Carbon-14 radiolabelling and tissue distribution evaluation of a potential anti-TB compound

Article abstract of DOI:10.1002/jlcr.3391

This paper describes a five-step synthesis of a carbon-14-labelled pyrazole compound (11). A total of 2.96 MBq of 11 was obtained with the specific activity of 2242.4 MBq/mmol. The radiochemical purity was >99%, and the overall radiochemical yield was 60% based on the [¹⁴C₆] 4-bromoaniline starting material. Biodistribution results showed that the radiotracer (administrated orally) has a high accumulation in the small intestine, large intestine and liver of both non-infected and tuberculosis (TB)-infected mice. Therefore, this suggests that compound 11 undergoes hepatobiliary clearance. The compound under investigation has been found to be slowly released from the liver between 2 and 8 h. The study revealed that 11 has no affinity for TB cells.

Full text of DOI:10.1002/jlcr.3391

Research article
Received 20 October 2015,
Revised 23 February 2016,
Accepted 25 February 2016
Published online 24 April 2016 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3391
Carbon-14 radiolabelling and tissue
distribution evaluation of a potential
anti-TB compound
a
b
c
*
Molahlehi S. Sonopo, Kobus Venter, Susan Winks,
Biljana Marjanovic-Painter,a Garreth L. Morgans,c and Jan R. Zeevaartd
This paper describes a five-step synthesis of a carbon-14-labelled pyrazole compound (11). A total of 2.96 MBq of 11 was
obtained with the specific activity of 2242.4 MBq/mmol. The radiochemical purity was >99%, and the overall radiochemical
yield was 60% based on the [14C6] 4-bromoaniline starting material. Biodistribution results showed that the radiotracer
(administrated orally) has a high accumulation in the small intestine, large intestine and liver of both non-infected and
tuberculosis (TB)-infected mice. Therefore, this suggests that compound 11 undergoes hepatobiliary clearance. The
compound under investigation has been found to be slowly released from the liver between 2 and 8 h. The study revealed
that 11 has no affinity for TB cells.
Keywords: pyrazole; C-14 labelling; tuberculosis; tissue distribution
pharmacokinetics (PK) studies carried out on the structural
Introduction
analogues of 11 on mouse model have shown that these types
Tuberculosis (TB) is a one of the leading cause of death among the
of compounds have good bioavailability and are well distributed
infectious diseases worldwide. This bacterium emerged as a disease
in the whole blood. The only concern was that all of these
threat more than one century ago, and it is estimated that 1.5
compounds showed a relatively short half-life in plasma, which
million people died from TB in 2013. Approximately nine million
new cases of TB were reported in 2013 alone, and 13% of this
was approximately 40 min. To support the PK studies, the tissue
distribution studies of compound 11 were conducted to evaluate
number were co-infected with human immunodeficiency virus
its TB affinity to the lungs, before embarking on improving the
(HIV).1–3 Although TB can be cured, the disease has been slowly
declining at a steady rate from the years 2000 and 2013, and it is
estimated that 37 million lives were saved through effective
diagnosis and treatment in those years.1 The current TB treatment
therapy consist of isoniazid, rifampicin, pyrazinamide and
ethambutol that results with the undesired side effects, which
among them, include hepatotoxicity. The treatment usually takes
a period of 6 to 12 months, increasing the chances for developing
multidrug-resistant and extensive drug-resistant infections of
Mycobacterium tuberculosis, the causative agent of TB.4–9 The
occurrence of TB in immunocompromised individuals with HIV
represents a serious health problem because the combination
increases resistance to the available antituberculosis drugs on the
market.10 Therefore, new drugs for TB, which will reduce the time
of treatment, exhibit good drug resistance against M. tuberculosis
and that will enhance TB treatment outcomes in individuals with
advanced HIV infection, are urgently needed.11,12
Compounds bearing a pyrazole moiety have shown to possess
interesting biological activities, such as anticancer, antibacterial,
anti-inflammatory, antiviral, antileishmanial and antimicrobial.13–19
In the current study, a compound bearing a pyrazol moiety from
iThemba Pharmaceuticals TB projects inventory with minimum
inhibitory concentration (MIC) against M. tuberculosis of 0.65 μM
has been radiolabelled with the carbon-14 isotope. Furthermore,
this compound was less toxic with the IC50 > 100 μg/mL. The
in vivo properties of these compounds.20 Compound 11 was
preferred over the others because of its simplicity to install the
radiolabelled molecule at the last step of the synthesis. To the best
of our knowledge, the carbon-14 radiosynthesis of pyrazole 11 has
never been reported before. Moreover, the biodistribution of this
compound has not been evaluated in experimental animals.
