Antileishmanial activity of a series of N2, N 4-disubstituted quinazoline-2,4-diamines

Article abstract of DOI:10.1021/jm5000408

A series of N²,N⁴-disubstituted quinazoline-2,4- diamines has been synthesized and tested against Leishmania donovani and L. amazonensis intracellular amastigotes. A structure-activity and structure-property relationship study was conducted in part using the Topliss operational scheme to identify new lead compounds. This study led to the identification of quinazolines with EC₅₀ values in the single digit micromolar or high nanomolar range in addition to favorable physicochemical properties. Quinazoline 23 also displayed efficacy in a murine model of visceral leishmaniasis, reducing liver parasitemia by 37% when given by the intraperitoneal route at 15 mg kg^-1 day^-1 for 5 consecutive days. Their antileishmanial efficacy, ease of synthesis, and favorable physicochemical properties make the N²,N ⁴-disubstituted quinazoline-2,4-diamine compound series a suitable platform for future development of antileishmanial agents.

Full text of DOI:10.1021/jm5000408

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Article
2
4
Antileishmanial Activity of a Series of N,N-
Disubstituted Quinazoline-2,4-diamines
Kurt S. Van Horn, Xiaohua Zhu, Trupti Pandharkar, Sihyung Yang, Brian Vesely, Manu Vanaerschot, Jean-
Claude Dujardin, Suman Rijal, Dennis E Kyle, Michael Zhuo Wang, Karl A Werbovetz, and Roman Manetsch
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 29 May 2014
Downloaded from http://pubs.acs.org on May 30, 2014
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Journal of Medicinal Chemistry
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Antileishmanial Activity of a Series of
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N ,N ꢀDisubstituted Quinazolineꢀ2,4ꢀdiamines
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Kurt S. Van Horn , Xiaohua Zhu , Trupti Pandharkar , Sihyung Yang , Brian Vesely , Manu
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Vanaerschot , Jean-Claude Dujardin , Suman Rijal , Dennis E. Kyle , Michael Zhuo Wang , Karl A.
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Werbovetz , Roman Manetsch
1
) Department of Chemistry, University of South Florida , Tampa, Florida 33620, United States
2
) Division of Medicinal Chemistry and Pharmacognosy, Ohio State University, Columbus, Ohio
43210, United States
3
) Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, United
States
4) Department of Global Health, University of South Florida, Tampa, Florida 33620, United States
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) Unit of Molecular Parasitology, Department of Parasitology, Institute of Tropical Medicine,
Antwerp, Belgium
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) Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
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) B.P. Koirala Institute of Health Sciences, Ghopa, Dharan, Nepal
Receieved TBD
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Abstract
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A series of N ,N ꢀdisubstituted quinazolineꢀ2,4ꢀdiamines has been synthesized and tested against
Leishmania donovani and L. amazonensis intracellular amastigotes. A structureꢀactivity and structureꢀ
property relationship study was conducted in part using the Topliss operational scheme to identify new
lead compounds. This study led to the identification of quinazolines with EC s in the single digit
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micromolar or high nanomolar range in addition to favorable physicochemical properties. Quinazoline
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3 also displayed efficacy in a murine model of visceral leishmaniasis, reducing liver parasitemia by
37% when given by the intraperitoneal route at 15 mg/kg/day for five consecutive days. Their
2
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antileishmanial efficacy, ease of synthesis, and favorable physicochemical properties make the N ,N ꢀ
disubstituted quinazolineꢀ2,4ꢀdiamine compound series a suitable platform for future development of
antileishmanial agents.
Keywords: leishmaniasis, protozoa, quinazoline, structureꢀactivity relationship, structureꢀproperty
relationship
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Introduction
Leishmaniasis is a debilitating disease that is prevalent across the globe with 350 million people in 88
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countries at risk of acquiring leishmaniasis.
A recent effort by the World Health Organization to
provide a current estimate of the incidence of leishmaniasis concluded that between 0.2 and 0.4 million
cases of visceral leishmaniasis occur each year, while from 0.7 to 1.2 million cases of cutaneous
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leishmaniasis occur each year. This study also estimated that from 20,000 to 40,000 deaths occur each
year due to leishmaniasis. More than twenty Leishmania species are known to cause the disease in
4
humans. Leishmaniasis manifests itself in numerous forms depending on which parasite species infects
the host. The parasites are transferred from host to host by about thirty species of female sandfly vectors
5
of the genera Phlebotomus and Lutzomyia that infect the host when taking a blood meal. Symptoms of
leishmaniasis include unsightly spontaneously healing ulcers on the skin when cutaneous leishmaniasis
presents, nonꢀhealing lesions in the mucosa when mucocutaneous leishmaniasis is the affliction, and
chronic, debilitating infection of the reticuloendothelial system which is fatal if left untreated due to
1
visceral leishmaniasis. The majority of cases of visceral leishmaniasis are caused by L. donovani in
6
East Africa and Asia, L. infantum in the Mediterranean region, and L. chagasi in Latin America. It
7
, 8
should be noted that the latter two are genetically identical. L. infantum and L. chagasi mainly affect
children and immunocompromised individuals and are zoonotic parasites with canines being a major
1
reservoir. L. donovani, on the other hand, is an anthroponotic parasite that affects a broad range of
1
ages.
For nearly one hundred years, antimonials have been the drug of choice to combat leishmaniasis. In
1
912 Gaspar Vianna first reported the use of the trivalent antimonial tartar emetic for the treatment of
9
cutaneous leishmaniasis caused by L. braziliensis in Brazil. Shortly thereafter McCombie Young and
Upendranath Brahmachari used trivalent and pentavalent antimonials to treat visceral leishmaniasis in
10
India with great success, decreasing the mortality rate of 95% to just 10% in ten years (Figure 1.A).
Pentavalent antimonials such as meglumine antimoniate and sodium stibogluconate are currently the
1, 11, 12
first line antileishmanial drugs in many areas.
Treatment involves daily injections for up to a thirty
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day period. Problems with this treatment include a high rate of resistance that has been encountered in
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India, especially the state of Bihar, where up to 60% of infected individuals do not improve with
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treatment.
The high rate of resistance to pentavalent antimonials in India has led to the increasing
14
use of amphotericin B and miltefosine against visceral leishmaniasis. Since the 1960’s, amphotericin
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B has been the second line treatment for visceral leishmaniasis. It has a cure rate of over ninety
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percent, but is often accompanied by severe side effects such as nephrotoxicity that require
7, 11
administration in a hospital setting.
Lipid formulations of amphotericin B have fewer side effects and
7
, 11
are safer to use with the same cure rate.
Depending on the dose and formulation, the treatment
6
, 11
regimen varies from 3ꢀ5 days to eight weeks of administration on alternate days.
Miltefosine is the
first oral drug to be released for leishmaniasis and is currently available in India, Germany, and
11
Colombia. Miltefosine is not recommended for women who are pregnant or may become pregnant
1, 11
because it is teratogenic.
Miltefosine resistance has been demonstrated in vitro, and its long halfꢀlife
in the body, the twentyꢀeight day treatment regimen, as well as it previously being available over the
1
, 11, 15
counter in India have led to concerns of clinical resistance.
A recent study of 567 individuals in
the Bihar state of India has been performed to determine the efficacy of miltefosine since its
1
6
introduction in 2002. The six month cure rate was found to be roughly 90% and gastrointestinal
1
6
intolerance was encountered in 64.5% of the cases with two deaths related to drug toxicity. Patients
who did not improve with treatment were cured using amphotericin B. The authors of this study
concluded that the failure rate of miltefosine has increased in the ten years since its introduction for the
treatment of visceral leishmaniasis in India. A recent study also showed that 20% of the visceral
17
leishmaniasis patients in Nepal who were treated with miltefosine relapsed 12 months after treatment.
Due to increased parasite resistance, toxicity issues, increasing failure rates of current treatments, and
the lack of effective clinical agents against cutaneous leishmaniasis, new drugs are needed to have an
effective strategy for treating leishmaniasis. Quinazolines are a class of compounds that have shown
potential as antileishmanials. Berman et al. reported a class of 2,4ꢀdiaminoquinazolines with EC as
50
low as 0.04 nM against L. major amastigotes in human monocyteꢀderived macrophages (A, Figure 1.B),
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however the development of this compound series was abandoned due to toxicity issues. Bhattacharjee
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et al. (B), Ram et al. (C), and Shakya and Gupta et al. (D and E) have also tested quinazolines as
1
9ꢀ22
antileishmanials.
This class of compounds has been reported as being dihydrofolate reductase
18, 23
(DHFR) inhibitors, although another mechanism of action may be involved with Leishmania.
Recently, we tested a small library of structurally diverse compounds, originally designed as potential
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anticancer probes, for antileishmanial activity in a L. mexicana axenic amastigote assay. Among this
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library were N ,N ꢀdisubstituted quinazolines 1 and 2 (Figure 1.B), which are structurally different from
quinazoline series AꢀE. Antileishmanial testing of quinazolines 1 and 2 against L. mexicana axenic
amastigotes revealed EC50 values in the single digit micromolar range, motivating us to investigate
whether quinazolines structurally related to the hits 1 and 2 have potential to display potent
2
4
antileishmanial activity. Herein, we report a detailed structureꢀactivity relationship (SAR) study
focusing on the 2ꢀposition, the 4ꢀposition, and the quinazoline’s benzenoid ring. All compounds were
initially examined in L. donovani and L. amazonensis intracellular amastigote assays to preselect
quinazoline candidates active against parasites responsible for causing visceral leishmaniasis and
cutaneous leishmaniasis, respectively. Promising compounds have subsequently been tested for efficacy
in a murine model of visceral leishmaniasis.
Figure 1
Results and Discussion
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5ꢀ27
Synthetic Chemistry. The compounds were synthesized following known procedures (Figure 2).
Commercially available anthranilic acids (a) were cyclized with urea and the resulting quinazolineꢀ2,4ꢀ
dione (b) was reacted with phosphorous oxychloride to give the 2,4ꢀdichloroquinazoline (c).
Substitution with amines occurred selectively at position 4 yielding 4ꢀaminoꢀ2ꢀchloroquinazoline (d)
followed by substitution at position 2 to give the 2,4ꢀdiaminosubstituted quinazoline. In this synthetic
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sequence, only 4ꢀaminoꢀ2ꢀchloroquinazoline (d) and the final N ,N ꢀdisubstituted quinazolineꢀ2,4ꢀ
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diamine have been purified and characterized.
