Design, synthesis and structure-activity relationships of 3,5-diaryl-1H-pyrazoles as inhibitors of arylamine N-acetyltransferase

Article abstract of DOI:10.1016/j.bmcl.2013.02.052

The synthesis and inhibitory potencies of a novel series of 3,5-diaryl-1H-pyrazoles as specific inhibitors of prokaryotic arylamine N-acetyltransferase enzymes is described. The series is based on hit compound 1 3,5-diaryl-1H-pyrazole identified from a high-throughout screen that has been carried out previously and found to inhibit the growth of Mycobacterium tuberculosis.

Full text of DOI:10.1016/j.bmcl.2013.02.052

Accepted Manuscript
Design, synthesis and structure-activity relationships of 3,5-diaryl-1H-pyra‐
zoles as inhibitors of arylamine N-acetyltransferase
Elizabeth Fullam, James Talbot, Areej Abuhammed, Isaac Westwood, Stephen
G. Davies, Angela J. Russell, Edith Sim
PII:
S0960-894X(13)00228-X
http://dx.doi.org/10.1016/j.bmcl.2013.02.052
BMCL 20170
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
11 December 2012
8 February 2013
12 February 2013
Please cite this article as: Fullam, E., Talbot, J., Abuhammed, A., Westwood, I., Davies, S.G., Russell, A.J., Sim,
E., Design, synthesis and structure-activity relationships of 3,5-diaryl-1H-pyrazoles as inhibitors of arylamine N-
acetyltransferase, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.
2013.02.052
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Design, synthesis and structure-activity relationships of 3,5-diaryl-1H-pyrazoles as
inhibitors of arylamine N-acetyltransferase†
Elizabeth Fullam,*abc James Talbot,abAreej Abuhammed,ad Isaac Westwood,abe Stephen G.
Davies,b Angela J. Russell,ab Edith Sim*ae
aDepartment of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK, OX1 3QT.
bDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield
Road, Oxford, UK, OX1 3TA.
*c
Current address School of Life Sciences, Gibbet Hill Campus, The University of Warwick,
Coventry, CV4 7AL, United Kingdom. E-mail e.fullam@warwick.ac.uk Tel +44 (0)24 765 74251
d Faculty of Pharmacy, University of Jordan, Queen Rania Street, Amman 11942, Jordan
eCurrent address Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road,
London SW3 6JB.
e Current address Faculty of Science, Engineering and Computing Kingston University, Penrhyn
Road, Kingston KT1 2EE, UK. E-mail e.sim@kingston.ac.uk
*Corresponding authors: Elizabeth Fullam: email e.fullam@warwick.ac.uk Tel +44 (0)24 765
74251 and Edith Sim: email e.sim@kingston.ac.uk Tel +44 (0)20 8417 2492
†Electronic Supplementary information (ESI) available: Experimental data for compounds 1 to 32
and materials and methods for in vitro assays
1

The synthesis and inhibitory potencies of a novel series of 3,5-diaryl-1H-pyrazoles as specific
inhibitors of prokaryotic arylamine N-acetyltransferase enzymes is described. The series is based
on hit compound 1 3,5-diaryl-1H-pyrazole identified from a high-throughout screen that has been
carried out previously and found to inhibit the growth of Mycobacterium tuberculosis.
Graphical Abstract
FIGURE UPLOADED SEPARATELY
A series of novel 3,5-diaryl-1H-pyrazoles inhibitors were synthesized and evaluated as inhibitors
of prokaryotic arylamine N-acetyltransferase enzymes.
2

