2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization

Article abstract of DOI:10.1021/acsinfecdis.7b00275

Mycobacterium tuberculosis (MTb) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose MTb whole-cell structure-activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the ndh encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in MTb was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by ndhA in MTb. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial MTb SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors.

Full text of DOI:10.1021/acsinfecdis.7b00275

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2-Mercapto-quinazolinones as inhibitors of NDH-2 and
Mycobacterium tuberculosis: Structure-activity relationships,
mechanism of action and ADME characterization.
Dinakaran Murugesan, Peter C. Ray, Tracy Bayliss, Gareth A. Prosser, Justin R. Harrison, Kirsteen Green,
Candice Soares de Melo, Tzu-Shean Feng, Leslie J. Street, Kelly Chibale, Digby F. Warner, Valerie Mizrahi,
Ola Epemolu, Paul Scullion, Lucy Ellis, Jennifer Riley, Yoko Shishikura, Liam Ferguson, Maria Osuna-Cabello,
Kevin D Read, Simon R. Green, Dirk A. Lamprecht, Peter M. Finin, Adrie J. C. Steyn, Thomas R. Ioerger,
Jim Sacchettini, Kyu Y. Rhee, Kriti Arora, Clifton E. Barry, III, Paul G. Wyatt, and Helena Ingrid M. Boshoff
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00275 • Publication Date (Web): 09 Mar 2018
Downloaded from http://pubs.acs.org on March 10, 2018
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Biological Chemistry and Drug Discovery College of Life Sciences
Boshoff, Helena; NIAID, TBRS
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2
-Mercapto-quinazolinones as inhibitors of NDH-2 and Mycobacterium tuberculosis: Structure-activity
relationships, mechanism of action and ADME characterization.
a,α
a,α
a
b
a
Dinakaran Murugesan , Peter C. Ray , Tracy Bayliss , Gareth A. Prosser , Justin R. Harrison , Kirsteen
0
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a
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d
d, c, e
f, c
Green , Candice Soares de Melo , Tzu-Shean Feng , Leslie J. Street , Kelly Chibale
, Digby F. Warner ,
f,
c
a
a
a
a
a
Valerie Mizrahi , Ola Epemolu , Paul Scullion , Lucy Ellis , Jennifer Riley , Yoko Shishikura , Liam
a
a
a
a
g
g,¤
Ferguson , Maria Osuna-Cabello , Kevin D. Read , Simon R. Green , Dirk A. Lamprecht , Peter M. Finin ,
g,h
i
i
j
b
b,c
Adrie J.C. Steyn , Thomas R. Ioerger , Jim Sacchettini , Kyu Y. Rhee , Kriti Arora , Clifton E. Barry III , Paul G.
,
a
,b
Wyatt* and Helena I.M. Boshoff*
a
Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,
University of Dundee, Sir James Black Centre, Dundee, DD1 5EH, UK
b
Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of
Allergy and Infectious Disease, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD, 20892, USA
c
Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, South Africa
d
Drug Discovery and Development Centre (H3D), Department of Chemistry, University of Cape Town,
Rondebosch 7701, South Africa
e
South African Medical Research Council Drug Discovery and Development Research Unit, Department of
Chemistry, University of Cape Town, Rondebosch 7701, South Africa
f
SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for
Biomedical TB Research, Dept. of Pathology, Faculty of Health Sciences, University of Cape Town,
Rondebosch 7701, South Africa
g
Africa Health Research Institute (AHRI), K-RITH Tower Building Level 3, 719 Umbilo Road, Durban 4001,
South Africa
h
Department of Microbiology, University of Alabama at Birmingham, 1720 2nd Avenue South, Birmingham,
Alabama 35294-2170, USA
i
Department of Computer Science and Engineering, Texas A&M University, College Station, TX, USA
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j
Division of Infectious Diseases, Weill Department of Medicine, Weill Cornell Medical College, NY, NY 10065,
USA
¤
Current address: Department of Internal Medicine, University of Pittsburgh, 1218 Scaife Hall 3550 Terrace
Street, Pittsburgh, Pennsylvania 15261, USA
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α
*
Corresponding authors, contributed equally
hboshoff@niaid.nih.gov; P.G.Wyatt@dundee.ac.uk
Mycobacterium tuberculosis (MTb) possesses two non-proton pumping type II NADH dehydrogenase (NDH-
2
) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the
structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based
design of small-molecule inhibitors. Herein, we disclose MTb whole-cell structure-activity relationships
(SAR) for a series of 2-mercapto-quinazolinones which target the ndh encoded NDH-2 with nanomolar
potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as
quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and
compound exposures. Resistance to the compounds in MTb was conferred by promoter mutations in the
alternative non-essential NDH-2 encoded by ndhA in MTb. Bioenergetic analyses revealed a decrease in
oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled
from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent
inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic
studies further demonstrated non-competitive inhibition, suggesting binding of this scaffold to an allosteric
site. In summary, while the initial MTb SAR showed limited improvement in potency, these results,
combined with structural information of the bacterial protein, will aid in the future discovery of new and
improved NDH-2 inhibitors.
KEYWORDS
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Mycobacterium tuberculosis, mercapto-quinazolinones, structure-activity relationship (SAR), type II NADH
dehydrogenase (NDH-2), small molecule NDH-2 inhibitors, respiration
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Tuberculosis (TB) is a major global health problem, resulting in significant morbidity each year.
Although mortality has fallen dramatically since 1990, TB now ranks alongside HIV as a leading cause of
death worldwide. While HIV-related deaths have been declining largely as a result of improved access to,
1
and availability of, better HIV treatments, this has not been the case for TB , the treatment of which
requires 6 months of chemotherapy with a combination of four agents (isoniazid, rifampicin, pyrazinamide
2
and ethambutol) to achieve durable cure of drug-sensitive TB . The large number of TB patients, coupled
with the chemotherapeutic burden, often leads to poor patient adherence and sub-optimal treatment
outcomes in the developing world, as well as the emergence of multi-drug resistant TB (MDR-TB, defined as
resistance to isoniazid (INH) and rifampicin (RIF)) and extensively drug-resistant tuberculosis (XDR-TB,
defined as MDR-TB plus resistance to any fluoroquinolone and one of three second-line injectable drugs,
capreomycin, kanamycin (Kan), and amikacin). To address this global TB health problem, an improved
treatment regimen is needed which will reduce treatment duration, and prevent relapse and the
3-8
development of TB drug resistance . However, achieving this goal will require discovery of multiple novel
and mechanistically distinct anti-mycobacterial agents possessing reduced liabilities for investigation of
new drug regimens that might shorten the duration of treatment, and simplify management of the disease
9
-12
by improving adherence and reducing costs
.
Identification of novel drug targets that will lead to treatment shortening is challenging. Targets of
drugs currently in use or phase 3 clinical evaluation for TB chemotherapy include cell wall biosynthesis,
translation, transcription, folate synthesis, ATP generation and maintenance of DNA topology as broad
categories, although the nitroimidazoles delaminid and pretomanid, generate reactive nitrogen
13
intermediates that inhibit several essential processes . Mechanistically novel drugs would conceptually
target distinct processes from the above and to date, successes in the field have all emerged from target
1
3
identification of hits discovered in MTb whole cell screens .
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In this work, we describe the identification of a 2-mercapto-quinazoline scaffold identified from a
MTb whole cell screen, which had been previously reported to inhibit the mycobacterial type II NADH
1
4
dehydrogenase ; providing further evidence for its inhibition of the MTb NDH-2. MTb encodes two NDH-2
1
5-17
genes of which the one encoded by ndh is play a critical role for growth both in vitro and in vivo
. NDH-2
1
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catalyses the transfer of electrons from NADH into the mycobacterial respiratory pathway and has been
16, 18-20
proposed to be targeted by a number of early stage inhibitors
In contrast, the proton pumping type I
15-17
NADH dehydrogenase can be deleted without apparent effects on growth both in vitro or in vivo
. As
the current series was considered to have a promising drug-like profile (Table 1), a focused optimization
program was initiated.
RESULTS AND DISCUSSION
Screening of a commercial diversity library, details of which will be published elsewhere, afforded a 2-
mercapto-quinazolinone cluster of hits (1, 2 and 3), with compounds 1 and 2 showing potent MTb whole-
cell minimum inhibitory concentration (MIC) in two distinct growth media. The compounds were assessed
in a series of early stage biology profiling assays to understand better the mechanism of action (MoA).
