93, 127.62, 124.83, 122.06, 121.49, 113.86,
55.25, 45.09, 44.97. HRMS: m/z calcd for C23H22ClN4O [M + H]+
405.1477; found 405.1486. Rf = 0.62 (9:1 dichloromethane to
methanol). Melting point 221−223 °C.
2,4,6-Trichloroquinazoline (69c). Commercially available 2-
amino-5-chlorobenzoic acid (1 g, 5.8 mmol) was reacted according
to general procedure A to give crude 69b. Without further purification,
69b was reacted according to general procedure B to give 0.55 g (2.7
1
mmol) of the crude title compound (47% over two steps). H NMR
(250 MHz, CDCl3) δ 8.17 (d, J = 8.9 Hz, 1H), 7.94 (d, J = 2.0 Hz,
1H), 7.66 (dd, J = 8.9, 2.0 Hz, 1H). 13C NMR (63 MHz, CDCl3) δ
163.85, 156.31, 152.68, 142.97, 130.45, 127.37, 127.07, 120.77. MS
(ESI): [M + H]+ 233; found 232.9. Rf = 0.68 (hexanes to ethyl acetate
4:1).
7-Chloro- N4-(4-chlorobenzyl)-N2-(4-methoxybenzyl)-
quinazoline-2,4-diamine (73). 71d (0.082 g, 0.24 mmol) was
reacted with 4-methoxybenzylamine and purified according to method
Da to furnish 0.044 g (0.10 mmol) of the title compound as a white
crystalline solid in 42% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.57
(s, 1H), 8.63 (s, 1H), 8.47 (d, J = 8.8 Hz, 1H), 7.51 (s, 1H), 7.40 (dd, J
= 8.8, 1.7 Hz, 1H), 7.27 (dd, J = 14.6, 8.3 Hz, 4H), 7.07 (d, J = 8.2 Hz,
2H), 6.73 (d, J = 8.3 Hz, 2H), 4.72 (d, J = 5.4 Hz, 2H), 4.49 (d, J = 5.4
Hz, 2H), 3.67 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 159.82,
158.80, 153.37, 140.77, 139.98, 137.19, 132.08, 130.51, 129.69, 129.10,
128.69, 127.06, 124.67, 116.54, 114.09, 109.05, 90.55, 55.42, 44.19,
43.88. HRMS: m/z calcd for C23H21Cl2N4O [M + H]+ 439.1087;
found 439.1085. Rf = 0.79 (9:1 dichloromethane to methanol).
Melting point 261−262 °C.
Physicochemical Property Assays. Permeability Assay. Perme-
ability Pe was determined by a standard parallel artificial membrane
permeability assay (PAMPA by pION) as reported previously.14b
Aqueous Solubility Assay. Solubility at pH 7.4 was determined
using a Biomek FX lab automation workstation with pION μSOL
evolution software as reported previously and at pH 2.0 using an in-
house HPLC assay.14b For pH 2.0, a calibration curve was made by
plotting the area under the curve at 254 nm (UV by HPLC) against
the concentration of each compound injected after performing a serial
dilution (25−0.781 μM) using a solvent in which the compound is
soluble (DMSO). A 100 μM solution was then made for each
compound in a buffer at pH 2.0 by performing a 1:100 serial dilution
using a 10 mM DMSO stock solution of each compound. This
solution was incubated at 21 °C for 18 h, filtered using a filter plate,
and injected into the HPLC to compare the area found at wavelength
254 nm with the previously made calibration curve.
Partition Coefficient Assay. Log D was also determined in-house
via an HPLC method adapted from the strategy reported by Donovan
and Pescatore.14a Two buffers were made at a concentration of 50 μM
each: ammonium acetate at pH 7.4 and ammonium formate at pH 3.0.
A set of compounds with known log D values between −0.36 and 5.68
were used to make a calibration curve at each pH by using a linear
gradient between 0 and 100% acetonitrile, with buffer at the specific
pH used as the second solvent. The curve was made by plotting the
log D value against the retention time. Quinazolines were then injected
into the HPLC and the retention time compared to the calibration
curve previously determined.
