Development of (E)-2-((1,4-Dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide, ML336: Novel 2-Amidinophenylbenzamides as Potent Inhibitors of Venezuelan Equine Encephalitis Virus

Article abstract of DOI:10.1021/jm501203v

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort wa

Full text of DOI:10.1021/jm501203v

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Article
Development of (E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-
nitro-N-phenylbenzamide, ML336: novel 2-amidinophenylbenzamides
as potent inhibitors of Venezuelan Equine Encephalitis Virus
Chad E. Schroeder, Tuanli Yao, Julie Sotsky, Robert A. Smith, Sudeshna Roy, Yong-Kyu Chu, Haixun
Guo, Nichole A. Tower, James W. Noah, Sarah McKellip, Melinda Sosa, Lynn Rasmussen, Layton H.
Smith, E. Lucile White, Jeffrey Aubé, Colleen B. Jonsson, Donghoon Chung, and Jennifer E. Golden
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501203v • Publication Date (Web): 22 Sep 2014
Downloaded from http://pubs.acs.org on September 28, 2014
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Development of (E)ꢀ2ꢀ((1,4ꢀdimethylpiperazinꢀ2ꢀ
ylidene)amino)ꢀ5ꢀnitroꢀNꢀphenylbenzamide,
ML336: novel 2ꢀamidinophenylbenzamides as
potent inhibitors of Venezuelan Equine Encephalitis
Virus
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Chad E. Schroeder,§ Tuanli Yao,§ Julie Sotsky,‡ Robert A. Smith,§ Sudeshna Roy,§ YongꢀKyu
Chu,‡ Haixun Guo,‡ Nichole A. Tower,ɸ James W. Noah,ɸ Sara McKellip,ɸ Melinda Sosa,ɸ Lynn
Rasmussen,ɸ Layton H. Smith
Chung,‡ Jennifer E. Golden§*
,
† E. Lucile White,ɸ Jeffrey Aubé,§ Colleen B. Jonsson,‡ Donghoon
§University of Kansas Specialized Chemistry Center, Lawrence, KS 66049
‡Department of Microbiology and Immunology and Center for Predictive Medicine for
Biodefense and Emerging Infectious Diseases, University of Louisville, Louisville, KY 40202
ɸSouthern Research Institute, Birmingham, AL 35205
†Conrad Prebys Center for Chemical Genomics, SanfordꢀBurnham Medical Research Institute at
Lake Nona, Orlando, FL, USA, 32827
*Corresponding author
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ABSTRACT: Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic
alphavirus that can cause significant disease in humans. Given the absence of therapeutic options
available and the significance of VEEV as a weaponized agent, an optimization effort was
initiated around a quinazolinone screening hit 1 with promising cellular antiviral activity (EC50 =
0.8 ꢀM), limited cytotoxic liability (CC50 > 50 ꢀM), and modest in vitro efficacy in reducing
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viral progeny (63ꢀfold at 5 ꢀM). Scaffold optimization revealed a novel rearrangement affording
amidines, specifically compound 45, which was found to potently inhibit several VEEV strains
in the low nanomolar range without cytotoxicity, (EC50 = 0.02ꢀ0.04 ꢀM, CC50 > 50 ꢀM) while
limiting in vitro viral replication (EC90 = 0.17 ꢀM). Brain exposure was observed in mice with
45, significant protection was observed in VEEVꢀinfected mice at 5 mg/kg/day, and viral
replication appeared to be inhibited through interference of viral nonꢀstructural proteins.
INTRODUCTION
Alphaviruses are a group of infectious pathogens found worldwide for which no
therapeutic agents have been successfully developed. Approximately 30 alphaviruses of the
Togaviridae family have been identified, and almost a third contribute significantly to human
disease.1 Most members of the alphavirus genus are transmitted by infected mosquitoes.
Depending on the virus, infection generally manifests in one of two forms.2 Arthritogenic
alphaviruses such as Chikungunya, Ross River, Mayaro, and Sindbis viruses cause fever, rash,
and persistent arthralgia. Eastern, Western, and Venezuelan equine encephalitis viruses (EEEV,
WEEV and VEEV, respectively) are endemic to the American continent and produce a fluꢀlike
illness in human populations that is characterized by fever, headache, myalgia, sore throat,
vomiting, and virusꢀdependent propensity to progress to fatal encephalitis.3,4 Encephalitic
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alphaviruses have garnered increased attention in recent years due to the ease with which they
can be weaponized as biothreats.5 For instance, VEEV was previously investigated as an
incapacitating bioterror agent given that low levels of viral exposure caused illness and that the
virus could be generated in high titer and stored indefinitely.6,7 These features, combined with
the discovery that the virus could be aerosolized, are responsible for VEEV, along with EEEV
and WEEV, being classified as Centers for Disease Control (CDC) and National Institute for
Allergy and Infectious Diseases (NIAID) category B bioterrorism agents.8,9
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VEEV provides an attractive platform to investigate intervention of alphavirus infection.
Virus is difficult to detect in blood and cerebralꢀspinal fluid (CSF) samples from patients
infected with EEEV or WEEV; however, VEEV can be easily found in these and other
biospecimens, thus facilitating the in vivo efficacy assessment of new antiviral agents.10
Additionally, VEEV has a better safety margin than that of EEEV. While some strains of EEEV
have a human mortality rate averaging 50%, fatality due to VEEV infection is rare (< 1%
overall).4,11 Nonetheless, VEEV is a serious pathogen that warrants specific attention in its own
right. Fatality rates in equines can reach up to 85%, and in human populations, up to 14% of
infected patients experience chronic neurological complications that manifest as depression,
behavioral disorders, convulsions, and impairment of coordinated muscular movement.12 Based
on epidemiologic data, the neurological disease is more prevalent and severe in children based.13
While there are no FDAꢀapproved human vaccines or therapeutics available for VEEV (or
any alphavirus), advances in the development of agents targeting encephalitic alphaviruses are
investigated and have recently been reviewed.4 To date, human VEEV vaccines have seen
limited success due to insufficient protection, transient immunity or the incidence of adverse
reactions.14ꢀ19 Several small moleculeꢀderived antiꢀVEEV compounds have been reported,
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though most have uncharacterized mechanisms of action, demonstrate only weak in vitro
inhibition and do not appear to have been pursued further.7,20ꢀ22 An exception includes the
diazodiimine compound, BIOder, an inhibitor of the GSKꢀ3β host protein that governs
proinflammatory responses.23 While this proofꢀofꢀconcept compound showed only partial
efficacy in preventing mice from succumbing to VEEV infection, it demonstrated the potential of
using a small molecule inhibitor in this capacity. Given the health threat posed by alphaviruses
and the absence of available countermeasures, a program aimed at identifying small moleculeꢀ
derived VEEV inhibitors was undertaken by our team.
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RESULTS AND DISCUSSION
Screening and Hit Identification. As part of the Molecular Libraries Probe Production Centers
Network (MLPCN),24 this team launched a screening campaign seeking new structural hits for
development as potent, small molecule inhibitors of VEEV. The high throughput screen (HTS), a
cellꢀbased assay that measured the ability of a compound to inhibit a VEEVꢀinduced cytopathic
effect (CPE), was developed and validated employing TCꢀ83 strain, an attenuated vaccine strain
of wild type Trinidad donkey VEEV.25,26,27 The use of the TCꢀ83 strain was advantageous due to
its high congruency to wild type virus, fewer regulatory restrictions in handling (BLꢀ2 and nonꢀ
select agent); however, it retained high similarity to wildtype with respect to genomic sequence,
replication mechanism, and ability to obtain high viral titer (> 10e9 pfu/mL) compared to that
observed with wild type virus.26 A total of 348,140 compounds from the Molecular Libraries
Small Molecule Repository (MLSMR) were evaluated at a single concentration of 20 ꢀM,
resulting in 3608 hits that inhibited a VEEVꢀinduced CPE by > 14%. Of these, 90 compounds
were identified through subsequent antiviral doseꢀresponse experiments and cellular cytotoxicity
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(Vero 76 cells) evaluation that resulted in > 30% inhibition, CPE EC50 < 12.5 ꢀM, and CC50 > 25
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9
ꢀM, respectively. The hit list was further refined to five compounds based on lack of reactive
functionality, synthetic feasibility, PubChem promiscuity28 analysis, acceptable aqueous
solubility in PBS buffer for the most promising scaffolds, and validation of activity from
resynthesized and purified powder samples. Validated hits were further assessed in an in vitro
viral titer reduction assay to quantitatively determine their effect on the production of infectious
progeny virus. The team drafted a set of project criteria that, if reached, would afford a soluble,
cellularꢀacting, small molecule probe that was efficacious in potently attenuating a VEEVꢀ
induced CPE and reduced viral plaques by at least 1000ꢀfold at a concentration of 5 ꢀM. These
properties, coupled with a sufficient window for cytotoxicity and suitable physiochemical
parameters, were expected to deliver an impactful probe compound worthy of in vivo efficacy
studies. The quinazolinone chemotype, represented by hit compound 1, was an attractive starting
point due to its preliminary activity and physiochemical profile. Improvements were explored by
tuning various structural components (shaded regions, Fig. 1).