Tuberculosis (TB) remains the single most important bacterial cause of morbidity and mortality
in the world. Mycobacterium tuberculosis is the causative agent of TB and in 2010 TB infection
resulted
in
an
estimated
1.4
million
deaths
worldwide
(http://www.who.int/tb/publications/global_report/2011/en/). The TB bacillus currently infects
one-third of the world’s population and in 2010 alone 8.8 million new cases of TB were reported
1, 2. Treatment for TB is complicated and requires that at least three drugs be taken concomitantly
for six to nine months, often with unpleasant side effects. The long treatment time, coupled with
patient non-compliance has led to the emergence of multi-drug resistant (MDR) and extensively
drug-resistant (XDR-TB) strains of TB that further complicates treatment. Staggeringly, the
number of reported MDR cases has increased from 150,000 in 2008 to 650,000 in 2010. More
recently, totally drug-resistant (TDR-TB) strains of TB have emerged for which there are no
successful chemotherapy regimens. The M. tuberculosis cell envelope structure is rich in
polysaccharides and lipids and accounts for its unusual low permeability towards commonly used
antibiotics, contributing to its resistance 3-5. In combination with immuno-compromised
individuals with HIV, TB represents a serious health problem. Consequently there is a dire need
for new drugs to treat TB 6.
Biosynthetic pathways that are present in the pathogen M. tuberculosis and not present in the
7
8
human host represent an approach to identifying specific targets for TB therapy , . A unique
pathway, comprising a five gene operon (Rv3570c-Rv3566c), that is involved in complex cell
wall lipid biosynthesis and cholesterol catabolism in M. tuberculosis has been identified and
12, 13
characterized 9-11. These genes have been shown to be upregulated in microarray studies
.
Single gene, genetic knockouts of hsaD, hsaA and nat have shown that these enzymes are
essential for the intracellular survival of M. tuberculosis in macrophages 10, 11, 14-19. The nat gene
in M. tuberculosis is the last gene in this cluster and the N-acetyl transferase (NAT) enzyme can
3

catalyse the hydrolysis of both n-propionyl Coenzyme A, a product of cholesterol catabolism, as
well as the cofactor acetyl Coenzyme A 9, 16. Deletion of nat from Mycobacterium bovis BCG
(Δnat) affects the cell wall composition and the biosynthesis of mycolic acids 10. Therefore NAT
10
has been implicated as a potential anti-tubercular target for the development of inhibitors
.
Importantly, X-ray structures of the NAT enzyme are available to assist in the design of inhibitors
20. Although there is no reported NAT structure from M. tuberculosis (TBNAT), the NAT enzyme
from M. marinum (MMNAT) is 73% identical and 83% similar to the TBNAT at the amino acid
21, 22
level. The MMNAT structure is known with cofactor and substrate bound
and whilst not
identical to the TBNAT enzyme, has been shown to serve as a suitable model 16, 23
.
In previous work, we undertook a high-throughput screen of 5,000 drug-like compounds to find
inhibitors of NAT enzymes 24. From this screen, compounds of distinct structural classes were
characterised that were specific inhibitors of either prokaryotic or eukaryotic NAT enzymes 25-27
.
In this study we focus on the hit compound 4-methoxy-2-(5-(4-methoxyphenyl)-1H-pyrazol-3-
yl)phenol 1 identified from the screen (Fig 1), a specific inhibitor of prokaryotic NAT enzymes.
Here we report on the synthesis of a focused sub-library of 3,5-diaryl-1H-pyrazoles that have
been designed and synthesised based on predicted interactions of pyrazole 1 with MMNAT. We
have evaluated the activities of the sub-library for inhibition of MMNAT enzymic activity. Our
data reveal insights into inhibitor-MMNAT interactions and have allowed us to identify key
structural motifs within the 3,5-diaryl-1H-pyrazole scaffold that play an essential role in
inhibition of NAT activity.
The 3,5-diaryl-1H-pyrazole moiety identified represents a novel scaffold that had not been
identified previously as an inhibitor of NAT enzymic activity. Pyrazole 1, Figure 1, was deemed
to be a promising starting point for lead discovery in terms of predicted physicochemical
28
properties, drug-likeness
and chemical tractability: retrosynthetic analysis of the target
4

