Identification of GSK3186899/DDD853651 as a Preclinical Development Candidate for the Treatment of Visceral Leishmaniasis

Article abstract of DOI:10.1021/acs.jmedchem.8b01218

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.

Full text of DOI:10.1021/acs.jmedchem.8b01218

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Identification of GSK3186899/DDD853651 as a Preclinical
Development Candidate for the Treatment of Visceral Leishmaniasis
‡
†
Michael G. Thomas,† Manu De Rycker,† Myr‡iam Ajakane, Sebastian Albrecht,
́
§
†
†
́
Ana Isabel Alvarez-Pedraglio, Markus Boesche, Stephen Brand, Lorna Campbell,
Juan Cantizani-Perez,§ Laura A.T. Cleghorn,† Royston C.B. Copley,∥ Sabrinia D. Crouch,§
Alain Daugan,⊥ Gerard Drewes,‡ Santiago Ferrer,§ Sonja Ghidelli-Disse,‡ Silvia Gonzalez,§
Stephanie L. Gresham,# Alan P. Hill,∥ Sean J. Hindley,∥ Rhiannon M. Lowe,# Claire J. MacKenzie,†
Lorna MacLean,† Sujatha Manthri,† Franck Martin,⊥ Juan Miguel-Siles,§ Van Loc Nguyen,⊥
Suzanne Norval,† Maria Osuna-Cabello,† Andrew Woodland,† Stephen Patterson,† Imanol Pena,§
Maria Teresa Quesada-Campos,§ Iain H. Reid,∥ Charlotte Revill,† Jennifer Riley,†
Jose Ramon Ruiz-Gomez,§ Yoko Shishikura,† Frederick R.C. Simeons,† Alasdair Smith,†
Victoria C. Smith,† Daniel Spinks,† Laste Stojanovski,† John Thomas,† Stephen Thompson,†
Tim Underwood,∥ David W. Gray,† Jose M. Fiandor,§ Ian H. Gilbert,† Paul G. Wyatt,*,†
Kevin D. Read,*,† and Timothy J. Miles*,§
†Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School
of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
‡Cellzome GmbH, A GlaxoSmithKline Company, Meyerhofstrasse 1, 69117 Heidelberg, Germany
§Global Health R&D, GlaxoSmithKline, Calle Severo Ochoa, 2, 28760 Tres Cantos, Madrid Spain
∥Platform Technology & Science, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1
2NY, U.K.
⊥
́
Centre de Recherche, GlaxoSmithKline, Les Ulis, 25,27 Avenue du Quebec, 91140 Villebon sur Yvette France
#Platform Technology & Science, GlaxoSmithKline, David Jack Centre for R&D, Park Road, Ware, Hertfordshire SG12 0DP, U.K.
S
* Supporting Information
ABSTRACT: The leishmaniases are diseases that affect
millions of people across the world, in particular visceral
leishmaniasis (VL) which is fatal unless treated. Current
standard of care for VL suffers from multiple issues and there
is a limited pipeline of new candidate drugs. As such, there is a
clear unmet medical need to identify new treatments. This
paper describes the optimization of a phenotypic hit against
Leishmania donovani, the major causative organism of VL. The
key challenges were to balance solubility and metabolic
stability while maintaining potency. Herein, strategies to
address these shortcomings and enhance efficacy are
discussed, culminating in the discovery of preclinical develop-
ment candidate GSK3186899/DDD853651 (1) for VL.
also the communities in which they live, contributing to a
perpetual cycle of poverty. The leishmaniases affect people
across Africa, the Middle East, South America, and Asia. There
are about 20 species or subspecies of Leishmania which cause
human disease. Visceral leishmaniasis (VL, also known as kala
azar), caused by Leishmania donovani and Leishmania infantum,
is typically fatal without treatment with 200000−400000 new
INTRODUCTION
■
Kinetoplastid diseases are part of the list of the neglected tropical
diseases (NTDs) as defined by the World Health Organisation
(WHO).1 These diseases are caused by a group of flagellated
protozoans that are characterized by the presence of a distinct
network of DNA (known as “kinetoplast”) in their single large
mitochondrion.2 The human diseases more commonly caused
by these parasites include, among others, various forms of
leishmaniasis (cutaneous, muco-cutaneous, and visceral). These
diseases affect millions of people, not only those infected, but
Received: August 2, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Reaching the biophase for visceral leishmania within macrophages. Reprinted (adapted) with permission from ref 47. Copyright 2002 Nature
Publishing Group.
