Antitubercular 2-Pyrazolylpyrimidinones: Structure-Activity Relationship and Mode-of-Action Studies

Article abstract of DOI:10.1021/acs.jmedchem.0c01727

Phenotypic screening of a Medicines for Malaria Venture compound library against Mycobacterium tuberculosis (Mtb) identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of Mtb suggested a novel mechanism of action. The compounds were bactericidal against replicating Mtb and retained potency against clinical isolates of Mtb. Although selected MmpL3 mutant strains of Mtb showed resistance to these compounds, there was no shift in the minimum inhibitory concentration (MIC) against a mmpL3 hypomorph, suggesting mutations in MmpL3 as a possible resistance mechanism for the compounds but not necessarily as the target. RNA transcriptional profiling and the checkerboard board 2D-MIC assay in the presence of varying concentrations of ferrous salt indicated perturbation of the Fe-homeostasis by the compounds. Structure-activity relationship studies identified potent compounds with good physicochemical properties and in vitro microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.

Full text of DOI:10.1021/acs.jmedchem.0c01727

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Article
Antitubercular 2‑Pyrazolylpyrimidinones: Structure−Activity
Relationship and Mode-of-Action Studies
Candice Soares de Melo, Vinayak Singh, Alissa Myrick, Sandile B. Simelane, Dale Taylor,
Christel Brunschwig, Nina Lawrence, Dirk Schnappinger, Curtis A. Engelhart, Anuradha Kumar,
Tanya Parish, Qin Su, Timothy G. Myers, Helena I. M. Boshoff, Clifton E. Barry, III, Frederick A. Sirgel,
Paul D. van Helden, Kirsteen I. Buchanan, Tracy Bayliss, Simon R. Green, Peter C. Ray, Paul G. Wyatt,
Gregory S. Basarab, Charles J. Eyermann, Kelly Chibale,* and Sandeep R. Ghorpade*
Cite This: J. Med. Chem. 2021, 64, 719−740
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ABSTRACT: Phenotypic screening of a Medicines for Malaria Venture compound library against Mycobacterium tuberculosis (Mtb)
identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of Mtb
suggested a novel mechanism of action. The compounds were bactericidal against replicating Mtb and retained potency against
clinical isolates of Mtb. Although selected MmpL3 mutant strains of Mtb showed resistance to these compounds, there was no shift
in the minimum inhibitory concentration (MIC) against a mmpL3 hypomorph, suggesting mutations in MmpL3 as a possible
resistance mechanism for the compounds but not necessarily as the target. RNA transcriptional profiling and the checkerboard board
2D-MIC assay in the presence of varying concentrations of ferrous salt indicated perturbation of the Fe-homeostasis by the
compounds. Structure−activity relationship studies identified potent compounds with good physicochemical properties and in vitro
microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.
existing drugs.4,5 One approach to identify compounds with a
INTRODUCTION
■
novel mode of action is to phenotypically screen compound
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb)
libraries against Mtb growing under different media conditions
continues to be one of the leading causes of death and morbidity
with different carbon sources followed by a biology triage
globally, claiming 1.2 million lives in 2018.1 The emergence and
process to weed out actives hitting frequently encountered
spread of multi drug-resistant (MDR) and extensively drug-
targets such as the respiratory system (e.g., QcrB) and cell−wall
resistant (XDR) strains of Mtb that are resistant to the first- and
synthesis (e.g., MmpL3 and DprE1), as well as DNA damaging
second-line drugs have further exacerbated the situation.2 The
agents. Herein, we describe structure−activity relationship
rise in anti-microbial resistance warrants the search for new
(SAR) and target identification studies of one novel chemical
drugs with unique modes of action that can bypass existing
series, 2-pyrazolylpyrimidinonesrepresented in Figure 1. This
modes of resistance or can be used as adjunctive therapy to
series was identified through high-throughput screening of a
compensate for those that are vulnerable to promoting
Medicines for Malaria Venture (MMV) compound library.
resistance. While considerable progress has been made toward
establishing a TB drug pipeline, the high attrition rate in clinical
development reinforces the need to continually replenish the
Received: October 1, 2020
Published: January 4, 2021
pipeline with high-quality leads that act through the inhibition of
novel targets.3
In light of the above, we have been engaged in early TB drug
discovery to identify and progress new chemical series with
potentially novel modes of action lacking cross-resistance with
© 2021 The Authors. Published by
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American Chemical Society
J. Med. Chem. 2021, 64, 719−740
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Figure 2. (a) Structure of PZP; and (b) depiction of metal chelation by
pyrazolylpyrimidinone 1.
Figure 1. Generic structure of pyrazolylpyrimidones.
RESULTS AND DISCUSSION
reported to chelate iron through the pyrimidinone carbonyl and
2-position pyrazole nitrogen. Similarly, bidentate iron chelation
by the pyrazolylpyrimidinones from the pyrimidinone nitrogen
and pyrazole nitrogen as depicted in Figure 2b may be
hypothesized. A single-crystal X-ray structure of the pyrazo-
lylpyridines−Fe complex is reported in the literature which
supports this hypothesis.8,9 This aspect of metal chelation by the
compounds was considered while planning further SAR
exploration and target identification studies with the series.
Synthesis. In order to explore the SAR profiles of the series,
various routes were explored toward the synthesis of diverse
range of analogues as represented by compounds 3−54 in
Tables 2−5. To facilitate exploration of a broad array of
substituents off the pyrazole ring and pyrimidinone core, a
concise and scalable synthesis leading to an advanced
intermediate was desired. As summarized in Scheme 1, this
was achieved by the ring formation reaction of 6-substituted-2-
thiomethyl-pyrimidin-4-one 55 via condensation of the
appropriate β-keto-ester and thiourea, and subsequent methyl-
ation of the thiol.10 Intermediates 55a−g were reacted with
hydrazine hydrate in refluxing ethanol to give the versatile 2-
hydrazinylpyrimidin-4(1H)-ones 56a−g, which gave access to
analogues for SAR exploration on the pyrazole ring and the R1
substituent on the pyrimidinone core. Similarly, substitution of a
phenyl group at the R2 position was achieved through
bromination at C5 of 6-methyl-2-thiomethyl-pyrimidin-4-one
(55, R1 = CH3) followed by a Suzuki cross-coupling reaction
with phenyl boronic acid to provide 55h, which was then treated
with hydrazine to form 56h.
The Knorr reaction was used to incorporate the 3,5-
dimethylpyrazole via an acid-catalyzed condensation of
hydrazine precursors 56a−h and acetylacetone. This led to hit
compound 1 and related analogues exploring the R1 (11, 12, 19,
20, and 21) and R2 (8−9) positions of the pyrimidinone core as
well as quinazolin-4(3H)-one analogue (10). In addition, 2-
hydrazinylpyrimidin-4(1H)-one 56a and 56d were reacted with
(Z)-3-aminocrotonitrile to afford 5-aminopyrazolylpyrimidi-
none analogues 14 and 33,11 which allowed for several
transformations to be carried out to expand SAR exploration
on the pyrazole ring later described in Scheme 7. To explore
polar modifications off the pyrazole ring, a mixture of 2-(5-
amino-3-methyl-1H-pyrazol-1-yl)-6-phenylpyrimidin-4(3H)-
one 14 and pyridine in dichloromethane was treated with acetyl
chloride to provide N-acetyl amide 15.2 In addition, hydrazine
56a was heated with ethyl acetoacetate in acidic ethanol to yield
pyrazolone 13.
■
Phenotypic Hits with a Novel Mode of Action. A high-
throughput screen of a ∼530,000 diverse set of compounds from
MMV compound library against Mtb was conducted at the
National Institute of Allergy and Infectious Diseases of the
National Institutes of Health (NIAID/NIH, U.S.). Reconfirmed
hits were followed up for determination of minimum inhibitory
concentration (MIC) on multiple media that identified a cluster
of pan-active pyrazolylpyrimidinones represented by com-
pounds 1 and 2 (Table 1). The biology triage of these actives
Table 1. Properties of the Hits from the MMV Library
a
Screen
Properties
1
2
MIC 7H9/ADC/Tw (μM)
MIC GAST-Fe (μM)
0.6
0.02
1.5
0.3
0.4
1.5
8
4/4
10
2
0.02
2
0.4
0.2
6
1.2
ND
<5
2.2
MIC 7H9/Glu/BSA/Tx (μM)
MIC 7H9/Glu/Cas/Tx (μM)
MIC 7H9/DPCC/Cas/Tx (μM)
MIC 7H9/DPCC/Chol/BSA/Tx (μM)
Vero IC50 (μM)
HepG2 IC50 (Glu/Gal μM)
SolubilitypH7.4 (μM)
LogDpH7.4
2.5
266.304
98/69/22
Molecular Weight
Microsomal stability H/R/Mo %remaining
295.346
73/76/78
a
MICminimum inhibitory concentration against Mtb H37Rv;
7H9Middlebrook 7H9; ADCalbumin−dextrose−catalase sup-
plement; TwTween 80; GAST-Feglycerol-alanine-salts with
Tween and iron salts; Gluglucose; BSAbovine serum albumin;
TxTyloxapol; CasCasitone; DPCCdipalmitoylphosphatidyl-
choline and cholesterol; Cholcholesterol; H/R/Mohuman/rat/
mouse; IC5050% inhibitory concentrations; solubilitypH7.4aque-
ous solubility at pH 7.4.
(described in the target identification section), suggestive of a
unique mechanism of action (MoA), generated further interest
in the exploration of these hits. We recognized that
pyrazolylpyrimidinones potentially possess iron-chelating prop-
erties by virtue of their two sp2 heteroatoms in a 1,4-relationship
on adjacent rings, which might play a crucial role in the SAR and
mode of action.6 During these studies, we came across the
related pyrazolopyrimidinone (PZP, Figure 2a) compound
reported as an intracellular iron chelator in Mtb.7 PZP is
Syntheses of compounds 3−7 (Table 2) with changes in the
central core ring are summarized in Scheme 2. Compounds
masking the pyrimidinone amide were synthesized from
compound 1 via a Mitsunobu reaction to form methyl ether 3
and N-methylation of 1 with iodomethane using potassium
carbonate in N,N-dimethylformamide (DMF) to afford N-
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a
Table 2. Pharmacophore Evaluation of
Pyrazolylpyrimidinones
Table 3. Polar Substitutions on Pyrimidinone
a
a
MICwere measured in Middlebrook 7H9/Glu/BSA/Tyloxapol
media at day 7; IC5050% inhibitory concentrations; solubility
aqueous solubility measured at pH 7.4; CHOan epithelial cell-line
derived from the ovary of the Chinese hamster; and SIselectivity
index between CHO IC50 and Mtb MIC.
respectively. Briefly, the three-step synthesis from 57 and 58
involved a Suzuki cross-coupling reaction with phenylboronic
acid to afford intermediates 59 and 60, respectively, followed by
a Buchwald Hartwig amination to introduce the pyrazole
(intermediates 61 and 62, respectively), and finally O-
demethylation with boron tribromide to afford compounds 5
and 6, respectively.
Pyrimidin-2(1H)-one 7 was synthesized by the cyclization of
ethyl benzoylacetate with urea to provide uracil 63, which was
then reacted with phosphorus oxychloride in the presence of a
catalytic amount of DMF to give 2,4-dichloropyrimidine 64
(Scheme 2).12 The activated 2,4-dichloropyrimidine readily
undergoes nucleophilic substitution with the pyrazole at the 4-
chloro position by heating in the microwave at 80 °C
(intermediate 65) and subsequent hydrolysis of 2-Cl afforded
a
MIC were measured in Middlebrook 7H9/Glu/BSA/Tyloxapol
media at day 7; IC5050% inhibitory concentrations; solubility
aqueous solubility measured at pH 7.4; CHOan epithelial cell-line
derived from the ovary of the Chinese hamster; and VVero cell-
line; SIselectivity index between CHO IC50 and Mtb MIC.
methyl pyrimidinone 4. The nucleophilic aromatic substitution
reaction of 2,6-dichloro-4-iodopyridine with sodium methoxide
gave intermediates 57 and 58 in a 2:1 ratio, which upon
separation allowed for the synthesis of compounds 5 and 6,
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a
a
Table 4. SAR at the Pyrazole R5 Position
Table 5. SAR at the Pyrazole R3 Position
a
MICwere measured in Middlebrook 7H9/Glu/BSA/Tyloxapol
media at day 7; IC5050% inhibitory concentrations; solubility
aqueous solubility at pH 7.4; CHOan epithelial cell-line derived
from the ovary of the Chinese hamster; and SIselectivity index
between CHO IC50 and Mtb MIC.
compound 7, with the carbonyl functionality between the ring
nitrogens.
Compounds 16 and 17 (Table 2), in which the pyrazole ring
was replaced with other 5-membered heterocycles, were
synthesized in a similar manner from 2,4-dichloro-6-phenyl-
pyrimidine 64 (Scheme 3). The alkaline hydrolysis of
intermediate 64 with 20% aqueous sodium hydroxide occurred
primarily at the 4-Cl position to provide advanced intermediate
66 (isomer ratio 70:30),13 which was reacted with the
corresponding 5-membered heterocycle to displace 2-Cl using
cesium carbonate under microwave heating at 80 °C (Scheme
3). Compound 18 with an N-methylimidazole was synthesized
by condensing 1-methyl-1H-imidazole-2-carboximidamide with
ethyl benzoylacetate in low yields.
In order to introduce groups with polar functionalities at the
R1 and R2 positions on the pyrimidinone core, 2,4,6-
trichloropyrimidine was a useful and accessible starting material
when the appropriate β-keto-ester was not readily available to
pursue synthetic routes, as shown in Schemes 1 and 3.
Treatment of 2,4,6-trichloropyrimidine in aqueous 1,4-dioxane,
brought to the alkaline reaction with NaOH, led to hydrolysis at
room temperature with the formation of 4,6-dichloro-2-
hydroxypyrimidine 67 and 2,6-dichloropyrimidin-4(3H)-one
68, with the former precipitating out as the sodium salt (Scheme
4).14 The filtrate was concentrated to approximately half the
volume and cooled to afford a precipitate which was filtered off
and acidified to pH 2 using 5 N HCl to give the required
a
MICwere measured in Middlebrook 7H9/Glu/BSA/Tyloxapol
media at day 7; IC5050% inhibitory concentrations; solubility
aqueous solubility measured at pH 7.4; CHOan epithelial cell-line
derived from the ovary of the Chinese hamster; and SIselectivity
index between CHO IC50 and Mtb MIC.
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a
Scheme 1. Synthetic Route to Explore R1 and R2 on the Pyrimidinone and Polar Modifications at R5 on the Pyrazole
a
Reagents and conditions: (i) (a) thiourea, KOH, EtOH, reflux (b) iodomethane, NaOH, H2O/EtOH; (ii) NH2NH2·H2O, EtOH, reflux; (iii) Br2,
AcOH, 60 °C; (iv) phenylboronic acid, Pd(PPh3)2Cl2, 1,4-dioxane, K2CO3; (v) acetylacetone, EtOH, AcOH, reflux; (vi) 3-aminocrotonitrile,
EtOH, reflux; (vii) acetyl chloride, pyridine, CH2Cl2; and (viii) ethyl acetoacetate, EtOH, AcOH, reflux.
a
Scheme 2. Synthetic Routes to Access Core Modifications
a
Reagents and conditions: (i) MeOH, PPh3, DIAD, THF, 25 °C; (ii) MeI, K2CO3, DMF, 100 °C; (iii) NaOCH3, MeOH, 80 °C; (iv)
phenylboronic acid, Pd(PPh3)2Cl2, K2CO3, 1,4-dioxane, reflux; (v) 3,5-dimethylpyrazole, xanphos, Pd(OAc)2, Cs2CO3, 1,4-dioxane, 130 °C; (vi)
BBr3, MeOH; (vii) BBr3, CH2Cl2; (viii) urea, 1,4-dioxane, 140 °C, microwave; (ix) POCl3, DMF; (x) 3,5-dimethylpyrazole, Cs2CO3, 1,4-dioxane,
80 °C, microwave; and (xi) 20% aq NaOH, 80 °C.