compound identified a synthetic route with the potential to modify the scaffold and provide
diversification on both aryl-rings around the heterocyclic core depending on the starting material
selected. Furthermore, importantly, hit compound 1 was found to have good anti-mycobacterial
activity and inhibited the growth of both M. tuberculosis and M. bovis BCG on solid agar with an
MIC <10μg/mL (34μM) 24.
Given the importance of verifying the chemical identity of lead compounds emerging from high-
throughput screening 29, pyrazole 1 was resynthesised prior to embarking on the synthesis of a
focused sub-library of pyrazole compounds. Pyrazole 1 was resynthesised from commercially
available 2’-hydroxy-5’-methoxyacetophenone via a crossed-Claisen-type reaction with
30, 31
subsequent condensation with hydrazine to form the required pyrazole moiety, Scheme 1
.
The intermediate 1,3-diketone was formed as a mixture with its corresponding enol tautomers.
This mixture was not purified and was reacted directly with hydrazine hydrate. Characterisation
1
13
of the resynthesised pyrazole by H and C NMR, along with mass spectrometry and elemental
analysis confirmed that pyrazole 1 was identical to the hit compound from the screening library
32. Furthermore resynthesised pyrazole 1 was tested against the NAT enzyme from P. aeruginosa
(PANAT) used in the initial screen and found to have an IC50 of 9.7 0.3μM as an inhibitor of the
PANAT enzyme. Hit compound 1 was thus verified as a starting point for further investigation.
The NAT enzyme from M. marinum is more similar in sequence to the TBNAT enzyme than the
NAT enzymes used in the initial high-throughput screen 22, 23. Therefore, pyrazole 1 was tested
for inhibition against MMNAT and found to be a competitive inhibitor with high affinity for
MMNAT with a Ki of 23μM (SI, Figure 1).
To guide the synthesis of a focused sub-library of 3,5-diaryl-1H-pyrazole compounds, docking
simulations were performed to obtain structural insights into the binding mode of 1 with
5

MMNAT, using GOLD 33. The modelled results suggest that pyrazole 1 binds in the active-site
pocket of MMNAT, within 4Å of the γS of the active-site Cys70, part of the Cys-His-Asp active-
site catalytic triad of NAT enzymes (Fig 2A). The predicted binding mode and location of 1 has a
high degree of similarity with solved NAT structures with substrate bound: MMNAT with CoA
22
21
(pdb 2vfc)
(Fig 2B), MMNAT with hydralazine (pdb 3ltw)
(Fig 2C) and MSNAT with
34
isoniazid (pdb 1wf6) (Fig 2D). An overlay of the modelled results of 1 with the solved HDZ-
MMNAT, INH-MSNAT and CoA-MMNAT structures indicates that the aryl substituent at C(3)
of pyrazole 1 is directly positioned in the same location as the arylhydrazine substrates: the
benzene
ring
of
HDZ
and
the
pyridine
of
isoniazid.
The
5’-methoxy group of the aryl substituent at C(3) of pyrazole 1 group points towards the γS of the
active-site Cys70 in a similar manner to the terminal nitrogen of the hydrazyl group of isoniazid
and hydralazine, which is acetylated by Cys70 in the acetyltransferase reaction. The aryl
substituent at C(5) of pyrazole 1 is predicted to extend by a distance of some 8.5Å beyond the
arylamine substrate binding site pocket towards the P-loop (residues Gly129, Phe130, Gly131,
Gly132) into the cofactor CoA binding site in MMNAT. Consequently, it is envisaged from the
modelling studies that inhibition of the MMNAT enzyme is achieved by precluding direct access
of NAT substrates and the cofactor acetyl CoA to the active-site of MMNAT thereby preventing
the NAT reaction from occurring (Fig 3). These modeling studies are in agreement with the
kinetic data that indicate pyrazole 1 is a competitive inhibitor of MMNAT.
Detailed analysis of the molecular contacts predicted from the modelling studies between
pyrazole 1 with MMNAT reveal that the majority of interactions are hydrophobic (Fig2A). The 3-
aryl substituent within pyrazole 1 is predicted to bind in the hydrophobic pocket formed by
residues Phe38, Phe130 and Phe204 that form a lid over the active-site Cys70 with π-stacking
interactions between the 3-aryl substituent and Phe130. There is an additional hydrophobic
6