Figure 2
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In vitro Antileishmanial Efficacy and Cytotoxicity. All target compounds were tested against L.
donovani and L. amazonensis intracellular amastigotes to identify molecules that are broadly active
against these medically important Leishmania species. The testing involved murine peritoneal
macrophages as host cells, since compounds with potential for clinical use must be able to penetrate the
infected macrophage in a human host. Antileishmanial activity was determined in an assay using
28, 29
transgenic parasites expressing a βꢀlactamase gene as outlined previously.
Concentration response
data for each compound was fitted by a nonlinear regression model and the concentration that induces
0% inhibition was calculated as the effective concentration EC (L. donovani or L. amazonensis.).
5
5
0
Additionally, cytotoxicity against the macrophage cell line J774A.1 was determined as the effective
concentration EC (J774A.1) and the selectivity index SI was calculated as the ratio of EC value for
50
50
J774A.1 and the value for L. donovani (SI = EC (J774A.1)/EC (L. donovani)).
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0
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StructureꢀActivity Relationship Studies. To validate and optimize the antileishmanial activity of
2
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N ,N ꢀdisubstituted quinazolineꢀ2,4ꢀdiamines, two compound subseries were prepared and tested. The
2
4
first subseries focused mainly on the optimization of the N ꢀ and N ꢀmoieties (Table 1), whereas the
second subseries was designed to investigate whether analogues being substituted at the quinazoline's
benzenoid ring display improved antileishmanial activity (Table 2).
4
2
Starting from hit compound 2, N ꢀfurfurylꢀanalogues 3 and 4 were prepared in which the N ꢀisoꢀ
propyl group was replaced by a short trifluoroalkylꢀ or hydroxyalkylꢀgroup. While the 2,2,2ꢀ
trifluoroethylꢀsubstituted quinazoline 3 was slightly less potent than compound 2, alcohol 4 lost potency
against L. donovani and L. amazonensis by a factor of 10 and >13, respectively. Testing of a small set of
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quinazolineꢀ2,4ꢀdiamines 5ꢀ9 with N ꢀmonosubstituted or N ꢀdisubstituted with alkyl groups differing in
size and polarity did not identify a particular structural motif improving the potency over 2.
Replacement of the furfuryl and isoꢀpropyl groups in 2 by two benzyl or two nꢀbutyl groups yielded
quinazolines 10 and 11, of which both analogues were more potent than reference 2. Bisꢀbenzylꢀ
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substituted quinazoline 10 displayed EC s of 667 nM against L. donovani and 1.41 ꢁM against L.
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amazonensis, whereas analogue 11 was approximately twofold less potent in comparison to compound
1
0. Consequently, a following set of six quinazolines 12ꢀ17 was designed in which one of the 2ꢀ and 4ꢀ
positions was substituted by one benzyl amine, while the remaining position was derivatized with an
4
2
aniline, nꢀbutylamine, or methylamine. Interestingly, with the exception of the N ꢀbenzylꢀN ꢀmethylꢀ
quinazolineꢀ2,4ꢀdiamine 12, all of these compounds demonstrated subꢀmicromolar EC s against L.
5
0
2
donovani. Among these six quinazolines tested, the N ꢀbenzylꢀquinazolineꢀ2,4ꢀdiamines 15ꢀ17 appeared
4
to be at least twofold more potent against L. donovani than the N ꢀbenzyl counterparts 12ꢀ14 suggesting
2
4
that an N ꢀbenzyl is more favorable than an N ꢀbenzyl for antileishmanial activity. This observation is
similar to previous results with quinazolines 2ꢀ11. Compound 15 was the most potent compound with
an EC of 149 nM against L. donovani. For L. amazonensis, the set of 12ꢀ17 displayed a similar activity
5
0
2
trend, with N ꢀbenzylꢀquinazolines 15 and 16 being the most potent compounds with submicromolar or
single digit micromolar EC s.
50
Table 1
4
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2
N FurfurylꢀN ꢀisoꢀpropylꢀsubstituted quinazoline 2, with EC s of 2.50 ꢀM against L. donovani and
50
3
.71 ꢁM against L. amazonensis, was considered to be well suited as the key scaffold for a SAR study
focusing on the benzenoid moiety of the quinazoline core. In a systematic approach following the
Topliss operational scheme, compound 2 was monoꢀsubstituted with either a chlorine atom, a methyl
group, or a methoxy group in the 5ꢀ, 6ꢀ, 7ꢀ, and 8ꢀpositions to probe the benzenoid ring for steric and
3
0
electronic effects. Overall, substitution on the benzenoid ring provided compounds with EC s in the
50
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single digit micromolar or subꢀmicromolar range against L. donovani. Among the chloroꢀsubstituted
subseries, 5ꢀ and 6ꢀsubstituted analogues 18 and 19 were less potent by a factor of two or more
compared to reference 2, while the 7ꢀ and 8ꢀsubstituted quinazolines 20 and 21 displayed an activity on
par with compound 2. In contrast, the majority of the methylꢀ and methoxyꢀsubstituted quinazolines
demonstrated a modest potency improvement over quinazoline 2. For the methoxyꢀsubstituted
quinazolines, potency increased according to substitutions in 8ꢀ < 7ꢀ < 5ꢀ ≈ 6ꢀposition, whereas a
potency dependence in the order of 8ꢀ ≈ 6ꢀ < 7ꢀ ≈ 5ꢀpositions was observed for the methylꢀsubstituted
compounds. 7ꢀMethoxyꢀquinazoline 28 with an EC50 of 740 nM against L. donovani was the most
potent analogue of the compound subseries substituted at the benzenoid ring. In contrast, the testing of
the benzenoid ring substituted quinazolines against L. amazonensis gave insight into a SAR, which
differed from the one observed for L. donovani. 8ꢀMethoxyꢀsubstituted quinazoline 29 was the only
analogue which was marginally more potent than reference compound 2, whereas all of the other
analogues were equally or less potent than quinazoline 2.
1
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9
1
1
1
1
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1
1
1
1
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2
2
2
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Table 2
Cytotoxicity. An important quality of the quinazolines is their selectivity to inhibit parasite growth
over mammalian cells. Generally, the majority of the quinazolines 2ꢀ29 exhibited EC s of 20 ꢁM or
5
0
higher against J774A.1 (Tables 1 and 2). Quinazolines 18ꢀ20 and 22ꢀ24, whose benzenoid ring is
substituted with one chloride or one methyl in 5ꢀ, 6ꢀ or 7ꢀposition, were significantly less toxic than
2
their reference 2. The quinazolines displaying potent antileishmanial activity, especially the N ꢀbenzylꢀ
quinazolineꢀ2,4ꢀdiamines 15ꢀ17, were shown to have respectable SI values of 10 and larger indicating
that they are relatively selective, nontoxic chemotypes. The SI of 100 for compound 15 was particularly
enticing and led to this compound being tested in vivo.
Activity against Antimonyꢀresistant L. donovani. The potency of selected quinazolines against
antimonyꢀresistant Leishmania was assessed using murine peritoneal macrophages infected with two L.
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donovani clinical strains, BPK206/0 and BPK164/1 (Table 3). Strain BPK164/1 was isolated from a
bone marrow aspirate taken from a Nepalese visceral leishmaniasis patient not responding to antimonial
therapy; the BPK164/1 strain was originally found to be 6ꢀfold resistant to sodium stibogluconate
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
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5
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5
5
5
5
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5
5
6
31
compared to the antimonyꢀsusceptible reference strain BPK206/0. Compounds 15, 16, and 23
displayed similar activity against the antimonyꢀsusceptible and antimonyꢀresistant parasites. The EC50s
0
1
2
3
4
5
6
7
8
9
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9
0
against the clinical strains were 5ꢀ to 19ꢀfold higher than their EC s against βꢀlactamase expressing L.
5
0
donovani (Table 1). There are several possible reasons for the differences in susceptibility observed
between the clinical L. donovani isolates and the βꢀlactamase expressing genetically modified lab strain:
1
) the former strains are from the Indian subcontinent while the lab strain is of African origin.
Antileishmanial drugs display differences in efficacy for treating Indian visceral leishmaniasis
3
2, 33
compared to African visceral leishmaniasis;
strain differences could account for such differential
susceptibility; 2) the clinical strains could have a greater fitness in an in vitro intracellular amastigote
model than the lab adapted strain which has been in axenic culture for many passages, leading to lower
compound susceptibility in the former; 3) the clinical strains were assayed using a different method
under different conditions compared to the βꢀlactamase expressing lab strain, potentially contributing to
distinctions in compound susceptibility. Nevertheless, these data confirm the in vitro activity of
compounds 15, 16 and 23 against clinical strains currently circulating in endemic regions of the Indian
subcontinent.
Table 3
Mechanism of Action. We investigated the possibility that quinazolines inhibit folate metabolism in
18, 23
the parasite since previous studies have shown DHFR inhibition with similar structures.
L. donovani
axenic amastigotes and J774A.1 (mouse macrophage cell line) were adapted to grow in media deficient
in pꢀaminobenzoic acid (PABA) and folic acid prior to susceptibility testing. Susceptibility to
quinazolines and miltefosine, as well as the known antifolate drugs methotrexate and pyrimethamine as
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3
4, 35
positive controls, were assessed in the presence or absence of folinic acid (Figure 3).
The presence
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6
of folinic acid dramatically increased the EC50 values of each of the quinazolines as well as the
methotrexate and pyrimethamine. For example, quinazoline 23 was 6.4 fold less potent in the presence
of 488 nM folinic acid than in completely deficient media. Conversely, efficacy of miltefosine was not
affected by addition of folinic acid to the media. Interestingly, we saw no antagonism of activity of
quinazolines 10, 12, 13, 15, 16 or 23 for the mammalian cell line (J774A.1) in the presence of folinic
acid. For example, the EC of quinazoline 10 was 84.8 ± 2.2 ꢁM and 84.2 ± 2.8 ꢁM in the presence or
0
1
2
3
4
5
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9
0
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9
0
50
absence of 488 nM folinic acid, respectively. In contrast, the efficacy of methotrexate and
pyrimethamine were antagonized significantly by folinic acid in J774A.1. These results are consistent
with the hypothesis that the quinazolines interfere with folate metabolism in L. donovani.