Bacterial Strains and Growth Conditions. The methicillin resistant
S. aureus strain CBD-635 used in this study has previously been
described and its genome sequenced.17 For murine models of infection
we used MRSA strain USA300 FPR.18 In addition, a trimethoprim
resistant strain of S. aureus (NRS106) was used to observe resistance
to lead quinazoline-based compounds. This strain was acquired from
the Network on Antimicrobial Resistance in Staphylococcus aureus
N-Benzyl-2,7-dichloroquinazolin-4-amine (69d). 69c (0.51 g,
2.2 mmol) was reacted with benzylamine and purified according to
method Cb to furnish 0.59 g (1.9 mmol) of the title compound in 89%
yield. 1H NMR (250 MHz, CDCl3) δ 7.61 (d, J = 2.0 Hz, 1H), 7.57 (d,
J = 8.8 Hz, 1H), 7.34−7.18 (m, 6H), 6.29 (s, 1H), 4.76 (d, J = 5.3 Hz,
2H). 13C NMR (63 MHz, CDCl3) δ 160.42, 158.82, 151.67, 139.79,
136.95, 128.95, 128.39, 128.14, 127.04, 126.91, 122.42, 111.58, 45.86.
MS (ESI): [M + H]+ 304; found 304.0. Rf = 0.47 (hexanes to ethyl
acetate 2:1).
N2,N4-Dibenzyl-7-chloroquinazoline-2,4-diamine (69). 69d
(0.086 g, 0.28 mmol) was reacted with benzylamine and purified
according to method Db to furnish 0.076 g (0.20 mmol) of the title
1
compound as a yellow crystalline solid in 72% yield. H NMR (250
MHz, CD3OD) δ 7.81 (d, J = 8.7 Hz, 1H), 7.36−7.05 (m, 11H), 6.99
(d, J = 8.5 Hz, 1H), 4.70 (s, 2H), 4.57 (s, 2H). 13C NMR (101 MHz,
CDCl3) δ 159.74, 159.66, 152.49, 139.62, 138.75, 138.25, 128.73,
128.47, 127.94, 127.60, 127.54, 127.04, 124.33, 122.32, 121.69, 45.44,
45.10. HRMS: m/z calcd for C22H20ClN4 [M + H]+ 375.1371; found
375.1390. Rf = 0.54 (9:1 dichloromethane to methanol). Melting point
146−149 °C.
N4-Benzyl-N2-butyl-7-chloroquinazoline-2,4-diamine (70).
69d (0.088 g, 0.29 mmol) was reacted with butylamine and purified
according to method Db to furnish 0.089 g (0.26 mmol) of the title
1
compound as a yellow crystalline solid in 90% yield. H NMR (250
MHz, CD3OD) δ 7.82 (d, J = 8.7 Hz, 1H), 7.40−7.06 (m, 6H), 6.97
(dd, J = 8.7, 1.9 Hz, 1H), 4.99 (s, 2H), 3.39−3.27 (m, 2H), 1.35 (ddd,
J = 26.5, 20.7, 9.5 Hz, 4H), 0.88 (t, J = 7.2 Hz, 3H). 13C NMR (63
MHz, CD3OD) δ 161.65, 161.51, 153.70, 140.72, 139.64, 129.43,
128.47, 127.97, 125.24, 123.56, 122.13, 110.85, 45.39, 42.09, 33.22,
21.25, 14.39. HRMS: m/z calcd for C19H21ClN4 [M + H]+ 341.1527;
found 341.1545. Rf = 0.39 (9:1 dichloromethane to methanol).
Melting point 121−123 °C.
2,7-Dichloro-N-(4-chlorobenzyl)quinazoline-4-amine (71d).
69c (0.32 g, 1.4 mmol) was reacted with 4-chlorobenzylamine and
purified according to method Cb to furnish 0.33 g (0.98 mmol) of the
title compound in 71% yield. 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J
= 8.7 Hz, 1H), 7.62 (d, J = 1.6 Hz, 1H), 7.36 (dd, J = 8.7, 1.8 Hz, 1H),
7.25 (dd, J = 16.8, 8.4 Hz, 4H), 6.64 (s, 1H), 4.78 (d, J = 5.3 Hz, 2H).
13C NMR (101 MHz, CDCl3) δ 160.46, 158.67, 151.57, 139.86,
135.41, 133.84, 129.67, 128.88, 127.11, 126.70, 122.56, 111.54, 45.04.