Results and discussion
Synthesis
To elucidate any potential unfavourable target organ early on in
the drug discovery process, it is important to incorporate a
aRadiochemistry, Necsa, P. O. Box 482, Pretoria0001, South Africa
bMedical Research Council of South Africa, Pretoria, South Africa
ciThemba Pharmaceuticals (Pty) Ltd, Johannesburg, South Africa
dDST/NWU, Preclinical Drug Development Platform, North West University,
Potchefstroom, South Africa
*Correspondence to: Molahlehi S. Sonopo, Radiochemistry, Necsa, P. O. Box 482,
Pretoria, 0001, South Africa.
E-mail: molahlehi.sonopo@necsa.co.za
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M. S. Sonopo et al.
radioisotope into the structure in order to determine the (LC-MS) that were not found to be a serious issue as further
absorption, distribution, metabolism, excretion toxicology purification by preparative HPLC was envisaged during the
studies of a compound. In this case, carbon-14 was preferred radiolabelled studies. This latter procedure was then taken
as a radiotracer because of its long half-life and the fact that through as the best protocol for synthesizing the radiolabelled
the addition of other isotopes such as fluorine-18 will most likely compound 11 (Scheme 2).
change the structure of the molecule under investigation and
Thus, the condensation of the radiolabelled C-14 4-
therefore alter its biological action.21 Because the synthesis of bromoaniline (10) with acid chloride 7 followed by acid workup
an amide side chain is straightforward, it was decided to and silica gel column chromatography gave radiolabelled 11 as
introduce the carbon-14 radiolabel through an amide formation the major product with minor impurities evident from LC-MS
reaction. [14C6] 4-bromoaniline was used as the amine precursor. analysis. Therefore, compound 11 was subsequently purified by
The carbon-14 radiosynthesis of pyrazole compound 11 was HPLC using a Phenomenex Max RP 25-cm column (0.1% acetic
accomplished in five steps starting from the commercially acid/methanol as mobile phase) (Figure 1). The radiolabelled
available
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H- product 11 was obtained in >99% radiochemical purity and
pyrazole (1) (Scheme 1). Initially, the pyrazole boronate ester 1 60% radiochemical yield (decay corrected) based on the [14C6]
was subjected to the N-alkylation reaction with ethyl 2-bromo- 4-bromoaniline starting material. The calculated specific activity
2-methylpropanoate (2) using caesium carbonate as the base was 2242.4 MBq/mmol.
