Novel pyrimidines as antitubercular agents

Article abstract of DOI:10.1128/AAC.02063-17

Mycobacterium tuberculosis infection is responsible for a global pandemic. New drugs are needed that do not show cross-resistance with the existing front-line therapeutics. A triazine antitubercular hit led to the design of a related pyrimidine family. Th

Full text of DOI:10.1128/AAC.02063-17

AAC Accepted Manuscript Posted Online 8 January 2018
Antimicrob. Agents Chemother. doi:10.1128/AAC.02063-17
Copyright © 2018 American Society for Microbiology. All Rights Reserved.
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Novel Pyrimidines as Antitubercular Agents
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,*
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Daigo Inoyama , Steven D. Paget
, Riccardo Russo , Srinivasan Kandasamy ,
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Pradeep Kumar , Eric Singleton , James Occi, Margareta Tuckman , Matthew D. Zimmerman ,
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Hsin Pin Ho , Alexander L. Perryman , Véronique Dartois , Nancy Connell , and Joel S.
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,2,#
Freundlich
1. Department of Pharmacology, Physiology and Neuroscience, Rutgers University – New Jersey
Medical School, Newark, New Jersey, USA.
2. Division of Infectious Disease, Department of Medicine and the Ruy V. Lourenço Center for
the Study of Emerging and Re-emerging Pathogens, Rutgers University - New Jersey Medical
School, Newark, New Jersey USA.
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3. Public Health Research Institute, Rutgers University – New Jersey Medical School, Newark,
New Jersey, USA.
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Address correspondence to Joel S. Freundlich, freundjs@rutgers.edu.
Running Head: Antitubercular pyrimidines
Keywords: Mycobacterium tuberculosis, antitubercular, pyrimidine, pharmacokinetics.
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D.I. and S.D.P. contributed equally to this work.
&
Present address: Venenum BioDesign, Hamilton, NJ, USA.
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ABSTRACT
Mycobacterium tuberculosis infection is responsible for a global pandemic. New drugs
are needed that lack cross-resistance with the existing front-line therapeutics. A triazine
antitubercular hit led to the design of a related pyrimidine family. The synthesis of a focused
series of these analogs facilitated exploration of their in vitro activity, in vitro cytotoxicity,
physiochemical, and Absorption-Distribution-Metabolism-Excretion properties. Select
pyrimidines were then evaluated for their mouse pharmacokinetic profiles. The findings suggest
a rationale for the further evolution of this promising series of antitubercular small molecules,
which appear to share some similarities with the clinical compound PA-824 in terms of
activation, while highlighting more general guidelines for the optimization of small molecule
antituberculars.
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INTRODUCTION
Infection with Mycobacterium tuberculosis is responsible for tuberculosis (TB), a disease
of global significance which infects nearly nine million people and kills 1.4 million people on an
annual basis.(1) Despite whole-cell high-throughput phenotypic screening of nearly a million
compounds from both commercial and pharmaceutical libraries, the TB drug discovery pipeline
still suffers from a dearth of validated therapeutic strategies comprised of novel biological targets
and high-quality small molecules that modulate them.(2) Our laboratory has been focused on
leveraging novel computational techniques to discover, and in some cases re-discover,
antitubercular chemotypes as growth inhibitors of M. tuberculosis.(3) We assert that growth
inhibitors with new chemical scaffolds should have a heightened probability of interfering with
the native function of M. tuberculosis proteins outside the sphere of inhibition of our current set
of antitubercular drugs and chemical tools.
Our use of naïve Bayesian models (heretofore referred to as Bayesian models) trained
with screening data for M. tuberculosis growth inhibition and Vero cell cytotoxicity from the
Southern Research Institute led to the re-discovery of diaminotriazine nitrofurylhydrazones as
potent growth inhibitors of in vitro cultured M. tuberculosis. Our initial profiling of TCMDC-
125802 (renamed, JSF-2019; Fig. 1) found the molecule to exhibit a favorable lack of
cytotoxicity to Vero cells and mouse bone marrow macrophages in addition to tolerability in
mice, but failed to demonstrate in vivo efficacy in a  interferon gene disrupted C57BL/6 mouse
model of M. tuberculosis infection.(4) Efforts in our laboratories to explore the structure-activity
relationships (SAR) pertinent to this series led to pyrimidine analogs. An exploratory set of
pyrimidines is described herein, detailing their SAR pertinent to in vitro activity, Vero cell
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cytotoxicity, physiochemical properties, in vitro Absorption-Distribution-Metabolism-Excretion
(ADME), and mouse pharmacokinetics (PK).
MATERIALS AND METHODS
Chemistry: The synthesis of all pyrimidine molecules in this study was adapted from published
methods and is detailed in the Supplementary Materials.
Determination of minimum inhibitory concentration (MIC) against M. tuberculosis: The M.
tuberculosis H37Rv strain was grown in 7H9 broth (Becton, Dickinson and Company 271310),
plus 0.2% glycerol (Sigma G5516), 0.25% Tween 80-20% (Sigma P8074) and 20% 5x
ADC. The 5x ADC solution was prepared using 25 g/L Bovine Serum Albumin (Sigma A9647),
10g/L dextrose (Sigma D9434), and 4.2 g/L NaCl (Sigma S5886). Each compound was dissolved
in DMSO at a final concentration of 12 mg/mL and serial dilutions were performed to generate
typical test concentrations ranging from 50 – 0.024 g/mL. As appropriate, further dilutions
were made to determine an accurate MIC. M. tuberculosis strain H37Rv at the mid-logarithmic
stage of growth (OD580 = 0.4) was diluted 1:100 and 0.1 mL was added to each well of a 96-well
plate along with 0.1 mL of test compound solution. After 6 days of incubation at 37 °C, Alamar
Blue (Invitrogen, Grand Island, NY) reagent was added along with 12.5 L of 20% Tween 80
(Sigma, St. Louis, MO) to evaluate bacterial cell viability. Plates were scanned 24 h later at 570
nm with a reference wavelength of 600 nm utilizing a Biotek Instruments ELX 808. Inoculum
control wells of untreated H37Rv were used to create a survival inhibition curve with each assay.
Rifampicin was used as a positive control (MIC = 0.0125 - 0.05 g/mL).
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Determination of minimum bactericidal concentration (MBC) against M. tuberculosis: To
determine the minimum bactericidal concentration (MBC) against M. tuberculosis, the bacterial
cultures from the 96-well plates used for the compound MIC determination instead of
undergoing Alamar Blue addition were re-suspended, serially diluted with sterile PBS and plated
on Middlebrook 7H11 plates. Colony Forming Units (CFUs) were enumerated following 21-day
incubation at 37 °C. The MBC was reported as the minimum compound concentration at which a
2 log10 reduction in CFUs was observed as compared to the no-compound control.
Determination of kinetics of cidality against M. tuberculosis: Determination of kinetics of
cidality against M. tuberculosis: A 10 mL culture of M. tuberculosis H37Rv was grown up to
an OD600 of 0.6-0.8 in Middlebrook 7H9 medium supplemented with 10% ADS (albumin,
dextrose, sodium chloride), 0.25% glycerol and 0.05% Tween-80. The cells were harvested by
centrifugation, washed twice with Middlebrook 7H9 plus ADS without any detergent and the
pellet was resuspended in 10 mL of the same media. The cells were then diluted (1:100) and 100
µL aliquots of cells were added to wells in a 96-well plate. Stock solutions of isoniazid, JSF-
2371, JSF-2245 and JSF-2019 were prepared in DMSO, and concentrations of 10X, 30X and
60X of the respective MIC for each compound were prepared in 7H9 plus ADS media. 100 µL
of drug solution was added to the appropriate wells. The plates were incubated at 37 °C, aliquots
were removed from each culture at different time points, serially diluted and plated on
Middlebrook 7H10 agar plates to determine the CFUs.
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Physiochemical and ADME profiling: This set of studies was performed by BioDuro,
