Novel Aryl Substituted Pyrazoles as Small Molecule Inhibitors of Cytochrome P450 CYP121A1: Synthesis and Antimycobacterial Evaluation

Article abstract of DOI:10.1021/acs.jmedchem.7b01562

Three series of biarylpyrazole imidazole and triazoles are described, which vary in the linker between the biaryl pyrazole and imidazole/triazole group. The imidazole and triazole series with the short -CH₂- linker displayed promising antimycobacterial activity, with the imidazole-CH₂- series (7) showing low MIC values (6.25-25 μg/mL), which was also influenced by lipophilicity. Extending the linker to -C(O)NH(CH₂)₂- resulted in a loss of antimycobacterial activity. The binding affinity of the compounds with CYP121A1 was determined by UV-visible optical titrations with K_D values of 2.63, 35.6, and 290 μM, respectively, for the tightest binding compounds 7e, 8b, and 13d from their respective series. Both binding affinity assays and docking studies of the CYP121A1 inhibitors suggest type II indirect binding through interstitial water molecules, with key binding residues Thr77, Val78, Val82, Val83, Met86, Ser237, Gln385, and Arg386, comparable with the binding interactions observed with fluconazole and the natural substrate dicyclotyrosine.

Full text of DOI:10.1021/acs.jmedchem.7b01562

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Article
Novel Aryl Substituted Pyrazoles as Small Molecule Inhibitors of
Cytochrome P450 CYP121A1: Synthesis and Antimycobacterial Evaluation
Ismail M. Taban, Hosam E. A. E. Elshihawy, Beyza Touran, Benedetta Zucchini, Clare J.
Williamson, Dania Altuwairigi, Adeline S. T. Ngu, Kirsty J. McLean, Colin W. Levy, Sakshi
Sood, Leonardo B. Marino, Andrew W. Munro, Luiz Pedro S. de Carvalho, and Claire Simons
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01562 • Publication Date (Web): 29 Nov 2017
Downloaded from http://pubs.acs.org on November 29, 2017
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Novel Aryl Substituted Pyrazoles as Small Molecule Inhibitors of
Cytochrome P450 CYP121A1: Synthesis and Antimycobacterial
Evaluation
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Ismail M. Taban,a Hosam E.A.E. Elshihawy,b Beyza Touran,a,c Benedetta Zucchini,a,d
Clare J. Williamson,a Dania Altuwairigi,a Adeline S. T. Ngu,a Kirsty J. McLean,e
Colin W. Levy,e Sakshi Sood,f Leonardo B. Marino,g Andrew W. Munro,e Luiz Pedro
S. de Carvalhof and Claire Simonsa*
aSchool of Pharmacy & Pharmaceutical Sciences, Cardiff University, King Edward VII
Avenue, Cardiff CF10 3NB, UK;
bDepartment of Organic Chemistry, Faculty of Pharmacy, Suez Canal University,
Egypt
cAnkara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100
Tandogan, Ankara, Turkey
dDepartment of Pharmaceutical Sciences, University of Perugia, Via del Liceo, 1-06123
Perugia, Italy
eManchester Institute of Biotechnology, School of Chemistry, The University of
Manchester, 131 Princess Street, Manchester M1 7DN, UK
f Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute,
1 Midland Road,London NW1 1AT, UK
gFaculty of Pharmaceutical Sciences, UNESP - Univ Estadual Paulista, Araraquara, São
Paulo 14801-902, Brazil
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ABSTRACT: Three series of biarylpyrazole imidazole and triazoles are described,
which vary in the linker between the biaryl pyrazole and imidazole/triazole group.
The imidazole and triazole series with the short –CH2ꢀ linker displayed promising
antimycobacterial activity, with the imidazole–CH2ꢀ series (7) showing low MIC
values (6.25ꢀ25 ꢀg/mL), which was also influenced by lipophilicity. Extending the
linker to –C(O)NH(CH2)2ꢀ resulted in loss of antimycobacterial activity. Binding
affinity of the compounds with CYP121A1 was determined by UVꢀvisible optical
titrations with KD values of 2.63, 35.6 and 290 ꢀM, respectively, for the tightest
binding compounds 7e, 8b and 13d from their respective series. Both binding affinity
assays and docking studies of the CYP121A1 inhibitors suggests type II indirect
binding through interstitial water molecules, with key binding residues Thr77, Val78,
Val82, Val83, Met86, Ser237, Gln385 and Arg386, comparable with the binding
interactions observed with fluconazole and the natural substrate dicyclotyrosine.
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INTRODUCTION
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Tuberculosis (TB) is endemic in numerous countries of the developing world, and
serious issues are caused by infection with drugꢀ (and multidrugꢀ) resistant strains of
the causative bacterium Mycobacterium tuberculosis (Mtb) and by the common
occurrence of HIV coꢀinfection as a result of the high susceptibility of
immunocompromised individuals to TB infection.1,2 The global impact of TB is
evident from World Health Organization data which reported that, in 2015, 1.8
million people died from TB and approximately 2 billion people, a third of the
world’s population, were infected with latent TB.2 This disease has a global impact
and the “deadly synergy” of coꢀinfection with TB and HIV has serious lifeꢀlimiting
consequences for individuals who do not receive the relevant therapeutics in a timely
manner.1,2
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Challenges associated with the current antibiotic therapy include drug
intolerances and toxicities; pharmacokinetic drugꢀdrug interactions, particularly with
ART drugs in patients coꢀinfected with HIV; and patient adherence given the lengthy
treatment regimens.2 The most effective drugs in clinical use have been isoniazid, the
first oral mycobactericidal drug, and rifampicin. The combined used of rifampicin and
pyrazinamide shortened TB chemotherapy to 9 and 6 months respectively.3,4 Drugꢀ
resistant strains of Mtb fall into three main categories: multidrug resistant (MDR) TB,
a form of TB caused by bacteria that do not respond to at least isoniazid and
rifampicin, the two most powerful firstꢀline antiꢀTB drugs; extremely drug resistant
(XDR) TB, a form of MDR tuberculosis that responds to even fewer available
medicines, including the most effective secondꢀline antiꢀTB drugs; and totally drug
resistant (TDR) TB, which describes strains of Mtb that are resistant to all available
first and secondꢀline antiꢀTB drugs.1,2,5
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MDRꢀTB is now a major problem globally and threatens the ability to treat
and control TB infection. In 2015, an estimated 480,000 people developed MDRꢀTB,
resulting in 190,000 deaths.1 Treatment should be “individualised”, based on drug
susceptibility testing (DST). However, this is not always possible and treatment is
usually based on the pattern of drug resistance in the local geographical region. MDRꢀ
TB requires longer treatment (18ꢀ24 months) with drugs that are more toxic and less
efficacious. Worldwide, MDRꢀTB treatment success rates are as low as 50%.5 XDRꢀ
TB requires thirdꢀline antiꢀTB drugs, which are expensive and are often associated
with more severe side effects. The emergence of MDRꢀ, XDRꢀ and TDRꢀTB strains
has led to intensified research to identify new antiꢀTB drugs over the past decade.
Mycobacterium tuberculosis encodes twenty cytochrome P450 enzymes
(CYPs or P450s). One of these is CYP121A1, which was shown to be essential for
microbial viability.6 CYP121A1 catalyses the formation of an unusual intraꢀmolecular
bond between carbon atoms in the orthoꢀpositions of two tyrosine groups in the
cyclodipeptide dicyclotyrosine (cYY), the natural substrate, to form the metabolite
mycocyclosin. cYY interacts indirectly with the heme iron through water molecules
(Figure 1A). Interestingly, this type of indirect binding mode was also observed in the
crystal structure of CYP121A1 bound to the azole antifungal drug fluconazole.7 The
crystal structure of the CYP121A1/fluconazole complex, PDB 2IJ7, exhibits dual
fluconazole conformations (I and II). In the indirect conformation (I), a triazole
nitrogen of fluconazole binds to the heme iron via a distal water molecule that
remains coordinated to the heme iron. However, in the direct conformation (II) the
fluconazole binds through a triazole nitrogen directly to the heme iron (Figure 1B).
While both binding modes are evident, the indirect binding (via water) mode appears
predominant in this structure.8
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A
B
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Figure 1. Crystal structures of CYP121A1 coꢀcrystallized with (A) cYY (PDB 3GH), and (B)
fluconazole (PDB 2IJ7). Both the natural substrate and fluconazole interact indirectly with the
heme via interstitial water molecules with key amino acids identified.
