Structure-Activity Relationships of Pyrazolo[1,5- a]pyrimidin-7(4 H)-ones as Antitubercular Agents

Article abstract of DOI:10.1021/acsinfecdis.0c00851

Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of actio

Full text of DOI:10.1021/acsinfecdis.0c00851

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pubs.acs.org/journal/aidcbc
Article
Structure−Activity Relationships of Pyrazolo[1,5‑a]pyrimidin-
7(4H)‑ones as Antitubercular Agents
Sangmi Oh, M. Daben J. Libardo, Shaik Azeeza, Gary T. Pauly, Jose Santinni O. Roma, Andaleeb Sajid,
Yoshitaka Tateishi, Caroline Duncombe, Michael Goodwin, Thomas R. Ioerger, Paul G. Wyatt,
Peter C. Ray, David W. Gray, Helena I. M. Boshoff, and Clifton E. Barry, III*
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ABSTRACT: Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified
through high-throughput whole-cell screening as a potential
antituberculosis lead. The core of this scaffold has been identified
several times previously and has been associated with various modes of
action against Mycobacterium tuberculosis (Mtb). We explored this
scaffold through the synthesis of a focused library of analogues and
identified key features of the pharmacophore while achieving
substantial improvements in antitubercular activity. Our best hits
had low cytotoxicity and showed promising activity against Mtb within
macrophages. The mechanism of action of these compounds was not
related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake
as has been found for other compounds sharing this core structure.
Resistance to these compounds was conferred by mutation of a flavin
adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that
promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering
without establishing mechanistic similarity of chemically related growth inhibitors.
KEYWORDS: pyrazolo[1,5-a]pyrimidin-7(4H)-one, structure−activity relationship, tuberculosis, antitubercular activity
uberculosis (TB) is a bacterial infection caused by
TMycobacterium tuberculosis (Mtb), which is spread
through coughing.1,2 The World Health Organization
(WHO) estimates that 1.7 billion people, almost one-quarter
of the world’s population, are infected with Mtb, including
both active and latent TB patients.3 The urgency of eradicating
TB is therefore compelling not only because it is the single
deadliest infectious disease but also due to its growing
resistance toward available drugs. In 2018, there were about
half a million new cases with resistance to rifampicin, of which
78% are multidrug-resistant TB (MDR-TB).3 Among cases of
MDR-TB in 2018, 6.2% was estimated to have extensively
drug-resistant TB (XDR-TB), requiring complex regimens and
an especially long duration of treatment.3 However, treatment
success remains low due to a limited selection of highly active
drugs.4 There is, therefore, an urgent need for the development
of new drug candidates exhibiting novel mechanisms of action
to address drug resistance and shorten treatment duration.
To discover new antitubercular agents, high-throughput
screening (HTS) against whole cells has produced most of the
existing TB drugs and clinical candidates.5 An in-house HTS
campaign of diverse small-molecule libraries yielded com-
pound 1 (Figure 1) as a confirmed hit. This compound
displays an embedded pyrazolo[1,5-a]pyrimidin-7(4H)-one
scaffold. Several previous studies have reported pyrazolo[1,5-
a]pyrimidin-7(4H)-one derivatives with moderate antituber-
cular activities with varying modes of action.6−8 From whole-
cell screens against Mtb H37Rv, compounds 2 and 3, which
have a similar pyrazolo[1,5-a]pyrimidin-7(4H)-one scaffold,
were identified as hits.6 However, the mode of action profiling
by selecting spontaneous resistant mutants revealed important
differences. Resistant clones selected against 2 had mutations
in eccB3 (Rv0283), which is a component of the ESX-3 type
VII secretion system in the Mtb genome.6 Further study of this
compound demonstrated that ESX-3 was likely not the real
target but, rather, the molecule perturbs iron metabolism in
which ESX-3 plays a crucial part.8 On the other hand, mutants
selected for resistance to 3 exhibited insertions of a transposon
(IS6110) in rv1685c, a transcription factor whose biological
role and regulatory targets are not known.6 Furthermore,
Received: December 2, 2020
Published: January 6, 2021
https://dx.doi.org/10.1021/acsinfecdis.0c00851
© 2021 American Chemical Society
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Figure 1. Chemical structure of the hit compound, 1, and analogues 2−4 that have been previously reported.6−8
another analogue 4 (Figure 1) was reported to be an inhibitor
of the Mtb 1-deoxy-D-xylulose 5-phosphate synthase (DXS)
with a moderate MIC value.7,9 Strikingly, although all three
compounds display a high degree of chemical similarity, each
molecule has been shown to have a different mode of action.
In this work, we undertook a structure−activity relationship
(SAR) study starting from pyrazolo[1,5-a]pyrimidin-7(4H)-
one analogue 1 to shine light on the confusing landscape of
potential targets and resistance mechanisms.
establish which tautomer we were studying. The crystalline
form of 4 generated by normal reaction conditions in this
study was verified to have the structure shown as 4a in Figure
2A by single crystal X-ray diffraction (Cambridge Structural
Database ID. CCDC 2034666). It was clear that the electron
density around the two nitrogen atoms N1 and N3, along with
the CO bond length of 1.23 0.01 Å, support the tautomer
4a as being dominant. This bond length is consistent with the
expected sp2 CO bond length observed in aldehydes and
ketones and comfortably different from the ar C−O bond
length of 1.36 Å expected for phenolic compounds.10
Therefore, we used this tautomer structure (4a) to depict
the synthetic derivatives.
O-Methylation (P1) and N-methylation (P2) of hit 1 were
performed to evaluate the importance of these different
tautomeric configurations of the pyrazolopyrimidine core.
Following conversion to the chlorinated intermediate by
POCl3, quenching with sodium methoxide afforded P1.11
Direct methylation of 1 with methyl iodide in the presence of
Cs2CO3 generated N-Me analogue P2 (Scheme 2).
RESULTS AND DISCUSSION
■
Synthesis of Analogues and Structural Elucidation by
X-ray Crystallography. To identify the essential structural
moieties for antitubercular activity, we designed and
synthesized a set of modified analogues. The general synthesis
of these fused heterocycle pyrazolo[1,5-a]pyrimidin-7(4H)-
ones (1, 2, 4, P6, P8−P27) relies on the one-step
cyclocondensation reaction of commercially available β-
ketoesters 5 with aminopyrazoles 6 (Scheme 1). The core
To validate substituent effects in the C2 position, the
replacement of methyl with a hydroxyl (P3) or an amino group
(P4) was performed as described in Scheme 3. P3 was
generated from methyl phenylcyanoacetate 9 through a key
intermediate, amino pyrazolone 10, by the addition of
hydrazine12 followed by intramolecular cyclization. Intermedi-
ate 9 in turn was prepared by coupling benzyl cyanide 7 with
dimethyl carbonate 8. On the other hand, the coupling
reaction between iodobenzene 11 and malononitrile 12
provided phenylmalononitrile 13,13 which generated the
diamino pyrazole 14 after the reaction with hydrazine.12 The
fused heterocyclic P4 was finally generated by an intra-
molecular condensation reaction after the reaction with β-
ketoester 5.
Scheme 1. General Synthetic Scheme of Pyrazolo[1,5-
a]pyrimidin-7(4H)-one Derivatives 1, 2, 4, and P6, P8−
a
P27
a
Reagents and conditions: (a) toluene, reflux, overnight; (b) AcOH,
reflux, overnight.
scaffold, pyrazolo[1,5-a]pyrimidin-7(4H)-one, has three plau-
sible tautomeric structures (Figure 2A). Different tautomeric
forms of small molecules are known to interact with different
biological targets in unique ways; therefore, we wanted to
The triazinone analogue P5 was synthesized for the
evaluation of the main core scaffold, pyrazolo pyrimidinone.
Benzonitrile 15 was reacted with methanol in acidic conditions
Figure 2. (A) Three probable tautomeric structures (4a−c) of the pyrazolo[1,5-a]pyrimidin-7(4H)-one scaffold in 4; (B) X-ray crystal structure of
4.
