Novel Amino-pyrazole Ureas with Potent in Vitro and in Vivo Antileishmanial Activity

Article abstract of DOI:10.1021/acs.jmedchem.5b01456

Visceral leishmaniasis is a severe parasitic disease that is one of the most neglected tropical diseases. Treatment options are limited, and there is an urgent need for new therapeutic agents. Following an HTS campaign and hit optimization, a novel series of amino-pyrazole ureas has been identified with potent in vitro antileishmanial activity. Furthermore, compound 26 shows high levels of in vivo efficacy (>90%) against Leishmania infantum, thus demonstrating proof of concept for this series.

Full text of DOI:10.1021/acs.jmedchem.5b01456

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Article
Novel Amino-pyrazole Ureas with Potent In
Vitro and In Vivo Antileishmanial Activity
Charles E. Mowbray, Stéphanie Braillard, William Speed, Paul Alan Glossop, Gavin A.
Whitlock, Karl R Gibson, James EJ Mills, Alan Daniel Brown, J. Mark F. Gardner, Yafeng
Cao, Wen Hua, Garreth L Morgans, Pim-Bart Feijens, An Matheeussen, and Louis J Maes
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01456 • Publication Date (Web): 16 Nov 2015
Downloaded from http://pubs.acs.org on November 18, 2015
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Novel Aminoꢀpyrazole Ureas with Potent In Vitro
and In Vivo Antileishmanial Activity.
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Charles E. Mowbray,∗,† Stéphanie Braillard,† William Speed,† Paul A. Glossop,▲,‡ Gavin A.
Whitlock,‡ Karl R. Gibson,‡ James E. J. Mills,‡ Alan D. Brown,§ J. Mark F. Gardner,║ Yafeng
Cao,╩ Wen Hua,╩ Garreth L. Morgans,╦,ꢀ PimꢁBart Feijens,# An Matheeussen,# and Louis J.
Maes#.
†Drugs for Neglected Diseases initiative (DNDi), 15 Chemin LouisꢀDunant, 1202 Geneva,
Switzerland.
‡Sandexis Medicinal Chemistry Ltd, Innovation House, Discovery Park, Ramsgate Road,
Sandwich, Kent, CT13 9ND, UK.
§Pfizer Worldwide Medicinal Chemistry, The Portway Building, Granta Park, Great Abington,
Cambridge, CB21 6GS, UK.
║AMG Consultants Ltd, Discovery Park House, Discovery Park, Ramsgate Road, Sandwich,
Kent, CT13 9ND, UK.
╩WuXi AppTec (Wuhan) Co. Ltd., 666 Gaoxin Road, East Lake HighꢀTech Development Zone,
Wuhan 430075, China P. R.
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╦iThemba Pharmaceuticals, Building T5, Pinelands Site, High Street, Modderfontein 1609,
Gauteng, SA.
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#Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, S7,
Universiteitsplein 1, 2610 Wilrijk, Antwerp, Belgium.
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KEYWORDS Visceral leishmaniasis, L. infantum, L. donovani, antileishmanial, antiparasitic,
aminoꢀpyrazole, pyrazole, urea.
ABSTRACT Visceral leishmaniasis is a severe parasitic disease that is one of the most neglected
tropical diseases. Treatment options are limited and there is an urgent need for new therapeutic
agents. Following an HTS campaign and hit optimization, a novel series of aminoꢀpyrazole ureas
has been identified with potent in vitro antileishmanial activity. Furthermore, compound 26
shows high levels of in vivo efficacy (>90%) against Leishmania infantum, thus demonstrating
proof of concept for this series.
INTRODUCTION
Visceral leishmaniasis (VL) is a systemic disease caused by the protozoan parasites Leishmania
donovani and L. infantum.1,2 The parasites (promastigotes) are transmitted through the bite of
female phlebotomine sand flies; in the human host, intracellular parasites (amastigotes) target the
reticuloendothelial system, surviving and multiplying in different macrophage populations.1–4
VL occurs in Asia, East Africa, Latin America, the Mediterranean Basin and the Middle East;1
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90% of global cases occur in just six countries: Bangladesh, Brazil, Ethiopia, India, South Sudan
and Sudan.5 It is estimated that there are 0.2–0.4 million new cases of VL per year and a case
fatality of >20,000 per year.5 Most infections are asymptomatic but in patients who develop
symptoms, initial presentation is insidious, with development of splenomegaly, hepatomegaly,
fever, anaemia, pancytopaenia, weight loss and weakness occurring progressively over a period
of weeks or even months; VL can be fatal if left untreated.1,4,5 Despite this, VL is one of the most
neglected tropical diseases (NTD).1–3
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Current treatment options for patients with VL include sodium stibogluconate (SSG; antimonial
therapy), liposomal amphotericin B (LAB; polyene antibiotic), paromomycin (PM;
aminoglycoside antibiotic) and miltefosine (phospholipid).1–4 All these drugs have deficiencies
in terms of safety, efficacy, easeꢀofꢀuse, cost or drug resistance.1–4 For example, SSG, LAB and
PM are all delivered by longꢀduration, intravenous infusions or intramuscular injections; only
miltefosine is an oral drug, and all are associated with adverse events or toxicity.1,3,4 Research
and development for the treatment of VL has evolved in recent years, including the discovery of
two nitroimidazole drugs, fexinidazole (Phase II) and (2R)ꢀ2ꢀmethylꢀ6ꢀnitroꢀ2ꢀ{[4ꢀ
(trifluoromethoxy)phenoxy]methyl}ꢀ2H,3Hꢀimidazo[2,1ꢀb][1,3]oxazole (VLꢀ2098, preclinical),
from the Drugs for Neglected Diseases initiative (DNDi).6–11 The DNDi has also recently
published the discovery of two new series of antileishmanial compounds: 2ꢀsubstituted
quinolines with potent in vitro activity and promising in vivo efficacy against VL,12,13 and
aminothiazoles with excellent in vitro potency and good physicochemical properties.14 Current
research towards VL is intensive, as judged by the volume and frequency of medicinal chemistry
publications and the extent of a recent inꢀdepth review.2 Many research groups have reported
new chemical scaffolds with promising activity against VL and potential for the future
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development of antileishmanial agents.15–21 Furthermore, a significant number of recent
publications have emerged from research groups in India and Brazil, highlighting the urgency
with which new medicines are being sought in countries where VL is most prevalent.1,12–14,22–28
Overall, however, many of the newlyꢀpublished scaffolds contain undesirable structural alerts
associated with adverse toxicity findings.29 Of note, several pyrazoleꢀbased series of
antileishmanial compounds have been reported by research groups in Brazil, but these generally
have modest–weak activity against Leishmania species (e.g. L. amazonensis) that cause
cutaneous leishmaniasis (CL), not VL.1,28,30–35 Consequently, there is an urgent need for
additional research into more effective therapeutic options for the treatment of VL.1–4
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The overall goal of this project is to deliver a shortꢀcourse oral treatment for VL that meets the
published target product profile (TPP) to address the significant deficiencies of current standardꢀ
ofꢀcare medications.3,36 In this preliminary disclosure, we report the discovery of a novel series
of pyrazoleꢀbased antileishmanial compounds with excellent in vitro potency against L.
infantum. In addition, examples from this series show equivalent in vitro potency against L.
donovani and high levels of in vivo efficacy (>90%) against L. infantum in a hamster model of
VL. Compounds from the series also demonstrate promising in vivo pharmacokinetics and
metabolic stability in human microsomes. Consequently, this series has the potential to deliver a
breakthrough in the search for safe and effective oral agents for the treatment of VL.