cases each year and approximately 20000−30000 deaths.3
Current therapies are not fit for use in resource-poor settings due
to a combination of issues such as difficult administration, length
of treatment, side effects, teratogenicity, cost, resistance, and/or
clinical relapse.4−7 At the time of this work, the external
development pipeline for VL was sparse with only two additional
new chemical entities (NCEs) just entering the early preclinical
phase.8 Therefore, there was a clear unmet medical need for a
new therapeutic class.
The primary objective of a collaboration between the Drug
Discovery Unit, University of Dundee and GSK (funded by
Wellcome), has been to identify safe, effective, oral, and short-
course (ideally ≤10 days) drug candidates for VL, in line with
the DNDi (Drugs for Neglected Disease initiative) target
product profile.9 Hence, the focus was to identify orally available
compounds that demonstrate both in vitro and in vivo parasite
suppression comparable to miltefosine (the only available oral
therapy) in preclinical models, with an appropriate safety profile.
This strategy of focusing on parasite suppression has been used
successfully for discovery of most of the current protozoan
chemotherapies.10 Because there are very few fully validated
druggable targets in the kinetoplastids, most groups have
focused on phenotypic approaches.11,12
slowly in this assay, and second, compounds need to cross three
membranes and navigate two changes in pH to demonstrate in
vitro potency (Figure 1). However, the assay only identifies
cytocidal, rather than cytostatic compounds, which is an
important consideration when identifying new compound
series. Notably, all compounds which are either in the clinic,
or in clinical development, exhibit activity in this assay.14
This publication details the lead optimization of a N1-(1H-
pyrazolo[3,4-d]pyrimidin-6-yl)cyclohexyl-1,4-trans-diamine
series to find an appropriate balance between potency, relevant
measures of solubility, and/or metabolic stability. This effort led
to the in vivo profiling of a number of analogues, and finally the
identification of precandidate asset GSK3186899/DDD853651,
1(Figure 2).16,17 Subsequent target deconvolution identified
that the principal target was Cdc2-related kinase 12 (CRK12),17
although this was unknown during the lead optimization
program.
Leishmania parasites have a complex life cycle, involving a
vector (sandfly) stage and a host (human) stage.13 Given the
differences in biology between the vector and the host stage, it is
important that compounds are screened against the correct
physiological stage (amastigotes). In the human host, the
parasites are found within macrophages, more specifically within
parasitophorous vacuoles where the pH is ∼5.5. A high-content
screening method for this stage was recently developed, where
the parasites are cultured in differentiated THP-1 cells,
representative of the human disease.14 In the experience of
this collaboration and others,15 the hit rate was very low using
this whole cell in vitro intracellular amastigote assay, which may
be for several reasons. First, the parasites are dividing relatively
Figure 2. Structure of GSK3186899/DDD853651, 1.
RESULTS AND DISCUSSION
■
We previously reported that diaminothiazole 2 demonstrated
activity against the related parasite Trypanosoma brucei,18 and
that a scaffold-hopping strategy identified pyrazolopyrimidine 3
as having very weak activity against L. donovani (Ld) axenic
amastigotes,17 albeit with no activity against intracellular
parasites. Several changes were made to the cyclohexyl ring of
3 to allow us to explore the SAR around this portion of the
B
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. In Vitro Profile of 2−4
a
b
Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes.14 Data are the result of three replicates. Cli is
mouse liver microsomal intrinsic clearance; nd is not determined.
molecule, and one such change, the introduction of a
functionalized 4-amino group, led to compound 4-trans with
in vitro potency against intracellular parasites and no effect on
the nondividing human host cells (THP-1) (Table 1).
Interestingly 4-cis was >10-fold less potent within the intra-
macrophage assay compared to 4-trans. This relationship was
general across the series, and as such, the cis-isomers will not be
discussed further.