to give the N-protected pyrazole ester 3 in 86% yield. The
Suzuki–Miyaura coupling of 2-bromopyridine (4) with N-
protected pyrazole ester 3 using PdCl2 (dppf) as the coupling
Tissue distribution study
catalyst furnished the pyrazole derivative 5 in 99% yield. This The tissue distribution study in both healthy and TB-infected
reaction was followed by ester hydrolysis of the pyrazole mice after the oral administration of 11 is shown in Figures 2
derivative 5 with 2.0 M lithium hydroxide solution for 12 h at and 3, respectively. Compound 11 was examined over the time
ambient temperature to deliver the expected carboxylic acid 6 course study to determine the drug distributions, that is, in the
in 78% yield. The carboxylic acid 6 was converted to the acid liver, lungs and kidneys. The compound under investigation
chloride 7 using oxalyl chloride and a catalytic amount of N,N- was produced with high enough specific radioactivity that was
dimethylformamide in dichloromethane for 4 h in high yield able to be quantified by the liquid scintillation counter. The
(99%). With acid chloride 7, in hand, the stage was set for the animals were sacrificed at 2 and 8 h, respectively, and the %ID/
Schotten–Bauman reaction. Initially, model reactions (cold organ was calculated. At 2 h, the highest uptake of the 14C-
experiments) were conducted prior to optimizing the reaction radiolabelled compound was detected in the small intestine in
conditions for the amidation coupling reaction of 2-methyl- both healthy and TB-infected mice, followed by the large
2-[4-(pyridin-2-yl)-1H-pyrazol-1-yl]propanoyl chloride (7) and intestines and the liver. The blood uptake of the radiotracer 11
the radiolabelled 4-bromoaniline (10). Thus, the condensation was generally low, reaching a maximum of 0.269 and 0.228%
of acid chloride 7 with 4-bromoaniline (8) using triethylamine at 8 h in both healthy and TB-infected mice, respectively. There
as a base at ambient temperature afforded the corresponding was no statistical difference (P > 0.2) between the results for
acetamide 9 in modest yields (50–60%) after silica gel column the TB and non-TB-infected animals.
chromatographic purification. Alternatively, better results (80%
Compound 11 exhibited high localization of radioequivalents in
yields) were obtained employing an acid washing step during the small intestines after 2 h (15%) decreasing to the 2% after 8 h
aqueous extraction followed by silica gel column in the healthy mice. However, in the large intestine, the uptake
chromatographic purification. Suffice to say, minor impurities changed from 0.73% at 2 h to 12.3% at 8 h after administration.
were detected by liquid chromatography–mass spectrometry The high uptake at 8 h indicates that radiotracer 11 migrated over
Scheme 1. Synthesis of the unlabelled targeted amide 9.
J. Label Compd. Radiopharm 2016, 59 264–269
Copyright © 2016 John Wiley & Sons, Ltd.
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M. S. Sonopo et al.
Scheme 2. Synthesis of the labelled targeted amide 11.
22
20
18
16
14
12
10
8
TB-infected 2 h
TB-infected 8 h
6
4
2
Figure 1. Radio chromatogram of the 14C-labelled amide 11.
0
Organs
Figure 3. Accumulation of 14C-labelled pyrazole 11 in various organs of the TB-
infected animals, n = 3, as measured by scintillation counting and expressed as
percentage of administrated dose per gram (%ID/g). Administration was oral.
time into this organ from the small intestine, which is expected. The
high standard deviations in the small and large intestine are
because of this natural migration that will not be as fast in all
animals.22 Only a small part of these large organs (in mass) is
solubilized and the C-14 content determined. Taking the same and TB-infected mice. The relative uptake of 11 in the kidneys
segment of these organs from each mouse helped to keep the suggested some renal clearance. The heart and the lungs showed
variation in %ID/g for these organs low; even between the mice lower tissue accumulation in both healthy and TB-infected animals
with and without TB is low. About 1.49% was accumulated in the (Figures 2 and 3). The uptake in decreasing order was small
stomach after 2 h of post administration. After 8 h, only 0.8% of intestine > large intestines > liver > stomach > kidneys > lungs >
the 14C-radiolabelled compound was accumulated in this organ. heart, etc. The radiotracer exhibited none or negligible activities
Because the animals were gavage fed, this was a clear indication at the brain, tail, muscle and bladder.