RESULTS AND DISCUSSION
Chemistry. The synthesis of aminoꢀpyrazole intermediates was straightforward (Scheme 1).
Commercially available 3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀamine was chlorinated with NCS to
provide chloroꢀsubstituted amino pyrazole 1. Methyl picolinate was treated with lithiated
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cyclopropylacetonitrile to give cyano ketone 2, which was cyclized with hydrazine to provide
cyclopropylꢀsubstituted amino pyrazole 3. Final compounds were synthesized from 3ꢀ(pyridinꢀ2ꢀ
yl)ꢀ1Hꢀpyrazolꢀ5ꢀamine, chloroꢀderivative 1 or cyclopropylꢀderivative 3, according to one of two
general methods. Amides 4–10 were synthesized using the required aminoꢀpyrazole intermediate
and corresponding acid chloride, according to Method A (Scheme 2). Ureas 14–26 were
synthesized from the required aminoꢀpyrazole intermediates, via their phenyl carbamates 11–13
(synthesized using Method B) and corresponding amine, according to Method C (Scheme 3).
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Scheme 1. Synthesis of aminopyrazole intermediates.a
H
N
H
N
N
i
N
NH2
NH2
N
N
N
Cl
1
H
N
O
O
N
ii
iii
NH2
N
CN
N
OMe
2
3
aReagents and conditions: (i) NCS, DMF, 0 °C, 0.5 h; (ii) 2ꢀcyclopropylacetonitrile, LDA, dry
THF, ꢀ78 °C to rt, 18 h; (iii) hydrazine hydrate, EtOH, reflux, 16 h.
Scheme 2. General synthesis of amides 4–10.a
R2
H
N
H
N
O
N
N
i
NH2
NH
N
N
R1
R1
R1 = H
R1 = cPr; 3
R1 = H; 4–6
R1 = cPr; 7–10
aReagents and conditions: (i) Method A: corresponding acyl chloride, MeCN, rt to reflux, 16 h.
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Scheme 3. General synthesis of ureas 14–26.a
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H
N
O
10
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R2
N
NH
NH
N
R1
R1 = H; 14
R1 = Cl; 15
R1 = cPr; 16
H
N
H
N
O
N
N
O
i
ii
NH2
NH
N
N
R1
R1
H
N
O
R1 = H
R1 = H; 11
R1 = Cl; 12
R1 = cPr; 13
N
N
X
Y
NH
R1 = Cl; 1
N
Z
R1 = cPr; 3
R3
17–26
aReagents and conditions: (i) Method B: phenyl chloroformate, pyridine, 0 °C to rt, 12.5 h; (ii)
Method C: corresponding amine, triethylamine, chloroform, reflux, 16 h.
This pyrazole series was initially identified from an HTS campaign with approximately 95,000
compounds from a diverse subset, selected to represent the broad chemical space of Pfizer’s
smallꢀmolecule compound collection.37 Compound 4 was a moderately potent hit (IC50 = 10 ꢀM)
with in vitro activity against L. donovani amastigotes in THPꢀ1 cells (human monocytic
leukaemia cells). The primary HTS assay employed this intracellular assay, but some early
characterisation of the hit and initial analogues also showed activity (e.g. 4; IC50 ≥10.9 ꢀM)
against axenic L. donovani amastigotes (Table 1). Initial simplification of the structure led to
additional hit compounds 5 and 6 with improved potency (IC50 = 3.71 ꢀM and 0.738 ꢀM,
respectively) against axenic L. donovani amastigotes. However, 4–6 only showed weak activity
(IC50 >40 ꢀM) when tested in an in vitro macrophage assay using intracellular L. infantum
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amastigotes (Table 1); nor was there any evidence of cytotoxicity (CC50 >40 µM) towards the
host cell, primary mouse macrophages (PMM). In contrast, the clinical agent miltefosine, used as
a reference drug, demonstrated good activity (IC50 = 7.26 ꢀM) in this in vitro macrophage assay.
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Table 1. Structure and antileishmanial activity of hits 4–6.
O
H
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H
N
O
R2
N
N
NH
NH
N
N
N
O
4
5, 6
Compound R2
Axenic L.
donovani
amastigote
IC50 (ꢀM)a
Intramacrophage PMMb
L. infantum
amastigote
IC50 (ꢀM)b
CC50
(ꢀM)
ꢀ
>42.4
>64c
>40.3
>64c
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6
≥10.9
Cl
3.71
OMe 0.738
>40.3
>40.3
Miltefosine ꢀ
n.d.
7.26
33.1
aGeometricꢀmean value of at least three independent tests. Geometricꢀmean value of at least
three independent tests in primary mouse macrophages (PMM). Single test value (n = 1). n.d.,
not determined.
b
c
Although axenic amastigotes have been used for HTS (resulting in the identification of some
starting points), occasional poor translation of activity from axenic to intracellular amastigotes
has been reported.3 The axenic amastigote assay has advantages in terms of speed and cost, but
the resourceꢀintensive intracellular amastigote assay is thought to be the most relevant as the
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parasite survives and divides in macrophages to cause the actual disease.2 Consequently,
additional compounds were designed, synthesized and tested in the intracellular L. infantum
amastigote assay to establish whether this scaffold represented a genuinely potent series
(comparable to miltefosine) worthy of lead optimization. Initial exploration of the rightꢀhand
side of the molecule did not identify any alternative amide groups or substituents with the
desired potency against intracellular amastigotes.
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However, subsequent investigation of the pyrazole core did result in compounds with muchꢀ
improved potency. In particular, addition of a cyclopropyl group at the 4ꢀposition of the pyrazole
ring provided 7 (IC50 = 2.02 ꢀM) (Table 2), which was significantly more potent than 6, its
unsubstituted counterpart. Furthermore, 7 also demonstrated similar antileishmanial activity
against L. donovani (IC50 = 2.63 ꢀM). The encouraging potency of 7 prompted us to reꢀexamine
a variety of benzamide substituents with this new core, especially as we envisaged that the
cyclopropyl group would sterically hinder the amide moiety and increase the likelihood of
identifying metabolicallyꢀstable compounds. Excellent potency was observed with 2ꢀmethoxy
benzamide 8 (IC50 = 0.064 ꢀM). A variety of other benzamides showed similar levels of potency
to 7, for example the 2ꢀfluoro (9; IC50 = 0.965 ꢀM) and 2,6ꢀdichloro (10; IC50 = 2.11 ꢀM)
analogues (Table 2). Importantly, compounds 7–10 showed no evidence of cytotoxicity (CC50
>51 ꢀM) in the counterꢀscreen using the same PMM as those used in the intracellular amastigote
assay (Table 2). In addition, encouraging stability (e.g. 7; Clint = 12.75 ꢀL/min/mg) was observed
in human liver microsomes (HLM). Compounds 7–10 were significantly more potent than
miltefosine, representing a key breakthrough for the series that prompted further hit optimization.
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Table 2. Structure and antileishmanial activity of amides 7–10 and ureas 14–16.
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6
7
8
9
H
N
O
H
N
O
R2
R2
N
N
NH
NH
NH
N
N
R1
R1
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7–10
14–16
Compound R1 R2
Intramacrophage PMMa
L. infantum
amastigote
IC50 (ꢀM)a
CC50
(ꢀM)
cPr 4ꢀOMe
2.02
>64
>51
>64
>64
>64
>64
>45
33.1
7
cPr 2ꢀOMe
cPr 2ꢀF
0.064
0.965
8
9
cPr 2,6ꢀdiCl 2.11
10
14
15
16
H
3ꢀCl
H
0.450
0.066
0.130
7.26
Cl
cPr
H
Miltefosine ꢀ
ꢀ
aGeometricꢀmean value of at least two independent tests in PMM.