Figure 3
StructureꢀProperty Relationship Studies. In parallel to testing the compounds for antileishmanial
activity in vitro, a structureꢀproperty relationship (SPR) study focusing on log D, aqueous solubility, and
permeability has been conducted with all compounds to assess potential physicochemical liabilities
(
Table 4). Log D3.0 and Log D , the distribution coefficient between octanol and water at pH 3.0 and
7.4
36
pH 7.4, were experimentally determined via a previously described HPLCꢀbased method. Solubility at
pH 7.4 was determined using Biomek FX lab automation workstation with pION µSOL evolution
37
software as reported previously and at pH 2.0 using an inꢀhouse HPLC assay based on UV absorption.
Passive transcellular permeability was assessed in a standard parallel artificial membrane permeability
assay (PAMPA) at pH 7.4 and pH 4.0. Generally, the aqueous solubility, the distribution coefficient Log
D, and the permeability of all quinazolines display a pH dependence (exemplified on Log D or
permeability in Table 4). The permeability is enhanced at neutral pH while the aqueous solubility and
Log D are better at lower pH ranges. However, since the aqueous solubility, the distribution coefficient,
ꢀ6
ꢀ1
and the permeability are within the acceptable ranges (solubility > 20 ꢀM, Pe > 10 x 10 cm·s , 1 <
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Journal of Medicinal Chemistry
Log D < 4), the quinazoline compound series is considered to be suitable for the development of
bioavailable antileishmanial compounds.
1
2
3
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
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Table 4
0
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9
0
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9
0
In Vivo Antileishmanial Efficacy Studies. For the selection of the candidates for in vivo efficacy
against L. donovani, only compounds which displayed submicromolar in vitro activity against L.
donovani with a SI value greater than ten and a balanced combination of good physicochemical
properties were chosen. Among the benzylꢀsubstituted quinazolines 12 ꢀ 17, 15 and 16 appeared to be
the best candidates for in vivo evaluation due to their combination of potency, selectivity, and favorable
physicochemical properties. From the compound subseries substituted at the benzenoid ring, the
physicochemical properties were not discriminatory and hence compound 23 was chosen as a viable
candidate due to its submicromolar EC against L. donovani as well as the outstanding SI value of this
50
compound (> 40).
The three compounds mentioned above were dissolved in an appropriate vehicle (15 and 23 dissolved
in 0.5% methyl cellulose and 0.1% Tween80 in water, 16 dissolved in 45% (w/v) (2ꢀhydroxypropyl)ꢀβꢀ
cyclodextrin solution (HPβCD)) and administered to uninfected BALB/c mice intraperitoneally to
determine a tolerated dose for in vivo efficacy studies. While 15 was well tolerated when given at 30
mg/kg ip for five consecutive days, 16 (slowed breathing) and 23 (hypoactivity) were toxic to animals
when given at the same dosing regimen. Considering the toxicity of 16 and 23 when given at 30 mg/kg
i.p., lower doses of these compounds were administered in subsequent in vivo efficacy studies in a
murine visceral leishmaniasis model (Figure 4). When tested at 5 × 15 mg/kg ip, 23 inhibited liver
parasitemia by 37% compared to the vehicle control. However, 15 did not show significant
antileishmanial efficacy when tested at 5 × 30 mg/kg i.p. There was also no significant difference in the
parasite burden between mice in the group treated with compound 16 (5 × 10 mg/kg i.p.) and the vehicle
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control group (P > 0.05). As expected, the 45% HPβCD vehicle used to solubilize 16 itself resulted in
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2
9
1
8% inhibition of liver parasitemia, consistent with our previous report.
Figure 4
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0
Pharmacokinetics of 16 and 23 After p.o. and i.p. Administration in Mice. Pharmacokinetic
studies were conducted to determine the systemic and target tissue exposures of 16 and 23. The mean
plasma and tissue concentration–time profiles of 16 and 23 after p.o. and i.p. administration are shown
in Figures 5 and 6, respectively. The relevant pharmacokinetic outcomes are listed in Table 5. After p.o.
administration at 100 µmol/kg (or approximately 30 mg/kg), both compounds were absorbed from the
gastrointestinal tract of mice and plasma concentration reached a Cmax of 0.44 and 0.25 ꢁM for 16 and
2
3, respectively. The systemic and tissue exposure of 16 (AUC and Cmax) were considerably greater than
those of 23 (Table 4). After i.p. administration, plasma concentration reached a Cmax of 5.2 and 2.7 ꢁM
for 16 and 23, respectively, before decreasing rapidly in the first 4 h, followed by a slower elimination
process until 24 h. The rapid decline in plasma concentration was likely due to extensive tissue
redistribution after absorption, as indicated by the high target tissue concentrations. The plasma and
tissue exposures after i.p. administration were markedly greater than those after p.o. administration
(Table 4), suggesting significant firstꢀpass metabolism and/or partial gastrointestinal absorption after
oral administration of 16 and 23. The terminal elimination halfꢀlife ranged from 5 to 20 h. Minor
reversible overt toxicity (hypoactivity) was observed after i.p. administration of 16, however more
severe toxicity was observed, unexpectedly, after a single i.p. administration of 23, which warranted
euthanization of some mice within 15 min post dose. As efficacy was evaluated at lower doses than 30
mg/kg to avoid toxicity and dose linearity for pharmacokinetic outcomes were unknown, it is not
possible to directly correlate drug exposure with antileishmanial activity in mice. In addition, it is worth
pointing out that the terminal halfꢀlife of 23 after i.p. administration was considerably shorter than that
after p.o. administration (5 h vs. 20 h; Table 5), and the liver concentration of 23 was detectable at 12
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Journal of Medicinal Chemistry
and 24 h after p.o. administration, whereas it was below detection limit after i.p. administration (Figure
). These observations suggest flipꢀflop pharmacokinetics, where the rate of gastrointestinal absorption
1
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9
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6
is slower than the rate of elimination due to dosage formulations (e.g. sustainedꢀrelease), excipients,
38, 39
physiological factors (e.g. intestinal mobility and pH), and/or drug characteristics.
The dramatic
decrease (~12ꢀfold) in the permeability of 23 from neutral to acidic pH (Table 4) could contribute to the
0
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0
slowed absorption of this compound in the upper gastrointestinal tract. In contrast, the permeability of
1
6 decreased only 3ꢀfold from neutral to acidic pH (Table 4). Furthermore, 23 was metabolized quickly
in the mouse liver microsomes (t1/2 = 9.4 min; Table 5), suggesting that it is possible that the rate of
elimination was greater than the rate of gastrointestinal absoption for 23.
Figure 5
Figure 6
Table 5
Conclusions
2
4
N ,N ꢀdisubstituted quinazolineꢀ2,4ꢀdiamines 1 and 2 were found to display antileishmanial activity
in the single digit micromolar range. Subsequently a total of 28 molecules have been synthesized
systematically by varying the substitutions in the 2ꢀ, 4ꢀ, 5ꢀ, 6ꢀ, 7ꢀ, and/or 8ꢀpositions. All quinazolines
have been tested with the aim to further optimize hit compounds 1 and 2 and to conduct a detailed SAR
study against L. donovani and L. amazonensis. The most potent activities with EC s in the
50
submicromolar range against L. donovani were obtained with quinazolineꢀ2,4ꢀdiamine scaffolds bearing
2
4
2
4
either a N ꢀbenzylꢀN ꢀalkyl/phenyl or a N ꢀispropylꢀN ꢀfurfuryl substituent combination. Furthermore,
although the benzenoid ring of the quinazolineꢀ2,4ꢀdiamine scaffold has been identified to play a
secondary role for efficacy, quinazolines substituted at the 5ꢀ or 6ꢀposition with one methyl or one
methoxy group have also been identified to possess submicromolar EC s against L. donovani. Although
5
0
the quinazolineꢀ2,4ꢀdiamines appear to display some cytotoxicity against the macrophage cell line
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J774A.1 yielding modest SI values, the SI values of the best antileishmanial quinazolines were larger
than 10. In addition, assessment of key physicochemical properties confirmed that the quinazoline's
aqueous solubility, distribution coefficient, and passive transcellular permeability were in acceptable
ranges. These promising results led to efficacy testing of the lead compounds 15, 16 and 23 in an in vivo
murine visceral leishmaniasis model. While compounds 15 and 16 did not have activity translate from in
vitro to in vivo, quinazoline 23 reduced parasitemia by 37% when 15 mg/kg/day were given via the
intraperitoneal route for five consecutive days. Pharmocokinetic studies of compound 23 revealed a
maximum plasma concentration that was threefold higher than the EC and a terminal halfꢀlife of 5
1
2
3
4
5
6
7
8
9
1
1
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0
hours after i.p. administration. Although a clear correlation between in vitro activity, in vitro
physicochemical properties, and in vivo activity is not clearly observed, the potencies of frontrunner
2
4
compounds 15, 16 and 23 in conjunction with favorable physicochemical properties make N ,N ꢀ
disubstituted quinazolineꢀ2,4ꢀdiamines a suitable platform for the future development of antileishmanial
agents. For future compound design to be successful, optimization will not only focus on improving
antileishmanial activity and key physicochemical properties, but also on improving the
pharmacokinetics and SI values for the entire quinazoline compound series.
Experimental Section
Chemistry
General. All reagents and solvents were obtained from Aldrich Chemical Co. and used without
further purification. Anthranilic acids were purchased from SigmaꢀAldrich, Oakwood Products, Inc. or
TCI America. NMR spectra were recorded at ambient temperature on a 250 MHz Bruker, 400 MHz
1
Varian or 500 MHz Varian NMR 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
13
for chloroform (7.26 ppm), methanol (3.31 ppm) and dimethylsulfoxide (2.50 ppm). All C NMR
spectra were reported in ppm relative to the signals for chloroform (77 ppm), methanol (49 ppm) and
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Journal of Medicinal Chemistry
1
1
dimethylsulfoxide (39.5 ppm) with H decoupled observation. Data for H NMR are reported as follows:
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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4
5
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5
5
5
5
5
5
5
5
6
chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, sext =
1
3
sextet, sept = septet, oct = octet m = multiplet), integration and coupling constant (Hz), whereas C
NMR analyses were reported in terms of chemical shift. NMR data was analyzed by using MestReNova
Software ver. 5.3.2ꢀ4936. The purity of the final compounds was determined to be ≥ 95% by high
pressure liquid chromatography (HPLC) using an Agilent 1100 LC/MSDꢀVL with electrospray
ionization. Melting points were determined using a MELꢀTEMP 3.0 instrument and are uncorrected.