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a
a
Scheme 2. Synthesis of P1 and P2
Scheme 4. Synthesis of Analogue P7
a
Reagents and conditions: (a) 1 (1.0 equiv), Cs2CO3 (3 equiv), CF3I
in DMSO, 365 nm UV irradiation during overnight rt stirring.
nary SAR studies to address important questions around the
hit scaffold were focused on determining key moieties or
functionalities that may play important roles in antitubercular
activity. Minimum inhibitory concentrations were determined
against Mtb strain H37Rv in the presence (ADC) or absence
(GCas) of albumin. Lower MICs in GCas generally mean
lower protein binding. A focused library containing 27
compounds (P1−P27) was constructed for the preliminary
SAR (Tables 1 and 2). For comparative purposes, we also
resynthesized the literature compounds 2 and 4. In order to
explore the core pyrazolo[1,5-a]pyrimidin-7(4H)-one scaffold,
the methylated analogues P1 with O-Me and P2 with N-Me
were synthesized, resulting in loss of activity. Among the three
different tautomeric configurations in Figure 2A, the structure
of the O-methylated P1 derivative was constrained to the
tautomer (4b) form, while the N-methylated P2 was confined
to the tautomer (4a) and lost its hydrogen bond donor
capability. Polar functional groups in the C2 position including
hydroxyl (P3) or amino (P4) groups can cause significant
changes in the original scaffold including conformational
differences and hydrogen bonding characteristics. All of these
a
Reagents and conditions: (a) POCl3 (2.5 equiv), DIPEA (0.2 equiv),
90 °C, overnight; (b) NaOMe in MeOH (2.0 equiv), rt, overnight;
(c) MeI (5.0 equiv), Cs2CO3 (3.0 equiv), rt, overnight.
to generate iminoester 16, and then, condensation with ethyl
chloroformate resulted in the formation of imidate 17. After
the reaction with aminopyrazole 6, the desired product, P5,
was obtained.14
The trifluoromethylated analogue P7 was synthesized by a
light-promoted reaction with trifluoromethyl iodide combined
with Cs2CO3 (Scheme 4). Instead of blue LEDs as a light
source,15 365 nm UV light was alternatively used for this
photoactivation reaction, and the efficient and practical
incorporation of a trifluoromethyl group into the α-carbonyl
carbon was confirmed.
Structure−Activity Relationship Study of Synthetic
Analogues and Their Antitubercular Activities. Prelimi-
a
Scheme 3. Synthesis of Analogues P3−P5
a
Reagents and conditions: (a) 7 (1.0 equiv), 8 (10 equiv), NaOMe (2.0 equiv), 90 °C, overnight; (b) N2H4·H2O (2.0 equiv), EtOH, reflux,
overnight; (c) 5 (1.0 equiv), EtOH, reflux, overnight; (d) 11 (1.0 equiv), 12 (2.0 equiv), K2CO3 (4.0 equiv), CuBr2 (0.1 equiv), DMSO, 120 °C,
overnight; (e) methanolic HCl, rt, overnight; (f) ethyl chloroformate (1.2 equiv), K2CO3 (1.0 equiv), CH2Cl2, rt, 36 h; (g) EtOH, AcOH, reflux, 5
h.
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the initial hit. The next series, P8−P20, were derivatized
specifically in the R2 position with diverse moieties and
substituents. Benzyl (P8) instead of phenyl retained its activity
in GCas media, but cyclohexylmethyl (P9), morpholinomethyl
(P10), cyclohexyl (P11), and isobutyl (P12) were not
tolerated at this position. However, P8 displayed a somewhat
large discrepancy in the MIC with or without protein in the
media as evidenced by the markedly increased MIC in 7H9/
ADC media. Some heteroaryl groups including 3-picolyl
(P13), 3-furyl (P14), and pyrazinyl (P15) also showed the
same pattern of relatively big MIC differences in the two
media. With the phenyl group at the R2 position, a fluoro
substituent as a small electron-withdrawing group was
confirmed to retain its original potency or generate slightly
better activities regardless of the substitution pattern (P16−
P18). Furthermore, the compounds embedding 3,4-difluoro
(P19) or 3,4-dichloro (P20) were favorable for activity in
7H9/ADC due to less protein binding as seen by the 5-fold
improvement in the MIC compared to the initial hit, 1.
To assess the importance of the phenyl group at the R3
position, P21 was synthesized, resulting in loss of anti-
tubercular activity. Therefore, an aromatic ring in positions R2
and R3 was confirmed to be necessary for antitubercular
activity in the main scaffold. To evaluate the effect of the
methyl group at the R4 position, several analogues were
designed (P22, P23, P26, and P27). The removal of the
methyl group at position R4 (P22) was detrimental to the
activity compared to 1. On the other hand, P26, the R4 des-
methyl analogue of P25, showed 4-fold better potency. In
addition, the isopropyl substituted P23 was inactive against
Mtb. P19 showed improved potency (2.73 μM in 7H9/ADC
and 1.56 μM in 7H9/GCas). Surprisingly, the R4 trifluor-
omethyl analogue of P19 and P27, completely lost activity,
suggesting the possible effects of this electron withdrawing
functionality on the properties of this heterocycle. After
modifications of R2 and R3, P24 and P25 were found to retain
their activities in GCas and were 8-fold more potent in the
presence of protein than the initial hit without serious
cytotoxicity. Most active derivatives were not cytotoxic against
HepG2 cells and additionally did not exert overt mitochondrial
toxicity as seen by the lack of cytotoxicity during the growth of
these cells on galactose as the carbon source. Specifically, P25
showed a selectivity index of >60 in glucose media and >30 in
galactose media, respectively (Table 2). In addition, our data
indicated that the compounds were not highly vulnerable to
host metabolism as seen by the moderate stability in the
microsomal stability assays (Table S1). Therefore, active
compounds without serious cytotoxicity against HepG2 cells
including P19, P24, and P25 were selected and used for
further biological studies.
Table 1. Structure and Antitubercular Activities of
Compounds 1, 2, 4, and P1−P5
c
a
MIC of compounds tested against Mtb H37Rv in Middlebrook 7H9/
b
ADC/Tween. MIC of compounds tested against Mtb H37Rv in
7H9/glucose/casitone/tyloxapol. See the Methods section.
In Vitro and Ex Vivo Antitubercular Activity of
Pyrazolo[1,5-a]pyrimidin-7(4H)-one Derivatives. Targets
in mycolyl-arabinogalactan biosynthesis as well as in the bc1
complex of the terminal respiratory oxidase have been reported
to be promiscuous targets on the basis of high hit rates of
compound libraries against these proteins.16 To generate an
early understanding of the potential mechanism of action,
compounds 1, 2, 4, and P19 were profiled using assays that
report on the inhibition of these targets using a pini-LUX strain
that informs on the inhibition of cell wall biosynthesis17 as well
as a cydC::aph mutant that is hypersusceptible to QcrB
inhibitors.18,19 These assays indicated that these compounds
did not inhibit these apparently promiscuous targets (Table 3).
c
changes resulted in the loss of antimicrobial activity. In
addition, it was found that the triazinone derivative, P5, was
almost inactive against whole cells (Table 1).
Compounds P6−P27 were used to evaluate four different
positions around the core, pyrazolo[1,5-a]pyrimidin-7(4H)-
one (Table 2). First, P6, which has a methyl group, and P7,
which has a trifluoromethyl group at the R1 position, retained
activities in ADC media but displayed slightly detrimental
effects in albumin containing media, suggesting strong protein
binding and no electronic effect in the R1 position compared to
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Table 2. Structural Aspects and Antitubercular Activities of Compounds P6−P27
a
b
MIC of compound tested against Mtb H37Rv in Middlebrook 7H9/ADC/Tween. MIC of compound tested against Mtb H37Rv in 7H9/
c
glucose/casitone/tyloxapol. See the Methods section. Cytotoxicity of compound tested against HepG2 cells in DMEM/10% FBS supplemented
with glucose. Cytotoxicity of compound tested against HepG2 cells in DMEM/10% FBS with galactose. Not determined.
d
e
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Table 3. Biological Evaluation of Pyrazolo[1,5-a]pyrimidin-7(4H)-one Derivatives
MIC (μM)
reporter gene assay
a
b
c
d
e
f
g
h
hit
7H9
GAST
GBSA/DBSA
nitrate/butyrate
Chol
CydKO
cell wall
DNA damage
1
2
4
P19
INH
12.5
19.0
12.5
3.13
0.39
N/D
N/D
2.3
12.5
4.70
9.40
3.13
0.30
N/D
N/D
12.5
6.25
6.25
1.56
N/D
N/D
N/D
9.40
12.5
12.5
3.13
0.20
12.5
25.0
9.40
3.13
0.30
N/D
N/D
no
no
no
no
N/D
yes
N/D
no
no
no
no
N/D
N/D
yes
19.0
3.13
1.20
0.10
N/D
N/D
i
SQ109
MOX
N/D
N/D
a
b
MIC of compound against Mtb strain H37Rv in Middlebrook 7H9/BSA containing glucose/glycerol/Tween 80. MIC of compound against Mtb
c
strain H37Rv in GAST/Fe. MIC of compound against Mtb strain H37Rv in Middlebrook 7H9/BSA/Tyloxapol with glucose (GBSA) or
dipalmitoylphosphatidylcholine (DBSA) as the carbon source. MIC of compound against Mtb strain H37Rv in Middlebrook 7H9/BSA/
d
e
Tyloxapol/butyrate/0.1 mM nitrite/pH 6.0. MIC of compound against Mtb strain H37Rv in Middlebrook 7H9/BSA/Tyloxapol/cholesterol.
f
g
MIC of compound against Mtb strain H37Rv in Middlebrook 7H9/ADC with cydC::aph. Induction of the cell wall responsive iniBAC promoter
h
as measured using the piniB-LUX reporter strain. Induction of the DNA damage responsive recA and radA reporters using the precA-LUX and
pradA-LUX reporter strains. Not determined.
i
In addition, the reporters for compounds that result in DNA
damage indicated that the primary mechanism of action did
not involve the modulation of DNA homeostasis.17 MIC
determination under a variety of carbon sources with in vivo
relevance including dipalmitoyl-phosphatidylcholine, choles-
terol, and glucose showed that these compounds retained
activity against Mtb under all these conditions. In addition,
these compounds were active against Mtb, slowly replicating
under the growth restriction imposed by low pH and
nitrosative stress, suggesting that these compounds inhibit
targets of functional importance under a wide diversity of
growth conditions.