However, one issue for the series concerned poor metabolic stability in hamster that precluded us
from evaluating compounds in the in vivo hamster model of VL, recognised to be the most
relevant model of VL as it closely mimics the pathology of the disease in human.3,38 Poor amide
stability exclusive to hamster plasma (e.g. 7; 0.3% remaining after 6 h) could be overcome with
the stericallyꢀhindered 2,6ꢀdichloro analogue 10 (96.9% remaining after 6 h) but despite this, all
analogues had poor stability (Clint >100 ꢀL/min/mg) in hamster liver microsomes (HamLM). We
concluded that even when stericallyꢀhindered with the bulky cyclopropyl group, the amide
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moiety was likely to be responsible for the high rate of metabolism in hamster and so decided to
investigate ureas as a potential, more polar alternative. Initial benzyl urea analogues such as 14–
16 (Table 2) had excellent, submicromolar potency (e.g. 16; IC50 = 0.130 ꢀM, AlogP = 3.15)
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with no evidence of cytotoxicity (CC50 >45 ꢀM), although high levels of instability in HamLM
(Clint >400 ꢀL/min/mg) remained. Interestingly though, and in contrast to the amide subꢀseries,
14 did show that it was possible to obtain highlyꢀpotent (IC50 = 0.450 ꢀM) ureas without a
substituent on the pyrazole core. This provided an opportunity to synthesize additional urea
analogues with reduced lipophilicity and potential for greater metabolic stability in HamLM
(Clint <100 ꢀL/min/mg) that would enable in vivo efficacy studies.
Phenyl piperidine urea 17 was quickly identified as a lead compound with excellent in vitro
potency (IC50 = 0.296 ꢀM), good selectivity index (SI) over cytotoxicity (CC50 >64 ꢀM; SI
>242) and good stability in HLM (Table 3). Furthermore, 17 showed equivalent antileishmanial
activity against L. donovani (IC50 = 0.078 ꢀM). Compound 17 did not show any instability in
hamster plasma (100% remaining after 6 h) but instability in HamLM was still high (Clint = 216
ꢀL/min/mg), prompting us to undertake in vitro metabolite identification studies in HLM and
HamLM to understand this in more detail. Whilst there was only one metabolite observed in
HLM, seven metabolites were observed in HamLM. Overall, these data indicated that the
majority of oxidative metabolism in both HLM and HamLM occurred on the rightꢀhand side of
the molecule, i.e. the phenyl piperidine. This gave us the impetus to develop a medicinal
chemistry design strategy that focused on retention of the pyridyl leftꢀhand side and
unsubstituted pyrazole urea, combined with substituted aryl piperidines and piperazines to block
metabolism and/or lower lipophilicity.
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In general, analogues 18–26 (which resulted from our design strategy) possessed excellent levels
of antileishmanial activity and SI, combined with good metabolic stability in HLM and,
importantly, evidence of an improvement in HamLM stability. Substituted piperidines 18–22
possessed excellent–good potency (IC50 = 0.224–2.862 ꢀM) and good HLM stability (Clint =
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25.3–42.5 ꢀL/min/mg) across an AlogP range of 3–4. Improved stability in HamLM was
observed for chlorophenyl analogues 20 (Clint = 43.8 ꢀL/min/mg) and 21 (Clint = 63.5
ꢀL/min/mg), and cyanophenyl analogue 22 (Clint = 80.8 ꢀL/min/mg). As 20 and 21 had a higher
AlogP (3.82) than 17 (3.15), it appeared that the improvement in metabolic stability was due to
these substituents successfully blocking sites of metabolism. However, the choice of substituent
seemed to be important as the fluorophenyl analogues 18 and 19 had poor stability in HamLM
(Clint = 272 and 207 ꢀL/min/mg, respectively) that was similar to 17, despite having a lower
AlogP (3.36) than 20 and 21. Arylꢀoxy piperidines 23 and 24 retained excellent antileishmanial
activity (IC50 = 0.614 and 0.853 ꢀM, respectively). Both compounds are more polar than 17 and
had improved metabolic stability in HamLM, especially 24 (Clint = 30.8 ꢀL/min/mg), perhaps
driven by a combination of lower AlogP (2.43) and addition of the 4ꢀcyano substituent. The most
polar compound (25; AlogP = 1.94) had excellent stability in HLM and moderate stability in
HamLM (Clint = <11.9 and 67.3 ꢀL/min/mg, respectively), although potency was approximately
5ꢀfold weaker than 23. It is interesting that although 25 was the most stable compound in HLM,
it was only the fifth most stable in HamLM, suggesting that different SAR exists for metabolic
stability in these two species. Aryl piperazine 26 demonstrated good potency against both L.
infantum and L. donovani (IC50 = 2.37 and 1.31 ꢀM, respectively), combined with good stability
in HLM and HamLM (Clint = 30.8 and 46.5 ꢀL/min/mg, respectively). The pairwise comparison
of 18 (AlogP = 3.36) and 26 (AlogP = 2.78) shows that piperazine is a suitable, more polar
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alternative to piperidine that is able to significantly improve HamLM stability. This may be due
to the presence of the extra piperazine nitrogen atom, which could block a site of oxidative
metabolism that is specific to hamster (HamLM).
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Table 3. Structure, antileishmanial activity, cytotoxicity, SI, HLM/HamLM stability and AlogP
for ureas 17–26.
H
N
O
N
N
X
Y
NH
N
Z
R3
17–26
Compound
X
Y
Z
R3
Intramacrophage PMMa MRC5b
HLM HamLM AlogP39
L. infantum
amastigote
IC50 (ꢀM)a
CC50
(ꢀM)
CC50
(ꢀM)
SIc
Clint
(ꢀL/min/mg)
CH — CH H
CH — CH 4ꢀF
CH — CH 3ꢀF
CH — CH 4ꢀCl
CH — CH 3ꢀCl
0.296
0.530
0.224
1.105
0.396
>64
>64
>45
>55
>22
>64
>40
>64
>64
>57
>64
33.1
>64
>64
>64
>62
>64
>64
>64
>63
>63
>64
>64
24.7
>242 15.3
>120 42.5
>286 25.3
216
3.15
3.36
3.36
3.82
3.82
3.03
2.55
2.43
1.94
2.78
2.99
3.73
17
18
19
20
21
22
23
24
25
26
14
272
207
>74
>161 30.8
>22 25.3
>109 27.3
30.0
43.8
63.5
80.8
98.5
30.8
CH — CH 4ꢀCN 2.862
CH O CH H 0.614
CH O CH 4ꢀCN 0.853
>74
>21
>44
14.7
CH O
N
H
2.884
2.370
0.450
7.26
<11.9 67.3
N
ꢀ
— CH 4ꢀF
30.8
46.5
434
n.d.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
>142 184
3.26 n.d.
Miltefosine ꢀ
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aGeometricꢀmean value of at least two independent tests in PMM. bGeometricꢀmean value of at
least two independent tests in human fetal lung fibroblasts. Selectivity index representing the
ratio of CC50/IC50; data are presented as the geometricꢀmean value of ratios calculated using
separate data from at least two independent tests. n.d., not determined.
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c
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Examples 20–26 combined good antileishmanial activity (and SI) with the desired improvement
in HamLM stability and provided a number of options to evaluate this series in a hamster in vivo
model of VL. Consequently, hamsters infected with L. infantum were treated with oral doses of a
representative example (26) at 50 mg/kg b.i.d. for 5 days. Treatment with 26 resulted in 92.7%
and 95% reduction in parasite burden in liver and spleen, respectively, without any obvious signs
of toxicity. For comparison, when dosed orally at 40 mg/kg q.d. for 5 days, miltefosine showed
97.8% (liver) and 99.6% (spleen) reduction in parasite burden. Pharmacokinetic studies in
hamsters with a single oral dose of 50 mg/kg demonstrated that 26 rapidly achieved good levels
of exposure (Tmax = 1.33 h, Cmax = 5,680 ng/mL, AUC0–24 h = 25,376 h*ng/mL, t = 4.2 h).
