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9
5.1.6. Scaffold Synthesis
5.1.6.1. General procedure for the synthesis of 7-chloro-5-
methylpyrazolo[1,5-a]pyrimidine
methylpyrazolo[1,5-a]pyridimidine (10b).
two steps. dH (400 MHz; DMSO-d6) 8.58 (d, J = 4.0 Hz, 1H), 8.0 (s,
1H), 7.73 (td, J = 8.0 and 4.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.28
(t, J = 8.0, 1H), 5.91 (s, 1H), 4.7 (s, 2H), 2.57 (s, 3H), 1.30 (s, 9H).
12b Yield = 75% dH (400 MHz; DMSO-d6) 8.58 (d, J = 8.0 Hz, 1H),
7.73 (td, J = 8.0 and 2.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.28 (t,
J = 8.0, 1H), 5.91 (s, 1H), 4.7 (s, 2H), 2.87 (s, 3H), 2.57 (s, 3H), 1.30
(s, 9H).
(10a)
and
7-chloro-5-
Methyl acetoac-
etate (1.1 equiv) or ethyl acetoacetate (1.1 equiv) was added drop-
wise to a stirred solution of 3-amino-1H-pyrazole (1 equiv) or 3-
amino-5-methyl-1H-pyrazole (1 equiv) in acetic acid and refluxed
at 120 °C for 16 h. The reaction mixture was cooled and poured
into cooled TBME and stirred at 0 °C for 1 h. The resulting precipi-
tate was filtered, washed with TBME and dried at 70 °C to obtain a
cream solid. (a) R = H, (Yield = 87%, 18.35 g, 123.13 mmol). dH
(400 MHz; CDCl3) 12.22 (br s, 1H), 7.79 (dd, J = 2.0 and 0.8 Hz,
1H), 6.06 (dd, J = 2.0 and 0.8 Hz, 1H), 5.54 (s, 1H), 2.27 (m, 3H).
(b) R = Me, (Yield = 82%, 16.68 g, 101.87 mmol). dH (400 MHz;
DMSO-d6) 12.01 (bs, 1H), 5.90 (d, J = 0.4 Hz, 1H), 5.49 (d,
J = 0.8 Hz, 1H), 2.27 (d, J = 0.4 Hz, 3H), 2.26 (d, J = 0.4 Hz, 3H).
5.1.9. Library production
5.1.9.1. General procedure B for synthesis of 3-substituted
aminopyrazolo[1,5-a]pyrimidines (13).
To a solution of 12
(1 equiv) in dioxane and water (5:1) was added boronic acid
(1.2 equiv) followed by addition of cesium carbonate (1 equiv)
and PdCl2(dffp) (0.1 equiv). The reaction mixture was degassed
and heated at 80 °C overnight. The reaction was diluted with EtOAc
and washed with water (2Â). After drying over Na2SO4, filtration
and concentration, the crude product was dissolved in DCM
(4 ml) and TFA (8 ml) was added. The reaction was stirred for 4 h
at rt followed by concentration. The residue was dissolved in
DCM and washed with 10% Na2CO3 (2Â), the organic layer was
dried (MgSO4), filtered and concentrated. The crude product was
purified by flash column chromatography (80% EtOAc/Hex) or
recrystallization from DCM/Hexane or by prep TLC (EtOAc) to give
the desired product.
N,N-Dimethylaniline (1.25 equiv) was added to
a stirred
solution of 5-methylpyrazolo[1,5-a]pyrimidin-7(4H)-one (a) or
2,5-dimethylpyrazolo[1,5-a]pyrimidin-7(4H)-one (b) (1 equiv) in
toluene, followed by the addition of phosphorus oxychloride
(3 equiv). The reaction mixture was heated at reflux for 4 h. The
reaction mixture was allowed to cool and was neutralised with
NaOH (2 M). The aqueous solution was extracted with DCM (3Â)
and the combined organic phase was washed with brine. The
organic phase was dried, concentrated and the crude product
was purified by column chromatography (10% EtOAc/hexane) to
yield the desired light yellow solid 10a Yield = 41%. dH (400 MHz;
CDCl3) 8.15 (d, J = 2.4 Hz, 1H), 6.83 (s, 1H), 6.65 (d, J = 2.4 Hz, 1H),
2.59 (s, 3H). MS: m/z 168 [M]+ 10b Yield = 46%. dH (400 MHz;
CDCl3) 6.74 (s, 1H), 6.43 (s, 1H), 2.56 (s, 3H), 2.54 (s, 3H). LCMS
(ESI): m/z 182 [M+H]+.