The natural substrate cYY binds to the CYP121A1 active site without
inducing any significant change in the P450 structure, consistent with it being a good
substrate for the enzyme.6 Through crystallization of ligand complexes it was
discovered that iodopyrazole binds with its pyrazole group in a channel between P450
helices F and G, and “stacked” between Trp182 and Phe168 side chains. The iodine
atom protrudes towards the active site and interacts with hydrophobic amino acids.7 In
designing potential new inhibitors of CYP121A1, we noted that ‘Y’ꢀshaped molecules
might provide a good “fit” within the P450 active site. This may be the result of a
high conformational energy barrier necessary for binding to CYP121A1.
Using this observation to help design novel CYP121A1 inhibitors, our
research explores the CYP121A1 binding and antiꢀmycobacterial activity of various
‘Yꢀshaped’ biaryl pyrazole imidazole or triazole derivatives (Figure 2). Using
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different linkers between the hydrophobic biaryl pyrazole moiety (blue, Figure 2) and
the heme binding azole group (red, Figure 2) the proximity of the heme binding
moiety will be extended to determine if this has an effect on indirect/direct binding of
new ligands to the heme iron.
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Figure 2. General structure of ‘Yꢀshaped’ biaryl pyrazole, imidazole and triazole derivatives
showing different linkers.
RESULTS AND DISCUSSION
Chemistry. The imidazole (7) or triazole (8) pyrazole compounds were obtained via
a fiveꢀstep synthetic route (Scheme 1) beginning with the preparation of the imines
(3) on reaction of acetophenones (1) with phenylhydrazine under acidic conditions.9,10
The aldehydes (4) were prepared as previously described9,10 by a VilsmeierꢀHaack
reaction of the imines (3). Reduction of the aldehydes (4) with NaBH4 resulted in high
yields of the corresponding alcohols (5), which were subsequently converted to the
o
chlorides (6) on treatment with thionyl chloride in toluene at 115 C for 2 h.11 The
chlorides were generally found to be unstable so were prepared immediately before
use in the following step. Reaction of the chlorides (6) with either the potassium salt
of imidazole or triazole, prepared in situ by treatment of imidazole or triazole with
o
o
potassium carbonate in acetonitrile at 45 C for 1 h, overnight at 70 C, gave the
required final imidazole (7) and triazole (8) pyrazole derivatives (Scheme 1).
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Scheme 1. Reagents and Conditions: (i) AcOH, r.t., 15 min (ii) AcOH, EtOH, 80 oC, 1 h (iii)
o
POCl3, DMF, 90 °C, 2 h (iv) NaBH4, EtOH, r.t. 1 h (v) SOCl2, toluene, 115 C, 2 h (vi)
imidazole, K2CO3, CH3CN, 45 oC, 1 h then 70 oC, overnight (vii) imidazole, K2CO3, CH3CN,
45 oC, 1 h then 70 oC, overnight. [a, R = H; b, R = 4ꢀF; c, R = 4ꢀCl; d R = 4ꢀCH3; e, R = 4ꢀ
OCH3; f, R = 4ꢀBr; g, R = 4ꢀI; h, R = 4ꢀCN].
The ‘extended’ pyrazole compounds were prepared from the aldehydes (4).
Oxidation to the carboxylic acids (9) was achieved on treatment of the aldehydes (4)
with potassium permanganate (Scheme 2).12 To prepare the aminoethylimidazole
dihydrochloride salt (12) the corresponding 2ꢀ(2ꢀ(1Hꢀimidazolꢀ1ꢀyl)ethyl)isoindolineꢀ
1,3ꢀdione (11) was required. The isoindolineꢀ1,3ꢀdione was prepared following the
procedure of Popkov and Skvortsova,13 which involved melted imidazole refluxed
with bromoethylphthalamide (10) and potassium iodide, resulting in the desired
product (11) in 49 % yield after recrystallisation from isopropanol. The isoindoleꢀ1,3ꢀ
dione was then treated with hydrazine following the IngꢀManske procedure to give
the required aminoethylimidazole dihydrochloride (12) in 82 % yield (Scheme 2).
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t
o
Scheme 2. Reagents and Conditions: (i) KMnO4, BuOHꢀH2O, 75 C, 45 min (ii) imidazole,
o
o
100 C, 3 h then toluene, 115 C, 20 h (iii) NH2NH2, EtOH, reflux, 4 h (iv) CDI, DMF, 1 h
then 12, Et3N, DMF, r.t., 20 h. [a, R = H; b, R = 4ꢀF; c, R = 4ꢀCl; d R = 4ꢀCH3; e, R = 4ꢀ
OCH3].
The final amides (13) were prepared by a carbonyldiimidazole (CDI) coupling
reaction through coupling of the carboxylic acids (9) with the free amine, generated in
situ on treatment of the aminoethylimidazole dihydrochloride salt (13) with
triethylamine in DMF. The final amide derivatives (13) were obtained in low to
moderate yields (Scheme 2).
CYP121A1 Ligand Binding Affinity. The CYP121A1 binding affinity (Kd) of the
various compounds was determined by UVꢀvis optical titration. Results shown in
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Tables 1, 2 and 3 reveal that most of the compounds show a red (type II) shift in the
Soret peak position (Figure 3, for example 7b), indicating that, in the solution state,
most of these compounds coordinate either (i) directly to the CYP121A1 heme iron or
(ii) indirectly to the heme iron through interstitial water molecule(s). Another
possibility is (iii) that a ligand binding distant from the heme does not induce a heme
spectral shift (but would block the active site).
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Table 1. KD and MIC values for series 7 against M. tuberculosis H37Rv
Compound
R
Soret peak shift
(nm)
MIC
(ꢀg/mL)
12.5
25
12.5
12.5
25
6.25
6.25
100
>100
20
KD (ꢀM)
H
F
Cl
2.63 ± 0.19
4.16 ± 0.47
7.42 ± 0.66
416 to 423.5
416 to 423.5
416 to 422
416 to 421.5
416 to 421
416 to 420
416 to 422
416 to 418
7a
7b
7c
7d
7e
7f
7g
CH3 5.36 ± 0.59
OCH3 11.4 ± 0.9
Br
I
19.5 ± 0.2
13.6 ± 0.2
45.9 ± 0.6
8.6 ± 0.2
0.07 ± 0.01
5.82 ± 0.16
CN
7h
Fluconazole
Clotrimazole
cYY
ꢀ
Series 7 (imidazole deriviatives) showed the highest affinity for CYP121A1,
with compounds 7aꢀe having d values < 12 M ( D = 2.63 ± 0.19, 4.16 ± 0.47, 7.42
± 0.66, 5.36 ± 0.59, and 11.35 ± 0.94 M, respectively) (Table 1). The values are
comparable to the KD values for the azole drugs ketoconazole (imidazole) and
fluconazole (triazole), which are 3.3 ± 0.3 and 8.6 ± 0.2 M, respectively.
K
ꢀ
K
ꢀ
ꢀ
Compounds 7a and 7b induce the most extensive Soret absorbance shifts (416 to
423.5 nm), suggesting that the CYP121A1 heme iron is predominantly coordinated by
a direct imidazole nitrogen bond in these cases. Compounds 7c to 7h show less
extensive Soret red shifts on binding the CYP121A1 heme, suggesting that ligation
modes in these cases may involve imidazole nitrogen interactions with heme iron
mediated through an interstitial water ligand, and that these could occur to a greater
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(e.g. compounds 7f and 7h) or lesser (e.g. compounds 7c and 7g) extent. The smallest
Soret shift occurs with compound 7h (416 to 418 nm) and it is likely that indirect
coordination though the retained distal water on the heme iron is predominant in this
case. However, alternative active site binding poses made by series 7 ligands that do
not impact on the heme iron or its distal water ligand cannot be ruled out at this stage,
and this phenomonen could also provide explanations for the less extensive Soret
shifts induced by a number of the series 7 molecules.