Incorporated. Published protocols were utilized to determine mouse and human liver microsomal
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stability,(5) kinetic solubility,(6) Caco-2 permeability,(7) mouse plasma protein binding,(8) and
human cytochrome P450 inhibition.(9)
Determination of efficacy in an intracellular infection model: The activity of JSF-2371
against intracellular bacteria was determined by infecting J774.1 mouse macrophages (ATCC
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TIB-67) with the mc 6206 leuCD panCD strain of M. tuberculosis transformed with an mLux
plasmid (kindly provided by Professor Jeffery Cox, UC Berkeley) based on published
protocols.(10, 11) The infected macrophages were exposed to JSF-2371, rifampicin, or no drug
for 72 h. Luminescence was measured every 24 h and viable bacteria inside the macrophages
were recovered at the end of the experiment. The macrophages were suspended in Dulbecco’s
Modified Eagle Medium (Sigma-Aldrich D6429) supplemented with 10% Fetal Bovine Serum
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(Sigma-Aldrich F6178) to a concentration of 1.0 – 2.0 x 10 cells/mL. 96-well white flat-
bottomed assay plates were seeded with 100 L of the macrophage suspension and incubated
overnight to allow cells to adhere to the plate. The assay plates were then inoculated with 100 L
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of luminescent mc 6206 M. tuberculosis at a multiplicity of infection of 1:1. The plates were
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to allow the bacteria to infect the macrophages and then
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washed twice with 100 L of DMEM supplemented with FBS, pantothenic acid and leucine.
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After the second wash, 100 L of DMEM with gentamicin was added to each well and the plates
were incubated for an additional 2 h to kill any remaining extracellular bacteria. The plates were
washed twice with 100 L of DMEM supplemented with FBS, pantothenic acid and leucine, and
then JSF-2371 or rifampicin was added to the plates at the desired concentrations. The plates
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were incubated at 37 °C and 5% CO for 72 h. Luminescence was measured every 24 h using the
GloMax® Detection System (Promega). Each treatment was done in triplicate and the
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experiment was repeated twice. To determine the number of live bacteria present in macrophages
after 72 h from infection, macrophages were washed with warm PBS and then lysed by adding
200 L of 0.02% Triton X-100 (Sigma-Aldrich X100) to the wells. The plates were incubated for
15 min at room temperature and then wells were mixed vigorously by pipetting to release
intracellular bacteria. Serial dilutions of the lysed macrophages were prepared and plated on
7H10 agar plates. The plates were incubated at 37 °C for 3 weeks and then counted for colonies.
Mouse pharmacokinetics (PK) studies
Animals and ethics assurance: Animal studies were carried out in accordance with the guide
for the care and use of Laboratory Animals of the National Institutes of Health, with approval
from the Institutional Animal Care and Use Committee (IACUC) of the New Jersey Medical
School, Rutgers University, Newark. All animals were maintained under specific pathogen-free
conditions and fed water and chow ad libitum, and all efforts were made to minimize suffering or
discomfort. In the 5 h PK studies, two female CD-1 mice received a single dose of experimental
compound administered orally at 25 mg/kg in 5% DMA/60% PEG300/35% D5W (5% dextrose
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in water), and blood samples were collected in K EDTA coated tubes pre-dose, 0.5, 1, 3 and 5 h
post-dose. In iv/po PK studies, groups of three female CD-1 mice received a single dose of
experimental compound administered orally at 25 mg/kg in 0.5% CMC/0.5% Tween 80
suspension, or intravenously at 5 mg/kg in 5% DMA/95% (4 % Cremophor EL). Blood samples
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were collected in K EDTA coated tubes 0.25, 0.5, 1, 3, 5 and 8 h post-dose in the oral arm, and
0.033, 0.25, 0.5, 1 and 3 h post dose in the intravenous arm. Blood was kept on ice and
centrifuged to recover plasma, which was stored at -80 °C until analyzed by HPLC coupled to
tandem mass spectrometry (LC-MS/MS).
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LC/MS-MS analytical methods: LC/MS-MS quantitative analysis for all molecules was
performed on a Sciex Applied Biosystems Qtrap 4000 triple-quadrupole mass spectrometer
coupled to an Agilent 1260 HPLC system, and chromatography was performed on an Agilent
Zorbax SB-C8 column (2.1x30 mm; particle size, 3.5 µm) using a reverse phase gradient elution.
Milli-Q deionized water with 0.1% formic acid (A) was used for the aqueous mobile phase and
0.1% formic acid in acetonitrile (B) for the organic mobile phase. The gradient was: 5-90% B
over 2 min, 1 min at 90% B, followed by an immediate drop to 5% B and 1 min at 5% B.
Multiple-reaction monitoring of parent/daughter transitions in electrospray positive-ionization
mode was used to quantify all molecules. Sample analysis was accepted if the concentrations of
the quality control samples and standards were within 20% of the nominal concentration (Quality
control statistics may be found in Supplementary Table 3). Data processing was performed using
Analyst software (version 1.6.2; Applied Biosystems Sciex). Neat 1 mg/mL DMSO stocks for all
compounds were first serial diluted in 50/50 acetonitrile/water and subsequently serial diluted in
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drug free CD-1 mouse plasma (K EDTA, Bioreclamation IVT, NY) to create standard curves
(linear regression with 1/x^2 weighting) and quality control (QC) spiking solutions. 20 µL of
standards, QCs, control plasma, and study samples were extracted by adding 200 µL of
acetonitrile/methanol 50/50 protein precipitation solvent containing the internal standard (10
ng/mL verapamil). Extracts were vortexed for 5 minutes and centrifuged at 4000 rpm for 5
minutes. 100 µL of supernatant was transferred for HPLC-MS/MS analysis and diluted with 100
µL of Milli-Q deionized water.
RESULTS
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Initial design considerations for interrogation of the SAR around JSF-2019 led to
consideration of pyrimidine analogs. The pyrimidine analogs initially featured the same
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substituents as the original hit (JSF-2019) at the R , R , and R positions to determine the effect
of replacing the triazine core with pyrimidine. The pyrimidine core can possess the 5-nitrofuryl
hydrazone at either the 2- or 6-position with respect to the heteroatoms of the pyrimidine ring
(Figure 1), giving rise to two regioisomers. The dual-event Bayesian model (TAACF-CB2) that
predicted JSF-2019 as an active made similar projections for both pyrimidines (Supplementary
Table 1) based on their Bayesian scores (1.93 and 1.55 for the 2- and 6-hydrazone substituted
pyrimidines, respectively) exceeding the established cutoff for an active (0.427).(3) Importantly,
all three compounds exhibited calculated closest distance values of 0.887 – 0.893, indicative that
they are structurally quite dissimilar from the Bayesian model’s training set.
The two regioisomeric pyrimidine analogs of JSF-2019 were synthesized following the
reaction schemes as outlined in Figures 2 and 3. Crucial to the approach was reliance on a
published strategy for control of the regioselectivity of amine additions to commercially
available 4,6-dichloro-2-(methylsulfonyl)pyrimidine.(12) This compound underwent successive
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additions of aniline to afford 2-(methylsulfonyl)-N ,N -diphenylpyrimidine-4,6-diamine.
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Displacement of the methanesulfonyl (Ms) group with hydrazine formed 2-hydrazinyl-N ,N -
diphenylpyrimidine-4,6-diamine (A). Reaction of A with the appropriate aldehyde led to 5-
nitrofurylhydrazone JSF-2245, furylhydrazone JSF-2246, 5-nitrothienylhydrazone JSF-2275, 4-
nitrophenylhydrazone, JSF-2325, and 3-nitrophenylhydrazone JSF-2326. A was reacted with 5-
nitro-2-furoyl chloride to prepare the hydrazide JSF-2247. Alternatively, 4,6-dichloro-2-