½
Consequently, these data illustrate that compounds (e.g. 26) from this novel series are able to
successfully treat VL infection in an in vivo setting.
CONCLUSION
We have identified two subsets of active compounds developed from a single HTS hit (4). Early
hitꢀexpansion activities identified potent amides (7−10) that seem to require a 4ꢀsubstituent on
the pyrazole ring for good activity against intracellular L. infantum amastigotes. Unfortunately,
these compounds also have poor metabolic stability in hamster liver microsomes, which
precluded their evaluation in our hamster model of VL. In contrast, the subsequent design of urea
analogues (e.g. 14, 17−26) provided potent activity against intracellular L. infantum amastigotes
without the need for a pyrazoleꢀ4ꢀsubstituent, combined with evidence of muchꢀimproved
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metabolic stability in hamster liver microsomes. Furthermore, this novel series of pyrazole ureas
has potent antileishmanial activity against both L. infantum and L. donovani, the two species of
Leishmania parasites that cause VL in humans. Examples from the series (e.g. 26) demonstrate
high levels of in vivo efficacy in a hamster model of VL, representing a positive in vivo proof of
concept. Furthermore, compounds from the series already meet a number of the published
criteria for the selection of a clinical drug candidate for the oral treatment of VL.3,36 Additional
efforts are ongoing to optimize the series (e.g. physicochemical properties, pharmacokinetics and
solubility) and will be described in future publications.
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EXPERIMENTAL SECTION
Biology.
Compounds and reagents. For in vitro assays, compound stock solutions were prepared in
100% DMSO at 20 mM. Compounds were serially preꢀdiluted (2ꢀfold or 4ꢀfold) in DMSO
followed by a further (intermediate) dilution in demineralized water to assure a final inꢀtest
DMSO concentration of <1%. For in vivo efficacy studies, compound 26 was formulated in
4.5% (w/v) methylcellulose (viscosity = 15 cps) and 5% (v/v) Tweenꢀ80 in water at 25 mg/mL.
Miltefosine was formulated in water at 20 mg/mL.
Cell cultures. Primary peritoneal mouse macrophages (PMM) were collected two days after
peritoneal stimulation with a 2% potato starch suspension. MRC5SV2 cells (diploid human
embryonic lung fibroblasts) were cultured in minimal essential medium (MEM) containing
Earle’s salts, supplemented with Lꢀglutamine, NaHCO3 and 5% inactivated foetal calf serum. All
cultures and assays were conducted at 37 °C under an atmosphere of 5% CO2.
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Parasites. L. infantum (MHOM/MA/67/ITMAP263) and L. donovani (MHOM/ET/67/L82) were
maintained in the golden (Syrian) hamster (Mesocricetus auratus). Ex vivo amastigotes were
collected from the spleen of an infected donor hamster using two centrifugation purification
steps: 230g for 10 min, keeping the supernatant layer, and 4,100g for 30 min, keeping the pellet.
The spleen parasite burden was assessed using the Stauber technique.40 For the in vitro assays,
the inoculum was prepared in RPMIꢀ1640 medium, supplemented with 200 mM Lꢀglutamine,
16.5 mM NaHCO3 and 5% inactivated foetal calf serum. For the in vivo model, an infection
inoculum containing 2 × 107 amastigotes/100 ꢀL was prepared in phosphate buffered saline
(PBS).
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Animals. Female golden hamsters for the in vivo model of visceral leishmaniasis were
purchased from Janvier, France (body weight 80–100 g). This study using laboratory rodents was
carried out in strict accordance to all mandatory guidelines (EU directives, including the Revised
Directive 2010/63/EU on the protection of Animals used for Scientific Purposes that came into
force on 01/01/2013, and the declaration of Helsinki in its latest version) and was approved by
the ethical committee of the University of Antwerp, Belgium (UAꢀECD 2011ꢀ74). Female
golden hamsters for the pharmacokinetic study were purchased from Vital River, Beijing, China.
This study was conducted following institutional review and in accordance with institutional and
national guidelines at WuXi AppTec (the Institutional Animal Care and Use Committee
(IACUC)).
In vitro axenic L. donovani amastigote assay. The assay was performed as previously
reported.41
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In vitro intramacrophage L. infantum amastigote assay. The assay was performed in sterile
96ꢀwell microtiter plates, each well containing 10 ꢁL of the compound dilution and 190 ꢁL of
the PMM/amastigote inoculum (3 × 104 cells/4.5 × 105 parasites per well). Parasite
multiplication was compared to untreated infected controls (100% growth) and uninfected
controls (0% growth). After fiveꢀday incubation, total parasite burdens were microscopically
assessed after staining the cells with a 10% Giemsa solution. The results were expressed as
percentage reduction in parasite burden compared to untreated control wells. IC50 values were
determined using an extended dose range (2ꢀfold compound dilutions, 8ꢀpoint concentration
curve). Miltefosine was used as the reference drug.42 For selected compounds, this assay format
was also run using the L. donovani inoculum.
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In vitro MRC5 and PMM cytotoxicity assays. Assays were performed in sterile 96ꢀwell
microtiter plates, each well containing 10 ꢁL of the compound dilution and 190 ꢁL of MRC5SV2
or PMM inoculum (3 × 104 cells/mL). Cell growth was compared to untreated controls (100%
growth) and assayꢀmedia controls (0% growth). After threeꢀday incubation, cell viability was
assessed fluorometrically by adding resazurin (50 ꢁL/well of a stock solution in phosphate buffer
(50 ꢀg/mL)), incubating for four hours and measuring fluorescence (λex 550 nm, λem 590 nm).
The results were expressed as percentage reduction in cell growth compared to untreated control
wells. IC50 values were determined using an extended dose range (2ꢀfold compound dilutions, 8ꢀ
point concentration curve) to a highest concentration of 64 ꢁM. Tamoxifen was included as the
reference drug.
In vivo hamster model of visceral leishmaniasis. Female golden hamsters were randomly
allocated to experimental groups of six animals each, based on body weight. At the start of the
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experiment (Day 0), each animal was infected with L. infantum inoculum, delivered by
intracardial injection. Six hamsters were assigned to the vehicleꢀtreated, infected control group.
Six hamsters were assigned per group (1 group/compound) for the evaluation of compound 26
and miltefosine. At Day 21 postꢀinfection (21 dpi), all animals in each group were dosed orally
for five consecutive days (21–25 dpi): compound 26 was dosed at 50 mg/kg b.i.d.; miltefosine
was dosed at 40 mg/kg q.d. At Day 35 (ten days after the final oral dose), all animals were
euthanized and autopsies were conducted. The study evaluated the following parameters:
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1. Adverse effects: all animals were observed daily for the occurrence/presence of adverse
effects.
2. Body weight: all animals were weighed twice per week to monitor general health.
3. Parasite burden: amastigote burdens in each target organ (liver, spleen and bone marrow)
were determined at Day 35. The organs of individual animals were weighed (except bone
marrow). Impression smears were stained with Giemsa for microscopic examination of
the total amastigote burden, defined as the mean number of amastigotes per cell
multiplied by the number of cells counted (minimum 500 nuclei); results were expressed
as a percentage reduction in amastigote burden compared to vehicleꢀtreated, infected
control animals.