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Figure 1. Shaded regions of hit compound 1 optimized by structureꢀactivity relationships.
Quinazolinone Chemistry and SAR. The optimization process was driven by the TCꢀ83 CPEꢀ
based dose response assay, cytotoxicity assessment, and titer reduction experiments. The
possibility of performing any future in vivo studies was expected to pivot on the ability of an
antiꢀVEEV compound to reach encephalitic brain tissue.29 As such, careful attention was paid to
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balancing potency in both CPEꢀbased and titer reduction assays with suitable physiochemical
properties such as cLogP, polar surface area (tPSA), molecular weight, and solubility such that
analogs were more likely to be CNSꢀpermeable.30,31 Generally, analogs of 1 were prepared in a
threeꢀstep procedure that involved treating substituted anthranilic acids 2 with chloroacetyl
chloride in the presence of triethylamine to afford 2ꢀ(2ꢀchloroacetamido)ꢀ5ꢀnitrobenzoic acid 3
(Scheme 1).32 Dehydrative amidation was carried out by treating intermediates 3 with a selected
aniline and POCl3.33 The resulting chloromethylquinazolinones 4 were aminated with
alkylpiperazines to give quinazolinones 5.34
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Scheme 1. Synthetic Route for Assembly of Quinazolinone Analogs
Reagents: a) ClCOCH2Cl, NEt3, CH2Cl2, 0 °C to rt, 95%; b) substituted aniline, POCl3, acetonitrile, MWI, 150 °C,
56ꢀ70%; c) KI, K2CO3, alkylpiperazine, acetonitrile, MWI, 80 °C, 25ꢀ80%.
Survey of the scaffold began with determining the necessity of the C6ꢀnitro functionality.
Migration of the C6ꢀnitro group to any other of the available C5ꢀ, C7ꢀ, or C8ꢀpositions afforded
analogs without significant CPE assay potency (> 25 ꢀM). Replacement of the nitro group was
initially investigated on the parent scaffold bearing a 2ꢀfluorophenyl moiety; however, once it
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was determined that removal of the fluorine substituent from the phenyl group improved potency
threeꢀfold and substantially enhanced reduction in viral titer, subsequent generations of analogs
were made with this alteration (Table 1, entries 1 vs. 4). Substitution of the nitro group with a
hydrogen atom or a trifluoromethyl group resulted in loss of CPE assay activity (entries 2ꢀ3).
Several replacements were studied, including a carboxylic acid, tetrazole, sulfonic acid, and
amide isosteres; however, these changes were not tolerated with the exception of nitrile analog
10 (entry 6) which lost some potency and was inferior in reducing viral titer compared to hit
compound 1.
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Table 1. Structure-Activity Relationships Involving the Quinazolinone C6-Nitro Group
VEEV
(TCꢀ83)
CPE
selectivity
index
(CC50/EC50)
VEEV
titer,c,d
log
cytotoxicity
entry compound
cLogPe
(CC50, ꢀM)b
(EC50,
reduction
ꢀM)a
R1
NO2
H
CF3
NO2
I
CN
R2
1
2
3
4
5
6
7
8
9
10
11
12
13
2ꢀFꢀphenyl
2ꢀFꢀphenyl
2ꢀFꢀphenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
0.8
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 62.5
NA
NA
> 166.7
> 2.9
> 45.5
NA
NA
NA
NA
NA
1.8
NT
NT
> 7.8
0.3
3.4
3.6
4.5
3.3
4.6
2.9
ꢀ1.6
1.2
3.0
2.4
2.2
4.1
1
6
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8
> 25.0
> 25.0
0.3
17.1
1.1
> 25.0
> 25.0
> 25.0
> 25.0
> 25.0
> 25.0
> 25.0
9
1.7
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17
SO3H
CO2H
NHꢀtetrazole
CONH2
CONMe2
2ꢀpyridyl
3ꢀpyridyl
NT
NT
NT
NT
NT
NT
NT
phenyl
phenyl
NA
phenyl
NA
3.9
aData were an average of > 3 experiments; Data were an average of >2 experiments; NT = not tested; Data were
collected using 5 ꢀM of compound, and data was analyzed using Microsoft Excel 2010; eData were calculated using
SYBYL 8.0, Tripos Associates, St. Louis MO, 2010.
b
c
d
Given the potency dependency on the presence of the C6 nitro functionality, a majority of the
SAR was investigated with this moiety preserved, and attention shifted to modifications of the Nꢀ
ethyl portion of the piperazine appendage (XꢀY, Table 2). Replacement of the Nꢀethyl group for
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Nꢀmethyl did not impact the CPE assay, but profoundly improved the reduction of viral plaques
(entry 1). The introduction of a larger Nꢀisopropyl substituent adversely affected potency,
suggesting a spacial constraint in the binding site (entry 2). The loss of potency associated with
the Nꢀphenyl analog 20 also supported a steric argument; however, the observed effect may have
been due, at least in part, to the preference of a more basic amine capable of having beneficial
interactions with proximal binding residues. To address this ambiguity, NꢀH analog 21 was
prepared and found to be superior to any of its predecessors in the study with respect to CPE
potency (EC50 = 0.2 ꢀM) and titer reduction efficacy (> 7.8 log). Furthermore, the analogs
bearing nonꢀbasic, Nꢀethyl replacements (e.g., morpholine 22, piperidine 23, and amide 24) were
determined to possess significantly inferior activity profiles compared to NꢀH analog 21. These
combined results suggested that there is a strong preference for a basic NꢀH moiety in this region
of the scaffold.
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In a parallel effort the effect of changing the 2ꢀfluorophenyl amide substituent was also surveyed
(Table 2, R2). Compared to the hit 1, a dramatic increase in titer reduction was observed by
incorporating a 3ꢀfluorophenyl group (25, entry 8); however, simplifying to a nonꢀsubstituted
phenyl ring advantageously produced analog 8 with similar overall activity to analog 21.
Exchange of the phenyl group for alkyl groups of various sizes or a hydrogen atom was not
tolerated (27-30, entries 11ꢀ14). Other significant findings included the hybrid R2ꢀphenylꢀ
containing, NꢀH piperazine 31 and 4ꢀmethoxyphenyl derivative 34 which both produced a
favorable reduction in CPE and viral plaques that was comparable to analog 21. Gratifyingly,
this endeavor revealed that several analogs could be generated with improved activity as
compared to the hit compound by modulating two distinct regions of the scaffold while
advantageously adjusting molecular weight and cLogP.
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Table 2. Quinazolinone Structure-Activity Relationships Involving the N-aryl and
Piperazine Groups
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9
VEEV
(TCꢀ83)
CPE
selectivity
index
VEEV
titer,d,e
log
cytotoxicity
entry compound
cLogPf
(CC50, ꢀM)b
(CC50/
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22
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(EC50,
EC50)c
reduction
ꢀM)a
R2
2ꢀFꢀphenyl
X
Y
CH2 Nꢀmethyl
1
2
0.8
4.4
> 50.0
> 50.0
> 62.5
> 11.4
7.1
NT
2.9
3.7
18
19
Nꢀiꢀ
propyl
2ꢀFꢀphenyl
CH2
2ꢀFꢀphenyl
2ꢀFꢀphenyl
2ꢀFꢀphenyl
2ꢀFꢀphenyl
2ꢀFꢀphenyl
3ꢀFꢀphenyl
4ꢀFꢀphenyl
phenyl
methyl
iꢀpropyl
benzyl
H
phenyl
2ꢀMeOꢀphenyl CH2
3ꢀMeOꢀphenyl CH2
4ꢀMeOꢀphenyl
3ꢀNMe2ꢀphenyl CH2
4ꢀNMe2ꢀphenyl CH2
3ꢀthiophene
CH2 Nꢀphenyl
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
> 25.0
0.2
8.7
4.7
> 25.0
0.4
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
NA
> 250.0
> 5.7
> 10.6
NA
> 125.0
> 62.5
> 166.7
NA
NA
NA
NA
> 250.0
NA
NT
> 7.8
0.3
0.4
NT
7.1
5.9
> 7.8
NT
NT
NT
NT
> 7.8
NT
NT
> 7.4
NT
NT
4.3
2.3
2.3
3.6
2.0
3.4
3.4
3.3
1.3
2.1
3.0
1.5
2.1
2.2
2.2
2.2
2.6
2.6
1.8
20
21
22
23
24
25
26
8
27
28
29
30
31
32
33
34
35
36
37
CH2
CH2
CH2
CO
CH2 Nꢀethyl
CH2 Nꢀethyl
CH2 Nꢀethyl
CH2 Nꢀethyl
CH2 Nꢀethyl
CH2 Nꢀethyl
CH2 Nꢀethyl
NꢀH
O
CH2
NꢀH
0.8
0.3
> 25.0
> 25.0
> 25.0
> 25.0
0.2
> 25.0
5.1
0.2
CH2
NꢀH
NꢀH
NꢀH
NꢀH
NꢀH
NꢀH
9.8
> 250
NA
NA
> 35.7
CH2
> 25.0
> 25.0
1.4
CH2
NꢀH
NT
aData were an average of > 3 experiments; Data were an average of >2 experiments;cNA = not applicable; NT =
not tested; eData were collected using 5 ꢀM of compound, and data was analyzed using Microsoft Excel 2010; fData
were calculated using SYBYL 8.0, Tripos Associates, St. Louis M, 2010.