(methylsulfonyl)pyrimidine reacted with lithium anilide to afford 4,6-dichloro-N-
phenylpyrimidin-2-amine. Sequential additions of aniline and hydrazine afforded 6-substituted
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hydrazine (B). Condensation of B with the appropriate aldehyde led to 5-nitrofurylhydrazone
JSF-2332, furylhydrazone JSF-2370, 4-nitrophenylhydrazone JSF-2327, and 5-
nitrothienylhydrazone JSF-2371. Reaction with 5-nitrofuran-2-carbonyl chloride led to the
corresponding hydrazide JSF-2372.
The synthesized pyrimidine analogs were initially assessed for their respective ability to
inhibit the growth of in vitro cultured M. tuberculosis (H37Rv strain) under standard liquid
culture conditions (media: Middlebrook 7H9 supplemented with albumin, dextrose, catalase, and
glycerol) at 37 °C via an Alamar blue assay (Table 1).(13) An MIC was determined, representing
the concentration of compound inhibiting 90% of the growth of the bacteria. To assess
cytotoxicity to a model mammalian cell line, we determined the CC50 (concentration of
compound required to inhibit 50% of the growth of the cells) for each compound versus Vero
cells – African Green monkey kidney cells (#ATCC CCL-81).(14) The selectivity index (SI)
may be defined as CC50/MIC, and one typically targets an SI ≥ 10.
A comparison of the SAR for JSF-2019, JSF-2245 and JSF-2332 demonstrated the 2-
hydrazone substituted pyrimidine (JSF-2245) to be essentially equipotent to JSF-2019, while the
6-substituted pyrimidine (JSF-2332) was threefold less active. The Vero cell-based SI values for
all three compounds were similar (within a factor of two). Replacement of the 5-nitro substituent
with a hydrogen led to a significant loss (140X) of whole-cell activity with 2-hydrazone
substituted JSF-2246, while in the 6-hydrazone case an 18-fold loss was realized. Little change
in Vero cell cytotoxicity in either series was noted. Similarly, replacements of the 5-nitrofuryl
with 5-nitrothienyl (JSF-2275), 4-nitrophenyl (JSF-2325), and 3-nitrophenyl (JSF-2326) led to
significant losses in whole-cell activity. Except for JSF-2275 (with a CC50 > 120 M), these
analogs also failed to demonstrate a substantial improvement in Vero cell cytotoxicity. The acyl
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hydrazide (JSF-2247) was slightly less active (MIC = 0.23 M), but exhibited significantly more
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Vero cell cytotoxicity (CC50 = 1.8 M).
The 6-substituted pyrimidine series displayed some similar trends to the 2-substituted
series such as the significant loss of whole-cell efficacy when replacing the 5-nitrofuryl group
with a 4-nitrophenyl (JSF-2327) or furyl (JSF-2370). Similarly, the hydrazone moiety may not
be substituted by a hydrazide (JSF-2372) without an unfavorable change in antitubercular
activity. The 5-nitrothienyl analog (JSF-2371), however, did not follow the trend in the 2-
substituted series. JSF-2371 exhibited enhanced activity (MIC = 0.11 M) and reduced
cytotoxicity as compared to JSF-2332, strikingly with an SI = 250.
Those compounds with an MIC ≤ 20 M were also profiled for their cidal activity
through determination of the minimum bactericidal concentration (MBC; minimum
concentration of compound resulting in a ≥2 log10 decrease in colony-forming units of bacteria
grown on solid culture medium) vs. M. tuberculosis. While JSF-2019 exhibited an MBC = 0.058
– 0.11 M, the two most cidal pyrimidines were JSF-2245 (MBC = 0.24 – 0.48 M) and JSF-
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2371 (MBC = 0.44 – 0.92 M). Thus, both analogs may be considered bactericidal given the
criteria that the MBC/MIC ≤ 4. The kinetics of cidality for these two compounds were also
determined and compared to JSF-2019. JSF-2245 at 30X MIC, somewhat similar to JSF-2019,
afforded a >6 log10 kill over 10 d. JSF-2371, at a similar multiple of its MIC, reduced the CFUs
by ~ 1 log10 over 10 – 14 d and then a rebound was observed through an outgrowth of persistent
and/or resistant bacteria.
The kinetic solubility of the original hit JSF-2019 in phosphate-buffered saline (PBS) at
pH 7.4 was determined to be less than 0.06 M (Table 2). The lack of in vivo efficacy of JSF-
2019 in a mouse model of M. tuberculosis may in part be attributed to its poor solubility. As
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expected based on their slightly increased clogP values as compared to JSF-2019, none of the
subset of pyrimidine 2- or 4-hydrazones displayed a significantly enhanced aqueous solubility.
However, the solubilities in pH 7.4 PBS of hydrazide analogs JSF-2247 and JSF-2372 were
determined to be 5.96 and 9.23 M, respectively.
In parallel, the metabolic stability of the pyrimidines was assessed in the presence of
mouse liver microsomal (MLM) preparations for the determination of half-life (t1/2) and intrinsic
clearance (Clint) (Table 2). The original hit, JSF-2019, displayed acceptable metabolic stability:
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1/2 = 63.6 min and Clint = 10.9 L/min/mg microsomal protein. Hydrazone JSF-2371 and
hydrazide JSF-2372 exhibited greater metabolic stability than JSF-2019. JSF-2245 and JSF-2332,
the most similar pyrimidine analogs of JSF-2019, both exhibited significantly diminished t1/2 and
increased Clint values as compared to the triazine hit. In an attempt to flag a potential liability
downstream of initial animal studies to be conducted in mice, human liver microsomal (HLM)
stabilities were also measured for these five pyrimidines and JSF-2019. As expected, JSF-2019
was sufficiently stable (t1/2 = 77.9 min) as were pyrimidines JSF-2372 (t1/2 = 100 min) and JSF-
2371 (t1/2 = 68.6 min).
The pharmacokinetic (PK) profile of select pyrimidine analogs in female CD-1 mice via
oral (po) administration was determined (Figure 4 and Table 3). Of significance were the
exposure of the compound in plasma, related to its area under the curve (AUC) value, and a
qualitative determination of how much exposure was above the MIC. In addition, since
tuberculosis is primarily a pulmonary disease, the ratio of Clung/Cplasma was determined at t = 5 h.
For all compounds tested except JSF-2247, this value was >1.0 and deemed acceptable. The
AUC of JSF-2019 was determined to be 869.1 h×ng/mL. As shown in Figure 4A, the compound
displayed good exposure as both mice had a Cplasma that was higher than the MIC (0.0625
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g/mL) over the course of the 5 h study. JSF-2245 and JSF-2371 also exhibited Cplasma levels
higher than their MIC for more than 3 and 5 h, respectively. Both compounds were determined to
have an AUC comparable to that of the original hit (788.5 and 726.4 h×ng/mL, respectively).
JSF-2332 demonstrated a similar AUC value, but its Cplasma was below the MIC within 1–2 h.
The exposure of JSF-2247 (AUC = 534.6 h×ng/mL) was slightly diminished compared to these
three pyrimidines and its Cplasma dropped below the MIC after 1.5 – 2.5 h. Lastly, the hydrazide
analog JSF-2372 had the lowest exposure with Cplasma never exceeding its MIC and an AUC of
162.7 h×ng/mL.
JSF-2245 exhibited a favorable PK profile characterized by a slightly greater exposure
than JSF-2371, consistent with its higher Caco-2 cell permeability (Table 2).(7) Significantly,
however, JSF-2371 exhibited a longer half-life in vivo, suggested by its larger MLM t1/2. In
addition, JSF-2371 exhibited minimal inhibition (IC50 values: 4.44 - >50 M) of human
cytochrome P450 enzymes (1A2, 2C9, 2C19, 2D6, and 3A4; Table 4). A concern, however, was
its high degree of mouse plasma protein binding (99.98%) and slightly low plasma stability at 5
h (70.0%) (Supplementary Table 2). It is interesting to note that both properties in the presence
of human plasma were acceptable (Supplementary Table 2). JSF-2371 was selected for PK
profiling via both intravenous (iv) and po administration to obtain the relative oral bioavailability
of the compound (Quality control statistics may be found in Supplementary Table 3). A
formulation of 5% DMA/4% Cremophor was used for iv administration of JSF-2371. Oral
administration involved a formulation of 0.5% CMC/0.5% Tween 80 and resulted in oral
bioavailability (F) of 0.5%. Finally, given the significance of M. tuberculosis infected
macrophages in the lung pathology in mouse models and actual human disease, we determined
2
that JSF-2371 was cidal to the M. tuberculosis leuCD panCD mc 6206 strain infecting J774.1
13