that the compound under investigation was well digested in the
When comparing the biodistribution in healthy versus TB-
stomach. At 2 h after oral administration, the liver contained infected mice at the two time points (Figures 4 and 5), no
approximately 3.6% and the kidneys 0.86% of the recovered statistically significant difference (P > 0.2) is observed at 2 and
activity. Lower accumulations (2.4 and 0.64%) on these organs were 8 h respectively, after injection. This is gratifying as it indicates
found at the 8-h time point. There was some evidence for the that the method followed is reliable although it indicates that
metabolism of the radiotracer in the liver because of the lowering the compound has no preference for TB in vivo. One would have
of uptake from 2 to 8 h. This trend is noticeable in both healthy expected higher lung uptake in the TB-infected animals if the
compound would have stayed intact in vivo.23 This is unexpected
22
Healthy animals 2 h
Healthy animals 8 h
20
18
16
14
12
10
8
22
20
18
16
14
12
10
8
Healthy animals 2 h
TB infected animals
6
6
4
4
2
2
0
0
Organs
Figure 2. Accumulation of 14C-labelled pyrazole 11 in various organs of the non-
TB-infected animals, n = 3, as measured by scintillation counting and expressed as
percentage of administrated dose per gram (%ID/g).
Organs
Figure 4. A comparison between healthy and TB-infected animals after 2 h.
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M. S. Sonopo et al.
removed in vacuo. The obtained residue was subjected to column
chromatography eluting with hexane : EtOAc (7:3) to give ethyl 2-methyl-
2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)propanoate
in a 38% yield as a pale brown solid (6.1 g, 19.79 mmol). δH (400 MHz, CDCl3)
7.88 (1 H, s), 7.83 (1 H, s), 4.16 (2 H, q, J = 7.1 Hz), 1.84 (6 H, s), 1.32 (12 H, s),
1.19 (3 H, t, J = 7.1 Hz). δC (100 MHz, CDCl3) 172.22, 145.44, 134.11, 107.85,
83.32, 64.19, 61.79, 25.50, 24.80, 13.97. MS (ESI) M + Na: 330.3 (15%), 331.2
(100%), 332.2 (10%)
22
20
18
16
14
12
10
8
Healthy animals
TB-infected 8 h
Ethyl 2-methyl-2-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)propanoate (5)
6
4
To a solution of ethyl 2-methyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)-1H-pyrazol-1-yl)propanoate (4.00 g, 12.98 mmol) in dioxane (100 mL)
and water (20 mL) was added 2-bromopyridine (1.24 mL, 12.98 mmol)
followed by addition of Cs2CO3 (8.46 g, 26.0 mmol) and PdCl2 (dppf)
(0.475 g, 0.65 mmol). The reaction mixture was heated at 80 °C for 12 h,
cooled to room temperature and filtered through a thick pad of Celite.
The solvents were evaporated, and the residue was dissolved into ethyl
acetate (100 mL) and washed with water (2 × 50 mL) and brine (30 mL).
The organic layer was dried over Na2SO4, filtered and evaporated to give a
residue that was subjected to silica gel column chromatography eluting with
EtOAc/hexane (30/70) to afford ethyl 2-methyl-2-(4-(pyridin-2-yl)-1H-pyrazol-
1-yl)propanoate in a 89% yield as a brown oil (3.00 g, 11.57 mmol). δH
2
0
Organs
Figure 5. A comparison between healthy and TB-infected animals after 8 h.
as the in vitro data suggest a higher MIC99 value for this
compound. The difference in vivo can possibly be explained by (400 MHz, CDCl3) 8.50 (1 H, d, J = 5.5 Hz), 8.15 (1 H, s), 7.96 (1 H, s), 7.61 (1 H,
ddd, J = 7.8, J = 5.9 and J = 1.8 Hz), 7.45 (1 H, d, J = 7.9 Hz), 7.06 (1 H, dd,
J = 7.4 and J = 5.0 Hz), 4.13 (2 H, q, J = 7.1 Hz), 1.85 (6 H, s), 1.16 (3 H, t,
J = 7.1 Hz). 13C NMR (100 MHz, CDCl3) δ 172.02, 151.77, 149.27, 137.40,
136.59, 126.09, 122.87, 120.99, 119.48, 64.39, 61.70, 25.28, 24.70, 13.83. MS
(ESI) M + H: 260 (100%).