These included screening the compounds against a cyd knockout strain which is known to be hyper-
2
1
1
susceptible to inhibitors of the cytochrome bc complex , which suggested that the series was unlikely to
target QcrB. Profiling also investigated the extent of upregulation of the promoter of the iniBAC gene
cluster, known to be induced by inhibitors of cell wall biosynthesis, such as isoniazid, ethionamide, SQ109
2
2-23
and ethambutol
, which suggested 1 and 2 did not have an effect on cell wall biosynthesis. These
1
4
biology profiling data were considered promising, especially in conjunction with a recent report in which
mutations in MTb mutants spontaneously resistant to compound 1 mapped to ndhA, the gene encoding the
non-essential type-II NADH dehydrogenase that is involved in NADH re-oxidation in the mycobacterial
14
oxidative phosphorylation pathway . There are two closely related non-proton pumping type II NADH
24-27
dehydrogenases in the MTb genome, of which only one (encoded by ndh, Rv1854c) is essential
. Ioerger
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et al. identified promoter mutations in ndhA which resulted in >40 fold upregulation of gene expression,
likely compensating for compound 1 inhibiting the essential NDH-2 homolog.
Table 1: Profiling of confirmed hits 1, 2 and 3
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Confirmed hits
1
2
3
43
>50
1.7
-
GAST MIC (µM)
H9-ADC MIC (µM)
GAST LLE
0.3
0.6
4.2
5.9
4.5
3.5
7
Cyd KO reporter
Not likely QcrB inhibitor
Not likely QcrB inhibitor
pini-LUC reporter
HepG2 IC50 (µM)
CHI-LogD
Not likely cell wall inhibitor
Not likely cell wall inhibitor
-
> 50
1.8
-
-
-
1.5
Microsomal Cli
Mouse 2.3
Human <0.5
317
2.3 / 2.3
75
Mouse 1.6
-
(
mL/min/g)
a
MW
325
1.7 / 1.7
75
337
2.7 / 2.7
75
a
a
clogP / clogD
a
TPSA
Kin. solubility
8
3
109
4.3
-
b
(µM)
aPFI
4.7
4.7
Calculated using StarDrop (http://www.optibrium.com).
b
Kinetic aqueous solubility was measured using laser nephelometry of compounds in 2.5% DMSO.
Compound 1 had a promising MTb MIC-derived ligand-lipophilicity efficiency (LLE) drug-likeness
28-29
profile, suggestive of a quality starting point for medicinal chemistry optimisation
. Compound 1 also
showed no noticeable cytotoxicity in a mammalian cell line (HepG2). Compounds 1 and 2 also had
moderate kinetic solubility and reasonable mouse hepatic microsomal stability, with 1 having excellent
human microsomal stability (Table 1). Herein, we report on the development of the structure-activity
relationship (SAR) for 1, as well as extended ADME characterisation of key compounds.
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Synthetic chemistry: Quinazolinone amides reported herein were synthesized utilizing known
procedures, which are detailed in Scheme 1. Commercially available anthranilic acids (28) were cyclized
with thiourea, and the resulting 2-mercapto quinazoline-4-diones (29) or commercially available 2-
mercapto-4(3H)-quinazolinone was reacted with 2-bromo acetic acid to form the 2-((4-oxo-3,4-
dihydroquinazolin-2-yl)thio)acetic acids (30). Primary and secondary amines (31) were coupled using
standard coupling reagents to afford compounds 1, 2, 4-11 and14.
1
1
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Scheme 1. General synthetic route for synthesis of quinazolinone amides
Reagents and conditions: General synthetic approach to quinazolinones 1, 2, 4–11 and 14. (a) neat
thiourea at 180°C, 3 h; (b) 2-bromoacetic acid, triethylamine, DMF, 80°C, 12 h; (c) primary and/or
secondary amine 31, EDC·HCl, HOAT, N,N-diisopropylethylamine, DMF:ACN 1:1, room temperature, 12 h; or
primary and/or secondary amine 31, HATU, N,N-diisopropylethylamine, DCM, room temperature, 12 h.
MTb whole-cell SAR: The initial medicinal chemistry plan focussed on developing the SAR with the
aim of better understanding the pharmacophore. Initial efforts were guided by mouse microsomal
metabolite identification (met-ID) studies on 1, which revealed significant oxidation of the cyclohexyl and
quinazolin-4(3H)-one rings as well as cleavage of the amide bond (Figure S1). To understand the scope,
cycloalkyls 4, 5 and 6 were prepared (Table 2). The large bulky lipophilic cyclohexyl 1 and cycloheptyl 4
were both equally favoured, with good whole-cell MIC potency. In contrast, the smaller ring-contracted
cyclopentyl 5 and cyclobutyl 6 were slightly less potent (Table 2), and the cyclopropyl and ring deletion
NHMe analogues (data not shown) resulted in a complete loss of whole-cell activity, suggesting that a bulky
hydrophobic group was required to obtain good whole-cell potency. As mouse met-ID of 1 showed
significant oxidation of the cyclohexyl group, the 2-, 3- and 4-hydroxylated cyclohexyl derivatives were
prepared with the aim of improving solubility as well as microsomal stability. Improvements in both kinetic
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solubility and mouse microsomal stability were indeed achieved, but no whole-cell activity was observed
(data not shown). An additional focussed set of polar saturated 4-, 5-, and 6-membered oxygen-containing
saturated heterocycles was prepared; however, whilst having good kinetic solubility and mouse microsomal
stability, they too lost all whole-cell activity (data not shown). There were also attempts to improve
0
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2
solubility with differentially functionalised 4-substituted piperidines (NAc, NSO Me, NMe and NBn) but,
once again, there was a loss of whole-cell activity (data not shown). As the cyclohexyl group within 1 did
not appear to tolerate polar solubilising groups, we turned our focus to introducing fluorine in an attempt
3
0-31
to improve microsomal stability
. We were encouraged to find that the 4, 4-difluorocyclohexan 7
retained good whole-cell potency, and also had slightly improved mouse microsomal stability versus 1. In
comparison to 1, mouse microsomal met-ID on 7 showed a small amount of amide bond cleavage as well as
oxidation of the quinazolin-4(3H)-one ring (Figure S2). The 2, 2- and 3, 3-difluorocyclohexan compounds 8
and 9 had moderate to poor whole-cell potency. Met-ID for both 1 and 7 revealed cleavage of the amide
bond, so attempts to hinder the amide through formation of the 1- methyl-, 1-methanol- and 1-cyano-
substituted cyclohexane were explored, all of which resulted in a complete loss of MIC potency as well as
increased mouse microsomal instability (data not shown). Addition of a methylene bridge in 1 and 7 to
afford 10 and 11 improved MIC potency, further emphasising the requirement for bulky lipophilic groups.
Table 2: Evaluation R1 (cyclohexyl) SAR.
a
H37Rv MIC
Mouse
Cli
(mL/min/g)
c
Solubility
b
ID
4
R
1
(µM)
cLogP
(
µM)
GAST
7H9-ADC
0.3
0.6
1.8
2.9
6.4
39
5
1.0
1.6
2.5
101
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2.3
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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
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
8
9
9.4
4.7
12.5
4.7
2.3
2.3
2.3
2.9
219
187
1
0
1
0.2
0.8
0.3
1.1
2.6
2.6
5.9
4.7
-
1
250
a
Minimum inhibitory concentration (MIC) is the minimum concentration required to inhibit >99% growth of M. tuberculosis in liquid culture.
b
Isoniazid was included as an internal control reference compound (MIC of 0.2 ± 0.1 µM). Intrinsic clearance (Cli) using CD1 mouse liver microsomes.
c
Kinetic aqueous solubility was measured using laser nephelometry of compounds in 2.5% DMSO.
There were concerns over the S-linker, based on previous experience from whole-cell screening
where confirmed hits with similar S-linker compounds were found to react with glutathione (GSH) both
with and without microsomal activation. GSH trapping on 7, with and without human liver microsomes
(
Figure S3) showed GSH adducts 12 and 13, without microsomal activation. It is presumed that GSH results
in cleavage of the sulphur-quinazolinone 7 linker, to afford 12, with GSH coupling to the displaced S-Linker
to afford 13. Human microsomal oxidation of the quinazolinone ring of 7 was also observed (see Figure 1).
Figure 1. Metabolite identification of 7 in a GSH trapping experiment.