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a
Scheme 3. Synthetic Route for Pyrazole Replacements
a
Reagents and conditions: (i) urea, 1,4-dioxane, 140 °C, microwave; (ii) POCl3, DMF; (iii) 20% aq NaOH, 80 °C (iv) 5-membered heterocycle,
Cs2CO3, 1,4-dioxane, 80 °C, microwave; and (v) EtOH, reflux, 1 h.
a
Scheme 4. Synthetic Route to Polar Substitutions on R1 of the Pyrimidinone
a
Reagents and conditions: (i) aqueous NaOH, dioxane; (ii) boronic acid, Cs2CO3, Pd(OAc)2, dppf, 1,4-dioxane, 70 °C or K2CO3, PdCl2(dppf)2,
1,4-dioxane, 70 °C; and (iii) 3,5-dimethylpyrazole, Cs2CO3, 1,4-dioxane, 130 °C, microwave.
a
Scheme 5. Synthetic Route to Amide 26
a
Reagents and conditions: (i) CH3ONa, THF, 0 °C; (ii) 3,5-dimethylpyrazole, Cs2CO3, 1,4-dioxane, 110 °C, microwave; (iii) 4-fluoroaniline,
HATU, Et3N, DMF; and (iv) BBr3, CH2Cl2.
regioisomer 68, in approximately 40% yield. The reactivity of
each position of the pyrimidine halides follows the general order
C6(4) > C2 ≫ C5.15 A strong preference for the C6 position has
been observed in Suzuki cross-coupling reactions.16−20 Coupled
with the use of a lower temperature for the Suzuki coupling, this
allowed for the sequential introduction of different substituents.
Thus, 2,6-dichloropyrimidin-4(3H)-one 68 underwent a
Suzuki cross-coupling with the appropriate boronic acid, using
Pd(OAc)2/dppf or PdCl2(dppf)2 as the catalyst to afford
compounds 69a−d. Coupling of intermediates 69a−d with 3,5-
dimethylpyrazole was achieved by heating to 130 °C under
microwave irradiation using 15 mol % Pd2(dba)3, xanphos, and
Cs2CO3 in 1,4-dioxane to afford the desired compounds 22−25
(Table 3). A slightly modified procedure, as shown in Scheme 5,
was used to prepare amide 26. Methyl 2,6-dichloropyrimidine-4-
carboxylate was treated with sodium methoxide at 0 °C to obtain
intermediate 70. Intermediate 70 was then reacted with 3,5-
dimethylpyrazole under microwave heating using Cs2CO3 as a
base to give acid 71, which was followed by subsequent in situ
hydrolysis of the methyl ester. Acid 71 was coupled with 4-
fluoroaniline using 1-[bis(dimethylamino)methylene]-1H-
1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
(HATU) to give amide intermediate 72, which was demethy-
lated using boron tribromide in dichloromethane to give
compound 26.
To determine if polarity at the R2 position on the
pyrimidinone core was tolerated, compounds 27−29 (Table
3) were synthesized using the route, as shown in Scheme 6. 4-
Fluorobenzaldehyde was condensed with thiourea and ethyl
cyanoacetate using potassium hydroxide in refluxing ethanol to
give 2-mecapto-5-cyanopyrimidinone intermediate 73.21 The
thiol of intermediate 73 was methylated using methyl iodide in
the presence of sodium hydroxide followed by displacement
with hydrazine in refluxing ethanol to give 2-hydrazinyl-5-
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a
Scheme 6. Synthetic Route to Polar Substitutions on R2 of the Pyrimidinone
a
Reagents and conditions: (i) ethyl 2-cyanoacetate, KOH, ethanol, reflux; (ii) (a) MeI, NaOH, H2O (b) NH2NH2·H2O, EtOH, reflux; (iii)
acetylacetone, AcOH, EtOH, 85 °C; (iv) H2SO4, 80 °C; and (v) tert-butyl nitrite, AcOH, 75 °C.
a
Scheme 7. Synthetic Routes Used to Explore the Pyrazole R5 Position
a
Reagents and conditions: (i) diketone, EtOH, AcOH, reflux; (ii) 3-aminocrotonitrile, EtOH, reflux; (iii) acetaldehyde, DMF, AcOH, NaCNBH4,
MeOH, 25 °C; (iv) N-(2-methoxyethyl)-3-oxobutanamide, Lawesson’s reagent, THF, 25 °C; and (v) Cs2CO3, Pd2(dba)3, XantPhos, 1,4-dioxane,
aryl halide, 120 °C
cyanopyrimidinone intermediate 74. This intermediate was then
condensed with acetylacetone by heating in acetic acid-ethanol
at 85 °C to obtain compound 27, which was hydrolyzed to
amide 28 by heating in concentrated sulfuric acid at 85 °C.22
The conversion of amide 28 to acid 29 was achieved via
nitrosation by heating with tert-butyl nitrite in acetic acid at 85
°C followed by hydrolysis.23
Compounds 30−47 (Table 4) were synthesized to explore
the SAR around the R5 position of the pyrazole ring, keeping the
C6-position on the pyrimidinone core fixed as trifluoromethyl.
Using the same methodology, as shown Scheme 1, the
condensation of 56d with the corresponding 1,3-dicarbonyl
compound afforded compounds 30−32 exclusively as one
regioisomer as confirmed by NOESY NMR (Scheme 7). In
addition, 56d provided access to compound 33, which served as
a versatile intermediate and allowed for several transformations
to form N-substituted pyrazole derivatives. The reductive
amination of acetaldehyde with 33 in the presence of sodium
cyanoborohydride in methanol gave ethylaminopyrazole 34.
Attempts to synthesize the N,N-dialkyl derivative from
compound 34 failed as the corresponding derivatives decom-
posed on standing. Compound 47 was synthesized in the same
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a
Scheme 8. Synthetic Route to Compounds 36−39
a
Reagents and conditions: (i) NH2-R, EtOH, reflux; (ii) 33, AcOH, EtOH, 100 °C.
a
Scheme 9. Synthesis of Compound 35
a
Reagents and conditions: (i) (a) thiourea, KOH, EtOH and (b) MeI, NaOH, H2O; (ii) mCPBA, CH2Cl2, 25 °C; (iii) N2H2. H2O, EtOH; (iv)
EtI, K2CO3, DMF, 70 °C; and (v) Cs2CO3, 1,4-Dioxane, 130 °C.
a
Scheme 10. Synthesis of Compounds with Pyrazole C4 Substitutions
a
Reagents and conditions: (i) Br2, AcOH, 100 °C, 2h; (ii) pyridine-3-boronic acid, K2CO3, Pd(dppf)Cl2, THF/water (10:1), 70 °C, 16h; (iii) N-
bromosuccinamide, p-toluene sulphonic acid, CH2Cl2; 0 °C; (iv) morpholine, Et3N, CH2Cl2; and (v) EtOH/AcOH reflux, 16 h.
manner by reductive amination of acetaldehyde with 2-
hydrazineyl-6-(3-pyridinyl)pyrimidinone 56g. The synthesis of
5-(aryl-amino)pyrazoles 40−46 was achieved via a Buchwald
Hartwig amination of 33 and the corresponding aryl halide.
Compound 38 was synthesized from hydrazine 56d and N-(2-
methoxyethyl)-3-oxobutanamide with Lawesson’s reagent,
which allows for the efficient installation of substituted-5-
amino groups on the pyrazole core in a single step.24 However,
this methodology failed in the case of the synthesis of
compounds 36, 37, and 39, with 2-(5-hydroxy-3-methyl-1H-
pyrazol-1-yl)-6-(trifluoromethyl)pyrimidin-4(3H)-one being
obtained as the major product. An alternate highly regioselective
route to 5-(substituted-amino)pyrazoles 36, 37, and 39 was
achieved following a modified literature procedure.25
As shown in Scheme 8, the displacement of one methylthio
group of the α-oxoketene dithioacetal 7526,27 by the appropriate
amine in the presence of acetic acid in ethanol afforded the N,S-
acetals 76a-c, which were useful three-carbon 1,3-electro-
philes.28 Cyclocondensation with hydrazine 56d in refluxing
ethanol, gave access to 5-(substituted-amino)pyrazoles 36, 37,
and 39. The regioisomeric products with 3-(substituted-
amino)pyrazoles could be isolated in minor quantities and
showed much less potent activity against Mtb (data not shown).
Compound 35, with an O-ethyl group at R5, was synthesized,
as shown in Scheme 9. The condensation of ethyl acetoacetate
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a
Scheme 11. Synthesis of Compounds with Pyrazole C3 Modifications
a
Reagents and conditions. (i) DMF, 130 °C, 2 h; (ii) Cs2CO3, 1,4-dioxane, 130 °C, microwave; (iii) N,N-dimethylamine, HATU, Et3N, DMF; (iv)
(a) DIBALH, CH2Cl2, −78 °C; (b) N,N-dimethylamine, NaCNBH3, THF, RT, 24 h; and (v) Cs2CO3, 1,4-dioxane, 130 °C, microwave.
and hydrazine delivered pyrazolone 77, which on subsequent O-
ethylation with ethyl iodide gave 78. Intermediate 78 was
coupled with 79, synthesized by the oxidation of thiomethyl-
pyrimidin-4-one 55d with mCPBA to yield 35.
Formal Hit Assessment to Determine the Pharmaco-
phore Critical for Antitubercular Activity. Although the
initial hits, 1 and 2 (Table 1), showed reasonably potent
antitubercular activity (MIC < 2 μM), they suffered from
unfavorable physicochemical properties including low solubility,
low to moderate microsomal metabolic stability (MS), and a low
selectivity index (SI) between Mtb MIC and mammalian cell
toxicity as determined by measuring 50% inhibitory concen-
trations (IC50) determined against a CHO cell-line (an epithelial
cell-line derived from the Chinese-hamster ovary). A formal hit
assessment study was undertaken to determine the minimum
pharmacophoric features critical for antitubercular potency and
to understand the SAR features needed to improve the SI.
Initially, compounds with single point modifications on the
pyrimidinone and pyrazole portions of the molecule were
synthesized and profiled in a first wave of assays comprising Mtb
MIC, CHO IC50, and aqueous solubility. The substituents on
the pyrimidinone and pyrazole rings were labeled R1-R5, as
shown Figure 1 (see Table 2). Masking of the pyrimidinone
amide as −OCH3 (3) or N-CH3 (4) abolished the Mtb activity,
indicating the essentiality of the NH, which might be involved in
the critical hydrogen bond (HB) donor−acceptor interactions
with the target. Each of the nitrogens of the pyrimidinone ring
were found to be essential for maintaining Mtb activity (5 and
6). Interestingly, compound 7 with the carbonyl functionality
between the ring nitrogens pyrimidin-2(1H)-one vs pyrimidin-
4(3H)-one as in 1 maintained similar Mtb activity. The
additional SAR explorations in this work were limited to the
pyrimidin-4(3H)-one core as in compound 1, considering the
ease of synthesis.
Compounds 48−50 with substitutions on the pyrazole C4
position were synthesized, as shown in Scheme 10. The
bromination of compound 12 gave compound 48, which on
Suzuki cross-coupling with pyridine-3-boronic acid gave
compound 49. Attempts to displace C4-Br of 48 with
morpholine to synthesize compound 50 were unsuccessful. In
an alternative route, the morpholine ring was preinstalled on
acetylacetone via bromination with N-bromosuccinimide
(intermediate 80) followed by the reaction with morpholine
in the presence of triethylamine to give intermediate 81. This
intermediate was then condensed with hydrazine 56d by
refluxing in ethanol-acetic acid to give compound 50.
Compound 51 was synthesized in a similar fashion to
compound 33, by condensing hydrazine 56d with 3-cyclo-
propyl-3-oxopropanenitrile (82) (Scheme 11). The synthesis of
compounds 52−54 was accomplished from 83, as shown in
Scheme 11. The reaction of 83 with 85 under microwave heating
gave acid 53. This compound was then coupled with
dimethylamine using HATU to give compound 54. Pyrazole
ester 83 was converted to the corresponding dimethylamino-
methyl derivative 84 via a standard two-step protocol involving
the reduction of the ester to the aldehyde followed by reductive
amination with dimethylamine. Intermediate 84 was reacted
with 2-methylsulphonylpyrimidinone 79 under microwave
heating to obtain compound 52.
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Compound 8 with a methyl group added at the pyrimidinone
C5 (R2) position, while keeping a phenyl substituent at C6
(R1), retained a good MIC value (2 μM) while improving
aqueous solubility (120 μM) and the SI (12.5) for cytotoxicity.
However, isomeric compound 9 with C5-phenyl (R2) and C6-
methyl (R1) showed a poor MIC value (9 μM) with a lower SI of
1, whereas the fusion of the phenyl ring with the pyrimidinone
ring to form the corresponding quinazolinone (compound 10)
retained potency but with a low SI of only 2. A phenyl
substituent at the R1 (pyrimidinone C6) position was necessary
to maintain Mtb activity. Compounds 11 and 12 with CH3 and
CF3 groups, respectively, at R1 instead of the phenyl substituent
showed a 10−15-fold loss in activity. As compound 12 with the
CF3 group at R1 showed higher solubility and SI relative to 1,
this modification was included in further explorations for
potency and selectivity. Overall, these SAR observations
suggested the requirement for hydrophobic substitutions on
the pyrimidinone ring for the retention of potent in vitro activity
against Mtb.
A few polar modifications on the pyrazole ring were attempted
in order to evaluate the scope of reducing lipophilicity and
improving drug-like properties. Changing the C5′-methyl (R5)
on the pyrazole to oxygen abolished the activity as observed for
compound 13, whereas an amino group at the C5′-position as in
compound 14 resulted in the retention of activity comparable to
compound 1 (3 μM) and significantly improved solubility (180
μM). Unfortunately, this polar modification did not improve the
SI. N-acetyl amide derivative 15 retained the activity and SI
profile similar to parent amine 14. Compound 16, with an
unsubstituted pyrazole, lost potency considerably (MIC 50
μM). A similar loss in potency was observed when the pyrazole
ring was replaced with other 5-membered rings like triazole or
imidazole, as in compounds 17 and 18, respectively. This
suggests the critical nature of an appropriately substituted
pyrazole moiety for maintaining antitubercular activity.
methyl group, which could influence HB-donor acceptor
interactions of the pyrimidinone ring amide. The 3,5-
disubstituted pyrazole ring was critical for maintaining activity,
but there was a clear scope to extend substituents from the R5
position to improve potency and physicochemical properties.
The scope of substitution at the R4-postion remained to be
explored.
SAR of Polar Groups at R1 and R2 Positions of the
Pyrimidinone Core. Several polar R1 and R2 substituents on
the pyrimidinone ring were incorporated in order to expand the
scope relative to activity (Table 3) and increase solubility and SI.