Low resolution mass spectra were performed on an Agilent 1100 LC/MSDꢀVL with electrospray
ionization. High 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 preꢀcoated
plates (0.25 mm) from EMD Chemical Inc. and components were visualized by ultraviolet light (254
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
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3
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0
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2
3
4
5
6
7
8
9
0
nm). Reported R was determined for TLC. Silicycle silica gel 230ꢀ400 (particle size 40ꢀ63 ꢁm) mesh
f
was used for all flash column chromatography.
General Procedure A: Cyclization of Anthranilic Acids to the Corresponding Quinazolineꢀ2,4ꢀ
diones: 1 equivalent of anthranilic acid and 3.5 equivalents of urea were mortar and pestled to a powder
o
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 Procedure B: Chlorination of Quinazolineꢀ2,4ꢀdiones to the Corresponding 2,4ꢀ
Dichloroquinazolines: 1 equivalent of quinazolineꢀ2,4ꢀdione and 1 equivalent of N,Nꢀdimethylaniline
were mixed in 12 equivalents of phosphorous oxychloride and the mixture refluxed under an argon
atmosphere until starting material was no longer present by TLC (3ꢀ16 hours). The mixture was then
cooled and added to ice in the amount of ten times the reaction volume. The solution was filtered to give
crude product.
General Procedure C: Amine Substitution of 2,4ꢀDichloroquinazolines to Yield 4ꢀAminoꢀ
substituted 2ꢀChloroquinazolines: 1.1 equivalents of amine and sodium acetate were mixed with 1
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equivalent of 2,4ꢀdichloroquinazoline at 0.1 M concentration in a 3 to 1 mix of tetrahydrofuran and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
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 Na SO . The crude was then purified by either method Ca or Cb:
2
4
Purification Method Ca: the compound was recrystallized with ethanol and water, filtered, and
0
1
2
3
4
5
6
7
8
9
0
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2
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4
5
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4
5
6
7
8
9
0
rinsed with cold ethanol to yield pure product.
Purification Method Cb: the crude was purified by flash chromatography using hexanes and
ethyl acetate.
General Procedure D: Amine Substitution of 4ꢀAminosubstitutedꢀ2ꢀchloroquinazolines to Yield
2
,4ꢀDiaminoꢀsubstituted Quinazolines: 1.5 equivalents of amine was mixed with 1 equivalent of 4ꢀ
aminoꢀsubstituted 2ꢀchloroquinazoline at 0.2 M concentration in ethanol in a sealed tube and heated to
o
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, was filtered, and
rinsed with cold ethanol to yield pure product.
Purification Method Db: solvent was evaporated and the crude mixture was purified by flash
chromatography using dichloromethane and methanol.
2
,4ꢀDichloroquinazoline (1c): commercially available benzoyleneurea (0.12 mol) and N,Nꢀ
dimethylaniline (0.12 mol) were mixed in 60 mL of phosphorous oxychloride and heated to reflux under
an atmosphere of argon. After 5 hours the solution was cooled and slowly added to 300 mL of ice. Once
quenching was finished, the compound was extracted with chloroform (4 x 125 mL) and purified by
flash chromatography using hexanes and ethyl acetate to yield the title compound in 61% yield (14.5 g,
1
7
3 mmol). H NMR (500 MHz, CDCl ) δ 8.29 – 8.24 (m, 1H), 8.02 – 7.99 (m, 2H), 7.74 (ddd, J = 6.3,
3
1
3
5.0, 3.2, 1H). C NMR (126 MHz, CDCl ) δ 164.25, 155.36, 152.60, 136.41, 129.61, 128.25, 126.42,
3
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Journal of Medicinal Chemistry
+
1
22.59. Mass (ESI): [M+H] 199, 201; found 198.9 (100%), 200.9 (64%). R = 0.56 (hexanes to ethyl
f
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
acetate 4:1).
2ꢀChloroꢀNꢀ(furanꢀ2ꢀylmethyl)quinazolinꢀ4ꢀamine (2d): 1g (5.0 mmol) of 1c was reacted with
furfurylamine and purified according to method Ca to furnish 1.21 g (4.7 mmol) of the title compound
1
in 93% yield. H NMR (250 MHz, CDCl ) δ 7.75 – 7.55 (m, 3H), 7.34 (ddd, J = 8.1, 5.4, 2.9 Hz, 1H),
0
1
2
3
4
5
6
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9
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9
0
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9
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8
9
0
1
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9
0
3
1
3
6
1
.48 (s, 1H), 6.30ꢀ6.22 (m, 2H), 4.77 (d, J = 5.1 Hz, 2H). C NMR (63 MHz, CDCl ) δ 160.57, 157.54,
3
50.82, 150.24, 142.56, 133.62, 127.66, 126.32, 121.09, 113.24, 110.65, 108.66, 38.43. Mass (ESI):
+
[M+H] 260; found 260.1. R = 0.14 (hexanes to ethyl acetate 4:1).
f
4
2
N ꢀ(Furanꢀ2ꢀylmethyl)ꢀN ꢀisopropylquinazolineꢀ2,4ꢀdiamine (2): 1.05 g (4.04 mmol) of 2d was
reacted with isopropylamine and purified according to method Da to furnish 0.47 g (1.66 mmol) of the
1
title compound as a light yellow solid in 41% yield. H NMR (500 MHz, CD OD) δ 7.94 (d, J = 8.1 Hz,
3
1
4
H), 7.53 – 7.46 (m, 1H), 7.44 – 7.40 (m, 1H), 7.31 (s, 1H), 7.17 – 7.09 (m, 1H), 6.38 – 6.31 (m, 2H),
13
.78 (s, 2H), 4.26 (sept, J = 6.5 Hz, 1H), 1.27 (d, J = 6.5 Hz, 6H). C NMR (126 MHz, CD OD) δ
3
160.14, 154.22, 151.24, 142.01, 141.97, 134.05, 123.14, 123.04, 118.38, 110.17, 109.87, 107.44, 43.29,
+
3
7.65, 21.62. HRMS: m/z calcd for C H N O [M+H] 283.1553; found 283.1558. R = 0.44 (9:1
1
6
19
4
f
o
dichloromethane to methanol). Melting point 190ꢀ192 C.
4
2
N ꢀ(Furanꢀ2ꢀylmethyl)ꢀN ꢀ(2,2,2ꢀtrifluoroethyl)quinazolineꢀ2,4ꢀdiamine (3): 0.045 g (0.17 mmol)
of 2d was reacted with 2,2,2ꢀtrifluoroethylamine and purified according to method Da to furnish 0.041 g
1
(0.13 mmol) of the title compound as a white crystalline solid in 76% yield. H NMR (500 MHz,
CD OD) δ 8.17 (d, J = 8.2, 1H), 7.79 (dd, J = 8.4, 7.3, 1H), 7.51 (d, J = 7.1, 1H), 7.48 – 7.45 (m, 1H),
3
13
7
.44 (dd, J = 7.3, 1.0, 1H), 6.39 (m, 2H), 4.89 (s, 2H), 4.38 (q, J = 9.0, 2H). C NMR (126 MHz,
CD OD) δ 160.64, 153.51, 150.23, 142.31, 138.72, 135.45, 125.29, 124.36 (q, J = 278.71), 123.64,
3
1
16.87, 110.16, 110.05, 107.89, 41.73 (q, J = 35.03), 38.11. HRMS: m/z calcd for C H F N O
15 14 3 4
+
o
[
M+H] 323.1114; found 323.1122. R = 0.14 (dichloromethane). Decomposed at 225 C.
f
2
ꢀ(4ꢀ(Furanꢀ2ꢀylmethylamino)quinazolinꢀ2ꢀylamino)ethanol (4): 0.12 g (0.46 mmol) of 2d was
reacted with 2ꢀaminoethanol and purified according to method Da to furnish 0.048 g (0.17 mmol) of the
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1
title compound in 37% yield. H NMR (500 MHz, CD OD) δ 8.11 (d, J = 7.6 Hz, 1H), 7.76 (ddd, J =
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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 –
1
3
6.36 (m, 1H), 3.77 (s, 2H), 3.70 (s, 2H). C NMR (101 MHz, CDCl ) δ 159.87, 159.63, 151.51, 150.07,
3
1
42.05, 132.90, 124.21, 121.55, 121.39, 110.88, 110.45, 107.64, 63.77, 44.88, 37.89. HRMS: m/z calcd
+
for C H N O [M+H] 285.1346; found 285.1350. R = 0.24 (dichloromethane to methanol 9:1).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
15 17
4
2
f
o
Melting point 219ꢀ221 C.
Nꢀ2,2,2ꢀTrifluoroethylꢀ2ꢀchloroquinazolinꢀ4ꢀamine (5d): 0.050 g (0.25 mmol) of 1c was reacted
with 2,2,2ꢀtrifluoroethylamine and purified according to method Cb to furnish 0.017 g (0.065 mmol) of
1
the title compound in 26% yield. H NMR (400 MHz, CD OD) δ 8.16 (d, J = 7.2 Hz, 1H), 7.82 (t, J =
3
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). Mass (ESI):
+
[M+H] 235, 237; found 235.0 (100%), 237.1 (33%). R = 0.11 (hexanes to ethyl acetate 4:1).
f
2
4
N ꢀIsopropylꢀN ꢀ(2,2,2ꢀtrifluoroethyl)quinazolineꢀ2,4ꢀdiamine (5): 0.017 g (0.065 mmol) of 5d
was reacted with isopropylamine and purified according to method Db to furnish 0.005 g (0.018 mmol)
1
of the title compound as a white crystalline solid in 28% yield. H NMR (500 MHz, CD OD) δ 7.91 (dd,
3
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),
13
4
1
.38 (q, J = 9.3, 2H), 4.24 (sept, J = 6.5, 1H), 1.26 (d, J = 6.5, 6H). C NMR (126 MHz, CD OD) δ
3
60.87, 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 C H F N [M+H] 285.1322; found 285.1325. Melting point
1
3
16
3
4
o
103ꢀ108 C.
