Discovery and Structure-Activity-Relationship Study of N-Alkyl-5-hydroxypyrimidinone Carboxamides as Novel Antitubercular Agents Targeting Decaprenylphosphoryl-β-D-ribose 2′-Oxidase

Article abstract of DOI:10.1021/acs.jmedchem.8b00883

Magnesium plays an important role in infection with Mycobacterium tuberculosis (Mtb) as a signal of the extracellular environment, as a cofactor for many enzymes, and as a structural element in important macromolecules. Raltegravir, an antiretroviral drug

Full text of DOI:10.1021/acs.jmedchem.8b00883

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
Discovery of N-Alkyl-5-hydroxypyrimidinone Carboxamides as Novel
Antitubercular Agents Targeting Decaprenylphosphoryl-#-D-ribose
2#-oxidase (DprE1) and Their Structure-activity Relationship Study
Sangmi Oh, Yumi Park, Curtis A. Engelhart, Joshua B. Wallach, Dirk Schnappinger, Kriti Arora,
Michelle Manikkam, Brian Gac, Hongwu Wang, Nicholas Murgolo, David B Olsen, Michael Goodwin,
Michelle Sutphin, Danielle M. Weiner, Laura E Via, Helena I. M. Boshoff, and Clifton E. Barry, III
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00883 • Publication Date (Web): 10 Oct 2018
Downloaded from http://pubs.acs.org on October 10, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery of N-Alkyl-5-hydroxypyrimidinone Carboxamides as
Novel Antitubercular Agents Targeting Decaprenylphosphoryl-β-
D-ribose 2′-oxidase (DprE1) and Their Structure-activity
Relationship Study
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
Sangmi Oh1, Yumi Park1, Curtis A. Engelhart2, Joshua B. Wallach2, Dirk Schnappinger2,
Kriti Arora1, Michelle Manikkam1, Brian Gac1, Hongwu Wang3, Nicholas Murgolo3, David
B. Olsen3, Michael Goodwin1, Michelle Sutphin1, Danielle M. Weiner1, Laura E. Via1,4,
Helena I. M. Boshoff1, Clifton E. Barry III1,4,*
1 Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute
of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, United
States
2Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York
10021, United States
3 Merck & Co., Inc., Discovery Research, 770 Sumneytown Pike, West Point, Pennsylvania 19486, United
States
4 Institute for Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town 7935,
South Africa
* Corresponding author.
Tel.: +1-301-435-7509; fax: +1-301-480-3506; e-mail: cbarry@niaid.nih.gov
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 39
1
2
3
4
ABSTRACT
5
6
7
8
Magnesium plays an important role in infection with Mycobacterium tuberculosis (Mtb) as a signal of the
extracellular environment, as a cofactor for many enzymes and as a structural element in important
macromolecules. Raltegravir, an antiretroviral drug that inhibit HIV-1 integrase is known to derive its
potency by selective sequestration of active site magnesium ions in addition to binding to a hydrophobic
pocket. In order to determine if essential Mtb related phosphoryl transfers could be disrupted in a similar
manner, a directed screened of known molecules with integrase inhibitor like pharmacophores (N-alkyl-5-
hydroxypyrimidinone carboxamides) was performed. Initial hits afforded compounds with low micromolar
potency against Mtb, acceptable cytotoxicity and PK characteristics and robust SAR. Elucidation of the
target of these compounds revealed that they lacked magnesium dependence and instead disappointingly
9
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
inhibited a known promiscuous target in Mtb DprE1 (Rv3790, decaprenylphosphoryl--D-ribose 2′-oxidase).
KEYWORDS
Pyrimidinedione
Structure-activity relationship
Tuberculosis
Anti-tubercular activity
ACS Paragon Plus Environment

Page 3 of 39
Journal of Medicinal Chemistry
1
2
3
4
INTRODUCTION
5
6
7
8
Tuberculosis (TB) remains a global threat despite more than a century of research on its etiologic agent
Mycobacterium tuberculosis (Mtb). According to the WHO, 10.4 million people were newly infected by TB,
and 1.3 million people died from this disease in 2016.1 Treatment of drug-sensitive TB requires at least 6
months of chemotherapy using drugs that were introduced into the market several decades ago whereas
treatment of multi-drug and extensively drug-resistant TB incorporate more drugs into an already complex
treatment regimen, thereby increasing duration of chemotherapy up to 24 months. The financial burden of
TB chemotherapy also increases dramatically for the diagnosis and treatment of drug-resistant TB and was
estimated to have incurred a global cost of US$ 2.0 billion in 2017, severely straining resource-constrained
TB control programs.1 New agents that inhibit novel targets are one strategy to strengthen the repertoire of
candidates in the drug development pipeline that may lead to simplified chemotherapy for drug-sensitive
TB and better options for drug-resistant therapy.2-3
9
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
Mg2+ is important in all bacteria serving, for example, as an important counterion for ATP in
numerous enzyme reactions as well as enabling formation of the tertiary structure of the bacterial ribosome
and contributing to the stability of lipid bilayers in cell envelopes.4-6 In Mtb, two systems have been
implicated in Mg2+ homeostasis, PhoPR and MgtC, and directly or indirectly exert broad control over various
aspects of mycobacterial metabolism. The PhoP system, which in Enterobacteriaceae regulates Mg2+ levels
and controls virulence,7 plays a role in controlling several families of virulence-associated methyl-branched
fatty acid containing acyl trehaloses that are unique to the pathogenic mycobacterial species.8 PhoP-
deficient mutants cannot grow in low concentrations of Mg2+, do not replicate in macrophages and fail to
establish or maintain infection in mouse lungs.9 MgtC is a Mg2+-responsive virulence factor thought to
function by interacting with the F1F0 ATP synthase,10 which was discovered in Mtb by homology to a well-
studied analog in Salmonella.11 Like mutants lacking PhoP, mgtC mutants are impaired for growth in low
concentrations of Mg2+ in a mildly acidic environment, do not replicate in macrophages, and fail to grow in
murine lungs and spleens.12 The reliance by Mtb on Mg2+ as both a signal and a cofactor spurred our
interest in potential therapeutic strategies that impact these systems.
Target-based approaches to TB drug discovery have not afforded any of the drugs in the pre-clinical
or clinical development pipeline. Whole cell screening has been most successful in identifying hits of interest
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 39
1
2
3
4
5
6
7
8
9
with subsequent efforts dedicated to the lead development and unraveling mechanism(s) of action.13
However, the hit rates of commercially available small-molecule libraries are often quite low and the hits
often possess low structural diversity and poor physicochemical properties.14 In contrast, small molecule
collections based on privileged scaffolds offer the potential of libraries encompassing attractive
physicochemical properties and containing core scaffolds known to target a diversity of cellular targets,
hereby increasing the likelihood of acceptable hit rates with compounds that inhibit specific targets.
Privileged structures are defined as chemical scaffolds which could afford potent and selective small-
molecule regulators for different kinds of biomolecules.14-16 Therefore, privileged structures typically display
favorable drug-like properties to become more high-quality leads. The dihydroxypyrimidine or
pyrimidinedione scaffold is considered a privileged scaffold found in many biologically active molecules.
The broad-ranging pharmacological roles generated by the pyrimidinedione moiety include antiviral
agents,17-18 antibacterial and antifungal agents,19-21 and kinase inhibitors.22 Among them, Raltegravir, which
is a Mg2+-dependent integrase inhibitor developed by MSD is a good example of an antiretroviral drug
embedded with the pyrimidinedione scaffold.23-25 In an effort to select hits that involve Mg2+-dependent
processes we were interested in exploring the activity of the Mg2+-chelating pyrimidinediones.
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
In this work, a novel series of N-alkyl-5-hydroxypyrimidinone carboxamides with antibacterial
activity against Mtb is reported. The initial hit compound, P01, was derived from high-throughput screening
of a library provided by St. Jude Children’s Research Hospital which contained diverse pyrimidinedione
derivatives as a privileged substructure.26 Preliminary structure-activity relationship (SAR) of the hit against
Mtb was explored as well as preliminary characterization of its biochemical target decaprenylphosphoryl--
D-ribose 2′-oxidase (DprE1), identified by resistance selection followed by whole genome sequencing.
RESULTS AND DISCUSSION
High-Throughput Screening of Pyrimidinedione Library. The pyrimidinedione scaffold is a core for a
diverse range of compounds inhibiting a multitude of potentially Mg2+-dependent targets based on the cation
dependence of Raltegravir binding to its target. As a result, we decided to screen under a growth condition
that supported robust in vitro growth with modest Mg2+ concentrations to exclude compounds that exert
toxicity simply by cation chelation. Thus, a pyrimidinedione library containing 6,207 compounds was
ACS Paragon Plus Environment