Compound 19 with a 4-fluorophenyl group at the R1 position
retained activity and cytotoxicity similar to compound 1. The
replacement of the phenyl ring with pyridine rings considerably
improved solubility, as observed for compounds 20 and 21,
although with some loss in Mtb activity. Compound 20, with a 4-
pyridyl ring, was more potent (MIC 9 μM) than compound 21
with a 3-pyridyl ring (MIC 50 μM) leading to the speculation of
the role of HB-interactions through the 4-pyridine nitrogen.
Hence, compounds 22 to 25 with various polar substitutions at
the para-position of the phenyl ring at the R1 position were
prepared. Unfortunately, all these compounds lost activity. Next,
we considered the option of inserting a polar linker between the
pyrimidinone and phenyl rings. Among the various groups
attempted, only compound 26 with a 4-flurophenyl group
extended by a carboxamide linker at the R1 position, retained
weak activity (25 μM).
Based on the encouraging data for compound 8 with a C5-
methyl group on the pyrimidinone, we explored adding a few
polar groups such as CN, CONH2, and CO2H at this position,
but unfortunately this led to the loss of activity (compounds 27,
28, and 29). Overall, these observations indicated very limited
scope for adding polar substitutions on the pyrimidinone ring.
Hence, we turned our attention to exploring possible
interactions from the pyrazole ring.
The SAR observations regarding the critical pharmacophoric
features required for maintaining and/or improving antituber-
cular activity along with the scope for further SAR studies are
summarized in Figure 3. In summary, the pyrimidin-4(3H)-one
SAR of the Pyrazole Ring. Further exploration on the
pyrazole ring was planned, maintaining the CF3 as the R1
substituent on the pyrimidinone, as it increased the solubility
and SI exemplified for compound 12. In addition, the smaller
CF3 group kept the molecular weight and lipophilicity on the
low side compared to the phenyl. The addition of bulkier alkyls
like ethyl and cyclopropyl (compounds 30 and 31) at the R5
position on the pyrazole were tolerated for Mtb activity relative
to 12 but with a deterioration of the SI, whereas a slight loss in
activity was observed for compound 32 with the phenyl group at
the R5 position. Isomers of these compounds with bulkier
groups at the R3 position could not be synthesized because of
the selectivity observed in the condensation reaction of
hydrazine with requisite diketones favoring the placement of
bulkier groups at the R5 position. Compound 33 with a NH2 at
the R5 position maintained a profile similar to compound 12,
whereas compound 34 with an aminoethyl at the R5 position
showed increased toxicity against CHO cell lines without a
significant improvement in activity. Compound 35 with an O-
ethyl group at the R5 position had a potency and selectivity
profile similar to compound 12. The extension of polar groups
like −NH2 or −OH via a short ethylamino linker from the R5
position as in compounds 36 and 37, respectively, was
detrimental to activity against Mtb. Compounds 38 and 39,
with the neutral ether substituents like methoxyethylamino and
pyranamino, respectively, at the R5 position, showed more
potent activity (MIC 1.5−2 μM) as compared to 12 while the SI
was about the same. Based on these observations, it is speculated
Figure 3. Essential pharmacophoric features and scope for further SAR
studies.
ring was retained for further SAR studies considering the critical
nature of the cyclic amide as well as the ring nitrogens for
maintaining the antitubercular activity. The SAR observations
suggest the important role of hydrophobic substituents at the R1
and R2 positions for potency. We explored the scope of polar
interactions further away from the main scaffold by extending
polar groups from the aryl ring at the R1 position. In addition, we
also explored a few polar groups at the R2 position instead of a
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d
Table 6. In Vitro ADME Properties of Selected Compounds
compound
8
12
39
40
45
47
0.66
1.09
50
a
clog P
1.82
1.82
120
1.36
1.36
200
1.30
1.09
50
2.52
2.52
1.46
1.46
25
1.54
1.40
160
a
clog D
b
solubility (μM)
15
90
c
MS H/R/Mo % remaining after 30 min
65/54/45
ND
>99/92/>99
0.006
>99/>99/93
0.014
98/98/96
0.013
>99/96/>99
ND
80/95/94
0.009
ND
0.03
d
PPB human fu
a
b
c
d
Calculated log P/log D (StarDrop). Kinetic solubility measured at pH 7.4. Microsomal stability H: human, R: rat Mo: mouse. PPB; fu
fraction unbound.
that the R5 position lies in a more hydrophobic region of the
compound binding site within the target in Mtb. Hence, we
decided to examine aryl groups, some with polarity, at R5 toward
maintaining activity while improving solubility with polar
substituents placed distant from the main scaffold.
to improve the SI against mammalian cell-line toxicity. The
potential impact of both these activities in vivo remains to be
determined.
Physicochemical, DMPK, and Safety Profiles. Presented in
Table 6 are physicochemical and in vitro DMPK properties of
representative compounds, including solubility, microsomal
MS, and human plasma protein binding (PPB). Compounds
showed moderate to high aqueous solubility with excellent in
vitro microsomal stability, with the exception of compound 8
that was only moderately stable in microsomes. A small number
of compounds that were tested for the inhibition of liver
cytochrome P450 enzymes did not show significant inhibition
up to 20 μM (data not shown), indicating minimal possibility of
metabolism-based drug−drug interactions of these compounds,
which is a desirable attribute of a TB drug.29,30 The compounds
tested in the series were highly bound to the human plasma most
likely due to the albumin binding of acidic pyrimidinone.31 The
safety profiles of compounds 1 and 12 were evaluated across a
panel of 21 liability targets (39 functional assays), which
included cell-based GPCRs and ion channels in both agonist and
antagonist readout, and biochemical functional assays for
nuclear hormone receptors and phosphodiesterases.32 Both
compounds showed no significant inhibition or activation of the
enzyme/receptors at 10 μM test concentration. Both com-
pounds showed hERG IC50 > 30 μM. In summary, the in vitro
safety profile of the compounds indicated no obvious safety
liability even though the reason for mammalian cytotoxicity
observed was not clear.
The pharmacokinetic parameters of representative com-
pounds, 12, 47, and 40 were measured from mouse blood at
intravenous doses of 2 mg/kg and oral doses of 20 mg/kg in the
mouse (Table 7). The compounds were well tolerated following
either route of administration, with no obvious effects noted in
the animals over the course of the exposure. Compounds 12 and
40 showed a low volume of distribution and low rate of clearance
with moderate oral bioavailability of approximately 40%.
Compound 47 showed rapid blood clearance and high volume
of distribution with an oral bioavailability of 33%. Further
analysis of mouse blood samples from the PK studies of 47
showed the glucuronide as the major metabolite. However,
further optimization of physicochemical properties to improve
pharmacokinetic properties, along with improved SI, is
warranted in order to identify a compound suitable for in vivo
efficacy studies in TB animal models.
Compound 40 with a 4-F-aniline at the R5 position showed
Mtb potency and a SI profile similar to 39, with an aminopyran at
position R5, but had lower aqueous solubility (15 μM) as
expected because of the overall increase in lipophilicity with an
additional aryl ring. Compound 41 with a 2,4-difluoroanilino
group at the R5 position was the most potent compound (MIC
0.2 μM) obtained so far in these studies but was poorly soluble
(<5 μM) in aqueous media. The addition of polar groups like
CN and dimethylcarboxamide (42 and 43) at the para-position
of the R5-anilino groups decreased activity (MIC 9 and >50 μM,
respectively). Similarly, compound 44 with a 4-pyridylamino at
the R5 position was inactive against Mtb up to the highest
concentration tested (MIC > 50 μM). Interestingly, compound
45 with a 2-pyridylamino group at the same R5 position was
quite potent with a MIC of ≤1 μM. The compound had
moderate aqueous solubility (25 μM) with a modest SI of 25 for
CHO cell-line toxicity, thus making it a valuable compound for
MoA studies (see below). A few compounds with polar
substituents para to the amino group on the 2-pyridyl ring
were synthesized and screened with a view to improving
physicochemical properties. For example, compound 46 with a
4-morpholino-2-pyridylamino group at the R5 position retained
good activity (MIC = 2 μM) with improved solubility (180 μM)
but was significantly more cytotoxic (SI 0.6).
Compound 47, with a 3-pyridyl at the R1 position and an
ethylamino group at the R5 position, showed modest activity
with an MIC of 6.25 μM and CHO IC50 of >50 μM.
A limited SAR exploration at the R4 position on the pyrazole
was conducted to evaluate the scope for further improvement in
potency and selectivity. Compound 48 with 4-bromopyrazole
showed only moderate activity (MIC 25 μM, CHO IC50 > 50
μM) and was used as an intermediate for further synthesis.
Replacing the bromine with a 3-pyridyl ring (49) abolished Mtb
activity (MIC >50 μM), whereas the morpholine ring at this
position (50) was well tolerated with an MIC = 6.25 μM and a
cleaner cytotoxicity profile (CHO IC50 > 50 μM). In
comparison, the R3 position on the pyrazole showed a very
limited scope for further modifications. In general, a cyclopropyl
group as in compound 51 was well tolerated for Mtb potency but
larger groups in general gave weakly active compounds. Polar
groups like dimethylaminomethyl, carboxylic acid, and amide as
in compounds 52, 53, and 54, respectively, led to considerable
loss of activity against Mtb (MICs > 50 μM).
Bactericidality and Activity against Clinical Isolates. The
compounds were found to be bactericidal against replicating
Mtb showing 2 log CFU reduction at 1−2× MIC over the time
period of 7 days (Fig. S1). Importantly, we also evaluated the
activity of 1, 12, 15, and 20 against a panel of clinical isolates
(Table S1). All the tested compounds retained MICs against
clinical isolates within the 4-fold range of MICs against the drug-
sensitive Mtb H37Rv strain.
In summary, various modification on the pyrimidinone and
pyrazole portions of the scaffold showed potential for improve-
ment in potency against Mtb in vitro and MICs as low as 200 nM
could be achieved. However, in general there was a narrow scope
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differently to the protein depending on the mutation. To
investigate whether or not compounds 1, 2, 12, 15, 47, 40, and
38 retain target selectivity for MmpL3 in Mtb cells, we asked
whether or not conditional depletion of MmpL3 in a mmpL3
hypomorph (Grover et al., manuscript under review) would
sensitize Mtb to the growth inhibitory effects of the compounds.
However, transcriptional silencing of mmpL3 resulted in no
significant change in the MICs of the compounds, confirming
that MmpL3 is not the direct target of this series (Table S2). We
hypothesize that MmpL3 acts as a transporter of these
compounds across the cell membrane as the compounds can
form heme-like iron-complexes, and MmpL3 is known to act as a
heme transporter.37,38 Hence, MmpL3 is likely to be responsible
for building resistance toward compounds through the impair-
ment of transport of compounds across the cell membrane
rather than being a direct target. It is to be noted that the
compounds were PiniB-LUX negativeindicating a non-cell
wall MoA.
Based on the structural similarity of the compounds with the
published thymidylate kinase inhibitors,39,40 we asked whether
this compound series can inhibit Mtb thymidylate kinase, which
is an essential target encoded by Rv3247c. To investigate this,
we tested the activity of the compounds 1, 2, and 12 against the
Rv3247c hypomorph. However, we did not observe any MIC
modulation of the compounds upon silencing of Rv3247c,
suggesting that the tested compounds are not the thymidylate
kinase inhibitors.
Treatment with 45 and 12 Affects Genes Involved in Fe
Homeostasis. In continuation of our efforts to explore MoA, the
effects of 45 (at 1 μM and 10 μM) and 12 (at 20 μM) treatment
on the gene expression of Mtb was investigated. Mtb cultures
were treated with the compound for 6 h, harvested, and the RNA
extracted for transcriptional profiling by microarray analysis.
The data obtained from the transcriptional profiling of Mtb
exposed to 45 and 12 were similar (Table S3). We performed in-
depth analysis of the data obtained from samples treated with
45, which was tested at two concentrations. The 50 most
upregulated (>4-fold) genes were selected in order to
understand their potential contribution in the cellular response
based on the MoA of 45 (Table S4). This indicated a
transcriptional signature related to iron-sequestration. The
upregulated genes included rv2377c-78c-79c-80c-81c-82c-83c-
84-85-2386c (mbtA-H)all of which are associated with
mycobactin biosynthesis or regulation,41 which are known to
be upregulated in response to iron deprivation.
Table 7. Pharmacokinetic Parameters in Male C57/BL6
Mouse Blood
a
route of
administration
i.v.
oral
i.v.
oral
i.v.
oral
12
47
40
dose (mg/kg)
2
20
2
20
2
20
apparent t1/2 (h)
CLtotal
(mL/min/kg)
3.1
2.7
4
5.6
80.9
4.4
4.0
0.4
6.1
CLint
(mL/min/kg)
476
>4000
34
Vd (L/kg)
0.7
29.1
37.4
2.4
0.1
33.1
Cmax (μM)
65.6
0.5
12004
2.9
0.5
313
104.5
1.0
65417
Tmax (h)
AUC0−∞
(min·μM)
2928
91
15373
oral
bioavailability
F (%)
41
33
44
a
i.v.: intravenous; t1/2: elimination half-life; CLtotal: total blood
clearance; CLint: intrinsic clearance; Vd: volume of distribution during
elimination phase; Cmax, maximum (peak) plasma concentration
following oral administration; and AUC: area under the curve.
Target Deconvolution Studies. Biology Triaging. In
order to explore the MoA, potent compounds were initially
screened against various tool strains to rule out the involvement
of known mechanisms and/or targets. The tested compounds
retained activity against a QcrB mutant (A396T) and did not
show hypersensitivity against a cytochrome-bd oxidase knock-
out mutant strain (cydKO),33 thereby eliminating them as
potential targets. The compounds did not show a positive signal
in two standard bioluminescence reporter assays: PiniB-
LUX34detects modulation in the iniB expression, if a
compound targets Mtb cell−wall biosynthesis, and PrecA-
LUX34detects modulation in the recA expression, an indicator
of genotoxic compounds. A strain carrying a mutation in DprE1
(C387S), which confers resistance to other DprE1 inhibitors,
was not resistant, suggesting DprE1 is not the target. However,
strains carrying mutations in MmpL3 were cross-resistant to
compounds (Table 8).35 The compounds showed an increase of
a
Table 8. Cross-Screening against mmpL3 Mutants
strain
1
2
12
15
27
47
40
1.2
38
H37Rv WT
MIC (μM)
1.7
4.8
15
5.8
1.2
Another upregulated gene-set rv1342-43-44-45-46-47-48-49
is also associated with the mycobactin biosynthesis or regulation
or with iron transport where rv1348 and rv1349 were annotated
as iron-regulated transporters, both being essential for growth in
vitro.42−44 The esx-3 gene cluster is composed of 11 genes
stretching from rv0282 to rv0292. All of these were upregulated
2−5-fold in response to the stress caused by the compound.
rv0287 and rv0288 are predicted to be essential for growth in
vitro. Interestingly, ESX-3 is known to be required for the
mycobactin-mediated siderophore iron uptake pathway,45−48
and it is essential in Mtb but not in Mycobacterium smegmatis, a
species which, in addition to mycobactins, produces exochelin
that functions independently of ESX-3. However, ESX-3 is still
required for mycobactin-mediated iron uptake in the latter
species.45,46,48 The nonessential nature of ESX-3 in M. smegmatis
has previously proven to be of a great advantage when resolving
mechanisms related to ESX-3 and mycobactin iron metabo-
lism.48 Some of the top upregulated genes are involved in lipid
MmpL3
F255L MIC
(μM)
MmpL3
F644L MIC
(μM)
MmpL3 V681 16
MIC (μM)
MmpL3
G596R
MIC (μM)
18
55
170
17
>200
24
41
>200
1
39
1.9
5.4
5.5
1.9
45
>200
>200
40
110
a
WT: wild-type.