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2
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2
2
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2
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3
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3
3
3
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3
4
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4
4
4
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5
5
5
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5
5
5
6
Whilst the level of GSH adduct formation for 7 was relatively low and no HepG2 cytotoxicity was
observed, this was considered a liability of the series as the reactivity did not require microsomal activation
3
2-33
and the ability to predict and quantify the risk of idiosyncratic adverse drug reactions is limited
attempted to reduce this liability by modifying the linker. N-methylation of the amide and/or the
methylene linker to afford 14, 15 and 16, was not tolerated. The NH-, O- and CH -linkers were readily
. We
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
2
prepared to afford 17, 18 and 19. However all resulted in a loss of MIC potency (see Table 3). Oxidation of
S-atom in compound 1 was attempted using a variety of conditions and oxidation reagents (for example 3-
chlorobenzoic acid, potassium permanganate and Oxone). However, all attempts failed to deliver the
desired sulfone, possibly as a result of increased reactivity.
Table 3: Evaluation of S-linker SAR.
a
H37Rv MIC
µM
Mouse
CLi
mL/min/g
c
Solubility
µM
b
ID
Structure
cLogP
GAST 7H9-ADC
1
1
4
5
50
50
50
50
50
50
2.4
13
250
250
250
2.6
2.9
7.5
50
16
1
7
50
25
50
50
50
50
1.9
1.2
2.1
2.1
-
250
-
18
19
1.6
250
a
Minimum inhibitory concentration (MIC) is the minimum concentration required to inhibit >99% growth of M. tuberculosis in liquid culture.
b
Isoniazid was included as an internal control reference compound (MIC of 0.2 ± 0.1 µM). Intrinsic clearance (Cli) using CD1 mouse liver microsomes.
c
Kinetic aqueous solubility was measured using laser nephelometry of compounds in 2.5% DMSO.
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Changes to the quinazolinone ring were then explored, starting with N-methylation to afford 20,
which was not tolerated. Removal of the carbonyl, by synthesis of quinazoline 21 and quinazolin-4-amine
22, was also not tolerated. Saturation of the quinazolinone core phenyl afforded 23, which retained good
MIC potency and also resulted in improved solubility; albeit microsomal stability was poor. The related 5-
membered saturated analogue 24 showed similar MIC potency, but with improved microsomal stability as
well as solubility. However, the di-methyl analogue 25 was less well tolerated, suggesting that a bulky
hydrophobic ring was preferred. The above SAR suggested that the pyrimidinone core was a key part of the
pharmacophore. As human-metID showed oxidation of the quinazolinone ring of 7, we synthesized the
presumably more stable fluorine analogue 26 which, whilst not as potent as 7, retained good MIC potency.
However, the solubility was poor and the mouse microsomal stability moderate. In an attempt to improve
solubility as well as microsomal stability, the pyridopyrimidinone 27 was prepared; whilst solubility and
microsomal stability were improved, the compound had moderate to weak MIC potency.
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
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
Table 4: Evaluation of quinazolinone core.
a
H37Rv MIC
Mouse
CLi
(mL/min/g)
c
(
µM)
Solubility
b
ID
Structure
cLogP
(
µM)
GAST 7H9-ADC
2
2
2
0
50
50
50
50
50
50
2.9
3.8
3.8
11
-
1
2
-
-
-
-
23
1.2
1.6
1.6
1.2
2.2
1.8
6.6
1.2
250
250
2
4
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1
1
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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
2
5
6.3
0.6
9.4
12.5
0.8
2.1
2.4
1.2
1.1
2.2
1.6
250
55
26
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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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
2
7
2.5
250
a
Minimum inhibitory concentration (MIC) is the minimum concentration required to inhibit >99% growth of M. tuberculosis in liquid culture.
b
Isoniazid was included as an internal control reference compound (MIC of 0.2 ± 0.1 µM). Intrinsic clearance (Cli) using CD1 mouse liver microsomes.
c
Kinetic aqueous solubility was measured using laser nephelometry of compounds in 2.5% DMSO.
A summary of the overall whole-cell SAR for 1, as well as the effects on both kinetic solubility and
microsomal stability, is shown in Figure 2 and in Tables 2-4.
Figure 2. Overview of structure-activity and property relationships of the mercapto-quinazolinones.
Pharmacokinetic studies were initiated in order to assess the potential for in vivo efficacy studies of
the 2-mercapto-quinazolinones. Compound 1, when dosed as the free base, had reasonable bioavailability,
consistent with its moderate Cli and solubility, and good permeability (Table 5). Compound 7 showed a
similar bioavailability and exposure profile to 1 (Table 5).
Table 5: Pharmacokinetic profiling of compounds 1, 7 and 11.
1
7
11
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GAST MIC (µM)
7H9-ADC MIC (µM)
HepG2 IC50 (µM)
Measured CHI-LogD
Microsomal clearance
(ml/min/g)
0.3 (95 ng/mL) 0.4 (141 ng/mL) 0.8 (294 ng/mL)
0.6 (190 ng/mL) 0.4 (141 ng/mL) 0.8 (294 ng/mL)
> 50
1.8
>50
1.5
>50
1.6
Mouse 2.3
Human <0.5
317
Mouse 4.8
Mouse 1.4
a
MW
clogP / clogD
353
2.3/2.3
75
367
2.7/2.7
75
a
a
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
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
2.3/2.3
75
a
TPSA
PAMPA
83 (nm/sec)
83 (free base)
Free base
748
64 (nm/sec)
111 (HCl salt)
65 (nm/sec)
>250
Free base
1112
b
Kinetic solubility (µM)
C57 Mouse PK at 3 iv & 10 po (mg/kg)
HCl salt
399
1.3
C
max (ng/ml)
T
1/2
h
1.5
AUC0-8h (ng.min/ml)
156800
23
67085
39
128309
Cl (ml/min/kg)
b
Vdss (L/kg)
0.8
1
%F
46
29
PPB (% unbound)
Calculated using StarDrop (http://www.optibrium.com).
14
13
21
a
b
Kinetic aqueous solubility was measured using laser nephelometry of compounds in 2.5% DMSO.
As compound 7 had comparable MIC potency, mouse microsomal stability and pharmacokinetic
profile to 1; a dose linearity study was conducted to evaluate if exposures above the MIC could be
achieved. However, no dose linearity was observed between the 30 and 100 mg/kg doses, determined by
the area under the curve (AUC) comparison (Figure S4). This may be a consequence of solubility limited
absorption at the highest dose. Compound 11 demonstrated better kinetic solubility and comparable MIC
to compounds 1 and 7, yet despite an improved Cmax and Tmax compared to compound 1, likely driven by its
improved kinetic solubility, it had a lower exposure (AUC) over time (Figure S5 and Table 5), likely a
consequence of it’s higher clearance (Mouse Cli 4.7 mL/min/g versus 2.3 or 1.4 for compound 1 and 7,
respectively). The modest compound exposures upon oral dosing, combined with the lack of ex vivo intra-
macrophage efficacy (vide infra), suggested that efficacy studies of these compounds in infected mice were
unlikely to validate the target/drug candidate pair in an animal model where the disease is macrophage-
based. As such an efficacy experiment was not performed.
Biological characterization of the mercapto-quinazolinones
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5
6
Previous work on a mercapto-quinazolinone had suggested that compound 1 might target NDH-2 as
evidenced by promoter mutations in the non-essential ndhA gene encoding an orthologue of the type II
1
4
NADH dehydrogenase . We similarly identified promoter mutations for ndhA but were not able to identify
polymorphisms in the apparently essential ndh (Rv1854c) (Table S1) suggesting that mutations were either
deleterious for enzyme function or that single amino acid mutations alone might not sufficiently decrease
affinity of this putative inhibitor. The upregulation of ndhA could serve to compensate for loss of NDH-2
function or could serve to bind excess inhibitor in the cell. It is intriguing that ndh promoter mutations were
not identified possibly because this gene is not readily upregulated by single nucleotide substitutions in its
promoter. We also identified mutations in Rv0678 (Table S1) encoding a transcriptional repressor that has
previously been demonstrated to control expression of the MmpS5-MmpL5 transporter and has been
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
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3
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0
1
2
3
4
5
6
7
8
9
0
3
4
implicated in resistance to bedaquiline and clofazimine . In accordance with the predicted essential role of
1
6
NDH-2 as complex I in oxidative phosphorylation , treatment of MTb with compound 1 resulted in
depletion of cellular ATP levels, a phenomenon also observed with bedaquiline (TM207), an inhibitor of the
3
5
ATP synthase, as well as EU306, an inhibitor of the cytochrome bc complex but not with inhibitors of
1
macromolecular biosynthesis including INH (cell wall), Rif (RNA polymerase), and Kan (protein
synthesis)(Figure 3).