On the basis of the data presented in Table 1, the initial
medicinal chemistry focus was to improve metabolic stability
(mouse microsomal intrinsic clearance <5 mL·min−1·g−1), with
the aim of rapidly identifying analogues suitable for testing in an
in vivo mouse efficacy model. Despite the mode of action
(MoA) of the series being unknown when the chemistry
program was initiated, 3 and 4 did closely resemble a set of
antitumor cyclin dependent kinase inhibitors.19 The closest
exemplar was N-(trans-4-((3-(5-fluoro-2-methoxyphenyl)-1H-
pyrazolo[3,4-d]pyrimidin-6-yl)amino)cyclohexyl)-
methanesulfonamide (example 26 from ref 19), which
demonstrated IC50 values of 0.199 and 0.114 μM against
Cdk4 and Cdk2, respectively. Repurposing of human kinase
inhibitors as a strategy for infectious and neglected diseases has
been explored by others,20−22 in which they all recognize
selectivity over the human orthologue as a potential challenge.
Therefore, monitoring against a panel of human kinases for this
series would be critical, particularly for compounds progressing
into in vivo efficacy experiments.
Interestingly, replacements for the sulphonamide (e.g. 5)
were inactive against intracellular parasites within this series
(Table 2). Exchanging the benzyl group with an iso-butyl group
led to an increase in activity (6), although both compounds were
metabolically unstable. Replacing the iso-butyl group (RHS)
with a trifluoropropyl group (7) gave greater stability when
incubated with mouse liver microsomes and only a small loss in
potency compared to 6. Switching focus to the LHS, it was noted
that replacing the iso-butyl group with either a 2-methoxyphenyl
or a 2-pyridyl caused a significant increase in potency (8 and 9),
the latter being noteworthy for also having improved mouse
metabolic stability. Additionally, this series demonstrated
increased stability in human vs mouse liver microsomes (see 5
and 8 in Table 2).
To facilitate the synthesis of the compounds described within
this paper, a set of key amines was synthesized according to
Scheme 1. N-Boc-trans-1,4-cyclohexanediamine was deproto-
nated with n-BuLi then reacted with the relevant sulphonyl
chloride, and the resulting Boc protected intermediates were
treated with TFA to give 10a−c.
Examples 3−9, 15, and 17 were synthesized according to
Scheme 2. Thus, 5-bromo-2-chloro-4-methoxypyrimidine was
treated with iso-propylmagnesium chloride and various
aldehydes at −40 °C. The resulting alcohols were oxidized
with Dess−Martin periodinane to yield ketones 11a−d.
Treatment of 11a with cyclohexylamine in the presence of
Hunig’s base led to aminopyrimidine 12. Similarly, 11a was
̈
treated with tert-butyl-(trans-4-aminocyclohexyl)carbamate,
C
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. In Vitro Profiles of 4−9
a
Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes.14 Data are the result of at least four replicates.
b
c
HepG2 is a human liver cancer cell line. Cli is liver microsomal intrinsic clearance.
a
Scheme 1. Synthesis of Intermediates 10a−c
highly solubilizing vehicle (10% (v/v) dimethyl sulfoxide
(DMSO), 60% polyethylene glycol 400, and 30% deionized
water) to maximize the chance of achieving sufficient exposure
in mice when 15 was progressed to in vivo experiments.
To monitor the effects of the series on human kinases,
chemoproteomic profiling on Kinobeads was performed in a K-
562 protein extract to identify potential human off-targets for 8
and 15 (Figure 3). The compounds were tested at a
concentration of 5 μM for their activity against 210 human
protein kinases, and while 8 showed competition of several
protein kinases, 15 did not affect any of the captured protein
kinases. This gave us confidence that a suitable kinase selectivity
profile could be achieved within this series. On the basis of this
observation and overall in vitro/DMPK profile (Table 3), 15
was progressed to an animal disease model for VL.