Prompted by the favorable selectivity indices obtained for
several of the synthesized derivatives, the efficacy of the
compounds against intracellular Mtb was determined. For that,
the cytotoxicity of selected analogues was first evaluated
against the murine macrophage cell line, J774. While largely
tolerated by HepG2 cells, the tested compounds P19, P24, and
P25 exhibited modest cytotoxicity against J774 cells (Figure
3A) with IC50 values of 29.6 1.2, 22.5 2.1, and 20.2 0.8
μM respectively.
J774 cells were then infected with Mtb H37Rv at a
multiplicity of infection of 5 (MOI 1:5) and treated with
P19 or P25 for 7 days. Taking into consideration both their in
vitro MICs and corresponding IC50 values against J774 cells,
we settled on a low dose of 7.5 μM and a high dose of 15 μM
for both compounds, concentrations above the MIC but below
the cytotoxic threshold for J774 cells. It was found that both
P19 and P25 possess cidal activity against intramacrophage
Mtb H37Rv (Figure 3B) at the tested concentrations. High
doses of P19 reduced the bacterial burden by ∼0.5 logs while
P25 reduced bacterial counts by 1.5 and 2 orders of magnitude
at 7.5 and 15 μM, respectively.
Figure 3. (A) Murine macrophage cytotoxicity was measured using a
standard luminescence-based assay. Curves represent the average of
three trials done in duplicates. (B) J774 cells were infected with Mtb
H37Rv (MOI 1:5) and treated with the corresponding compounds
for 7 days. Survival of intracellular bacteria was measured by plating
serial dilutions following J774 lysis. Bars: mean SEM obtained from
two trials done in duplicates.
Biological Evaluation of Pyrazolo[1,5-a]pyrimidin-
7(4H)-ones. Compound 2, which was previously shown to
interfere with iron homeostasis in mycobacteria by directly
binding to intracellular Fe2+, shares a similar scaffold with 1, 4,
P19, and P24.8 To deplete the iron levels in the medium, we
added 100 μM desferrioxamine (DFO; MIC = 250 μM), a
siderophore produced by Streptomyces that cannot be utilized
by mycobacteria.20 While siderophores selectively bind to Fe3+,
the loss of extracellular Fe3+ due to chelation would lead to the
depletion of intracellular Fe2+ during growth. We also
supplemented the iron levels by the addition of 250 μM
FeCl3, half the concentration of the inorganic salt’s MIC (500
μM). We utilized in-house controls, the antitubercular
benzoxazoles (593 and 596), compounds that interfere with
iron metabolism as evidenced by transcriptional upregulation
of mycobactin biosynthetic genes and the esx3 operon
(unpublished results), as positive controls to qualify the
assay. The benzoxazoles 593 and 596 were 8- to 16-fold more
potent under iron-depleted conditions (Figure 4A). Similarly,
it was confirmed that 2 had greater efficacy under iron-limited
conditions in accordance to previously published results.8 In
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1 did not inhibit iron uptake unlike DFO, which completely
inhibited uptake. Taken together with the result of our iron
limitation experiments, the antitubercular activities of these
pyrazolo[1,5-a]pyrimidin-7(4H)-one analogues based on hit 1
were not related to the perturbations in iron homeostasis.
To determine the mechanism of action of the most active
analogues, we independently raised mutants against P19 and
P24 at 5× and 10× of their in vitro MIC. Despite having
minimal structural differences, we were only able to raise
resistant mutants against P19. These clones arose at a
frequency of 3 × 10−8 at 5× MIC and 4 × 10−9 at 10× MIC.
We measured resistance of the mutants (P19R) against P19,
P24, 1, 2, and 4. We found that mutants raised against 5×
MIC of P19 were 4- to 7-fold more resistant than the WT, and
those that were raised against 10× MIC were highly resistant
(≥8-fold) to P19. Unsurprisingly, the resistant mutants
exhibited similar resistance profiles against P24. P19R strains
were also highly resistant to the original hit compound, 1. On
the other hand, the resistant mutants exhibited relatively small
increased MICs against 2 or 4, values that are within the
margin of error of typical MIC measurements, indicating a lack
of cross-resistance of P19R strains against 2 and 4. This data
suggested that 1, P19, and P24 had distinct targets or
resistance mechanisms from those of 2 and 4 (Table 4).
The genomic DNA of the isolated P19R strains was extracted
and sequenced, and it was found that all single-nucleotide
polymorphisms (SNPs) mapped onto rv1751, a nonessential
gene annotated as a probable oxidoreductase but whose
function has not been definitively demonstrated. A closer
examination of the SNPs and their corresponding MICs
showed that the level of resistance can be classified into three
categories: (1) mutations in P152, L250, and A434 resulting in
low-level resistance, (2) mutations in V96 and H97 imparting
intermediate resistance, and (3) mutations in L399 conferring
high-level resistance to P19 and P24. Sequence similarity
searching showed primarily flavin adenine dinucleotide (FAD)-
dependent hydroxylases and monooxygenases from different
organisms (a logical result considering Rv1751 is also
predicted to have a FAD-binding domain) whose known
substrates are aromatic compounds (e.g., phenols and
hydroxybenzoates).21−23 One of the more similar proteins
was Rox, a rifampicin monooxygenase from Streptomyces
venezuelae (PDB ID: 5VQB).24 We modeled Rv1751 using
the crystal structure of Rox as a template (Figure 5A). In this
modeled structure, the SNPs that conferred intermediate- and
high-level resistance were located proximal to the RIF binding
Figure 4. (A) Effect of iron levels on the potency of test compounds.
MICs were measured in the presence of 100 μM desferrioxamine
(DFO) or 250 μM FeCl3. The increase in potency of the compounds
is expressed as MIC ratios equal to MIC in pure 7H9 media divided
by MIC in 7H9 supplemented with either DFO or FeCl3. Bars
represent mean SEM (n = 2). (B) Effect of 1 in the iron uptake of
Mtb in vitro. Cells treated with the specified compound were fed 55Fe,
and the radioactivity inside H37Rv was measured over a 72 h time
course. Data represent mean SD (n = 3). CPM, counts per minute.
contrast, neither 4 nor the newly described pyrazolo[1,5-
a]pyrimidin-7(4H)-ones (P19 and P24) were affected by iron
depletion, producing at best, a negligible 2-fold decrease in
MIC. Excess iron in the medium led to neither activation nor
attenuation of any of the drugs tested, indicating that none of
these compounds act as ionophores that shuttle iron into the
cell.