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cells (Supplementary Figure 1). At 1.0 g/mL, JSF-2371 afforded an ca. two log10 reduction in
bacterial colony-forming units (CFUs) as compared to the no-drug control, similar to the
rifampicin (5 g/mL) positive control. A 10 g/mL concentration of JSF-2371 reduced the
bacterial load to below the limit of quantification (~ 0 CFUs).
Efforts to continue the optimization of the pyrimidines are currently in progress,
bolstered by these initial results as well as preliminary studies to probe their mechanism of action.
Similar to JSF-2019, JSF-2371 largely maintains its in vitro activity versus clinical M.
tuberculosis strains with resistance to one or more of the following approved tuberculosis drugs:
isoniazid, rifampicin, ethambutol, kanamycin, streptomycin, and capreomycin (Supplementary
Table 4) with 4x resistance in two multi drug-resistant strains. Interestingly, JSF-2371 does not
significantly inhibit the growth of the ESKAPE bacteria while JSF-2019 demonstrates a MIC =
2.3 M versus a clinical methicillin-resistant strain of S. aureus (Supplementary Table 5). JSF-
2019 and JSF-2245 exhibit cross-resistance (8 – 32 X MIC shift) with laboratory PA-824–
resistant strains (Supplementary Table 6) harboring mutations in F420 biosynthesis (fbiB, fbiC)(15,
16) or the F420-dependent glucose-6-phosphate dehydrogenase fgd1.(17) Expectedly, JSF-2246,
lacking a nitro group, is not cross-resistant with these strains. JSF-2371 intriguingly only exhibits
low-level cross-resistance (two-fold MIC shift) with these strains. Additionally, a mutant was
generated with a transposon insertion in the PA-824 activating gene ddn,(18, 19) and JSF-2019,
JSF-2245 and JSF-2371 exhibit different losses in activity (5-, 8-, and 16-fold, respectively)
while JSF-2246 lacks cross-resistance.
DISCUSSION
14