a
Reagents and conditions: (a) n-BuLi, R1Cl, THF, −78°C, 1−1.5 h;
(b) TFA, DCM, RT, overnight, 80−99% over 2 steps
There are a number of preclinical in vivo animal protocols
available for assessing the efficacy of compounds intended to
treat VL, including mouse, rat, hamster, dog, and monkey
models.24 Of these, mouse and hamster are the most common
models for testing drug efficacy.25,26 However, there is a marked
lack of clinical validation of these models and the absence of
clinical PharmacoKinetic/PharmacoDynamic (PK/PD) data
makes it impossible to suggest which model is more predictive of
the clinical outcome in human. That said, after reviewing the
available clinical data27,28 and comparing to our nonclinical in
vivo data for miltefosine, it was observed that the mouse
exposure data was predictive for humans. In addition, both
liposomal amphotericin B and miltefosine achieve a >95%
suppression in parasite load within this mouse model.26 Mouse
was therefore selected as our primary model to evaluate in vivo
efficacy.
and the resulting compound treated with TFA to remove the
Boc group, yielding 13. This intermediate could then be either
acylated or sulphonylated to give 14a,b. Alternatively, a more
direct route was used for synthesis of intermediates 14c−h,
whereby coupling of the appropriate combinations of 10a−c
with 11a−d led to the relevant intermediates. Finally, treatment
of either 12 or 14a−h with hydrazine hydrate generated the
desired target compounds (3−9, 15, and 17).
On the basis of the SAR learnings summarized in Table 2
(notably 7 and 9), compound 15 was prepared with the aim of
improving potency and metabolic stability over 4 (Table 3). The
in vitro profile of 15 supported progression into a mouse
pharmacokinetic (PK) study. However, the compound showed
poor solubility when measured using chemiluminescent nitro-
gen detection (CLND, a technique used to measure kinetic
aqueous solubility).23 It would therefore be necessary to use a
D
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2. Synthesis of Compounds 3−9, 15, and 17
a
Reagents and conditions: (a) iPrMgCl (2M in Et2O), THF, −40°C, 30 min; (b) Dess−Martin periodinane, DCM, RT, 1 h, 39−67% over two
steps; (c) cyclohexylamine (for 12), tert-butyl-(trans-4-aminocyclohexyl)carbamate (for 13) or 10a−c (for 14c−h), DIPEA, EtOH, 120 °C
(microwave, 2 h); (d) TFA, DCM, 1 h, 80% over two steps; (e) phenylacetyl chloride, trimethylamine, DCM, overnight (for 14a), benzylsulphonyl
chloride, Cs2CO3, DMF, overnight (for 14b); (f) hydrazine hydrate, EtOH, 150 °C (microwave), 30 min to 1 h, 30−72% over two steps.
Table 3. In Vitro Profile of 15
a
b
Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes.14 Data are the result of 18 replicates. HepG2 is a
c
d
human liver cancer cell line. Cli is liver microsomal intrinsic clearance. CLND is kinetic aqueous solubility measured using chemiluminescent
nitrogen detection.23 AUC0−last is the area under the curve until the last measurement. %CV is the % of coefficient of variation. Cmax is the
e
f
g
h
i
j
maximum concentration reached. Clb is mouse clearance in blood. Vss is volume of distribution at steady state. Fpo is oral bioavailability.
Female Balb/c mice were infected with L. donovani
amastigotes, and the infection was allowed to establish for 7
days. These mice (groups of 5) were treated with either vehicle
(oral), sodium stibogluonate (pentostam, 15 mg/kg, which is
the ED30, subcutaneously), miltefosine (12 mg/kg, which is the
ED70, orally) as standards, or 15 (50 mg/kg, orally). Pentosam
and miltefosine were dosed once daily for 5 days with vehicle
and 15 dosed twice daily over same period.
Fourteen days, after infection, all animals were humanely
euthanized and parasite burden was determined microscopi-
cally.29 Parasite burden was expressed in Leishman−Donovan
units (LDU), the number of amastigotes per 500 nucleated cells
multiplied by the organ weight in grammes.30
Pleasingly, 15 demonstrated an 85% suppression in parasite
load (Figure 4) within the liver with 50 mg/kg b.i.d. dosing for 5
days, while the controls performed as expected. This result
established excellent proof of concept for the series. Despite 15
demonstrating promising efficacy and oral bioavailability (44%),
marked variability in exposure was observed (%CV = 79, Table
3) and it was also noted that first pass metabolism could only
account for 3% of the remaining noncirculating parent
compound (56%). Because 15 had a high artificial membrane
permeability (4.4 × 10−5 cm/s) and low CLND solubility
(Table 3), it was hypothesized that increasing the solubility
should increase exposure and reduce variability.