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Figure 3. UV-Vis optical binding titration for compound 7b binding to CYP121A1. The
left hand panel shows data from a compound 7b titration with CYP121A1 (~4.7 ꢀM) with the
ligandꢀfree spectrum as a thick black line, spectra following progressive additions of 7b as
thin solid lines, and the final nearꢀsaturated protein spectrum shown as a thick red line. The
inset shows overlaid difference spectra generated by the subtraction of the starting spectrum
from each consecutive ligandꢀbound spectrum collected in the titration. The right hand panel
shows a plot of compound 7bꢀinduced absorbance change – calculated as the difference
between the peak and trough in the difference spectra in the left hand panel, using the same
wavelength pair (429 and 392 nm, respectively) throughout. Data were fitted using the Hill
equation to give a 7b KD value of 4.16 ± 0.47 ꢀM.
Series 8 (triazole derivatives) has affinities ranging from KD = 35.6 ± 2.7 ꢀM
(compound 8b) to 193 ± 21 ꢀM (compound 8f) (Table 2). Series 13 (pyrazole amide
derivatives) had weaker affinity, with moderate KD values between 290 and 400 ꢀM
(290 ± 27, 314 ± 43 and 394 ± 39 ꢀM for compounds 13d, 13e and 13a, respectively).
No evidence of binding was observed for halogenated derivatives of this compound
class (Table 3). As was also observed for series 7, the extent of the CYP121A1 heme
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Soret shift observed at apparent saturation with the series 8 compounds varied
considerably. In series 8, a similar pattern of Soret shifts was observed to that seen
with series 7, with peak shifts as small as 416 to 417.5 nm (compound 8f), and as
large as 416 to 423 nm (compound 8b). Again, we infer that the most extensive
absorbance changes occur for those ligands that show predominantly direct
coordination of heme iron through a triazole nitrogen (i.e. compounds 8a and 8b),
while less extensive Soret shifts (i.e. compounds 8d and 8f) are likely indicative of
either indirect (through the heme distal water) coordination of the CYP121A1 heme
iron, or possibly result from mixed binding poses that include modes that do not
influence the heme spectrum. For series 13, ligand binding curves were obtained for
the compounds 13a, 13d and 13e. However, the extents of Soret band shift were
modest (416 to 417.5/418 nm). Once again, these data suggest either indirect
coordination of heme iron through an imidazole nitrogen or a combination of this type
of ligand binding mode with other pose(s) that do not impact on the heme spectrum.
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Table 2. KD and MIC values for series 8 against M. tuberculosis H37Rv
Compound
R
Soret peak shift
(nm)
MIC
(ꢀg/mL)
50
25
25
>100
25
25
KD (ꢀM)
H
F
Cl
73.5 ± 0.7
35.6 ± 2.7
83.8 ± 7.4
416 to 422
416 to 423
416 to 421
416 to 418
416 to 420
416 to 417.5
No binding
No binding
8a
8b
8c
8d
8e
8f
CH3 187 ± 13
OCH3 96.5 ± 8.4
Br
I
CN
193 ± 21
ꢀ
ꢀ
50
>100
8g
8h
Azole antifungal drugs (clotrimazole, econazole, fluconazole, ketoconazole
and miconazole) bind tightly to Mtb CYP121A1, inducing a Soret peak shift to
between 421ꢀ423.5 nm. The KD values for clotrimazole, econazole and miconazole
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are all < 0.2 ꢁM, indicating very tight binding, and as determined from data fitting
using the Morrison (quadratic) equation.14
6
7
8
9
Table 3. KD and MIC values for series 13 against M. tuberculosis H37Rv
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Compound
R
Soret peak shift
(nm)
MIC
(ꢀg/mL)
>100
>100
>100
KD (ꢀM)
H
F
Cl
394 ± 39
416 to 417.5
No binding
No binding
416 to 417.5
416 to 418
13a
13b
13c
13d
13e
ꢀ
ꢀ
CH3 290 ± 27
OCH3 314 ± 43
>100
100
MIC determination against Mycobacterium tuberculosis. The derivatives were
screened against M. tuberculosis H37Rv by the REMA (Resazurin Microtiter Assay)
method.15 In the imidazole (7) and triazole (8) pyrazole compounds, the imidazoles
(7) were generally more inhibitory to M. tuberculosis growth and displayed better
activity than the triazoles (8) (Tables 1 and 2), with the 4ꢀbromo (7f) and 4ꢀiodo (7g)
derivatives displaying good activity (MIC 6.25 ꢀg/mL). All the imidazoles (7), with
the exception of the 4ꢀnitrile derivative (7h, MIC90 = 100 ꢀg/mL), were significantly
more active than the azole drug fluconazole. Compounds 7a-d, 7f and 7g are more
active than clotrimazole (MIC90 = 20 ꢀg/mL) (Table 1). The ‘extended’ pyrazole
compounds 13aꢀe had little antimicrobial activity (MIC90 ≥100 ꢀg/mL) (Table 3).
Molecular Modelling and Crystallography
The Molecular Operating Environment (MOE) program16 was used to perform
molecular docking and was found to closely replicate the position and binding
interactions of cYY and fluconazole, as observed in the crystal structures PDB 3G5H
and PDB 2IJ7 respectively.
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A
B
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C
Figure 4. Molecular modelling of selected compounds. (A) The triazole group of 8b interacts
with the heme indirectly via an interstitial water molecule and binds in a similar
conformation, and with the same key amino acids, as that observed for cYY and fluconazole.
(B) The nitrile derivative 7h binds in a different conformation with the nitrile bound to water
molecules and held in a position further away from the heme active site. (C) The amide
imidazole 13b also interacts with the heme via an interstitial water molecule, but its greater
flexibility also resulted in various other conformations.
The imidazole (7) and triazole (8) compounds were all found to interact with
the heme through an interstitial water molecule via hydrogen bonding interactions
with Ser237, Gln 385 and Arg386 on one side of the structure, whilst on the other side
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of the structure interactions are through hydrophobic amino acid residues including
Thr77, Val78, Val82, Val83, Met86 (Figure 4A). The docking results were consistent
with the type II binding indicated from the binding affinity assays. The only exception
was the nitrile substituted derivatives 7h and 8h. In both cases the nitrile group
interacted with Ala167 and Trp182 via interstitial water molecules, holding the
compounds away from the active site with the imidazole/triazole ring positioned away
from the heme group (Figure 4B).
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The amide imidazole compounds (13) were positioned in a similar manner
(Figure 4C) to 7 and 8. However, whereas the imidazole (7) and triazole (8) were
consistently positioned within the active site, numerous conformations were observed
for the amide imidazole series (13), possibly owing to their increased conformational
flexibility, which may correlate with the moderate binding affinity observed.
A
B
Figure 5. Xꢀray crystal structures of (A) 7b and (B) 7e binding to CYP121A1.
Two compounds were successful coꢀcrystallized with CYP121A1, 7b and 7e
(Figure 5). However, the crystallized structures did not correlate with the binding
affinity results as the imidazole group was positioned away from the heme group and
in both cases the compounds were located in positions too far away to form direct or
indirect binding interactions with the heme iron. However, the observed binding poses
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of 7b and 7e are similar and both clearly result in effective blockage of the active site
and thus inhibition of binding of the cYY substrate. These data illustrate how
different P450 ligand binding modes can be adopted by molecules bound to proteins
in the solution state or in the more constrained environment of protein crystals. In the
case of the compound series investigated in this study, there is clear evidence from
structural, molecular modelling and UVꢀvisible ligand titration studies for the
adoption of binding modes that involve direct or indirect (via the distal water ligand)
coordination of the heme iron by imidazole or triazole nitrogens, as well as for modes
in which the bound ligands are distant from the heme and are unlikely to influence the
CYP121A1 heme spectrum.
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Conclusion
The data presented demonstrate the successful binding to CYP121A1 of a range of
different compounds inspired by the structure of CYP121A1’s natural substrate (the
cyclic dipeptide cYY). Series 7 produced the most effective antimycobacterial agents
with the tightest binding constants, and with KD values for the eight compounds in
this series in the range between 2.63ꢀ45.9 ꢀM, with an average (mean) KD value of
~13.7 ꢀM. The series 7 compounds with the best MIC values were those with bromo
(7f) and iodo (7g) substituents (MIC = 6.25 ꢀg/mL), Hansch analysis suggested that
increased lipophilicity (Figure 6) may correlate with improved antimycobacterial
activity in this series, possibly through facilitating increased drug uptake across the
Mtb lipidꢀrich cell wall (Table 1).