2
ꢀChloroꢀNꢀ(2ꢀ(methylthio)ethyl)quinazolinꢀ4ꢀamine (6d): 0.20 g (1.0 mmol) of 1c was reacted
with 2ꢀ(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, CD OD) δ 7.93 (dd, J = 8.3, 0.8 Hz, 1H), 7.65
3
(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
2
.5, 6.6 Hz, 2H), 2.75 – 2.65 (m, 2H), 2.08 (s, 3H). Mass (ESI): [M+H] 254, 256; found 254.0 (100%),
56.0 (33%). R = 0.17 (hexanes to ethyl acetate 4:1).
f
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Journal of Medicinal Chemistry
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4
N ꢀIsopropylꢀN ꢀ(2ꢀ(methylthio)ethyl)quinazolineꢀ2,4ꢀdiamine (6): 0.12 g (0.47 mmol) of 6d was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reacted with isopropylamine and purified according to method Db to furnish 0.056 g (0.20 mmol) of the
1
title compound as a white solid in 43% yield. H NMR (500 MHz, CD OD) δ 7.81 (d, J = 8.1 Hz, 1H),
3
7
.47 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 8.2 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 4.20 (sept, J = 6.5 Hz, 1H),
13
3
.76 – 3.71 (m, 2H), 2.79 – 2.74 (m, 2H), 2.11 (s, 3H), 1.22 (d, J = 6.5 Hz, 6H). C NMR (126 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CD OD) 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,
3
+
1
4.11. HRMS: m/z calcd for C H N S [M+H] 277.1481; found 277.1488. R = 0.52 (dichloromethane
1
4
21
4
f
o
to methanol 9:1). Melting point 87ꢀ90 C.
Methylꢀ2ꢀ(2ꢀchloroquinazolinꢀ4ꢀylamino)acetate (7d): 0.198 g (0.99 mmol) of 1c was reacted with
glycine methyl ester and purified according to method Ca to furnish 0.18 g (0.72 mmol) of the title
1
compound in 73% yield. H NMR (400 MHz, CD OD) δ 7.85 (d, J = 8.1 Hz, 1H), 7.62 (dd, J = 11.3, 4.1
3
13
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). C NMR (101
MHz, CD OD) δ 170.77, 161.67, 157.12, 150.04, 133.76, 126.38, 125.93, 122.25, 113.24, 51.72, 42.35.
3
Rf = 0.17 (hexanes to ethyl acetate 2:1).
Ethylꢀ2ꢀ(2ꢀ(isopropylamino)quinazolinꢀ4ꢀylamino)acetate (7): 0.12 g (0.47 mmol) of 7d was
reacted with isopropylamine and purified according to method Db to furnish 0.060 g (0.21 mmol) of the
title compound as a white crystalline solid in 44% yield. A transesterification reaction occurred leading
1
to the ethylester product. H NMR (400 MHz, CD OD) δ 7.84 (d, J = 8.2 Hz, 1H), 7.57 – 7.51 (t, J = 7.8
3
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
3
1
1
.21 (d, J = 6.5 Hz, 6H). C NMR (101 MHz, CD OD) δ 172.23, 162.13, 159.29, 150.86, 134.31,
3
23.71, 123.57, 122.61, 111.84, 62.22, 43.87, 43.66, 23.25, 14.52. HRMS: m/z calcd for C H N O
2
1
5
21
4
+
[M+H] 289.1659; found 289.1668. R = 0.34 (dichloromethane to methanol 9:1).
f
2
ꢀChloroꢀNꢀcyclohexylquinazolinꢀ4ꢀamine (8d): 0.16 g (0.80 mmol) of 1c was reacted with
cyclohexylamine and purified according to method Cb to furnish 0.15 g (0.55 mmol) of the title
1
compound in 69% yield. H NMR (250 MHz, CD OD) δ 8.05 – 7.99 (m, 1H), 7.60 (ddd, J = 8.4, 7.0,
3
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), 1.97ꢀ1.84
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3
(
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). C
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NMR (63 MHz, CD OD) δ 161.94, 159.05, 151.35, 134.67, 127.25, 126.90, 123.83, 114.81, 51.68,
3
+
33.32, 26.71, 26.48. Mass (ESI): [M+H] 262, 264; found 262.1 (100%), 264.1 (33%). R = 0.43
f
(hexanes to ethyl acetate 2:1).
4
2
N ꢀCyclohexylꢀN ꢀisopropylquinazolineꢀ2,4ꢀdiamine (8): 0.10 g (0.40 mmol) of 8d was reacted
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with isopropylamine and purified according to method Db to furnish 0.060 g (0.21 mmol) of the title
1
compound as a white crystalline solid in 53% yield. H NMR (400 MHz, CD OD) δ 7.95 (d, J = 8.0 Hz,
3
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,
2
H), 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,
13
J = 6.5 Hz, 6H). C NMR (101 MHz, CD OD) δ 159.82, 157.34, 147.57, 133.16, 122.65, 121.83,
3
+
121.25, 110.84, 50.46, 42.93, 32.26, 25.37, 21.93. HRMS: m/z calcd for C H N [M+H] 285.2074;
1
7
25
4
o
found 285.2077. R = 0.38 (dichloromethane to methanol 9:1). Decomposed at 345 C.
f
N,NꢀDimethylꢀ2ꢀchloroquinazolinꢀ4ꢀamine (9d): 0.054 g (0.27 mmol) of 1c was reacted with
dimethylamine and purified according to method Ca to furnish 0.037 g (0.18 mmol) of the title
1
compound in 69% yield. H NMR (400 MHz, CD OD) δ 8.06 (d, J = 8.5 Hz, 1H), 7.68 – 7.62 (m, 1H),
3
13
7
.52 (d, J = 8.4 Hz, 1H), 7.39 – 7.33 (m, 1H), 3.35 (s, 6H). C NMR (101 MHz, CD OD) δ 163.76,
3
1
56.03, 152.57, 133.10, 126.41, 125.89, 124.83, 114.07, 41.20. R = 0.35 (hexanes to ethyl acetate 4:1).
f
2
4
4
N ꢀIsopropylꢀN N ꢀdimethylquinazolineꢀ2,4ꢀdiamine (9): 0.030 g (0.14 mmol) of 9d was reacted
,
with isopropylamine and purified according to method Db to furnish 0.015 g (0.065 mmol) of the title
1
compound as a white solid in 46% yield. H NMR (400 MHz, CD OD) δ 8.11 (d, J = 8.3 Hz, 1H), 7.69
3
(t, J = 8.2 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.31 (t, J = 8.2 Hz, 1H), 4.31 – 4.19 (m, 1H), 3.48 (s, 6H),
1
3
1.29 (d, J = 6.5 Hz, 6H). C NMR (101 MHz, CD OD) δ 162.97, 152.20, 143.16, 134.25, 127.59,
3
+
1
22.97, 117.82, 110.60, 43.69, 41.45, 21.46. HRMS: m/z calcd for C H N [M+H] 231.1604; found
1
3
19
4
o
2
31.1608. R = 0.33 (dichloromethane to methanol 9:1). Melting point 110ꢀ115 C.
f
NꢀBenzylꢀ2ꢀchloroquinazolinꢀ4ꢀamine (10d): 0.30g (1.5 mmol) of 1c 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.
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H NMR (400 MHz, CDCl ) δ 7.70 (ddd, J = 11.3, 8.4, 4.9 Hz, 3H), 7.48 – 7.26 (m, 5H), 6.27 (s, 1H),
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
4
.83 (d, J = 5.3 Hz, 2H). C NMR (101 MHz, CDCl ) δ 160.93, 157.93, 151.09, 137.48, 133.76,
3
129.11, 128.51, 128.21, 128.00, 126.46, 121.07, 113.38, 45.94. R = 0.21 (hexanes to ethyl acetate 4:1).
f
2
4
N ,N ꢀDibenzylquinazolineꢀ2,4ꢀdiamine (10): 0.10 g (0.37 mmol) of 10d was reacted with
benzylamine and purified according to method Da to furnish 0.101 g (0.297 mmol) of the title
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
compound as a white crystalline solid in 80% yield. H NMR (500 MHz, DMSOꢀd6) 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
1
3
= 7.8, 1.3 Hz, 1H), 7.36ꢀ7.12 (m, 10H), 4.75 (d, J = 5.5 Hz, 2H), 4.63 (d, J = 4.8 Hz, 2H). C NMR
(
126 MHz, DMSOꢀd6) δ 160.26, 153.49, 139.44, 138.85, 138.29, 135.67, 129.93, 128.85, 128.80,
1
28.24, 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 C H N [M+H] 341.1761; found 341.1751. R = 0.45 (dichloromethane to methanol 9:1).
2
2
21
4
f
o
Melting point 237ꢀ240 C.
NꢀButylꢀ2ꢀchloroquinazolinꢀ4ꢀamine (11d): 0.48 g (2.4 mmol) of 1c was reacted with nꢀbutylamine
and purified according to method Ca to furnish 0.49 g (2.1 mmol) of the title compound in 86% yield.
1
H NMR (250 MHz, CDCl ) δ 7.77 – 7.53 (m, 3H), 7.44 – 7.31 (m, 1H), 5.82 (s, NH), 3.61 (td, J = 7.1,
3
1
3
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). C NMR (101
MHz, CDCl ) δ 161.14, 150.99, 133.58, 128.10, 126.29, 121.19, 120.79, 113.42, 41.57, 31.47, 20.35,
3
+
1
4.02. Mass (ESI): [M+H] 236, 238; found 236.0 (100%), 238.0 (34%). R = 0.21 (hexanes to ethyl
f
o
acetate 4:1). Melting point 103ꢀ105 C.