interaction between this 3-aryl substituent and Val95. The docking studies suggest that the 5’-
methoxy group of the 3-aryl substituent is in close proximity (3.7Å) to potentially form a
hydrogen bond with the backbone carbonyl of Thr109. Thr109 has been demonstrated previously
to be important in substrate binding and recognition of NAT substrates 21, 34. The backbone
carbonyl group of Thr109 forms a hydrogen-bond with the hydrazyl group of HDZ or INH in the
MMNAT-HDZ and MSNAT-INH structures respectively 34, 35. Interestingly, only one hydrogen-
bond is predicted between pyrazole 1 and MMNAT, between the 2’-hydroxy group of the 3-aryl
substituent and the backbone carbonyl group of P-loop residue Gly131. Although the MMNAT-
CoA structure (pdb 2vfc) shows that this P-loop residue is not directly involved in binding to
CoA (P-loop residues Phe130 and Gly132 are involved), binding of pyrazole 1 at this site would
block the binding of the cofactor acetyl CoA. The 5-aryl substituent extends beyond the P-loop
and binds in a second hydrophobic pocket formed by Trp97 and Met209 and Met222. The side-
chain of Trp97 is involved in the recognition of the bridging phosphate group of CoA and forms a
hydrogen-bond from the indole nitrogen to a phosphate moiety in CoA, therefore this interaction
potentially further interrupts binding of CoA to MMNAT. Therefore, the docking studies suggest
a number of binding interactions between pyrazole 1 and MMNAT that prevent NAT substrates
and the cofactor from binding.
Based on these docking results R1, R2, R3 and R4 (Table 1) were selected for rational systematic
modification to incorporate -H/-F/-OH/-OMe structural modifications into the design of the
inhibitor with the aim of investigating the predicted role of the electrostatics, polar moieties and
hydrogen bonding between pyrazole 1 and MMNAT. The focused array of pyrazoles was
synthesized following an optimization of the synthetic route to target compound 1: the mixed
Claisen-type condensation reactions of arylmethylketones were heated in order to drive formation
31,
of the diketone intermediate
36. Intermediate 1,3-diketones were synthesized from
commercially available arylmethylketones and arylcarbonyl chlorides and the reactions
7

proceeded in good yields. The attempted synthesis of pyrazoles 3 and 4 via treatment of the
corresponding dihydroxy-substituted ketones with hydrazine hydrate gave a complex mix of
products from which 3 and 4 could not be isolated. This was postulated to be due to deprotonation
of the acidic phenolic-OH groups under the reaction conditions. Therefore pyrazoles 3 and 4 were
synthesised by an alternative route involving protection of the phenol groups within the starting
arylmethylketones as the corresponding TBDMS ethers. The O-TBDMS protected
arylmethylketones were next treated with LiHMDS and the requisite acyl chloride, deprotected
via treatment with tetrabutylammonium fluoride and the crude reaction mixtures subsequently
treated with hydrazine hydrate (Supplementary information, Scheme 1). This resulted in the
parallel production of an array of fifteen pyrazoles 2-16 in good overall yields.
To determine SARs for the 3,5-diaryl pyrazole class of heterocycles, the fifteen compounds that
formed the sub-library were tested for their inhibitory properties against the MMNAT enzyme.
Initially, the compounds were tested at a final concentration of 30μM as inhibitors in a reaction
where MMNAT catalysed the acetylation of isoniazid as measured by the hydrolysis of 5,5'-
dithiobis-(2-nitrobenzoic acid) (DTNB), as described previously 37. The synthesized sub-library
showed a range of inhibitory activities against MMNAT, from 15% inhibition of MMNAT
enzymic activity up to 66% inhibition at 30μM, Table 1. Half maximal inhibitory concentrations
(IC50) were determined for the five most potent inhibitors. Testing the analogues of 1 for
inhibition of MMNAT has allowed for the derivation of key structural features and preliminary
SARs for the library of 3,5-diaryl-1H-pyrazole compounds.
Of the fifteen compounds tested, seven compounds were found to have reduced potency at 30μΜ
compared to the initial hit pyrazole 1 at the same concentration: 2 (29%), 3 (21%), 4 (15%), 6
(26%), 9 (26%), 15 (18%) and 16 (29%). Comparison between the initial hit compound 1 and its
8