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Figure 3. Mercapto-quinazolinones deplete cellular ATP levels in MTb. ATP was measured using the
BacTiter Glo assay after 24 hours of drug exposure and expressed as a fraction of the drug-free vehicle
control levels.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
6
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
We predicted that inhibition of NDH-2 would result in a more severe phenotype on a mutant lacking the
second copy of the type II NADH dehydrogenase. Indeed, while treatment of wild-type MTb H37Rv at MIC
concentrations could not fully suppress growth of cells over 7 days of treatment, similar concentrations of
compound resulted in an almost 2-log kill of a ΔndhA mutant (Figure 4). Despite the higher vulnerability of
the ndhA mutant to killing by compound 1, the MIC to this strain as well as a nuoG deletion mutant lacking
a functional type I NADH dehydrogenase, was indistinguishable from WT cells (results not shown).
Inhibition of anaerobic non-replicating cells with compound 1 did not affect viability (Figure S6) which
1
6
contrasts with the anaerobic cidal activity of other NDH-2 inhibitors including the phenothiazines and a
1
9
recently described quinolone scaffold , possibly due to differences in compound access to the target
under anaerobic conditions or to growth dependent differences in compound modification by MTb. In
addition, this compound lacked activity against MTb growing in infected macrophages (Figure S7) possibly
due GSH-catalysed compound inactivation or inability to access the mycobacterial phagosome.
Figure 4. Mtb lacking the alternative type II NADH dehydrogenase encoded by ndhA was more
susceptible to compound 1. CFU after 7 days of treatment as compared to CFU at start of drug treatment
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1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
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(
inoculum). Unpaired t test comparison between WT and ΔndhA at 0.5µM compound 1: two-tailed P value
= 0.0023.
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Mercapto-quinazolinones target Complex I of the MTb ETC. At the times indicated by the
dotted vertical lines, either glucose (A) or palmitate (B) was added to MTb as carbon source (CS), followed
by the mercapto-quinazolinones (Compound) and lastly, the uncoupler CCCP to induce maximal respiration.
The mercapto-quinazolinones diminished MTb’s uncoupling capacity significantly compared to that of the
untreated (UT) control. The oxygen consumption rate (OCR) is reported as a percentage of baseline values.
Mercapto-quinazolinones inhibit ATP production in the presence of NADH, but not succinate (C, D). ATP
production in inverted membrane vesicles (IMVs) in the presence of NADH or succinate as electron donors
after 30 mins (C) and over 60 mins (D). P-values were calculated by one-way ANOVA using GraphPad Prism
7.02.
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The effect of the mercapto-quinazolinones on MTb bioenergetics in whole cells was further demonstrated
by analysis of the mycobacterial oxygen consumption rate (OCR) (Figure 5). Addition of compounds 1 and 7
had a minimal effect on basal MTb OCR levels over ~45 min. The same results were obtained in the
presence of glucose or palmitate as carbon sources (Figure 5A and B). Uncoupling of MTb oxidative
phosphorylation (OXPHOS) with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) significantly
diminished maximal respiration in the presence of the two inhibitors compared to the untreated control.
These findings suggest an energy generation pathway common to both fatty acid and glucose oxidation as
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
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
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3
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9
0
1
2
3
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9
0
2
1, 36
the target of the compounds. Previous studies
have shown that MTb can rapidly reroute electron flux
to overcome inhibition of cytochrome bc -aa
1
3
, cytochrome bd, or ATP synthase, resulting in enhanced
respiration. Therefore, we speculated that the target of compounds 1 and 7 was at the point of entry into
the ETC - that is, Complex 1 (NADH dehydrogenase) or Complex II (succinate dehydrogenase). To test this
prediction, ATP production was measured in MTb inverted membrane vesicles (IMVs) in the presence of
the mercapto-quinazolinones (Figure 5C and D). Notably, ATP production was significantly inhibited when
NADH was provided as electron donor but not when succinate was the source of reducing equivalents,
suggesting that the site of inhibition was NADH dehydrogenase. The mechanism by which the compounds
interfere with NADH dehydrogenase function differs from clofazimine and the quinolinequinones, which
have been proposed to interfere with electron transport by activating NDH-2 resulting in production of
1
8, 20
reactive oxygen species
.
To verify that NDH-2 was indeed the target for this class of mercapto-quinazolinones, we expressed MTb
NDH-2 (MtNdh) encoded by the predicted essential ndh in E. coli as a recombinant MBP-fusion protein, and
purified the protein to near homogeneity via amylose-resin affinity chromatography and gel filtration. The
recombinant enzyme was highly active with coenzyme Q2 (ubiquinone Q-2) and NADH as substrates,
3
7
delivering steady-state kinetic parameters similar to previously published results . Quinazolinones 1-3, 7
and 11 were found to have sub-micromolar IC50 values against the MtNdh (Figure 6). Compounds 1, 7, and
1
1 showed the highest potency, with IC50 values from 7-26 nM; values 3-orders of magnitude lower than
24
that for the previously characterised NDH-2 inhibitor chlorpromazine (CPZ; 10-25 µM; Figure 6) but
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
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4
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4
4
4
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5
5
5
5
5
6
superior to the quinolinyl pyrimidines discovered in a target-based screening effort against the MTb NDH-2
3
8
. No time-dependent inhibition was observed for these compounds. The mode of inhibition against
MtNdh was further investigated using compound 1: double reciprocal plots of enzyme activity under
varying concentrations of 1 and either Q2 or NADH suggested patterns indicative of a non-competitive
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
mode of inhibition with both substrates (K values 30-40 nM), although the effect on slope was less evident
i
when varying Q2 (Figure 7). This is similar to previous studies with a thiophenazine-based NDH-2 inhibitor
39
,
and suggests that inhibition occurs through binding of the compound at an allosteric site, independent
of the main substrate binding pockets.
Figure 6. Inhibition of recombinant MBP-MtNdh by select quinazolinones. Steady-state enzyme activity
was measured by monitoring absorbance changes at 340 nm dues to NADH oxidation, as described in
Materials and Methods. NADH and Q2 were fixed at 250 µM and 40 µM, respectively. Each data point is the
mean ± SD of at least 3 independent measurements. Inset table shows calculated IC50 values.
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Figure 7. Double-reciprocal plot of inhibition kinetics of MBP-MtNdh by 1. NADH (left) or Q2 (right)
concentrations were varied, in the presence of fixed concentrations of 40 µM Q2 (A) or 250 µM NADH (B).
Data points are the average of at least 3 independent measurements. Solid lines represent best-fit values of
the data to the general Michaelis-Menten reversible inhibition equation, using non-linear regression
analysis.
In conclusion, the non-proton pumping type II NADH dehydrogenase (NDH-2) has been shown to play a
critical role in the respiratory metabolism of bacteria. The 2-mercapto-quinazolinone scaffold is a potent
inhibitor of the mycobacterial NDH-2 and although in this study MTb whole-cell SAR only obtained limited
improvement in potency, this information, together with the recently disclosed structural information of
40
the enzyme from the bacterium Caldalkalibacillus thermarum , will aid in the future rational structure
based development of new and improved NDH-2 inhibitors. Critical for any structure-guided inhibitor
design is an understanding of the ligand-binding pocket of the NDH-2 protein. The low amino acid identity
between the C. thermarum and MTb NDH-2 proteins (30%) combined with the finding that this inhibitor
likely binds to an allosteric site with no allosteric ligand-binding sites identified on the existing bacterial
4
0
NDH-2 structure , suggests that co-crystallization of the compound with the MTb NDH-2 protein will be
essential to facilitate rational drug design. During revision of this paper, the SAR of a similar mercapto-
4
1
quinazolinone scaffold
was reported with a complementary paper describing the importance of the
mycobacterial type II NADH dehydrogenase despite the non-essentiality of the individual ndh and ndhA
4
2
genes .
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EXPERIMENTAL
Chemistry summary: All commercially available reagents, solvents and starting materials were purchased
from Aldrich Chemical Co. (UK). Where necessary a Biotage FLASH 25+ column chromatography system was
used to purify mixtures; reagent-grade solvents used for chromatography were purchased from Fisher
Scientific (UK) and flash column chromatography silica cartridges were obtained from Biotage (UK).