(1) that the compound is digested (broken down) in vivo and
therefore only a part of the molecule (which no longer has
anti-TB properties) is followed via the C-14 tracer or (2) the
compound has no binding affinity for TB cells even if it has a
high toxicity towards the cells. This illustrates the necessity of
biodistribution studies early in the drug discovery process.
2-Methyl-2-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)propanoic acid (6)
To a solution of ethyl 2-methyl-2-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)propanoate
(5.00 g, 19.28 mmol) in methanol (75 mL) was added a solution of 5.0 M
lithium hydroxide aqueous solution (15.43 mL, 77 mmol). The reaction
mixture was stirred at ambient temperature overnight. Most of the methanol
was evaporated, and the residue was taken to pH = 3 with 2 M hydrochloric
acid. The aqueous layer was extracted with ethyl acetate (3 × 30 mL), and the
organic layer was dried over Na2SO4, filtered and evaporated in vacuo. The
desired compound (2-methyl-2-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)propanoic
acid) was obtained in a 45% yield (2.00 g, 8.65 mmol). δH (400 MHz, DMSO)
8.50 (1 H, d, J = 4.7 Hz), 8.46 (1 H, s), 8.04 (1 H, s), 7.76 (1 H, td, J = 7.6 and
J = 1.8 Hz), 7.71 (1 H, d, J = 7.9 Hz), 7.21–7.14 (1 H, m), 1.79 (6 H, s). 13C NMR
(100 MHz, DMSO) δC 173.57, 151.67, 149.25, 137.10, 136.80, 127.02, 122.50,
121.10, 119.44, 63.97, 25.08. MS (ESI) M + H: 232.2 (100%).
Experimental section
General
All reactions requiring inert atmosphere were performed under nitrogen
in oven-dried glassware, unless otherwise stated. Tetrahydrofuran was
distilled under nitrogen from sodium wire and benzophenone. All the
required chemicals and reagents were purchased from Sigma-Aldrich
(St. Louis, MO, USA) or Merck (Merck KGaA, Darmstadt, Germany) and
were used without further purification. Carbon-14 bromoaniline was
purchased from American Radiolabeled Chemicals, Inc. (ARC, St. Louis,
MO 63146, USA). Qualitative thin-layer chromatography was performed
on pre-coated Merck aluminium sheets (silica gel 60 PF254, 0.25 mm).
After development, the plates were visualized by using UV254 light
followed by the use of iodine, anisaldehyde or vanillin-based stains.
Normal and flash chromatography was performed using Merck Kieselgel
60 (230–400 mesh) and on columns with a 1 or 4 cm diameter. Nuclear
magnetic resonance (NMR) spectra of synthesized compounds were
recorded on a Bruker AVANCE DRX400 spectrometer (Bruker Biospin
Corporation, Germany), and chemical shifts were referenced to the
solvent shift. The solvents used more often were deuterated chloroform
(CDCl3, δH 7.26) and deuterated dimethyl sulfoxide (DMSO-d6, δH 2.50) for
1H NMR. Radioactivity measurements were carried out using a triathler
liquid scintillation counter (Hidex, Turku, Finland) or Perkin-Elmer Tri-Carb
3100 TR scintillation spectrophotometer (Canberra Packard, Canada).
2-Methyl-2-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)propanoyl chloride (7)
2-Methyl-2-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)propanoic acid (0.300 g,
1.29 mmol) was dissolved in DCM (40 mL) and cooled to 0 °C in an ice
bath. Oxalyl chloride (1.070 mL, 2.14 mmol) (2 M solution in DCM) was
added dropwise (evolution of gas noted), followed by a catalytic amount
of DMF (5.02 μL), and the ice bath was removed. The reaction was stirred
at room temperature for 4 h. On completion of addition, the solvent and
excess oxalyl chloride were removed by rotary evaporation, followed by
further drying under high vacuum to remove the final traces of HCl
and oxalyl chloride. The acid chloride (0.310 g, 1.242 mmol) was obtained
as an oil. This product was immediately used in the next reaction without
further analysis.