5.1.10. Synthesis of 2,5-dimethyl-3-phenyl-N-(pyridin-2-ylme-
thyl)pyrazolo[1,5-a]pyrimidin-7-amine (1)
A representative experimental for General Procedure B. Charac-
terization data of 2–6 and 31–52 is provided in the Supplementary
data. The title compound was synthesized from 12b (400 mg,
0.925 mmol) using general procedure B to afford (84 mg, 27%) as
an off white solid after purification by flash column chromatogra-
phy (80% EtOAc/Hex) followed by recrystallization from DCM/Hex-
ane and finally by prep TLC (EtOAc). dH (400 MHz; DMSO-d6) 8.68
(ddd, J = 4.0, 1.6 and 0.8 Hz, 1H), 7.78–7.71 (m, 3H), 7.45 (t,
J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 1H), 7.30–7.26 (m, 2H), 5.86 (s,
1H), 4.75 (d, J = 8.0 Hz, 2H), 2.63 (s, 3H), 2.53 (s, 3H). dC
(101 MHz; CDCl3) 159.5, 155.5, 151.5, 149.6, 145.9, 145.8, 137.0,
133.0, 128.8 (x2), 128.4 (x2), 125.8, 122.8, 121.2, 107.5, 85.9,
46.9, 25.2, 14.4. LCMS (ESI): m/z 330.1 [M+H]+; HPLC purity 96.3%.
5.1.7. General procedure for the synthesis of 3-bromo-7-chloro-
5-methylpyrazolo (11a) and 3-bromo-7-chloro-2,5-
dimethylpyrazolo[1,5-a]pyrimidine (11b)
A solution of 10a or 10b (1 equiv) in dichloromethane was
cooled to 0 °C using an ice bath. N-bromosuccinimide (1.1 equiv)
was added slowly to the solution that was stirred for 16 h at room
temperature. The reaction mixture was washed diluted with water
and the reaction mixture extracted with dichloromethane (3Â).
The organic phase was dried over Na2SO4 and concentrated to yield
the desired product as a yellow solid. 11a Yield = quantitative. dH
(400 MHz; CDCl3) 8.13 (s, 1H), 6.88 (s, 1H), 2.63 (s, 3H). LCMS
(ESI): m/z 247 [M+H]+ 11b Yield = quantitative. dH (400 MHz;
CDCl3) 8.13 (s, 1H), 2.85 (s, 3H), 2.63 (s, 3H). LCMS (ESI): m/z 261
[M+H]+.
5.2. M. tuberculosis H37Rv minimum inhibitory concentration
(MIC) assays
The broth microdilution method27 allows a range of antibiotic
concentrations to be tested on a single 96-well microtitre plate
in order to determine the minimum inhibitory concentration
(MIC). MICs were determined according to the broth microdilution
method in liquid 7H9 medium supplemented with ADC (bovine
5.1.8. Intermediate synthesis
5.1.8.1. General procedure for the synthesis of tert-butyl (3-
bromo-5-methylpyrazolo[1,5-a]pyrimidin-7-yl)(pyridin-2-ylm-
albumin fraction V [5 mg/ml], D-Glucose [2 mg/ml], sodium chlo-
ethyl)carbamate
dimethylpyrazolo[1,5-a]pyrimidin-7-yl)(pyridin-2-ylmethyl)-
carbamate (12b). To a solution of 11a or 11b (1 equiv) and
(12a)
and
tert-butyl
(3-bromo-2,5-
ride [0.81 mg/ml]) and/or in the glycerol–alanine–salts (GAST-Fe)
medium pH 6.6, supplemented with [per liter: 0.3 g of Bacto Casi-
tone (Difco), 0.05 g of ferric ammonium citrate, 4.0 g of dibasic
diisopropylethylamine (1.5 equiv) in 2-propanol was added pyri-
din-2-ylmethanamine (1.2 equiv). After heating for 16 h at 80 °C,
the precipitate that formed was collected by filtration. The product
was washed with water and 2-propanol and dried to yield the pro-
duct as a white solid, which was sufficiently pure to be used in the
next step. Di-tert-butyl dicarbonate (1.5 equiv) was added to a stir-
red solution of the free amine (1 equiv) in dichloromethane, fol-
lowed by the addition of dimethylaminopyridine (0.1 equiv), and
diisopropylamine (1.5 equiv). After stirring for 24 h, the reaction
mixture was concentrated and partitioned between EtOAc and
water. After separation, the organic phase was washed with 10%
citric acid and brine, dried and concentrated. The crude product
was purified by column chromatography.12a Yield = 78% over
potassium phosphate, 2.0 g of citric acid, 1.0 g of L-alanine, 1.2 g
of magnesium chloride hexahydrate, 0.6 g of potassium sulfate,
2.0 g of ammonium chloride, 1.80 ml of 10 g sodium hydroxide,
and 10.0 ml of glycerol, Tween80 was added to 0.05%].33 Low iron
GAST medium is normal GAST medium but without the ferric
ammonium citrate.
Briefly, a 10 ml culture of Mycobacterium tuberculosis (Ma)29 is
grown to an OD600 of 0.6–0.7. The culture is then diluted with
1:500 in liquid 7H9 or diluted 1:100 in BSA-free GAST/Fe medium.
In a 96-well microtitre plate, 50
from Rows 2–12. The compounds to be tested are added to Row 1
in duplicate, at a final concentration of 640 M (stocks are made up
to a concentration of 12.8 mM in DMSO, and diluted to 640 M in
ll of medium is added to all wells
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