Analytical thin-layer chromatography (TLC) was performed on precoated TLC plates (layer 0.20 mm silica gel
0
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0 with fluorescent indicator UV254, from Merck). Developed plates were air-dried and analyzed under a
®
UV lamp (UV 254/365 nm). Microwave irradiation was conducted using a BIOTAGE INITIATOR unit. The
machine consists of a continuous focused microwave power delivery system with operator-selectable
1
13
power output (0-400 W at 2.45 GHz). All H and C NMR spectra were recorded on a Bruker ARX-500
1
13
spectrometer (500 and 300 MHz for H and C NMR, respectively). Chemical shifts (δ) are reported in ppm
relative to the residual solvent peak or internal standard (tetramethylsilane), and coupling constants (J) are
reported in hertz (Hz). Data are reported as follows: chemical shift, multiplicity (br=broad, s=singlet,
d=doublet, t=triplet, m=multiplet), integration. LC−MS analyses were performed with either an Agilent
HPLC 1100 series connected to a Bruker Daltonics MicrOTOF or an Agilent Technologies 1200 series HPLC
connected to an Agilent Technologies 6130 quadrupole spectrometer, where both instruments were
connected to an Agilent diode array detector. LC−MS chromatographic separations were conducted with a
Waters X bridge C18 column, 50 mm × 2.1 mm, 3.5 μm particle size; mobile phase, water/acetonitrile +
0
.1% HCOOH, or water/acetonitrile + 0.1% NH ; linear gradient from 80:20 to 5:95 over 3.5 min and then
3
−1
held for 1.5 min; flow rate of 0.5 mL.min . All assay compounds had a measured purity of ≥95% (by TIC and
UV) as determined using this analytical LC−MS system. High resolution electrospray measurements were
performed on a Bruker Daltonics MicrOTOF mass spectrometer.
General method for the preparation of amide coupling (1, 4-11, 14-16, 19-27) To a solution of 2-((4-oxo-
3
,4-dihydroquinazolin-2-yl)thio)acetic acid (1.0 equ) in a 1:1 ratio of acetonitrile (1.5 mL) and DMF (1.5 mL)
were added EDC.HCl (1.0 equ), HOAT (1.2 equ) or HATU (1.0 equ), and primary/secondary amine (1.0 equ)
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stirred for 30 min, DIPEA (2.0 equ) was added to the reaction mixture and stirred at room temperature
overnight. If a precipitate had formed this was filtered off, washed with acetonitrile and air dried. If the
reaction mixture had remained homogenous the solution was purified directly by preparative HPLC under
acidic conditions, using 0.1 % HCOOH in water:acetonitrile (5-95%) gradient elution to afford the product.
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N-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (1). To a solution of acid of 2-((4-oxo-
3
,4-dihydroquinazolin-2-yl)thio)acetic acid (30) (500 mg, 2.1 mmol, 1.0 equ) in 1:1 ratio of acetonitrile (1.5
mL) and DMF (1.5 mL) were added EDC.HCl (1.2 equ), HOAT (1.2 equ) or HATU (804 mg, 2.1 mmol, 1.0 equ)
in DCM (3 mL) and Cyclohexylamine (209 mg, 2.1 mmol, 1.0 equ) stirred for 30 min, DIPEA (0.73 mL, 2.1
mmol, 2.0 equ) was added to the reaction mixture and stirred at room temperature overnight. The reaction
mixture was purified by preparative HPLC under acidic conditions, using 0.1% HCOOH in water:acetonitrile
(5-95%) gradient elution to afford a white solid of N-cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-
yl)thio)acetamide as a white powder (450 mg, 66% yield), C H N O S, MW 317.41; ¹H NMR (500 MHz,
1
6
19
3
2
DMSO) δ 12.66 (brs, 1H), 8.14 (d, J=7.9 Hz, 1H), 8.04 (dd, J = 1.3, 7.9 Hz, 1H), 7.80 - 7.76 (m, 1H), 7.50 (d, J =
7
1
3
.9 Hz, 1H), 7.45 - 7.41 (m, 1H), 3.92 (s, 2H), 3.59 - 3.51 (m, 1H), 1.76 - 1.64 (m, 4H), 1.55 (dd, J = 3.5, 9.1 Hz,
+
+
20 3 2
H), 1.30 - 1.12 (m, 5H). LCMS m/z 318.12 [M+1] 100%. HRMS (m/z): [M + H] calcd for C16H N O S,
18.1270; found 318.1284.
N-Cycloheptyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (4). White powder (21 mg, 23% yield),
C H N O S, MW 331.43; ¹H NMR (500 MHz, DMSO) δ 12.65 (brs, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.02 (dd, J =
17 21
3
2
1.3, 7.9 Hz, 1H), 7.77 - 7.72 (m, 1H), 7.48 - 7.37 (m, 2H), 3.88 (s, 2H), 3.73 (ddd, J = 4.2, 8.6, 16.9 Hz, 1H),
+
+
1.60 - 1.34 (m, 12H). LCMS m/z 332.14 [M+1] 100%. HRMS (m/z): [M + H] calcd for C H N O S, 332.1427;
1
7
22
3
2
found 332.1439.
N-Cyclopentyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (5). White powder (39 mg, 24% yield),
15 17 3 2
C H N O S, MW 303.38; ¹H NMR (500 MHz, DMSO) δ 8.23 (d, J = 7.1 Hz, 1H), 8.04 (dd, J = 1.3, 8.0 Hz, 1H),
7
.79 - 7.75 (m, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.45 - 7.41 (m, 1H), 4.03 - 3.98 (m, 1H), 3.91 (s, 2H), 1.82 - 1.76
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+
+
(
m, 2H), 1.67 - 1.62 (m, 2H), 1.53 - 1.36 (m, 4H). LCMS m/z 304.12 [M + H] 100%. HRMS (m/z): [M + H]
calcd for C15 S, 304.1114; found 304.1107.
18 3 2
H N O
N-Cyclobutyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (6). White powder (76 mg, 66% yield),
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8
9
0
1
2
3
4
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C
14 15 3 2
H N O S, MW 289.35; ¹H NMR (400 MHz, DMSO) δ 12.67 (s, 1H), 8.54 (d, J = 7.7 Hz, 1H), 8.05 - 8.02 (m,
1
H), 7.81 - 7.75 (m, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 4.19 (dd, J = 8.1, 16.1 Hz, 1H), 3.91
+
(
s, 2H), 2.20 - 2.10 (m, 2H), 1.97 - 1.85 (m, 2H), 1.67 - 1.57 (m, 2H). LCMS m/z 290.04 [M + H] 100%. HRMS
+
(m/z): [M + H] calcd for C H N O S, 290.0957; found 290.0962.
1
4
16
3
2
N-(4,4-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (7). To a solution of 2-((4-
oxo-3,4-dihydroquinazolin-2-yl)thio)acetic acid 30 (108 mg, 0.4 mmol, 1.0 equ) in DCM (3 mL) were added
HATU (160 mg, 0.4 mmol, 1.0 equ) and 4,4-difluorocyclohexan-1-amine (57 mg, 0.4 mmol, 1.0 equ) and
stirred for 30 min, DIPEA (0.074 mL, 0.4 mmol, 2.0 equ) was added to the reaction mixture and stirred at
room temperature overnight. The reaction mixture was purified by preparative HPLC under acidic
conditions, using 0.1% HCOOH in water:acetonitrile (5-95%) gradient elution to afford a white solid of N-
(
4,4-difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide as a white powder 7 (115 mg,
7
8
3
17 2 3 2
5% yield), C16H F N O S, MW 353.39; ¹H NMR (500 MHz, DMSO) δ 12.64 (s, 1H), 8.39 (d, J = 6.9 Hz, 1H),
.02 (dd, J = 1.2, 7.9 Hz, 1H), 7.77-7.72 (m, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 3.90 (s, 2H),
+
.77 (t, J = 4.0 Hz, 1H), 2.04 - 1.75 (m, 6H), 1.56 - 1.47 (m, 2H). LCMS m/z 354.14 [M + H] 100%. HRMS
+
(m/z): [M + H] calcd for C H F N O S, 354.1082; found 354.1091.
16 18
2
3
2
N-(4,4-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide HCl Salt preparation (7).