Synthesis
N-(4-bromophenyl)-2-methyl-2-(4-(pyridine-2-yl)-1H-pyrazol-1-yl)pro-
Ethyl 2-methyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
panamide (9)
1H-pyrazol-1-yl)propanoate (3)
To the crude mixture of the acyl chloride (0.300 g, 1.15 mmol) in DCM was
To a solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole added triethylamine (0.643 mL, 4.61 mmol) followed by 4-bromoaniline
(10.00 g, 51.50 mmol) in DMF (100 mL) at room temperature was added (0.397 g, 2.31 mmol). The reaction was stirred overnight and monitored by
Cs2CO3 (25.2 g, 77.0 mmol) followed by ethyl 2-bromo-2-methylpropanoate
(7.87 mL, 53.60 mmol). The reaction mixture was refluxed at 90 °C for 18 h
and then cooled to room temperature. The resulting solids were filtered
and washed with DMF. The filtrate was extracted with EtOAc (3 × 50 mL).
The combined organic layer was washed with H2O, dried over Na2SO4 and
LC-MS to confirm that the reaction had gone to completion. On completion,
ammonium chloride (10 mL) was added to quench the reaction. The
resulting mixture was extracted with EtOAc (3 × 20 mL). The combined
organic layers were washed with 2 M HCl (2 × 20 mL), with H2O and brine,
and dried over Na2SO4 and removed in vacuo. The obtained residue was
J. Label Compd. Radiopharm 2016, 59 264–269
Copyright © 2016 John Wiley & Sons, Ltd.
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M. S. Sonopo et al.
subjected to column chromatography eluting with hexane : EtOAc (6:4) to
give N-(4-bromophenyl)-2-methyl-2-(4-(pyridine-2-yl)-1H-pyrazol-1-yl)pro-
earmarked and assigned at random to three groups: uninfected
treatment group (12 mice), a TB-infected treatment group (12 mice)
and an untreated TB-infected group (8 mice).
panamide (9) in 80% yield. IR νmax (cmÀ1): 3307, 2989, 2928, 2853, 1683,
1595, 1520, 1487, 1393, 1372, 1307, 1286, 1240, 1223, 1153, 1072, 10 008,
975, 962, 822, 777, 728, 646, 503. δH (400 MHz, CDCl3) 8.74 (1H, s), 8.57
(1 H, d, J = 4.7 Hz), 8.25 (1 H, s), 8.18 (1 H, s), 7.69 (1 H, td, J = 7.6 and
J = 1.8 Hz), 7.50 (1 H, d, J = 7.6 Hz),7.38 (2H, d, J = 8.8 Hz), 7.33 (2H, d,
J = 8.8 Hz), 7.14 (1 H, m), 1.98 (6 H, s). 13C NMR (100 MHz, DMSO) δC 170.35,
151.20, 149.73, 138.91, 136.76, 136.64, 131.82, 127.30, 124.06, 121.63,
121.51, 119.67, 116.97, 65.86, 25.99. HRMS calculated for C18H17BrN4O
(M-H) 383.0513, found 383.0535.
TB infection of mice
M. tuberculosis (MTB) H37Rv (ATCC 27294) were cultivated on 7H11 agar
plates (BD Diagnostics, Johannesburg, South Africa) supplemented with
10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC)
enrichment (BD Diagnostics, Johannesburg, South Africa) for 3 weeks at
37 °C, harvested, resuspended in 15% glycerol/Dubos broth and kept
frozen in 1 mL aliquots at À80 °C. A random selection of three tubes were
used for quantification by plating out 10-fold serial dilutions on 7H11
agar plates supplemented with OADC, and colony forming units per
millilitre aliquot were ≈108.