2
4
N ,N ꢀDibutylquinazolineꢀ2,4ꢀdiamine (11): 0.080 g (0.34 mmol) of 11d was reacted with nꢀ
butylamine and purified according to method Da to furnish 0.031 g (0.114 mmol) of the title compound
1
as a white solid in 34% yield. H NMR (500 MHz, DMSOꢀd6) δ 9.78 (s, NH), 8.36 (d, J = 8.5 Hz, 1H),
8
3
7
.14 (s, NH), 7.76 (t, J = 8.0 Hz, 1H), 7.42 (d, J = 6.6 Hz, 1H), 7.37 (t, J = 6.2 Hz, 1H), 3.58 (m, 2H),
.45 (m, 2H), 1.65 (p, J = 7.5 Hz, 2H), 1.57 (p, J = 7.5, 2H), 1.36 (sept, J = 7.3 Hz, 4H), 0.92 (td, J =
1
3
.3, 4.7 Hz, 6H). C NMR (101 MHz, DMSOꢀd6) δ 160.10, 153.50, 139.26, 135.43, 124.83, 124.36,
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16.97, 110.02, 41.37, 40.72, 31.49, 30.69, 20.16, 19.93, 14.12, 14.07. HRMS: m/z calcd for C H N
16 25 4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
o
[
M+H] 273.2074; found 273.2072. Melting point 151ꢀ154 C.
4
2
N ꢀBenzylꢀN ꢀmethylquinazolineꢀ2,4ꢀdiamine (12): 0.088 g (0.326 mmol) of 10d was reacted with
2
.0 M methylamine in methanol and purified according to method Da to furnish 0.085 g (0.321 mmol)
1
of the title compound as a cream colored solid in 98% yield. H NMR (400 MHz, CDCl ) δ 8.29 (s, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7
.42 (d, J = 7.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 9.0 Hz, 1H), 7.20 – 7.11 (m, 3H), 7.06 (t,
13
J = 7.6 Hz, 1H), 4.80 (s, 2H), 2.93 (s, 3H). C NMR (101 MHz, CDCl ) δ 160.11, 156.03, 142.69,
3
138.07, 133.96, 128.35, 127.94, 127.26, 123.74, 123.10, 119.20, 110.28, 45.06, 28.01. HRMS: m/z
+
calcd for C H N [M+H] 265.1448; found 265.1454. R = 0.47 (dichloromethane to methanol 9:1).
1
6
17
4
f
4
2
N ꢀBenzylꢀN ꢀbutylquinazolineꢀ2,4ꢀdiamine (13): 0.10 g (0.37 mmol) of 10d was reacted with nꢀ
butylamine and purified according to method Da to furnish 0.047 g (0.15 mmol) of the title compound
1
as a white solid in 41% yield. H NMR (400 MHz, CDCl ) δ 9.54 (s, 1H), 8.43 (d, J = 8.1 Hz, 1H), 7.82
3
(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
3
1.49 (p, J = 7.3 Hz, 2H), 1.31 (sext, J = 7.3 Hz, 2H), 0.85 (t, J = 7.3 Hz, 3H). C NMR (101 MHz,
CDCl ) δ 160.08, 153.46, 137.43, 134.65, 128.41, 127.83, 127.40, 124.41, 124.12, 116.62, 109.99,
3
+
1
09.68, 45.31, 41.02, 31.30, 19.95, 13.66. HRMS: m/z calcd for C H N [M+H] 307.1917; found
19 23
4
o
3
07.1926. R = 0.33 (dichloromethane to methanol 9:1). Melting point 210ꢀ212 C.
f
4
2
N ꢀBenzylꢀN ꢀphenylquinazolineꢀ2,4ꢀdiamine (14): 0.10 g (0.37 mmol) of 10d was reacted with
aniline and purified according to method Da to furnish 0.117 g (0.358 mmol) of the title compound as a
1
white crystalline solid in 97% yield. H NMR (500 MHz, DMSOꢀd6) δ 10.46 (s, 2H), 8.46 (d, J = 8.3
Hz, 1H), 7.84 (ddd, J = 8.4, 7.3, 1.1 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.48 (td, J = 7.8, 1.0 Hz, 1H),
7.45 – 7.41 (m, 2H), 7.37 – 7.29 (m, 6H), 7.29 – 7.22 (m, 1H), 7.18 (t, J = 7.4 Hz, 1H), 4.76 (d, J = 5.9
13
Hz, 2H). C NMR (126 MHz, DMSOꢀd6) δ 160.71, 151.94, 139.36, 138.02, 137.13, 135.93, 129.41,
1
28.89, 127.78, 127.67, 125.45, 125.31, 124.81, 122.71, 117.94, 110.70, 45.14. HRMS: m/z calcd for
+
C H N [M+H] 327.1604; found 327.1613. R = 0.56 (dichloromethane to methanol 9:1).
21
19
4
f
o
Decomposed at 310 C.
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Journal of Medicinal Chemistry
NꢀMethylꢀ2ꢀchloroquinazolinꢀ4ꢀamine (15d): 1.0 g (5.0 mmol) of 1c was reacted with methylamine
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and purified according to method Ca to furnish 0.89 g (4.6 mmol) of the title compound in 92% yield.
1
H NMR (400 MHz, CDCl ) δ 7.74ꢀ7.66 (m, 3H), 7.41 (t, J = 6.9 Hz, 1H), 6.28 (s, NH), 3.20 (d, J = 4.7
3
13
Hz, 3H). C NMR (101 MHz, CDCl ) δ 161.82, 158.02, 150.80, 133.64, 127.90, 126.41, 121.02,
3
+
1
13.60, 28.74. Mass (ESI): [M+H] 194; found 194. R = 0.12 (hexanes to ethyl acetate 4:1).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
f
2
4
N ꢀBenzylꢀN ꢀmethylquinazolineꢀ2,4ꢀdiamine (15): 0.287 g (1.48 mmol) of 15d was reacted with
benzylamine and purified according to method Da to furnish 0.193 g (0.730 mmol) of the title
1
compound as a white crystalline solid in 49% yield. H NMR (250 MHz, CD OD) δ 8.06 (d, J = 8.1 Hz,
3
1
3
1
H), 7.73 (t, J = 8.4 Hz, 1H), 7.50 – 7.20 (m, 7H), 4.75 (s, 2H), 3.14 (s, 3H). C NMR (63 MHz,
CD OD) δ 162.17, 154.54, 140.08, 139.51, 136.27, 129.70, 128.69, 128.59, 125.92, 124.85, 117.86,
3
+
111.30, 45.91, 29.04. HRMS: m/z calcd for C H N [M+H] 265.1453; found 265.1457. R = 0.31
1
6
17
4
f
o
(
dichloromethane to methanol 9:1). Decomposed at 240 C.
2
4
N ꢀBenzylꢀN ꢀbutylquinazolineꢀ2,4ꢀdiamine (16): 0.48 g (2.0 mmol) of 11d was reacted with
benzylamine and purified according to method Da to furnish 0.40 g (1.3 mmol) of the title compound as
1
a white crystalline solid in 65% yield. H NMR (500 MHz, DMSOꢀd6) δ 9.84 (s, 1H), 8.64 (s, 1H), 8.37
(
d, J = 7.7 Hz, 1H), 7.76 (ddd, J = 8.4, 7.3, 1.1 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.4ꢀ7.3 (m, 5H), 7.24
(
t, J = 7.1 Hz, 1H), 4.66 (d, J = 4.4 Hz, 2H), 3.49 (d, J = 6.1 Hz, 2H), 1.61ꢀ1.43 (m, 2H), 1.33ꢀ1.17 (m,
13
2
H), 0.80 (t, J = 6.9 Hz, 3H). C NMR (126 MHz, DMSOꢀd6) δ 160.11, 153.48, 139.24, 139.11,
135.49, 128.83, 127.61, 127.51, 124.83, 124.54, 117.14, 110.15, 44.46, 41.46, 30.62, 20.11, 14.09.
+
HRMS: m/z calcd for C H N [M+H] 307.1923; found 307.1921. R = 0.48 (dichloromethane to
19
23
4
f
o
methanol 9:1). Melting point 194ꢀ196 C.
2ꢀChloroꢀNꢀphenylꢀquinazolinꢀ4ꢀamine (17d): 0.30 g (1.5 mmol) of 1c 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
1
9
3% yield. H NMR (400 MHz, CD OD) δ 8.33 (d, J = 8.3 Hz, 1H), 7.80 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H),
3
7
.78 – 7.73 (m, 2H), 7.64 (dd, J = 8.4, 0.9 Hz, 1H), 7.56 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.40 – 7.34 (m,
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Journal of Medicinal Chemistry
Page 24 of 56
1
3
2
1
H), 7.17 (t, J = 7.4 Hz, 1H). C NMR (101 MHz, CD OD) δ 159.61, 156.96, 150.58, 137.99, 133.70,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
28.28, 126.50, 126.00, 124.67, 122.55, 122.40, 113.65. R = 0.19 (hexanes to ethyl acetate 4:1).
f
2
4
N ꢀBenzylꢀN ꢀphenylquinazolineꢀ2,4ꢀdiamine (17): 0.10 g (0.39 mmol) of 17d was reacted with
benzylamine and purified according to method Da to furnish 0.010 g (0.031 mmol) of the title
1
compound as a white crystalline solid in 10% yield. H NMR (500 MHz, DMSOꢀd6) δ 9.56 (s, 1H), 9.13
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
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), 7.39 (dd, J = 8.4, 7.4 Hz, 2H), 7.27 (ddd, J = 8.1, 7.0, 1.2 Hz,
13
1H), 7.22 (dd, J = 8.4, 7.4 Hz, 2H), 7.13 (t, J = 7.4 Hz, 1H), 6.89 (t, J = 7.3 Hz, 1H), 1.89 (s, 2H). C
NMR (101 MHz, DMSOꢀd6) δ 159.01, 157.09, 152.33, 141.80, 140.10, 133.64, 129.12, 128.92, 126.26,
+
1
24.14, 123.77, 123.10, 122.52, 121.45, 119.58, 112.43, 21.76. HRMS: m/z calcd for C H N [M+H]
21 19 4
327.1604; found 327.1614. R = 0.53 (dichloromethane to methanol 9:1).
f
2
,4,8ꢀTrichloroquinazoline (18c): 0.5 g (2.9 mmol) of commercially available 2ꢀaminoꢀ3ꢀ
chlorobenzoic acid was reacted according to general procedure A to give crude 18b. Without further
purification, 18b was reacted according to general procedure B to give 0.15 g (0.6 mmol) of the crude
+
title compound (20% over two steps). Mass (ESI): [M+H] 233, 235; found 232.9 (100%), 235.0 (86%).