Page 5 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
screened for inhibition of growth of Mtb in a single-point assay at 10 µM final concentration. In medium
containing glucose/glycerol/Tween 80 as the carbon sources where the final Mg2+ concentration is 0.2mM,
38 hits (hit rate of 0.6%) giving >50% growth inhibition over a 3-day incubation period were generated.
These hits were further validated in dose titrations by full MIC determination. To observe any Mg2+-
dependence of the hits and to diminish the effects of protein binding, these assays were performed in
minimal medium (GAST) containing either 0.06, 6 or 60 mM MgCl2, representing the lowest concentrations
that still supports Mtb growth, standard or 10-fold higher in vitro Mg2+ concentrations, respectively. Three
compounds having similar scaffolds exerted sub-micromolar inhibition of Mtb growth for up 19 days of
incubation with little evidence of rescue in response to increasing extracellular Mg2+ concentrations. One of
these (P01) was chosen for further follow-up after consideration of structural aspects. As shown in Figure
1, P01 is a N-methyl-5-hydroxypyrimidinone derivative conjugated with a substituted benzyl group on the
left-hand side and benzyl substituted carboxamide moiety on the right-hand side. Preliminary biological
profiling of resynthesized P01 was performed to verify that the compound was active independent of carbon
source and to determine whether the compound inhibited targets in mycolyl-arabinogalactan biosynthesis
or induced DNA damage as previously described.27 The effect of the compound on inhibition of respiratory
metabolism was also assessed using the cyd::aph strain.28 The compound was found to be active under all
replicating conditions with decreased activity in the slow growth assay wherein Mtb is subjected to the sub-
optimal environment of acidic pH and nitrosative stress (Table 1). The cell wall reporter assay hinted that
the compound might affect a target in cell wall biosynthesis as evidenced by the upregulation of the iniBAC
promoter.
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
Figure 1. Hit compound, P01, and its derivatization for SAR study
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 39
1
2
3
4
Table 1. Biological aspects of hit compound, P01
5
6
7
8
MIC (µM)
Reporter Gene Assay
Cell wallg DNA damageh
Hit
7H9 /
ADCa
GBSA /
Nitrate /
GASTb
Chole
1.6
CydKOf
6.2
DBSAc
Butyrated
9
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
P01
4.7 ± 1.5
0.4 ± 0.2
3.1
12.5
Positive
No induction
b
af MIC of compound against Mtb strain H37Rv in: a Middlebrook 7H9/BSA containing glucose/glycerol/Tween 80,
GAST, c Middlebrook 7H9/BSA/Tyloxapol with glucose (GBSA) or dipalmitoyl phosphatidylcholine (DBSA) as carbon
source, d Middlebrook 7H9/BSA/Tyloxapol/butyrate/0.1 mM nitrite/pH 6.0,e Middlebrook 7H9/BSA/Tyloxapol/cholesterol,
and f Middlebrook 7H9/ADC with cydC::aph. g Induction of the cell wall responsive iniBAC promoter as measured using
the piniB-LUX reporter strain. h Induction of the DNA damage responsive recA and radA reporters using the precA-LUX
and pradA-LUX reporter strains.
Synthesis of Derivatives and Their Structure-activity Relationship. To understand the structural
aspects of the initial hit that were critical for antitubercular activity, a series of pyrimidinedione derivatives,
modified in each moiety of the original structure, were designed and prepared. The general synthesis of N-
alkyl-5-hydroxypyrimidinone carboxamides P1P13 is described in Scheme 1. For the formation of
dihydroxypyrimidine ring, previously reported procedures starting from nitrile derivatives (1) were used.18,
29-31
Briefly, amidoximes 2 were prepared from nitriles 1 after the reaction with salt-free hydroxylamine.
Next, Michael-type addition reaction of 2 with dimethyl acetylenedicarboxylate followed by intramolecular
cyclization were performed to generate dihydroxypyrimidine intermediates 46. After benzoyl protection of
5-hydroxyl group, alkylation on the N3 position mediated by Cs2CO3 followed by a thermal amidation
reaction, produced the desired products P1P13.
Scheme 1. General synthetic scheme of N-alkyl-5-hydroxypyrimidinone carboxamide derivativesa
ACS Paragon Plus Environment

Page 7 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
OH
N
CO2Me
CO2Me
OH
N
NH2
1
a
b
c
R1 CN
R1
OH
N
N
R1
O
R1
NH2
CO2Me
1
1
4 6
~
1
2
3
(R )
(R )
(R )
R1 of = o-CF3Ph, = Ph, = H
4
5
6
9
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
O
N
O
N
O
OBz
CO2Me
R2
OBz
R2
OH
d
e
f
HN
N
R1
R1
R1
N
CO2Me
N
COR3
7 9
10 13
~
P01 P13
~
~
R1 of = o-CF3Ph, = Ph, = H
R1 of
R2 of
= o-CF3Ph,
10 12 13 11
= Et
7
8
9
10, 11
12
13
= Ph, = H
,
,
= Me,
aReagents and conditions: (a) HONH2HCl, KOH, MeOH, reflux, 6 h; (b) dimethyl acetylenedicarboxylate, MeOH, rt,
overnight; (c) xylene, reflux, overnight; (d) BzCl, pyridine, rt, overnight; (e) R2I, Cs2CO3, THF; (f) amine or aniline (R3-
H), MeOH, reflux, overnight.
To validate the importance of the alkyl group at the N3 position, P14 without the methyl substituent
was synthesized by direct thermal amidation from the intermediate 4 (Scheme 2). During N3-methylation
in the presence of MeI and Cs2CO3, the addition of excess amounts of reagents, higher temperature and a
longer reaction time gave rise to the formation of over-methylated compound 14 which was the precursor
of P15. In addition, the O-Me regioisomer 15 was also produced as a by-product during methylation.
Structurally, the O-Me group in the pyrimidine ring was easily substituted by benzylamine during the thermal
amidation reaction allowing the reaction time for the synthesis of P16 to be shortened to avoid over-
substitution by benzylamines. To incorporate another methyl group in the acidic 5-hydroxyl position, 16
generated from 10 by debenzoylation was methylated by trimethylsilyldiazomethane to afford the
methylated compound, P17.17
The synthesis of P18 which has the ketone functionality at the position of C6 normally occupied by
the amide, is shown in Scheme 3. The 5-hydroxyl group in 16 was benzylated, and subsequent basic
hydrolysis afforded 18.32 The carboxylic moiety of 18 was transformed into Weinreb amide 19 which
underwent a selective mono-addition of Grignard reagent produced by (2-chloroethyl)benzene to generate
the corresponding ketone 20, with P18 obtained after debenzylation.33
All final compounds (P1P18) were fully characterized by LC / MS, 1H and 13C NMR. In addition,
HRMS and melting point analyses were performed for some active compounds. Antitubercular activities of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 39
1
2
3
4
5
6
synthetic compounds in two different media and their cytotoxicity against HepG2 cells are summarized in
Table 2.
7
8
9
Scheme 2. Synthesis of pyrimidinedione derivatives, from P14 to P17a
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
OH
O
OH
OH
N
HN
a
O
N
CO2Me
N
CF3
CF3
HN
4
P14
b
O
O
OH
OBz
N
N
a
c
O
N
N
CO2Me
O
CF3
HN
CF3
OBz
14
P15
HN
N
CO2Me
O
N
O
N
O
CF3
7
OBz
CO2Me
e
OH
d
OBz
N
N
N
+
O
N
CO2Me
CF3
CF3
HN
CF3
10
15
P16
f
O
O
O
O
O
OH
a
g
N
N
N
O
N
CO2Me
N
N
CO2Me
CF3
CF3
HN
CF3
16
17
P17
aReagents and conditions: (a) benzylamine (3 eq), MeOH, reflux, overnight; (b) BzCl, pyridine, rt, overnight; (c) MeI (5
eq), Cs2CO3 (3 eq), THF, 40 C, overnight; (d) MeI (3 eq), K2CO3 (2 eq), DMF, rt, 4 h; (e) benzylamine (2.2 eq),
reflux, 30 min; (f) N-benzylmethylamine, MeOH, reflux, 3 h; (g) TMSCHN2, CHCl3 / MeOH, rt, overnight.
Scheme 3. Synthesis of pyrimidinedione derivative, P18a
O
O
OBn
OBn
O
c
a, b
N
N
16
N
COOH
N
CF3
CF3
N
O
18
19
O
O
N
OBn
OH
N
N
d
e
O
O
N
CF3
CF3
P18
20
aReagents and conditions: (a) BnBr, KI, K2CO3, DMF, 80 C, overnight; (b) NaOH, MeOH / H2O, rt, 3 h; (c) CDI,
CH2Cl2, rt, 1 h, and then N,O-dimethylhydroxylamineHCl, TEA, rt, overnight; (d) generation of the Grignard reagent
using (2-chloroethyl) benzene, Mg, and 1,2-dibromethane in THF, and then rt, 2 h; (e) 10% Pd/C, H2, MeOH / THF, rt,
1 h.
ACS Paragon Plus Environment

Page 9 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Initial structure-activity relationship (SAR) studies around the hit compound P01 focused on
identifying key moieties or functionalities which play important roles in antitubercular activity. This focused
library of 18 compounds (P01P18) constructed for preliminary SAR, guided the next steps for hit to lead
9
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
modification, despite its small size. Compounds P02P03 and P15 were synthesized to evaluate the left-
hand side modification of R1. The benzene ring was necessary for antitubercular activity as seen by
complete loss of potency in the absence of this moiety (P03) with the o-CF3 substitution (P01) further
improving potency. A methyl substitution in the benzylic position was detrimental for activity as seen by the
5-fold increase in MIC of P15 compared to the initial hit, P01.
To assess the importance of the methyl group in the N3 position, two compounds including P04
with an ethyl group and P14 without an alkyl substituent were synthesized. Replacement of the methyl
(P01) by the ethyl group (P04) showed good potency with improved MICs (2.3 µM in 7H9/ADC, 0.4 µM in
GAST), but deletion of the methyl at this position (P14) was not tolerated. Not surprisingly, the O-Me
regioisomer (P16) was inactive against Mtb. Methyl protection of the acidic 5-hydroxyl group also resulted
in loss of antitubercular activity. These results demonstrate that the N-alkyl-5-hydroxypyrimidinone core
was critical for retaining potency against this pathogen.
The compounds P05P13 and P18 were synthesized to determine SAR of the right-hand side R3
position. As shown in Table 2, the benzyl group at the R3 position was important for antitubercular activity
because its replacement by a phenyl group (P05) reduced potency around 4-fold over P01 and the
saturated cyclohexyl methyl group (P06) was also less potent with likely increased protein binding as
evidenced by its lack of activity in BSA-containing Middlebrook 7H9-based medium. Replacement of the
phenyl with certain heteroaryl groups such as the 2-picolyl moiety (P07) was tolerated at the R3 position
but its regioisomer, 3-picolyl (P08), showed a decrease in activity. Initial investigations into substituent
effects on the benzene ring revealed that o-F (P09) was tolerated with a potency similar to that of P01 and
>4-fold more potent than its regioisomer P10 with p-F. Interestingly, the compound with the benzodioxole
moiety (P11) replacing the benzene ring was completely inactive against Mtb indicating that substituent
effects in the benzene ring were important for the antitubercular activity of this scaffold. Conformational
restriction of amide linkage by N-methylation (P12) as well as methylation of the benzylic position (P13)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 39
1
2
3
4
5
6
7
8
9
resulted in loss of activity. In addition, the amide functionality was important because P18 with a ketone
replacing the amide at R3 was inactive. None of the derivatives were cytotoxic against HepG2 cells and
additionally did not exert overt mitochondrial toxicity as seen by lack of cytotoxicity during growth of these
cells on galactose as carbon source (Table 2).
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
The pharmacokinetic (PK) parameters of C57BL/6 mice administered a single 10 mg/kg oral
suspension of P01 indicated absorption was fairly low (Cmax 0.25 µg/mL, AUC of 1.06 µgh/mL), but the
half-life of 4 h and Vd of 33.7 L/kg were acceptable (Table 3). Even though PK parameters are encouraging
as a starting point of in-depth medicinal chemistry, efficacy studies in mice were not pursued with this hit
compound because PK modeling suggested a much higher dose would be needed to achieve an average
plasma concentration above the MIC of P01 (~1.9 µg/mL in 7H9/ADC) because of its high metabolic rate.
Table 2. Structural aspects and antitubercular activities of compounds P01P18
Chemical Structure
Cytotoxicity
(IC50, µM)
O
N
MIC (µM)
R2
OH
R3
N
Compound
R1
O
R1
R2
R3
7H9/ADCa
GASTb
Glucosec
Galactosed
H
N
P01
P02
P03
o-CF3Ph
Me
Me
Me
4.7
9.4
0.59
3.1
>100
>100
>100
>100
>100
>100
H
N
Ph
H
H
N
>100
>100
H
N
P04
P05
P06
o-CF3Ph
o-CF3Ph
o-CF3Ph
Et
2.3
0.39
1.6
>100
>100
>100
>100
>100
>100
H
N
Me
Me
18.8
>100
H
12.5
N
H
N
P07
P08
o-CF3Ph
o-CF3Ph
Me
Me
3.1
1.2
4.7
>100
>100
>100
>100
N
H
N
18.8
N
F
H
N
P09
o-CF3Ph
Me
4.7
0.59
>100
>100
F
H
N
P10
P11
P12
o-CF3Ph
o-CF3Ph
o-CF3Ph
Me
Me
Me
18.8
>100
>100
2.3
>100
>100
>100
>100
>100
>100
O
O
H
N
37.5
>100
N
H
N
P13
o-CF3Ph
Me
>100
>100
>100
>100
ACS Paragon Plus Environment