MS (ESI): [M + H]+ 338; found 337.9. Rf = 0.51 (hexanes to ethyl
acetate 2:1).
7-Chloro-N2,N4-bis(4-chlorobenzyl)quinazoline-2,4-diamine
(71). 71d (0.082 g, 0.24 mmol) was reacted with 4-chlorobenzylamine
and purified according to method Da to furnish 0.038 g (0.086 mmol)
Q
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(NARSA). Bacterial strains were grown in tryptic soy broth (TSB) at
37 °C, and when necessary, agar was added to a final concentration of
1.5% (w/v) for growth plates and 0.7% (w/v) for overlay media.
Disk Diffusion Assay. These were performed as described
previously.19 Briefly, bacterial strains were streaked onto plates and
incubated overnight. A single colony was used to inoculate broth
media and allowed to grow overnight. The overnight culture was
diluted 1:1000 into 5 mL of molten overlay agar and mixed before
being used to inoculate growth plates. After plates were allowed to dry,
three sterile filter disks were added per plate and inoculated with 10 μL
of test compound. The stock concentration for each test compound
was 5 mg mL−1 suspended in DMSO. Bacterial plates were incubated
for 24 h. Zones of inhibition were measured in millimeters, with the
assay being performed in triplicate.
Microtiter MIC and MBC Determination Assays. Both assays were
performed as described in our previous publications.19 Briefly,
minimum inhibitory concentrations (MIC) were determined for
compounds which displayed antimicrobial activity against S. aureus
CBD-635. Broth cultures were prepared as described above. These
overnight cultures were diluted 1:1000 into fresh media, and 200 μL
was transferred to a sterile 96-well plate. Relevant concentrations of
test compounds were applied to each well and samples mixed by
pipetting before incubation at 37 °C overnight. MICs were determined
by visual inspection of the minimum concentration of drug that
produced no bacterial growth (as determined by a lack of turbidity).
The assay was performed in triplicate to verify the MIC. The
minimum bactericidal concentrations (MBC) were determined from
MIC samples by serially diluting and plating onto TSA. Plates were
incubated overnight at 37 °C and the MBC determined by calculating
CFU per mL for each of the concentrations of compound tested
compared to the initial inoculum. The data is represented as the
percent killing by comparison of drug containing samples to samples
that contain no drug.
Derivations of Spontaneous Mutation Frequencies. TSA was
prepared containing 32, 50, 53, and 62 at a concentration of 2.5× the
MIC. Overnight broth cultures of S. aureus CBD-635 were prepared as
described above before 1 mL aliquots were extracted and harvested by
centrifugation. The pellet was then resuspended in 100 μL of fresh
TSB and used to inoculate the quinazoline containing media. Plates
were incubated overnight at 37 °C. Assays were performed a total of
10 times using fresh overnight cultures. The CFU/mL of each
overnight culture was determined by serial dilution and plating. The
spontaneous mutation frequency for each quinazoline compound was
determined by dividing the number of colonies obtained by the total
bacterial inoculum.
Sequence Analysis of QRDR and DHFR for Spontaneously
Resistant Mutants. DNA was extracted from wild-type and
spontaneously resistant CBD-635 mutants using a DNeasy kit
(Qiagen), according to the manufactures protocol. The QRDR of
gyrAB and grlAB was PCR amplified using primers described by us
previously.15 The dfrA gene was PCR amplified using primers
G C T C C A A T A G C A G T G T A G T C / G G T C A A C G T A T -
GAAGCTCCTTC. Samples were then sent to MWG to undergo
sequencing analysis.
if the lead quinazolines work synergistically with sulfamethoxazole, we
used a checkerboard microtitration method to determine the fractional
inhibitory concentration (FIC) and ΣFIC index of paired combina-
tions of quinazoline and sulfamethoxazole. The ΣFIC index was then
used to determine if synergy, antagonism, or indifference occurred as a
result of interactions between antibacterial agents. Briefly, the MIC of
sulfamethoxazole and quinazolines individually and combined were
determined. From this we determined the FIC as follows. The FIC
value was calculated from the MIC of sulfamethoxazole alone divided
by the MIC of sulfamethoxazole in combination with lead agents. The
FIC of lead quinazolines was determined from the MIC of each
compound alone divided by the MIC of each compound in
combination with sulfamethoxazole. The sum of these two values
yields the ΣFIC index. This value is then used to determine whether
synergism (ΣFIC ≤ 0.5), indifference (0.5 < ΣFIC ≤ 4), or
antagonism (ΣFIC > 4) occurred between the two compounds.