unsubstituted analogue 6 shows a significant decrease in inhibition of MMNAT indicating that
the functional groups of hit compound 1 play a positive role in the binding of 3,5-diaryl-1H-
pyrazole compounds to MMNAT. Intriguingly, the docking studies predicted that the 2’-hydroxy
group of the aryl substituent at C(3) of pyrazole 1 forms a hydrogen bond to the backbone
carbonyl group of Gly131. Altering the 2’-hydroxy group to a 2’-methoxy group, whilst keeping
the remaining functionalities the same (pyrazole 2) results in a dramatic decrease in the inhibition
of MMNAT suggesting the importance of the –OH hydrogen bond donor properties of the 2’-
hydroxy group of 1 for MMNAT inhibition. In addition, the hydroxy group is required to be
positioned at the 2’-position of the 3-aryl substituent. When the hydroxyl group is orientated at
the 3’-position (pyrazole 4) a decrease in inhibition of MMNAT is also observed (Table 1). The
modelling studies predict a number of electrostatic interactions between the 3-aryl substituent
within 1 and a hydrophobic pocket formed by three phenylaniline residues: Phe38, Phe130 and
Phe204. When the 5’-methoxy group is replaced by a 5’-hydroxy group (pyrazole 3) the
inhibition of MMNAT is greatly reduced with an IC50 greater than 30μM. Furthermore, when R1
and R3 are substituted to F groups: pyrazole 15, the same reduction in IC50 value is observed. This
suggests that the pyrazole compounds do bind in a hydrophobic pocket in MMNAT as predicted
from the docking studies and that more hydrophobic groups on the 3-aryl substituent are preferred
when R4 remains a methoxy group. This is consistent with previous studies that have shown that
NAT enzymes prefer lipophilic substrates 37.
Of the fifteen compounds synthesised, it was very encouraging that five compounds inhibited the
activity of the MMNAT enzyme at 30μM by greater than 50%: compounds 7, 8, 10, 11, and 14,
Table 1, with IC50 values of 12.9μM, 21.3μM, 17.6μM, 11.7μM and 18.8μM respectively. A wide-
range of functionality at R4 can be well tolerated and cause good inhibition of MMNAT.
Interestingly, two of the most potent inhibitors of MMNAT enzymic activity: 7 and 11 (IC50 of
9

12.9μM and 11.7μM respectively) both have unfunctionalised 5-phenyl substituents, suggesting
that functionality on the 3-aryl substituent is more important than functionalisation of the 5-aryl
substituent for improving inhibition of MMNAT enzymic activity. It is possible that pyrazoles 7
and 11 have a different mode of binding to MMNAT as a result of the unfunctionalised 5-phenyl
substituent and docking studies suggest that pyrazoles 7 and 11 can bind in a ‘flipped’ orientation
to MMNAT (Fig 4) compared to the hit compound 1, in which the 5-aryl substituent is located in
the active-site pocket in close proximity to Cys70. As a result of the predicted “flipped”
orientation of 7 and 11 the pyrazole moiety is orientated to point towards the P-loop of MMNAT
with the potential to form an additional hydrogen-bond from N(2) of the heterocyclic pyrazole
core to the backbone carbonyl group of Gly131 (Fig 5) and may go some way to understanding
the improved inhibitory properties of these compounds in the first instance.
In conclusion we have proposed binding modes for a focused library of 3,5-diarylpyrazoles to the
mycobacterial NAT enzyme, MMNAT. In silico docking studies and analysis of the inhibition
data from the sub-library of 1 with MMNAT has allowed for the elucidation of key structural
features for the 3,5-diaryl pyrazoles, which are most important for inhibition of MMNAT
enzymic activity. Analysis of the binding mode of this library of compounds suggest that the 3,5-
diarylpyrazoles are able to bind in the active-site, hydrophobic pocket of MMNAT, stabilised by
a number of hydrophobic interactions in a manner that precludes substrate and cofactor from
binding. Key hydrogen bonding interactions are predicted to occur between the 3,5-
diarylpyrazole scaffold and Gly131 and Thr109, which is further supported by the kinetic data
and preliminary SAR analyses. We have identified that the hydrophobic groups on the 3-aryl
substituent improve potency and that the most potent inhibitors in the sub-library have
unfunctionalised 5-phenyl substituents. This study provides an important insight into the key
structural features of this class of compound that inhibits NAT activity that will contribute to
10