The property forecast index (PFI) provides a probabilistic
score of the likely developability risks and solubility of
compounds.31 15 had a PFI of 7.2 and the impact of modulating
PFI by either introducing polarity (reducing ChromLogD) or
incorporating saturated isosteres (reducing ∼ #Ar) are high-
lighted in Table 4. A collateral benefit of reducing the PFI was
that 15 and subsequent molecules all displayed excellent
metabolic stability.
16 was synthesized according to Scheme 3, where
commercially available 3-bromo-6-(methylthio)-1H-pyrazolo-
[3,4-d] pyrimidine was oxidized to sulphone 22. The pyrazole
N−H of 22 was then protected with THP to yield 23, and the
sulphone was subsequently displaced with 10b. The resulting
E
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Chemoproteomic affinity profiling of compounds 8 and 15. Kinobeads were incubated with K-562 cell extract either in the presence of
vehicle (DMSO) or compound 15 or compound 8 (5 μM). Proteins captured by the beads in both conditions were quantified by LC−MS/MS
analysis. Labeled proteins show more than 2-fold reduced binding to the Kinobead matrix due to competition with compounds 15 or 8 against vehicle
respectively and represent potential human off-targets.
Figure 4. In vivo efficacy profile of pentostam, miltefosine, and 15. The mean LDU values from individual animal livers were compared to the control
to give the percentage (%) suppression of parasite load in Balb/c mice (that were infected with L. donovani). Error bars represent the standard
deviation (SD). Lead optimization criteria was 70% suppression in liver parasite burden, while preclinical candidate was >95%.
bromide 24 was reacted with 5-pyrimidylboronic acid using
Suzuki coupling conditions, and subsequent THP deprotection
led to 16.
treatment with hydrazine gave compounds 1, 18−21, and
36−38. In the case of 29f, the Boc group was deprotected with
TFA following the reaction with hydrazine to give 20. Chiral
separation of 29c furnished the enantiomers 36 and 37.
Several LHS heteroaromatic analogues were synthesized.
However, as demonstrated by 16, none showed any advantage
over 15 in terms of either solubility or potency (Table 4). Hence,
the focus was to identify saturated isosteres to replace the pyridyl
LHS in 15, thus reducing the number of aromatic rings. Initial
compounds 17 and 18, where the pyridyl was replaced by a
saturated ring to reduce PFI, showed an improvement in
solubility, although both were less potent than 15. Pyrrolidine
19 demonstrated similar in vitro potency to piperidine 18, albeit
17 was synthesized according to Scheme 2, whereas an
alternative route was required for compounds 18−21. Synthesis
of these derivatives, along with 1 and 36−38, was accomplished
according to Scheme 4. Commercially available 2,4-dichloro-5-
pyrimidinecarbonyl chloride was treated with a suitable amine to
give amides 26a−g, which were subsequently treated with
sodium methoxide to selectively displace the pyrimidinyl 4-
chloro yielding 27a−g. The pyrimidinyl 2-chloro group could
then be displaced with amine 10a or 10b to give 28a−g. Finally,
reaction of 28a−g with Lawesson’s reagent followed by
F
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 4. PFI and in Vitro Profiles of 16−21
a
Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes.14 Data are the result of at least three replicates.
CLND is kinetic aqueous solubility measured using chemiluminescent nitrogen detection.23 PFI is the property forecast index.31
b
c
d
ChromLogDpH7.4 is a measure lipophilicity at pH7.4.31
a
Scheme 3. Synthesis of Compound 16
a
Reagents and conditions: (a) oxone, MeCN, H2O, RT, 3 h, 62%; (b) 3,4-dihydro-2H-pyran, p-toluenesulfonic acid monohydrate, THF, reflux,
overnight, 79%; (c) 10b, DIPEA, EtOH, 100°C, overnight, 68%; (d) Pd(PPh3)4, K2CO3, DMF, H2O, 150°C, 20 min; (e) conc HCl, MeOH,
100°C, 30 min, 85% over two steps.
lower solubility. Interestingly, piperazine 20 was less potent but
had excellent CLND solubility, presumably due to increased
basicity and reduced ChromLogD. Interrogation of the data
(Figure 5) suggested targeting a PFI between 4.2 and 6 would
give a high probability of achieving a desirable CLND solubility
while maintaining intracellular potency and good absorption.