N-(4,4-difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (60 mg, 0.17 mmol, 1.0
o
equ) in ethyl acetate (10 mL) and ethanol (3 mL) was heated up to 80 C , until dissolved (90%). The
solution was cooled immediately, followed by the addition of 2M HCl in diethyl ether (1 mL, 747 mg, 20.49
mmol, 120 equ) and stirred for a further 15 min, where white precipitates were formed. The precipitate
was filtered and dried to afford the N-(4,4-difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-
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yl)thio)acetamide HCl Salt (50 mg, 75% yield), C H F N O S.HCl, MW 389.85; ¹H NMR (500 MHz, DMSO) δ
16 17
2
3
2
8.32 (d, J = 7.6 Hz, 1H), 8.04 (dd, J = 1.2, 7.9 Hz, 1H), 7.80-7.76 (m, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.45-7.41 (m,
1H), 3.95 (s, 2H), 2.52-2.50 (m, 1H), 2.05 - 1.99 (m, 2H), 1.97 - 1.76 (m, 4H), 1.57 - 1.48 (m, 2H). LCMS m/z
+
+
18 2 3 2
354.14 [M + H] 100%. HRMS (m/z): [M + H] calcd for C16H F N O S, 354.1082; found 354.1091.
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N-(2,2-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (8). White powder (93
mg, 62% yield), C16 , MW 353.39; ¹H NMR (500 MHz, DMSO) δ 12.64 (s, 1H), 8.46 (d, J = 9.0 Hz,
H), 8.04 (d, J = 7.9 Hz, 1H), 7.79 - 7.75 (m, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.43 (dd, J = 7.5 Hz, 1H), 4.03 - 4.01
17 2 3 3 2
H F N O S
1
+
(
m, 3H), 2.08 - 2.03 (m, 1H), 1.86 - 1.63 (m, 4H), 1.49 - 1.38 (m, 3H). LCMS m/z 354.10 [M + H] 100%. HRMS
+
(
m/z): [M + H] calcd for C16
H
18
F
2
N
3 3
O S
2
, 354.1082; found 354.1094.
N-(3,3-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9). White powder (83
mg, 55% yield), C H F N O S , MW 353.39; ¹H NMR (500 MHz, DMSO) δ 12.61 (brs, 1H), 8.31 (d, J = 7.9 Hz,
1
6
17
2
3
3 2
1
2
H), 8.04 (dd, J = 1.3, 7.9 Hz, 1H), 7.80 - 7.76 (m, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 3.94 (s,
H), 3.91 - 3.75 (m, 1H), 2.21 - 2.15 (m, 1H), 1.98 - 1.95 (m, 1H), 1.81 - 1.68 (m, 4H), 1.45 - 1.24 (m, 2H).
+
+
18 2 3 3 2
LCMS m/z 354.10 [M + H] 100%. HRMS (m/z): [M + H] calcd for C16H F N O S , 354.1082; found 354.1095.
N-(Cyclohexylmethyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (10). White powder (34 mg,
2
1
5% yield), C17
21 3 2
H N O S, MW 331.43; ¹H NMR (500 MHz, DMSO) δ 12.64 (s, 1H), 8.16 (s, 1H), 8.05 - 8.02 (m,
H), 7.79 - 7.75 (m, 1H), 7.50 (dd, J = 3.1, 8.1 Hz, 1H), 7.44 - 7.40 (m, 1H), 3.93 (d, J = 2.5 Hz, 2H), 2.95 - 2.92
(m, 2H), 1.67 - 1.58 (m, 5H), 1.38 - 1.34 (m, 1H), 1.12 - 1.07 (m, 3H), 0.87 - 0.79 (m, 2H). LCMS m/z 332.21
+
+
[M + H] 100%. HRMS (m/z): [M + H] calcd for C H N O S, 332.1427; found 332.1431.
1
7
22
3
2
N-((4,4-Difluorocyclohexyl)methyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (11).
powder (120 mg, 77% yield), C17 S, MW 367.41; ¹H NMR (500 MHz, DMSO) ¹H NMR (500 MHz,
DMSO) δ 12.64 (s, 1H), 8.27 (t, J = 5.8 Hz, 1H), 8.04 (dd, J = 1.3, 7.9 Hz, 1H), 7.80 - 7.76 (m, 1H), 7.50 (d, J =
White
19 3 2 2
H N F O
8
.0 Hz, 1H), 7.45 - 7.42 (m, 1H), 3.95 (s, 2H), 3.02 (t, J = 6.4 Hz, 2H), 1.96 - 1.87 (m, 2H), 1.74 - 1.52 (m, 5H),
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5
5
5
6
+
+
1
.17 - 1.08 (m, 2H). LCMS m/z 368.01 [M + H] 100%. HRMS (m/z): [M + H] calcd for C H N F O S,
17 20
3
2
2
368.1238; found 368.1255.
N-Cyclohexyl-N-methyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (14). White powder (35 mg,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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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
2
1
5% yield), C17
21 3 2
H N O S, MW 331.43; ¹H NMR (500 MHz, DMSO) δ 12.60 (brs, 1H), 8.04 (dd, J = 1.6, 7.9 Hz,
H), 7.80 - 7.74 (m, 1H), 7.49 - 7.40 (m, 2H), 4.34 (s, 1H), 4.23 (s, 1H), 3.80 – 3.76 (m, 1H), 3.01 (s, 2H), 2.74
(
s, 1H), 1.76 (t, J = 12.0 Hz, 3H), 1.62 - 1.52 (m, 2H), 1.52 - 1.43 (m, 2H), 1.38 - 1.23 (m, 2H), 1.15 - 1.05 (m,
+
+
1
H). LCMS m/z 332.15 [M + H] . HRMS (m/z): [M + H] calcd for C H N O S, 332.1427; found 332.1440.
17 22 3 2
2
-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)propanoic acid (56).
To a solution of 2-thioxo-2,3-
dihydroquinazolin-4(1H)-one 29 (500 mg, 2.8 mmol, 1.0 equ), were added 2-bromopropanoic acid (557 mg,
3.6 mmol, 1.3 equ) and triethylamine (2.3 mL, 16.8 mmol, 6.0 equ) in DMF (5 mL), and stirred for 12 hr at
o
75 C. Once the reaction has completed, it was poured onto crushed ice and acidified with 1N HCl, where
the product precipitated out of the solution. The precipitate was filtered and dried to yield 2-((4-oxo-3,4-
dihydroquinazolin-2-yl)thio)propanoic acid 56 (548 mg, 78% yield), C H N O S, MW 250.27; ¹H NMR (500
1
1
10
2
3
MHz, DMSO) δ 12.66 (s, 1H), 8.04 (q, J = 3.10 Hz, 1H), 7.70 (m, 1H), 7.50 (d, J = 8.00 Hz, 1H), 7.44 (m, 1H),
+
4
.57 (q, J = 7.3 Hz, 2H), 1.55 (d, J = 7.30 Hz, 3H). LCMS m/z 251.00 [M + H] 100%.
N-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)propanamide (15). White powder (45 mg, 35%
yield), C H N O S, MW 331.43; ¹H NMR (500 MHz, DMSO) δ 12.59 (brs, 1H), 8.17 (d, J = 7.7 Hz, 1H), 8.04
17 21
3
2
(dd, J = 1.4, 7.9 Hz, 1H), 7.81 - 7.77 (m, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.45 - 7.42 (m, 1H), 4.54 (q, J = 7.0 Hz,
1H), 3.56 - 3.48 (m, 1H), 1.76 - 1.61 (m, 4H), 1.53 - 1.50 (m, 4H), 1.29 - 1.10 (m, 5H). LCMS m/z 332.10 [M +
+
+
22 3 2
H] 100%. HRMS (m/z): [M + H] calcd for C17H N O S, 332.1427; found 332.1432.
N-Cyclohexyl-N-methyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)propanamide (16). White powder (40
mg, 29% yield), C18 S, MW 345.46; ¹H NMR (500 MHz, DMSO) δ 12.60 (brs, 1H), 8.07 - 8.02 (m, 1H),
.82 - 7.75 (m, 1H), 7.50 - 7.41 (m, 2H), 4.24 – 4.18 (m, 1H), 3.05 (s, 2H), 2.75 (s, 1H), 1.80 - 1.70 (m, 3H),
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.64 - 1.54 (m, 1H), 1.54 - 1.47 (m, 6H), 1.36 - 1.20 (m, 2H), 1.14 - 1.02 (m, 2H). LCMS m/z 346.10 [M + H]
+
100%. HRMS (m/z): [M + H] calcd for C18
H
24
N
3
O
2
S, 346.1584; found 346.1608.