future improvement of potency that can be accomplished by structural modifications to optimize
these identified key interactions. A number of pyrazole compounds have been reported to have
38, 39
anti-tubercular activity
and here we report that hit compound 1 inhibits the growth of M.
tuberculosis and M. bovis BCG with an MIC of less than 10μg/mL and also inhibits NAT
enzymic activity. Anti-tubercular agents are urgently needed and the resulting low-molecular
weight, drug like pyrazole compounds presented here represent promising candidates to be
further optimized for TB therapy.
Supporting information
Experimental details can be found in the Supporting Information.
Acknowledgements
We would like to thank Hilary Long for excellent technical assistance. We are also grateful to the
University of Jordan for a studentship (A.A.).
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Spectroscopic data for 1: Chemical shifts (δ) are reported in parts per million (p.p.m.)
1
and are referenced to the residual solvent peak. Coupling constants (J) are measured in Hz. H
12

NMR (400 MHz, CDCl3), 3.84 (3H, s), 3.88 (3H, s), 6.80 (1H, s), 6.85 (1H, dd, J1=8.84, J2=3.03), 6.98
(1H, d, J=8.84), 7.01 (2H, d, J=8.59), 7.17 (1H, d, J=3.03), 7.55 (2H, d, J=8.59); 13C NMR (400 MHz,
CDCl3): δ=55.4, 56.0, 98.9, 111.5, 114.5, 114.7, 115.3, 117.6, 127.1, 152.6; mp: 146-150oC; IR vmax
(NaCl) 1508.4 cm-1; MS (ES+) m/z 297.05 (MH+, 100%); HRMS (ES+) C17H17N2O3 calcd: 297.1234,
obsd: 297.1233.
33.
Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D. Proteins 2003,
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13

Figure Legends
Fig. 1 Lead compound 4-methoxy-2-(5-(4-methoxyphenyl)-1H-pyrazol-3-yl)phenol 1
Fig. 2 Predicted interactions of 1 with the NAT enzyme from M. marinum (MMNAT) and
comparison of the binding site of 1 with the binding site of CoA, hydralazine and isoniazid in
MMNAT. The protein is depicted in cartoon format and MMNAT, the residues that are predicted
to interact with 1 are shown in stick format, the lead compound 1, substrate and cofactor are
shown in stick format. (a) Compound 1 was docked using GOLD and the residues that are
predicted to interact with MMNAT are shown. The carbon atom’s of 1 are depicted in magenta
(b) The docking result of 1 is overlaid on the CoA-MMNAT structure (pdb 2vfc). The carbon
atom’s of CoA are depicted in yellow (c) The docking result of 1 is overlaid on the HDZ-
MMNAT structure (pdb 3ltw). The carbon atom’s of HDZ are depicted in cyan (d) The docking
result of 1 is overlaid on the INH-MMNAT structure (pdb 1wf6). The carbon atom’s of INH are
depicted in grey.
Fig. 3 Surface representation of MMNAT docked with 1. The molecular surface of the MMNAT
protein is displayed and coloured to show hydrophobicity (red being most hydrophobic through
to white to blue being most hydrophilic). The molecular surface of 1 is shown and carbon atom
(green), oxygen (red), nitrogen (blue).
Fig. 4 Space filled representation of compounds 1, 7, 8, 10 and 11. The inhibitors are in the
predicted docking orientation found in the active site pocket of MMNAT. (Carbon atoms (grey),
oxygen atoms (red), nitrogen atoms (dark blue), fluorine atoms (light blue),
Fig. 5 Predicted binding interactions of compounds 7, 8, 10 and 11. Compounds 7 (A), 8 (B), 10
(C) and 11 (D) were docked into the MMNAT protein (pdb code 2vfb) using the GOLD docking
program. The compounds are shown in ball-and-stick format with the carbon atoms in magenta,
nitrogen atoms in dark blue, oxygen atoms in red and the residues of MMNAT that are predicted
to interact with the ligands are shown in stick format.
14

Scheme 1. Synthesis of 1. Reagents and conditions: a) (i-iv): (i) LiHMDS, THF, -78oC, (ii) -
10oC, (iii) 4-methoxybenzoychloride, RT, (iv) aq. work up, b) (v-vi): (v) NH2NH2.xH2O, RT, (vi)
H2O).
15