Promisingly, the morpholine analogue 21 in Table 4 showed
similar in vitro potency to piperidine 18 with reduced PFI and
increased CLND solubility. Knowing that an improvement in
potency (in vitro) between compounds 9 and 15 was observed,
the iso-butyl-sulphonamide version of morpholine analogue 21
was of interest. This compound (30) was synthesized following
Scheme 4, i.e., displacing the chloro of 27g with amine 10a. As
shown in Table 5, 30 demonstrated excellent in vitro potency
and CLND solubility, additionally showing a similar profile to
G
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 4. Synthesis of Compounds 1, 18−21, 30, and 36−38
a
Reagents and conditions: (a) R1H, DIPEA, DCM, 0°C, 3 h, 53−92%; (b) NaOMe, THF, −40°C, 3 h, 48−86%; (c) 10a or 10b, DIPEA, 1,4-
dioxane, 100°C, overnight, 54−71%; (d) Lawesson’s reagent, THF, 45°C, 2 h; (e) hydrazine hydrate, 1,4-dioxane, 90 °C, overnight, 47−58% over
two steps.
Figure 5. CLND solubility vs PFI profile within this series of compounds. Ld InMac is the intramacrophage assay carried out in THP-1 cells with L.
donovani amastigotes.14 Number of biological replicates for all active compounds ≥2; PFI is the property forecast index;31 CLND is kinetic aqueous
solubility measured using chemiluminescent nitrogen detection.23
H
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 5. In Vitro Profile of 30
a
Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes.14 Data are the result of at least eight replicates.
b
c
d
HepG2 is a human liver cancer cell line. Cli is liver microsomal intrinsic clearance. CLND is kinetic aqueous solubility measured using
chemiluminescent nitrogen detection.23 PFI is the property forecast index.31
e
15 (Figure 3) against a panel of 210 human protein kinases at 5
μM (data not shown).
30 was selected for progression to the mouse in vivo efficacy
model, and the greater solubility of 30 compared to 15 now
allowed use of a vehicle that was suitable for subsequent
toxicology studies (i.e., 0.5% HPMC, 0.4% Tween 80 and 0.5%
benzyl alcohol) and investigation of higher doses within the
Table 8. Profile of 15, 21, 30, and 31
15
21
30
31
a
CLND solubility (μM)
8
259
7
5.0
3.7
273
328
9
4.9
nd
68
b
FaSSIF (μg/mL)
<1
7.2
3.2
nd
130
8.0
4.1
135
c
PFI
pKa
d
melting point (°C)
218
model.
a
CLND is kinetic aqueous solubility measured using chemilumines-
Analogue 30 demonstrated in vivo efficacy very close to our
cent nitrogen detection.23 FaSSIF is fasted state simulated intestinal
b
preclinical candidate target (i.e., >95% parasite suppression) at
100 mg/kg b.i.d. for 5 days, and a dose response study utilizing a
10 day regimen showed that a dose of 50 mg/kg b.i.d. achieved
the same level of efficacy (Table 6).
fluid solubility.32 PFI is the property forecast index.31 pK a is the
c
d
negative log10 of acid dissociation constant.35 nd is not determined.
FaSSIF solubility as variability between batches is known.34
However, within this series of compounds, minimal FaSSIF
variability was observed within the same batch and for 31 there
was little variation between the lead optimization batches (see
Table S20 in Supporting Information). Also, these compounds
demonstrated that there was little correlation between FaSSIF
and PFI or CLND within this series (see Table 8). Although
different pH conditions were used to measure FaSSIF (pH 6.5)
and PFI or CLND (pH 7.4), the measured pKa values35 of these
compounds would suggest that the degree of protonation should
have been similar under these different pH conditions.
a
Table 6. Mouse in Vivo Efficacy Profile of 30
% suppression of parasite load
compared to the control group
standard
oral regimen of 30
deviation (SD)
100 mg/kg b.i.d.
for 5 days
10 mg/kg b.i.d. for
10 days
25 mg/kg b.i.d. for
10 days
50 mg/kg b.i.d. for
10 days
94
36
74
97
5.24
27.31
4.49
0.95
Reviewing the general solubility equation (GSE, Yalkowsky
and co-workers)36 which relates lipophilicity and melting point
to aqueous solubility (see eq 1), highlighted the importance of
the melting point of the compound on its solubility.
a
The vehicle was 0.5% HPMC, 0.4% Tween 80 and 0.5% benzyl
alcohol using 5 mice per dose level.