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-0.7
-0.8
-0.9
-1
I
Br
R² = 0.7258
9
Me
Cl
H
-1.1
-1.2
-1.3
-1.4
-1.5
-1.6
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F
OMe
3
3.4
3.8
4.2
4.6
5
LogP
Figure 6. Hansch analysis illustrating correlation between MIC and calculated LogP17 values
for the imidazole compounds (7) (the nitrile 7h is excluded as an outlier).
The 4ꢀnitrile derivative 7h is a relatively weak binder (KD = 45.9 ꢀM), a property
which may be explained from molecular docking (Figure 4B) where the nitrile
interacts with Ala167 and Trp182 via interstitial water molecules, holding the
compound at a distance too far from the active site to attain indirect or direct
interaction with the heme.
From the antimicrobial MIC data, it is clear that extending the “linker” region of the
pyrazole derivatives from –CH2ꢀ (series 7 and 8) to ꢀC(O)NHꢀ(CH2)2ꢀ (series 13) does
not translate to improved MIC values. Computational docking studies suggested that
these extended compounds (series 13) were able to interact with the heme indirectly
and fit within the hydrophobic CYP121A1 pocket. However, only moderate KD
values between 290 and 400 ꢀM were observed for compounds 13d, 13e and 13a,
respectively, with only partial type II heme shifts (from 416 nm to 417.5/418 nm).
There was also no evidence of binding for halogenated derivatives of this compound
class.
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EXPERIMENTAL SECTION
7
8
General Procedures. All reagents and solvents were of general purpose or
9
analytical grade and purchased from SigmaꢀAldrich Ltd, Fisher Scientific, Fluka and
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1
Acros. H and 13C NMR spectra were recorded with a Bruker Avance DPX500
spectrometer operating at 500 and 125 MHz, with Me4Si as internal standard. Mass
spectra (HRMS) were determined by the EPSRC mass spectrometry centre (Swansea,
UK). Elemental analysis was performed by MEDAC Ltd (Chobham, Surrey, UK).
Flash column chromatography was performed with silica gel 60 (230ꢀ400 mesh)
(Merck) and TLC was carried out on precoated silica plates (kiesel gel 60 F254, BDH).
Compounds were visualised by illumination under UV light (254 nm) or by the use of
vanillin stain followed by heating. Melting points were determined on an
electrothermal instrument and are uncorrected. All solvents were dried prior to use
and stored over 4 Å molecular sieves, under nitrogen. All the compounds were ≥ 95%
pure.
The imines (3), aldehydes (4), alcohols (5), chlorides (6), carboxylic acids (9) and 1ꢀ
(2ꢀammonioethyl)ꢀ1Hꢀimidazolꢀ3ꢀium chloride (12) were prepared as previously
described.9ꢀ13 All compounds were more than 95% pure.
General method for the preparation of imidazole and triazole derivatives
(7) and (8). To a stirred suspension on K2CO3 (3 mmol) in dry acetonitrile (20 mL)
was added imidazole (3 mmol) or triazole (3 mmol). The reaction mixture was
refluxed at 45 °C for 1 h. After cooling to room temperature the chloromethyl
pyrazole compound (6) (0.76 mmol) was added and the reaction mixture refluxed at
70 °C overnight. The solvent was evaporated under reduced pressure and the resulting
mixture diluted with EtOAc (50 mL) and washed with H2O (3 x 20 mL). The EtOAc
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layer was dried (MgSO4) and evaporated under reduced pressure to give the crude
imidazole (7) or triazole (8) which was was further purified by recrystallisation.
(1,3-Diphenyl)-4-((1,3-imidazol-1-yl)methyl)-1H-pyrazole (7a). Prepared
from 4ꢀ(chloromethyl)ꢀ1,3ꢀdiphenylꢀ1Hꢀpyrazole (6a) (0.35 g, 1.3 mmol). Product
obtained as a yellow crystalline solid after recrystallization from methanol, yield 0.12
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o
1
g (32 %). M.p. 124ꢀ128 C. TLC (3:1 petroleum ether/EtOAc), Rf = 0.06. H NMR
(CDCl3): δ 7.85 (s, 1H, pyrazole), 7.74 (d, J = 7.7 Hz, 2H, triazole), 7.68 (s, 1H, Ar),
7.61 (d, J = 7.0 Hz, 2H, Ar), 7.42ꢀ7.50 (m, 5H, Ar), 7.33 (t, J = 7.5 Hz, 1H, Ar), 7.12
(s, 1H, Ar), 6.96 (s, 1H, Ar), 5.26 (s, 2H, CH2). 13CꢀNMR (CDCl3): δ 151.4, 116.2 (2
x pyrazole), 139.6, 132.4 (2 x Ar), 136.9, 129.5, 129.3, 128.9, 128.6, 127.9, 127.7,
126.4, 119.1, 119.0 (12 x CH, Ar), 41.9 (CH2). Anal. Calcd for C19H16N4 (300.36): C,
75.98; H, 5.37; N, 18.64. Found: C, 76.10; H, 5.39; N, 18.58.
4-((1H-Imidazol-1-yl)methyl)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole
(7b). Prepared from 4ꢀ(chloromethyl)ꢀ3ꢀ(4ꢀfluorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole (6b)
(0.22 g, 0.76 mmol). Product obtained as a yellow crystalline solid after
recrystallization from EtOHꢀH2O 1:1 v/v, yield 0.07 g (29 %). M.p. 128ꢀ130 oC. TLC
1
(3:1 petroleum ether/EtOAc), Rf = 0.06. H NMR (CDCl3): δ 7.89 (s, 1H, pyrazole),
7.78 (s, 1H, Ar), 7.73 (d, J = 7.8 Hz, 2H, Ar), 7.55ꢀ7.58 (m, 2H, Ar), 7.49 (t, J = 7.7
Hz, 2H, Ar), 7.34 (t, J = 7.5 Hz, 1H, Ar), 7.13ꢀ7.18 (m, 3H, Ar), 6.95 (s, 1H, Ar), 5.25
(s, 2H, CH2). 13CꢀNMR (CDCl3): δ 164.0, 150.6, 139.5 (3 x C, Ar), 162.0, 128.5 (2 x
C, pyrazole), 136.8, 129.7, 129.6, 128.9, 128.0, 127.0, 117.1, 117.0, 116.0, 115.8 (13
x CH, Ar), 41.9 (CH2). Anal. Calcd for C19H15FN4 (318.35): C, 71.68; H, 4.75; N,
17.60. Found: C, 71.51; H, 4.98; N, 17.62.
4-((1H-Imidazol-1-yl)methyl)-3-(4-chlorophenyl)-1-phenyl-1H-pyrazole
(7c). Prepared from 4ꢀ(chloromethyl)ꢀ3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole (6c)
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(0.91 g, 3 mmol). Product obtained as a yellow crystalline solid after recrystallization
from ethanol, yield 0.52 g (52 %). M.p. 155ꢀ158 oC. TLC (3:1 petroleum
ether/EtOAc), Rf = 0.05. 1H NMR (CDCl3): δ 7.88 (s, 1H, pyrazole), 7.78 (s, 1H, Ar),
7.73 (d, J = 8.4 Hz, 2H, Ar), 7.54 (d, J = 8.0 Hz, 2H, Ar), 7.49 (d, J = 7.6 Hz, 2H,
Ar), 7.45 (d, J = 8.1 Hz, 2H, Ar), 7.35 (t, J = 7.4 Hz, 1H Ar), 7.14 (s, 1H, Ar), 6.96 (s,
1H, Ar), 5.26 (s, 2H, CH2). 13CꢀNMR (CDCl3): δ 150.3, 116.0 (2 x C, pyrazole),
139.5, 134.6, 130.8 (3 x C, Ar), 136.8, 129.6, 129.1, 129.0, 128.1, 127.1, 119.1, 119.0
(12 x CH, Ar), 41.9 (CH2). Anal. Calcd for C19H15ClN4 (334.80): C, 68.16; H, 4.52;
N, 16.73. Found: C, 68.09; H, 4.17; N, 16.58.
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4-((1
H
-Imidazol-1-yl)methyl)-1-phenyl-3-(
p
-tolyl)-1
H
-pyrazole
(7d).