Figure 1

Figure 2
Phe38
Phe38
A
B
Phe204
Phe204
Met209
Gly131
Met209
Gly131
Cys70
Cys70
Met222
Met222
Thr109
Thr109
His110
His110
Phe130
Phe130
Val95
Val95
Trp97
Trp97
Phe38
Phe38
C
D
Phe204
Phe204
Met209
Gly131
Met209
Gly131
Cys70
Cys70
Met222
Met222
Thr109
Thr109
His110
His110
Phe130
Phe130
Val95
Val95
Trp97
Trp97
FIGURE 2

Figure 3
Substrate
binding pocket
P-loop and CoA
binding pocket
Figure 3

Figure 4
1
8
7
10
11
Figure 4

Figure 5
Cys70
Cys70
A
B
Thr109
His110
Thr109
Phe204
Gly131
Phe204
Gly131
His110
Phe38
Phe38
Phe130
Val95
Val95
Met209
Met209
Met222
Trp97
Trp97
Cys70
Cys70
C Thr109
D
Thr109
Phe204
Phe204
Gly131
His110
His110
Phe38
Gly131
Met209
Val95
Val95
Phe130
Met209
Met222
Met222
Trp97
Trp97
FIGURE 5

Scheme 1
OH
O
OH
O
OH
OH
O
O
OH
NH
N
a)
b)
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
1
Scheme 1: reagents and condiꢀons: i) LiHMDS, THF, -78oC, 1 hour, ii) -10oC, 2 hours, iii) 4-methoxybenzoylchloride, RT, 16
hours, iv) aq. work-up, v) NH2NH2.xH2O MeOH, RT, 15 minutes, vi) H2O

Table 1. Biological activities of 3,5-diaryl-1H-pyrazoles with MMNAT
R1
N
NH
R2
R4
R3
Compound R1
R2
R3
R4
Overall
clogPb
tPSAb
% Inhibition
IC50 (µM)d
Yield
(%)a
86
at 30µMc
1
2
3
-OH
-H
-H
-H
-OH
-H
-H
-H
-H
-OMe
-OMe
-H
-OMe
-OMe
-OMe
-OMe
-OMe
-H
2.47
2.79
2.27
1.84
4.43
4.46
3.6
3.84
2.13
2.34
2.75
3.94
4.16
3.64
4.79
4.93
63.1
52.1
74.1
73.8
24.4
33.6
63.1
53.9
42.9
33.6
42.9
53.9
44.6
24.4
33.6
24.4
65.8±10.4
29.2±6.1
21.5±4.3
15.6±4.8
45.2±2.6
26.5±5.9
53.9±5.5
59.2±1.3
26.5±6.4
61.2±4.3
50.7±3.0
46.5±4.6
32.7±2.4
53.5±4.6
18.9±5.1
29.7±4.8
18.5 ± 1.0
>30
-OMe
-OH
-H
-OMe
-OH
-OH
-H
85
31
24
87
91
58
45
98
40
89
63
21
10
89
81
>30
>30
>30
>30
4
5
-H
6
-H
-H
-OMe
-H
7
8
9
-OH
-OH
-H
-OMe
-OMe
-H
-H
-OMe
-F
-F
-F
-F
-F
12.9±1.0
21.3±1.0
>30
17.6±1.0
11.7±1.0
>30
>30
18.8±.9
>30
-F
-OMe
-F
10
11
12
13
14
15
16
-H
-OMe
-OH
-OH
-F
-F
-F
-H
-H
-H
-H
-H
-OMe
-F
-H
-OMe
-F
-H
>30
aOverall yield calculated over total synthetic steps starting from arylmethylketones. All compounds are
>95% pure as determined by RP-HPLC.
bThe calculated octanol/water partition coefficient: clogP and topological polar surface area: tPSA
parameters were calculated in CS Chemdraw Ultra v12.0.2.
cMean % inhibition ± standard deviation (n = 3), relative to a control as described in Materials and
Methods.
d IC50: Half-maximal inhibitory concentration against MMNAT

Phe38
Phe204
Met209
Gly131
Cys70
Met222
Thr109
His110
Phe130
Val95
Trp97