We further determined the effect of the hit compound 1
directly on iron uptake by Mtb, on the basis of the reported
accumulation of intracellular iron in M. smegmatis following
treatment with 2.8 We treated Mtb cells with 1 in a growth
medium labeled with 55Fe and tracked the influx of
radioactivity into the cells. As shown in Figure 4B, compound
Table 4. Spontaneous Resistant Mutants Raised against P19 and Their Corresponding MICs
SNPs identified in P19 resistant mutants in rv1751 (# of independent mutants isolated)
Cpd
MIC (μM)
WT
0.78
L399W (7)
V96L (6)
H97Y/N (2)
L250Q (2)
P152S (1)
A434T (1)
P19
MIC
fold change
MIC
fold change
MIC
fold change
MIC
fold change
MIC
fold change
MIC
12.5
16
18.8
12
12.5
10
0.39
1
2.3
3
7.82
5
2.3
2
1.2
3
1.56
2
6.25
4
1.56
1.3
0.39
1
1.56
1
2.3
3
6.26
4
3.13
2.6
1.2
3
4.7
3
1.56
2
3.90
2.5
2.3
2
1.2
3
2.3
1.5
0.04
1.56
2
3.12
2
1.56
1.3
1.2
3
2.3
1.5
0.04
P24
1
1.56
1.2
2
0.39
1.56
0.04
4
3.13
2
0.04
2.3
1.5
0.04
INH
0.04
0.04
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Figure 5. (A) Calculated structure of Rv1751 (red ribbons) superimposed on crystal structure of rifampicin monooxygenase (Rox, green ribbons)
from S. venezuelae. Rifampicin (RIF) is shown in magenta, and FAD is shown in cyan. (B) Co-crystal structures of RIF and FAD are superimposed
onto the calculated structure of Rv1751 (red ribbons). Residues mutated in P19R are highlighted in yellow (conferring high-level resistance) and
cyan (conferring low-level resistance).
Figure 6. (A) Degree of compound degradation upon exposure to WT H37Rv and the indicated P19R strains was measured via LC/MS. Bars
represent mean
SEM (n = 3). (B) The correlation between compound degradation by Mtb and in vitro MIC shows the dependence of
antimycobacterial activity to compound metabolism.
site, while those that lead to low-level resistance were distal to
this site (Figure 5B). H97 and L399 both point directly toward
the RIF binding site, suggesting that mutations in these
positions will likely lead to a change in the substrate binding
affinity, in this case, a change in the binding affinity of P19 to
Rv1751 (Figure 5B). Taken together, these results suggested
that the resistant clones have mutations that affected the
catabolism of P19, resulting in decreased susceptibility.
To determine the potential role of Rv1751 in the resistance
toward P19, the degree of P19 degradation when exposed to
Mtb was measured via a LC/MS-based assay. Under conditions
that saturated the compound with Mtb cells (P19 at 10× MIC
added to 109 CFUs of H37Rv), it was found that there was
about 10% degradation of P19 by Mtb after 24 h of exposure.
When we performed a similar experiment using the P19R−
intermediate Rv1751 V96L mutant, almost 30% of the
compound was degraded, and when the L399W mutant was
used, nearly 50% of P19 was metabolized (Figures 6A and S1).
Interestingly, the degree to which P19 was catabolized by the
various forms of Rv1751 matches the level of resistance that
these polymorphisms impart to cells. That is, the higher level
of resistance (higher MICs) of specific strains are associated
with their greater catabolic capability (Figure 6B). Finally, in
accordance with the slight cross-resistance of the P19R strains
to 4, there were only small differences in the degree of
degradation of 4 when it was exposed to either the WT or the
P19R strains. To identify the major metabolites of P19
generated in mutant cells, a heavy oxygen (18O) stable isotope
labeling experiment coupled with MS-based analysis was
performed. On the basis of the preliminary LC/MS analysis
of the metabolites (Figure S1), we speculated that one or two
oxygen atoms were incorporated into some of the major
metabolites. Due to the fact that the incorporation test with
18O-enriched water (H218O) did not provide any meaningful
information, P19 was incubated with the L399W mutant cells
under an 18O2 environment for 2 days, and then, the media was
analyzed by a triple quadrupole tandem MS fragmentation
technique to characterize the degradation pattern. As shown in
Figures S2−S4, L399W mutant cells accelerated the catabolic
degradation of P19 by the hydroxylation of R1, hydroxylation
of the phenyl ring in the R3 position, and/or N-methylation of
the pyrimidinone ring.
CONCLUSIONS
■
Although the pyrazolo[1,5-a]pyrimidin-7(4H)-one scaffold has
been found in several antitubercular agents with different mode
of actions, there are no reports regarding the exploration of the
SAR based on the evaluation of activity against Mtb cells. We
confirmed the exact tautomeric structure by small-molecule X-
ray crystallography and explored the SAR from 1 by the design
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and synthesis of a focused library containing 26 analogues in
which we modified each moiety in the original pyrazolo[1,5-
a]pyrimidin-7(4H)-one scaffold to understand its contribution
to whole-cell activity. The compounds were found to be active
against Mtb under a variety of in vitro growth conditions that
reflect the carbon sources thought to be important for in vivo
pathogenesis of the organism. The compounds were found to
have favorable selectivity indices against Mtb compared to
eukaryotic cells with greater than 10-fold selectivity for the
pathogen than HepG2 cells. The most promising analogues
including P19, P24, and P25 were found to exert growth
inhibition of Mtb during parasitism of host macrophages.
These compounds were selected and evaluated for further
biological studies, which indicated that the compounds did not
inhibit the targets in cell wall biosynthesis of respiration that
have previously been reported to be apparently promiscuous
targets. Despite being nearly identical in structure to a known
iron chelator, these compounds did not exert their
antitubercular effect by perturbation of iron homeostasis. We
were, however, unable to decipher the intracellular target of
this scaffold since resistant mutants mapped to rv1751
encoding a putative FAD-dependent hydroxylase, which we
could confirm, led to increased catabolism of the compound.
These studies suggest that structural similarity does not
necessarily imply mechanistic similarity. Highly similar
scaffolds with completely different targets have been reported
previously; for example, very closely related indazole
sulfonamides have been reported to be potent inhibitors of
inosine monophosphate dehydrogenase25 and the β-ketoacyl-
ACP synthase involved in mycolic acid biosynthesis.26 Such
profound changes in the target with closely related structures
suggests that the chemical clustering of the hit series, as is
commonly done in Mtb whole-cell-based screening programs,
must be done with additional care when selecting a hit series
and interpreting the SAR in the absence of mechanistic
information.
the previously reported procedure with some modifica-
tions.27−29 The synthetic procedures of P111,29 and P230
were followed by the previously reported procedure with some
modifications. The key intermediate 10 for the synthesis of
P3,12,31 intermediate 14 for the precursor of P4,12,13 and
intermediate 17 for the P514 were prepared using the
previously reported procedure with some modifications.
2-Methyl-3,5-diphenylpyrazolo[1,5-a]pyrimidin-7(4H)-
1
one (1). Off-white solid: mp = 293 °C; H NMR (300 MHz,
DMSO-d6) δ 12.16 (s, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.51−
7.42 (m, 7H), 7.35−7.30 (m, 1H), 5.92 (s, 1H), 2.32 (s, 3H);
13C NMR (75 MHz, DMSO-d6) δ 155.9, 151.0, 150.4, 138.8,
132.8, 130.8, 130.4, 129.5, 128.35, 128.31, 127.9, 126.5, 104.0,
94.9, 13.1; LRMS (ESI) m/z 302 [M + H]+.
3-(4-Chlorophenyl)-5-(cyclohexylmethyl)pyrazolo[1,5-a]-
pyrimidin-7(4H)-one (2). Off-white solid: 1H NMR (300
MHz, DMSO-d6) δ 11.54 (bs, 1H), 7.82 (s, 1H), 7.40−7.31
(m, 4H), 5.53 (s, 1H), 2.43 (d, J = 6.6 Hz, 2H), 1.68−1.58 (m,
6H), 1.19−1.06 (m, 3H), 0.99−0.92 (m, 2H); 13C NMR (75
MHz, DMSO-d6) δ 156.4, 153.2, 141.5, 137.4, 131.6, 129.1,
128.9, 128.3, 103.5, 95.9, 40.0, 37.0, 32.2, 25.6, 25.3; LRMS
(ESI) m/z 342 [M + H]+.
3-(4-Fluorophenyl)-5-(4-methoxyphenyl)-2-
methylpyrazolo[1,5-a]pyrimidin-7(4H)-one (4). White solid:
1
mp = 271 °C; H NMR (300 MHz, DMSO-d6) δ 11.97 (s,
1H), 7.73 (d, J = 8.7 Hz, 2H), 7.54−7.50 (m, 2H), 7.28 (t, J =
9.0 Hz, 2H), 7.06 (d, J = 9.0 Hz, 2H), 5.91 (s, 1H), 3.82 (s,
3H), 2.27 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 162.8,
161.3, 159.6, 155.9, 150.6, 150.2, 139.0, 131.7, 131.6, 129.6,
127.31, 127.27, 124.8, 115.4, 115.1, 114.0, 103.0, 94.0, 55.4,
13.0; LRMS (ESI) m/z 350 [M + H]+.