Prepared from 4ꢀ(chloromethyl)ꢀ1ꢀphenylꢀ3ꢀ(pꢀtolyl)ꢀ1Hꢀpyrazole (6d) (0.35 g, 1.24
mmol). Product obtained as a yellowꢀorange crystalline solid after recrystallization
from EtOHꢀH2O 1:1 v/v, yield 0.065 g (17 %). M.p. 145ꢀ148 oC. TLC (3:1 petroleum
1
ether/EtOAc), Rf = 0.05. H NMR (CDCl3): δ 7.87 (s, 1H, pyrazole), 7.8 (s, 1H, Ar),
7.74 (d, J = 8.3 Hz, 2H, Ar), 7.48ꢀ7.50 (m, 4H, Ar), 7.28ꢀ7.34 (m, 4H, Ar), 7.13 (s,
1H, Ar), 6.95 (s, 1H, Ar), 5.27 (s, 2H, CH2), 2.43 (s, 3H, CH3). 13CꢀNMR (CDCl3):
δ 151.5, 115.9 (2 x C, pyrazole), 139.7, 138.5, 129.4 (3 x C, Ar), 136.8, 129.6, 129.5,
128.6, 127.8, 127.7, 126.8, 119.1 (13 x CH, Ar), 42.0 (CH2), 21.3 (CH3). Anal. Calcd
for C20H18N4 (314.38): C, 76.41; H, 5.77; N, 17.81. Found: C, 76.40; H, 5.79; N,
17.77.
4-((1H-Imidazol-1-yl)methyl)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole
(7e). Prepared from 4ꢀ(chlorophenyl)ꢀ3ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole
(6e) (0.88g, 2.9 mmol). Product obtained as a white crystalline solid after
recrystallization from EtOHꢀH2O 1:1 v/v, yield 0.42 g (43 %). M.p. 102ꢀ106 oC. TLC
1
(3:1 petroleum ether/EtOAc), Rf = 0.05. H NMR (CDCl3): δ 7.89 (s, 1H, pyrazole),
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7.85 (s, 1H, Ar), 7.74 (d, J = 7.6 Hz, 2H, Ar), 7.52 (d, J = 8.8 Hz, 2H, Ar), 7.48 (d, J
= 7.5 Hz, 2H, Ar), 7.32 (t, J = 7.5 Hz, 1H, Ar), 7.14 (s, 1H, Ar), 7.01 (d, J = 8.8 Hz,
13
2H, Ar), 6.96 (s, 1H, Ar), 5.27 (s, 2H, CH2), 3.88 (s, 3H, OCH3). CꢀNMR (CDCl3):
9
δ 159.9, 139.7, 124.8 (3 x C, Ar), 151.4, 115.6 (2 x C, pyrazole), 136.8, 129.5, 129.1,
128.5, 127.5, 126.8, 119.0, 114.4 (13 x CH, Ar), 55.4 (CH3), 42.1 (CH2). Anal. Calcd
for C20H18N4O (330.39): C, 71.68; H, 4.75; N, 17.60. Found: C, 71.66; H, 4.76; N,
17.54.
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4-((1H-Imidazole-1-yl)methyl)-3-(4-bromophenyl)-1-phenyl-1H-pyrazole
(7f). Prepared from (4ꢀ(chloromethyl)ꢀ3ꢀ(4ꢀbromophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole) (6f)
(0.75 g, 2.2 mmol). Product obtained as a white crystalline solid after recrystallization
from EtOHꢀH2O 1:1 v/v, yield 0.45 g (56 %). M.p. 161ꢀ163 oC. TLC (9:1
1
CH2Cl2/MeOH), Rf = 0.83. H NMR (CDCl3): δ 8.60 (s, 1H, pyrazole), 8.15 (s, 1H,
imidazole), 7.75 (d, J = 7.6 Hz, 2H, Ar), 7.60 (d, J = 8.4 Hz, 2H, Ar), 7.5ꢀ.47 (m, 4H,
Ar), 7.35 (t, J = 7.4 Hz, 1H, Ar), 7.20 (s, 1H, imidazole), 6.90 (s, 1H, imidazole), 5.43
(s, 2H, CH2). 13CꢀNMR (CDCl3): δ 150.2 (C, pyrazole), 139.5 (C, Ar), 137.0 (CH,
imidazole), 132.0 (2 x CH, Ar), 131.3 (C, Ar), 130.0 (CH, Ar), 129.6 (C, Ar), 129.3
(CH, imidazole), 127.9 (2 x CH, Ar), 127.1 (2 x CH, Ar), 122.8 (CH, pyrazole), 119.1
(2 x CH, Ar), 118.9 (CH, imidazole), 116.4 (C, pyrazole), 41.7 (CH2). Anal. Calcd for
C19H15N4Br (379.25): C, 60.17; H, 3.99; N, 14.77. Found: C, 59.92; H, 4.02; N,
14.67.
4-((1H-Imidazole-1-yl)methyl)-3-(4-iodophenyl)-1-phenyl-1H-pyrazole
(7g). Prepared from (4ꢀ(chloromethyl)ꢀ3ꢀ(4ꢀiodophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole) (6g)
(0.75 g, 1.9 mmol). Product obtained as a pale yellow crystalline solid after
recrystallization from EtOHꢀH2O 1:1 v/v, yield 0.45 g (55 %). M.p. 169ꢀ173 oC. TLC
1
(3:2 petroleum ether/EtOAc), Rf = 0.08. H NMR (CDCl3): δ 8.10 (s, 1H, pyrazole),
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7.90 (s, 1H, imidazole), 7.80 (d, J = 8.5 Hz, 2H, Ar), 7.74 (m, 3H, Ar) 7.16 (s, 1H,
imidazole), 6.95 (s, 1H, imidazole), 5.32 (s, 2H, CH2). 13CꢀNMR (CDCl3): δ 150.3 (C,
pyrazole), 139.5 (C, Ar), 138.0 (2 x CH, Ar), 137.0 (CH, imidazole), 131.9 (C, Ar),
130.0 (CH, Ar), 129.6 (CH, imidazole), 129.5 (2 x CH, Ar), 127.9 (2 x CH, Ar), 127.1
(CH, pyrazole), 119.1 (2 x CH, Ar), 118.9 (CH, imidazole), 116.4 (C, pyrazole), 94.4
(C, CꢀI), 41.7 (CH2). Anal. Calcd for C19H15N4I (426.25): C, 53.54; H, 3.55; N, 13.14.
Found: C, 53.74; H, 3.23; N, 13.15.
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4-(4-((1H-Imidazol-1-yl)methyl)-1-phenyl-1H-pyrazol-3-yl)benzonitrile
(7h). Prepared from 4ꢀ(4ꢀ(chloromethyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ3ꢀyl)benzonitrile (6h)
(0.75 g, 2.5 mmol). Product obtained as a pale yellow crystalline solid after
recrystallization from EtOHꢀH2O 1:1 v/v, yield 0.46 g (57 %). M.p. 197ꢀ200 oC. TLC
(9:1 CH2Cl2/MeOH), Rf = 0.63. 1H NMR (CDCl3): δ 7.83 (s, 1H, pyrazole). 7.76ꢀ7.72
(m, 6H, Ar), 7.58 (s, 1H, imidazole), 7.50 (t, J = 8.0 Hz, 2H, Ar), 7.40 (t, J = 7.4 Hz,
1H, Ar), 7.14 (s, 1H, imidazole), 6.90 (s, 1H, imidazole), 5.27 (s, 2H, CH2). 13CꢀNMR
(CDCl3): δ 149.1 (C, pyrazole), 139.3 (C, Ar), 137.0 (CH, imidazole), 132.8 (CH,
imidazole), 132.6 (2 x CH, Ar), 130.2 (2 x CH, Ar), 129.7 (CH, imidazole), 128.3 (2 x
CH, Ar), 128.1 (CH, pyrazole), 127.4 (2 x CH, Ar), 119.2 (CN), 118.8 (C, Ar), 116.8
(C, pyrazole), 112.0 (C, Ar), 41.7 (CH2). Anal. Calcd for C20H15N5 (325.37): C,
73.83; H, 4.65; N, 21.52. Found: C, 73.76; H, 4.69; N, 21.51.