Page 11 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
H
P14
P15
P16
P17
P18
o-CF3Ph
H
>100
>100
3.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
N
N/Ae (see Scheme 2)
N/A (see Scheme 2)
N/A (see Scheme 2)
N/A (see Scheme 3)
18.3
>100
>100
100
37.5
>100
12.5
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
a MIC of compound tested against Mtb H37Rv in Middlebrook 7H9/ADC. b MIC of compound tested against Mtb H37Rv
c
in GAST. See experimental section. Cytotoxicity of compound tested against HepG2 cells in DMEM/10% FBS
supplemented with glucose. d Cytotoxicity of compound tested against HepG2 cells in DMEM/10% FBS with galactose.
e Not available. Chemical structures described in Scheme 2 and 3.
Table 3. Oral exposure of P01 in mice after a single dose
PK propertya
unit
P01
Cmax
Tmax
T1/2
AUC
Vd
µg/mL
h
0.25
1.0
h
4.1
µgh/mL
L/kg
µg/h
1.06
33.7
9.48
Cl
a Pharmacokinetic parameters of P01 in C57Bl/6 mice dosed orally with 10 mg/kg.
Activity Validation in vitro and ex vivo and Target Protein Identification using Whole Genome
Sequencing. The hit compound P01 was bactericidal to actively replicating Mtb in vitro with a 12 log
reduction in colony forming units observed over 7 days with weak dose-dependence across a wide range
of concentrations (Figure 2A) but lacked bactericidal activity against anaerobic non-replicating Mtb cells
generated in the Wayne model of hypoxic adaptation (Figure 2B).34 The parental hit compound (P01) and
three of its in vitro active analogues (P04, P07, P09) were also active against Mtb replicating in J774 murine
macrophages indicating that the compound retained bactericidal activity under these conditions and was
also able to reach the pathogen in its phagosomal niche (Figure 2C).
To identify the putative target of P01, resistant mutants were generated by plating of Mtb on solid
medium plates containing 1, 2, 5, 10 MIC concentrations of the compound with a mutation frequency
of 7  10-9 obtained at 10 MIC. Twenty-four P01-resistant mutants were tested for their level of resistance
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 39
1
2
3
4
by MIC determination against P01 and all were found to be more than 10-fold resistant compared to the
5
6
parental strain. Four of these resistant strains were submitted for whole genome sequencing. All four were
7
8
found to contain the same Y314H mutation in DprE1 (Rv3790, decaprenylphosphoryl--D-ribose 2 ′ -
9
oxidase). DprE1 together with DprE2 catalyzes the epimerization of decaprenylphosphoryl ribose (DPR) to
decaprenylphosphoryl arabinose (DPA) in the synthesis of cell wall linked arabinan.35 DprE1 has already
been established as a highly vulnerable target in mycobacterial cell wall biosynthesis as seen by the high
number of chemically diverse scaffolds that inhibit this essential target, leading to cellular growth inhibition
both in vitro and in vivo.36-37 The high frequency of hits against this target is likely also driven by its extra-
cytoplasmic localization in the periplasmic face of the plasma membrane. The validity of inhibiting this target
as a viable strategy in TB chemotherapy will be tested in the on-going clinical trial of a benzothiazinone
compound (PBTZ169).38 To further confirm that the N-alkyl-5-hydroxypyrimidinone carboxamides inhibited
DprE1, we tested the vulnerability of a panel of diverse dprE1 mutants to P01. Strains with the Y314C and
P116S substitution but not the N346S mutation in DprE1 were found to be cross-resistant to P01 (Table 4).
Y314C has previously been observed to confer resistance to the DprE1 inhibitor TCA139, but not to the
benzothiazinone scaffold.40
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
ACS Paragon Plus Environment

Page 13 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 2. Activity of the hydroxypyrimidinone analogues in vitro and ex vivo. (A) Kill kinetics of P01 at 150
fold MIC under replicating condition for 7 days; (B) P01 lacks anaerobic bactericidal activity over for 7 days
of exposure indicated at fold concentrations of their aerobic MIC; (C) Intracellular killing assay of
hydroxypyrimidinone analogues in murine macrophage J774. Infected cells were treated at 150 fold MIC
of analogues for 4 or 7 days followed by colony enumeration.
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 4. Different mutations in DprE1 confer different levels of resistance to P01
DprE1 Mutation
Fold Change
MIC (M)
Wild type
Y314H
3.13
>50
25
-
>16
8
Y314C
N364Sa
P116Sa
2.34
18.8
0.75
6
a Mutants raised against an undisclosed DprE1 inhibitor.
Target Validation using a Regulated Expression of the dprE1-dprE2 Locus. The on-target activity of
the N-alkyl-5-hydroxypyrimidinone carboxamides was further confirmed using a strain where the level of
DprE1-DprE2 epimerase complex was transcriptionally regulated by the tetracycline repressor TetR. In this
strain, dprE1-dprE2 transcription is dependent on the presence of tetracycline. Removal of tetracycline
results in repression of dprE1-dprE2 and a concomitant increase in susceptibility to known DprE1 inhibitors
such as TCA139 and the benzothiazinone, BTZ04341 (Figure 3A & B) whereas ethambutol which inhibits the
downstream arabinosyltransferases showed equipotent efficacy during dprE1-dprE2 repression (Figure 3C).
As expected, active compounds P01 and P04, exerted more growth inhibition of the mycobacterial cells
during dprE1-dprE2 transcriptional repression (Figure 3D & E), whereas the inactive homolog P13 did not
result in growth inhibition of this regulated mutant (Figure 3F). The higher resistance of the tetracycline-
regulated cells during treatment with TCA1, P01 and P04 inhibitors is likely driven by higher basal levels
of protein of one or both of the DprE subunits. The clear shift in susceptibility during regulated expression
of the subunits of this epimerase complex establishes the on-target activity of the N-alkyl-5-
hydroxypyrimidinone carboxamides in whole cells.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 39
1
2
3
4
5
6
7
8
9
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
Figure 3. The hydroxypyrimidinone analogues target the DprE1-DprE2 enzymes in whole cells as
evidenced by increased vulnerability of a transcriptionally down-regulated dprE1-dprE2 mutant. Removal
of anhydrotetracycline (atc) results in transcriptional repression of the dprE1-dprE2 operon. Growth in the
presence of the DprE1 inhibitor TCA1 (A), the covalent DprE1 inhibitor BTZ043 (B), the
arabinosyltransferase inhibitor ethambutol (C) and the P01 (D), P04 (E) and P13 (F) analogues was
recorded relative to the DMSO-only control. Data are representative of two independent experiments.
Molecular Docking Study of N-Alkyl-5-hydroxypyrimidinone Carboxamides in DprE1. Molecular
modeling was used to understand the potential interactions of synthetic analogues with DprE1. Figure 4A
shows the model of P01 in the binding site of DprE1. An extensive hydrogen bond (HB) network was formed
between P01 and the polar residues lining the interior of the binding site. The carbonyl oxygen atom of the
pyrimidinone core formed a HB with the sidechain amino group of residue N385. The 5-hydroxyl group,
with a pKa of 4.5 as calculated by ACD Labs pKa method,42 was deprotonated, and the negatively charged
oxygen atom formed HBs with both H132 and Q336 sidechains. Methylation of this oxygen atom as in P17
disrupted the HBs, hence, rendered the compound inactive. The exocyclic amide oxygen on the right-hand
side formed an HB with the K418 sidechain. The amide –NH group, on the other hand, formed an
intramolecular HB with one nitrogen atom of the pyrimidinone core, stabilizing the ligand bound
ACS Paragon Plus Environment