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The potential for pyrimidine compounds as novel antitubercular agents has been
demonstrated through preliminary SAR studies taking into consideration in vitro efficacy, in
vitro cytotoxicity, physiochemical properties, in vitro ADME parameters, and mouse PK profiles.
The original hit compound JSF-2019 was discovered through a virtual screening of the GSK set
of antimalarials using a dual-event Bayesian machine learning model that simultaneously
predicts whole-cell in vitro efficacy against M. tuberculosis and a relative lack of Vero cell
cytotoxicity.(3, 20) Our SAR studies of the compound to improve its activity and PK profiles led
to pyrimidine analogs. From our preliminary SAR investigations, it was apparent that the in vitro
antitubercular efficacy, Vero cell-based SI, and MLM stability of JSF-2019 could be mostly
maintained if not improved with analogs JSF-2371 and JSF-2245. However, we were unable
concomitantly to improve aqueous solubility; further studies will need to build on the single-digit
micromolar solubilities of hydrazides JSF-2247 and JSF-2372. Insight from mouse PK studies
assessing compound exposure (two mice judged over a five hour window post a single oral dose
of 25 mg/kg) afforded a further critical evaluation of these pyrimidines and established JSF-2371
as the preferred candidate for a more extensive PK profiling. Unfortunately, JSF-2371
demonstrated poor oral bioavailability, which halted its progression to dose tolerability and
proportionality studies. Also concerning were its in vitro killing kinetic profile demonstrating
limited cidality and mouse plasma profile (very high protein binding and borderline stability).
The process of attempting to evolve the triazine JSF-2019 via the pyrimidine series also
afforded an opportunity to begin to explore their respective mechanisms of action. A common
feature amongst JSF-2019 and pyrimidines such as JSF-2245 and JSF-2371 is their
nitroheterocycle (i.e., furan or thiophene). At this juncture, through studies with spontaneous
drug-resistant mutants exhibiting PA-824 cross-resistance, we can state that JSF-2019, JSF-2245,
15