b
d
More extensive study of the piperazine appendage included excision of the methylene linkage
between the quinazolinone core to provide piperazine 38 and NHꢀpiperidine analog 39 (Table 3),
neither of which led to impressive CPE inhibition. Trading the piperazine group for a
hexahydropyrimidine attachment or ring expanded 1,4ꢀdiazepane moiety only marginally
affected CPE potency as compared to hit 1 (analogs 40 and 41, respectively). Nonetheless, the
team pursued a series of openꢀchain, diamino analogs that are represented by nꢀpropylamine 42.
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Interestingly, 42 showed respectable CPE inhibition (EC50 = 0.6 ꢀM), but it was the only one of
5
its kind that was isolated.
6
7
8
9
Table 3. Quinazolinone Structure-Activity Relationships Involving the Alkylpiperazine
Appendage
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
VEEV
(TCꢀ83) cytotoxicity
CPE
(EC50,
ꢀM)a
selectivity
index
(CC50/EC50)
VEEV
titer,c,d
log
entry compound
cLogPe
(CC50,
ꢀM)b
reduction
R3
1
2
> 25.0
3.6
> 50.0
> 50.0
NA
NT
NT
3.5
3.6
38
39
> 13.9
3
4
5
1.1
0.9
0.6
> 50.0
> 50.0
> 50.0
> 45.5
> 55.6
> 83.3
NT
NT
NT
2.2
2.1
2.4
40
41
42
aData were an average of > 3 experiments; Data are an average of >2 experiments; NT = not tested; Data were
collected using 5 ꢀM of compound, and data was analyzed using Microsoft Excel 2010; eData were calculated using
SYBYL 8.0, Tripos Associates, St. Louis M, 2010.
b
c
d
2-Amidinephenylbenzamide Chemistry. Examination of reactions employing a two carbonꢀ
tethered, acyclic diamine revealed the formation of a rearranged (E)ꢀamidine 45 that was
structurally confirmed by 1H and 13C NMR, NOESY, and 2D NMR spectroscopy (Scheme 2).35
The reaction was initially carried out with monoꢀBOC protected diamines, thus providing a
snapshot of the reaction pathway leading to the amidine product by proceeding through
intermediate 44; however, a oneꢀpot conversion was achieved with symmetrically substituted
diamines to afford 45 in a reasonable 69% yield. Amidines were the only isolated products from
the reaction, and the exclusive isolation of the (E)ꢀconfigured double bond amidine was
presumed to result from electronic and steric contributions in the transition state. Interestingly,
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amidines were generated when chloromethylquinolinone starting materials were treated with
diamines separated by a twoꢀcarbon linkage, but a threeꢀcarbon tethered diamine produced the
expected quinazolinone 42. It is reasoned that amidine formation is favored when the reaction
proceeds through a sixꢀmembered, spirocyclic intermediate such as 46, resulting from an
intramolecular attack of the terminal amine, followed by carbonꢀnitrogen bond cleavage. To our
knowledge, this rearrangement has not been previously reported, and as such, a more
comprehensive examination of the scope of this transformation is underway and will be
disclosed in due course.35
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22
23
24
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Formation of Rearranged Amidines
Reagents: (a) CH3NHCH2CH2NCH3BOC, KI, K2CO3, CH3CN, MWI, 80 °C, 10 min, 35%; (b) TFA, CH2Cl2, rt, 45
min, then aq. NaHCO3, 50%; (c) CH3NHCH2CH2NHCH3, K2CO3, CH3CN, 50 °C, 2 h, 69%.
2-Amidinephenylbenzamide SAR. The conversion of quinazolinone intermediate 43 to amidine
45 represented a fortuitous scaffold modification, resulting in a nearly a 7ꢀfold improvement in
antiviral potency over the best quinazolineꢀbased analogs while preserving the viral titer
reduction capability and nonꢀcytotoxic attributes (45, Table 4). Functional groups that were
optimized for the quinazolinone scaffold showed a correlative enhancement of antiviral potency
for the amidine series, though at an improved, lowꢀnanomolar threshold. While the importance of
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the quinazolinone C6ꢀnitro group to retaining potency translated to the corresponding C5ꢀ
position of the amidine chemotype, it was notable that the nitro group could be migrated to the
amidine C4 position (compound 58) or replaced with a fluorine atom or nitrile group (60 and 63,
respectively) without obliterating activity.
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11
12
13
14
15
16
17
18
19
20
21
22
23
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35
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Amidine Structure-Activity Relationships
VEEV
VEEV
titer,d,e
selectivity
indexc
(TCꢀ83) cytoꢀ
CPE toxicityb
potencya
entry compound
cLogPf
EC50,
ꢀM
CC50,
ꢀM
log
reduction
R4
R5
R6
R7
CC50/EC50
H
H
H
NO2
NO2
NO2
1
2
3
phenyl
phenyl
CH3
0.03
> 50.0 > 1666.7
> 7.2
> 7.2
NT
3.4
4.0
3.1
45
47
48
CH2CH3 0.05
> 50.0 > 1000.0
3ꢀthiophene
CH3
CH3
0.5
> 50.0
> 50.0
100.0
> 1.5
2ꢀCH3Oꢀ
phenyl
H
H
H
NO2
NO2
NO2
4
5
6
33.7
NT
NT
3.4
3.4
3.4
49
50
51
3ꢀCH3Oꢀ
phenyl
CH3
CH3
8.1
> 50.0
> 6.2
4ꢀCH3Oꢀ
phenyl
0.04
> 50.0 > 1250.0
> 7.2
H
H
NO2
NO2
NO2
NO2
NO2
H
7
2ꢀFꢀphenyl
3ꢀFꢀphenyl
4ꢀFꢀphenyl
iꢀpropyl
benzyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
0.2
0.1
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 250.0
> 500.0
> 555.6
NA
> 3.8
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
3.6
3.6
3.6
2.8
3.8
3.3
3.4
3.6
3.6
3.8
4.5
52
53
54
55
56
57
58
59
60
61
62
8
H
9
0.09
> 25.0
3.7
H
10
11
12
13
14
15
16
17
H
> 13.5
NA
H
phenyl
> 25.0
8.4
NO2
F
H
phenyl
> 6.0
NA
H
phenyl
> 25.0
6.4
H
F
phenyl
> 7.8
> 11.4
> 2.2
F
F
phenyl
4.4
H
CF3
phenyl
22.3
H
CN
CN
18
19
phenyl
phenyl
CH3
CH3
0.4
0.1
> 50.0
> 50.0
> 125.0
> 500.0
> 5.8
NT
3.2
3.8
63
64
Cl
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F
CN
CN
20
21
22
23
phenyl
CH3
CH3
CH3
CH3
0.4
3.7
0.8
2.0
> 50.0
> 50.0
> 50.0
> 50.0
> 125.0
> 13.5
> 62.5
> 25.0
NT
NT
3.4
3.3
3.3
3.6
65
66
67
68
H
H
H
2ꢀFꢀphenyl
3ꢀFꢀphenyl
CN
> 1.4
NT
CO2CH3
phenyl
aData were an average of > 3 experiments; Data were an average of > 2experiments;cNA = not applicable; NT =
not tested; eData were collected using 5 ꢀM of compound, and data was analyzed using Microsoft Excel 2010; fData
were calculated using SYBYL 8.0, Tripos Associates, St. Louis M, 2010.
b
d
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46
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50
51
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59
60
The result of this effort revealed a set of amidineꢀderived VEEV inhibitors with exceptional, low
nanomolar antiviral potency, a robust selectivity window and significant titer reduction efficacy.