Page 15 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
conformation. Methylation of this group as in P12 abolished this intramolecular HB, and the size of the
methyl group forces the amide to rotate out of the plane of the pyrimidinone core leading to an inactive P12.
Replacing the amide –NH group with a methylene as in P18 had a similar impact that reduced its binding
affinity. Furthermore, the methyl group in N3 position made hydrophobic contact with the sidechain carbon
atoms of K367, demethylation here as in P16 was likely the reason for its reduced activity.
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
Hydrophobic interactions with the DprE1 active site contributed substantially to the ligand binding
affinity. The trifluoromethyl group on phenyl ring of the left-hand side made hydrophobic contacts with the
sidechain of Y314, while the phenyl ring itself engaged in hydrophobic interaction with the sidechain of
V365 at the bottom of the binding site. Deleting the o-CF3 group lowered the binding affinity several-fold as
shown for P02, while removing the whole o-CF3Ph moiety as in P03 made the compound completely
inactive. The phenyl ring on the right-hand side made π-cation interaction with the R325 sidechain.
Replacing this group with a cyclohexyl moiety reduces this favorable interaction and leads to the low
potency of P06. In addition, this phenyl group made hydrophobic contacts with the sidechains of residues
L317, L363 and V365. The size of the binding pocket around this phenyl group was not large enough to
accommodate P11, hence, its much lower potency.
The model of P01 was compared to the crystal structure of a known DprE1 inhibitor TCA139 (Figure
4B). The pyrimidinedione core of P01 binds at the same sub pocket as the thiophene core, both forming
strong interaction with the H132 sidechain. The amide carbonyl group in both compounds forms hydrogen
bond with the sidechain of K418. The distal benzyl group off the amide binds in the same hydrophobic
region as the benzothiazole group of TCA1. Additionally, the other side of both molecules engages the
Y314 sidechain. This docking model and the similar interaction pattern with the DprE1 binding site in
comparison with TCA1 provide further evidence that these compounds target Mtb DprE1.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 39
1
2
3
4
5
6
7
8
9
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
Figure 4. Molecular docking study of P01 in DprE1. (A) Docking model of P01 in the binding site of Mtb
DprE1 (PDB code: 4P8N). The protein structure is shown as cyan colored cartoon with the key residues
involved the ligand binding displayed. The protein sidechain carbon atoms are colored cyan, and the ligand
(P01) carbon atoms colored brown. Oxygen atoms are colored red, nitrogen blue, fluorine light cyan and
polar hydrogen atom on the ligand white. Hydrogen bonds between the ligand and the protein are depicted
as yellow dashed lines; (B) Overlay the P01 model with the crystal structure of TCA1 in complex with DprE1
(PDB code: 4KW5). The carbon atoms of TCA1 are colored magenta. The TCA1 bound protein structure
and the carbon atoms of the key residues are also colored magenta.
CONCLUSIONS
In order to discover a novel candidate for the treatment of TB, we screened a pyrimidinedione library which
contains this embedded privileged structure associated with magnesium-chelating properties, and
generated a new hit, N-alkyl-5-hydroxypyrimidinone carboxamide P01. The SAR of P01 was examined
through the construction of 17 analogues in which we comprehensively modified each moiety in the original
scaffold. Even though the MICs of the active compounds were not dependent on Mg2+ concentration, they
showed significant growth inhibition in each media without notable cytotoxicity. Through whole genome
sequencing of resistant mutants, we identified that this series targets DprE1 which is an essential enzyme
for pathogen-specific cell wall biogenesis. Several molecules that target DprE1 are in preclinical and clinical
development43 but no drug targeting this enzyme has been approved for clinical use to date. The N-alkyl-
5-hydroxypyrimidinone carboxamides represent a potentially viable alternative scaffold should current
candidates fail to complete clinical development. These compounds have favorable solubility, toxicity and
PK properties that should provide adequate opportunity for lead optimization. Although it was disappointing
ACS Paragon Plus Environment

Page 17 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
to not identify scaffolds that potentiated Mg2+-dependent processes in the cell, alternative screening
procedures may still identify candidate molecules within this privileged core structure that do.
6
7
8
9
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
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 39
1
2
3
4
EXPERIMENTAL SECTION
5
6
7
8
Chemistry. H and 13C NMR spectra were recorded on a Varian Mercury-300 NMR Spectrometer, and
chemical shifts were measured in ppm relative to specific solvent signal. Routine mass and purity analyses
(LRMS) were performed on HP Agilent LC/MS series 1100 system equipped with a reverse phase column
(Agilent Poroshell 120 EC-C18, 2.7 μm, 50  2.1 mm) and photodiode array detector using electrospray
ionization (ESI). The gradient mobile phase consisting of acetonitrile / water with 0.1% formic acid and UV
detection at 254 and 210 nm were used to confirm all final products (P01P18) to be ≥95%. Melting point
was measured on Electrothermal 9100 apparatus. Accurate masses (HRMS) were obtained using Waters
LCT Premiere time-of-flight mass spectrometer. The instrument was operated in the W-mode at a resolution
of 10,000 and in positive ion mode. Ions were generated with the Z-Spray electrospray ionization (ESI) with
a capillary voltage of 3.4 kV. Accurate masses were determined using the internal standard method. Most
reagents used in the synthetic procedure were purchased from Sigma-Aldrich, Alfa Aesar, and TCI. The
progress of reaction was monitored using thin-layer chromatography (TLC) (silica gel 60 F254 0.25 mm),
and the products were visualized by UV light (254 and 365 nm). SiliaFlash® P60 (40–60 µm) used in flash
column chromatography was purchased from Silicycle Inc. Other solvents were purchased from commercial
vendors and used without further purification unless otherwise mentioned.
1
9
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
Synthesis of P01P17
1. General procedure for pyrimidinedione formation starting from substituted acetonitrile. Synthesis
of pyrimidinedione intermediates 46 were performed using the previously reported procedure.18, 29-31
1-1.
Methyl
5,6-dihydroxy-2-(2-(trifluoromethyl)benzyl)pyrimidine-4-carboxylate
(4).
Synthesis from the starting material, 2-(trifluoromethyl)phenylacetonitrile, was followed by known procedure
(overall 26%, brown solid): 1H NMR (300 MHz, (CD3)2SO)  7.71 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.6 Hz,
1H), 7.47 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 7.5 Hz, 1H), 4.07 (s, 2H), 3.73 (s, 3H); 13C NMR (75 MHz,
(CD3)2SO)  168.3, 166.0, 159.3, 148.1, 145.2, 134.6, 132.6, 131.8, 127.4, 127.1, 126.0 (q), 122.6, 52.2,
36.4; LRMS (ESI) m/z 329 [M + 1]+.
ACS Paragon Plus Environment