8−100-fold in MIC values against the strains with either
MmpL3F255L or MmpL3V681I or MmpL3G596R, whereas, there
was no change in activity against the MmpL3F644L mutant. Most
of these mutations lie within the region required for proton
translocation.36 Variability in the MICs against these mutant
strains is suggestive of the fact that the compounds may bind
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Figure 4. Fe2+ plays a critical role in the MoA of 45. (A) Fe2+ rescue of Mtb from growth inhibition mediated by 45. (B) Spectroscopic titration of 45
with Fe2+. The data are representative of two biological replicates performed in duplicate.
metabolism, for example, rv3249-51-52 potentially a transcrip-
tional response to correct damage to fatty acids. Fe has been
reported to affect lipid metabolism, specifically because of Fe-
catalyzed lipid oxidation. In line with this, we observed that
genes, including rv3741-42, (oxidoreductase) were upregulated.
The observed upregulation of the rv3269-70 operon encoding a
stress response protein and a metal cation transporter,
respectively, further supports the notion that the cellular
response to 45 is one of the disruption of iron homeostasis.
Rv0285PE5 which was upregulated in our study, is down-
regulated in the presence of iron.49 Importantly, most of the
upregulated genes of our data set were found to be down-
regulated when Mtb was exposed to an excess of iron.49 Our data
set also complements the recently published work by Kurthkoti
et al.,50 where authors performed transcriptomics in iron-starved
cultures and identified similar upregulated clusters of genes. This
strongly suggests that the disruption of Fe homeostasis is an
important component of the MoA of 45.
In summary, our data suggest that upon exposure to 2-
pyrazolylpyrimidinones, Mtb goes into an iron-starvation state.
As expected, it upregulates genes involved in the mycobactin
(siderophore) synthesis pathway and its regulation. Side-
rophores are small, high-affinity iron-chelating compounds
secreted by microorganisms such as bacteria and fungi. In the
process of cellular uptake, the Fe3+-siderophore complex is
subsequently reduced to Fe2+ to release the iron.
Iron Plays a Significant Role in the MoA. To confirm the role
of iron in the MoA of this series, an Fe rescue experiment was
performed by supplementing standard Middlebrook 7H9/Glu/
CAS/Tx media with 200 μM ferric ammonium citrate. The
supplementation of Fe3+ resulted in a 4-fold increase in the MIC
(Table S5). Under physiological conditions, iron can exist in
either the reduced ferrous (Fe2+) form or the oxidized ferric
(Fe3+) form. The redox potential of Fe2+/Fe3+ makes iron
extremely versatile when it is incorporated into proteins as a
catalytic center or as an electron carrier. Thus, iron is important
for numerous biological processes, which include the tricarbox-
ylic acid cycle, gene regulation, DNA biosynthesis, and so forth.
Although iron is abundant in nature, it does not normally occur
in its biologically relevant but aerobically unstable ferrous form.
To evaluate this, we supplemented the Mtb culture with 200 μM
ferrous sulfate, resulting in an 8-fold increase in the MIC for 45
(Table S5). Next, we confirmed the ability of Fe2+ to rescue the
Mtb from 45 toxicity in a checkerboard (2D) assay using GAST
(glycerol-alanine-salts-Tween 80) media which is devoid of Fe
unlike Middlebrook 7H9/Glu/CAS/Tx (Figure 4A). The
inhibition of Mtb growth by 45 in GAST medium supplemented
with 12.5 μM Fe+2 (MIC 0.12 μM) decreased in a dose-
dependent manner with increasing concentration of Fe2+ and
resulted in a ≥32-fold increase in MIC at 100/200 μM Fe2+. To
determine that 45 forms a complex with Fe2+, we performed
UV−vis scanning of 45 with increasing amounts of Fe2+ (Figure
4B). The band intensity at 270, which was observed for 45 alone,
decreased after the addition of Fe2+, whereas the absorbance
around 285 is shifted to 300 with the increase in intensity,
indicating the formation of the 45-Fe2+ complex.
In line with the observation of better rescue with ferrous than
ferric iron and a preference of heme for the former, there is a
strong possibility that the compound binds iron to form a heme-
like complex that enters Mtb via MmpL3. This also supports the
reason for resistance with some of the MmpL3 mutants as
described previouslyheme prefers ferrous over ferric.
Involvement of a heme-like structure could also explain the
associated cytotoxicity of the series, as described earlier. This is
because of the toxic nature of heme to eukaryotic cells, which
require specific heme-binding proteins to maintain a nontoxic
homeostasis.
CONCLUSIONS
■
Whole-cell phenotypic screening of MMV compound library led
to the identification of 2-pyrazolylpyrimidinones with potent
antitubercular activity and a novel mode of action. Detailed SAR
studies identified several compounds with potent activity against
Mtb with moderate to high aqueous solubility and excellent in
vitro microsomal stability. The compounds were bactericidal
against replicating Mtb and retained potency against clinical
isolates. Transcriptional profiling suggested that upon exposure
to 2-pyrazolylpyrimidinones, Mtb goes into an iron-starvation
state that may account for lethality. Iron supplementation using
a high concentration of ferrous salts showed shifts in MICs of the
compounds, further confirming that 2-pyrazolylpyrimidinones
perturbs the Fe-homeostasis in Mtb. Further optimization of
pharmacokinetic properties, along with improved SI between
MIC and mammalian cytotoxicity, is needed to identify a
compound suitable for in vivo efficacy studies in mouse TB
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6120 quadrupole (single) mass spectrometer, equipped with APCI and
ESI multimode ionization sources. Using a Kinetex Core C18 2.6 μm
column (50 mm × 3 mm); mobile phase B of 0.4% acetic acid, 10 mM
ammonium acetate in a 9:1 ratio of HPLC grade methanol and type 1
water, mobile phase A of 0.4% acetic acid in 10 mM ammonium acetate
in HPLC grade (type 1) water, with a flow rate of 0.9 mL/min, DAD; or
an Agilent UPLC−MS was used: Agilent Technologies 6150
quadrupole, ES ionization, coupled with an Agilent Technologies
1290 Infinity II series UPLC system Agilent 1290 series HPLC at two
wavelengths 254 and 290 nm using the following conditions: Kinetex
1.7 μm Evo C18 100A, LC column 50 mm × 2.1 mm, solvent A of 0.1%
(formic acid) water, and solvent B of 0.1% (formic acid) acetonitrile.
The structures of the intermediates and end products were confirmed
by 1H NMR and mass spectrometry. Proton magnetic resonance
spectra were determined in an appropriate deuterated solvent on a
Varian Mercury spectrometer at 300 MHz or a Varian Unity
spectrometer at 400 MHz.
models and eventual development as a viable drug useful for the
treatment of TB in humans.
EXPERIMENTAL SECTION
■
MIC Testing and Triage Assays. Unless otherwise indicated, an
Alamar Blue fluorescence-based broth microdilution assay was used to
assess MIC of compounds against Mtb strains, as described
previously.51,52 Rifampicin was included as a control. Biology triage
assays were carried out as described by Naran et al.34
MICs against Mutant Strains. MICs were determined after 5 days
of growth in Middlebrook 7H9 medium plus OADC supplement and
0.05% w/v Tween 80 as described previously.53 Growth was monitored
by OD. Data were fit using the Levenberg−Marquardt least-squares
plot. MIC was defined as the concentration required to inhibit growth
by 90%.
RNA Extraction and Transcriptional Profiling. Total RNA was
extracted from Mtb H37Rv that had been treated for 6 h with 1× and
10× MIC of the compound or vehicle control as previously described.54
RNA quality was confirmed by UV spectral analysis and Agilent 2100
Bioanalyzer returning a RNA integrity number of 8 or higher.
Fluorescent-tagged cDNA was prepared via a direct random-primed
labeling method as follows. To 4.0 μg of RNA, 4.5 μg of a random
hexamer (Invitrogen# 48190-011) was added, a final volume of 14.5 μL,
heat denatured at 70 °C for 5 min then immediately cooled to 0 °C on
ice. cDNA synthesis and fluorophore (Cy3 or Cy5) incorporation were
carried out using the following reverse transcription reaction
components 5 μL of 5× First-Strand buffer, 1.25 μL of 0.1 M DTT,
2.5 μL of dNTP mix (made by 5 mM each of dATP, dGTP), dTTP, 0.5
mM dCTP, plus 1 μL of 200 U/μL SuperScriptIII (Invitrogen# 18080-
044), 1 μL of 40 U/μL RNAseOut, and 1 μL of Cy3 or Cy5-dCTP
(GE# PA55321) were added for incubation at 25 °C for 5 min and at 48
°C for 90 min. Template RNA was chemically hydrolyzed by 5 μL of 1
M NaOH and heated at 70 °C for 15 min. The hydrolysis reaction was
neutralized with 5 μL of 1 M HCl. The labeled cDNA was purified on an
Amicon Ultra-0.5 column (Millipor# C82301) following the
manufacturer’s recommendations for PCR purification. cDNA yields
and dye incorporation were measured with a Nanodrop ND-1000. A
mixture of equal amounts (0.7 μg) of Cy3- and Cy5-labeled cDNA was
loaded into hybridization chambers for incubation with an Agilent
SurePrint G3 4x44K custom oligonuclotide microarray (design number
021966, 021362).
Microarrays were hybridized robotically using a TECAN HS Pro
4800 hybridization station, Agilent 2× gene expression hybridization
HI-RPM buffer, and 10× blocking reagent at 65 °C for 17 h and washed
with Agilent Gene Expression Wash Buffer 1 at room temperature and
Gene Expression Wash Buffer 2 at 37 °C. Then, slides were dried under
a nitrogen gas for 3 min at 30 °C. The slides were imaged using an
Agilent high-resolution DNA microarray scanner (model G2505C) at 5
μm resolution and 100/10% PMT dual scanning for XDR extended
dynamic range. Agilent Feature Extraction software was used for image
analysis.
DMPK. All protocols for in vitro DMPK studies and mouse PK
studies are available in the supplementary document. Animal studies
were conducted following guidelines and policies as stipulated in the
UCT Research Ethics Code for Use of Animals in Research and
Teaching, after review and approval of the experimental protocol by the
UCT Senate Animal Ethics Committee (protocol FHS-AEC 013/032).
Chemistry. All commercial reagents were purchased from Sigma-
Aldrich, Combi-Blocks, Enamine, or Fluorochem and were used
without further purification. Solvents were used as received unless
otherwise stated. Analytical thin-layer chromatography was performed
on SiO2 plates on aluminum backing. Visualization was accomplished
by UV irradiation at 254 and 220 nm. Flash column chromatography
was performed using a Teledyne ISCO flash purification system with
SiO2 60 (particle size 0.040−0.055 mm, 230−400 mesh). Purity of all
final derivatives for biological testing was confirmed to be >95% as
determined using an Agilent 1260 Infinity binary pump, Agilent 1260
Infinity diode array detector (DAD), Agilent 1290 Infinity column
compartment, Agilent 1260 Infinity standard autosampler, and Agilent
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-phenylpyrimidin-4(3H)-one
(1). A mixture of 2-hydrazinyl-6-phenylpyrimidin-4(3H)-one (56a 0.2
g, 0.989 mmol) and pentane-2,4-dione (0.121 mL, 1.187 mmol) was
heated in a mixture of ethanol (0.5 mL) and acetic acid (1.5 ml) at 100
°C for 16 h. After cooling to room temperature, the reaction mixture
was poured into ice cooled water and stirred for 30 min. Solids formed
were filtered, washed with water, and dried to yield 1 as a white solid
(200 mg, 0.744 mmol, 75% yield). HPLC purity: >99%. LC−MS APCI:
1
calcd for C15H14N4O, 266.304; observed m/z [M + H]+, 267.1. H
NMR (300 MHz, CDCl3): δ 8.01−7.97 (m, 2H), 7.53−7.50 (m, 3H),
6.73 (s, 1H), 6.12 (s, 1H), 2.55 (s, 3H), 2.32 (s, 3H).
6-Methyl-2-(3-methyl-5-(p-tolylamino)-1H-pyrazol-1-yl)-
pyrimidin-4(3H)-one (2). Compound was received from the MMV
screening library. For synthesis and analytical data, please refer
WO2012154880.2
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-4-methoxy-6-phenylpyrimi-
dine (3). DIAD (0.110 mL, 0.563 mmol) was added dropwise to a
solution of 2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-phenylpyrimidin-
4(3H)-one (1) (100 mg, 0.376 mmol), triphenylphosphine (148 mg,
0.563 mmol) in methanol (12.03 mg, 0.376 mmol), and tetrahydrofur-
an (100 mL), and the reaction mixture was stirred at room temperature
for 4 h. The mixture was concentrated under reduced pressure, the
residue partitioned between dichloromethane and water, and the layers
separated. The aqueous phase was extracted with dichloromethane, the
combined organic solutions washed consecutively with water, 2 N
aqueous sodium hydroxide, water, and finally brine. The solution was
then dried over sodium sulfate and evaporated under reduced pressure.
The crude product was purified on an ISCO system using a 4 g silicycle
column and eluting with a gradient 0 to 70% ethyl acetate in hexane
over 20 min to yield 3 as a white solid (27 mg, 0.09 mmol, 24% yield).
HPLC purity: 95%. LC−MS APCI: calcd for C16H16N4O, 280.33;
observed m/z [M + H]+, 281.1. 1H NMR (300 MHz, CDCl3): δ 7.99−
7.96 (m, 2H), 7.49−7.47 (m, 3H), 6.92 (s, 1H), 6.31 (br s, 1H), 6.08 (s,
1H), 3.56 (s, 3H), 2.50 (s, 3H), 2.33 (s, 3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-3-methyl-6-phenylpyrimidin-
4(3H)-one (4). To a stirred solution of 2-(3,5-dimethyl-1H-pyrazol-1-
yl)-6-phenylpyrimidin-4(3H)-one (1) (50 mg, 0.188 mmol) in DMF
(2 mL) was added potassium carbonate (52 mg, 0.563 mmol). The
reaction mixture was stirred for 5 min and iodomethane (35 μL, 0.563
mmol) was added. The reaction mixture was stirred at 100 °C
overnight. The reaction mixture was extracted with ethyl acetate (3 ×
10 mL). The combined organic layer was washed with LiCl solution (1
× 10 mL) and dried over sodium sulfate, and the solvent was
evaporated. The obtained residue was purified by flash chromatography
by using 15% ethyl acetate in hexane obtained 4 as a white solid (39 mg,
0.141 mmol, 75% yield). HPLC Purity: 96%. LC−MS APCI: calcd for
1
C16H16N4O, 280.33; observed m/z [M + H]+, 281.1. H NMR (300
MHz, CDCl3): δ 8.01−7.97 (m, 2H), 7.53−7.50 (m, 3H), 6.73 (s, 1H),
6.18 (s, 1H), 3.52 (s, 3H), 2.55 (s, 3H), 2.32 (s, 3H).
6-(3,5-Dimethyl-1H-pyrazol-1-yl)-4-phenylpyridin-2(1H)-one (5).
To a cooled solution of crude 61 (0.40 g, 1.43 mmol) in methanol (7
mL) was added boron tribromide (1 M solution in dichloromethane)
(4.2 mL, 4.29 mmol) and stirred for 16 h at room temperature. Water
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was added to the reaction mixture and extracted with dichloromethane.