N-Cyclohexylglycine (53). Cyclohexylamine 51 (100 mg, 1.0 mmol, 1.0 equ) and ethyl 2-hydroxyacetate 52
104.9 mg, 1.0 mmol, 61.0 equ) were stirred neat in a round-bottom flask for 12 h. The solvent of the crude
mixture was evaporated completely, and the residue purified via flash column chromatography eluting with
0% DCM in methanol to afford N-Cyclohexylglycine 53 (100 mg, 63% yield), C , MW 157.21 ; ¹H
NMR (500 MHz, DMSO) δ 6.34 (brs, 1H), 4.05 – 4.04 (m, 2H), 3.82 – 3.77 (m, 1H), 3.77-3.69 (m, 1H), 1.86-
1
1
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4
4
4
5
5
5
5
5
5
5
5
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5
6
0
1
2
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9
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8
9
0
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7
8
9
0
(
1
8 2
H15NO
1
.83 (q, J = 5.28 Hz, 2H), 1.67-1.63 (m, 2H), 1.57-1.53 (m, 1H), 1.35-1.26 (m, 2H), 1.15-1.07 (m, 3H). LCMS
+
m/z 158.21 [M + H] 100%.
2
-(Methylthio)quinazolin-4(3H)-one (54). A solution of iodomethane (398 mg, 2.8 mmol, 1.0 equ) in
methanol (8 mL) was added slowly to a closed vessel containing a solution of 2-thioxo-2,3-
dihydroquinazolin-4(1H)-one 29 (500 mg, 2.8 mmol, 1.0 equ) in 1% aqueous NaOH (8 mL) under N
2
atmosphere and stirred for 1 h at room temperature. The reaction mixture was then cooled to 0°C, and the
pH adjusted to 7 with an aqueous solution of 1N HCl. The solvent was removed under reduced pressure,
and the solid was filtered, washed with water/methanol and dried to afford 2-(methylthio)quinazolin-
4
9 8 2
(3H)-one 54 (532 mg, 98% yield), C H N OS, MW 192.24; ¹H NMR (500 MHz, DMSO) δ 12.62 (s, 1H), 8.09
(
q, J = 3.13 Hz, 1H), 7.81 (m, 1H), 7.59 (d, J = 8.10 Hz, 1H), 7.47 (m, 1H), 2.63 (s, 3H). LCMS m/z 193.18 [M +
+
H] 100%.
N-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)amino)acetamide
(17).
2-amino-N-cyclohexyl-
acetamide 55 (40.63 mg, 0.26 mmol, 1.0 equ), 2-(methylthio)quinazolin-4(3H)-one 54 (50 mg, 0.26 mmol,
1
.0 equ) were suspended in ethyl acetate (2 mL) in a sealed microwave vial and irradiated (0-400 W at 2.65
o
Ghz) at 140 C for 2 h. Upon completion the reaction was filtered and the filtrate was extracted with diethyl
ether to afford N-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)amino)acetamide as a white powder 17
(4.4 mg, 4.4% yield), C16
H
20
N
4
O
2
, MW 300.36; ¹H NMR (500 MHz, DMSO) δ 8.10 (d, J = 7.7 Hz, 1H), 7.58 -
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.56 (m, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.22 - 7.18 (m, 1H), 3.95 (s, 2H), 1.76-1.66 (m, 4H), 1.56 - 1.53 (m, 1H),
+
+
21 4 2
1.31 - 1.07 (m, 6H). LCMS m/z 301.0 [M + H] 100%. HRMS (m/z): [M + H] calcd for C16H N O , 301.1659;
found 301.1665.
0
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N-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)oxy)acetamide (18). 2-(methylthio)quinazolin-4(3H)-
one 54 (50 mg, 0.26 mmol, 1.0 equ) and N-cyclohexylglycine 53 (21.7 mg, 0.12 mmol, 1.0 equ) were
o
dissolved in THF (2 mL) and heated to 80 C. Upon reaction completion the crude product was directly
purified via prep-HPLC Gilson purification using 0.1% ammonia in water:acetonitrile 5-95% gradient
elution.to afford the product as a white powder 18 (10 mg, 24% yield), C H N O , MW 301.34; ¹H NMR
1
6
19
3
3
(500 MHz, DMSO) δ 12.36 (brs, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.9 (d, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.40-7.33 (m,
1H), 3.77 (s, 2H), 3.60 – 3.56 (m, 1H), 1.76-1.66 (m, 4H), 1.57-1.53 (m, 1H), 1.31 - 1.10 (m, 6H). LCMS m/z
+
+
20 3
302.1 [M + H] 100%. HRMS (m/z): [M + H] calcd for C16H N O32, 302.1499; found 302.1487.
N-Cyclohexyl-3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanamide (19). White powder (110 mg, 80% yield),
, MW 299.37; ¹H NMR (500 MHz, DMSO) δ 12.18 (s, 1H), 8.08 (dd, J = 1.3, 7.9 Hz, 1H), 7.85 - 7.76
17 21 3 2
C H N O
(m, 2H), 7.56 (d, J = 8.0 Hz, 1H), 7.48 - 7.45 (m, 1H), 3.55 - 3.48 (m, 1H), 2.83 (t, J = 7.4 Hz, 2H), 2.58 (t, J =
+
7
.4 Hz, 2H), 1.73 - 1.64 (m, 4H), 1.55 - 1.52 (m, 1H), 1.28 - 1.09 (m, 5H). LCMS m/z 300.10 [M + H] 100%.
+
HRMS (m/z): [M + H] calcd for C17
H
22
N
3
O
2
, 300.1707; found 300.1721.
2
-((3-Methyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic acid (50). 2-mercapto-3-methylquinazolin-
4(3H)-one 49 (220 mg, 1.14 mmol, 1.0 equ), 2-bromoacetic acid (206 mg, 1.48 mmol, 1.3 equ) and
o
triethylamine (0.95 mL, 6.8 mmol, 6.0 equ) were stirred in DMF (5 mL) for 12 hr at 75 C. Upon reaction
completion the reaction mixture was poured onto crushed ice, acidified with 1N HCl. The precipitate
formed was collected and dried to afford the product 2-((3-Methyl-4-oxo-3,4-dihydroquinazolin-2-
10 2 3
yl)thio)acetic acid 50 (250 mg, 87% yield), as a white powder. C11H N O S, MW 250.27; ¹H NMR (500 MHz,
DMSO) δ 12.89 (s, 1H), 8.08 (q, J = 3.05 Hz, 1H), 7.79 (m, 1H), 7.46 (m, 2H), 4.11 (s, 2H), 3.55 (s, 3H). LCMS
+
m/z 251.04 [M + H] 100%.
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N-Cyclohexyl-2-((3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (20). White powder (20 mg,
15% yield), C17
21 3 2
H N O S, MW 331.42; ¹H NMR (500 MHz, DMSO) δ 8.15 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 7.9 Hz,
1H), 7.82 - 7.78 (m, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.47 - 7.43 (m, 1H), 3.98 (s, 2H), 3.54 (s, 4H), 1.76 - 1.65 (m,
1
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0
1
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9
0
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8
9
0
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8
9
0
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4
5
6
7
8
9
0
+
+
4
H), 1.55 (d, J = 12.8 Hz, 1H), 1.28 - 1.11 (m, 5H). LCMS m/z 332.15 [M + H] 100%. HRMS (m/z): [M + H]
calcd for C17 S, 332.1427; found 332.1436.
22 3 2
H N O
2
-(Quinazolin-2-ylthio)acetic acid (44). 2-chloroquinazoline 43 (0.2 g, 1.22 mmol) and sodium thioglycolic
acid 39b (1 equiv) were dissolved in DMF and triethylamine (6.0 equiv) added. The reaction mixture was
heated to 100°C overnight. The resulting precipitate was filtered and used without any further purification
to afford the product 2-(Quinazolin-2-ylthio)acetic acid 44 (0.153 g, 57% yield) as a white powder.
C
10
H
N
8 2
2
O S, MW 220.25, ¹H NMR (500 MHz, DMSO) δ 9.41 (d, J = 1.5 Hz, 1H), 8.09 (m, 1H), 7.95-7.93 (m,
+
1H), 7.79-7.77 (m, 1H), 7.66-7.61 (m, 1H), 4.05 (s, 2H). LCMS m/z 221.1 [M + H] 100%.