Page 19 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1-2. Methyl 2-benzyl-5,6-dihydroxypyrimidine-4-carboxylate (5). Synthesis from the starting
material, benzyl cyanide, was followed by known procedure (overall 40%): 1H NMR (300 MHz, (CD3)2SO)
 10.24 (bs, 1H), 7.347.21 (m, 5H), 3.81 (s, 2H), 3.80 (s, 3H); 13C NMR (75 MHz, (CD3)2SO)  166.1,
159.1, 149.3, 145.0136.6, 132.5, 128.6, 128.5, 126.7, 52.2, 40.7: LRMS (ESI) m/z 261 [M + 1]+.
1-3. Methyl 2-ethyl-5,6-dihydroxypyrimidine-4-carboxylate (6). Synthesis from the starting
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
1
material, propionitrile, was followed by known procedure (overall 11%): H NMR (300 MHz, (CD3)2SO) 
10.13 (bs, 1H), 3.80 (s, 3H), 2.47 (q, J = 7.5 Hz, 2H), 1.14 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz, (CD3)2SO)
 166.3, 159.1, 151.8, 144.8, 128.7, 52.1, 27.0, 11.5; LRMS (ESI) m/z 199 [M + 1]+.
2. General procedure of benzoylation in 5-OH group (79). To a solution of 4 (1.0 eq) in pyridine (0.5
M) was added benzoyl chloride (1.0 eq) at rt. After stirring overnight at rt, the reaction mixture was diluted
with EtOAc and washed with 1N HCl, sat. NaHCO3, and brine. The organic layer was dried over Na2SO4
and evaporated in vacuo. The crude solid was suspended in EtOAc and filtered, washed with Et2O to obtain
the desired product.
2-1. Methyl 5-(benzoyloxy)-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-dihydropyrimidine-4-
1
carboxylate (7). Off-white solid (69%): Rf = 0.43 (EtOAc / hexane = 1 / 1); H NMR (300 MHz, CDCl3) 
8.16 (d, J = 7.2 Hz, 2H), 7.66 (t, J = 7.2 Hz, 1H), 7.567.48 (m, 4H), 7.387.33 (m, 2H), 4.26 (s, 2H), 3.83
(s, 3H); 13C NMR (75 MHz, CDCl3)  163.5, 163.1, 159.2, 156.3, 143.4, 138.2, 134.2, 132.7, 131.9, 130.7,
129.1, 128.8, 128.3, 128.2, 126.7 (q), 125.9, 122.3, 53.4, 38.4; LRMS (ESI) m/z 433 [M + 1]+.
2-2. Methyl 5-(benzoyloxy)-2-benzyl-6-oxo-1,6-dihydropyrimidine-4-carboxylate (8). Off-white
solid (72%): Rf = 0.29 (EtOAc / hexane = 1 / 1); 1H NMR (300 MHz, CDCl3)  8.24 (d, J = 8.4 Hz, 2H), 7.69
(t, J = 7.5 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.317.27 (m, 5H), 4.03 (s, 2H), 3.90 (2, 3H); 13C NMR (75 MHz,
CDCl3)  163.5, 163.2, 160.2, 157.9, 143.9, 137.9, 134.3, 134.0, 130.7, 129.5, 129.2, 128.9, 128.3, 128.0,
53.4, 42.0; LRMS (ESI) m/z 365 [M + 1]+.
2-3. Methyl 5-(benzoyloxy)-2-ethyl-6-oxo-1,6-dihydropyrimidine-4-carboxylate (9). Off-white
solid (55%): Rf = 0.19 (EtOAc / hexane = 1 / 1); 1H NMR (300 MHz, CDCl3)  8.19 (d, J = 8.4 Hz, 2H), 7.67
(t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.5 Hz, 2H), 2.73 (q, J = 7.5 Hz, 2H), 1.29 (t, J = 7.5 Hz, 3H); 13C NMR (75
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 39
1
2
3
4
5
6
MHz, CDCl3)  163.7, 163.3, 160.8, 160.7, 144.2, 137.5, 134.2, 130.6, 128.8, 128.4, 53.4, 28.9, 11.6; LRMS
(ESI) m/z 303 [M + 1]+.
7
8
9
3. General procedure of alkylation mediated by Cs2CO3 (1013). To a solution of starting material (1.0
eq) in anhydrous THF (0.2 M) was added Cs2CO3 (2.0 eq) and alkyl iodide (2.5 eq). The reaction mixture
was stirred at rt for about 5 h in the case of methylation, or stirred overnight at 40°C in the case of ethylation.
The reaction mixture was diluted with EtOAc and washed with 1N HCl and brine. The organic layer was
dried over Na2SO4 and evaporated in vacuo. The crude mixture was purified by flash column
chromatography (EtOAc / hexane) to obtain the desired product.
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
3-1.
Methyl
5-(benzoyloxy)-1-methyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
dihydropyrimidine-4-carboxylate (10). Off-white solid (79%): Rf = 0.53 (EtOAc / hexane = 1 / 1); 1H NMR
(300 MHz, CDCl3)  8.17 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.9 Hz, 1H), 7.48 (t, J =
7.7 Hz, 2H), 7.39 (t, J = 7.5 Hz, 1H), 7.07 (d, J = 7.2 Hz, 1H), 4.43 (s, 2H), 3.81 (s, 3H), 3.37 (s, 3H); 13
C
NMR (75 MHz, CDCl3)  163.6, 163.1, 158.6, 156.6, 140.5, 137.7, 134.1, 132.7, 132.6, 130.6, 128.9, 128.7,
128.4, 128.3, 128.0, 127.8, 126.7 (q), 126.1, 122.4, 53.1, 38.8, 31.9; LRMS (ESI) m/z 447 [M + 1]+.
3-2.
Methyl
5-(benzoyloxy)-1-ethyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
dihydropyrimidine-4-carboxylate (11). Off-white solid (45%): Rf = 0.53 (EtOAc / hexane = 1 / 1); 1H NMR
(300 MHz, CDCl3)  8.20 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.8 Hz, 1H), 7.65 (t, J = 7.9 Hz, 1H), 7.51 (t, J =
7.2 Hz, 2H), 7.42 (t, J = 7.3 Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 4.44 (s, 2H), 3.89 (q, J = 7.2 Hz, 2H), 3.84 (s,
3H), 1.23 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3)  163.8, 163.3, 158.3, 156.2, 140.7, 138.1, 134.2,
133.2, 132.7, 130.7, 129.3, 128.8, 128.4, 128.0, 127.9, 126.7 (q), 126.2, 122.6, 53.3, 41.2, 38.3, 13.2;
LRMS (ESI) m/z 461 [M + 1]+.
3-3. Methyl 5-(benzoyloxy)-2-benzyl-1-methyl-6-oxo-1,6-dihydropyrimidine-4-carboxylate
(12). Off-white solid (61%): Rf = 0.35 (EtOAc / hexane = 1 / 1); 1H NMR (300 MHz, CDCl3)  8.18 (d, J =
7.8 Hz, 2H), 7.63 (t, J = 7.8 Hz, 1H), 7.49 (t, J = 7.8 Hz, 2H), 7.367.21 (m, 5H), 4.25 (s, 2H), 3.85 (s, 3H),
3.44 (s, 3H); 13C NMR (75 MHz, CDCl3)  163.7, 163.3, 158.7, 157.7, 140.7, 137.5, 134.1, 133.7, 130.6,
129.3, 128.7, 128.4, 127.7, 53.2, 42.5, 32.0; LRMS (ESI) m/z 379 [M + 1]+.
3-4. Methyl 5-(benzoyloxy)-2-ethyl-1-methyl-6-oxo-1,6-dihydropyrimidine-4-carboxylate (13).
Off-white solid (97%): Rf = 0.25 (EtOAc / hexane = 3 / 2); 1H NMR (300 MHz, CDCl3)  8.16 (J = 7.8 Hz,
ACS Paragon Plus Environment

Page 21 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
2H), 7.61 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.5 Hz, 2H), 3.80 (s, 3H), 3.57 (s, 3H), 2.82 (q, J = 7.3 Hz, 2H),
1.35 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, CDCl3)  163.76, 163.5, 160.3, 158.6, 140.9, 136.7, 134.0,
130.6, 128.6, 128.4, 53.1, 31.4, 28.9, 10.8; LRMS (ESI) m/z 317 [M + 1]+.
9
4. Synthesis of methyl 5-(benzoyloxy)-1-methyl-6-oxo-2-(1-(2-(trifluoromethyl)phenyl)ethyl)-1,6-
dihydropyrimidine-4-carboxylate (14). To a solution of 7 (1.0 eq) in anhydrous THF (0.2 M) was added
Cs2CO3 (3.0 eq) and MeI (5.0 eq). The reaction mixture was stirred overnight at 40 °C. The reaction mixture
was diluted with EtOAc, and washed with 1N HCl and brine. The organic layer was dried over Na2SO4 and
evaporated in vacuo. The crude mixture was purified by flash column chromatography (EtOAc / hexane =
2 / 3) to obtain the desired product as an off-white solid (58%): Rf = 0.65 (EtOAc / hexane = 1 / 1); 1H NMR
(300 MHz, CDCl3)  8.19 (d, J = 7.8 Hz, 2H), 7.74 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 7.2 Hz, 1H), 7.50 (t, J =
7.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 1H), 7.27 (d, J = 7.2 Hz, 1H), 4.60 (q, J = 6.6 Hz, 1H), 3.86 (s, 3H), 3.35 (s,
3H), 1.72 (d, J = 6.6 Hz, 3H); LRMS (ESI) m/z 461 [M + 1]+.
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
5. Synthesis of methyl 5-(benzoyloxy)-6-methoxy-2-(2-(trifluoromethyl)benzyl)pyrimidine-4-
carboxylate (15). To a solution of 7 (1.0 eq) in anhydrous DMF (0.2 M) was added K2CO3 (2.0 eq) and MeI
(3.0 eq). The reaction mixture was stirred at rt for 4 h. The reaction mixture was diluted with EtOAc, and
washed with 1N HCl and brine. The organic layer was dried over Na2SO4 and evaporated in vacuo. The
crude mixture was purified by flash column chromatography (EtOAc / hexane = 1 / 9  1 / 4  2 / 3) to
obtain two different isomers, the desired product 15 (O-Me, 11%) and 10 (N-Me, 83%): Rf = 0.31 (EtOAc /
hexane = 1 / 4); 1H NMR (300 MHz, CDCl3)  8.18 (d, J = 7.8 Hz, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.557.46
(m, 3H), 7.407.33 (m, 2H), 4.50 (s, 2H), 3.87 (s, 3H), 3.84 (s, 3H); 13C NMR (75 MHz, CDCl3)  165.5,
163.9, 163.8, 163.3, 146.9, 136.2, 134.2, 132.3, 132.1, 131.8, 130.6, 128.9, 128.4, 126.9, 126.5, 126.1 (q),
122.8, 55.0, 53.3, 41.6; LRMS (ESI) m/z 447 [M + 1]+.
6.
Synthesis
of
methyl
5-hydroxy-1-methyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
dihydropyrimidine-4-carboxylate (16). To a solution of 10 (1.0 eq) in anhydrous MeOH (0.2 M) was added
N-benzylmethylamine (2.0 eq). The reaction mixture was refluxed for 2 h. The reaction mixture was diluted
with EtOAc and washed with 1N HCl and brine. The organic layer was dried over MgSO4 and evaporated
in vacuo. The crude mixture was suspended in EtOH and stirred for about 2 h. The solid was filtered, and
washed with EtOH and Et2O to obtain the desired product as an off-white solid (84%): Rf = 0.28 (MeOH /
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 39
1
2
3
4
5
6
7
8
9
CH2Cl2 = 5 / 95); 1H NMR (300 MHz, CDCl3)  10.58 (bs, 1H), 7.71 (d, J = 7.2 Hz, 1H), 7.45 (t, J = 7.5 Hz,
1H), 7.38 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 7.8 Hz, 1H), 4.36 (s, 2H), 4.01 (s, 3H), 3.33 (s, 3H); 13C NMR (75
MHz, CDCl3)  169.7, 158.7, 149.1, 148.9, 133.5, 132.7, 128.5, 128.1, 127.7, 126.8 (q), 126.2, 124.4, 122.5,
53.6, 38.7, 31.9; LRMS (ESI) m/z 343 [M + 1]+.
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
7.
Synthesis
of
methyl
5-methoxy-1-methyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
dihydropyrimidine-4-carboxylate (17). To a solution of 16 (1.0 eq) in anhydrous CHCl3 / MeOH (2 / 1, 0.2
M) was added trimethylsilyldiazomethane (5.5 eq, 1.1 eq  5 times) potionwise for about 1 h. The reaction
mixture was stirred overnight at rt. The reaction mixture was evaporated in vacuo, and suspended in EtOH.
After stirring for 2 h, the solid was filtered, and washed with EtOH and Et2O to obtain the desired product
as an off-white solid (80%): Rf = 0.24 (EtOAc / hexane = 2 / 3); 1H NMR (300 MHz, CDCl3)  7.70 (d, J =
7.8 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.00 (d, J = 7.5 Hz, 1H), 4.35 (s, 2H), 4.01 (s,
3H), 3.92 (s, 3H), 3.32 (s, 3H); 13C NMR (75 MHz, CDCl3)  164.6, 160.1, 153.7, 144.1, 139.9, 133.1, 132.7,
128.8, 128.5, 128.1, 127.7, 126.7 (q), 126.1, 122.5, 60.7, 53.0, 38.8, 31.5; LRMS (ESI) m/z 357 [M + 1]+.
8. General Procedure of thermal addition and elimination reaction (P1P17). To a solution of starting
material (1.0 eq) in anhydrous MeOH (0.2 M) was added each amine (3.0 eq or 2.2 eq in the case of P16).
The reaction mixture was refluxed until completion of reaction (TLC or LC/MS). In most cases, overnight
reflux was enough to complete the reaction except P05 (3 days), P12 (5 days), and P16 (30 min). The
reaction mixture was diluted with EtOAc, and washed with 1N HCl and brine. The organic layer was dried
over MgSO4 and evaporated in vacuo. The crude mixture was suspended in EtOH or Et2O for crystallization
of each desired product.
8-1.
N-Benzyl-5-hydroxy-1-methyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
dihydropyrimidine-4-carboxamide (P01). Off-white solid (83%): mp = 131.7 ºC; Rf = 0.54 (MeOH / CHCl3
1
= 1 / 9); H NMR (300 MHz, CDCl3)  12.00 (s, 1H), 7.67 (d, J = 7.2 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H),
7.357.29 (m, 4H), 7.237.21 (m, 2H), 7.03 (d, J = 7.5 Hz, 1H), 4.49 (d, J = 6.0 Hz, 2H), 4.21 (s, 2H), 3.41
(s, 3H); 13C NMR (CDCl3, 75 MHz)  167.9, 158.8, 148.4, 146.9, 136.9, 133.3, 132.3, 130.1, 128.8, 128.7,
128.3, 127.83, 127.78, 127.6, 126.5 (q), 126.1, 125.1, 122.5, 43.1, 38.1, 31.3; HRMS (ESI) m/z calc’d for
C21H19F3N3O3 [M + H]+ 418.1379, found 418.1382.
ACS Paragon Plus Environment