Chemistry. Unless otherwise indicated, all reactions were magnetically stirred under an inert
atmosphere. All reagents, including solvents, were used as received. Anhydrous solvents were
dried inꢀhouse by passing through activated alumina. Thinꢀlayer chromatography was performed
on glassꢀbacked precoated silica gel 60 plates, and compounds were visualized using UV light or
iodine. Silica gel column chromatography was performed using 200−300ꢀmesh silica gel.
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Preparative HPLC was performed using Gilsonꢀ281 liquid handlers equipped with one of four
columns, chosen from: 1) Phenomenex Synergi C18 150 × 30 mm, 4 µm, 2) YMCꢀpack ODSꢀ
AQ 150 × 30 mm, 5 µm, 3) Agella Venusil ASB C18 150 × 21.2 mm, 5 µm, or 4) Boston
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Symmetrix C18 ODSꢀR 150 × 30 mm, 5 µm; elution was performed with 0.225% (by volume) of
formic acid in water (solvent A), and acetonitrile (solvent B); fractions containing product were
lyophilized. NMR spectra were recorded on a Bruker AVANCE 400 MHz spectrometer in the
solvents specified. LCMS spectra were recorded on an Agilent 1200 or Shimadzu 2020
spectrometer equipped with electrospray ionization, quadruple MS detector and Chromolith
Flash RPꢀ18e 25 × 2.0 mm column, eluting with 0.0375% (by volume) of TFA in water (solvent
A), and 0.01875% (by volume) of TFA in acetonitrile (solvent B). Analytical HPLC were
performed using a Shimadzu LC20AB machine and one of two columns, chosen from: 1)
Xtimate C18 2.1 × 30 mm, 3 µm, or 2) CHROMꢀMATRIX Innovation C18 2.1 × 30 mm, 2.6
µm; elution was performed with 0.0375% (by volume) of TFA in water (solvent A), and
0.01875% (by volume) of TFA in acetonitrile (solvent B). Purity of final compounds was ≥96%,
as determined by HPLC. Unless otherwise stated, final compounds were isolated as amorphous
solids without collection of melting point data.
4-Chloro-3-(pyridin-2-yl)-1H-pyrazol-5-amine (1). To a solution of 3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀamine (1.6 g, 10.0 mmol) in DMF (16 mL) at 0 ºC was added NCS (1.47 g, 11.0
mmol) in portions. The mixture was then stirred at 0 ºC for 0.5 h. Saturated aqueous NH4Cl
solution (150 mL) was then added and the resulting precipitate was collected by filtration and
dried under vacuum to yield 1 as a grey solid (1.3 g, 67%). 1H NMR (400 MHz, DMSOꢀd6): δ
12.45 (m, 1H), 8.63 (d, J = 4.8 Hz, 1H), 7.90 (s, 2H), 7.36 (s, 1H), 4.94 (m, 2H).
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2-Cyclopropyl-3-oxo-3-(pyridin-2-yl)propanenitrile (2). To a solution of 2ꢀ
5
6
7
8
cyclopropylacetonitrile (5.6 g, 76.7 mmol) in dry THF (70 mL) at −78 ºC was added LDA (80.6
mmol, 40.3 mL) dropwise and the mixture stirred for 1 h. A solution of methyl picolinate (10 g,
73.0 mmol) in dry THF (30 mL) was then added dropwise, maintaining the temperature below
−60 ºC. After addition, the mixture was warmed slowly to room temperature and stirred for 18 h.
The mixture was quenched with 10% citric acid (150 mL), extracted with ethyl acetate (100 mL
× 2), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give 2 as a
yellow oil (14.8 g, crude). 1H NMR (400 MHz, CDCl3): δ 8.73 (m, 1H), 8.17 (td, J = 7.9, 1.0 Hz,
1H), 7.93 (dt, J = 7.7, 1.8 Hz, 1H), 7.58 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H), 4.92 (d, J = 7.9 Hz, 1H),
1.48 (m, 1H), 0.69 (m, 4H).
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4-Cyclopropyl-3-(pyridin-2-yl)-1H-pyrazol-5-amine (3). To a solution of 2ꢀcyclopropylꢀ3ꢀ
oxoꢀ3ꢀ(pyridinꢀ2ꢀyl)propanenitrile (2) (14.8 g, 73.0 mmol) in ethanol (200 mL) was added
hydrazine hydrate (4.3 mL, 79.6 mmol) and the mixture stirred at reflux for 16 h. The mixture
was cooled to room temperature and concentrated under reduced pressure. The residue was
washed with petroleum ether/ethyl acetate (10/1, 50 mL) to give 3 as a yellow solid (11.5 g,
crude). 1H NMR (400 MHz, DMSOꢀd6): δ 11.86 (s, 1H), 8.59 (td, J = 4.6, 1.3 Hz, 1H), 7.88 (m,
2H), 7.30 (m, 1H), 4.38 (s, 2H), 1.69 (m, 1H), 0.86 (m, 2H), 0.36 (m, 2H).
Method A: Amide formation (4–10). To a solution of the corresponding amine (1.0 equiv) in
acetonitrile at room temperature was added dropwise a solution of the corresponding acyl
chloride (1.0 equiv) in acetonitrile. The reaction mixture was then stirred at reflux for 16 h. After
cooling to room temperature, the mixture was concentrated under reduced pressure to give the
crude product, which was purified by HPLC.
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3-Isopropoxy-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)-5-(pyrrolidine-1-carbonyl) benzamide
(4). To a suspension of dimethyl 5ꢀhydroxyisophthalate (21.0 g, 0.1 mol) and anhydrous
potassium carbonate solution (27.6 g, 0.2 mol) in DMF (100 mL) at room temperature was added
2ꢀbromopropane (25.5 g, 0.15 mol) and the mixture stirred at 80 ºC for 16 h. After cooling to
room temperature, the mixture was diluted with water (250 mL) and extracted with ethyl acetate
(150 mL × 2). The combined organic extracts were washed with brine (100 mL × 3), dried over
anhydrous Na2SO4 and concentrated under reduced pressure to give dimethyl 5ꢀ
isopropoxyisophthalate as a white solid (25.0 g, crude). LCꢀMS (ESI) m/z = 253.1 [M + H]+, t =
1.250 min.
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To a solution of dimethyl 5ꢀisopropoxyisophthalate (20.0 g, 79.4 mmol) in methanol (400 mL)
was added aqueous NaOH solution (71 mL, 1 M, 71 mmol) and the mixture stirred at 60 ºC for
16 h. The methanol was removed under reduced pressure and the residue diluted with water (150
mL). The aqueous phase was adjusted to pH 6 and extracted twice with ethyl acetate (100 mL).
The combined organic extracts were washed with brine (100 mL), dried over anhydrous Na2SO4
and concentrated under reduced pressure to give 3ꢀisopropoxyꢀ5ꢀ(methoxycarbonyl)benzoic acid
as a white solid (14.0 g, 74%). LCꢀMS (ESI) m/z = 239.1 [M + H]+, t = 1.010 min. 1H NMR (400
MHz, DMSOꢀd6): δ 8.05 (t, J = 1.2 Hz, 1H), 7.63 (m, 2H), 4.74 (m, 1H), 3.87 (s, 1H), 1.29 (d, J
= 6.0 Hz, 6H).