Evaluation of the Antimicrobial Efficacy and Toxicity Using a G.
mellonella Model of Infection. To determine the in vivo efficacy of
compounds, a G. mellonella model of S. aureus infection was used as
previously described.20 Briefly, 1 mL aliquots of overnight cultures of
S. aureus CBD-635 were pelleted by centrifugation and washed in
sterile PBS before being resuspended in 100 μL of PBS. G. mellonella
larvae (N = 10) that weighed 200−300 mg were then inoculated with
5 μL of S. aureus (1 × 109 CFU) into the last left proleg. Larvae were
then treated with compounds at 1 h post-inoculation. Treatments were
performed in the same manner as infection, except that injections were
into the next left proleg moving toward the head of the larvae. Larvae
were then incubated at 37 °C and mortality rates were monitored for
78 h; larvae were considered dead if they did not respond to physical
stimuli. Data was analyzed for statistical significance using a log rank
and χ square test with 1 degree of freedom.
In Vivo Efficacy Testing Using a Murine Model of Lethal
Peritonitis. Six mice per treatment were IP inoculated with a lethal
dose of MRSA strain FPR (1 × 108 CFU/mL) in 5% mucin. After 1 h,
we then injected mice with either 5× MIC or 10× MIC of vancomycin
(positive controls), 125 μL of 2-(hydroxypropyl)-β-cyclodextrin
(vehicle, negative control), 5 mg kg−1 compound 50 or 53, or 1 mg
kg−1 compound 53 via tail vein. Upon inoculation, mice were
monitored twice daily for 5 days. The clinical end point of this study
was when infected animals reached a premoribund state. The criteria
for determining this was if mice develop a hunched posture, rapid,
shallow, and/or labored breathing, ruffled fur, lethargy, failure to
respond to stimuli, soiled anogenital area, paralysis, paresis, head tilt,
circling, vocalizations, nonpurposeful movements, and/or were unable
to eat or drink. Any animal which displayed these signs was
euthanized. We then compared the number of animals that reach a
premoribund state between the control (vehicle or known antibiotic
treated) and test groups. Data was analyzed for statistical significance
using a log rank and χ squared test with 1 degree of freedom.
AUTHOR INFORMATION
■
Corresponding Authors
*For R.M.: phone, 813-974-7306; E-mail, manetsch@usf.edu.
*For L.N.S.: phone, 813-974-2087; E-mail, shaw@usf.edu.
Investigating the Mechanism of Action of Quinazoline-Based
Compounds. To evaluate the effect quinazolines have on DHFR
reduction of dihydrofolic acid, a tetrahydrofolic acid assay was
performed. An overnight culture of S. aureus CDB-635 was diluted
1:1000 into fresh TSB followed by the addition of compound 53 at 5×
the MIC. This culture was then seeded into a sterile 96-well plate and
exogenous tetrahydrofolic acid added at concentrations ranging from 0
to 224 μM. The cultures were then incubated at 37 °C for 18 h. The
samples were visually inspected to determine if the addition of
exogenous tetrahydrofolic acid rescued quinazoline mediated cell
killing. This assay was repeated in triplicate in order to verify effects,
with trimethoprim and vancomycin controls run in parallel.
Trimethoprim is a known DHFR inhibitor, while vancomycin is a
glycopeptide antibiotic that inhibits cell wall biosynthesis.
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
CA-MRSA community-associated methicillin resistant S. aureus;
HAI hospital-acquired infection; MIC minimum inhibitory
concentration; MRSA methicillin resistant S. aureus; SAR
structure−activity relationship; VRSA vancomycin resistant S.
aureus
Fractional Inhibitory Concentration Index of Quinazoline-Based
Compounds in Combination with Sulfamethoxazole. To determine
R
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3-carboxyl-4(1H)-quinolones to deliver orally bioavailable antima-
larials. J. Med. Chem. 2012, 55, 4205−4219.