7-Methoxy-2-methyl-3,5-diphenylpyrazolo[1,5-a]-
pyrimidine (P1). Pale-yellow solid: 1H NMR (300 MHz,
DMSO-d6) δ 8.28−8.25 (m, 2H), 7.86−7.83 (m, 2H), 7.59−
7.47 (m, 5H), 7.32−7.27 (m, 1H), 7.15 (s, 1H), 4.31 (s, 3H),
2.57 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 157.8, 156.3,
152.6, 147.7, 138.0, 133.2, 131.0, 129.5, 129.1, 128.9, 128.0,
126.5, 108.2, 85.2, 58.4, 58.3, 15.2; LRMS (ESI) m/z 316 [M
+ H]+.
METHODS
■
1
Chemistry. H and 13C NMR spectra were recorded on a
2,4-Dimethyl-3,5-diphenylpyrazolo[1,5-a]pyrimidin-
7(4H)-one (P2). Pale-yellow solid: 1H NMR (300 MHz,
DMSO-d6) δ 8.06−8.02 (m, 2H), 7.71 (d, J = 7.8 Hz, 2H),
7.53 (t, J = 7.8 Hz, 2H), 7.48−7.37 (m, 4H), 6.50 (s, 1H), 4.27
(s, 3H), 2.52 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 160.3,
158.0, 150.9, 147.8, 138.1, 130.8, 130.5, 130.2, 129.2, 129.1,
127.8, 127.5, 110.0, 96.9, 36.7, 11.3; LRMS (ESI) m/z 316 [M
+ H]+.
Varian Mercury-300 NMR spectrometer or a Bruker AVANCE
III HD NanoBay 400 MHz spectrometer, and chemical shifts
were measured in ppm relative to the specific solvent signal.
Routine mass and purity analyses (LRMS) were performed on
an HP Agilent LC/MS series 1100 system equipped with a
reverse phase column (Agilent Poroshell 120 EC-C18, 2.7 μm,
50 × 2.1 mm) and photodiode array detector coupled to an
Agilent 1946 DSL quadrupole mass selective detector using
electrospray ionization (ESI). The gradient mobile phase
consisting of acetonitrile/water with 0.1% formic acid and UV
detection at 254 and 210 nm were used to confirm all final
products to be ≥95%. The melting point was measured on an
Electrothermal 9100 apparatus. Most reagents used in the
synthetic procedure were purchased from Sigma-Aldrich, Alfa
Aesar, and TCI. The progress of the reaction was monitored
using thin-layer chromatography (TLC) (silica gel 60 F254
0.25 mm), and the products were visualized by UV light (254
and 365 nm). SiliaFlash P60 (40−60 μm) used in flash column
chromatography was purchased from Silicycle Inc. Other
solvents were purchased from commercial vendors and used
without further purification unless otherwise mentioned.
Synthesis for the Compounds and Their Characterization
Data. General syntheses of 1, 2, 4, and P6−P27 and the final
cyclocondensation reactions for P3−P5 were performed using
3,5-Diphenylpyrazolo[1,5-a]pyrimidine-2,7(1H,4H)-dione
1
(P3). Pale-yellow solid: H NMR (300 MHz, DMSO-d6) δ
12.0 (s, 1H), 7.77 (s, 2H), 7.64 (d, J = 7.2 Hz, 2H), 7.55 (m,
3H), 7.43 (t, J = 7.4 Hz, 2H), 7.26 (t, J = 6.9 Hz, 1H), 5.95 (s,
1H); 13C NMR (75 MHz, DMSO-d6) δ 161.9, 155.2, 150.4,
138.7, 132.9, 130.7, 130.2, 128.6, 128.5, 128.3, 128.0, 125.9,
95.8, 90.7, 56.1, 18.6; LRMS (ESI) m/z 304 [M + H]+.
2-Amino-3,5-diphenylpyrazolo[1,5-a]pyrimidin-7(4H)-one
(P4). Off-white solid: 1H NMR (300 MHz, DMSO-d6) δ 11.79
(s, 1H), 7.74 (s, 2H), 7.52−7.43 (m, 7H), 7.30 (t, J = 7.0 Hz,
1H), 5.85 (s, 1H), 5.29 (bs, 2H); 13C NMR (75 MHz, DMSO-
d6) δ 156.0, 155.3, 149.5, 138.8, 133.0, 130.7, 130.4, 128.7,
128.5, 127.8, 126.2; LRMS (ESI) m/z 303 [M + H]+.
7-Methyl-2,8-diphenylpyrazolo[1,5-a][1,3,5]triazin-4(1H)-
one (P5). Pale-yellow solid: 1H NMR (300 MHz, DMSO-d6) δ
8.11 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 7.8 Hz, 2H), 7.60−7.43
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(m, 5H), 7.33−7.28 (m, 1H), 2.45 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ 153.0, 151.8, 145.4, 144.3, 132.0, 131.34,
131.27, 128.7, 128.5, 127.9, 126.6, 111.4, 14.4; LRMS (ESI)
m/z 303 [M + H]+.
151.9, 151.2, 149.3, 139.7, 136.6, 131.4, 130.2, 129.3, 127.5,
124.1, 104.9, 96.4, 13.8; LRMS (ESI) m/z 303 [M + H]+.
5-(Furan-3-yl)-2-methyl-3-phenylpyrazolo[1,5-a]-
1
pyrimidin-7(4H)-one (P14). Pale-yellow solid: H NMR (300
2,6-Dimethyl-3,5-diphenylpyrazolo[1,5-a]pyrimidin-
7(4H)-one (P6). Off-white solid: mp = 270−280 °C
MHz, DMSO-d6) δ 11.78 (s, 1H), 8.47 (s, 1H), 7.81 (t, J = 1.8
Hz, 1H), 7.46−7.45 (m, 4H), 7.37−7.32 (m, 1H), 7.10 (s,
1H), 6.08 (s, 1H), 2.26 (s, 3H); 13C NMR (75 MHz, DMSO-
d6) δ 156.6, 151.1, 145.2, 144.5, 143.9, 139.2, 131.6, 130.3,
129.3, 127.5, 120.0, 110.0, 104.5, 94.3, 13.9; LRMS (ESI) m/z
292 [M + H]+.
1
(decomposition); H NMR (300 MHz, DMSO-d6) δ 12.03
(s, 1H), 7.49 (s, 5H), 7.40−7.38 (m, 3H), 7.31−7.24 (m, 1H),
2.29 (s, 3H), 1.86 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ
157.0, 150.3, 147.8, 138.2, 133.7, 130.9, 129.6, 129.5, 129.1,
128.4, 128.2, 126.5, 102.6, 101.8, 13.2, 11.9; LRMS (ESI) m/z
316 [M + H]+.
2-Methyl-3-phenyl-5-(pyrazin-2-yl)pyrazolo[1,5-a]-
1
pyrimidin-7(4H)-one (P15). Pale-yellow solid: H NMR (300
MHz, CDCl3) δ 9.24 (s, 1H), 8.76 (d, J = 2.4 Hz, 1H), 8.66 (t,
J = 1.8 Hz, 1H), 7.57−7.38 (m, 6H), 6.58 (s, 1H), 2.51 (s,
3H); 13C NMR (75 MHz, CDCl3) δ 156.7, 152.6, 146.8,
143.7, 143.1, 142.8, 142.0, 137.8, 130.9, 129.8, 128.6, 127.7,
105.2, 95.1, 13.6; LRMS (ESI) m/z 304 [M + H]+.
5-(2-Fluorophenyl)-2-methyl-3-phenylpyrazolo[1,5-a]-
pyrimidin-7(4H)-one (P16). White solid: 1H NMR (300 MHz,
DMSO-d6) δ 12.35 (s, 1H), 7.69−7.56 (m, 2H), 7.47−7.45
(m, 4H), 7.40−7.32 (m, 3H), 5.84 (s, 1H), 2.32 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) δ 160.8, 157.5, 155.6, 150.4,
145.9, 138.7, 132.6, 132.5, 131.0, 130.6, 129.5, 128.6, 126.8,
124.64, 124.60, 121.4, 116.1, 115.8, 130.8, 97.2, 13.2; LRMS
(ESI) m/z 320 [M + H]+.
2-Methyl-3,5-diphenyl-6-(trifluoromethyl)pyrazolo[1,5-a]-
pyrimidin-7(4H)-one (P7). Off-white solid: H NMR (400
1
MHz, DMSO-d6) δ 12.91 (bs, 1H), 7.55−7.47 (m, 5H), 7.44−
7.41 (m, 4H), 7.37−7.32 (m, 1H), 2.31 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 153.5, 152.8, 151.6, 137.3, 132.9,
129.94, 129.85, 128.4, 128.2, 127.9, 127.1, 125.2, 122.5, 105, 5,
96.8, 96.5, 13.0; LRMS (ESI) m/z 370 [M + H]+.