4
2
,8ꢀDichloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀquinazolinꢀ4ꢀamine (18d): 0.10 g (0.43 mmol) of 18c was
reacted with furfurylamine according to general procedure Cb to give 0.11 g (0.37 mmol) of 18d in 87%
1
yield. H NMR (250 MHz, CD OD) δ 7.89 (dd, J = 8.3, 1.2 Hz, 1H), 7.69 (dd, J = 7.7, 1.2 Hz, 1H), 7.33
3
1
3
(dd, J = 1.7, 1.0 Hz, 1H), 7.30 – 7.21 (m, 1H), 6.28 – 6.21 (m, 2H), 4.67 (s, 2H). C NMR (63 MHz,
CD OD) δ 162.61, 159.88, 152.48, 148.51, 143.44, 134.77, 132.04, 127.17, 122.63, 116.31, 111.47,
3
+
1
09.13, 39.00. Mass (ESI): [M+H] 294, 296; found 294.0 (100%), 296.0 (64%).
4
2
8ꢀChloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀN ꢀisopropylquinazolineꢀ2,4ꢀdiamine (18): 0.047 g (0.16 mmol)
of 18d was reacted with isopropylamine according to general procedure Db to give 0.030 g (0.095
1
mmol) of the title compound as a yellow solid in 59% yield. H NMR (400 MHz, DMSOꢀd6) δ 8.38 (s,
NH), 7.97 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.56 (s, 1H), 6.95 (t, J = 7.8 Hz, 1H), 6.65 (s,
NH), 6.42 – 6.36 (m, 1H), 6.32 (s, 1H), 4.70 (d, J = 5.5 Hz, 2H), 4.28ꢀ4.14 (m, 1H), 1.17 (d, J = 5.5 Hz,
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Page 25 of 56
Journal of Medicinal Chemistry
1
3
6
1
H). C NMR (101 MHz, CDCl ) δ 159.87, 158.98, 151.43, 148.83, 142.16, 132.55, 128.98, 119.93,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
19.78, 111.79, 110.46, 107.66, 42.93, 38.15, 22.99.HRMS: m/z calcd for C H ClN O [M+H]
1
6
17
4
o
317.1164; found 317.1173. Decomposed at 150 C.
2
,4,7ꢀTrichloroquinazoline (19c): 0.75 g (4.4 mmol) of commercially available 2ꢀaminoꢀ4ꢀ
chlorobenzoic acid was reacted according to general procedure A to give crude 19b. Without further
purification, 19b was reacted according to general procedure B to give 0.64 g (2.7 mmol) of the crude
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
title compound (61% over two steps). Mass (ESI): [M+H] 233, 235; found 232.9 (100%), 235.0 (94%).
Rf = 0.68 (hexanes to ethyl acetate 4:1).
4
2
,7ꢀDichloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀquinazolinꢀ4ꢀamine (19d): 0.30 g (1.3 mmol) of 19c was
reacted with furfurylamine according to general procedure Cb to give 0.35 g (1.2 mmol) of 19d in 92%
1
yield. H NMR (250 MHz, CD OD) δ 8.13 – 8.06 (m, 1H), 7.62 – 7.58 (m, 1H), 7.49 (d, J = 2.1 Hz,
3
13
1
H), 7.47 – 7.42 (m, 1H), 6.37 – 6.35 (m, 2H), 4.80 (s, 2H). C NMR (63 MHz, CD OD) δ 163.24,
3
1
60.01, 156.93, 152.44, 143.49, 140.90, 128.01, 126.34, 125.69, 113.46, 111.47, 109.11, 38.87. Mass
+
(ESI): [M+H] 294, 296; found 294.0 (100%), 296.0 (63%). R = 0.27 (hexanes to ethyl acetate 4:1).
f
4
2
7
ꢀChloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀN ꢀisopropylquinazolineꢀ2,4ꢀdiamine (19): 0.113 g (0.38 mmol)
of 19d was reacted with isopropylamine according to general procedure Db to give 0.085 g (0.27 mmol)
1
of the title compound in 71% yield. H NMR (500 MHz, DMSOꢀd6) δ 8.37 (s, NH), 8.03 (d, J = 8.7 Hz,
1
H), 7.56 – 7.55 (m, 1H), 7.23 (s, 1H), 7.02 (dd, J = 8.7, 1.8 Hz, 1H), 6.57 (s, NH), 6.46 – 6.37 (m, 1H),
1
3
6.34 (s, 1H), 4.70 (d, J = 5.6 Hz, 2H), 4.16 (oct, J = 6.6 Hz, 1H), 1.15 (d, J = 6.5 Hz, 6H). C NMR
(
126 MHz, DMSOꢀd6) δ 159.87, 153.82, 152.94, 142.36, 142.32, 137.40, 125.34, 123.78, 120.26,
+
1
10.95, 110.90, 107.56, 42.22, 37.32, 23.15. HRMS: m/z calcd for C H ClN O [M+H] 317.1164;
1
6
18
4
found 317.1161. R = 0.63 (dichloromethane to methanol 9:1).
f
2
,4,6ꢀTrichloroquinazoline (20c): 1 g (5.8 mmol) of commercially available 2ꢀaminoꢀ5ꢀ
chlorobenzoic acid was reacted according to general procedure A to give crude 20b. Without further
purification, 20b was reacted according to general procedure B to give 0.55 g (2.7 mmol) of the crude
1
title compound (47% over two steps). H NMR (250 MHz, CDCl ) δ 8.17 (d, J = 8.9 Hz, 1H), 7.94 (d, J
3
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Journal of Medicinal Chemistry
Page 26 of 56
1
3
=
2.0 Hz, 1H), 7.66 (dd, J = 8.9, 2.0 Hz, 1H). C NMR (63 MHz, CDCl ) δ 163.85, 156.31, 152.68,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
42.97, 130.45, 127.37, 127.07, 120.77. Mass (ESI): [M+H] 233, 235; found 232.9 (100%), 235.0
(98%). R = 0.68 (hexanes to ethyl acetate 4:1).
f
4
2
,6ꢀDichloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀquinazolinꢀ4ꢀamine (20d): 0.30 g (1.3 mmol) of 20c was
reacted with furfurylamine according to general procedure Cb to give 0.072 g (0.24 mmol) of 20d in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
8% yield. H NMR (400 MHz, DMSOꢀd6) δ 9.23 (t, J = 5.1 Hz, NH), 8.44 (d, J = 2.1 Hz, 1H), 7.78
13
(
dd, J = 8.9, 2.1 Hz, 1H), 7.65 – 7.54 (m, 2H), 6.42 – 6.34 (m, 2H), 4.69 (d, J = 5.4 Hz, 2H). C NMR
(63 MHz, CDCl ) δ 160.42, 158.82, 151.67, 139.79, 136.95, 128.95, 128.39, 128.14, 127.04, 126.91,
3
+
1
22.42, 111.58, 45.86
.
Mass (ESI): [M+H] 294, 296; found 294.0 (100%), 296.0 (67%). R = 0.27
f
(hexanes to ethyl acetate 4:1).
4
2
6ꢀChloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀN ꢀisopropylquinazolineꢀ2,4ꢀdiamine (20): 0.041 g (0.14 mmol)
of 20d was reacted with isopropylamine according to general procedure Db to give 0.026 g (0.082
1
mmol) of the title compound in 59% yield. H NMR (400 MHz, DMSOꢀd6) δ 8.36 (s, NH), 8.15 (d, J =
2.3 Hz, 1H), 7.57 (d, J = 0.7 Hz, 1H), 7.46 (dd, J = 8.9, 2.3 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 6.56 (s,
NH), 6.39 (dd, J = 3.0, 1.9 Hz, 1H), 6.34 (s, 1H), 4.68 (d, J = 5.4 Hz, 2H), 4.14 (oct, J = 6.6 Hz, 1H),
1
3
1
1
.14 (d, J = 6.5 Hz, 6H). C NMR (101 MHz, CDCl ) δ 159.22, 158.96, 151.64, 150.51, 142.01,
3
33.02, 126.36, 125.48, 120.96, 111.58, 110.43, 107.59, 42.72, 37.84, 23.16. HRMS: m/z calcd for
+
C H ClN O [M+H] 317.1164; found 317.1165.
16
17
4
2,4,5ꢀTrichloroquinazoline (21c): 0.36 g (2.1 mmol) of commercially available 2ꢀaminoꢀ6ꢀ
chlorobenzoic acid was reacted according to general procedure A to give crude 21b. Without further
purification, 21b was reacted according to general procedure B to give 0.12 g (0.51 mmol) of the crude
+
title compound (24% over two steps). Mass (ESI): [M+H] 233, 235; found 235.0 (100%), 232.9 (84%).
Rf = 0.68 (hexanes to ethyl acetate 4:1).
4
2
5
ꢀChloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀN ꢀisopropylquinazolineꢀ2,4ꢀdiamine (21): 0.045 g (0.15 mmol)
of 21c was reacted with furfurylamine according to general procedure Cb. 21d was reacted with
isopropylamine according to general procedure Db to give 0.006 g (0.019 mmol) of the title compound
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Journal of Medicinal Chemistry
1
in 13% yield over two steps. H NMR (400 MHz, CDCl ) δ 7.78 (s, 1H), 7.35 (dd, J = 1.8, 0.8 Hz, 1H),
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
.28 – 7.24 (m, 2H), 6.98 – 6.88 (m, 1H), 6.31 (dd, J = 3.2, 1.9 Hz, 1H), 6.26 (dd, J = 3.2, 0.8 Hz, 1H),
1
3
4.93 (s, 1H), 4.73 (d, J = 5.0 Hz, 2H), 4.22 (oct, J = 6.5 Hz, 1H), 1.21 (d, J = 6.5 Hz, 6H). C NMR (101
MHz, CDCl ) δ 159.17, 158.25, 155.04, 151.58, 142.07, 131.75, 128.92, 124.92, 122.93, 110.40,
3
+
1
08.71, 107.17, 42.66, 38.54, 23.17. HRMS: m/z calcd for C H ClN O [M+H] 317.1164; found
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16 18
4
3
17.1169.
,4,ꢀDichloroꢀ8ꢀmethylquinazoline (22c): 1.0 g (6.6 mmol) of commercially available 2ꢀaminoꢀ3ꢀ
2
methylbenzoic acid was reacted according to general procedure A to give crude 22b. Without further
purification, 22b was reacted according to general procedure B to give 1.1 g (5.2 mmol) of the crude
+
title compound (78% over two steps). Mass (ESI): [M+H] 213, 215; found 213.0 (100%), 215.0 (73%).