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and JSF-2371 may not share the same mechanism of activation as PA-824. From growth
inhibition assays with PA-824–resistant strains harboring a mutation in F420 biosynthesis (fbiB or
fbiC) or in F420 reduction to F420
H
2
(fgd1), we believe that nitrofurans JSF-2019 and JSF-2245
biosynthesis.
share a similar requirement for the activation of a nitroreductase through F420
H
2
Mutations in the biosynthesis of F420 (fbiB or fbiC) lead to significant losses of activity of JSF-
2019 (MIC shift of 32-fold) and JSF-2245 (MIC shift of 8–16 fold). However, nitrothiophene
JSF-2371 only exhibits a twofold loss of activity. This appears to run counter to its 16-fold loss
of activity versus the tn::ddn strain. JSF-2019 and JSF-2245 exhibit 5- and 8-fold reductions in
activity against the tn:ddn strain, suggesting that Ddn may not be the primary nitroreductase
pertinent to their activation. Ddn appears to be the primary nitroreductase for PA-824 (MIC shift
of >62-fold). Further studies are in progress to probe the mechanism/s of activation of JSF-2019,
JSF-2245, and JSF-2371. The opportunity clearly exists to purposefully design antitubercular
nitroheterocyclic small molecules with a distinct mode of activation through further optimization
of the pyrimidines disclosed herein.
While the optimization to address liabilities of the pyrimidines continues, we assert that
lessons are to be learned from our efforts to date. This reported effort has focused on
antituberculars, but we suggest that generalization of these lessons to other antibacterials and
antimicrobials should be straightforward. From its inception, a program to evolve a chemical tool
to validate a mechanism and/or to advance a drug discovery effort should focus on demonstrating
in vivo activity in the first-line animal model. For tuberculosis research, this model typically
involves the mouse, which has been utilized for over seventy years with recognition of its
strengths and weaknesses.(21, 22) Workman and Collins have written about the ideal properties
for a chemical probe and been guided principally by experiences in the oncology field.(23) We
16