The combined organic phase was dried over sodium sulfate,
concentrated under vacuum, and purified by preparative HPLC to
give 5 as a white solid (0.08 g, 21%). HPLC purity: 98.6%. LC−MS
APCI: calcd for C16H15N3O, 265.122; observed m/z [M + H]+, 266.2.
1H NMR (400 MHz, CDCl3): δ 9.38 (br s, 1H), 7.64−7.62 (m, 2H),
crystallized from ethanol to give 13 as a white solid (39 mg, 0.148
mmol, 20% yield). HPLC purity 93%. LC−MS APCI: calcd for
C14H12N4O2, 268.27; observed m/z [M + H]+, 269.1. 1H NMR (400
MHz, DMSO-d6): δ 8.22−8.20 (m, 2H), 7.54−7.51 (m, 2H), 6.74 (s,
1H), 5.31 (s, 1H), 2.29 (s, 3H).
2-(5-Amino-3-methyl-1H-pyrazol-1-yl)-6-phenylpyrimidin-4(1H)-
one (14). 2-Hydrazineyl-6-phenylpyrimidin-4(1H)-one 56a (0.1 g,
0.495 mmol) and 3-aminocrotonitrile (0.042 g, 0.519 mmol) were
stirred in ethanol (5 mL) and refluxed for 5 h. The solvent was removed,
and the crude product was recrystallized from methanol to afford 14 as a
white solid (55 mg, 0.742 mmol, 42% yield). HPLC purity: 97%. LC−
MS APCI: calcd for C14H13N5O, 267.29; observed m/z [M + H]+,
268.1. 1H NMR (300 MHz, DMSO-d6): δ 8.04−7.92 (m, 2H), 7.60−
7.49 (m, 3H), 6.91 (s, 2H), 6.73 (s, 1H), 5.35 (s, 1H), 2.13 (s, 3H). 13C
NMR (101 MHz, DMSO-d6): δ 192.03, 152.67, 151.14, 136.44, 131.20,
129.40, 127.22, 89.28, 14.37.
N-(3-Methyl-1-(4-oxo-6-phenyl-1,4-dihydropyrimidin-2-yl)-1H-
pyrazol-5-yl)acetamide (15). A mixture of 2-(5-amino-3-methyl-1H-
pyrazol-1-yl)-6-phenylpyrimidin-4(1H)-one (14, 100 mg, 0.374
mmol) and pyridine (0.04 mL, 0.442 mmol) in dichloromethane (2
mL) was treated with a solution of acetyl chloride (0.04 mL, 0.0448
mmol) in dichloromethane (1 mL). The reaction mixture was stirred at
room temperature for 24 h before it was diluted with dichloromethane
(5 mL). The mixture was washed with 2 N HCl (10 mL), 5% aqueous
solution of NaHCO3, and brine. The organic phase was dried over
sodium sulfate, filtered, and concentrated in vacuo to give 15 as a white
solid (45 mg, 0.145 mmol, 39% yield). HPLC purity: 98%. LC−MS
APCI: calcd for C16H15N5O2, 309.33; observed m/z [M + H]+, 310.1.
1H NMR (300 MHz, DMSO-d6): δ 11.55 (s, 1H), 8.13−8.03 (m, 2H),
7.51−7.47 (m, 3H), 6.76 (s, 1H), 6.70 (s, 1H), 6.07 (s, 1H), 2.58 (s,
3H), 2.3 (s, 3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-phenylpyridin-4(1H)-one (6).
Synthesized from intermediate 62 with a protocol similar to the one
described for compound 5. Yield 25% (white solid); HPLC purity:
98.9%. LC−MS APCI: calcd for C16H15N3O, 265.122; observed m/z
[M + H]+, 266.2. 1H NMR (400 MHz, CDCl3): δ 10.49 (br s, 1H), 7.96
(d, J = 6.4 Hz, 2H), 7.46−7.40 (m, 4H), 7.11 (s, 1H), 6.07 (s, 1H), 2.70
(s, 3H), 2.35 (s, 3H).
6-(3,5-Dimethyl-1H-pyrazol-1-yl)-4-phenylpyrimidin-2(1H)-one
(7). To the solution of 2-chloro-4-(3,5-dimethyl-1H-pyrazol-1-yl)-6-
phenylpyrimidine (intermediate 65, 0.16 g, 0.56 mmol) in THF (2 mL)
was added 2 M aqueous NaOH solution (2 mL) at room temperature,
and the mixture was heated at 80 °C for 4 h. The reaction mixture was
then cooled in ice-bath and acidified with glacial acetic acid. The
precipitated solid was filtered under vacuum, washed with chilled water,
and dried to get 6-(3,5-dimethyl-1H-pyrazol-1-yl)-4-phenylpyrimidin-
2(1H)-one (7, 0.12 g, 80% yield) as a white crystalline powder. HPLC
purity: 99.2%. LC−MS APCI: calcd for C15H14N4O, 266.117; observed
1
m/z [M + H]+, 267.2. H NMR (400 MHz, DMSO-d6): δ 8.11 (m,
2H), 7.53 (m, 3H), 6.92 (s, 1H), 6.26 (s, 1H), 2.71 (s, 3H), 2.23 (s,
3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-5-methyl-6-phenylpyrimidin-
4(3H)-one (8). Synthesized from intermediate 56b following a protocol
similar to compound 1. Yield 10% (white solid). HPLC purity: 97%.
LC−MS APCI: calcd for C16H16N4O, 280.331; observed m/z [M +
H]+, 281.1. 1H NMR (300 MHz, CDCl3): δ 7.68−7.58 (m, 2H), 7.55−
7.39 (m, 3H), 6.05 (s, 1H), 2.69 (s, 3H), 2.30 (s, 3H), 2.19 (s, 3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-methyl-5-phenylpyrimidin-
4(3H)-one (9). Synthesized from intermediate 56h following a protocol
similar to compound 1. Yield 10% (white solid). HPLC purity: 98.9%.
LC−MS APCI: calcd for C16H16N4O, 280.132; observed m/z [M +
H]+, 281.0. 1H NMR (400 MHz, CDCl3): δ 10.59 (s, 1H), 7.27−7.44
(m, 5H), 6.05 (s, 1H), 2.71 (s, 3H), 2.26 (s, 3H), 2.22 (s, 3H). 13C
NMR (100 MHz, CDCl3): 13.63, 15.08, 22.99, 111.38, 122.18, 127.70,
128.36, 130.17, 133.72, 143.40, 145.66, 151.82, 161.01, 161.19.
2-(3,5-Dimethyl-1H-pyrazol-1-yl)quinazolin-4(3H)-one (10). Syn-
thesized from 2-hydrazineylquinazolin-4(3H)-one following a protocol
similar to compound 1. Yield 47% (white solid). HPLC purity: 96.3%.
LC−MS APCI: calcd for C13H12N4O, 240.10; observed m/z [M + H]+,
241.1. 1H NMR (400 MHz, DMSO-d6): δ 11.92 (s, 1H), 8.11 (dd, J =
7.9, 1.5 Hz, 1H), 7.81 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 7.65 (d, J = 8.1 Hz,
1H), 7.53−7.43 (m, 1H), 6.27 (s, 1H), 2.69 (s, 3H), 2.26 (s, 3H). 13C
NMR (101 MHz, DMSO-d6): δ 160.68, 151.04, 147.19, 143.23, 139.59,
135.19, 126.65, 126.46, 118.79, 115.59, 111.34, 14.74, 13.85.
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-methylpyrimidin-4(3H)-one
(11). Synthesized from intermediate 56c following a protocol similar to
compound 1. Yield 78% (white solid). HPLC purity: 96%. LC−MS
APCI: calcd for C10H12N4O, 204.23; observed m/z [M + H]+, 205.1 1H
NMR (300 MHz, CDCl3): δ 6.73 (s, 1H), 6.12 (d, 1H), 2.55 (s, 3H),
2.32 (s, 3H), 2.23 (d, 1H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(trifluoromethyl)pyrimidin-
4(3H)-one (12). Synthesized from intermediate 56d following a
protocol similar to compound 1. Yield 70% (white solid). HPLC
purity: 98.9%. LC−MS APCI: calcd for C10H9F3N4O, 258.07; observed
m/z [M + H]+, 259.0. 1H NMR (400 MHz, DMSO-d6): δ 6.79 (s, 1H),
6.27 (s, 1H), 2.56 (s, 3H), 2.23 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): δ 13.83, 14.77, 111.89, 119.62, 122.34, 143.71, 152.04.
2-(3-Methyl-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)-6-phenylpyrimi-
din-4(1H)-one (13). A mixture of 2-hydrazineyl-6-phenylpyrimidin-
4(1H)-one (56a, 150 mg, 0.742 mmol) and ethyl acetoacetate (0.114
mL, 0.890 mmol) was heated in a mixture of ethanol: acetic acid, 1:3,
under reflux for 5 h. The solid product obtained after cooling and
pouring onto ice-cold water was filtered off, washed with water, and
7.63−7.52 (m, 3H), 6.95 (s, 1H), 6.64 (s, 1H), 2.22 (d, J = 15.1 Hz,
6H).
6-Phenyl-2-(1H-pyrazol-1-yl)pyrimidin-4(3H)-one (16). In a 10 mL
microwave vial, 2-chloro-6-phenylpyrimidin-4(3H)-one (66, 0.1 g,
0.484 mmol), 1H-pyrazole (0.066 g, 0.968 mmol), and cesium
carbonate (0.473 g, 1.452 mmol) were mixed in 1,4-dioxane (1 mL).
The vial was capped and microwaved at 100 °C for 1 h. LCMS indicated
two products of the same mass (isomers from 70:30 ratio of the starting
material). The reaction mixture was concentrated and purified by
preparative HPLC to afford 16 as a white solid (29 mg, 0.121 mmol,
25% yield). HPLC purity: 96%. LC−MS APCI: calcd for C13H10N4O,
238.25; observed m/z [M + H]+, 239.1. 1H NMR (300 MHz, DMSO-
d6): δ 8.78 (d, 1H, J = 3 Hz), 8.24−8.20 (m, 2H), 7.89 (d, 1H, J = 1.2
Hz), 7.56−7.53 (m, 3H), 7.07 (s, 1H), 6.65 (dd, 1H, J = 3 and 1.8 Hz).
2-(3,5-Dimethyl-1H-1,2,4-triazol-1-yl)-6-phenylpyrimidin-4(3H)-
one (17). Synthesized from intermediate 66 and 3,5-dimethyl-1H-
1,2,4-triazole following a protocol similar to compound 16. Yield 28%
(white solid). HPLC purity: 99%. LC−MS APCI: calcd for
1
C14H13N5O, 267.29; observed m/z [M + H]+, 268.1. H NMR (300
MHz, DMSO-d6): δ 7.95−7.93 (m, 2H), 7.63−7.57 (m, 3H), 7.22 (s,
1H), 2.80 (s, 3H), 2.32 (s, 3H).
2-(1-Methyl-1H-imidazole-2-yl)-6-phenylpyrimidin-4(1H)-one
(18). 1-methyl-1H-imidazole-2-carboximidamide (0.075 g, 0.61 mmol)
and ethyl benzoylacetate (0.12 g, 0.61 mmol) were refluxed in EtOH for
2 h. The reaction was cooled, solvent was evaporated under vacuo, and
the residue was purified on preparative HPLC to give 2-(1-methyl-1H-
imidazol-2-yl)-6-phenylpyrimidin-4(1H)-one (18, 15 mg, Yield 10%)
as a brown solid. HPLC purity: 94.2%. LC−MS APCI: calcd for
C14H12N4O, 252.101; observed m/z [M + H]+, 253.4, 1H NMR (400
MHz, DMSO-d6): δ 7.99 (t, J = 3.6 Hz, 2H), 7.52−7.49 (m, 3H), 7.21
(s, 1H), 7.14 (s, 1H), 6.83 (s, 1H), 4.29 (s, 3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-fluorophenyl)pyrimidin-
4(3H)-one (19). Synthesized from intermediate 56e following a
protocol similar to compound 1. Yield 33% (white solid). HPLC
purity: 93%. LC−MS APCI: calcd for C15H13FN4O, 284.29; observed
m/z [M + H]+, 285.1. 1H NMR (300 MHz, DMSO-d6): δ 8.18−8.14
(m, 2H), 7.58−7.32 (m, 2H), 6.89 (s, 1H), 6.26 (s, 1H), 2.71 (s, 3H),
2.24 (s, 3H), 2.09 (br s, 1H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(pyridin-4-yl)pyrimidin-
4(3H)-one (20). Synthesized from intermediate 56f following a
protocol similar to compound 1. Yield 45% (white solid). HPLC
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purity: 96%. LC−MS APCI: calcd for C14H13N5O, 267.29; observed
m/z [M + H]+, 268.1. 1H NMR (300 MHz, DMSO-d6): δ 8.76 (d, 1H, J
= 1.5 Hz), 8.74 (d, 1H, J = 1.5 Hz), 8.04 (d, 1H, J = 1.8 Hz), 8.02 (d, 1H,
J = 1.8 Hz), 7.07 (s, 1H), 6.28 (s, 1H), 2.72 (s, 3H), 2.25 (s, 3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(pyridin-3-yl)pyrimidin-
4(3H)-one (21). Synthesized from intermediate 56g following a
protocol similar to compound 1. Yield 56% (white solid). HPLC
purity: 99%. LC−MS APCI: calcd for C14H13N5O, 267.29; observed
m/z [M + H]+, 268.1. 1H NMR (300 MHz, DMSO-d6): δ 9.27 (dd, 1H,
J = 3 and 1.5 Hz), 8.72 (dd, 1H, J = 6 and 3 Hz), 8.45 (dt, 1H, J = 9 and 3
Hz), 7.56 (ddd, 1H, J = 9, 6 and 1.5 Hz), 7.02 (s, 1H), 6.27 (s, 1H), 2.71
(s, 3H), 2.24 (s, 3H).
N-(4-(2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-oxo-1,6-dihydropyrimi-
din-4-yl)phenyl) Methanesulfonamide (22). In 1,4-dioxane (2 mL)
solution of N-(4-(2-chloro-6-oxo-1,6-dihydropyrimidin-4-yl)phenyl)-
methanesulfonamide (69a 0.09 g, 0.300 mmol), under a nitrogen
atmosphere, cesium carbonate (0.147 g, 0.450 mmol), 3,5-Dimethyl-
pyrazole (0.035 g, 0.360 mmol), XantPhos (0.052 g, 0.090 mmol), and
Tris(dibenzylideneacetone)dipalladium(0) (0.041 g, 0.045 mmol)
were added, and the mixture was heated at 130 °C for 18 h in a
sealed tube. The reaction mixture was cooled to room temperature,
concentrated in vacuo, filtered, and washed with DCM/MeOH,
(80:20) and the filtrate concentrated to yield the crude product. The
crude product was dissolved in 1 mL of water and acidified with 1 M
HCl, then concentrated and purified by preparative HPLC to afford 22
as an off-white solid (3 mg, 8.01 μmol, 3% yield). HPLC purity: 96%.
LC−MS APCI: calcd for C16H17N5O3S, 359.40; observed m/z [M +
H]+, 360.1. 1H NMR (300 MHz, DMSO-d6): δ 8.00 (d, J = 8.4 Hz, 2H),
7.27 (d, J = 8.3 Hz, 2H), 6.57 (s, 1H), 6.14 (s, 1H), 3.03 (s, 3H), 2.65 (s,
3H), 2.21 (s, 3H).