Page 23 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
8-2. N,2-Dibenzyl-5-hydroxy-1-methyl-6-oxo-1,6-dihydropyrimidine-4-carboxamide (P02).
Off-white solid (87%): Rf = 0.32 (MeOH / CHCl3 = 1 / 9); 1H NMR (300 MHz, CDCl3)  12.01 (s, 1H), 7.89
(bs, 1H), 7.367.24 (m, 8H), 7.10 (d, J = 6.9 Hz, 2H), 4.58 (d, J = 6.0 Hz, 2H), 4.04 (s, 2H), 3.42 (s, 3H);
9
13C NMR (75 MHz, CDCl3)  168.3, 159.0, 149.4, 147.1, 137.2, 134.6, 129.1, 129.0, 128.3, 127.9, 127.6,
125.3, 43.2, 41.8, 31.7; LRMS (ESI) m/z 350 [M + H]+.
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
8-3. N-Benzyl-2-ethyl-5-hydroxy-1-methyl-6-oxo-1,6-dihydropyrimidine-4-carboxamide (P03).
Off-white solid (80%): Rf = 0.44 (MeOH / CHCl3 = 1 / 9); 1H NMR (300 MHz, CDCl3)  11.93 (s, 1H), 7.98
(bs, 1H), 7.407.31 (m, 5H), 4.62 (d, J = 6.3 Hz, 2H), 3.54 (s, 3H), 2.68 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2
Hz, 3H); 13C NMR (75 MHz, CDCl3)  168.5, 158.9, 151.8, 146.7, 137.5, 129.0, 127.9, 127.7, 125.2, 43.1,
31.0, 27.8, 10.7; LRMS (ESI) m/z 288 [M + H]+.
8-4. N-Benzyl-1-ethyl-5-hydroxy-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-dihydropyrimidine-
4-carboxamide (P04). Off-white solid (73%); Rf = 0.57 (MeOH / CHCl3 = 1 / 9); 1H NMR (300 MHz, CDCl3)
 11.93 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.52 (bs, 1H), 7.447.30 (m, 5H), 7.247.21 (m, 2H), 7.06 (d, J =
7.8 Hz, 1H), 4.50 (d, J = 6.0 Hz, 2H), 4.23 (s, 2H), 3.97 (q, J = 7.5 Hz, 2H), 1.28 (t, J = 7.5 Hz, 3H); 13C
NMR (75 MHz, CDCl3)  168.1, 158.6, 148.1, 147.4, 137.1, 133.9, 132.3, 130.7, 129.1, 128.8, 128.5, 128.1,
128.0, 127.8, 126.7 (q), 126.3, 152.2, 122.7, 43.3, 40.4, 37.7, 13.5; HRMS (ESI) m/z calc’d for C22H21N3O3F3
[M + H]+ 432.1535, found 432.1540.
8-5.
5-Hydroxy-1-methyl-6-oxo-N-phenyl-2-(2-(trifluoromethyl)benzyl)-1,6-
1
dihydropyrimidine-4-carboxamide (P05). Off-white solid (59%): Rf = 0.45 (MeOH / CHCl3 = 1 / 9); H
NMR (300 MHz, CDCl3)  11.78 (s, 1H), 9.04 (s, 1H), 7.79 (d, J = 7.5 Hz, 1H), 7.617.45 (m, 4H), 7.35 (t,
J = 7.9 Hz, 2H), 7.217.13 (m, 2H), 4.30 (s, 2H), 3.53 (s, 3H); 13C NMR (75 MHz, CDCl3)  166.1, 158.7,
148.7, 147.4, 136.4, 133.5, 132.4, 131.0, 129.3, 127.9, 126.8 (q), 126.2, 125.3, 125.2, 122.6, 119.8, 38.2,
31.5; LRMS (ESI) m/z 404 [M + H]+.
8-6.
N-(Cyclohexylmethyl)-5-hydroxy-1-methyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
1
dihydropyrimidine-4-carboxamide (P06). Off-white solid (89%): Rf = 0.57 (MeOH / CHCl3 = 1 / 9); H
NMR (300 MHz, CDCl3)  12.11 (s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.44 (t, J = 7.5
Hz, 1H), 7.30 (bs, 1H), 7.12 (d, J = 7.5 Hz, 1H), 4.25 (s, 2H), 3.49 (s, 3H), 3.15 (t, J = 6.5 Hz, 2H), 1.731.58
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 39
1
2
3
4
5
6
7
8
(m, 5H), 1.501.35 (m, 1H), 1.291.10 (m, 3H), 0.920.81 (m, 2H); 13C NMR (75 MHz, CDCl3)  168.1,
159.0, 148.3, 146.9, 133.6, 132.3, 130.8, 129.1, 128.7, 127.7, 126.6 (q), 126.2, 125.3, 122.6, 45.2, 38.3,
37.8, 31.4, 30.8, 26.4, 25.9; LRMS (ESI) m/z 424 [M + H]+.
9
8-7.
5-Hydroxy-1-methyl-6-oxo-N-(pyridin-2-ylmethyl)-2-(2-(trifluoromethyl)benzyl)-1,6-
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
1
dihydropyrimidine-4-carboxamide (P07). Off-white solid (82%): Rf = 0.26 (MeOH / CHCl3 = 1 / 9); H
NMR (300 MHz, CDCl3)  12.09 (bs, 1H), 8.54 (d, J = 4.2 Hz, 1H), 8.44 (bs, 1H), 7.737.66 (m, 2H), 7.49
(t, J = 7.5 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.287.22 (m, 2H), 7.09 (d, J = 7.5 Hz, 1H), 4.66 (d, J = 5.7 Hz,
2H), 4.27 (s, 2H), 3.43 (s, 3H); 13C NMR (75 MHz, CDCl3)  168.4, 159.0, 155.5, 149.2, 148.4, 147.2, 137.2,
133.5, 132.5, 130.1, 128.8, 128.4, 127.7, 126.6 (q), 126.2, 125.4, 122.8, 122.6, 122.3, 44.1, 38.3, 31.5;
HRMS (ESI) m/z calc’d for C20H18N4O3F3 [M + H]+ 419.1331, found 419.1324.
8-8.
5-Hydroxy-1-methyl-6-oxo-N-(pyridin-3-ylmethyl)-2-(2-(trifluoromethyl)benzyl)-1,6-
1
dihydropyrimidine-4-carboxamide (P08). Off-white solid (79%): Rf = 0.22 (MeOH / CHCl3 = 1 / 9); H
NMR (300 MHz, CDCl3)  11.82 (bs, 1H), 8.568.51 (m, 2H), 7.66 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.8 Hz,
1H), 7.43 (t, J = 7.2 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.307.26 (m, 1H), 7.01 (d, J = 7.8 Hz, 1H), 4.52 (d,
J = 6.0 Hz, 2H), 4.22 (s, 2H), 3.42 (s, 3H); 13C NMR (75 MHz, CDCl3)  168.2, 158.8, 149.4, 149.3, 148.6,
147.1, 135.7, 133.4, 132.8, 132.4, 130.0, 128.8, 128.4, 127.8, 126.6 (q), 126.1, 125.0, 123.8, 122.5, 40.7,
38.2, 31.5; LRMS (ESI) m/z 419 [M + H]+.
8-9.
N-(2-Fluorobenzyl)-5-hydroxy-1-methyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
1
dihydropyrimidine-4-carboxamide (P09). Off-white solid (86%): Rf = 0.40 (MeOH / CHCl3 = 1 / 9); H
NMR (300 MHz, CDCl3)  11.91 (s, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.63 (bs, 1H), 7.46 (t, J = 7.2 Hz, 1H),
7.38 (t, J = 7.2 Hz, 1H), 7.337.28 (m, 2H), 7.147.04 (m, 3H), 4.55 (d, J = 6.3 Hz, 2H), 4.23 (s, 2H), 3.44
(s, 3H); 13C NMR (75 MHz, CDCl3)  168.1, 162.8, 159.5, 158.9, 148.5, 147.0, 133.4, 132.3, 130.4, 130.3,
129.9, 129.8, 128.8, 128.4, 127.7, 126.6 (q), 126.2, 125.1, 124.5 (d), 124.2, 124.1, 122.6, 115.8, 115.5,
38.2, 37.3, 31.5; HRMS (ESI) m/z calc’d for C21H18N3O3F4 [M + H]+ 436.1284, found 436.1282.
8-10.
N-(4-Fluorobenzyl)-5-hydroxy-1-methyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
1
dihydropyrimidine-4-carboxamide (P10). Off-white solid (79%): Rf = 0.46 (MeOH / CHCl3 = 1 / 9); H
NMR (300 MHz, CDCl3)  11.93 (s, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.59 (bs, 1H), 7.43 (t, J = 7.2 Hz, 1H),
ACS Paragon Plus Environment