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choose to more generally focus on what we term chemical tools, which may or may not exhibit
polypharmacology,(24, 25) and may seed a drug discovery effort in addition to increasing our
basic understanding of fundamental biology. Rather than relying solely on Lipinski’s Rule of
Five,(26) which while based on orally bioavailable drugs across many therapeutic areas may best
be applied to filtering large libraries for initial screens and/or prioritizing the resulting set of
hits,(23) we will suggest guidelines for compound profile parameters for antituberculars that
should help usher an optimization towards in vivo active compounds. The focus, taking a cue
from prior efforts in the pharmaceutical industry,(27) is in profiling activity, cytotoxicity,
physiochemical, ADME, and PK properties early to identify and then address chemical tool
shortcomings more efficiently.
In vitro efficacy, which may be measured in a number of assays,(28) is typically a critical
requirement for in vivo activity and should be present along with an absence of significant in
vitro and in vivo toxicity pertinent to the infected host. We assert that most typically an MIC ≤ 1
M is sufficient, although caveats involve the specifics of the growth inhibition assay (bacterial
sub-population and what aspects of the host environment are taken into account).(29) The MIC
should be at least tenfold smaller than the CC50 (SI ≥ 10). Since both intracellular and
extracellular bacteria characterize an M. tuberculosis infection in humans, the results from an
infected macrophage assay should be weighed carefully. The active compound must be present
in vivo at a sufficient concentration for an adequate time window to maintain coverage to inhibit
the growth of, or preferably be cidal to, M. tuberculosis (MBC/MIC ≤ 4; where MBC is the
minimum bactericidal concentration equal to the amount of compound resulting in a 2 log10
reduction in CFUs, as compared to the no-drug control).(30, 31)
17

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A set of molecular properties has been evolved and shown to correlate with oral
bioavailability, given the strong preference for oral administration of tuberculosis drugs.(30) It is
critical to work with soluble compounds (pH 7.4 PBS solubility of at least 1 M if not ≥100 M)
to avoid issues with formulation for in vivo studies, as well as with enzyme and cellular
assays.(32) MLM stability is crucial (t1/2 ≥ 1 h and Clint ≤ 10 L/min/mg protein) such that the
active molecule is not metabolized rapidly in the model host.(33) These microsomal preparations
represent the membrane-bound fraction from hepatocytes and predominantly exhibit Phase I
metabolic reactivity to provide more soluble and hydrophilic compounds for eventual excretion.
Promising compounds in these aforementioned assays should also exhibit favorable Caco-2 cell
permeability, prior to study in a mouse infection model. Oral absorption correlates with
permeability to these human colon adenocarcinoma cells that form tight junctions and express
transporters on the apical (A) and basolateral (B) surfaces.(34) Compounds are considered
-6
sufficiently permeable if PA-B > 10 x 10 cm/s and PB-A/PA-B should be ≤ 3, indicating a lack of
active efflux.(30) While these in vitro ADME assays are critical, the significance of the mouse
PK studies cannot be understated. The two mice/five h studies with a single oral dose of
compound allow a rough determination of compound exposure and help prioritize compounds by
their AUC0-t, Cmax, and t1/2. The goal is to generally maximize these parameters unless they are
associated with toxicity. While the reduction of M. tuberculosis burden in mice does not
correlate with one specific PK or PK/PD index, it certainly would seem to require a basal level of
oral exposure.(29)
Thus, the evolution of the pyrimidines and other concurrent programs in our laboratories
have helped clarify key in vitro activity and cytotoxicity, physiochemical, in vitro ADME, and in
vivo PK parameters and their target values to increase the likelihood of arriving at small
18

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molecule antituberculars with sufficient exposure when dosed orally in the mouse model. This
represents a multiple-objective optimization.(35) While JSF-2371 fell short of this target oral
exposure, it has reinforced the need to probe structural variations of the compound to meet this
goal. Upon achieving significant oral exposure, the compound will then offer the opportunity to
demonstrate that its mechanism of action does modulate one or more novel mycobacterial targets
of therapeutic relevance, as seen by a significant in vitro reduction in bacterial load post-dosing
regimen.
ACKNOWLEDGEMENTS
Professor William R. Jacobs, Jr. (Albert Einstein College of Medicine) is thanked for the kind
2
donation of the mc 6206 strain and Professor Jeffrey S. Cox (UC Berkeley) is acknowledged for
the gracious donation of the mLux plasmid. Mr. Xin Wang (Rutgers University) and Dr. Sean
Ekins (Collaborations Pharmaceuticals) is thanked for his critical comments during the course of
this project.
ETHICS STATEMENT
All animal studies were ethically reviewed and carried out in accordance with the Guide for the
Care and Use of Laboratory Animals of the National Institutes of Health, with approval from the
Institutional Animal Care and Use Committee (IACUC) of the New Jersey Medical School,
Rutgers University, Newark.
FUNDING INFORMATION
This work was supported by the NIH grant U19AI109713.
19

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22

O N
2
O N
2
O N
2
O
O
O
N
N
N
N
N
NH
NH
NH
6
6
2
5
N1
1
N3
1
N
3
3
N
N
N
N
N
N
2
N
N
H
H
H
H
H
H
JSF-2019
JSF-2245
JSF-2332
5
5
29
30
5
5
31
32
FIG 1 Structure of JSF-2019 and its two pyrimidine analogs.
23

5
5
33
34
2 2
FIG 2 Synthetic route to JSF-2245: (a) PhNH , 2,6-lutidine, DMSO, rt; (b) PhNH , 2,6-lutidine,
5
5
35
36
DMSO, rt; (c) NH NH , DMSO, 70 °C; (d) 5-nitro-2-furaldehyde, MeOH, rt.
2
2
24