5-Benzyl-2-methyl-3-phenylpyrazolo[1,5-a]pyrimidin-
7(4H)-one (P8). Pale-yellow solid: 1H NMR (300 MHz,
DMSO-d6) δ 12.00 (s, 1H), 7.51−7.34 (m, 9H), 7.30−7.24
(m, 1H), 5.47 (s, 1H), 3.96 (s, 3H), 2.26 (s, 3H); 13C NMR
(75 MHz, DMSO-d6) δ 155.9, 153.2, 150.0, 138.5, 137.1,
130.7, 129.4, 128.8, 128.7, 128.6, 126.9, 126.8, 103.1, 95.8,
37.5, 13.0; LRMS (ESI) m/z 316 [M + H]+.
5-(3-Fluorophenyl)-2-methyl-3-phenylpyrazolo[1,5-a]-
1
pyrimidin-7(4H)-one (P17). Pale-yellow solid: H NMR (300
5-(Cyclohexylmethyl)-2-methyl-3-phenylpyrazolo[1,5-a]-
1
MHz, DMSO-d6) δ 12.17 (bs, 1H), 7.69−7.33 (m, 9H), 6.02
(s, 1H), 2.32 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 163.4,
160.2, 155.7, 150.5, 149.7, 138.8, 135.0, 130.8, 130.7, 130.6,
129.5, 128.5, 126.7, 124.3, 117.5, 117.2, 115.3, 115.0, 104.3,
95.4, 13.1; LRMS (ESI) m/z 320 [M + H]+.
pyrimidin-7(4H)-one (P9). White solid: H NMR (300 MHz,
DMSO-d6/CDCl3) δ 11.64 (s, 1H), 7.48−7.33 (m, 5H), 5.49
(s, 1H), 2.43 (d, J = 4.8 Hz, 2H), 2.27 (s, 3H), 1.68−1.65 (m,
6H), 1.22−1.15 (m, 3H), 1.00−0.94 (m, 2H); 13C NMR (75
MHz, DMSO-d6/CDCl3) δ 155.8, 152.8, 149.8, 138.4, 130.7,
129.3, 128.4, 126.5, 102.9, 95.6, 95.5, 36.9, 32.2, 25.7, 25.4,
12.9; LRMS (ESI) m/z 322 [M + H]+.
5-(4-Fluorophenyl)-2-methyl-3-phenylpyrazolo[1,5-a]-
pyrimidin-7(4H)-one (P18). White solid: 1H NMR (300 MHz,
DMSO-d6) δ 12.13 (s, 1H), 7.81 (bs, 2H), 7.47−7.42 (m,
4H), 7.37−7.32 (m, 3H), 5.92 (s, 1H), 2.29 (s, 3H); 13C NMR
(75 MHz, DMSO-d6) δ 165.2, 161.9, 155.8, 150.3, 150.1,
138.9, 130.9, 130.6, 130.5, 129.5, 128.5, 126.7, 115.6, 115.3,
104.1, 95.0, 13.1; LRMS (ESI) m/z 320 [M + H]+.
5-(3,4-Difluorophenyl)-2-methyl-3-phenylpyrazolo[1,5-a]-
pyrimidin-7(4H)-one (P19). Off-white solid: mp = 255 °C; 1H
NMR (300 MHz, DMSO-d6) δ 12.13 (bs, 1H), 7.94 (t, J = 9.3
Hz, 1H), 7.73−7.45 (m, 6H), 7.37−7.32 (m, 1H), 6.00 (bs,
1H), 2.32 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 155.6,
152.5, 152.4, 150.8, 150.6, 149.2, 149.1, 147.5, 147.4, 138.7,
130.8, 129.5, 128.5, 126.8, 125.5, 117.8, 117.6, 104.2, 95.4,
13.1; LRMS (ESI) m/z 338 [M + H]+.
5-(3,4-Dichlorophenyl)-2-methyl-3-phenylpyrazolo[1,5-
a]pyrimidin-7(4H)-one (P20). Pale-yellow solid: mp = 280 °C;
1H NMR (300 MHz, DMSO-d6) δ 12.17 (bs, 1H), 8.08 (bs,
1H), 7.78 (bs, 2H), 7.52−7.46 (m, 4H), 7.38−7.33 (m, 1H),
6.05 (bs, 1H), 2.32 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ
155.6, 150.6, 148.6, 138.8, 133.3, 131.2, 130.7, 130.6, 130.1,
129.5, 128.6, 128.4, 126.8, 104.2, 95.7, 13.1; LRMS (ESI) m/z
370 [M + H]+.
2-Methyl-5-(morpholinomethyl)-3-phenylpyrazolo[1,5-a]-
1
pyrimidin-7(4H)-one (P10). Pale-green solid: H NMR (300
MHz, CDCl3) δ 7.41−7.31 (m, 6H), 5.70 (bs, 1H), 3.68 (bs,
4H), 3.52 (bs, 2H), 2.55 (bs, 4H), 2.40 (bs, 3H); 13C NMR
(75 MHz, CDCl3) δ 157.0, 151.2, 148.6, 138.3, 131.2, 129.2,
128.2, 127.1, 103.8, 95.5, 67.0, 58.4, 53.5, 29.8, 13.4; LRMS
(ESI) m/z 325 [M + H]+.
5-Cyclohexyl-2-methyl-3-phenylpyrazolo[1,5-a]-
pyrimidin-7(4H)-one (P11). White solid: 1H NMR (300 MHz,
DMSO-d6/CDCl3) δ 11.60 (s, 1H), 7.47−7.30 (m, 5H), 5.52
(s, 1H), 2.65−2.58 (m, 1H), 2.27 (s, 3H), 1.91−1.70 (m, 5H),
1.44−1.23 (m, 5H); 13C NMR (75 MHz, DMSO-d6/CDCl3) δ
158.8, 156.4, 149.9, 138.4, 130.8, 129.4, 128.4, 126.5, 102.9,
92.1, 40.4, 31.5, 25.7, 25.1, 12.9; LRMS (ESI) m/z 308 [M +
H]+.
5-Isobutyl-2-methyl-3-phenylpyrazolo[1,5-a]pyrimidin-
7(4H)-one (P12). Off-white solid: 1H NMR (300 MHz,
DMSO-d6) δ 11.69 (s, 1H), 7.51−7.33 (m, 5H), 5.56 (s, 1H),
2.44 (d, J = 7.5 Hz, 2H), 2.26 (s, 3H), 2.04−1.91 (m, 1H),
0.91 (d, J = 6.6 Hz, 6H); 13C NMR (75 MHz, DMSO-d6) δ
155.8, 153.3, 149.8, 138.6, 130.8, 129.4, 128.7, 126.8, 102.9,
95.7, 95.6, 40.8, 27.8, 21.9, 13.0; LRMS (ESI) m/z 282 [M +
H]+.
2,3-Dimethyl-5-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-
one (P21). Off-white solid: 1H NMR (300 MHz, DMSO-d6) δ
11.90 (s, 1H), 7.76−7.74 (m, 2H), 7.56−7.54 (m, 3H), 5.81
(s, 1H), 2.22 (s, 3H), 2.10 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) δ 156.7, 152.3, 150.7, 139.9, 133.6, 131.4, 129.5,
128.5, 97.2, 94.52, 94.47, 13.0, 7.4; LRMS (ESI) m/z 240 [M
+ H]+.
2-Methyl-3-phenyl-5-(pyridin-3-yl)pyrazolo[1,5-a]-
1
pyrimidin-7(4H)-one (P13). Off-white solid: H NMR (300
MHz, DMSO-d6) δ 12.26 (bs, 1H), 8.95 (s, 1H), 8.72 (d, J =
4.5 Hz, 1H), 8.18 (d, J = 7.5 Hz, 1H), 7.57−7.46 (m, 5H),
7.38−7.30 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ 156.3,
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3,5-Diphenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (P22).
Off-white solid: H NMR (300 MHz, DMSO-d6) δ 12.26
refinement of the XYZ-centroids of 165 reflections above
20σ(I) with 11.33° < 2θ < 118.2°. Data were corrected for
absorption effects using the Multi-Scan method (SADABS).
The ratio of minimum to maximum apparent transmission was
0.904. The calculated minimum and maximum transmission
coefficients (based on crystal size) are 0.5500 and 0.9200.
The structure was solved and refined using the Bruker
SHELXTL Software Package, using the space group P121/c1,
with Z = 4 for the formula unit, C20H16FN3O2. The final
anisotropic full-matrix least-squares refinement on F2 with 238
variables converged at R1 = 3.76% for the observed data and
wR2 = 10.14% for all data. The goodness-of-fit was 1.167. The
largest peak in the final difference electron density synthesis
was 0.545 e−/Å3, and the largest hole was −0.395 e−/Å3 with
an RMS deviation of 0.133 e−/Å3. On the basis of the final
model, the calculated density was 1.432 g/cm3 and F(000),
728 e−.