1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-1H-[1,2,4]triazole (8a). Prepared
from 4ꢀ(chloromethyl)ꢀ1,3ꢀdiphenylꢀ1Hꢀpyrazole (6a) (0.40 g, 1.5 mmol). Product
obtained as a light yellow crystalline solid after recrystallization from methanol, yield
o
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0.115 g (26 %). M.p. 101ꢀ104 C. TLC (3:1 petroleum ether/EtOAc), Rf = 0.06. H
NMR (CDCl3): δ 8.29 (s, 1H, pyrazole), 8.07 (m, 2H, Ar), 7.77 (d, J = 7.7 Hz, 2H,
Ar), 7.65 (d, J = 7.0 Hz, 2H, Ar), 7.42ꢀ7.50 (m, 5H, Ar), 7.34 (t, J = 7.5 Hz, 1H, Ar),
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5.50 (s, 2H, CH2). 13CꢀNMR (CDCl3): δ 151.7, 114.1 (2 x C, pyrazole), 139.6, 132.1
(2 x C, Ar), 151.1, 142.6, 129.5, 128.9, 128.7, 128.6, 128.0, 127.0, 119.2 (13 x CH,
Ar), 44.7 (CH2). Anal. Calcd for C19H15N5 (301.35): C, 71.74; H, 5.02; N, 23.23.
Found: C, 71.97; H, 5.06; N, 23.35.
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1-((3-(4-Fluorophenyl)-1-phenyl-1H-pyrazole-4-yl)methyl)-1H-1,2,4-
triazole (8b). Prepared from 4ꢀ(chloromethyl)ꢀ3ꢀ(4ꢀfluorophenyl)ꢀ1ꢀphenylꢀ1Hꢀ
pyrazole (6b) (0.15 g, 0.52 mmol). Product obtained as a pale yellow crystalline solid
after recrystallization from EtOHꢀH2O 1:1 v/v, yield 0.039 g (24 %). M.p. 114ꢀ118
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oC. TLC (3:1 petroleum ether/EtOAc), Rf = 0.06. H NMR (CDCl3): δ 8.09 (s, 1H,
pyrazole), 8.03 (d, J = 7.7 Hz, 2H, triazole), 7.75 (d, J = 7.6 Hz, 2H, Ar), 7.61ꢀ7.64
(m, 2H, Ar), 7.49 (t, J = 7.7 Hz, 2H, Ar), 7.35 (t, J = 7.4 Hz, 1H, Ar), 7.17 (t, J = 8.7
Hz, 2H, Ar), 5.45 (s, 2H, CH2). 13CꢀNMR (CDCl3): δ 164.0, 150.8, 139.5 (3 x C, Ar),
150.8, 114.2 (2 x C, pyrazole), 152.1, 142.7, 129.8, 129.7, 129.5, 128.5, 127.1, 119.2,
116.0, 115.8 (12 x CH, Ar), 44.4 (CH2). Anal. Calcd for C18H14FN5 (319.34): C,
67.70; H, 4.42; N, 21.92. Found: C, 67.98; H, 4.00; N, 22.05.
1-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazole-4-yl)methyl)-1H-1,2,4-
triazole (8c). Prepared from 4ꢀ(chloromethyl)ꢀ3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀphenylꢀ1Hꢀ
pyrazole (6c) (0.61 g, 2 mmol). Product obtained as a pale yellow crystalline solid
o
after recrystallization from ethanol, yield 0.27 g (40 %). M.p. 119ꢀ122 C. TLC (3:1
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petroleum ether/EtOAc), Rf = 0.05. H NMR (CDCl3): δ 8.10 (s, 1H, pyrazole), 8.02
(s, 2H, triazole), 7.74 (d, J = 7.8 Hz, 2H, Ar), 7.60 (d, J = 8.5 Hz, 2H, Ar), 7.44ꢀ7.51
(m, 4H, Ar), 7.35 (t, J = 7.5 Hz, 1H, Ar), 5.55 (s, 2H, CH2). 13CꢀNMR (CDCl3):
δ 150.5, 114.4 (2 x C, pyrazole), 139.5, 134.7, 120.7(3 x C, Ar), 150.2, 142.7, 129.6,
129.2, 129.1, 128.6, 127.1, 119.2 (12 x CH, Ar), 44.3 (CH2). Anal. Calcd for
C18H14ClN5 (335.79): C, 64.38; H, 4.20; N, 20.85. Found: C, 64.63; H, 3.81; N,
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1((1-Phenyl-3-(
p
-tolyl)-1H-pyrazol-4-yl)methyl)-1
H
-1,2,4-triazole
(8d).
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Prepared from 4ꢀ(chloromethyl)ꢀ1ꢀphenylꢀ3ꢀ(pꢀtolyl)ꢀ1Hꢀpyrazole (6d) (0.35 g, 1.24
9
mmol). Product obtained as a yellowꢀorange crystalline solid after recrystallization
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from EtOHꢀH2O 1:1 v/v, yield 0.11 g (29 %). M.p. 128ꢀ130 C. TLC (3:1 petroleum
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ether/EtOAc), Rf = 0.14. H NMR (CDCl3): δ 8.14 (s, 1H, pyrazole), 8.04 (d, J = 5.6
Hz, 2H, Ar), 7.76 (d, J = 8.1 Hz, 2H, Ar), 7.52 (d, J = 7.9 Hz, 2H, Ar), 7.48 (t, J = 7.8
Hz, 2H, Ar), 7.33 (t, J = 7.2 Hz, 1H,Ar), 7.28ꢀ7.30 (m, 2H, Ar), 5.47 (s, 2H, CH2),
2.43 (s, 3H, OCH3). 13CꢀNMR (CDCl3): δ 151.8, 114.1 (2 x C, pyrazole), 139.7,
138.6, 129.3 (3 x C, Ar), 151.6, 142.2, 129.6, 129.5, 128.4, 127.8, 126.9, 119.1 (12 x
CH, Ar), 44.6 (CH2), 21.3 (CH3). Anal. Calcd for C19H17N5 (315.37): C, 72.36; H,
5.43; N, 22.20. Found: C, 72.43; H, 5.43; N, 22.41.
1-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)-1H-1,2,4-
triazole (8e). Prepared from 4ꢀ(chlorophenyl)ꢀ3ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀphenylꢀ1Hꢀ
pyrazole (6e) (0.45 g, 1.5 mmol). Product obtained as an orange crystalline solid after
recrystallization from EtOHꢀH2O 1:1 v/v, yield 0.21 g (43 %). M.p. 106ꢀ108 oC. TLC
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(3:1 petroleum ether/EtOAc), Rf = 0.05. H NMR (CDCl3): δ 8.21 (s, 1H, pyrazole),
8.05 (d, J = 7.7 Hz, 2H, Ar), 7.76 (d, J = 8.0 Hz, 2H, Ar), 7.57 (d, J = 8.7 Hz, 2H,
Ar), 7.48 (d, J = 8.0 Hz, 2H, Ar), 7.33 (t, J = 7.4 Hz, 1H,Ar), 7.01 (d, J = 8.7 Hz, 2H,
Ar), 5.47 (s, 2H, CH2), 3.88 (s, 3H, OCH3). 13CꢀNMR (CDCl3): δ 160.0, 139.7, 124.7
(3 x C, Ar), 151.6, 114.0 (2 x C, pyrazole), 151.9, 142.7, 129.6, 129.5, 129.2, 128.4,
126.8, 119.2, 114.2 (12 x CH, Ar), 55.4 (CH3), 44.5 (CH2). Anal. Calcd for
C19H17N5O (331.37): C, 68.87; H, 5.17; N, 21.12. Found: C, 68.87; H, 4.87; N,
21.15.
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1-((3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)-1H-1,2,4-
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triazole (8f). Prepared from (4ꢀ(chloromethyl)ꢀ3ꢀ(4ꢀbromophenyl)ꢀ1ꢀphenylꢀ1Hꢀ
pyrazole) (6f) (0.7 g, 2 mmol). Product obtained as a white crystalline solid after
recrystallization from EtOHꢀH2O 1:1 v/v, yield 0.36 g (47 %). M.p. 144ꢀ146 oC. TLC
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(9:1 CH2Cl2/MeOH), Rf = 0.68. H NMR (CDCl3): δ 8.04 (s, 1H, pyrazole), 8.02 (s,
2H, triazole), 7.74 (dd, J = 1.1, 8.7 Hz, 2H, Ar), 7.6 (dd, J = 1.9, 6.6 Hz, 2H, Ar), 7.54
(s, 1H, Ar), 7.52ꢀ7.48 (m, 2H, Ar), 7.35 (t, J = 7.4 Hz, 1H, Ar), 5.45 (s, 2H, CH2).