5
5
37
38
5
5
39
40
5
5
5
5
41
42
43
44
FIG 3 Synthetic route to JSF-2332: (a) PhNH
2 2
, LiHMDS, THF, -78 ˚C; (b) PhNH , 2,6-lutidine,
DMSO, 60 °C; (c) NH NH , DMSO, 70 °C; (d) 5-nitro-2-furaldehyde, MeOH, rt.
2
2
25

5
45
(A)
JSF-2019
(B)
JSF-2245
Time (h)
Time (h)
(C)
JSF-2247
(D)
JSF-2332
Time (h)
Time (h)
(E)
JSF-2371
(F)
JSF-2372
Time (h)
Time (h)
5
5
5
5
5
46
47
48
49
50
FIG 4 The concentration of compound (Cplasma) plotted as a function of time in mouse #1 (◊) and
mouse #2 (□) for (A) JSF-2019, (B) JSF-2245, (C) JSF-2247, (D) JSF-2332, (E) JSF-2371, and
(F) JSF-2372. The dashed lines indicate the approximate MIC of each compound.
26

5
5
5
51
52
53
Table 1 Chemical structure, minimum inhibitory concentration (MIC) against the H37Rv
M. tuberculosis strain, minimum bactericidal concentration (MBC) against the H37Rv M.
tuberculosis strain, and Vero cell cytotoxicity (CC50) of pyrimidine compounds
Vero
M. tuberculosis M. tuberculosis
cell
CC50 in
g/mL
M)
a
Compound
Structure
MIC in g/mL
M)
MBC in
(
g/mL (M)
(
TCMDC-125802
0.062
(0.15)
0.024 – 0.049
(0.058 – 0.11)
4.0
(9.6)
(JSF-2019)
0
.049
0.098 – 0.20
(0.24 – 0.48)
3.1
(7.5)
JSF-2245
(0.12)
6
.2
25 – 50
(67 – 130)
6.2
(17)
JSF-2246
JSF-2247
JSF-2275
(17)
0.098
0.39 – 0.78
(0.90 – 1.8)
0.78
(1.8)
(
0.23)
>
50
> 50
(>120)
N.D.
(
(
>120)
>
50
6.2
(15)
JSF-2325
JSF-2326
JSF-2327
N.D.
N.D.
N.D.
>120)
1
2
1.6
(3.8)
(28)
>
50
25
(59)
(
>120)
0
.19
0.39 – 0.78
(0.94 – 1.9)
6.2
(15)
JSF-2332
JSF-2370
JSF-2371
(
0.46)
3
.1
>50
(>130)
12
(32)
(8.4)
0.048
0.19 – 0.39
(0.44 – 0.92)
12
(28)
(
0.11)
27

0
.78
1.6 – 3.1
(3.7 – 7.2)
12
(28)
JSF-2372
(
1.8)
a
5
54
N.D. = Not determined
5
5
55
56
28

5
5
57
58
Table 2 Physiochemical properties and in vitro Absorption-Distribution-Metabolism-
Excretion (ADME) profile of select compounds
JSF-
019
JSF-
2245
JSF-
2247
JSF-
2332
JSF-
2371
JSF-
2372
2
Kinetic
solubility in
pH 7.4 PBS
<
0.06
0.0605
25.1
5.96
26.6
< 0.06
42.5
< 0.06
65.4
9.23
70.0
(
M)
MLM
Stability t1/2
63.6
10.9
77.9
8.90
(min)
MLM
Stability
Clint
27.6
35.4
19.6
26.1
59.2
11.7
16.3
55.5
12.5
10.6
68.6
10.1
9.9
100
6.90
(
L/min/mg
protein)
HLM
Stability t1/2
min)
(
HLM
Stability
Clint
(
L/min/mg
protein)
Caco-2 cell
1
4.10
.53 /
15.1 /
9.31
a
2.27 /
0.297
0.0279 /
0.0152
a
P
A-B / PB-A
N.D.
N.D.
-
6
(
x 10 cm/s)
a
5
5
59
60
Not determined
29

5
5
61
62
Table 3 Mouse PK data for select compounds
JSF-
019
JSF-
2245
JSF-
2247
JSF-
2332
JSF-
2371
JSF-
2372
2
a
AUC
h*ng/mL)
8
69.1
17
788.5
535
49
534.6
456
98
704.1
281
190
3.7
726.4
196
48
162.7
109
780
0.21
0.14
0
(
a
C
max
3
(ng/mL)
MIC
6
2
(ng/mL)
a
AUC/MIC
14
16
5.5
15
a
C
max/MIC
5.1
11
4.7
1.5
4.1
a
T > MIC
h)
5
2
.0
.6
4.0
1.5
2.0
5.0
(
Clung/Cplasma
2.6
0.3
6.0
1.0
1.0
at 5 h
b
t
1/2 (h)
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.86
1436
1.34
n.d.
n.d.
n.d.
b
Clearance
b
d
V (L/kg)
a
5
5
5
5
5
63
64
65
66
67
Following administration of a single 25 mg/kg po dose
b
Following administration of a single 5 mg/kg iv dose
n.d. = not determined
30