1
(bs, 1H), 8.22 (s, 1H), 7.84 (d, J = 6.3 Hz, 2H), 7.69 (d, J =
6.3 Hz, 2H), 7.58−7.53 (m, 3H), 7.49−7.43 (m, 2H), 7.33−
7.29 (m, 1H), 6.05 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ
156.9, 152.4, 143.1, 138.7, 133.6, 131.5, 129.3, 128.8, 128.3,
127.2, 106.5, 95.8; LRMS (ESI) m/z 288 [M + H]+.
2-Isopropyl-3,5-diphenylpyrazolo[1,5-a]pyrimidin-7(4H)-
one (P23). Off-white solid: 1H NMR (300 MHz, DMSO-d6) δ
12.15 (s, 1H), 7.73 (d, J = 7.8 Hz, 2H), 7.56−7.44 (m, 7H),
7.38−7.33 (m, 1H), 5.92 (s, 1H), 3.10−3.01 (m, 1H), 1.18 (d,
J = 6.9 Hz, 6H); 13C NMR (75 MHz, DMSO-d6) δ 159.2,
156.1, 151.1, 139.0, 132.9, 130.9, 130.6, 130.2, 128.5, 128.1,
127.0, 103.2, 94.9, 26.0, 22.1; LRMS (ESI) m/z 330 [M + H]+.
5-(3,4-Dichlorophenyl)-3-(4-fluorophenyl)-2-
methylpyrazolo[1,5-a]pyrimidin-7(4H)-one (P24). Off-white
solid: 1H NMR (300 MHz, DMSO-d6) δ 12.14 (bs, 1H), 8.07
(s, 1H), 7.77 (s, 2H), 7.56 (bs, 2H), 7.30 (t, J = 8.7 Hz, 2H),
6.06 (s, 1H), 2.29 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ
162.9, 159.7, 155.5, 150.3, 148.7, 139.1, 133.3, 131.7, 131.6,
131.3, 130.7, 130.0, 128.9, 128.3, 128.2, 127.1, 127.0, 125.3,
115.5, 115.2, 103.5, 95.6, 12.9; LRMS (ESI) m/z 388 [M +
H]+.
Crystallographic data has been deposited in the Cambridge
Structural Database under CCDC 2034666. These data can be
obtained free of charge from FIZ Karlsruhe via www.ccdc.cam.
ac.uk/structures.
Biology. Efficacy and Validating Inhibition of Pyrazolo-
[1,5-a]pyrimidin-7(4H)-one Series against Mtb In Vitro and
Ex Vivo. The antimicrobial susceptibility testing against Mtb
H37Rv and the P19R strains was performed in either 7H9/
ADC (4.7 g/L 7H9 base, 5 g/L albumin fraction V, 2 g/L
glucose, 0.81 g/L NaCl, 0.02% glycerol, and 0.05% Tween 80)
or 7H9/GCas (4.7 g/L 7H9 base, 4 g/L glucose, 0.81 g/L
NaCl, 0.3 g/L Casitone, and 0.05% Tyloxapol). Bacteria were
grown in the corresponding media up to an OD650nm of 0.2−
0.4. A 2-fold serial dilution series of the test compounds was
placed in each well of a sterile 96-well round-bottom plate, and
then, 50 μL of H37Rv diluted to an OD650nm of 0.0002 was
added. Plates were incubated at 37 °C for 2 weeks prior to
determining the MIC. The MIC is defined here as the drug
concentration that completely inhibits the growth of the cells.
The MICs under iron-replete or -deplete conditions were done
by supplementing the growth medium with either 100 μM of
DFO (deplete) or 250 μM FeCl3 (replete).
3-(4-Chlorophenyl)-5-(3,4-difluorophenyl)-2-
methylpyrazolo[1,5-a]pyrimidin-7(4H)-one (P25). Pale-yel-
1
low solid: H NMR (300 MHz, DMSO-d6) δ 12.11 (bs,
1H), 7.95 (t, J = 9.6 Hz, 1H), 7.67−7.50 (m, 6H), 6.06 (s,
1H), 2.32 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 155.6,
152.6, 152.4, 150.8, 150.7, 149.2, 149.1, 147.6, 147.4, 139.2,
131.5, 131.3, 129.7, 128.5, 125.5, 117.9, 117.8, 117.6, 117.5,
103.2, 95.4, 13.0; LRMS (ESI) m/z 372 [M + H]+.
3-(4-Chlorophenyl)-5-(3,4-difluorophenyl)pyrazolo[1,5-a]-
1
pyrimidin-7(4H)-one (P26). Off-white solid: H NMR (300
MHz, DMSO-d6) δ 12.18 (bs, 1H), 8.25 (s, 1H), 8.08−8.01
(m, 1H), 7.75 (bs, 2H), 7.69−7.60 (m, 2H), 7.51 (d, J = 6.9
Hz, 2H), 6.15 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ
155.9, 152.7, 152.5, 150.9, 150.8, 149.4, 149.2, 147.7, 147.5,
142.1, 138.6, 131.1, 129.6, 129.3, 128.6, 125.6, 117.9, 117.7,
105.0, 95.6; LRMS (ESI) m/z 358 [M + H]+.
For the ex vivo efficacy test, J774 cells (2 × 104 cells/well)
were seeded in flat-bottom 24 well plates (Corning Inc.) in
DMEM GlutaMAX (Gibco/ThermoFisher Scientific) supple-
mented with 10% fetal bovine serum, 20 mM HEPES + 0.5
mM sodium pyruvate (hereafter abbreviated DMEM/FBS).
Cells were infected with Mtb (1 × 105 cells/well, MOI 1:5) for
24 h, followed by medium removal and washing (2×) with an
equal volume of Dulbecco’s PBS. Infected cells were fed
DMEM/FBS and exposed to test compounds at the specified
concentrations in fresh growth medium (1000 μL/well). Cells
were incubated at 37 °C and 95% humidity in 5% CO2
incubator for 7 days. The medium was changed every 2
days, and the infected cells were treated with drugs each time.
After 7 days of incubation, 0.1% SDS was added in each well to
lyse the J774 cells. After 5 min, the lysate was thoroughly
mixed and diluted in 7H9/ADC, and the appropriate dilutions
were plated in duplicate on 7H11/OADC plates to calculate
most probable number of bacteria. Colonies were counted
manually in each plate following 6−8 weeks of incubation at 37
°C.
5-(3,4-Difluorophenyl)-3-phenyl-2-(trifluoromethyl)-
pyrazolo[1,5-a]pyrimidin-7(4H)-one (P27). Pale-yellow solid:
1H NMR (300 MHz, DMSO-d6) δ 7.94−7.87 (m, 1H), 7.63−
7.60 (m, 2H), 7.51−7.44 (m, 4H), 6.21 (s, 1H); 13C NMR (75
MHz, DMSO-d6) δ 155.4, 152.8, 150.8, 150.6, 149.4, 149.3,
147.5, 147.3, 141.6, 141.1, 140.6, 130.4, 129.9, 128.5, 128.2,
128.0, 126.0, 119.6, 118.2, 117.9, 117.7, 104.1, 96.3; LRMS
(ESI) m/z 392 [M + H]+.
X-ray Crystallography. A crystal of compound 4 of
approximate dimensions 0.100 × 0.200 × 0.800 mm was
used for X-ray crystallographic analysis at T = 100 K. The X-
ray data were collected on a Bruker D8 QUEST system
equipped with a multilayer mirror monochromator using a Cu
Kα microfocus sealed tube. A total of 3013 frames were
collected. The total exposure time was 3.20 h. The frames were
integrated with the Bruker SAINT software package using a
narrow-frame algorithm. The integration of the data using a
monoclinic unit cell yielded a total of 55 109 reflections to a
maximum θ angle of 61.24° (0.88 Å resolution), of which 2473
were independent (average redundancy of 22.284, complete-
ness = 99.4%, Rint = 5.22%, Rsig = 1.59%) and 2299 (92.96%)
were greater than 2σ(F2). The final cell constants of a =
12.9908(5) Å, b = 10.2770(4) Å, c = 12.2626(5) Å, β =
98.196(2)°, and volume = 1620.41(11) Å3 are based upon the
Cytotoxicity Testing against Mammalian Cells. The in
vitro cytotoxicity of the pyrazolopyrimidinones were measured
in DMEM supplemented with 10% FBS using either glucose (5
mM) or galactose (10 mM) as the carbon source. Briefly, 1 ×
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Article
104 cells (HepG2 or J774) were seeded onto each well of a
sterile 96-well tissue culture treated flask and left to attach
overnight. The next day, media was aspirated and replaced
with a 2-fold serial dilution of compounds in the same media.