Twenty mice were subsequently infected by aerosol route using a Glas-
Col Inhalation Exposure Chamber (Glas-Col, Terre Haute, USA). One millilitre
aliquot of MTB was thawed and passed three times through a 26-gauge
needle to prepare a single-cell suspension that was added to 10 mL of sterile
deionized water and transferred to a nebulizer after 1 mL was removed for
quantification. The viable MTB in nebulizer solution (used for aerosol
infection) and lungs of mice were quantified by bacterial cultivation. Mice were
sacrificed 1 day after infection (n = 4) and 4 weeks after infection (n = 4); lungs
were removed aseptically, and each lung was suspended in 2 mL of 0.005%
Tween 80/phosphate-buffered saline (pH = 7.4) and homogenized by
N-(4-bromophenyl-14C6)-2-methyl-2-(4-(pyridine-2-yl)-1H-pyrazol-1-
yl)propanamide (11)
To the crude mixture of the acyl chloride (4.402 mg, 0.0176 mmol) in
DCM was added triethylamine (3.0717 μL, 0.0220 mmol) followed by 4-
bromoaniline (0.459 mg, 0.0022 mmol, 6.25 MBq). The reaction was
stirred overnight and monitored by LC-MS to confirm that the reaction
had gone to completion. On completion, the resulting mixture was
extracted with CH2Cl2 (2 mL × 3), washed with aqueous K2CO3 solution,
water, 1 N HCl and brine, dried and concentrated under vacuum to afford
the product 11 in 60% yield (0.51 mg, 0.00132 mmol of 2.96 MBq) as a
yellow oil. The specific activity of 11 was found to be 2242.4 MBq/mmol.
Analytical HPLC method
means of
a motorized dispersing instrument (Heidolph DIAX 900,
Germany). Ten-fold dilutions of nebulizer solution and lung homogenates
were plated out in triplicate on 7H11 agar plates supplemented with OADC
enrichment and Polymyxin B sulphate salt (0.0333 g/L, SIGMA, South Africa),
amphotericin B sulphate (0.0222 g/L, SIGMA, South Africa), carbenicillin
disodium salt (0.112 g/L, SIGMA, South Africa) and trimethoprim lactate salt
(0.0226 g/L, SIGMA, South Africa) to make it selective for cultivation of MTB.
Inoculated agar plates were incubated at 37 °C for weeks, and CFU counts
were recorded. CFU counts were transformed to logarithms. The mean
logarithms and standard deviation of viable MTB CFU counts were 7.17
0.64 (nebulizer solution), 2.05 0.20 (whole mouse lung at 1 day post
infection) and 6.68 0.66 (whole mouse lung at 4 weeks post infection).
An agilent HPLC instrument (Agilent Technologies, Wilmington, DE, USA)
coupled to a β detector Ramona star (Raytest, Benzstraβe 4,D-75334,
Straubenhardt, Germany) was used for the separation and identification
of 14C pyrazole 11. The instrument was equipped with a 1200 series
quaternary pump, thermostated column compartment, a single quad
mass spectrometer, a 1200 series diode array UV detector and fraction
collector. Gradient elution with 0.1% acetic acid (A) and methanol (B)
as mobile phase was used with a flow of 1 mL/min, by the following
steps: at 1 min 30% A/70% B, in 10 min, methanol was increased from
70 to 90%, and from 10 to 12 min, methanol was decreased back to
70%. A Phenomenex MaxRP 4.6 × 250 mm 5 μm column, ultrapure water
(18 MΩ) and HPLC grade methanol (Sigma Aldrich) were used. Analyses
were carried out on 255 nm wavelength and column temperature of
40 °C. Under these conditions, N-(4-bromophenyl)-2-methyl-2-(4-
(pyridine-2-yl)-1H-pyrazol-1-yl)propanamide elutes at retention time (RT)
≈7.2 min. Radiometric detection was carried out using 600 μL liquid cell
and scintillation liquid: Ultima Flo-M (PerkinElmer Inc., Waltham,
MA02451 USA) with the flow of 2 mL/min.