(15) Cormier, R.; Burda, W. N.; Harrington, L.; Edlinger, J.;
Kodigepalli, K. M.; Thomas, J.; Kapolka, R.; Roma, G.; Anderson, B.
E.; Turos, E.; Shaw, L. N. Studies on the antimicrobial properties of N-
acylated ciprofloxacins. Bioorg. Med. Chem. Lett. 2012, 22 (20), 6513−
6520.
(16) Ghose, A. K.; Crippen, G. M. Quantitative structure−activity
relationship by distance geometry: quinazolines as dihydrofolate
reductase inhibitors. J. Med. Chem. 1982, 25 (8), 892−899.
(17) Carroll, W. A.; Agrios, K. A.; Altenbach, R. J.; Buckner, S. A.;
Chen, Y.; Coghlan, M. J.; Daza, A. V.; Drizin, I.; Gopalakrishnan, M.;
Henry, R. F.; Kort, M. E.; Kym, P. R.; Milicic, I.; Smith, J. C.; Tang, R.;
Turner, S. C.; Whiteaker, K. L.; Zhang, H.; Sullivan, J. P. Synthesis and
structure−activity relationships of a novel series of tricyclic
dihydropyridine-based KATP openers that potently inhibit bladder
contractions in vitro. J. Med. Chem. 2004, 47 (12), 3180−3192.
(18) Diep, B. A.; Gill, S. R.; Chang, R. F.; Phan, T. H.; Chen, J. H.;
Davidson, M. G.; Lin, F.; Lin, J.; Carleton, H. A.; Mongodin, E. F.;
Sensabaugh, G. F.; Perdreau-Remington, F. Complete genome
sequence of USA300, an epidemic clone of community-acquired
meticillin-resistant Staphylococcus aureus. The Lancet 2006, 367 (9512),
731−739.
REFERENCES
■
(1) Kuehnert, M. J.; Kruszon-Moran, D.; Hill, H. A.; McQuillan, G.;
McAllister, S. K.; Fosheim, G.; McDougal, L. K.; Chaitram, J.; Jensen,
B.; Fridkin, S. K.; Killgore, G.; Tenover, F. C. Prevalence of
Staphylococcus aureus nasal colonization in the United States, 2001−
2002. J. Infect. Dis. 2006, 193 (2), 172−179.
(2) MRSA may kill more US citizens than HIV. BMJ (Br. Med. J.)
2007, 335 (7625), 850−850.
(3) Stephens, J.; Davies, L. The war on superbugs. Scrip Mag. 2008,
2401, 44−47.
(4) Hayden, M. K.; Rezai, K.; Hayes, R. A.; Lolans, K.; Quinn, J. P.;
Weinstein, R. A. Development of daptomycin resistance in vivo in
methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 2005, 43
(10), 5285−5287.
(5) Public Health Dispatch: Vancomycin-Resistant Staphylococcus
aureusPennsylvania. In Mortality Morbidity Wkly. Rep. 2002, 51, 902.
(6) Lam, T.; Hilgers, M. T.; Cunningham, M. L.; Kwan, B. P.;
Nelson, K. J.; Brown-Driver, V.; Ong, V.; Trzoss, M.; Hough, G.;
Shaw, K. J.; Finn, J. Structure-based design of new dihydrofolate
reductase antibacterial agents: 7-(benzimidazol-1-yl)-2,4-diaminoqui-
nazolines. J. Med. Chem. 2014, 57 (3), 651−668.
(7) Huband, M. D.; Cohen, M. A.; Zurack, M.; Hanna, D. L.; Skerlos,
L. A.; Sulavik, M. C.; Gibson, G. W.; Gage, J. W.; Ellsworth, E.; Stier,
M. A.; Gracheck, S. J. In vitro and in vivo activities of PD 0305970 and
PD 0326448, new bacterial gyrase/topoisomerase inhibitors with
potent antibacterial activities versus multidrug-resistant gram-positive
and fastidious organism groups. Antimicrob. Agents Chemother. 2007,
51 (4), 1191−1201.
(8) Rohini, R.; Muralidhar Reddy, P.; Shanker, K.; Hu, A.; Ravinder,
V. Antimicrobial study of newly synthesized 6-substituted indolo[1,2-
c]quinazolines. Eur. J. Med. Chem. 2010, 45 (3), 1200−1205.