A subset of the most active representatives from the amidine series and the quinazolinone series
(generally, those with CPE EC50 values ≤ 1 ꢀM), were also evaluated in a CPE assay using wild
type VEEV, Trinidad Donkey (TrD) strain, to confirm activity against a clinically relevant virus.
Gratifyingly, strong agreement was observed across TCꢀ83 and TrD strains for both chemotypes,
especially for the most promising amidines of interest (entries 1ꢀ3, Table 5).
Table 5. Comparison of Cellular Data and Physiochemical Properties of Key Compounds
parameter
compound
comparative data
entry
1
45
51
1
2
3
CPE EC50, TCꢀ83 strain
0.8 ꢀM
0.3 ꢀM
0.6 ꢀM
0.03 ꢀM
0.04 ꢀM
0.02 ꢀM
CPE EC50, V3526 strain
CPE EC50, TrD strain
0.02 ꢀM
0.04 ꢀM
0.05 ꢀM
1.8 log
(69ꢀfold)
0.5 log
(3ꢀfold)
7.2 log
7.2 log
5 ꢀM compound
concentration
4
(16 million fold) (16 million fold)
titer
reduction,
TCꢀ83
7.2 log 7.2 log
1 ꢀM compound
concentration
0.5 ꢀM compound
concentration
5
6
7
8
(16 million fold) (16 million fold)
5.8 log
NT
NT
NT
strain
(630,000 fold)
dose response, EC90
0.17 ꢀM
0.17 ꢀM
no measurable no measurable
viral replication viral replication viral replication
no measurable
5 ꢀM compound
concentration
1 ꢀM compound
concentration
titer
reduction,
TrD strain
2.4 log
(251ꢀfold)
no measurable
viral replication viral replication
no measurable no measurable
no measurable
9
10
NT
0.5 ꢀM compound
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concentration
viral replication viral replication
5
6
7
8
11
12
13
Vero 76 cellular toxicity, CC50
aqueous solubility, PBS buffer
molecular weighta
> 50.0 ꢀM
> 122.0 ꢀg/mL
411.4 g/mol
> 50.0 ꢀM
40.4 ꢀg/mL
367.4 g/mol
99.8 Å2
3.4
1
> 50.0 ꢀM
105.6 ꢀg/mL
397.4 g/mol
14 topological polar surface area, tPSAa
91.0 Å2
3.4
0
109.0 Å2
9
10
11
12
13
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18
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44
45
46
47
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49
50
51
52
53
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57
58
59
60
15
16
17
18
19
20
cLogPb
3.4
1
hydrogen bond donorsc
hydrogen bond acceptorsc
heavy atomsc
7
5
6
30
27
29
0.36
4.1
ligand efficiency, LE
0.28
0.39
lipophilic ligand efficiency, LLE
2.7
4.1
NT = not tested; aData were generated using CambridgeSoft ChemBioDraw, version 12. bData were calculated using
SYBYL 8.0, Tripos Associates, St. Louis M, 2010. cPubChem calculated chemical and physical parameters.
Pharmacology of Compound 45. Compared to the hit quinazolinone 1, analogs 45 and 51
showed the best enhancement of CPE potency while also benefitting from decreased molecular
weight and maintained cLogP. These compounds were also assessed in plaque reduction assays
employing TCꢀ83 or TrD strains at lower compound concentrations than the 5 ꢀM concentration
used during the SAR evaluation phase (entries 4ꢀ10, Table 5). While all three compounds
blocked detectable levels of viral replication of wildtype TrD strain at 5 ꢀM, only the amidines
were effective at 0.5 ꢀM, the lowest concentration tested initially. Assessment of compounds 45
and 51 in a dose response titer reduction assay revealed an EC90 = 0.17 ꢀM for both compounds,
demonstrating that in the 150 nM range, only 1/10 of virus was produced compared to the
control, indicating efficient inhibition of virus replication. While rearrangement of the structural
architecture from quinazolinones to amidines altered the number of hydrogen bond donors and
acceptors, the cLogP values remained constant and improvements in ligand efficiency36 and
lipophilic ligand efficiency37 were due to enhancements in potency alone. Analogs 45 and 51
were relatively evenly matched with respect to in vitro potency and efficacy; however, amidine
45 was slightly favored in terms of reduced molecular weight and topological polar surface area.
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Consequently, amidine 45 was selected as the lead probe compound, ML336, and the compound
was characterized further in terms of its in vitro pharmacology to establish a baseline profile
against which future analogs could be optimized (Table 6). Diminished solubility was observed
in CPE assay medium compared to an assessment in PBS, however, the solubility concentrations
were still well above (~ 1200 to 3700ꢀfold) the observed EC50 of the compound in the CPE assay
(entries 1ꢀ2, table 6). The compound showed exceptional chemical stability in the presence of an
excess of dithiotreitol (DTT). Aqueous stability, assessed initially in PBS, showed some liability,
though this was shown to be an effect of some solubility limitation in that particular medium
(entries 4ꢀ5, Table 6). In mice, compound 45 demonstrated limited liabilities in plasma stability
and moderate plasma protein binding (entries 6ꢀ8). A parallel artificial membrane permeability
assay (PAMPA) was used as an in vitro model of passive, transcellular bloodꢀbrain barrier
(BBB) permeability at biological pH. Compound 45 registered at the lower end of the
moderately permeable range of this assay.
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17
18
19
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21
22
23
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 6. Summary of In Vitro Pharmacology Data for Compound 45 (ML336)
entry
1
assessment
aqueous solubility, 1x PBS, pH 7.4a
aqueous solubility, VEEV CPE assay mediumb
chemical stability with 5x DTT, 8 hc
result
40.4 ꢀg/mL, 110.0 ꢀM
13.1 ꢀg/mL, or 35.7 ꢀM
99.0% parent remaining
82.9% parent remaining
95.9% parent remaining
65.4% parent remaining
85.0%
2
3
4
PBS, pH 7.4
1:1 PBS:acetonitrile
aqueous
stability, 48 hc
5
6
plasma stability, % remaining after 3 h (mouse)a
7
1 ꢀM (mouse)
10 ꢀM (mouse)
plasma protein binding, % bounda
8
77.0%
9
BBB PAMPA, pH 7.4a,d
13 x 10ꢀ6 cm/s
10
11
12
13
14
hepatic microsomal stability, after 1 hour (mouse)a
hepatocyte toxicity (Fa2Nꢀ4), LD50 (human)a
42.9% parent remaining
> 25.0 ꢀM
1.5 %
7 %
1A2 (human)
CYP450 inhibition profile,
2C9 (human)
2C19 (human)
percent inhibition at 10 ꢀMa,e
17 %
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16
2D6 (human)
3A4 (human)
28 %
18 %
6
7
8
9
PanLabs profiling panel: assessment of percent inhibition of
norepinephrine transporter,
91%
17
67 host derived targets at a 10 ꢀM compound concentrationf
aData collected by Ms. Arianna MangravitaꢀNovo at the Conrad Prebys Sanford Burnham Medical Research Institute; CPE
b
assay medium = high glucose DMEM with 10% FBS and 1x Pen/Strep; cData collected by Mr. Patrick Porubsky at the
10
11
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16
17
18
19
20
21
22
23
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28
29
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
d
University of Kansas Analytical Chemistry Core, Specialized Chemistry Center; Donor and Acceptor pH: 7.4; Controls:
VerapamilꢀHCl (highly permeable): 138; Corticosterone (moderately permeable): 14; Theophylline (poorly permeable): 0.32;
eControl inhibitors: For CYP3A4, ketoconazole, 99% inhibition; for CYP2C9, sulfaphenazole, 96% inhibition; for CYP1A2, αꢀ
naphtoflavone, 84% inhibition; for CYP2C19, lansoprazole, 86% inhibition; for CYP2D6, quinidine, 88ꢀ98% inhibition;
fEurofins PanLabs data, all targets were assessed in duplicate and complete profile is included in the supplemental material.
Cytochrome P450 induction was not assessed; however, only insignificant inhibition of 1A2,
2C9, 2C19, 2D6, and 3A4 was observed (< 30% at 10 ꢀM, entries 12ꢀ16). Some metabolic
liability was detected after 1 hour exposure to mouse liver homogenates (~43% of 45 remained),
though the compound showed no toxicity (> 25 ꢀM LD50) toward human hepatocytes. Lastly,
compound 45 was evaluated in radioligand binding assays against a panel of 67 GPCRs, ion
channels and transporters.38 At a single concentration of 10 ꢀM, 91% inhibition of the human
norepinephrine transporter was observed; however, inhibition of all other targets in the collection
was < 36%.39 Overall, this pharmacological profile obtained of compound 45 was encouraging in
combination with the potency and toxicity data that the prototype compound may be useful in
establishing baseline optimization parameters for in vivo assessment.