(m, 2H), 7.24 (t, J = 9 Hz, 2H), 6.94 (s, 1H), 6.28 (s, 1H), 2.72 (s, 3H),
(s, 3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-4-(4-fluorophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile (27). A mixture of 4-(4-fluorophen-
yl)-2-hydrazinyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 74 (50
mg, 0.204 mmol) and pentane-2,4-dione (0.025 mL, 0.245 mmol)
was heated in a mixture of ethanol (0.5 mL) and acetic acid (1.5 mL) at
85 °C for 3 h. The reaction was cooled, and a yellow crystalline
precipitate formed was filtered under vacuum, triturated with a small
amount of ethanol, and dried to afford 27 as a light-yellow solid (37 mg,
0.120 mmol, 59% yield). HPLC purity: 95.8%. LC−MS ESI: calcd for
1
C16H12FN5O, 309.103; observed m/z [M − H]+, 308.10. H NMR
(300 MHz, DMSO-d6): δ 8.05−8.10 (m, 2H), 7.46 (t, J = 9 Hz, 2H),
6.35 (s, 1H), 2.65 (s, 3H), 2.27 (s, 3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-4-(4-fluorophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carboxamide (28). 2-(3,5-Dimethyl-1H-pyra-
zol-1-yl)-4-(4-fluorophenyl)-6-oxo-1,6-dihydropyrimidine-5-carboni-
trile 27 (50 mg, 0.162 mmol) was heated with H2SO4 (0.5 mL, 9.38
mmol) for 2 h at 80 °C. The reaction mixture was then poured slowly on
ice with vigorous stirring. A white precipitate was formed which was
filtered, washed with cold water and cold ethanol, and dried to give 28
as a light-yellow solid (32 mg, 0.098 mmol, 60% yield). HPLC purity:
>98% LC−MS ESI: calcd for C16H14FN5O2, 327.113; observed m/z
[M − H]+, 326.10. 1H NMR (300 MHz, DMSO-d6): δ 7.87−7.92 (m,
2H), 7.83 (br s, 1H), 7.50 (br s, 1H), 7.32 (t, J = 9 Hz, 2H), 6.25 (s,
1H), 2.62 (s, 3H), 2.24 (s, 3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-4-(4-fluorophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carboxylic Acid (29). In a 7 mL reaction vial, 2-
(3,5-dimethyl-1H-pyrazol-1-yl)-4-(4-fluorophenyl)-6-oxo-1,6-dihy-
dropyrimidine-5-carboxamide 28 (20 mg, 0.061 mmol) and tert-butyl
nitrite (0.022 mL, 0.183 mmol) were stirred in acetic acid (0.5 mL) a 75
°C for 3 h. A white precipitate was formed. It was centrifuged out,
washed with cold ethanol, and dried in an oven to give 29 as a white
solid (12 mg, 0.037 mmol, 60% yield). HPLC purity: >99% LC−MS
ESI: calcd for C16H13FN4O3, 328.097; observed m/z [M − H]+, 327.10.
1H NMR (300 MHz, DMSO-d6): δ 7.77−7.81 (m, 2H), 7.35 (t, J = 9
4-(2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-oxo-3,6-dihydropyrimi-
din-4-yl)-N-methylbenzamide (23). Synthesis protocol was the same
as for compound 22, starting from intermediate 4-(2-chloro-6-oxo-1,6-
dihydropyrimidin-4-yl)-N-methylbenzamide (69b). Yield: 38% (white
solid). HPLC purity: 97%; LC−MS APCI: calcd for C17H17N5O2,
323.128; observed m/z [M + H]+, 324.1. 1H NMR (400 MHz, DMSO-
d6): δ 8.27 (d, J = 8.5 Hz, 2H), 7.96 (d, J = 8.5 Hz, 2H), 6.67 (s, 1H),
6.18 (s, 1H), 2.80 (s, 3H), 2.73 (s, 3H), 2.20 (s, 3H).
Hz, 2H), 6.27 (s, 1H), 2.62 (s, 3H), 2.24 (s, 3H).
2-(5-Ethyl-3-methyl-1H-pyrazol-1-yl)-6-(trifluoromethyl)-
pyrimidin-4(3H)-one (30). Synthesized from 56d and hexane-2,4-
dione following a protocol similar to compound 1. Yield: 35% (white
solid). HPLC purity: 99%. LC−MS APCI: calcd for C11H11F3N4O,
272.088; observed m/z [M + H]+, 272.8. 1H NMR (400 MHz, DMSO-
d6): δ 6.71 (s, 1H), 6.27 (s, 1H), 3.04 (q, J = 1.60 Hz, 2H), 2.58 (s, 1H),
2.24 (s, 2H), 1.22 (t, J = 7.20 Hz, 3H); 13C NMR (100 MHz, DMSO-
d6): δ 13.22, 13.38, 13.89, 21.53, 108.02, 110.15, 110.32, 149.74,
151.87.
2-(5-Cyclopropyl-3-methyl-1H-pyrazol-1-yl)-6-(trifluoromethyl)-
pyrimidin-4(3H)-one (31). Synthesized from 56d and 1-cyclo-
propylbutane-1,3-dione following a protocol similar to compound 1.
Yield: 30% (white solid). HPLC purity: 98.4%. LC−MS APCI: calcd
for C12H11F3N4O, 284.088; observed m/z [M + H]+, 285.4. 1H NMR
(400 MHz, DMSO-d6): δ 6.75 (s, 1H), 6.09 (s, 1H), 2.20 (s, 3H),
0.94−0.99 (m, 2H), 0.70−0.72 (m, 2H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-(methylsulfonyl)phenyl)-
pyrimidin-4(1H)-one (24). Synthesis protocol was the same as for
compound 22, starting from intermediate 2-chloro-6-(4-
(methylsulfonyl)phenyl)pyrimidin-4(3H)-one (69c). Yield: 32%
(white solid). HPLC purity: 98%; LC−MS APCI: calcd for
C16H16N4O3S, 344.39; observed m/z [M + H]+, 345.1. 1H NMR
(300 MHz, DMSO-d6): δ 8.52−8.42 (m, 2H), 8.09 (d, J = 8.3 Hz, 2H),
6.77 (s, 1H), 6.20 (s, 1H), 3.29 (s, 3H), 2.75 (s, 3H), 2.22 (s, 3H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-morpholinophenyl)-
pyrimidin-4(1H)-one (25). Synthesis protocol was the same as for
compound 22, starting from intermediate 2-chloro-6-(4-
morpholinophenyl)pyrimidin-4(3H)-one (69d). Yield: 3% (white
solid). HPLC purity: 95%; LC−MS APCI: calcd for C19H21N5O2,
351.41; observed m/z [M + H]+, 352.1.1H NMR (300 MHz, Methanol-
d4): δ 8.07 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 6.63 (s, 1H),
6.14 (s, 1H), 3.86 (s, 6H), 2.76 (s, 3H), 2.27 (s, 3H), 1.91 (s, 2H).
2-(3,5-Dimethyl-1H-pyrazol-1-yl)-N-(4-fluorophenyl)-6-oxo-1,6-
dihydropyrimidine-4-carboxamide (26). 2-(3,5-dimethyl-1H-pyra-
zol-1-yl)-N-(4-fluorophenyl)-6-methoxypyrimidine-4-carboxamide 72
(40 mg, 0.117 mmol) was dissolved in dichloromethane (2 mL) under
N2 and BBr3, 1 M in dichloromethane (0.352 mL, 0.352 mmol) was
added and stirred under N2 at room temperature for 48 h. The reaction
was quenched with dilute HCl and diluted with dichloromethane. The
dichloromethane layer was separated, and the aqueous layer was again
extracted with dichloromethane. Organic layers were combined,
washed with brine, and concentrated under vacuum. The residue was
purified by column chromatography using 0−20% methanol in
dichloromethane to give 26 as an off-white solid (12 mg, 0.035
mmol, 30% yield). HPLC purity: 96.5% LC−MS ESI: calcd for
2-(3-Methyl-5-phenyl-1H-pyrazol-1-yl)-6-(trifluoromethyl)-
pyrimidin-4(3H)-one (32). Synthesized from 56d and 1-phenylbutane-
1,3-dione following a protocol similar to compound 1. Yield: 39%
(white solid). HPLC purity: 98.5%. LC−MS APCI: calcd for
1
C15H11F3N4O, 320.088; observed m/z [M + H]+, 320.8. H NMR
(400 MHz, CDCl3): δ 13.59 (s, 1H), 7.34−7.42 (m, 5H), 6.84 (s, 1H),
6.59 (s, 1H), 2.51 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 13.82,
108.53, 111.52, 119.39, 121.94, 128.40, 128.81, 129.18, 130.59, 130.59,
146.20, 151.83, 153.0.
2-(5-Amino-3-methyl-1H-pyrazol-1-yl)-6-(trifluoromethyl)-
pyrimidin-4(3H)-one (33). To a solution of 56d (0.70 g, 1.28 mmol) in
DMF was added 3-aminocrotonitrile (0.70 g, 8.43 mmol) and heated to
100 °C for 16 h. Water was added, extracted with ethyl acetate, and the
combined organic layer was washed with saturated sodium bicarbonate
solution, water, and brine solution, dried over sodium sulfate, and
concentrated under vacuum. The crude product was purified by
preparative HPLC to yield 33 as a white solid (0.076 g, 23%). HPLC
1
C16H14FN5O2, 327.113; observed m/z [M + H]+, 328.10. H NMR
(300 MHz, DMSO-d6): δ 12.80 (br s, 1H), 10.15 (br s, 1H), 7.81−7.86
734
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purity: 95.5%. LC−MS APCI: calcd for C9H8F3N5O, 259.068;
(400 MHz, DMSO-d6): δ 7.71 (s, 1H), 6.45 (s, 1H), 5.37 (s, 1H), 3.52
(t, J = 5.20 Hz, 2H), 3.27−3.33 (m, 5H), 2.13 (s, 3H). 13C NMR (100
MHz, DMSO-d6): δ 14.44, 44.66, 58.52, 70.51, 87.07, 106.26, 120.03,
122.75, 151.28, 152.24, 154.78, 166.47.
1
observed m/z [M + H]+, 259.8. H NMR (400 MHz, DMSO-d6): δ
7.15 (s, 2H), 6.30 (s, 1H), 5.23 (s, 1H), 2.08 (s, 3H). 13C NMR (100
MHz, DMSO-d6): δ 14.34, 88.68, 105.72, 120.24, 122.97, 151.14,
151.32, 151.55, 151.88, 156.03, 168.05.
2-(3-Methyl-5-((tetrahydro-2H-pyran-4-yl)amino)-1H-pyrazol-1-
yl)-6-(trifluoromethyl)pyrimidin-4(3H)-one (39). A protocol similar to
compound 36. Yield: 20% (white solid). HPLC purity: 97%. LC−MS
APCI: calcd for C14H16F3N5O2, 343.126; observed m/z [M + H]+,
344.1. 1H NMR (400 MHz, CDCl3): δ 10.35 (s, 1H), 7.54 (s, 1H), 6.53
(s, 1H), 5.23 (s, 1H), 3.95−4.00 (m, 2H), 3.55−3.61 (m, 2H), 0.42−
3.44 (m, 1H), 2.22 (s, 3H), 2.06−2.09 (m, 2H), 1.60−1.68 (m, 2H).
2-(5-((4-Fluorophenyl)amino)-3-methyl-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one (40). In a sealed tube 33 (0.25
g, 0.96 mmol), 1-bromo-4-fluoro benzene (0.33 g, 1.92 mmol) and
cesium carbonate (0.93 g, 2.88 mmol) were taken in dioxane (10 mL).
The mixture was purged with nitrogen gas for 20 min. To this
tris(dibenzylideneacetone)dipalladium(0) (0.079 g, 0.08 mmol) and
XantPhos (0.07 g, 0.13 mmol) was added and sealed. The reaction
mixture was stirred at 120 °C for 4 h. The reaction mixture filtered
through Celite and filtrate was concentrated under vacuum. The crude
product was purified by preparative HPLC to obtain 40 as a white solid
(0.09 g, 0.51 mmol, 26% yield). HPLC purity: 95.4%; LC−MS APCI:
calcd for C15H11F4N5O, 353.090; observed m/z [M + H]+, 354.0. 1H
NMR (400 MHz, CD3OD): δ 7.22−7.34 (m, 2H), 6.97−7.09 (m, 2H),
6.40 (s, 1H), 5.87 (s, 1H), 2.24 (s, 3H). 13C NMR (400 MHz,
CD3OD): δ 14.00, 90.34, 105.40, 118.74, 121.42, 122.71, 124.14,
130.36, 138.72, 148.73, 152.72, 158.26, 160.06, 160.64, 176.68.
2-(5-((2,4-Difluorophenyl)amino)-3-methyl-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one (41). A protocol similar to
compound 36. Yield: 8% (white solid). HPLC purity: 97.5%; LC−MS
APCI: calcd for C15H10F5N5O, 371.081; observed m/z [M + H]+,
2-(5-(Ethylamino)-3-methyl-1H-pyrazol-1-yl)-6-(trifluoromethyl)-
pyrimidin-4(3H)-one (34). To a solution of 33 (0.20 g, 0.77 mmol),
acetic acid (0.14 g, 2.30 mmol) in DMF (5 mL) and acetaldehyde (0.10
g, 2.31 mmol) were added and the reaction was allowed to stir at room
temperature for 16 h. Sodium cyanoborohydride (0.24 g, 3.86 mmol) in
methanol (2 mL) was added and stirred at room temperature overnight.
The reaction mixture was concentrated, water was added, and extracted
with ethyl acetate. The organic layer was concentrated and purified by
preparative HPLC to give 34 as a white solid (0.07 g, 32%). HPLC
purity: 95.2%; LC−MS APCI: calcd for C11H12F3N5O, 287.099;
observed m/z [M + H]+, 288.0. 1H NMR (400 MHz, CDCl3): δ 7.14 (s,
1H), 6.48 (s, 1H), 5.19 (s, 1H), 3.23 (q, J = 6.40 Hz, 2H), 2.19 (s, 3H),
1.32 (t, J = 7.20 Hz, 3H); 13C NMR (100 MHz, CD3OD): δ 12.56,
13.40, 39.09, 104.82, 119.62, 122.34, 152.30, 152.51, 152.85, 153.14,
155.01.
2-(5-Ethoxy-3-methyl-1H-pyrazol-1-yl)-6-(trifluoromethyl)-
pyrimidin-4(3H)-one (35). To solution of sulphonyl intermediate 79
(0.50 g, 2.06 mmol) and cesium carbonate (1.6 g, 5.16 mmol) in 1,4-
dioxane was added 78 (0.52 g, 4.12 mmol) in sealed tube and heated to
100 °C for 16 h. Solids were filtered through Celite and the filtrate
concentrated under vacuum. The crude product was purified by
preparative HPLC to afford 35 as a white solid (0.07 g, 12%). HPLC
purity: 95%; LC−MS APCI: calcd for C11H11F3N4O2, 288.083;
1
observed m/z [M + H]+, 288.8. H NMR (400 MHz, CDCl3): δ
10.43 (s, 1H), 6.53 (s, 1H), 5.80 (s, 1H), 4.28 (q, J = 7.20 Hz, 2H), 2.67
(s, 3H), 1.43 (t, J = 7.20 Hz, 3H).
1
372.4. H NMR (400 MHz, CDCl3): δ 10.46 (s, 1H), 9.81 (s, 1H),
7.31−7.34 (m, 1H), 6.92−6.99 (m, 2H), 6.58 (s, 1H), 5.81 (s, 1H),
2.27 (s, 3H).