Page 25 of 39
Journal of Medicinal Chemistry
1
2
3
4
7.35 (t, J = 7.2 Hz, 1H), 7.247.19 (m, 2H), 7.057.00 (m, 3H), 4.47 (d, J = 6.3 Hz, 2H), 4.23 (s, 2H), 3.43
5
6
7
8
(s, 3H); 13C NMR (75 MHz, CDCl3)  168.1, 164.2, 160.9, 158.9, 148.6, 147.2, 133.6, 133.0 (d), 132.4,
130.2, 129.8, 129.7, 128.9, 128.5, 127.8, 126.7 (q), 126.3, 125.2, 122.6, 116.1, 115.8, 42.6, 38.4, 31.6;
LRMS (ESI) m/z 436 [M + H]+.
9
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
8-11.
N-(Benzo[d][1,3]dioxol-5-ylmethyl)-5-hydroxy-1-methyl-6-oxo-2-(2-
(trifluoromethyl)benzyl)-1,6-dihydropyrimidine-4-carboxamide (P11). Pale yellow solid (61%): Rf =
0.36 (MeOH / CHCl3 = 1 / 9); 1H NMR (300 MHz, CDCl3)  7.68 (d, J = 7.5 Hz, 1H), 7.54 (bs, 1H), 7.44 (t,
J = 7.5 Hz, 1H), 7.37 (t, J = 7.2 Hz, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.786.69 (m, 3H), 5.96 (s, 2H), 4.40 (d,
J = 6.0 Hz, 2H), 4.22 (s, 2H), 3.43 (s, 3H); 13C NMR (75 MHz, CDCl3)  167.9, 158.9, 148.4, 148.1, 147.4,
147.1, 133.5, 132.4, 130.8, 130.1, 128.8, 128.4, 127.7, 126.6 (q), 126.2, 125.1, 122.5, 121.3, 108.5, 108.4,
101.3 (t), 43.1, 38.2, 31.5; LRMS (ESI) m/z 462 [M + H]+.
8-12.
N-Benzyl-5-hydroxy-N,1-dimethyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
dihydropyrimidine-4-carboxamide (Rotameric mixture, P12). Pale yellow solid (43%): Rf = 0.54 (MeOH
1
/ CHCl3 = 1 / 9); H NMR (300 MHz, (CD3)2CO)  11.48 (s, 0.5H) c, 10.84 (s, 0.5H), 7.78 (d, J = 7.8 Hz,
1H), 7.647.56 (m, 1H), 7.497.26 (m, 6H), 7.107.09 (m, 1H), 4.76 (s, 1H), 4.64 (s, 1H), 4.39 (s, 1H), 4.32
(s, 1H), 3.61 (s, 1.5H), 3.58 (s, 1.5H), 2.79 (s, 3H); 13C NMR (75 MHz, (CD3)2CO)  168.4, 159.6, 149.6,
149.5, 146.1, 145.2, 138.2, 137.9, 135.5, 135.0, 133.3, 133.2, 132.8, 130.9, 129.4, 129.2, 128.5, 128.4,
128.3, 128.2, 128.1, 126.8, 122.5, 54.4, 51.8, 38.6, 38.4, 36.6, 34.1, 31.2; LRMS (ESI) m/z 432 [M + H]+.
8-13.
5-Hydroxy-1-methyl-6-oxo-N-(1-phenylethyl)-2-(2-(trifluoromethyl)benzyl)-1,6-
dihydropyrimidine-4-carboxamide (P13). Pale yellow solid (67%): Rf = 0.49 (MeOH / CHCl3 = 1 / 9); 1H
NMR (300 MHz, CDCl3)  7.71 (d, J = 7.5 Hz, 1H), 7.49 (t, J = 7.8 Hz, 2H), 7.437.26 (m, 4H), 7.227.19
(m, 2H), 7.10 (d, J = 7.2 Hz, 1H), 5.10 (quint, J = 6.9 Hz, 1H), 4.24 (s, 2H), 3.47 (s, 3H), 1.47 (d, J = 6.9 Hz,
3H); 13C NMR (75 MHz, CDCl3)  167.2, 158.9, 148.4, 146.9, 142.3, 133.6, 132.2, 130.7, 129.0, 128.9,
128.6, 127.8, 127.7, 126.6 (q), 126.2, 126.0, 125.2, 122.6, 48.9, 38.2, 31.4, 22.1; LRMS (ESI) m/z 432 [M
+ H]+.
8-14.
N-Benzyl-5-hydroxy-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-dihydropyrimidine-4-
carboxamide (P14). Off-white solid (78%): Rf = 0.23 (MeOH / CHCl3 = 1 / 9); 1H NMR (300 MHz, (CD3)2SO)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 39
1
2
3
4
 12.35 (bs, 1H), 8.87 (t, J = 5.8 Hz, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.7
5
6
7
8
Hz, 1H), 7.357.24 (m, 6H), 4.43 (d, J = 6.3 Hz, 1H), 4.10 (s, 2H); 13C NMR (75 MHz, (CD3)2SO)  168.3,
158.1, 148.0, 147.7, 138.3, 134.7, 132.7, 130.9, 128.4, 127.4, 127.3, 127.2, 127.1, 126.8, 126.2, 125.9 (q),
122.6, 42.2, 36.4; LRMS (ESI) m/z 404 [M + H]+.
9
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
8-15.
N-Benzyl-5-hydroxy-1-methyl-6-oxo-2-(1-(2-(trifluoromethyl)phenyl)ethyl)-1,6-
0.63 (MeOH
dihydropyrimidine-4-carboxamide (P15). Off-white solid (80%): Rf
=
/
= 1 / 9); 1H NMR (300 MHz, CDCl3)  12.05 (s, 1H), 7.90 (bs, 1H), 7.72 (d, J = 7.2 Hz, 1H), 7.447.33 (m,
7H), 7.06 (d, J = 7.5 Hz, 1H), 4.65 (q, J = 3.1 Hz, 2H), 4.53 (q, J = 6.6 Hz, 1H), 3.31 (s, 3H), 1.61 (d, J =
6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3)  168.5, 159.0, 151.7, 147.3, 140.2, 137.3, 132.9, 129.1, 128.0,
127.9, 127.8, 127.5, 127.0 (q), 126.4, 124.7, 122.7, 43.3, 40.2, 30.8, 21.8; LRMS (ESI) m/z 432 [M + H]+.
8-16.
N-Benzyl-5-hydroxy-6-methoxy-2-(2-(trifluoromethyl)benzyl)pyrimidine-4-
carboxamide (P16). Pale yellow solid (60%): Rf = 0.64 (only CH2Cl2); 1H NMR (300 MHz, CDCl3)  11.77
(s, 1H), 8.17 (bs, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.467.26 (m, 8H), 4.58 (d, J = 6.0 Hz, 2H), 4.29 (s, 2H),
3.98 (s, 3H); 13C NMR (75 MHz, CDCl3)  168.0, 160.9, 156.4, 142.2, 137.1, 136.8, 133.2, 132.1, 131.6,
129.0, 127.9, 127.7, 126.7, 126.4, 126.0 (q), 122.8, 54.7, 43.1, 41.0; LRMS (ESI) m/z 418 [M + H]+.
8-17.
N-Benzyl-5-methoxy-1-methyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
dihydropyrimidine-4-carboxamide (P17). Pale yellow solid (91%): Rf = 0.69 (MeOH / CHCl3 = 1 / 9); 1H
NMR (300 MHz, CDCl3)  7.61 (d, J = 7.5 Hz, 1H), 7.51 (bs, 1H), 7.397.26 (m, 5H), 7.18 (d, J = 7.5 Hz,
2H), 7.05 (d, J = 7.8 Hz, 1H), 4.45 (d, J = 5.7 Hz, 2H), 4.24 (s, 2H), 4.01 (s, 3H), 3.42 (s, 3H); 13C NMR (75
MHz, CDCl3)  162.3, 160.7, 152.6, 144.6, 138.7, 137.9, 133.0, 132.2, 130.5, 128.7, 128.4, 127.9, 127.7,
127.6, 126.5 (q), 126.1, 122.5, 61.0, 43.5, 38.4, 31.3; LRMS (ESI) m/z 432 [M + H]+.
Synthesis of P18
1. Synthesis of 5-(benzyloxy)-1-methyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-dihydropyrimidine-4-
carboxylic acid (18). To a solution of 9 (1.0 eq) in anhydrous DMF (0.2 M) was added K2CO3 (3.0 eq), KI
(0.3 eq), and benzyl bromide (3.0 eq). The reaction mixture was heated overnight at 80 C. The reaction
mixture was diluted in EtOAc, and washed with 1N HCl and brine. The organic layer was dried over MgSO4
and evaporated in vacuo. The crude mixture was dissolved in MeOH (0.1 M). 5 M of NaOH solution (20%
ACS Paragon Plus Environment