To that end, preliminary in vivo mouse pharmacokinetic (PK) data was collected for
compound 45 in order to assess plasma and brain exposure relative to time and dose (Table 7).40
The concentration of compound 45, delivered by intraperitoneal (IP) administration, was
determined in plasma at 20 and 120 minutes postꢀadministration for each of three doses (1, 5 and
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10 mg/kg) with 3 mice in each cohort. Additionally, the concentration of compound 45 in brain
tissue was determined at the terminal endpoint (120 min) for each group. In accordance with the
in vitro PK profile, the compound was readily depleted from circulation over the experimental
window; however, significant exposure was observed for each dose in plasma and brain tissue.
Moreover, > 10ꢀfold concentrations over the in vitro CPE assay EC50 values were observed in
both plasma and brain for the 5 mg/kg and 10 mg/kg doses after 120 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 7. In Vivo Exposure Data for Compound 45 in Mice
plasma data (mouse)
averaged concentration
± standard deviation (ꢀM)
brain data (mouse)
averaged concentration
± standard deviation (ꢀM)
time
number
compound
(min) of mice
45 dosea
CV
(%)
CV
(%)
b
per groupc
20
120
20
3
3
3
3
3
3
0.20 ± 0.03
0.023 ± 0.007
1.45 ± 0.26
0.29 ± 0.01
2.68 ± 0.36
0.49 ± 0.25
15.1
30.8
17.6
4.7
ND
0.19 ± 0.22
ND
ND
115.8
ND
1 mg/kg
5 mg/kg
10 mg/kg
120
20
0.21± 0.02
ND
10.7
ND
13.5
49.9
120
0.35 ± 0.06
17.0
b
c
aIP administration; Each time point is an average of 2 LC/MC injections (< 20% difference); C57BL/6 mice were
d
used; Data was an average of three independent experiments for each dose and timepoint (i.e., 3 mice in each
cohort, including vehicle control – not shown); CV = coefficient of variation; ND = not determined.
These preliminary in vivo studies encouraged us to evaluate 45 in a lethal mouse model of VEEV
infection.20 Single and multiple dose range finding studies were performed to define any
potential toxicity of compound 45 in mice prior to conducting efficacy studies. The compound
was administered IP twice daily to C3H/HeN mice in doses of 5 mg/kg/day and 40 mg/kg/day
for four days. For all mice, no apparent toxicity or adverse effects were observed in the mice as
measured by body weight loss, lethargy, hunched posture and ruffled fur. To test the in vivo
efficacy of compound 45 in a 21ꢀday lethal mouse model, compound 45 was administered IP at
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2.5 mg/kg twice daily for four days to one group of VEEV (TCꢀ83)ꢀinfected mice (Fig. 2).41
Mice receiving treatment were dosed with compound four hours prior to intranasal exposure to
VEEV TCꢀ83. Compared to the infected, untreated group, significant survival rates (71%) were
observed with VEEVꢀinfected mice treated with compound 45.
3
4
5
6
7
8
9
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40
41
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43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
Figure 2. Survival data for VEEV infected C3H/HeN mice treated with compound 45 over a 21
day study compared to controls. Survival curves were generated using the KaplanꢀMeier
method42 and were compared using the Logꢀrank (MantelꢀCox) Test; X2 value = 11.70, pꢀvalue =
0.0029 (2 degrees of freedom). The error bars show the standard error of the mean.
Mechanism of Action Studies. To understand the regions of the viral protein that are important
to the molecular action of the compound, an assay using hit quinazolinone 1-resistant mutant
viruses was performed. Previously we have reported the isolation of mutant virus strains that are
resistant to hit quinazolinone 1 with mutations of Y102C and D116N in nonꢀstructural protein
2.27 More recently, using the same method, we have isolated additional quinazolinone 1-resistant
mutant VEEV TCꢀ83 strains with a mutation of Q120K in nsP4 (Table 8). The alphavirus
genome comprises two open reading frames, a structural and nonꢀstructural segment. The
structural segment encodes for proteins constituting the virion: (1) envelope proteins E1 and E2,
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and (2) core nucleocapsid protein C. The nonstructural segment encodes nonꢀstructural proteins
1ꢀ4 (nsP1–nsP4) which are necessary for transcription and replication of viral RNA.43
7
8
9
Mutant viruses obtained from those studies44 were used to examine the resistance profile of
amidine 45. To confirm that each of these sites conferred resistance, the amino acids were
changed in VEEV V3526 by siteꢀdirected mutagenesis, and the EC50 value for compound 45 was
measured for each mutant virus (Table 8). The activity of compound 45 significantly decreased
when it was tested against these mutant viruses compared to the parental VEEV strains,
suggesting that compound 45 may target a critical function of nsp2/nsp4 in the VEEV replicase
complex, resulting in inhibition of viral RNA synthesis. At this point in time, a function has not
been ascribed to the domain in the nsP2 or nsP4 that compound 45 targets, but more advanced
experiments are underway to elucidate their role in the potent inhibition exhibited by these
compounds.
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45
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48
49
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60
Table 8. Effect of Compound 45 on Wildtype and Mutant VEE Viruses
compound 45 CPE
potency EC50 (ꢀM)
VEEV protein segment of
fold loss of potency
(mutant EC50/parental EC50)
mutation
strain
located mutation
none
(parental
strain)
nsP2
0.03
ꢀ
nsP2
nsP2
nsP4
Y102C
D116N
Q210K
17
19
567
633
TCꢀ83
> 50
> 1667
none
(parental
strain)
nsP2
0.05
ꢀ
V3526
nsP2
nsP4
Y102C
Q210K
19
66
380
1320
CONCLUSION
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With the emergence of alphavirus infections and the biohazards associated with their misuse has
evolved an urgent need for effective countermeasures that have yet to be realized. Our efforts,
directed at the identification and development of VEEV inhibitors, revealed a quinazolinone hit
scaffold that was optimized through structural modification to afford novel, rearranged amidines
with tremendous potential for further lead development and utility in dissecting novel
mechanistic elements associated with alphavirus replication. The probe compound 45 showed
potent inhibition of viral CPE for several strains of VEEV in the low nanomolar range without
inducing cytotoxicity. These antiviral effects translated to impressive cellular efficacy in plaque
reduction assays at submicromolar concentrations of compound, thus demonstrating the ability of
amidine 45 to potently arrest viral replication. Gratifyingly, in vitro phamacokinetic data
collected on amidine 45 predicted moderate brain exposure which was corroborated by
preliminary pharmacokinetic studies in mice. These results helped to establish a dosing strategy
for efficacy studies in which amidine 45 was shown to impart significant protection to VEEVꢀ
infected mice in a 21ꢀday study. Mechanistically, these compounds appear to inhibit viral
replication through a unique pathway involving interference with viral nonꢀstructural proteins 2
and 4. Ultimately, the discovery of amidine 45 and its analogs represent an exciting advance in
alphavirus research in that these agents offer a distinctive mechanistic intervention and a
potential therapeutic and biodefense opportunity for VEEV. Future work centers on optimizing
the structural amidine architecture to refine in vivo pharmacokinetic parameters, evaluating these
and improved compounds in advanced animal efficacy models for VEEV, determining specific
proteins of mechanistic relevance, and profiling these compounds against other viruses of
therapeutic interest.
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EXPERIMENTAL SECTION
5
6
7
Chemistry. Purity of all final compounds was confirmed by HPLC/MS analyis and determined
8
9
1
to be > 95%. H and 13C NMR spectra were recorded on a Bruker AM 400 spectrometer
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(operating at 400 and 101 MHz respectively) or a Bruker AVIII spectrometer (operating at 500
and 126 MHz respectively) in CDCl3 (residual internal standard CHCl3 = δ 7.26), DMSOꢀd6
(residual internal standard CD3SOCD2H = δ 2.50), or acetoneꢀd6 (residual internal standard
CD3COCD2H = δ 2.05). The chemical shifts (δ) reported are given in parts per million (ppm) and
the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, br
s = broad singlet, d = doublet, t = triplet, q = quartet, p = pentuplet, dd = doublet of doublet, dt =
doublet of triplet, td = triplet of doublet, tt = triplet of triplet, and m = multiplet. The LCꢀMS
analysis was performed on an Agilent 1200 RRL HPLC system with photodiode array UV
detection and an Agilent 6224 TOF mass spectrometer. The chromatographic method utilized the
following parameters: a Waters Acquity BEH Cꢀ18 2.1 x 50 mm, 1.7 ꢁm column; UV detection
wavelength = 214 nm; flow rate = 0.4 mL/min; gradient = 5ꢀ100% MeCN over 3 minutes with a
hold of 0.8 minutes at 100% MeCN; the aqueous mobile phase contained 0.15% NH4OH. The
mass spectrometer utilized the following parameters: an Agilent multimode source which
simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and
hexakis(1H, 1H, 3Hꢀtetrafluoropropoxy) phosphazine; and a makeꢀup solvent of 90:10:0.1
MeOH/H2O/HCO2H which was introduced to the LC flow prior to the source to assist ionization.