Rf = 0.67 (hexanes to ethyl acetate 4:1).
4
2
ꢀChloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀ8ꢀmethylquinazolinꢀ4ꢀamine (22d): 0.50 g (2.3 mmol) of 22c was
reacted with furfurylamine according to general procedure Cb to give 0.43 g (1.6 mmol) of 22d in 70%
1
yield. H NMR (400 MHz, DMSOꢀd6) δ 9.08 (t, J = 5.5 Hz, NH), 8.07 (d, J = 8.2 Hz, 1H), 7.58 – 7.53
(m, 2H), 7.33 (t, J = 7.7 Hz, 1H), 6.39 – 6.35 (m, 1H), 6.32 (d, J = 3.0 Hz, 1H), 4.70 (d, J = 5.5 Hz, 2H),
1
3
2
1
2
.45 (s, 3H). C NMR (101 MHz, DMSOꢀd6) δ 161.92, 156.75, 152.08, 149.93, 142.85, 135.39,
+
34.26, 126.11, 121.26, 113.88, 111.21, 108.30, 38.10, 17.88. Mass (ESI): [M+H] 274, 276; found
74.1 (100%), 276.1 (35%). R = 0.33 (hexanes to ethyl acetate 4:1).
f
4
2
N ꢀ(Furanꢀ2ꢀylmethyl)ꢀN ꢀisopropylꢀ8ꢀmethylquinazolineꢀ2,4ꢀdiamine (22): 0.13 g (0.47 mmol)
of 22d was reacted with isopropylamine according to general procedure Db to give 0.10 g (0.34 mmol)
1
of the title compound as a yellow solid in 74% yield. H NMR (400 MHz, CDCl ) δ 8.87 (s, 1H), 8.53
3
(s, 1H), 7.94 (s, 1H), 7.35 (d, J = 7.3 Hz, 1H), 7.22 – 7.21 (m, 1H), 7.04 (t, J = 7.7 Hz, 1H), 6.27 – 6.19
13
(m, 2H), 4.80 (d, J = 5.2 Hz, 2H), 4.22 (oct, J = 6.7 Hz, 1H), 1.25 (d, J = 6.6 Hz, 6H). C NMR (101
MHz, CDCl ) δ 160.37, 153.04, 150.27, 142.20, 138.12, 135.91, 126.12, 123.57, 121.16, 110.46,
3
+
1
09.19, 108.09, 43.89, 38.50, 22.34, 18.07. HRMS: m/z calcd for C H N O [M+H] 297.1710; found
1
7
21
4
o
2
97.1712. R = 0.50 (dichloromethane to methanol 9:1). Decomposed at 150 C.
f
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Journal of Medicinal Chemistry
Page 28 of 56
2
,4ꢀDichloroꢀ7ꢀmethylquinazoline (23c): 0.5 g (3.3 mmol) of commercially available 2ꢀaminoꢀ4ꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
methylbenzoic acid was reacted according to general procedure A to give crude 23b. Without further
purification, 23b was reacted according to general procedure B to give 0.34 g (1.6 mmol) of the crude
+
title compound (48% over two steps). Mass (ESI): [M+H] 213, 215; found 213.0 (100%), 215.0 (63%).
Rf = 0.55 (hexanes to ethyl acetate 4:1).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
2
ꢀChloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀ7ꢀmethylquinazolinꢀ4ꢀamine (23d): 0.20 g (0.94 mmol) of 23c
was reacted with furfurylamine according to general procedure Cb to give 0.091 g (0.33 mmol) of 23d
1
in 35% yield. H NMR (400 MHz, CD OD) δ 7.91 (d, J = 8.4 Hz, 1H), 7.40 (s, 1H), 7.34 (s, 1H), 7.27
3
1
3
(
d, J = 8.5 Hz, 1H), 6.32 (d, J = 1.2 Hz, 2H), 4.74 (s, 2H), 2.43 (s, 3H). C NMR (101 MHz, CD OD) δ
3
1
61.28, 157.55, 151.56, 150.46, 144.94, 142.11, 128.03, 125.11, 122.27, 111.37, 110.21, 107.66, 37.53,
+
20.59. Mass (ESI): [M+H] 274, 276; found 274.1 (100%), 276.1 (36%). R = 0.32 (hexanes to ethyl
f
acetate 4:1).
4
2
N ꢀ(Furanꢀ2ꢀylmethyl)ꢀN ꢀisopropylꢀ7ꢀmethylquinazolineꢀ2,4ꢀdiamine (23): 0.068 g (0.25 mmol)
of 23d was reacted with isopropylamine according to general procedure Db to give 0.030 g (0.10 mmol)
1
of the title compound in 40% yield. H NMR (250 MHz, DMSOꢀd6) δ 10.00 (s, NH), 8.21 (d, J = 7.6
Hz, 1H), 8.04 (s, 1H), 7.60 (d, J = 1.3 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.43ꢀ6.32 (m, 2H), 4.77 (d, J =
1
3
4
1
.9 Hz, 2H), 4.32ꢀ4.16 (m, 1H), 2.41 (s, 3H), 1.22 (d, J = 6.5 Hz, 6H). C NMR (63 MHz, DMSOꢀd6) δ
59.59, 151.02, 145.75, 142.30, 138.22, 135.88, 125.02, 124.02, 116.51, 112.23, 110.54, 107.67, 42.88,
+
37.55, 22.21, 21.34. HRMS: m/z calcd for C H N O [M+H] 297.1710; found 297.1716. R = 0.44
1
7
21
4
f
(
dichloromethane to methanol 9:1).
2
,4,ꢀDichloroꢀ6ꢀmethylquinazoline (24c): 1 g (6.6 mmol) of commercially available 2ꢀaminoꢀ5ꢀ
methylbenzoic acid was reacted according to general procedure A to give crude 24b. Without further
purification, 24b was reacted according to general procedure B to give 0.34 g (1.6 mmol) of the crude
+
title compound (24% over two steps). Mass (ESI): [M+H] 213; found 213.
4
2
ꢀChloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀ6ꢀmethylquinazolinꢀ4ꢀamine (24d): 0.32 g (1.5 mmol) of 24c was
reacted with furfurylamine according to general procedure Cb to give 0.19 g (0.69 mmol) of 24d in 46%
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Page 29 of 56
Journal of Medicinal Chemistry
1
yield. H NMR (400 MHz, CD OD) δ 7.79 (s, 1H), 7.50 (dd, J = 8.5, 1.3 Hz, 1H), 7.41 (d, J = 8.3 Hz,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
2
H), 6.33 (d, J = 1.0 Hz, 2H), 4.74 (s, 2H), 2.41 (s, 3H). C NMR (101 MHz, CD OD) δ 161.06,
3
156.69, 151.55, 148.37, 142.11, 136.74, 135.25, 125.55, 121.59, 113.35, 110.21, 107.68, 37.56, 20.30.
+
Mass (ESI): [M+H] 274, 276; found 274.1 (100%), 276.1 (33%).
4
2
N ꢀ(Furanꢀ2ꢀylmethyl)ꢀN ꢀisopropylꢀ6ꢀmethylquinazolineꢀ2,4ꢀdiamine (24): 0.114 g (0.42 mmol)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of 24d was reacted with isopropylamine according to general procedure Db to give 0.105 g (0.35 mmol)
1
of the title compound as a yellow solid in 83% yield. H NMR (400 MHz, CDCl ) δ 7.51 (s, 1H), 7.40 –
3
7.29 (m, 3H), 6.33 (dd, J = 3.2, 1.9 Hz, 1H), 6.31 (d, J = 3.1 Hz, 1H), 4.82 (s, 2H), 4.27 (oct, J = 6.6 Hz,
13
1
H), 2.36 (s, 3H), 1.27 (d, J = 6.5 Hz, 6H). C NMR (101 MHz, CDCl ) δ 159.79, 154.12, 150.67,
3
1
42.21, 135.81, 133.04, 122.06, 119.30, 110.46, 109.44, 107.96, 43.45, 38.27, 22.63, 21.05.HRMS: m/z
+
calcd for C H N O [M+H] 297.1710; found 297.1712. R = 0.43 (dichloromethane to methanol 9:1).
1
7
21
4
f
o
Melting point 200ꢀ204 C.
2
,4,ꢀDichloroꢀ5ꢀmethylquinazoline (25c): 1 g (6.6 mmol) of commercially available 2ꢀaminoꢀ6ꢀ
methylbenzoic acid was reacted according to general procedure A to give crude 25b. Without further
purification, 25b was reacted according to general procedure B to give 0.60 g (2.8 mmol) of the crude
+
title compound (42% over two steps). Mass (ESI): [M+H] 213, 215; found 213.0 (100%), 215.0 (71%).
Rf = 0.77 (hexanes to ethyl acetate 1:1).
4
2
ꢀChloroꢀN ꢀ(furanꢀ2ꢀylmethyl)ꢀ5ꢀmethylquinazolinꢀ4ꢀamine (25d): 0.30 g (1.4 mmol) of 25c was
reacted with furfurylamine according to general procedure Cb to give 0.055 g (0.20 mmol) of 25d in
1
1
4% yield. H NMR (400 MHz, CD OD) δ 7.59 – 7.54 (m, 1H), 7.45 – 7.34 (m, 2H), 7.25 (d, J = 7.1
3
1
3
Hz, 1H), 6.36 (s, 2H), 4.79 (s, 2H), 2.82 (s, 3H). C NMR (101 MHz, CD OD) δ 162.25, 152.30,
3
142.08, 134.99, 134.03, 132.97, 129.84, 129.37, 126.90, 124.28, 110.25, 107.61, 38.32, 22.20. Mass
+
(
ESI): [M+H] 274, 276; found 274.1 (100%), 276.1 (33%). R = 0.26 (hexanes to ethyl acetate 4:1).
f
4
2
N ꢀ(Furanꢀ2ꢀylmethyl)ꢀN ꢀisopropylꢀ5ꢀmethylquinazolineꢀ2,4ꢀdiamine (25): 0.10 g (0.37 mmol)
of 25d was reacted with isopropylamine according to general procedure Db to give 0.066 g (0.22 mmol)
1
of the title compound as a yellow solid in 60% yield. H NMR (400 MHz, CD OD) δ 7.41 (d, J = 1.0
3
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