Tissue distribution of [14C] pyrazole study
Twelve MTB infected and 12 uninfected mice were gavage fed with a
dose of 2 mg/0.111 MBq/kg. For each of the MTB infected and uninfected
groups, four mice were euthanized by cervical dislocation per time point
after 1 and 2 h post injection to allow for adequate 14C-labelled pyrazole
uptake. Blood, brains, hearts, kidneys, livers, lungs, small intestine, large
intestine, bladder, stomach, tail, muscle and spleens were pooled from
each animal by gross dissection. The pooled tissues were stored at
À80 °C for the preparation of the 14C assay. Total radioactivity of the
collected tissue samples was quantified in duplicate. Weighed tissue
samples (~0.2 g) were digested overnight in 1 mL of Biosol (National
Diagnostics Laboratories, USA) tissue solubilizer at 50 °C. Coloured
samples were decolourized by adding a maximum of 0.2 mL of 30%
H2O2 for 60 min at 50 °C. Scintillation cocktail (Bioscint, National
Diagnostics Laboratories, USA) was added to each sample, and the
radioactivity concentration was determined in a Perkin-Elmer Tri-Carb
3100 TR scintillation spectrophotometer, and the samples were counted
for 10 min. Standards of known activity were prepared by adding a 14C-
labelled compound of known activity to different organ samples
covering a range of spectral quench parameter of the isotope values.
The recovery of the known activity was 8% providing credibility to the
measured activities in the unknown samples.
Animals and animal ethics
The study was performed with the approval of the ethics committee of
the Medical Research Council of South Africa (MRC Ethics Committee
for Research on Animals, ECRA Reference No.: 02/12.) in accordance with
the guidelines of the National Code for Animal Use in Research,
Education, Diagnosis and Testing of Drugs and related substances in
South Africa. Specific pathogen-free male BALB/C mice (8 weeks old;
body mass between 21 and 25 g) were obtained from South African
Vaccine Producers (Sandringham, South Africa). On arrival, the mice were
allowed to acclimatize for 2 weeks. A total of 32 mice were used in this
study. Mice were housed in groups of 12 in TECHNIPLAST (Techniplast
USA, West Chester, PA) individually ventilated polypropylene cages with
0.22 μ filtered cage top covers and with overall dimensions of 45 cm
(l) × 35 cm (w) × 21.0 cm (h) and floor area of 1600 cm2 at a room
temperature of 22 2 °C, relative humidity of 55%, a light/dark cycle of
12 h, ventilation of 23 supplied air changes per hour and 47 extracted
air changes per hour within the biosafety level 3 laboratory animal unit
at the Medical Research Council building in Pretoria. Mice were provided
ad libitum with sterile deionized water, sterilized mouse feed (Epol,
Kempton Park, South Africa), non-toxic exfoliated vermiculate chips
(Mandoval Vermiculate CC, 8 Johnson Street, Alrode, South Africa) and
Conclusions
Pyrazole 11 was successfully radiolabelled with the carbon-14
isotope. Although a modest radiochemical yield of 60% was
achieved during the synthesis, the radiochemical purity was over
sterile Erigrostis hay (SAVP, Sandringham, South Africa). Mice were 99% after HPLC purification. 14C-labelled pyrazole 11 was
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J. Label Compd. Radiopharm 2016, 59 264–269

M. S. Sonopo et al.
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administered orally to healthy and TB-infected mice. Compound 11
was found to have high accumulation in the following organs: small
intestine, large intestine and liver at 2 h post administration. At 8 h
post oral administration, the activity accumulation was lower in the
blood in both healthy and TB-infected animals. The study showed
that compound 11 has no in vivo affinity for the lung.
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The project was supported by funding from the Strategic Health
Innovation Partnerships of the South African Medical Research
Council with funds received from the South African Department
of Science and Technology. The National Research Foundation
through its PDP programme and Necsa through its program
Nuclear Technologies in Medicine and Biosciences Initiative.
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Conflict of interest
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[20] H. I. Boshoff, R. Gordon, C. E. Barry, unpublished data (nd).
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The authors did not report any conflict of interest.
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J. Label Compd. Radiopharm 2016, 59 264–269
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