N-Cyclohexyl-2-(quinazolin-2-ylthio)acetamide (21). White powder (21 mg, 10% yield), C H N OS, MW
1
6
19
3
3
3
01.41; ¹H NMR (300 MHz, DMSO) δ 9.36 (m, 1H), 8.05-8.0 (m, 2H), 7.79-7.76 (m, 1H), 3.95 (s, 2H), 3.52-
+
.48 (m, 1H), 2.49-2.48 (m, 2H), 1.72-1.32 (m, 5H), 1.25-1.21 (m, 3H). LCMS m/z 302.1 [M + H] 100%.
2
-((4-Aminoquinazolin-2-yl)thio)acetic acid (46). 2-Chloroquinazolin-4-amine 45 (0.2 g, 1.11 mmol, 1.0
equ), sodium thioglycolic acid 39b (0.103 g, 1.11 mmol, 1.0 equ) and triethylamine were dissolved in DMF
(2ml) (0.93 ml, 6.68 mmol, 6.0 equ). The reaction mixture was heated to 100°C overnight. The resulting
precipitate was filtered to afford a white powder (0.2 g, 76% yield), and used without further purification
+
LCMS (ESI) m/z 236.1 [M + H]
2
-((4-Aminoquinazolin-2-yl)thio)-N-cyclohexylacetamide (22). White powder (21 mg, 10% yield),
C
16
H
20
N
4
OS, MW 316.42; ¹H NMR (300 MHz, DMSO) δ 8.13 (dd, J = 3.0, 6.0 Hz, 1H), 7.97 (d, J = 6 Hz, 1H),
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.87 (brs, 2H), 7.77-7.69 (m, 1H), 7.51-7.49 (m, 1H), 7.38-7.34 (m, 1H), 3.75 (s, 2H), 3.55-3.45 (m, 1H), 1.76-
+
1.47 (m, 5H), 1.23-1.09 (m, 5H). LCMS m/z 317.1 [M + H] 100%.
2-((4-Oxo-3,4,5,6,7,8-hexahydroquinazolin-2-yl)thio)acetic acid (40).
2-sulfanyl-5,6,7,8-tetrahydro-4-
0
1
2
3
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8
9
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1
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quinazolinol 38 (0.1 g, 5.49 mmol, 1.0 equ), chloroacetic acid 39a (1.3 equiv.) and triethylamine (6 equiv.)
were stirred in DMF at room temperature overnight. Once the reaction was complete, the reaction mixture
was poured over crushed ice and acidified with 1N HCl. The white precipitate was collected by filtration and
washed with water to afford the product as a white powder (750 mg, 58% yield) which was used without
further purification. C10
H
12
N
2
O
3
S, MW 240.28; ¹H NMR (300 MHz, DMSO) δ 12.59 (brs, 1H), 3.93 (s, 2H), 2.45
+
(t, J = 3.0 Hz, 2H), 2.29 (t, J = 3.0 Hz, 2H), 1.70-1.63 (m, 4H). LCMS (ESI): m/z 239.1 [M - H] 100%.
N-Cyclohexyl-2-((4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-yl)thio)acetamide (23). White powder (43 mg,
17% yield), C H N O S, MW 321.44; ¹H NMR (300 MHz, DMSO) δ 12.47 (brs, 1H), 8.00 (brs, 1H), 3.80 (s,
1
6
23
3
2
2
H), 3.58-3.49 (m, 1H), 2.47 (t, J = 3.0 Hz, 2H), 2.31 (t, J = 3.0 Hz, 2H), 1.75-1.54 (m, 9H), 1.29-1.13 (m, 5H).
+
LCMS m/z 320.1 [M - H] 100%.
2
-((4-Oxo-4,5,6,7-tetrahydro-3H-cyclopenta[d]pyrimidin-2-yl)thio)acetic acid (42). 2-thioxo-1,2,3,5,6,7-
hexahydro-4H-cyclopenta[d]pyrimidin-4-one 41 (1.0 g, 5.94 mmol, 1.0 equ), chloroacetic acid 39a (1.3
equiv.) and triethylamine (6 equiv.) were stirred in DMF (2 mL) at room temperature overnight. Once the
reaction was complete it was poured over crushed ice and acidified with 1N HCl. The white precipitate was
collected by filtration and washed with water to afford the product as a white powder (800 mg, 59% yield),
which was used without further purification. C H N O S, MW 226.05; ¹H NMR (300 MHz, DMSO) δ 12.65
9
10
2
3
(brs, 1H), 3.96 (s, 2H), 2.71 (t, J = 3.0 Hz, 2H), 2.58 (t, J = 3.0 Hz, 2H), 1.97-1.93 (m, 2H). LCMS m/z 227.1 [M
+
+
H] 100%.
N-Cyclohexyl-2-((4-oxo-4,5,6,7-tetrahydro-3H-cyclopenta[d]pyrimidin-2-yl)thio)acetamide(24).
powder (59 mg, 22% yield), C15 S, MW 307.41; ¹H NMR (300 MHz, DMSO) δ 12.59 (brs, 1H), 8.03 (s,
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H), 3.84 (s, 2H), 3.58-3.49 (m, 1H), 2.71 (t, J = 3.0 Hz, 2H), 2.60 (t, J = 3.0 Hz, 2H), 2.0-1.92 (m, 2H), 1.75-
+
1.52 (m, 5H), 1.29-1.16 (m, 5H). LCMS (ESI): m/z 308.1 [M + H] 100%.
2-((4,5-Dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio)acetic acid (48). 5,6-Dimethyl-2-thiouracil 47 (0.5 g,
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
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
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2
3
4
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8
9
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1
2
3
4
5
6
7
8
9
0
3
.20 mmol), chloroacetic acid 39a (0.39 g, 4.16 mmol) and triethylamine (2.62 mL, 1.94 mmol) were stirred
in DMF (2 mL) at room temperature overnight. Once the reaction was complete, it was poured over
crushed ice and acidified with 1N HCl. The white precipitate was collected by filtration and washed with
water to afford the product as a white powder (0.36 g, 54% yield) which was used without further
purification.C
8 10 2 3
H N O S, MW 214.24, ¹H NMR (300 MHz, DMSO) δ 12.56 (brs, 1H), 3.89 (s, 2H), 2.14 (s, 3H),
1
.85 (s, 3H). LCMS (ESI): m/z 213.1 [M - H]; HPLC purity 98%.
N-Cyclohexyl-2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio)acetamide (25). White powder (0.047
g, 17% yield). C H N O S, MW 295.40; ¹H NMR (400 MHz, DMSO-d6) δ 7.99 (dd, J = 20.3, 8.1 Hz, 1H), 3.80
1
4
21
3
2
(s, 2H), 3.11 – 2.97 (m, 1H), 2.20 (s, 3H), 1.89 (s, 3H), 1.78 – 1.64 (m, 5H), 1.33 – 1.12 (m, 5H). LCMS (ESI):
+
m/z 296.1 [M + H] 100%.
6
-Fluoro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (33). 2-Amino-5-fluorobenzoic acid 32 (1 g, 6.5 mmol,
o
1
.0 equ) and thiourea (2.1 g, 25.8 mmol, 6.0 equ) were heated neat in a round-bottom flask to 180 C for 3h
with stirring. The reaction was cooled and 25 ml of water added. After stirring for 10 min the precipitate
formed was filtered off, washed with cold water and dried to afford 6-Fluoro-2-thioxo-2,3-
8 5 2
dihydroquinazolin-4(1H)-one 33 (0.45 g, 35.9% yield) as a yellow powder. C H FN OS, MW 196.2; ¹H NMR
(500 MHz, DMSO) δ 9.72 (s, 1H), 7.51-7.39 (m, J = 8.5 Hz, 1H), 7.37-7.30 (m, J = 8.6 Hz, 1H), 7.19-7.16 (m, J
+
= 8.85 Hz, 1H). LCMS m/z 197.78 [M + H] 100%.
2
-((6-Fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic
acid
(34).
6-Fluoro-2-thioxo-2,3-
dihydroquinazolin-4(1H)-one 33 (414 mg, 2.1 mmol, 1.0 equ), 2-bromoacetic acid (586.39 mg, 4.2 mmol,
o
2
.0 equ) and triethylamine (1.76 mL, 12.6 mmol, 6.0 equ) were stirred in DMF (5 mL) at 75 C for 12 hr.
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