To a solution of 3ꢀisopropoxyꢀ5ꢀ(methoxycarbonyl)benzoic acid (14.0 g, 58.8 mmol) in DMF
(140 mL) was added HATU (27.0 g, 71.0 mmol), triethylamine (16.4 mL, 117.6 mmol) and
pyrrolidine (5.0 g, 70 mmol). The mixture was stirred at room temperature for 16 h then
partitioned between ethyl acetate (200 mL) and water (200 mL). The aqueous phase was
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separated and extracted with further ethyl acetate (200 mL). The combined organic extracts were
washed with saturated aqueous NH4Cl solution (200 mL) and brine (200 mL), dried over
anhydrous Na2SO4 and concentrated under reduced pressure to give methyl 3ꢀisopropoxyꢀ5ꢀ
(pyrrolidineꢀ1ꢀcarbonyl)benzoate as a white solid (16.5 g, 96%). LCꢀMS (ESI) m/z = 292.2 [M +
H]+, t = 1.020 min.
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To a solution of methyl 3ꢀisopropoxyꢀ5ꢀ(pyrrolidineꢀ1ꢀcarbonyl)benzoate (16.5 g, 56.7 mmol) in
methanol (200 mL) was added aqueous NaOH solution (56.7 mL, 2 M, 113.4 mmol) and the
mixture stirred at room temperature for 16 h. The solvent was removed under reduced pressure
and the residue diluted with water (150 mL). The aqueous phase was adjusted to pH 5 with
aqueous 2 M HCl solution and extracted twice with ethyl acetate (100 mL). The combined
organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to
give 3ꢀisopropoxyꢀ5ꢀ(pyrrolidineꢀ1ꢀcarbonyl)benzoic acid as a white solid (13.5 g, 86%). LCꢀ
MS (ESI) m/z = 278.1 [M + H]+, t = 0.932 min. 1H NMR (400 MHz, DMSOꢀd6):
δ 7.55 (s, 1H),
7.44 (d, J = 0.8 Hz, 1H), 7.24 (s, 1H), 4.71 (m, 1H), 3.46 (t, J = 6.0 Hz, 4H), 3.87 (s, 1H), 1.84
(m, 4H), 1.28 (d, J = 6.0 Hz, 6H).
A solution of 3ꢀisopropoxyꢀ5ꢀ(pyrrolidineꢀ1ꢀcarbonyl)benzoic acid (800 mg, 2.9 mmol) in
thionyl chloride (10 mL) was stirred at reflux for 2 h. The mixture was cooled to room
temperature and concentrated under reduced pressure. The residue was diluted with toluene (10
mL) and reꢀconcentrated under reduced pressure to give 3ꢀisopropoxyꢀ5ꢀ(pyrrolidineꢀ1ꢀ
carbonyl)benzoyl chloride as a white solid (860 mg, crude).
The title compound was synthesized according to Method A using 3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ
5ꢀamine (462 mg, 2.9 mmol) and 3ꢀisopropoxyꢀ5ꢀ(pyrrolidineꢀ1ꢀcarbonyl)benzoyl chloride (860
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mg, 2.9 mmol) in acetonitrile (40 mL), to yield 4 as a white solid (705 mg, 47%). HPLC: 99%
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pure. LCꢀMS (ESI) m/z = 420.2 [M + H]+, t = 0.850 min. 1H NMR (400 MHz, DMSOꢀd6):
δ
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13.20 (m, 1H), 11.00 (m, 1H), 8.63 (d, J = 3.6 Hz, 1H), 7.88 (m, 2H), 7.66 (m, 2H), 7.36 (s, 1H),
7.24 (s, 1H), 7.17 (s, 1H), 4.78 (m, 1H), 3.48 (t, J = 6.4 Hz, 2H), 3.40 (t, J = 6.4 Hz, 2H), 1.86
(m, 4H), 1.29 (m, 6H).
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4-Chloro-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)benzamide (5). The title compound was
synthesized according to Method A using 3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀamine (100 mg, 0.63
mmol) and 4ꢀchlorobenzoyl chloride (109 mg, 0.63 mmol) in acetonitrile (10 mL), to yield 5 as a
white solid (76 mg, 41%). HPLC: 99% pure. MS (ESI) m/z = 299 [M + H]+. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.06 (s, 1H), 8.64 (d, J = 4.4 Hz，1H), 8.04 (d, J = 8.8 Hz, 2H), 7.94 (m, 2H),
7.59 (d, J = 8.4 Hz, 2H), 7.40 (m, 1H), 7.17 (s, 1H).
4-Methoxy-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)benzamide (6). The title compound was
synthesized according to Method A using 3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀamine (850 mg, 5.3
mmol) and 4ꢀmethoxybenzoyl chloride (895 mg, 5.3 mmol) in acetonitrile (100 mL), to yield 6
as a white solid (1.04 g, 67%). HPLC: 99% pure. MS (ESI) m/z = 295 [M + H]+. 1H NMR (400
MHz, DMSOꢀd6): δ 13.15 (s, 1H), 10.75 (s, 1H), 8.63 (d, J = 3.6 Hz, 1H), 8.04 (d, J = 8.4 Hz,
2H), 7.88 (m, 2H), 7.35 (s, 1H), 7.24 (s, 1H), 7.04 (d, J = 8.2 Hz, 2H), 3.84 (s, 3H).
N-(4-Cyclopropyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)-4-methoxybenzamide (7). The title
compound was synthesized according to Method A using 4ꢀcyclopropylꢀ3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀamine (3) (300 mg, 1.5 mmol) and 4ꢀmethoxybenzoyl chloride (255 mg, 1.5 mmol) in
acetonitrile (20 mL), to yield 7 as a white solid (80.7 mg, 16%). HPLC: 98% pure. MS (ESI) m/z
= 335 [M + H]+. 1H NMR (400 MHz, DMSOꢀd6): δ 13.07 (m, 1H), 9.92 (s, 1H), 8.67 (d, J = 4.8
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Hz, 1H), 8.00 (m, 4H), 7.39 (m, 1H), 7.07 (m, 2H), 3.84 (s, 3H), 1.82 (m, 1H), 0.75 (m, 2H),
0.40 (m, 2H).
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N-(4-Cyclopropyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)-2-methoxybenzamide (8). The title
compound was synthesized according to Method A using 4ꢀcyclopropylꢀ3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀamine (3) (200 mg, 1.0 mmol) and 2ꢀmethoxybenzoyl chloride (170 mg, 1.0 mmol) in
acetonitrile (15 mL), to yield 8 as a white solid (121.5 mg, 36%). HPLC: 99% pure. MS (ESI)
m/z = 335 [M + H]+. 1H NMR (400 MHz, DMSOꢀd6): δ 9.77 (s, 1H), 8.65 (d, J = 4.8 Hz, 1H),
7.95 (m, 2H), 7.83 (m, 1H), 7.54 (m, 1H), 7.37 (m, 1H), 7.22 (m, 1H), 7.09 (m, 1H), 3.96 (s,
3H), 1.88 (m, 1H), 0.82 (m, 2H), 0.46 (m, 2H).
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N-(4-Cyclopropyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)-2-fluorobenzamide (9). The title
compound was synthesized according to Method A using 4ꢀcyclopropylꢀ3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀamine (3) (100 mg, 0.5 mmol) and 2ꢀfluorobenzoyl chloride (79 mg, 0.5 mmol) in
acetonitrile (10 mL), to yield 9 as a white solid (40.4 mg, 25% ). HPLC: 96% pure. MS (ESI) m/z
= 323 [M + H]+. 1H NMR (400 MHz, DMSOꢀd6): δ 13.10 (s, 1H), 10.04 (s, 1H), 8.67 (d, J = 4.0
Hz, 1H), 7.97 (m, 2H), 7.71 (dd, J = 8.0, 8.0 Hz, 1H), 7.56 (m, 1H), 7.37(m, 3H), 1.85 (s, 1H),
0.82 (d, J = 7.2 Hz, 2H), 0.44 (d, J = 4.4 Hz, 2H).