Page 27 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of MeOH) was added in the reaction mixture, and then it was stirred at rt for 5 h. The reaction mixture was
evaporated in vacuo and then diluted in EtOAc, washed with 1N HCl and brine. The separated organic layer
was dried under MgSO4 and evaporated in vacuo. The crude mixture was suspended in EtOH. After stirring
for 2 h, the solid was filtered, and washed with EtOH and Et2O to obtain the desired product as an off-white
solid (55%): Rf = 0.06 (only EtOAc); 1H NMR (300 MHz, CDCl3)  7.73 (d, J = 7.8 Hz, 1H), 7.547.42 (m,
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
4H), 7.387.31 (m, 3H), 7.05 (d, J = 7.5 Hz, 1H), 5.47 (s, 2H), 4.29 (s, 2H), 3.45 (s, 3H); 13C NMR (75 MHz,
CDCl3)  161.6, 160.1, 153.4, 144.8, 135.9, 135.0, 132.6, 132.4, 130.3, 129.0, 128.9, 128.8, 128.6, 128.5,
128.2, 126.9 (q), 126.1, 122.5, 75.1, 38.3, 31.6; LRMS (ESI) m/z 419 [M + H]+.
2. Synthesis of 5-(benzyloxy)-N-methoxy-N,1-dimethyl-6-oxo-2-(2-(trifluoromethyl)benzyl)-1,6-
dihydropyrimidine-4-carboxamide (19). To a solution of 18 (1.0 eq) in anhydrous DMF (0.2 M) was added
dropwise CDI (1.1 eq) solution in anhydrous CH2Cl2 (0.5 M) at 0~5 C. After stirring for 1 h at 0~5 C, N,O-
dimethylhydroxylamine hydrochloride (1.1 eq) and TEA (1.2 eq) were added to the reaction mixture
sequentially at the same temperature. After overnight stirring at rt, the reaction mixture was diluted in EtOAc,
and washed with 1N HCl and brine. The organic layer was dried over Na2SO4 and evaporated in vacuo.
The crude mixture was purified by flash column chromatography (EtOAc / hexane = 4 / 1) to obtain the
desired product as an off-white solid (61%): Rf = 0.26 (EtOAc / hexane = 1 / 1); 1H NMR (300 MHz, CDCl3)
 7.70 (d, J = 7.5 Hz, 1H), 7.517.29 (m, 7H), 7.08 (d, J = 7.5 Hz, 1H), 5.24 (s, 2H), 4.32 (s, 2H), 3.51 (s,
3H), 3.42 (s, 3H), 3.27 (s, 3H); 13C NMR (75 MHz, CDCl3)  165.7, 159.8, 154.3, 145.6, 139.7, 137.0, 133.2,
132.5, 129.8, 128.9, 128.5, 128.4, 128.2, 127.7, 126.5 (q), 126.2, 122.5, 74.3, 61.8, 38.6, 31.8, 31.2; LRMS
(ESI) m/z 462 [M + H]+.
3.
Synthesis
of
5-(benzyloxy)-3-methyl-6-(3-phenylpropanoyl)-2-(2-
(trifluoromethyl)benzyl)pyrimidin-4(3H)-one (20). Mg turnings (2.5 eq) was suspended into anhydrous
THF (1 M) and then heated to 40 °C. A few drops of 1,2-dibromoethane was added to activate Mg. To a
solution of (5-chloro-1-pentynyl)trimethylsilane (2.0 eq) in anhydrous THF (1 M) was added 1,2-
dibromoethane (total 0.2 eq). This solution was added slowly to Mg solution, and then the reaction mixture
was stirred overnight at 40 °C (Grignard reagent). To a solution of 19 (1.0 eq) in anhydrous THF (0.5 M)
was added dropwise Grignard reagent at 0~5 C. After stirring at rt for 2 h, the reaction mixture was
quenched with aq. NH4Cl and then extracted with EtOAc. The organic layer was washed with brine, dried
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 39
1
2
3
4
5
6
over Na2SO4 and evaporated in vacuo. The crude mixture was purified by flash column chromatography
(EtOAc / hexane = 1 / 4  1 / 3) to obtain the desired product as an off-white solid (75%): Rf = 0.63 (EtOAc
/ hexane = 1 / 1); 1H NMR (300 MHz, CDCl3)  7.70 (d, J = 7.5 Hz, 1H), 7.477.18 (m, 10H), 7.10 (d, J =
6.9 Hz, 2H), 7.06 (d, J = 7.5 Hz, 1H), 5.30 (s, 2H), 4.27 (s, 2H), 3.43 (s, 3H), 2.97 (t, J = 6.9 Hz, 2H), 2.86
(t, J = 6.9 Hz, 2H); 13C NMR (75 MHz, CDCl3)  199.8, 160.6, 153.3, 144.7, 140.9, 136.4, 133.2, 132.3,
130.2, 129.0, 128.9, 128.6, 128.5, 128.4, 128.3, 127.6, 126.5 (q), 126.1, 126.0, 122.5, 74.4, 42.2, 38.4,
31.2, 29.4; LRMS (ESI) m/z 507 [M + H]+.
7
8
9
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
4. Synthesis of 5-hydroxy-3-methyl-6-(3-phenylpropanoyl)-2-(2-(trifluoromethyl)benzyl)pyrimidin-
4(3H)-one (P18). To a solution of 20 in MeOH / THF (3 / 1, 0.2 M) was added 10% Pd/C. After stirring at rt
for 1 h under H2 atmosphere, the reaction mixture was filtered through celite pad and evaporated in vacuo.
The crude mixture was purified by flash column chromatography (MeOH / CH2Cl2 = 2 / 98  5 / 95) to
obtain the desired product as a pale yellow solid (71%): Rf = 0.54 (MeOH / CHCl3 = 1 / 9); 1H NMR (CDCl3,
300 MHz)  7.71 (d, J = 7.2 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.38 (t, J = 7.2 Hz, 1H), 7.297.16 (m, 3H),
7.117.07 (m, 3H), 4.26 (s, 2H), 3.45 (s, 3H), 3.18 (t, J = 7.5 Hz, 2H), 2.89 (t, J = 7.5 Hz, 2H); 13C NMR
(CDCl3, 75 MHz)  208.1, 159.6, 148.3, 140.7, 133.7, 132.3, 130.4, 129.3, 128.7, 128.5, 128.4, 127.6, 126.6
(q), 126.3, 122.6, 39.6, 38.3, 31.5, 29.8; LRMS (ESI) m/z 417 [M + H]+.
Biology.
Strains and media. Mtb H37Rv or mutants thereof were used for all experiments. Liquid media were either
Middlebrook 7H9-based or GAST whereas solid agar medium consisted of Middlebrook 7H11
supplemented with bovine serum albumin fraction V (5ꢀg/L)/dextrose (2ꢀg/L)/NaCl (0.81ꢀg/L)/0.5%
glycerol/0.06% oleic acid. Middlebrook 7H9/ADC consisted of Middlebrook 7H9 broth (Becton Dickinson)
supplemented with bovine serum albumin fraction V (5ꢀg/L)/dextrose (2ꢀg/L)/NaCl (0.81ꢀg/L)/0.2%
glycerol/0.05% Tween 80. GAST (glycerol-alanine-salts with Tween 80) was used as the defined liquid
medium.44 Glucose medium (GBSA) contained bovine serum albumin fraction V (5ꢀg/L)/dextrose
(4ꢀg/L)/NaCl (0.81ꢀg/L)/0.05% Tyloxapol. Dipalmitoyl phosphatidyl choline (DPPC) medium (DBSA)
contained bovine serum albumin fraction V (5ꢀg/L)/DPPC (5ꢀmg/L)/cholesterol (24 mg/L)/NaCl
(0.81ꢀg/L)/0.05% Tyloxapol. Cholesterol medium (Chol) contained bovine serum albumin fraction V
ACS Paragon Plus Environment

Page 29 of 39
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(5ꢀg/L)/cholesterol (97 mg/L)/NaCl (0.81ꢀg/L)/0.05% Tyloxapol. The low pH nitrosative stress medium
contained bovine serum albumin fraction V (5ꢀg/L)/butyric acid (2.5 mM)/NaNO2 (6.9 mg/mL)/NaCl
(0.81ꢀg/L)/0.05% Tyloxapol, pH adjusted to 6.0. MIC determination was performed as previously
described.45
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
Screening of the St. Jude’s Pyrimidinedione Library. The 6,207 compound pyrimidinedione library was
kindly provided St. Jude Children’s Research Hospital, Inc. and screened in a single-point assay at 10 µM
final concentration Middlebrook 7H9/ADC in 384-well plates (Greiner plates cat no. 781091) containing 25
µL Mtb expressing GFP from pMSP12 (Addgene plasmid # 30167). Rifampicin and isoniazid were used as
positive controls. Growth was measured by fluorescence after 3 days’ incubation. Thirty-eight hits were
selected for follow-up based on >50% inhibition of Mtb growth and z-scores of <-3. The selected hits were
followed up in duplicate twelve-point MIC determination in GAST with Mg2+ concentrations adjusted to 0.06,
6 and 60 mM. Growth was measured using an inverted enlarging mirror at 1, 2 and 3 weeks with MIC taken
as the concentration that completely inhibited all visible growth.
Efficacy and validating inhibition of N-alkyl-5-hydroxypyrimidinone carboxamides against Mtb in
vitro and ex vivo. In vitro efficacy was performed in aerobic and anaerobic condition. For aerobic
conditions, logarithmically growing Mtb (OD650 0.2) was diluted 1,000-fold in 1 mL of 7H9/ADC media and
exposed 1, 5, 10, 20, and 50 MIC P01 up to 7 days in duplicates. After 1, 2, 4, and 7 days of treatment
appropriate cell dilutions were plated on 7H11/OADC plates for CFU enumeration. For anaerobic conditions,
Mtb was cultured in the self-generated oxygen-depletion model as previously described.46 One milliliter of
anaerobic Mtb culture was exposed to 1, 5, 10, 20, and 50 MIC P01 in an anaerobic chamber for 7
days. Diluted cells were plated on 7H11/OADC plates for CFU enumeration.
For the ex vivo efficacy test, J774 cells (5  105 cells/well) were seeded in flat-bottom 24 well plates
(Corning Inc.) in DMEM GlutaMAX (Gibco/ThermoFisher Scientific) supplemented with 10% fetal bovine
serum, 20 mM HEPES + 0.5 mM sodium pyruvate (hereafter abbreviated DMEM/FBS). Cells were infected
with Mtb (5  105 cells/well, MOI 1) for 24 hours, followed by medium removal and washing (2) with an
equal volume of Dulbecco’s PBS. Infected cells were fed DMEM/FBS and exposed to test compounds at
ACS Paragon Plus Environment