Melting points were determined on a Stanford Research Systems OptiMelt apparatus. Microwave
irradiated reactions were carried out using a Biotage Initiator Classic synthesizer. Flash
chromatography separations were carried out using a Teledyne Isco CombiFlash Rf 200
purification system with silica gel columns. Massꢀdirected fractionation separations were carried
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out using an Agilent 1200 HPLC system with photodiode array UV detection and an Agilent
6120 mass spectrometer. The chromatographic method utilized a Waters XBridge Cꢀ18, 19 x 150
mm, 5 ꢁm column; UV detection wavelength = 214 nm; flow rate = 20 mL/min; focused
gradient = 5ꢀ100% MeCN; the aqueous mobile phase contained 0.15% NH4OH. Quinazolinones
were generally prepared according to Scheme 1 and by the protocols detailed for compound 1,
below, unless otherwise specified. Amidines were prepared by either of two methods depicted in
Scheme 2 and by the protocols detailed for compound 45, below, unless otherwise specified.
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Synthesis of 2-((4-Ethylpiperazin-1-yl)methyl)-3-(2-fluorophenyl)-6-nitroquinazolin-4(3H)-
one (1). Step 1, Synthesis of 2ꢀ(2ꢀchloroacetamido)ꢀ5ꢀnitrobenzoic acid (3). Triethylamine (2
mL, 14.4 mmol) was added to a solution of 2ꢀaminoꢀ5ꢀnitroꢀbenzoic acid (2.280 g, 12.5 mmol)
in dry CH2Cl2 (34 mL) under an atmosphere of nitrogen. The mixture was lowered to 0 °C and a
solution of chloroacetyl chloride (1.09 mL, 13.7 mmol) in dry CH2Cl2 (16 mL) was slowly
added. The mixture was allowed to reach rt over 2 h. The solvent was removed in vacuo and
water (20 mL) was added. The product was filtered, rinsed with water (2 x 20 mL), and then
1
rinsed with 5% ether/hexanes (3 x 30 mL) to give 3 (3.08 g, 95%) as a lightꢀbrown solid. H
NMR (400 MHz, acetoneꢀd6) δ 12.26 (s, 1H), 8.93–8.87 (m, 2H), 8.45 (dd, J = 9.3, 2.8 Hz, 1H),
4.42 (s, 2H). 13C NMR (101 MHz, acetoneꢀd6) δ 168.5, 166.9, 146.8, 143.1, 129.9, 127.7, 121.0,
117.2, 44.1.
Step 2: Synthesis of 2ꢀ(chloromethyl)ꢀ3ꢀ(2ꢀfluorophenyl)ꢀ6ꢀnitroquinazolinꢀ4(3H)ꢀone. A MW
vial was charged with 2ꢀ(2ꢀchloroacetamido)ꢀ5ꢀnitrobenzoic acid (1.93 g, 7.5 mmol) and capped.
The atmosphere was evacuated under reduced pressure and flushed with Ar. Dry MeCN (14 mL)
was added to the vial, followed by POCl3 (1.5 mL, 16.1 mmol) and a solution of 2ꢀfluoroaniline
(0.97 mL, 10.1 mmol) in dry MeCN (4 mL). The mixture was heated in a MW reactor at 150 °C
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for 15 min. The mixture was transferred to a larger flask and slowly quenched with saturated aq.
NaHCO3 (80 mL). The resulting solid was filtered and rinsed with water (3 x 50 mL) to give 2ꢀ
(chloromethyl)ꢀ3ꢀ(2ꢀfluorophenyl)ꢀ6ꢀnitroquinazolinꢀ4(3H)ꢀone (1.39 g, 56%) as a burntꢀorange
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1
solid. H NMR (400 MHz, CDCl3) δ 9.14 (d, J = 2.5 Hz, 1H), 8.60 (dd, J = 8.9, 2.6 Hz, 1H),
7.92 (d, J = 8.9 Hz, 1H), 7.64–7.57 (m, 1H), 7.48–7.31 (m, 3H), 4.40 (d, J = 12.0 Hz, 1H), 4.23
(d, J = 12.0 Hz, 1H).
Step 3: Synthesis of 2ꢀ((4ꢀethylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(2ꢀfluorophenyl)ꢀ6ꢀnitroquinazolinꢀ
4(3H)ꢀone (1). To a microwave vial, 2ꢀ(chloromethyl)ꢀ3ꢀ(2ꢀfluorophenyl)ꢀ6ꢀnitroquinazolinꢀ
4(3H)ꢀone (560 mg, 1.7 mmol) was added and dissolved in dry MeCN (9 mL). Potassium
carbonate (464 mg, 3.4 mmol), Nꢀethylpiperazine (0.30 mL, 2.4 mmol), and KI (106 mg, 0.64
mmol) were successively added, and the vial was capped. The mixture was heated in a MW
reactor at 80 °C for 10 min. The mixture was concentrated and the product was purified by flash
1
chromatography (0ꢀ10% MeOH/CH2Cl2) to give 1 (333 mg, 48%) as a paleꢀorange solid. H
NMR (400 MHz, CDCl3) δ 9.15 (d, J = 2.6 Hz, 1H), 8.58 (dd, J = 8.9, 2.7 Hz, 1H), 7.88 (d, J =
8.9 Hz, 1H), 7.55–7.49 (m, 1H), 7.37–7.28 (m, 3H), 3.44 (d, J = 13.7 Hz, 1H), 3.27 (d, J = 13.7
13
Hz, 1H), 2.44–2.16 (m, 10H), 1.04 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl3) δ 160.8,
159.4, 157.1, 156.9, 151.1, 146.2, 131.7, 131.6, 129.8, 129.4, 128.9, 124.7, 124.6, 124.4, 124.3,
t
123.9, 121.5, 116.8, 116.6, 61.8, 53.1, 52.6, 52.3, 12.1. LCꢀMS: R = 3.18 min, purity = 98%.
HRMS (m/z): calcd for C21H23FN5O3 (M + H)+ 412.1779; found 412.1808.
2-((4-Ethylpiperazin-1-yl)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one (8). Following the
same three step procedure used to synthesize 1, 2ꢀ(chloromethyl)ꢀ6ꢀnitroꢀ3ꢀphenylquinazolinꢀ
4(3H)ꢀone (168 mg, 0.53 mmol) was used to produce 8 (101 mg, 48%) as a paleꢀyellow solid.
mp 179ꢀ182 °C (decomp.). 1H NMR (400 MHz, CDCl3) δ 9.11 (d, J = 2.6 Hz, 1H), 8.55 (dd, J =
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9.0, 2.6 Hz, 1H), 7.87 (d, J = 9.0 Hz, 1H), 7.58–7.50 (m, 3H), 7.34–7.29 (m, 2H), 3.27 (s, 2H),
2.48–2.26 (m, 10H), 1.06 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 161.5, 157.1, 151.2,
146.0, 136.1, 129.7, 129.5, 129.3, 128.8, 128.6, 123.8, 121.6, 61.3, 53.0, 52.7, 52.4, 12.0. LCꢀ
MS: tR = 2.90 min, purity = 98%. HRMS (m/z): calcd for C21H24N5O3 (M + H)+ 394.1874; found
394.1896.
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2-((4-Ethylpiperazin-1-yl)methyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile
(10). Following the same procedure used to synthesize 1, 2ꢀ(chloromethyl)ꢀ4ꢀoxoꢀ3ꢀphenylꢀ3,4ꢀ
dihydroquinazolineꢀ6ꢀcarbonitrile (25 mg, 0.085 mmol) was used to produce 10 (20 mg, 63%) as
1
a yellow solid. mp 136ꢀ142 °C (decomp.). H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 2.0 Hz,
1H), 7.95 (dd, J = 8.5, 2.0 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.59–7.46 (m, 3H), 7.34–7.27 (m,
2H), 3.26 (s, 2H), 2.56–2.22 (m, 10H), 1.06 (t, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
161.2, 156.5, 149.7, 136.5, 136.1, 132.6, 129.7, 129.6, 129.0, 128.8, 121.9, 118.1, 110.7, 61.2,
t
52.7, 52.6, 52.4, 11.8. LCꢀMS: R = 2.91 min, purity = 97%. HRMS (m/z): calcd for C22H24N5O
(M + H)+ 374.1975; found 374.1996.
3-(2-Fluorophenyl)-2-((4-methylpiperazin-1-yl)methyl)-6-nitroquinazolin-4(3H)-one (18).