2,6-Dichloro-N-(4-cyclopropyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)benzamide (10). The title
compound was synthesized according to Method A using 4ꢀcyclopropylꢀ3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀamine (3) (200 mg, 1.0 mmol) and 2,6ꢀdichlorobenzoyl chloride (273 mg, 1.3 mmol)
in acetonitrile (15 mL), to yield 10 as a white solid (23.1 mg, 6%). HPLC: 98% pure. MS (ESI)
m/z = 373 [M + H]+. 1H NMR (400 MHz, DMSOꢀd6): δ 13.08 (m, 1H), 10.33 (s, 1H), 8.67 (d, J =
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4.8 Hz, 1H), 7.96 (m, 2H), 7.57 (m, 2H), 7.50 (m, 1H), 7.48 (m, 1H), 1.86 (s, 1H), 0.86 (m, 2H),
0.44 (m, 2H).
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Method B: Phenyl carbamate formation (11–13). To a solution of the corresponding amine
(1.0 equiv) in pyridine at 0 °C was added phenyl chloroformate (1.0 equiv) dropwise over a
period of 0.5 h. The reaction mixture was then stirred at room temperature for 12 h. The mixture
was quenched with water, filtered and the resulting solid washed with water to give the product,
which was dried under vacuum.
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Phenyl (3-(pyridin-2-yl)-1H-pyrazol-5-yl)carbamate (11). The title compound was
synthesized according to Method B using 3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀamine (25.0 g, 0.156
mol) and phenyl chloroformate (24.4 g, 0.156 mol) in pyridine (250 mL), to yield 11 as a grey
solid (30 g, 69%). 1H NMR (400 MHz, DMSOꢀd6): δ 13.09 (br s, 1H), 10.61 (m, 1H), 8.61 (m,
1H), 7.86 (m, 2H), 7.44 (m, 2H), 7.35 (m, 1H), 7.27 (m, 1H), 7.22 (d, J = 7.8 Hz, 2H), 6.89 (m,
1H).
Phenyl (4-chloro-3-(pyridin-2-yl)-1H-pyrazol-5-yl)carbamate (12). The title compound was
synthesized according to Method B using 4ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀamine (1) (200
mg, 1.0 mmol) and phenyl chloroformate (160 mg, 1.0 mmol) in pyridine (5 mL), to yield 12 as
a brown oil (300 mg, crude) that was used directly in the next step (to make 15).
Phenyl (4-cyclopropyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)carbamate (13). The title compound
was synthesized according to Method B using 4ꢀcyclopropylꢀ3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀ
amine (3) (200 mg, 1.0 mmol) and phenyl chloroformate (160 mg, 1.0 mmol) in pyridine (5 mL),
to yield 13 as a brown oil (300 mg, crude) that was used directly in the next step (to make 14).
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Method C: Urea Formation (14–26). A mixture of the corresponding phenyl carbamate (1.0
equiv), the corresponding amine (1.0 equiv) and triethylamine (2.0 equiv) in chloroform was
stirred at reflux for 16 h. After cooling to room temperature, the mixture was concentrated under
reduced pressure to give the crude product, which was purified by HPLC.
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1-(3-Chlorophenyl)methyl-3-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)urea (14). The title compound
was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀ
yl)carbamate (11) and 3ꢀchlorobenzylamine, to yield 14 (4 mg). HPLC: 100% pure. MS (ESI)
m/z = 328 [M + H]+, 350 [M + Na]+. 1H NMR (400 MHz, DMSOꢀd6): δ 12.83 (br s, 1H), 8.97 (br
s, 1H), 8.59 (m, 1H), 7.82 (m, 2H), 7.32 (m, 6H), 6.71 (br s, 1H), 4.36 (d, 2H).
1-Benzyl-3-(4-chloro-3-(pyridin-2-yl)-1H-pyrazol-5-yl)urea (15). The title compound was
synthesized according to Method C using phenyl (4ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀ
yl)carbamate (12) (300 mg, 0.95 mmol) and benzylamine (153 mg, 1.43 mmol) in chloroform
(10 mL), to yield 15 as a white solid (129 mg, 41%). HPLC: 100% pure. MS (ESI) m/z = 328 [M
+ H]+. 1H NMR (400 MHz, DMSOꢀd6): δ 13.38 (m, 1H), 8.67 (m, 2H), 7.95 (s, 2H), 7.41 (m,
1H), 7.34 (m, 4H), 7.26 (m, 2H), 4.34 (d, J = 6.0 Hz, 2H).
1-Benzyl-3-(4-cyclopropyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)urea (16). The title compound
was synthesized according to Method C using phenyl (4ꢀcyclopropylꢀ3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)carbamate (13) (150 mg, 0.47 mmol) and benzylamine (76 mg, 0.70 mmol) in
chloroform (5 mL), to yield 16 as a light yellow solid (45.8 mg, 29%). HPLC: 97% pure. MS
(ESI) m/z = 334 [M + H]+. 1H NMR (400 MHz, DMSOꢀd6): δ 12.80 (m, 1H), 8.63 (d, J = 4.8 Hz,
1H), 8.01 (m, 3H), 7.35 (m, 7H), 4.35 (d, J = 5.6 Hz, 2H), 1.77 (m, 1H), 0.84 (m, 2H), 0.35 (m,
2H).
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4-Phenyl-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)piperidine-1-carboxamide (17). The title
compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀ
yl)carbamate (11) (3.0 g, 10.7 mmol), 4ꢀphenylpiperidine (1.9 g, 11.8 mmol) and triethylamine
(3.7 mL, 26.8 mmol) in chloroform (80 mL) to yield the crude product, which was precipitated
from ethyl acetate (100 mL), filtered and recrystallized from DMSO (20 mL) to yield 17 as a
white solid (1.09 g, 29%). Mp = 237 °C. HPLC: 99% pure. MS (ESI) m/z = 348 [M + H]+. 1H
NMR (400 MHz, CDCl3): δ 12.66 (m, 1 H), 8.61 (d, J = 4.8 Hz, 1H), 7.74 (m, 1H), 7.68 (m, 2H),
7.50 (s, 1H), 7.32 (m, 2H), 7.27 (m, 4H), 7.09 (m, 1H), 4.24 (d, J = 13.6 Hz, 2H), 3.00 (td, J =
12.8, 2.4 Hz, 2H), 2.72 (m, 1H), 1.91 (d, J = 11.2 Hz, 2H), 1.72 (m, 2H).
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4-(4-Fluorophenyl)-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)piperidine-1-carboxamide (18). The
title compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)carbamate (11) and 4ꢀ(4ꢀfluorophenyl)piperidine, to yield 18 (4 mg). HPLC: 100%
pure. MS (ESI) m/z = 366 [M + H]+, 388 [M + Na]+. 1H NMR (400 MHz, CDCl3): δ 10.77 (br s,
1 H), 8.60 (m, 1H), 7.77 (t, 1H), 7.67 (d, 1H), 7.26 (m, 4H), 7.10 (m, 3H), 4.25 (d, 2H), 3.03 (t,
2H), 2.75 (m, 1H), 1.93 (d, 2H), 1.71 (m, 2H).
4-(3-Fluorophenyl)-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)piperidine-1-carboxamide (19). The
title compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)carbamate (11) and 4ꢀ(3ꢀfluorophenyl)piperidine, to yield 19 (4.5 mg). HPLC:
100% pure. MS (ESI) m/z = 366 [M + H]+, 388 [M + Na]+. 1H NMR (400 MHz, CDCl3): δ 11.16
(br s, 1H), 8.62 (d, J = 4.4 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.45 (s,
1H), 7.26 (m, 2H), 7.09 (s, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.93 (m, 2H), 4.26 (d, J = 12.8 Hz,
2H), 3.02 (t, J = 12.4 Hz, 2H), 2.75 (m, 1H), 1.94 (d, J = 12.8 Hz, 2H), 1.70 (m, 2H).