(9) DeGraw, J. I.; Brown, V. H.; Colwell, W. T.; Morrison, N. E.
Potential antileprotic agents. 3. Inhibition of mycobacterial dihy-
drofolic reductase by 2,4-diamino-5-methyl-6-alkylquinazolines. J. Med.
Chem. 1974, 17 (7), 762−764.
(19) Burda, W. N.; Fields, K. B.; Gill, J. B.; Burt, R.; Shepherd, M.;
Zhang, X. P.; Shaw, L. N. Neutral metallated and meso-substituted
porphyrins as antimicrobial agents against Gram-positive pathogens.
Eur. J. Clin. Microbiol. 2012, 31 (3), 327−335.
(20) Bionda, N.; Fleeman, R. M.; Shaw, L. N.; Cudic, P. Effect of
ester to amide or N-methylamide substitution on bacterial membrane
depolarization and antibacterial activity of novel cyclic lipopeptides.
ChemMedChem 2013, 8 (8), 1394−1402.
(10) Kung, P.; Casper, M. D.; Cook, K. L.; Wilson-Lingardo, L.;
Risen, L. M.; Vickers, T. A.; Ranken, R.; Blyn, L. B.; Wyatt, J. R.; Cook,
P. D.; Ecker, D. J. Structure−activity relationships of novel 2-
substituted quinazoline antibacterial agents. J. Med. Chem. 1999, 42
(22), 4705−4713.
̌
́ ́ ́
(11) Gottasova, R.; Kubíkova, J.; Cipak, L. Antibacterial effect of
some 2,6-disubstituted 4-aniloquinazolines. Folia Microbiol. 1998, 43
(6), 679−682.
(12) Van Horn, K. S.; Zhu, X.; Pandharkar, T.; Yang, S.; Vesely, B.;
Kyle, D. E.; Wang, M. Z.; Werbovetz, K. A.; Manetsch, R., unpublished
results.
(13) (a) Feng, J.; Zhang, Z.; Wallace, M. B.; Stafford, J. A.; Kaldor, S.
W.; Kassel, D. B.; Navre, M.; Shi, L.; Skene, R. J.; Asakawa, T.;
Takeuchi, K.; Xu, R.; Webb, D. R.; Gwaltney, S. L., II. Discovery of
alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of
dipeptidyl peptidase IV. J. Med. Chem. 2007, 50 (10), 2297−2300.
(b) Ife, R. J.; Brown, T. H.; Blurton, P.; Keeling, D. J.; Leach, C. A.;
Meeson, M. L.; Parsons, M. E.; Theobald, C. J. Reversible inhibitors of
the gastric (H+/K+)-ATPase. 5. Substituted 2,4-diaminoquinazolines
and thienopyrimidines. J. Med. Chem. 1995, 38 (14), 2763−2773.
(c) Kanuma, K.; Omodera, K.; Nishiguchi, M.; Funakoshi, T.; Chaki,
S.; Semple, G.; Tran, T.-A.; Kramer, B.; Hsu, D.; Casper, M.;
Thomsen, B.; Sekiguchi, Y. Lead optimization of 4-(dimethylamino)-
quinazolines, potent and selective antagonists for the melanin-
concentrating hormone receptor 1. Bioorg. Med. Chem. Lett. 2005,
15 (17), 3853−3856.
(14) (a) Donovan, S. F.; Pescatore, M. C. Method for measuring the
logarithm of the octanol−water partition coefficient by using short
octadecyl-poly(vinyl alcohol) high-performance liquid chromatogra-
phy columns. J. Chromatogr., A 2002, 952, 47−61. (b) Zhang, Y.;
Clark, J. A.; Connelly, M. C.; Zhu, F.; Min, J.; Guiguemde, W. A.;
Pradhan, A.; Iyer, L.; Furimsky, A.; Gow, J.; Parman, T.; El, M. F.;
Phillips, M. A.; Kyle, D. E.; Mirsalis, J.; Guy, R. K. Lead optimization of
S
dx.doi.org/10.1021/jm500039e | J. Med. Chem. XXXX, XXX, XXX−XXX