Following the same procedure used to synthesize 1 (except the reaction was heated at 150 °C for
15 min), 2ꢀ(chloromethyl)ꢀ3ꢀ(2ꢀfluorophenyl)ꢀ6ꢀnitroquinazolinꢀ4(3H)ꢀone (83 mg, 0.25 mmol)
and 1ꢀmethyl piperazine (0.04 mL, 0.36 mmol) were used to produce 18 (31 mg, 31%) as a lightꢀ
orange solid. mp 218ꢀ221 °C. 1H NMR (400 MHz, CDCl3) δ 9.11 (d, J = 2.6 Hz, 1H), 8.55 (dd, J
= 8.9, 2.6 Hz, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.55–7.47 (m, 1H), 7.36–7.24 (m, 3H), 3.42 (d, J =
13.7 Hz, 1H), 3.25 (d, J = 13.7 Hz, 1H), 2.38–2.18 (m, 11H). 13C NMR (101 MHz, CDCl3) δ
160.7, 159.4, 157.0, 156.9, 151.1, 146.1, 131.6, 131.6, 129.7, 129.3, 128.8, 124.7, 124.6, 124.4,
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124.2, 123.8, 121.4, 116.7, 116.5, 61.7, 54.9, 53.0, 46.1. LCꢀMS: R = 2.20 min, purity = 96%.
5
6
HRMS (m/z): calcd for C20H21FN5O3 (M + H)+ 398.1623; found 398.1654.
7
8
9
3-(2-Fluorophenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one (21). Step 1,
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synthesis
of
tertꢀbutylꢀ4ꢀ((3ꢀ(2ꢀfluorophenyl)ꢀ6ꢀnitroꢀ4ꢀoxoꢀ3,4ꢀdihydroquinazolinꢀ2ꢀ
roundꢀbottom flask, 2ꢀ(chloromethyl)ꢀ3ꢀ(2ꢀ
yl)methyl)piperazineꢀ1ꢀcarboxylate. In
a
fluorophenyl)ꢀ6ꢀnitroquinazolinꢀ4(3H)ꢀone (250 mg, 0.75 mmol) was dissolved in dry MeCN
(3.5 mL) under a nitrogen atmosphere. Potassium carbonate (124 mg, 0.90 mmol), 1ꢀBOCꢀ
piperazine (279 mg, 1.50 mmol), and KI (87 mg, 0.52 mmol) were added to the flask and the
mixture was thermally heated at 80 °C for 4 hours. The solvent was removed in vacuo and water
(10 mL) was added. The product was extracted with EtOAc (3 x 10 mL), washed with saturated
aq. NH4Cl (10 mL) and saturated aq. NaHCO3 (10 mL), and then dried with Na2SO4. The
product was purified by flash chromatography (0ꢀ50% EtOAc/hexanes) to give the title
1
compound (201 mg, 56%) as a white solid. H NMR (400 MHz, CDCl3) δ 9.09–9.04 (m, 1H),
8.51 (dd, J = 8.9, 2.7 Hz, 1H), 7.83–7.77 (m, 1H), 7.50–7.42 (m, 1H), 7.29–7.20 (m, 3H), 3.39
(d, J = 13.7 Hz, 1H), 3.24–3.09 (m, 5H), 2.25–2.16 (m, 2H), 2.11–2.02 (m, 2H), 1.36 (s, 9H).
Step 2, synthesis of 3ꢀ(2ꢀfluorophenyl)ꢀ6ꢀnitroꢀ2ꢀ(piperazinꢀ1ꢀylmethyl)quinazolinꢀ4(3H)ꢀone
(21). In
a
roundꢀbottom flask, tertꢀbutyl 4ꢀ((3ꢀ(2ꢀfluorophenyl)ꢀ6ꢀnitroꢀ4ꢀoxoꢀ3,4ꢀ
dihydroquinazolinꢀ2ꢀyl)methyl)piperazineꢀ1ꢀcarboxylate (71 mg, 0.15 mmol) was dissolved in
dry CH2Cl2 (2 mL) and TFA (0.07 mL, 0.91 mmol) was slowly added dropwise. The reaction
mixture was stirred at rt for 2 hours, then diluted with water (4 mL) and CH2Cl2 (4 mL). The
reaction was quenched to pH 10 using saturated aq. Na2CO3 (1 mL). The product was extracted
with CH2Cl2 (3 x 10 mL), dried with Na2SO4 and purified by flash chromatography (0ꢀ10%
1
MeOH/CH2Cl2) to give 21 (25 mg, 44%) as a white solid. mp 202ꢀ204 °C (decomp.). H NMR
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(400 MHz, CDCl3) δ 9.15 (d, J = 2.0 Hz, 1H), 8.64–8.53 (m, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.59–
7.49 (m, 1H), 7.41–7.25 (m, 3H), 3.52–3.37 (m, 2H), 3.27 (d, J = 13.6 Hz, 1H), 2.81–2.59 (m,
4H), 2.37–2.10 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 160.7, 159.4, 157.0, 156.9, 151.1, 146.1,
131.7, 131.6, 129.7, 129.3, 128.8, 124.7, 124.6, 124.4, 124.3, 123.8, 121.4, 116.7, 116.5, 62.4,
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t
54.3, 45.8. LCꢀMS: R = 2.22 min, purity = 100%. HRMS (m/z): calcd for C19H19FN5O3 (M +
H)+ 384.1466; found 384.1471.
2-((4-Ethylpiperazin-1-yl)methyl)-3-(3-fluorophenyl)-6-nitroquinazolin-4(3H)-one
(25).
Following the same procedure used to synthesize 1, 2ꢀ(chloromethyl)ꢀ3ꢀ(3ꢀfluorophenyl)ꢀ6ꢀ
nitroquinazolinꢀ4(3H)ꢀone (157 mg, 0.47 mmol) was used to produce 25 (155 mg, 80%) as a
1
white solid, mp 187ꢀ190 °C (decomp.). H NMR (400 MHz, CDCl3) δ 9.10 (d, J = 2.6 Hz, 1H),
8.56 (dd, J = 9.0, 2.6 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.58–7.50 (m, 1H), 7.29–7.22 (m, 1H),
13
7.18–7.12 (m, 2H), 3.36–3.26 (m, 2H), 2.51–2.24 (m, 10H), 1.07 (t, J = 7.2 Hz, 3H). C NMR
(101 MHz, CDCl3) δ 164.0, 161.6, 161.3, 156.6, 151.0, 146.1, 137.3, 137.2, 130.6, 130.5, 129.4,
128.8, 124.6, 124.6, 123.7, 121.5, 117.2, 117.0, 116.9, 116.7, 61.4, 53.1, 52.7, 52.3, 12.0. LCꢀ
MS: R = 2.93 min, purity = 98%. HRMS (m/z): calcd for C21H23FN5O3 (M + H)+ 412.1779;
t
found 412.1808.
2-((4-Ethylpiperazin-1-yl)methyl)-3-(4-fluorophenyl)-6-nitroquinazolin-4(3H)-one
(26).
Following the same procedure used to synthesize 1, 2ꢀ(chloromethyl)ꢀ3ꢀ(4ꢀfluorophenyl)ꢀ6ꢀ
nitroquinazolinꢀ4(3H)ꢀone (169 mg, 0.51 mmol) was used to produce 26 as a white solid (160
1
mg, 77%), mp 196ꢀ199 °C (decomp.). H NMR (400 MHz, CDCl3) δ 9.10 (d, J = 2.6 Hz, 1H),
8.56 (dd, J = 9.0, 2.6 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.37–7.31 (m, 2H), 7.28–7.21 (m, 2H),
3.29 (s, 2H), 2.52–2.24 (m, 10H), 1.07 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 164.2,
161.7, 161.6, 156.9, 151.0, 146.0, 131.9, 131.8, 130.9, 130.8, 129.3, 128.7, 123.7, 121.5, 116.5,
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116.3, 61.5, 53.1, 52.8, 52.3, 12.1. LCꢀMS: R = 2.98 min, purity = 98%. HRMS (m/z): calcd for
5
6
C21H23FN5O3 (M + H)+ 412.1779; found 412.1802.
7
8
9
6-Nitro-3-phenyl-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one (31). Following the same
procedure used to synthesize 21, compound 31 was obtained as a white solid (49 mg, 86%), mp
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190ꢀ195 °C. H NMR (400 MHz, DMSOꢀd6) δ 8.82 (d, J = 2.5 Hz, 1H), 8.61 (dd, J = 9.0, 2.7
Hz, 1H), 7.93 (d, J = 9.0 Hz, 1H), 7.58–7.49 (m, 5H), 3.25 (s, 2H), 2.70–2.62 (m, 4H), 2.23–2.15
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(m, 4H). C NMR (126 MHz, CDCl3) δ 161.6, 157.0, 151.2, 146.0, 136.1, 129.7, 129.5, 129.3,
128.8, 128.7, 123.9, 121.6, 62.0, 54.2, 45.9. LCꢀMS: tR = 2.61 min, purity = 99%. HRMS (m/z):
calcd for C19H20N5O3 (M + H)+ 366.1561; found 366.1567.