Cells were treated for 24 h, prior to viability determination
using CellTiter-Glo (Promega) according to the manufac-
turer’s instructions.
Iron Uptake Studies. To determine the role of the test
compounds in the iron uptake of Mtb, logarithmically growing
cells were labeled with 55Fe metabolically by supplementing
the growth medium with 0.75 μM 55FeCl3. The cells were then
treated with either DFO (39 μM) or 1 (10 μM) for 24 h prior
to collecting the cells and washing exhaustively in PBS. The
cells were pelleted and resuspended in 200 μL of scintillation
fluid, and counts per minute (CPM) was counted by a
scintillation counter (Beckman Coulter LS6500).
phase consisting of MeOH/acetonitrile = 1/1 with 0.2%
formic acid and 0.1% trifluoroacetic acid added. The flow rate
was 0.2 mL/min. The MS was operated at a resolution of at
least 25 000, and accurate mass assignments were determined
using the internal standard method.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00851.
Microsomal stability data for key compounds; metabo-
lite identification including QQQ mass spectra; proton
and carbon NMR spectra for all molecules (PDF)
SMILES and MIC data (XLSX)
Generation and Characterization of Pyrazolo[1,5-a]-
pyrimidin-7(4H)-one Resistant Mutants. To generate mutants
against the pyrazolo[1,5-a]pyrimidin-7(4H)-one series, 107,
108, and 109 cells of Mtb H37Rv were plated on 7H11/OADC
plates containing with 5× and 10× MIC of the drugs, with
drug-free plates used to enumerate bacterial load. The plates
were incubated at 37 °C for 4−6 weeks. After 4 weeks,
colonies on both the 5× and 10× MIC of P19 were picked and
inoculated in 7H9/ADC/Tween medium and the MIC was
tested to confirm their resistance against P19. Genomic DNA
of mutants was isolated by the cetyl trimethyl ammonium
bromide method. Whole genome sequencing was performed
and analyzed as described.25,32
AUTHOR INFORMATION
■
Corresponding Author
Clifton E. Barry, III − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20892, United
States; Institute for Infectious Disease and Molecular
Medicine, University of Cape Town, Cape Town 7935, South
Africa; orcid.org/0000-0002-2927-270X; Phone: +1-
301-435-7509; Email: cbarry@niaid.nih.gov; Fax: +1-301-
480-3506
Determination of Drug Metabolism in Live Bacteria. To
determine the degree of drug metabolism in live Mtb and P19R
strains, the LC/MS-based protocol following compound
exposure was employed. Cells were grown to an OD of 1.0,
washed twice with PBS, and resuspended using PBS at 1/3 the
original culture volume (final OD ∼ 3.0). Then, 1 mL of the
cell suspension was added to enough compound in DMSO to
create a final compound concentration of 10× MIC. A 200 μL
aliquot was removed for the 0 h time point and mixed with an
equal volume of acetonitrile and kept at 4 °C. After 24 and 48
h, another 200 μL aliquot was removed and mixed with
acetonitrile. The cells in these aliquots were pelleted, and the
supernatant was filtered through a 0.2 μm centrifuge filter prior
to injecting directly into the LC/MS. The LC gradient used to
separate the metabolites was 5−95% acetonitrile in H2O over
20 min. Extracted ion chromatograms were used to determine
the % degradation, which was calculated by comparing 0, 24,
and 48 h time points. The structural evaluation of the P19
catabolism into the metabolites was performed on an Agilent
1290 Infinity HPLC with a 6460C triple quadrupole mass
selective detector in product ion mode. Separation was
achieved with an Agilent EclipsePlus C18 column (2.1 × 50
mm, 1.8 μm) with a linear gradient of 95% aq. 0.1% formic
acid to 95% acetonitrile (with 0.1% formic acid) over 5 min
with a 0.8 mL/min flow rate. Energy resolved mass
spectrometry profiles were created in product ion mode with
collision energy ranging from 0 to 80 V (x-axis is the center-of-
mass calculation with nitrogen gas) with electro-spray
ionization in positive mode. For high resolution mass
spectrometry (HRMS) by Waters Xevo-G XS QTof, ions
were produced using positive ion electrospray at a capillary
voltage of 2.8 kV. The ESI source temperature was 280 °C.
The LC system was a Waters Acquity Class I UPLC. Samples
were stream injected using the loop method into the mobile
Authors
Sangmi Oh − Tuberculosis Research Section, Laboratory of
Clinical Immunology and Microbiology, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892, United States; orcid.org/
0000-0003-0895-1110
M. Daben J. Libardo − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20892, United
States
Shaik Azeeza − Tuberculosis Research Section, Laboratory of
Clinical Immunology and Microbiology, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892, United States
Gary T. Pauly − Chemical Biology Laboratory, National
Cancer Institute, Frederick, Maryland 21702, United States
Jose Santinni O. Roma − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20892, United
States
Andaleeb Sajid − Tuberculosis Research Section, Laboratory of
Clinical Immunology and Microbiology, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892, United States
Yoshitaka Tateishi − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20892, United
States
Caroline Duncombe − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases, National
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Article
(5) Lechartier, B., Rybniker, J., Zumla, A., and Cole, S. T. (2014)
Tuberculosis drug discovery in the post-post-genomic era. EMBO
Mol. Med. 6, 158−168.
Institutes of Health, Bethesda, Maryland 20892, United
States
Michael Goodwin − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20892, United
States
Thomas R. Ioerger − Department of Computer Science and
Engineering, Texas A&M University, College Station, Texas
77843, United States
Paul G. Wyatt − Drug Discovery Unit, Division of Biological
Chemistry and Drug Discovery, School of Life Sciences,
University of Dundee, Dundee DD1 5EH, United Kingdom;
orcid.org/0000-0002-0397-245X
Peter C. Ray − Drug Discovery Unit, Division of Biological
Chemistry and Drug Discovery, School of Life Sciences,
University of Dundee, Dundee DD1 5EH, United Kingdom
David W. Gray − Drug Discovery Unit, Division of Biological
Chemistry and Drug Discovery, School of Life Sciences,
University of Dundee, Dundee DD1 5EH, United Kingdom
Helena I. M. Boshoff − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20892, United
States; orcid.org/0000-0002-4333-206X
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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(13) Puente, A., Ofial, A. R., and Mayr, H. (2017) Nucleophilic
This work was funded in part by the Intramural Research
Program of NIAID (AI000693-25) and by grants from the
Foundation for the National Institutes of Health (BAR-
RY11HTB0) with support from the Bill & Melinda Gates
Foundation (OPP1024021).
Reactivities of Bis-Acceptor-Substituted Benzyl Anions. Eur. J. Org.
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Li, X. F., Zhang, X. G., and He, C. Y. (2018) Catalyst-free and visible
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ABBREVIATIONS
■
MIC, minimum inhibitory concentration; AcOH, acetic acid;
DIPEA, N,N-diisopropylethylamine; DMSO, dimethyl sulf-
oxide; Me, methyl; Ph, phenyl; Bn, benzyl; isoPr, isopropyl;
INH, isoniazid; MOX, moxifloxacin; ETH, ethionamide;
NMR, nuclear magnetic resonance; LRMS, low resolution
mass spectrometry; HRMS, high resolution mass spectrome-
try; LC/MS, liquid chromatography/mass spectrometry; ESI,
electrospray ionization; BSA, bovine serum albumin
(16) Cole, S. T. (2016) Inhibiting Mycobacterium tuberculosis
within and without. Philos. Trans. R. Soc., B 371, 20150506.
(17) Naran, K., Moosa, A., Barry, C. E., 3rd, Boshoff, H. I., Mizrahi,
V., and Warner, D. F. (2016) Bioluminescent Reporters for Rapid
Mechanism of Action Assessment in Tuberculosis Drug Discovery.
Antimicrob. Agents Chemother. 60, 6748−6757.
(18) Moosa, A., Lamprecht, D. A., Arora, K., Barry, C. E., 3rd,
Boshoff, H. I. M., Ioerger, T. R., Steyn, A. J. C., Mizrahi, V., and
Warner, D. F. (2017) Susceptibility of Mycobacterium tuberculosis
Cytochrome bd Oxidase Mutants to Compounds Targeting the
Terminal Respiratory Oxidase, Cytochrome c. Antimicrob. Agents
Chemother. 61, No. e01338-17.
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ACS Infect. Dis. 2021, 7, 479−492