4-((3-Methyl-1-(6-oxo-4-(trifluoromethyl)-1,6-dihydropyrimidin-
2-yl)-1H-pyrazol-5-yl)amino)benzonitrile (42). Protocol similar to
compound 36. Yield 29% (white solid). HPLC purity: 99.9%; LC−MS
APCI: calcd for C16H11F3N6O, 360.30; observed m/z [M + H]+, 361.0.
1H NMR (400 MHz, DMSO-d6): δ 12.43 (s, 1H), 7.73 (d, J = 8.80 Hz,
2-(5-((2-(Dimethylamino)ethyl)amino)-3-methyl-1H-pyrazol-1-
yl)-6-(trifluoromethyl)pyrimidin-4(3H)-one (36). To a solution of 76a
(0.10 g, 0.49 mmol) in ethanol (2.5 mL) and acetic acid (7.5 mL) was
added 2-hydrazinyl-6-(trifluoromethyl)pyrimidin-4(3H)-one (56d,
0.09 g, 0.49 mmol) and heated to reflux for 5 h. The solvent was
evaporated under vacuum. The crude product was purified with
preparative HPLC to yield 36 as a white solid (0.06 g, 0.181 mmol, 37%
yield). HPLC purity: 99.2%. LC−MS APCI: calcd for C13H17F3N6O,
330.142; observed m/z [M + H]+, 331.0. 1H NMR (300 MHz, DMSO-
d6): δ 7.65 (s, 1H), 6.48 (s, 1H), 5.17 (s, 1H), 3.21 (q, J = 6.00 Hz, 2H),
2.60 (t, J = 6.00 Hz, 2H), 2.31 (s, 6H), 2.19 (s, 3H).
2H), 7.17 (d, J = 8.80 Hz, 2H), 6.17 (s, 1H), 6.06 (s, 1H), 2.21 (s, 3H).
13C NMR (100 MHz, DMSO-d6): δ 14.46, 92.36, 101.50, 105.83,
116.12, 120.01, 123.59, 134.42, 143.47, 145.22, 149.10, 159.53, 171.58.
N,N-Dimethyl-4-((3-Methyl-1-(6-oxo-4-(trifluoromethyl)-1,6-di-
hydropyrimidin-2-yl)-1H-pyrazol-5-yl)amino)benzamide (43). Pro-
tocol similar to compound 36. Yield 30% (white solid). HPLC purity:
97.9%. LC−MS APCI: calcd for C18H17F3N6O2, 406.137; observed m/
z [M + H]+, 407.0. 1H NMR (400 MHz, DMSO-d6): δ 11.89 (s, 1H),
7.40 (d, J = 8.00 Hz, 2H), 7.09 (d, J = 8.00 Hz, 2H), 6.07 (s, 1H), 6.06
(s, 1H), 2.97 (s, 6H), 2.19 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ
14.47, 91.18, 115.60, 128.27, 129.59, 142.49, 144.92, 149.29, 170.44,
171.90.
2-(3-Methyl-5-(pyridin-4-ylamino)-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one (44). Protocol similar to
compound 36. Yield 12% (white solid). HPLC purity 99.8%; LC−
MS APCI: calcd for C14H11F3N6O, 336.28; observed m/z [M + H]+,
337.0. 1H NMR (400 MHz, DMSO-d6): δ 12.29 (s, 1H), 8.33 (d, J =
6.40 Hz, 2H), 6.98 (d, J = 6.40 Hz, 2H), 6.18 (s, 1H), 6.05 (s, 1H), 5.32
(s, 1H), 2.21 (s, 3H).
2-(3-Methyl-5-(pyridin-2-ylamino)-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one (45). Protocol similar to
compound 36. Yield 38% (white solid). HPLC purity: 98.2%. LC−
MS APCI: calcd for C14H11F3N6O, 336.095; observed m/z [M + H]+,
336.8. 1H NMR (400 MHz, CDCl3): δ 10.55 (s, 1H), 8.36 (d, J = 3.20
Hz, 1H), 7.64 (t, J = 7.20 Hz, 1H), 6.91−6.94 (m, 2H), 6.75 (d, J = 7.48
Hz, 1H), 6.59 (s, 1H), 2.32 (s, 3H); 13C NMR (100 MHz, CDCl3): δ
14.3, 96.13, 108.63, 111.78, 116.90, 137.89, 148.0, 151.95.
2-(3-Methyl-5-((5-morpholinopyridin-2-yl)amino)-1H-pyrazol-1-
yl)-6-(trifluoromethyl)pyrimidin-4(3H)-one (46). A protocol similar to
compound 36. Yield 15% (white solid). HPLC purity: 99.6%. LC−MS
APCI: calcd for C18H18F3N7O2, 421.147; observed m/z [M + H]+,
2-(5-((2-Hydroxyethyl)amino)-3-methyl-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one (37). A protocol similar to
compound 36. Yield: 16% (white solid). HPLC purity: 97.28%; LC−
MS APCI: calcd for C11H12F3N5O2, 303,094; observed m/z [M + H]+,
304.0. 1H NMR (400 MHz, CDCl3): δ 7.45 (s, 1H), 6.68 (s, 1H), 5.44
(s, 1H), 3.58 (t, J = 5.20 Hz, 2H), 3.21 (t, J = 5.20 Hz, 2H), 2.16 (s, 3H).
2-(5-((2-Methoxyethyl)amino)-3-methyl-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one (38). Step 1: To a −5 °C cooled
solution of 4-methyleneoxetan-2-one (1.0 g, 11.90 mmol) in
tetrahydrofuran (10 mL) was added 2-methoxyethylamine (0.90 g,
11.30 mmol) and stirred for 30 min. The solvent was evaporated under
vacuum. The crude product was purified by column chromatography
using 3−5% methanol in dichloromethane to afford N-(2-methox-
yethyl)-3-oxobutanamide (1.2 g, 7.50 mmol, 63%). LC−MS APCI:
calcd for C7H13NO3, 159.19; observed m/z [M + H]+, 160.2. 1H NMR
(300 MHz, DMSO-d6): δ 8.13 (s, 1H), 3.29−3.36 (m, 4H), 3.20−3.27
(m, 5H), 2.13 (s, 3H). Step 2: To a solution of N-(2-methoxyethyl)-3-
oxobutanamide (0.50 g, 3.14 mmol) in tetrahydrofuran (10 mL) was
added Lawesson’s reagent (1.40 g, 3.45 mmol) and stirred for 30 min at
room temperature under a nitrogen atmosphere. Hydrazine 33 (0.61 g,
3.45 mmol) was added to the reaction mixture and stirred at room
temperature for 24 h. The reaction was quenched with saturated
sodium bicarbonate solution and extracted with ethyl acetate. The
combined organic phase was washed with water and brine, dried over
sodium sulfate, and concentrated under vacuum. The crude product
was purified by preparative HPLC to yield 38 as a white solid (0.065 g,
0.22 mmol. 7% yield). HPLC purity: 98.9%. LC−MS APCI: calcd for
1
C12H14F3N5O2, 317.110; observed m/z [M + H]+, 318.0. H NMR
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422.4. 1H NMR (400 MHz, CDCl3): δ 10.37 (s, 1H), 8.03 (s, 1H), 7.32
(s, 1H), 6.79 (s, 1H), 6.73 (d, J = 8.00 Hz, 1H), 6.57 (s, 1H), 3.91 (t, J =
4.80 Hz, 4H), 3.13 (t, J = 4.40 Hz, 4H), 2.30 (s, 3H).
NMR (400 MHz, DMSO-d6): δ 13.10 (s, 1H), 6.79 (s, 1H), 3.69 (d, J =
2.80 Hz, 4H), 2.96 (d, J = 3.60 Hz, 4H), 2.51 (s, 3H), 2.30 (s, 3H).
2-(5-Amino-3-cyclopropyl-1H-pyrazol-1-yl)-6-(trifluoromethyl)-
pyrimidin-4(3H)-one (51). To a solution of 2-hydrazinyl-6-
(trifluoromethyl)pyrimidin-4(3H)-one (56d, 0.25, 1.28 mmol) in
DMF (5 mL) was added 3-cyclopropyl-3-oxopropanenitrile (0.15 g,
1.41 mmol) and heated at 130 °C in a microwave for 1 h. The reaction
mixture was poured into water and extracted with ethyl acetate. The
organic layer washed with plenty of water and brine solution, dried over
sodium sulfate, and concentrated in vacuo. The crude mass was purified
by prep HPLC to give 2-(5-amino-3-cyclopropyl-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one 51 as a white solid (51 mg,
0.179 mmol, 14% yield). HPLC purity: 96%; LC−MS APCI: calcd for
C11H10F3N5O, 285.084; observed m/z [M + H]+, 286.0. 1H NMR (400
MHz, CDCl3): δ 6.54 (s, 1H), 5.79 (br s, 2H), 5.25 (s, 1H), 1.82 (t, J =
4.40 Hz, 1H), 1.00 (d, J = 8.00 Hz, 2H), 0.82 (d, J = 2.00 Hz, 2H).
2-(3-((Dimethylamino)methyl)-5-methyl-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one (52). Step 1: ethyl 2,4-dioxo-
pentanoateTo a solution of sodium metal (0.86 g, 37.67 mmol)
dissolved in ethanol (15 mL) was added a mixture of diethyl oxalate
(5.0 g, 34.24 mmol) and acetone (2.0 g, 34.24 mmol) and heated at 60
°C for 16 h. The reaction mass was concentrated under vacuum, diluted
with water, and the product was extracted with ethyl acetate. The
organic phase was washed with water and brine, dried over sodium
sulfate, and concentrated. The crude product was then purified by
column chromatography using silica gel (230−400 mesh) with 10−
20% ethyl acetate in petroleum ether as an eluent to yield ethyl 2,4-
dioxopentanoate as yellow oil (2.1 g, 13.93 mmol, 37%). 1H NMR (300
MHz, CDCl3): δ 14.40 (s, 1H), 6.39 (s, 1H), 4.37 (q, J = 7.20 Hz, 2H),
2.28 (s, 3H), 1.40 (t, J = 7.20 Hz, 3H). Step 2: Ethyl 5-methyl-1H-
pyrazole-3-carboxylate (83)To a solution of ethyl 2,4-dioxopenta-
noate (2.0 g, 12.65 mmol) in ethanol (10 mL) and acetic acid (20 mL)
was added hydrazine hydrate (0.75 g, 15.18 mmol). The reaction
mixture was heated at the reflux for 16 h. Solvents were evaporated
under vacuum. Water was added, and the product was extracted with
ethyl acetate. The organic layer washed with saturated sodium
bicarbonate solution, water, and brine, dried over sodium sulfate, and
concentrated under vacuum to afford 83 an off-white solid (1.3 g, 8.6
mmol, 68%). LC−MS APCI: calcd for C7H10N2O2, 154.17; observed
2-(5-(Ethylamino)-3-methyl-1H-pyrazol-1-yl)-6-(pyridin-3-yl)-
pyrimidin-4(3H)-one (47). To a solution of 2-(5-amino-3-methyl-1H-
pyrazol-1-yl)-6-(pyridin-3-yl)pyrimidin-4(3H)-one (0.10 g, 0.37
mmol), acetic acid (0.07 g, 1.11 mmol) in DMF (5 mL) and
acetaldehyde (0.05 g, 1.11 mmol) were added, and reaction mixture
stirred at room temperature for 16 h. Sodium cyanoborohydride (0.11
g, 1.86 mmol) in methanol (2 mL) was added, and the reaction mixture
stirred at room temperature overnight. The reaction mixture was
concentrated, water was added, and extracted with ethyl acetate. The
organic layer was concentrated and purified by preparative HPLC to
give 47 as a white solid (0.03 g, 0.299 mmol, 27%). HPLC purity
97.53%; LC−MS APCI: calcd for C15H16N6O, 296.139; observed m/z
[M + H]+, 297.2. 1H NMR (400 MHz, CD3OD): δ 9.42 (s, 1H), 8.77−
8.82 (m, 2H), 7.82 (q, J = 5.20 Hz, 1H), 7.15 (s, 1H), 3.42 (q, J = 7.20
Hz, 2H), 2.40 (s, 3H), 1.39 (t, J = 7.20 Hz, 3H).
2-(4-Bromo-3,5-dimethyl-1H-pyrazol-1-yl)-6-(trifluoromethyl)-
pyrimidin-4(3H)-one (48). To a solution of 12 (1.0 g, 3.87 mmol) in
acetic acid (20 mL) was added bromine (0.24 mL, 4.65 mmol) at 20 °C
and stirred at room temperature for 3 days. Water was added to the
reaction mixture. Solids formed were filtered, washed with water, and
dried. The crude product was purified by preparative HPLC to yield 48
as a white solid (0.3 g, 1.07 mmol, 23.0%). HPLC purity: 99.3%; LC−
MS APCI: calcd for C10H8BrF3N4O, 335.983; observed m/z [M − H]+,
337.0; 1H NMR (400 MHz, DMSO-d6): δ 13.58 (s, 1H), 6.93 (s, 1H),
2.58 (s, 3H), 2.24 (s, 3H).
2-(3,5-Dimethyl-4-(pyridin-3-yl)-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one (49). A solution of 48 (0.50 g,
1.48 mmol), pyridine-3-boronic acid (0.22 g, 1.78 mmol) and
potassium carbonate (0.41 g, 2.96 mmol) in tetrahydrofuran/water
(20/2 mL) was repeatedly purged with nitrogen. Pd(dppf)Cl2 (0.18 g,
0.22 mmol) was added and heated at 70 °C for 16 h. The reaction
mixture was cooled, water was added, and extracted with ethyl acetate.
Combined organic layers were washed with water and brine solution,
dried over sodium sulfate, and concentrated under vacuum. The crude
product was purified by preparative HPLC to yield 49 as a white solid
(0.10 g, 0.36 mmol, 20% yield). HPLC purity: 99.8%; LC−MS APCI:
calcd for C15H12F3N5O, 335.099; observed m/z [M − H]+, 336.1. 1H
NMR (400 MHz, CDCl3): δ 400 MHz, CDCl3: δ 8.68 (d, J = 4.88 Hz,
1H), 8.58 (s, 1H), 7.66 (d, J = 7.60 Hz, 1H), 7.48 (t, J = 7.28 Hz, 1H),
6.64 (s, 1H), 2.68 (s, 3H), 2.29 (s, 3H).