13CꢀNMR (CDCl3): δ 152.4 (CH, triazole), 150.5 (C, pyrazole), 142.8 (CH, triazole),
139.5 (C, Ar), 132.2 (2 x CH, Ar), 129.7 (2 x CH, Ar), 129.6 (CH, Ar), 129.4 (2 x
CH, Ar), 129.3 (CH, Ar), 128.6 (CH, pyrazole), 128.0 (C, pyrazole). 127.2 (2 x CH,
Ar), 114.4 (C, Ar), 44.3 (CH2). Anal. Calcd for C18H14N5Br (380.24): C, 56.86; H,
3.71; N, 18.42. Found: C, 56.48; H, 3.99; N, 18.43.
1-((3-(4-Iodophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)-1H-1,2,4-triazole
(8g). Prepared from (4ꢀ(chloromethyl)ꢀ3ꢀ(4ꢀiodophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole) (6g)
(0.8 g, 2 mmol). Product obtained as a white crystalline solid after recrystallization
from EtOHꢀH2O 1:1 v/v, yield 0.4 g (46 %). M.p. 151ꢀ155 oC. TLC (9:1
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CH2Cl2/MeOH), Rf = 0.13. H NMR (CDCl3): δ 8.03 (s, 1H, pyrazole), 8.01 (s, 2H,
triazole), 7.82 (dd, J = 1.8, 6.6 Hz, Ar), 7.74 (dd, J = 1.2, 8.7 Hz, 2H, Ar), 7.50 (t, J =
8.0 Hz, 2H, Ar), 7.40 (d, J = 8.4 Hz, 2H, Ar), 7.35 (t, J = 7.4 Hz, 1H, Ar), 5.45 (s, 2H,
CH2). 13CꢀNMR (CDCl3): δ 152.4 (CH, triazole), 150.6 (C, pyrazole), 142.8 (CH,
triazole), 139.5 (C, Ar), 138.0 (2 x CH, Ar), 131.7 (CH, Ar), 129.6 (2 x CH, Ar),
129.4 (2 x CH, Ar), 128.7 (CH, Ar), 127.4 (CH, pyrazole), 119.2 (2 x CH, Ar), 114.4
(C, pyrazole), 94.6 (CꢀI), 44.3 (CH2). Anal. Calcd for C18H14N5I (427.24): C, 50.60;
H, 3.30; N, 16.39. Found: C, 50.87; H, 3.27; N, 16.38.
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(8h). Prepared from 4ꢀ(4ꢀ(chloromethyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ3ꢀyl)benzonitrile (6h)
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(0.5 g, 1.7 mmol). Product obtained as a white crystalline solid after recrystallization
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from EtOHꢀH2O 1:1 v/v, yield 0.23 g (42 %). M.p. 162ꢀ165 C. TLC (1:1 petroleum
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ether/EtOAc), Rf = 0.08. H NMR (DMSOꢀd6): δ 8.65 (s, 1H, triazole), 8.59 (s, 1H,
triazole), 8.01ꢀ7.94 (m, 3H), 7.89 (d, J = 7.7 Hz, 2H, Ar), 7.53 (t, J = 8.0 Hz, 2H, Ar),
7.37 (t, J = 7.4 Hz, 1H, Ar), 5.58 (s, 2H, CH2). 13CꢀNMR (CDCl3): δ 152.1 (CH,
triazole), 149.1 (C, pyrazole), 144.5 (CH, triazole), 139.5 (C), 137.3 (C), 133.2 (2 x
CH), 130.9 (CH), 130.1 (2 x CH), 128.7 (2 x CH), 127.4 (CH), 119.2 (C), 119.1 (2 x
CH), 116.8 (C), 111.2 (C), 43.5 (CH2).Anal. Calcd for C19H14N6 (326.36): C, 69.93;
H, 4.32; N, 25.74. Found: C, 69.80; H, 4.23; N, 25.78.
General method for the preparation of 1H-pyrazole-4-carboxamide.
Carboxylic acid (9) (1 mmol) was dissolved in anhydrous DMF (4 mL/mmol), then a
solution of 1,1’ꢀcarbonyldiimidazole (1.1 mmol) in dry DMF (1 mL/mmol) was added
and the reaction stirred for 1 h. The reaction was cooled to 0 oC and a mixture of 1ꢀ(2ꢀ
ammonioethyl)ꢀ1Hꢀimidazolꢀ3ꢀium chloride (12) (1 mmol) with triethylamine (3
mmol) in dry DMF (1 mL/mmol) was added and the mixture stirred at room
temperature for 20 h. Once complete, ice cold H2O (50 mL/mmol) was added and the
resulting yellow precipitate collected by filtration and washed with cold water and
dried [15].
N
-(2-(1H-Imidazol-1-yl)ethyl)-1,3-diphenyl-1H-pyrazole-4-carboxamide
(13a). Prepared from 1,3ꢀdiphenylꢀ1Hꢀpyrazoleꢀ4ꢀcarboxylic acid (9a) (0.42, 1.6
mmol). Product obtained as a white solid after purification by gradient flash column
chromatography, product eluted with EtOAc/MeOH 9:1 v/v, yield 0.24 g (40 %).
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M.p. 136ꢀ138 C. TLC (9:1 EtOAc/MeOH), Rf = 0.42. H NMR (DMSOꢀd6): δ 8.90
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(s, 1H, pyrazole), 8.37 (s, 1H, NH), 7.88 (d, J = 7.3 Hz, 2H, Ar), 7.79 (d, J = 6.5 Hz,
2H, Ar), 7.65 (s, 1H, imidazole), 7.58 (t, J = 6.8 Hz, 2H, Ar), 7.43 (t, J = 8.2 Hz, 4H,
Ar), 7.21 (s, 1H, imidazole), 6.92 (s, 1H, imidazole), 4.16 (s, 2H, CH2), 3.57 (d, J =
4.6 Hz, 2H, CH2). 13CꢀNMR (DMSOꢀd6): δ 162.9 (CO), 150.9 (C, pyrazole), 138.9,
132.2 (2 x C, Ar), 129.9 (CH, pyrazole), 129.7 (CH, Ar), 129.6 (CH, Ar), 129.1 (CH,
Ar), 128.4 (CH, Ar), 128.2 (CH, Ar), 128.2 (CH, Ar), 128.0 (CH, Ar), 127.9 (CH,
Ar), 127.7 (CH, pyrazole), 126.9 (CH, Ar), 126.6 (CH, Ar), 118.8 (CH, Ar), 117.4 (C,
pyrazole), 45.3, 40.0 (2 x CH2). Anal. Calcd for C21H19N5O (357.42): C, 70.57; H,
5.36; N, 19.59. Found: C, 70.28; H, 5.19; N, 19.29.
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N
-(2-(1H-Imidazol-1-yl)ethyl)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-
4-carboxamide (13b). Prepared from 3ꢀ(4ꢀfluorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazoleꢀ4ꢀ
carboxylic acid (9b) (0.25 g, 1.4 mmol). Product obtained as a white solid after
purification by gradient flash column chromatography, product eluted with
EtOAc/MeOH 9:1 v/v, yield 0.175 g (29 %). M.p. 158ꢀ160 oC. TLC (9:1
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CH2Cl2/MeOH), Rf = 0.2. H NMR (CD3OD): δ 8.49 (s, 1H, pyrazole), 7.83 (d, J =
7.7 Hz, 2H, Ar), 7.85 – 7.73 (m, 3H, Ar), 7.56 ꢀ 7.52 (Ψt, J = 7.6, 8.4 Hz, 2H, Ar),
7.41 (t, J = 7.0 Hz, 1H, imidazole), 7.20 (s, 1H, imidazole), 7.19 ꢀ 714 (m, 2H, Ar),
7.05 (s, 1H, imidazole), 4.28 (t, J = 6.0 Hz, 2H, CH2), 3.72 (t, J = 6.0 Hz, 2H, CH2).
13CꢀNMR (CD3OD): δ 166.1 (CO), 165.4 (FꢀC), 163.5 (C, Ar), 152.4 (C, pyrazole),
140.7 (C, Ar), 131.8, 131.7, 131.1, 129.9, 130.7, 128.9, 128.5 (13 x CH, Ar), 129.8
(C, Ar), 118.1 (C, pyrazole), 47.2, 41.4 (2 x CH2). Anal. Calcd for C21H18FN5O: 0.3
H2O (380.81): C, 66.60; H, 4.90; N, 18.31. Found: C, 66.57; H, 4.82; N, 17.99.