3-(4-Methoxyphenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one (34). Following
the same procedure used to synthesize 21, compound 34 was obtained as a white solid (146 mg,
1
90%), mp 180ꢀ183 °C (decomp.). H NMR (500 MHz, DMSOꢀd6) δ 8.80 (d, J = 2.5 Hz, 1H),
8.59 (dd, J = 9.0, 2.7 Hz, 1H), 7.92 (d, J = 9.0 Hz, 1H), 7.41–7.37 (m, 2H), 7.10–7.06 (m, 2H),
3.82 (s, 3H), 3.20 (s, 2H), 2.58–2.52 (m, 4H), 2.16–2.10 (m, 4H). 13C NMR (126 MHz, DMSOꢀ
d6) δ 161.2, 159.3, 157.9, 150.9, 145.2, 130.2, 129.1, 128.8, 128.5, 122.5, 121.2, 113.9, 61.7,
55.5, 53.5, 45.2. LCꢀMS: tR = 2.73 min, purity = 99%. HRMS (m/z): calcd for C20H22N5O4 (M +
H)+ 396.1666; found 396.1670.
6-Nitro-2-(piperazin-1-ylmethyl)-3-(thiophen-3-yl)quinazolin-4(3H)-one (37). Following the
same procedure used to synthesize 21 except during purification 5% NH4OH was added to
MeOH in the mobile phase, 37 was obtained as a white solid (79 mg, 58%), mp 205ꢀ208 °C
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(decomp.). H NMR (400 MHz, CDCl3) δ 9.12 (d, J = 2.6 Hz, 1H), 8.55 (dd, J = 8.9, 2.6 Hz,
1H), 7.86 (d, J = 8.9 Hz, 1H), 7.49 (dd, J = 5.1, 3.2 Hz, 1H), 7.42 (dd, J = 3.2, 1.3 Hz, 1H), 7.09
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(dd, J = 5.1, 1.3 Hz, 1H), 3.33 (br s, 2H), 2.86–2.75 (m, 4H), 2.42–2.31 (m, 4H). 13C NMR (101
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MHz, CDCl3) δ 161.4, 157.2, 150.8, 145.9, 133.3, 129.2, 128.6, 127.0, 125.8, 123.7, 123.5,
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t
121.3, 62.0, 54.5, 46.0. LCꢀMS: R = 2.62 min, purity = 100%. HRMS (m/z): calcd for
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C17H18N5O3S (M + H)+ 372.1125; found 372.1138.
2-(4-Ethylpiperazin-1-yl)-6-nitro-3-phenylquinazolin-4(3H)-one (38). Step 1, synthesis of 6ꢀ
nitroꢀ3ꢀphenylquinazolineꢀ2,4(1H,3H)ꢀdione. To a stirred mixture of 2ꢀaminoꢀ5ꢀnitrobenzanilide
(1.97 g, 7.66 mmol) in dry toluene (25 mL) was carefully added a solution of 20% (w/v)
phosgene in toluene (15 mL, 30.3 mmol). CAUTION! Phosgene is highly toxic and should be
handled with care. The mixture was heated at 95 °C for 19.5 h, then concentrated and purified by
flash column chromatography (0–90% EtOAc/hexanes) to give the title compound (1.45 g, 67%)
as a colorless solid. mp 297–299 °C (decomp.). 1H NMR (400 MHz, acetoneꢀd6) δ 10.93 (s, 1H),
8.82 (d, J = 2.6 Hz, 1H), 8.53 (dd, J = 9.0, 2.6 Hz, 1H), 7.58–7.45 (m, 4H), 7.45–7.39 (m, 2H).
Step 2, synthesis of 2ꢀ(4ꢀethylpiperazinꢀ1ꢀyl)ꢀ6ꢀnitroꢀ3ꢀphenylquinazolinꢀ4(3H)ꢀone (38).
Phosphorus pentachloride (44 mg, 0.21 mmol), 6ꢀnitroꢀ3ꢀphenylquinazolineꢀ2,4(1H,3H)ꢀdione
(44 mg, 0.16 mmol), and phosphorus oxychloride (1.5 mL, 16 mmol) were heated in a sealed
vial at 120 °C for 16 hours. The mixture was concentrated and redissolved in dry CH2Cl2 (5 mL).
Nꢀethylpiperazine (142 mg, 1.24 mmol) was added and the mixture was briefly stirred at rt. The
mixture was reꢀconcentrated and the product was purified by massꢀdirected fractionation to give
38 (19 mg, 31%) as a yellow solid. mp 179–182 °C (decomp.). 1H NMR (400 MHz, DMSOꢀd6) δ
8.71 (d, J = 2.6 Hz, 1H), 8.46 (dd, J = 9.0, 2.8 Hz, 1H), 7.61–7.44 (m, 6H), 3.24–3.13 (m, 4H),
2.26–2.16 (m, 2H), 2.16–2.05 (m, 4H), 0.92 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSOꢀd6)
δ 161.6, 156.0, 152.6, 142.4, 137.1, 128.9, 128.7, 128.6, 128.4, 126.7, 123.1, 117.9, 51.3, 51.2,
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48.2, 11.7. LCꢀMS: tR = 3.29 min, purity = 100%. HRMS (m/z): calcd for C20H22N5O3 (M + H)+
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380.1717; found 380.1732.
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6-Nitro-3-phenyl-2-(piperidin-4-yl)quinazolin-4(3H)-one (39). Step 1, synthesis of tertꢀbutyl
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4ꢀ((4ꢀnitroꢀ2ꢀ(phenylcarbamoyl)phenyl)carbamoyl)piperidineꢀ1ꢀcarboxylate. To
a
stirred
mixture of 1ꢀ(tertꢀbutoxycarbonyl)piperidineꢀ4ꢀcarboxylic acid (213 mg, 0.93 mmol) in dry
CH2Cl2 (10 mL) was added a catalytic amount of DMF (2 drops) at rt followed by the dropwise
addition of oxalyl chloride (479 mg, 3.77 mmol). The mixture was stirred at rt for 2 hours and
concentrated. The remaining residue was redissolved in dry THF (5 mL) and MeCN (5 mL) and
slowly added to a mixture of 2ꢀaminoꢀ5ꢀnitroꢀNꢀphenylbenzamide (211 mg, 0.82 mmol) and
95% (w/w) NaH (128 mg, 5.07 mmol) in dry THF (10 mL) at ꢀ78 °C. The mixture was allowed
to warm to rt and stirred for 16 hours. The reaction was quenched with water, extracted with
EtOAc (3x), washed with brine, and dried with Na2SO4. The product was purified by reverseꢀ
phase chromatography (Cꢀ18, 10ꢀ100% MeCN/H2O) to give the title compound (38 mg, 10%) as
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an offꢀwhite solid. H NMR (400 MHz, acetoneꢀd6) δ 11.41 (s, 1H), 10.17 (s, 1H), 8.84 (d, J =
9.3 Hz, 1H), 8.73 (d, J = 2.6 Hz, 1H), 8.35 (dd, J = 9.3, 2.7 Hz, 1H), 7.77–7.71 (m, 2H), 7.38 (tt,
J = 7.6, 2.0 Hz, 2H), 7.18 (tt, J = 7.1, 1.1 Hz, 1H), 4.08 (d, J = 12.7 Hz, 2H), 2.91–2.72 (m, 2H),
2.63 (tt, J = 11.5, 3.8 Hz, 1H), 1.97–1.88 (m, 2H), 1.67–1.53 (m, 2H), 1.40 (s, 9H).
Step 2, synthesis of 6ꢀnitroꢀ3ꢀphenylꢀ2ꢀ(piperidinꢀ4ꢀyl)quinazolinꢀ4(3H)ꢀone (39). Triethylamine
(10 mg, 0.10 mmol), chlorotrimethylsilane (9 mg, 0.08 mmol), and tertꢀbutylꢀ4ꢀ((4ꢀnitroꢀ2ꢀ
(phenylcarbamoyl)phenyl)carbamoyl)piperidineꢀ1ꢀcarboxylate (38 mg, 0.08 mmol) were
dissolved in dry MeCN (1.5 mL) and heated in a MW reactor at 150 °C for 10 min. The mixture
was purified by flash chromatography (0ꢀ60% EtOAc/hexanes) and reꢀdissolved in 3:1
CH2Cl2/TFA (2 mL). The reaction mixture was stirred at rt for 16 hours and concentrated. The
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