Table 7. PK Dose Escalation in Rats for 30
log S = 0.5 − 0.01(MP − 25) − log P
a
(1)
dose of 30 (mg/kg)
mean AUC0−last h·ng/mL (%CV )
10
100
300
162 (41)
2960 (33)
2383 (183)
Equation 1: The general solubility equation (GSE), where S is
solubility, MP is melting point in Celsius, and P is octanol−water
partition coefficient. In practice, the GSE is commonly used
a
where log P is replaced by log DpHx, meaning log S is strictly log
%CV is the % of coefficient of variation. The vehicle was 1%
37
SpHx
.
methylcellulose in aqueous buffer using three rats per dose level.
Data in Table 8 indicates a reciprocal trend between melting
point and FaSSIF solubility. To explore the potential means of
modulating the high melting points, a small molecule crystal
structure of 15 (Figure 6, as a representative example of the
series) was obtained.
The crystal structure of 15 was shown to contain a single, fully
ordered molecule in the centrosymmetric space group, P1. As
can be seen in Figure 6, all three N−H groups act as hydrogen
bond donors. Molecules of 15 form hydrogen-bonded dimers
through reciprocal N20−H20···N19 interactions between
inversion symmetry-related fused ring systems. These dimers
are linked together by further hydrogen bonds between the
sulfonamide and aminopyrimidine groups to form hydrogen-
bonded columns within the structure. Additionally, the fused
ring system and the pyridine in each molecule of 15 are
A rat PK dose escalation study with 30 (Table 7)
disappointingly showed greater than proportional increase in
exposure between 10 and 100 mg/kg and then a saturation of
absorption between 100 and 300 mg/kg limiting the maximum
exposure that could be explored. This saturation of absorption at
higher doses, together with high variability between animals,
implied that solubility might still be problematic. Although 30
showed reasonable CLND solubility data (Table 5), the
solubility profile in fasted state simulated intestinal fluid
(FaSSIF)32 was poor (9 μg/mL). Other compounds within
this series all showed a similar poor level of FaSSIF solubility
(see 15, 21, and 30 in Table 8) except for 3133 (see Table 9 for
structure), which had an improved profile (more discussion on
31 to follow). Care should always be taken when considering
I
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 9. In Vitro and FaSSIF Solubility Profiles of 1 and 31−40
a
Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes.14 Data are the result of at least three replicates.
CLND is kinetic aqueous solubility measured using chemiluminescent nitrogen detection.23 FaSSIF is fasted state simulated intestinal fluid
b
c
solubility.32 PFI is the property forecast index.31 nd is not determined.
d
approaching a coplanar arrangement [having a dihedral angle of
6.18(8)°], and this allows a π−π interaction between adjacent
molecules within the columns. As such, 15 exhibited a highly
stacked crystal lattice, therefore potentially explaining the low
FaSSIF solubility. A number of analogues were therefore
designed that would disrupt some of the hydrogen bonding
seen in this crystal structure. From the past SAR, removal of N19
or N20−H (Figure 6) meant significant in vitro potency
reduction (i.e., the N20−Me version of compound 15
demonstrated Ld InMac pEC50 < 4.3 and THP pEC50 < 4.3),
so focusing on disruption of the other hydrogen bonds via
introduction of strategically placed substituents was prioritized.
A second strategy involved introducing substituents to reduce
the overall planarity of this series, through either chiral centers or
J
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
selectively displaced with sodium methoxide to give 42a.