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4-(4-Chlorophenyl)-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)piperidine-1-carboxamide (20).
The title compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)carbamate (11) (2.6 g, 9.28mmol), 4ꢀ(4ꢀchlorophenyl)piperidine (2.2 g, 9.52 mmol)
and triethylamine (3.2 mL, 23.2mmol) in chloroform (70 mL), to yield the crude product, which
was precipitated from ethyl acetate (100 mL), filtered and recrystallized from DMSO (25 mL) to
yield 20 as a white solid (0.998 g, 28%). Mp = 231 °C. HPLC: 98% pure. MS (ESI) m/z = 382
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[M + H]+. 1H NMR (400 MHz, DMSOꢀd6, t = 80 °C): δ 12.54 (m, 1H), 8.73 (m, 1H), 8.58 (d, J =
4.0 Hz, 1H), 7.82 (m, 2H), 7.30 (m, 5H), 6.87 (m, 1H), 4.27 (d, J = 13.2 Hz, 2H), 2.91 (t, J =
12.0 Hz, 2H), 2.81 (m, 1H), 1.82 (d, J = 12.0 Hz, 2H), 1.59 (m, 2H).
4-(3-Chlorophenyl)-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)piperidine-1-carboxamide (21).
The title compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)carbamate (11) and 4ꢀ(3ꢀchlorophenyl)piperidine, to yield 21 (3 mg). HPLC: 98.8%
pure. MS (ESI) m/z = 382 [M + H]+, 404 [M + Na]+. 1H NMR (400 MHz, CDCl3): δ 10.78 (br s,
1H), 8.60 (m, 1H), 7.77 (m, 1H), 7.67 (m, 1H), 7.24 (m, 5H), 7.10 (m, 2H), 4.27 (m, 2H), 3.03
(m, 2H), 2.75 (m, 1H), 1.95 (m, 2H), 1.72 (m, 2H).
4-(4-Cyanophenyl)-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)piperidine-1-carboxamide (22). The
title compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)carbamate (11) and 4ꢀ(4ꢀpiperidinyl)benzonitrile, to yield 22 (3.6 mg). HPLC:
98.7% pure. MS (ESI) m/z = 373 [M + H]+, 395 [M + Na]+. 1H NMR (400 MHz, CDCl3): δ
11.13 (br s, 1H), 8.60 (d, J = 4.0 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.61
(d, J = 8.0 Hz, 2H), 7.45 (s, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.25 (m, 1H), 7.08 (s, 1H), 4.26 (d, J =
13.6 Hz, 2H), 3.00 (t, J = 12.4 Hz, 2H), 2.79 (m, 1H), 1.91 (d, J = 12.4 Hz, 2H), 1.71 (m, 2H).
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4-Phenoxy-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)piperidine-1-carboxamide (23). The title
compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀ
yl)carbamate (11) and 4ꢀ(phenoxy)ꢀpiperidine, to yield 23 (393 mg). HPLC: 100% pure. MS
(ESI) m/z = 364 [M + H]+, 386 [M + Na]+. 1H NMR (400 MHz, CDCl3): δ 11.19 (br s, 1H), 8.60
(d, J = 4.4 Hz, 1H), 7.75 (m, 1H), 7.67 (m, 1H), 7.42 (s, 1H), 7.28 (m, 3H), 7.08 (br s, 1H), 6.97
(t, J = 7.2 Hz, 1H), 6.93 (d, J = 8.0 Hz, 2H), 4.56 (m, 1H), 3.73 (m, 2H), 3.52 (m, 2H), 1.99 (m,
2H), 1.89 (m, 2H).
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4-(4-Cyanophenoxy)-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)piperidine-1-carboxamide (24).
The title compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)carbamate (11) (3.2 g, 11.4 mmol), 4ꢀ(piperidinꢀ4ꢀyloxy)benzonitrile (2.6 g, 12.9
mmol) and triethylamine (3.96 mL, 28.6 mmol) in chloroform (80 mL), to yield the crude
product, which was purified by silica gel chromatography (dichloromethane/methanol, 50:1 to
10:1) and recrystallized from acetonitrile/methanol (4:1, 25 mL) to yield 24 as a white solid
(1.05 g, 24%). Mp = 223 °C. HPLC: 98% pure. MS (ESI) m/z = 389 [M + H]+. 1H NMR (400
MHz, CDCl3): δ 11.37 (m, 1H), 8.62 (d, J = 5.2 Hz, 1H), 7.76 (ddd, J = 8.0, 8.0, 1.6, 1H), 7.60
(m, 4H), 7.25 (d, J = 6.0 Hz, 1H), 7.08 (m, 1 H), 6.94 (d, J = 8.8 Hz, 2H), 4.61 (td, J = 6.40, 2.8
Hz, 1H), 3.70 (m, 2H), 3.51 (m, 2H), 2.01 (m, 2H), 1.85 (td, J = 6.4, 4.0 Hz, 2H).
N-(3-(Pyridin-2-yl)-1H-pyrazol-5-yl)-4-(pyridin-2-yloxy)piperidine-1-carboxamide (25).
The title compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)carbamate (11) (2.8 g, 10.0 mmol), 2ꢀ(piperidinꢀ4ꢀyloxy)pyridine hydrochloride
(2.5 g, 11.7 mmol) and triethylamine (3.47 mL, 25.0 mmol) in chloroform (80 mL) to yield the
crude product, which was purified by silica gel chromatography (dichloromethane/methanol,
50:1 to 10:1) and recrystallized from acetonitrile/methanol (4:1, 25 mL) to yield 25 as a white
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solid (1.46 g, 40%). Mp = 216 °C. HPLC: 98% pure. MS (ESI) m/z = 365 [M + H]+. 1H NMR
(400 MHz, CDCl3): δ 11.07 (m, 1H), 8.60 (d, J = 4.8 Hz, 1H), 8.14 (d, J = 4.8 Hz, 1H), 7.76 (m,
1H), 7.67 (m, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.37 (s, 1H), 7.21−7.26 (m, 1H), 7.09 (m, 1H), 6.87
(t, J = 6.0 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 5.31 (m, 1H), 3.79 (m, 2H), 3.47 (m, 2H), 2.08 (m,
2H), 1.86 (m, 2H).
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4-(4-Fluorophenyl)-N-(3-(pyridin-2-yl)-1H-pyrazol-5-yl)piperazine-1-carboxamide (26).
The title compound was synthesized according to Method C using phenyl (3ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)carbamate (11) and 1ꢀ(4ꢀfluorophenyl)ꢀpiperazine, to yield 26 (842 mg). HPLC:
100% pure. MS (ESI) m/z = 389 [M + Na]+. 1H NMR (400 MHz, DMSOꢀd6): δ 12.77 (br s, 1 H),
9.24 (br s, 1H), 8.60 (m, 1H), 7.78 (m, 2H), 7.30 (m, 1H), 7.05 (m, 4H), 6.82 (br s, 1H), 3.62 (m,
4H), 3.10 (m, 4H).
AUTHOR INFORMATION
Corresponding Author
*Charles E. Mowbray. Eꢀmail: cmowbray@dndi.org. Phone: + 41 (0)22 906 9220.
Present Addresses
ꢂMojozi Consulting (Pty) Ltd., 88 Tenth Ave, Sydenham, Johannesburg, 2092, Gauteng, SA.
Author Contributions
▲Paul A. Glossop prepared this manuscript on behalf of all the authors. All authors have given
approval to the final version of the manuscript.
Funding Sources
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