1
m/z [M + H]+, 154.8. H NMR (400 MHz, DMSO-d6): δ 13.15 (s,
1H), 6.46 (s, 1H), 4.25 (q, J = 7.20 Hz, 2H), 2.26 (s, 3H), 1.27 (t, J =
6.80 Hz, 3H). Step 3: 5-methyl-1H-pyrazole-3-carbaldehydeTo a
solution of 83 (4.0 g, 25.97 mmol) in dichloromethane (50 mL) at −78
°C was added di-isobutylaluminiumhydride (1 M in dichloromethane,
52.0 mL, 51.94 mmol) dropwise under a nitrogen atmosphere. The
reaction mixture was stirred at −78 °C for 2 h. It was then quenched
with methanol at −78 °C, warmed to room temperature, and washed
with water. The organic phase was dried over sodium sulfate and
concentrated under vacuum to yield 5-methyl-1H-pyrazole-3-carbalde-
hyde. This material was taken as such for the next step without
purification. Step 4: N,N-Dimethyl-1-(5-methyl-1H-pyrazol-3-yl)-
methanamine (84)To a solution of 5-methyl-1H-pyrazole-3-
carbaldehyde (1.0 g, 9.09 mmol) in methanol was added N,N-dimethyl
amine (2a M in tetrahydrofuran, 23 mL, 45.45 mmol) and stirred for 24
h at room temperature. Sodium cyanoborohydride (1.7 g, 27.27 mmol)
was added to the reaction and stirred at room temperature for another 4
h. The reaction mixture was concentrated, the crude material diluted
with water, and the product was extracted with ethyl acetate. The
organic layer washed with water and brine solution, dried over sodium
sulfate, and concentrated under vacuum to afford 84 as a white solid
(0.60 g, 4.31 mmol, 48%). LC−MS APCI: calcd for C7H13N3, 139.20;
observed m/z [M + H]+, 139.8. Step 5: 2-(3-((Dimethylamino)methyl)-
5-methyl-1H-pyrazol-1-yl)-6-(trifluoromethyl) pyrimidin-4(3H)-one
(52)A solution of 84 (0.60 g, 4.31 mmol), sulfonyl intermediate
79 (0.48 g, 2.15 mmol) and cesium carbonate (2.0 g, 6.37 mmol) in 1,4-
dioxane was heated in sealed tube at 130 °C for 16 h. The reaction
mixture was filtered through Celite and the filtrate was concentrated
under vacuum. The crude product was then purified by preparative
HPLC to yield the free base as a gum. It was dissolved in
dichloromethane and stirred with hydrogen chloride in 1,4-dioxane
2-(3,5-Dimethyl-4-morpholino-1H-pyrazol-1-yl)-6-
(trifluoromethyl)pyrimidin-4(3H)-one (50). Step 1: 3-Bromopentane-
2,4-dione (80)To a solution of acetylacetone (1.0 g, 10.0 mmol) in
acetic acid (15 mL) was added bromine (1.9 g, 12.0 mmol) at 0 °C. The
reaction was heated at 60 °C for 3 h. Saturated sodium bicarbonate
solution was added, and the layer was separated. The combined organic
layer was washed with water, dried over sodium sulfate, and
concentrated under vacuum to yield 80 as a light-yellow solid (1.2 g,
67.0%). This was taken as such to the next step without further analysis.
Step 2: 3-Morpholinopentane-2,4-dione (81)To a solution of morpho-
line (0.58 g, 6.70 mmol) and triethylamine (1.1 g, 11.17 mmol) in
dichloromethane (10 mL) was added 80 (1.0 g, 5.58 mmol) dissolved
in dichloromethane (10 mL) at 0 °C. The reaction was stirred at room
temperature for 2 h. Water was added, and the layers separated. The
combined organic layers were washed with water, dried over sodium
sulfate, and concentrated under vacuum to yield 81 (0.80 g, 78%) as
viscous brown oil. LC−MS APCI: calcd for C9H15NO3, 185.22;
1
observed m/z [M + H]+, 186.2. H NMR (400 MHz, DMSO-d6): δ
4.31 (s, 1H), 3.57−3.62 (m, 4H), 2.84 (d, J = 4.40 Hz, 2H), 2.60 (d, J =
4.80 Hz, 2H), 2.18 (s, 6H). Step 3: 2-(3,5-Dimethyl-4-morpholino-1H-
pyrazol-1-yl)-6-trifluoromethylpyrimidin-4(3H)-one (50)A solution
of 81 (0.30 g, 1.62 mmol) and 56d (0.31 g, 1.62 mmol) in ethanol/
acetic acid mixture (15/5 mL) was refluxed for 16 h. The solvent was
evaporated under vacuum. Crude product was purified by preparative
HPLC to yield 50 (0.07 g, 13%). HPLC purity: 99.9%; LC−MS APCI:
calcd for C14H16F3N5O2, 343.126; observed m/z [M + H]+, 344.1.1H
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(in excess) for 2 h. Solvents were evaporated under vacuum, formed
solids were washed with diethyl ether, and dried under vacuum to afford
52 (0.06 g, 9.38%) as a white hydrochloride salt. HPLC purity: 99.8%.
LC−MS APCI: calcd for C12H14F3N5O, 301.115; observed m/z [M +
H]+, 302.2. 1H NMR (400 MHz, CD3OD): δ 6.83 (s, 1H), 6.59 (s, 1H),
4.43 (s, 2H), 2.98 (s, 6H), 2.75 (s, 3H).
1-(4-(4-Fluorophenyl)-6-oxo-1,6-dihydropyrimidin-2-yl)-5-meth-
yl-1H-pyrazole-3-carboxylic Acid (53). To solution of 6-(4-fluoro-
phenyl)-2-(methylsulfonyl)pyrimidin-4(3H)-one (0.3 g, 1.118 mmol)
85 (Synthesized according to protocol for compound 79) and cesium
carbonate (0.911 g, 2.80 mmol) in DMF was added 83 (0.172 g, 1.118
mmol) in a sealed tube and heated to 130 °C in a microwave for 2 h.
The reaction mixture was concentrated under vacuum and then was
purified by preparative HPLC to yield 53 as an off-white solid (60 mg,
0.179 mmol, 16% yield). HPLC purity: 94%. LC−MS APCI: calcd for
C15H11FN5O3, 314.27; observed m/z [M + H]+, 315.1. 1H NMR (300
MHz, Methanol-d4): δ 8.19−8.08 (m, 2H), 7.26 (t, J = 8.8 Hz, 2H),
6.77 (s, 1H), 6.66 (d, J = 1.1 Hz, 1H), 2.93−2.82 (m, 3H).
1-(4-(4-Fluorophenyl)-6-oxo-1,6-dihydropyrimidin-2-yl)-N,N,5-
trimethyl-1H-pyrazole-3-carboxamide (54). To a solution of HATU
(0.036 g, 0.095 mmol), 53 (0.02 g, 0.064 mmol) and dimethylamine
(0.017 mL, 0.191 mmol) in DMF was added triethylamine (0.018 mL,
0.127 mmol) and reaction mixture was stirred at 35 °C overnight. Upon
completion, the reaction mixture was concentrated in vacuo, washed
with 10% LiCl, and the resulting precipitate was filtered to yield 54 as a
white solid (5 mg, 0.014 mmol, 22% yield). HPLC purity: 96%. LC−
MS APCI: calcd for C17H16FN5O2, 341.34; observed m/z [M + H]+,
342.1.1H NMR (300 MHz, DMSO-d6): δ 8.21 (dd, J = 8.9, 5.6 Hz, 2H),
7.37 (t, J = 8.9 Hz, 2H), 7.08 (s, 1H), 6.64 (d, J = 1.0 Hz, 1H), 3.27 (s,
3H), 3.01 (s, 3H), 2.74 (d, J = 0.9 Hz, 3H).
Vinayak Singh − Drug Discovery and Development Centre
(H3D) and South African Medical Research Council Drug
Discovery and Development Research Unit, Department of
Chemistry and Institute of Infectious Disease and Molecular
Medicine, University of Cape Town, Rondebosch 7701, South
Africa; orcid.org/0000-0001-9002-2489
††Alissa Myrick − Drug Discovery and Development Centre
(H3D), University of Cape Town, Rondebosch 7701, South
Africa
Sandile B. Simelane − Drug Discovery and Development Centre
(H3D), Department of Chemistry, University of Cape Town,
Rondebosch 7701, South Africa
Dale Taylor − Drug Discovery and Development Centre (H3D),
Division of Clinical Pharmacology, Department of Medicine,
University of Cape Town, Observatory 7925, South Africa
Christel Brunschwig − Drug Discovery and Development
Centre (H3D), Division of Clinical Pharmacology,
Department of Medicine, University of Cape Town,
Observatory 7925, South Africa
Nina Lawrence − Drug Discovery and Development Centre
(H3D), Division of Clinical Pharmacology, Department of
Medicine, University of Cape Town, Observatory 7925, South
Africa
Dirk Schnappinger − Department of Microbiology and
Immunology, Weill Cornell Medical College, New York 10065,
United States
Curtis A. Engelhart − Department of Microbiology and
Immunology, Weill Cornell Medical College, New York 10065,
United States
Anuradha Kumar − Infectious Disease Research Institute,
Seattle, Washington 98102, United States
Tanya Parish − Infectious Disease Research Institute, Seattle,
Washington 98102, United States; orcid.org/0000-0001-
7507-0423
Qin Su − Genomic Technologies Section, Research Technologies
Branch, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20892,
United States
Timothy G. Myers − Genomic Technologies Section, Research
Technologies Branch, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda,
Maryland 20892, United States
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01727.
■
sı
Synthesis and analytical data of intermediates, micro-
biology assays, MIC data against clinical isolates,
screening data against MmpL3 under-expressing strain
of Mtb, in vitro DMPK assays, and mouse pharmacoki-
netic studies (PDF)
LCMS HPLC and NMRs of lead compounds (PDF)
Data from RNA microarray experiment (XLSX)
Data from RNA microarray experiment (XLSX)
Molecular formula strings and some data (CSV)
Helena I. M. Boshoff − Tuberculosis Research Section,
Laboratory of Clinical Infectious Diseases, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892, United States; orcid.org/
0000-0002-4333-206X
Clifton E. Barry, III − Tuberculosis Research Section,
Laboratory of Clinical Infectious Diseases, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892, United States
Frederick A. Sirgel − South African Medical Research Council
Centre for Tuberculosis Research/DST/NRF Centre of
Excellence for Biomedical Tuberculosis Research, Division of
Molecular Biology and Human Genetics, Faculty of Medicine
and Health Science, Stellenbosch University, Tygerberg 7505,
South Africa
Paul D. van Helden − South African Medical Research Council
Centre for Tuberculosis Research/DST/NRF Centre of
Excellence for Biomedical Tuberculosis Research, Division of
Molecular Biology and Human Genetics, Faculty of Medicine
and Health Science, Stellenbosch University, Tygerberg 7505,
South Africa
AUTHOR INFORMATION
Corresponding Authors
■
Kelly Chibale − Drug Discovery and Development Centre
(H3D), Department of Chemistry and South African Medical
Research Council Drug Discovery and Development Research
Unit, Department of Chemistry and Institute of Infectious
Disease and Molecular Medicine, University of Cape Town,
Rondebosch 7701, South Africa; orcid.org/0000-0002-
1327-4727; Phone: +27 21 650 2553;
Email: kelly.chibale@uct.ac.za
Sandeep R. Ghorpade − Drug Discovery and Development
Centre (H3D), Department of Chemistry, University of Cape
Town, Rondebosch 7701, South Africa; orcid.org/0000-
0002-9311-1572; Phone: +27 21 650 1250;
Email: Sandeep.ghorpade@uct.ac.za
Authors
Candice Soares de Melo − Drug Discovery and Development
Centre (H3D), Department of Chemistry, University of Cape
Town, Rondebosch 7701, South Africa
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Kirsteen I. Buchanan − Drug Discovery Unit, School of Life
Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Tracy Bayliss − Drug Discovery Unit, School of Life Sciences,
University of Dundee, Dundee DD1 5EH, U.K.
Town, SAMRC, and South African Research Chairs Initiative of
the Department of Science and Technology, administered
through the South African National Research Foundation are
gratefully acknowledged for support (K.C.).
Simon R. Green − Drug Discovery Unit, School of Life Sciences,
University of Dundee, Dundee DD1 5EH, U.K.; orcid.org/
0000-0001-5054-4792
Peter C. Ray − Drug Discovery Unit, School of Life Sciences,
University of Dundee, Dundee DD1 5EH, U.K.
Paul G. Wyatt − Drug Discovery Unit, School of Life Sciences,
University of Dundee, Dundee DD1 5EH, U.K.; orcid.org/
0000-0002-0397-245X
Gregory S. Basarab − Drug Discovery and Development Centre
(H3D), Department of Chemistry and Drug Discovery and
Development Centre (H3D), Division of Clinical
Pharmacology, Department of Medicine, University of Cape
Town, Rondebosch 7701, South Africa; orcid.org/0000-
0001-5684-6046
Notes
The authors declare no competing financial interest.
††AM deceased in November 2019.
ACKNOWLEDGMENTS
■
MMV, Meyrin, Switzerland for donating the compound library.
Dr. Sridevi Bashayam, Dr. Jayan Joseph, Dr. Jayanth
Thiruvellore, and Rajkumar Dhinakaran, Syngene, India for
profound chemistry support. Ronnett Seldon from TB biology,
H3D for performing MIC and biology triage assays. Dr. Nicole
Cardoso from TB biology, H3D for helping in Fe-chelation
experiment. Prof. Digby Warner, University of Cape Town for
useful discussions on biology triage assays, Technical staff at in
vitro DMPK and parasitology divisions of H3D for excellent
technical assistance in generating all in vitro DMPK and
cytotoxicity data, Marianna de Kock and Claudia Spies,
SAMRC and Stellenbosch University, South Africa for excellent
technical assistance in generating MICs against clinical TB
isolates. AbbVie Incorporation, North Chicago, IL, USA for in
vitro safety screening. Lina Castro, Shilah Bonnett and Megha
Gupta from Infectious Disease Research Institute for technical
assistance and support.
Charles J. Eyermann − Drug Discovery and Development
Centre (H3D), Department of Chemistry, University of Cape
Town, Rondebosch 7701, South Africa
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01727
Author Contributions
C.S.d.M. and V.S. contributed equally to the work. Chemical
synthesis, compound design, and SAR expansion were
performed by C.S.d.M., S.B.S., and S.R.G. Computational and
compound design support was provided by C.J.E. Iron
complementation experiment and the analysis was performed
by V.S. Analysis and interpretation of the biology data from
target deconvolution studies was performed by V.S. and A.M. In
vivo PK experiments including MetID studies were performed
by D.T. and C.B. In vitro DMPK data were analyzed by N.L. The
cytochrome P450 inhibition experiment was performed by N.L.
Screening against MmpL3 conditional knockdown mutant was
conducted by C.E. and the data were analyzed by C.E. and D.S.
Screening against MmpL3 mutant strains was carried out by
A.K. with data analysis by A.K. and T.P. The screening of MMV
compound library and RNA microarray experiment were
performed by H.I.M.B. Screening conceptualization and funding
support by C.E.B. Agilent microarray protocols were developed
by Q.S. and T.G.M. The synthesis of initial hits was performed
by K.I.B. Data analysis and compound acquisition performed by
T.B. Compound design and data analysis performed by P.C.R.
data analysis and manuscript review by S.R.G.. Scientific concept
and funding acquisition by P.G.W. G.S.B. provided scientific and
strategic guidance for the work and edited the manuscript.
Introduction and target deconvolution sections of the manu-
script were written by V.S. SAR, DMPK and part of synthesis
and experimental sections were written by S.R.G. Synthesis and
chemistry experimental sections were written by C.S.d.M, and
K.C. provided inputs on compound design, SAR analysis, and
edited the manuscript. All the authors have given approval to the
final version of the manuscript.
ABBREVIATIONS
■
AUC, area under the curve; BuOH, butanol; CS2, carbon
disulfide; DIAD, diisopropyl azodicarboxylate; DIPEA, diiso-
propylethylamine; DMF, N,N-dimethylformamide; EtOAc,
ethyl acetate; EtOH, ethanol; fu, fraction unbound; HATU, 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxid hexafluorophosphate; MIC, minimum
inhibitory concentration; mCPBA, meta-chloroperbenzoic
acid; NaBH4, NaCNBH3, sodium cyanoborohydride; POCl3,
phosphorus(V) oxychloride; RT, room temperature; SAR,
structure−activity relationship; SOCl2, thionyl chloride; TB,
tuberculosis; THF, tetrahydrofuran
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