N
-(2-(1H-Imidazol-1-yl)ethyl)-3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-
4-carboxamide (13c). Prepared from 3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazoleꢀ4ꢀ
carboxylic acid (9c) (0.42 g, 1.4 mmol). Product obtained as a white crystalline solid
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after recrystallization from acetonitrile, yield 0.11 g (19 %). M.p. 184ꢀ186 C. TLC
(9:1 CH2Cl2/MeOH), Rf = 0.13. 1H NMR (CD3OD): δ 8.53 (s, 1H, pyrazole), 7.83 (d,
J = 7.7 Hz, 2H, Ar), 7.72 (d, J = 8.5 Hz, 2H, Ar), 7.68 (s, 1H, imidazole), 7.55 (Ψt, J
= 7.6, 8.4 Hz 2H, Ar), 7.44 – 7.39 (m, 3H, Ar), 7.18 (s, 1H, imidazole), 7.03 (s, 1H,
imidazole), 4.25 (t, J = 6.0 Hz, 2H, CH2), 3.71 (t, J = 6.0 Hz, 2H, CH2). 13CꢀNMR
(CD3OD): δ 166.1 (CO), 152.2 (C, pyrazole), 140.7, 135.6, 132.3(3 x C, Ar), 131.2
(CH, pyrazole), 131.1, 130.8, 129.4, 128.6, 120.5 (12 x CH, Ar), 118.3 (C, pyrazole),
47.1, 41.4 (2 x CH2). Anal. Calcd for C21H18ClN5O: 0.1 H2O (393.66): C, 64.07; H,
4.65; N, 17.79. Found: C, 63.92; H, 4.33; N, 17.76.
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N
-(2-(1H-Imidazol-1-yl)ethyl)-1-phenyl-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (13d). Prepared from 1ꢀphenylꢀ3ꢀ(pꢀtolyl)ꢀ1Hꢀpyrazoleꢀ4ꢀcarboxylic
acid (9d) (0.47 g, 1.7 mmol). Product obtained as a white solid after purification by
gradient flash column chromatography, product eluted with EtOAc/MeOH 9:1 v/v,
yield 0.034 g (6 %). M.p. 76ꢀ80 C. TLC (9:1 CH2Cl2/MeOH), Rf = 0.20. 1H NMR
o
(CD3OD): δ 8.50 (s, 1H, pyrazole), 7.81 (d, J = 7.7 Hz, 2H, Ar), 7.69 (s, 1H,
imidazole), 7.59 (d, J = 8.1 Hz, 2H, Ar), 7.53 (Ψt, J = 7.7, 8.3 Hz, 2H, Ar), 7.39 (t, J
= 7.5 Hz, 1H, Ar), 7.24 (d, J = 8.0 Hz, 2H, Ar), 7.17 (s, 1H, imidazole), 7.03 (s, 1H,
imidazole), 4.23 (t, J = 6.0 Hz, 2H, CH2), 3.68 (t, J = 6.0 Hz, 2H, CH2), 2.45 (s, 3H,
CH3). 13CꢀNMR (CD3OD): δ 165.6 (CO), 152.50 (C, pyrazole), 139.98, 139.1 (2 x C,
Ar), 130.4 (CH, pyrazole), 129.9, 129.2, 128.3, 119.7 (12 x CH, Ar), 129.8 (C, Ar),
117.4 (C, pyrazole), 46.4, 40.6 (2 x CH2), 20.6 (CH3). Anal. Calcd for C22H21N5O
(371.44): C, 71.14; H, 5.70; N, 18.85. Found: C, 70.87; H, 5.74; N, 18.67.
N
-(2-(1H-Imidazol-1-yl)ethyl)-3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazole-4-carboxamide (13e). Prepared from 3ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀphenylꢀ1Hꢀ
pyrazoleꢀ4ꢀcarboxylic acid (9e) (0.4 g, 1.4 mmol). Product obtained as a yellow solid
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after purification by gradient flash column chromatography, product eluted with
EtOAc/MeOH 9:1 v/v, yield 0.18 g (32 %). M.p. 108ꢀ112 oC. TLC (9:1
CH2Cl2/MeOH), Rf = 0.60. 1H NMR (CD3OD): δ 8.51 (s, 1H, pyrazole), 7.83 (d, J =
7.7 Hz, 2H, Ar), 7.66 (s, 1H, imidazole), 7.55 (Ψt, J = 7.6, 8.4 Hz 2H, Ar), 7.42 –
7.27 (m, 4H, Ar/imidazole), 7.16 (s, 1H, imidazole), 7.01 – 6.98 (m, 2H, Ar), 4.25 (t,
J = 6.0 Hz, 2H, CH2), 3.86 (s, 3H, OCH3), 3.71 (t, J = 6.0 Hz, 2H, CH2). 13CꢀNMR
(CD3OD): δ 166.3 (CO), 161.1 (C, Ar), 152, 9 (C, pyrazole), 140.8, 134.8 (2 x C, Ar),
131.2 (CH, pyrazole), 130.7, 130.5, 129.2, 128.5, 122.1, 120.9, 115.4, 115.2 (12 x
CH, Ar), 118.5 (C, pyrazole), 55.8 (OCH3), 47.1, 41.5 (2 x CH2). Anal. Calcd for
C22H21N5O2: 0.2 H2O (391.04): C, 67.57; H, 5.51; N, 17.91. Found: C, 67.21; H, 5.25;
N, 17.56.
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CYP121A1 Spectral binding assays for ligand KD determination.
CYP121A1 protein was expressed and purified as described previously.14 Ligand
binding assays were performed by spectrophotometric titration using a Cary 60 UVꢀ
visible scanning spectrophotometer (Agilent, UK) and a 1 cm path length quartz
cuvette, recording spectra between 250 and 800 nm. Titrations were typically done
o
with 3ꢀ5 ꢁM CYP121A1 at 28 C in 100 mM potassium phosphate (KPi) buffer, 200
mM KCl, pH 7.85 with 0.004% Triton Xꢀ100. Ligand stock solutions were prepared
in dimethylsulfoxide (DMSO). Ligands were added in small volumes (typically 0.05ꢀ
0.2 µL aliquots) from concentrated stock solutions to the protein in a 1 mL final
volume. Spectral measurements were taken before ligand addition, and following
addition of each aliquot of ligand until no further spectral change occurred. Difference
spectra at each stage in the titration were obtained by subtraction of the initial ligandꢀ
free enzyme spectrum from subsequent spectra collected after each addition of ligand.
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From the difference spectra, a pair of wavelengths were identified and defined as the
absorbance maximum (Apeak) and minimum (Atrough). The overall absorbance change
(ꢁAmax) was calculated by subtracting the Atrough value from the Apeak value for each
spectrum collected after a ligand addition. Graphs of ꢁAmax against [ligand] were
plotted for each titration. Titrations were done in triplicate and the final Kd value
presented was determined as the average value across the three sets. The Kd values
were determined by fitting the data using either a standard hyberbolic function
(Equation 1) or the Hill equation (Equation 2) using Origin software (OriginLab,
Northampton, MA).
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Aobs = (Amax*L/(Kd+L))
(Equation 1)
In Equation 1 (the standard hyperbolic function, the MichaelisꢀMenten function
adapted for ligand binding), Aobs is the observed absorbance change at ligand
concentration L, Amax is the maximal absorbance change observed at apparent ligand
saturation, and Kd is dissociation constant for the binding of the ligand (the substrate
concentration at which Aobs = 0.5 x Amax).
A
obs = (Amax × Ln)/(Kn+Ln)
(Equation 2)
In Equation 2 (the sigmoidal Hill equation), Aobs is the observed absorbance change at
ligand concentration L, Amax is the absorbance change at apparent ligand saturation, K
is the apparent dissociation constant, and n is the Hill coefficient, a value describing
the apparent extent of cooperativity observed in ligand binding.
5.5 Antimycobacterial Activity Assay
M. tuberculosis H37Rv was grown in 7H9 liquid medium with 10 % Middlebrook
OADC Growth Supplement enrichment (BBL/BectonꢀDickinson, Sparks, MD, USA).
The bacterial growth occurred at 37 °C until the midꢀlog phase was reached (OD600nm
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