Treatment of 42a with the appropriate amine 10a−c in the
̈
presence of Hunigs base yielded 43a−c. Suzuki coupling of 43a
with 2-methoxyphenylboronic acid or Buchwald−Hartwig
coupling of 43b,c with morpholine, followed by deprotection
of the THP group, furnished compounds 31−35. In the case of
44d, THP deprotection was followed by separation of the
enantiomers by chiral chromatography to give 34 and 35. To
generate the more challenging 3-methylmorpholine analogues
39 and 40, a range of protecting groups, SNAr conditions, and
Buchwald−Hartwig coupling conditions were investigated. The
conditions that gave the best results involved the use of SEM as
the protecting group, which gave improved yields compared to
THP. Subsequently, use of RuPhos as catalyst, tris-
(dibenzylideneacetone)dipalladium(0) as the palladium source,
and potassium hexamethyldisilazide as base gave reasonable
yields in the Buchwald−Hartwig coupling. Thus, treatment of
43d with the appropriate enantiomer of 3-methylmorpholine
under these conditions gave 44e−f, with subsequent SEM
deprotection yielding 39 and 40.
As mentioned earlier, 31 (Tables 8 and 9) proved to be very
interesting as it demonstrated that it was possible to identify
compounds with improved FaSSIF solubility while maintaining
intracellular potency. It was postulated that the additional
methoxy group in the core could disrupt at least two or possibly
four hydrogen bonds and/or force the phenyl ring to be out of
the plane (Figure 6); in particular, it should disrupt the H-bonds
formed by N17 and N24. As such, the addition of this OMe into
the core of 21 (7 μg/mL) and 30 (9 μg/mL) was investigated in
order to improve the FaSSIF solubility (see analogues 32 and 33
in Table 9). Pleasingly, both examples (32 and 33)
demonstrated an increase in potency (in vitro), although only
32 showed an improvement in FaSSIF solubility compared to its
corresponding parent compound (21).
Figure 6. View of part of the crystal structure of 15. The numbering
scheme for the non-hydrogen atoms of the central molecule (depicted
with the solid bond type) is shown in full. The hydrogen bonds
associated with the central molecule are depicted as dashed lines. For
clarity, only fragments containing the hydrogen bond donors and
acceptors are shown for the surrounding molecules (with the open
bond type). Anisotropic atomic displacement ellipsoids for the non-
hydrogen atoms are shown at the 50% probability level. Hydrogen
atoms are displayed with an arbitrarily small radius.
causing steric clashes. Indeed, some substituents could serve
both of these desired functions and potentially improve the
FaSSIF solubility (see Table 9).
The 4-methoxy-1H-pyrazolo[3,4-d]pyrimidin-6-amino deriv-
atives 31−35 were synthesized according to Scheme 5.
Commercially available 3-bromo-4,6-dichloro-1H-pyrazolo-
[3,4-d]pyrimidine was THP-protected and the 4-chloro
Comparing 32 and 33 in more detail, it was postulated that
variations to the sulphonamide could affect the distance between
a
Scheme 5. Synthesis of Compounds 31−35, 39, and 40
a
Reagents and conditions: (a) For THP, 3,4-dihydro-2H-pyran, para-toluenesulfonic acid monohydrate, THF, 70°C, 2 h, 79% (41a) or for SEM, 2-
(trimethylsilyl)ethoxymethyl chloride, DIPEA, DCM, 0 °C, 1.5 h, 100% (41b); (b) NaOMe, MeOH, RT, 30 min, 100% (PG = THP, 42a) or 98%
(PG = SEM, 42b); (c) 10a−c, DIPEA, 1,4-dioxane, 110°C, 3 days, 40−51%; (d) 2-methoxyphenylboronic acid, Pd(PPh3)4, K2CO3, DMF, 130°C,
30 min for 44a (87%) or morpholine, NaOtBu, xantphos, Pd2 dba3, dioxane, 110°C, overnight for 44b−d 45−70%, or relevant 3-
methylmorpholine, KHMDS, RuPhos, Pd2 dba3, 1,4-dioxane, 110°C, 2 h for 44e,f 47−50%; (e) HCl, MeOH, 60°C, 30 min to 1 h (THP
deprotection of 44a−d) or AcCl, MeOH, RT, 30 min to 1 h (SEM deprotection of 44e,f) 32−83%.
K
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 7. Hydrogen bonded columns in the crystal structures of 1 and 15: (a) a view down a column in 1; (b) an orthogonal view to (a); (c) a view
down a column in 15; (d) an orthogonal view to (c). Hydrogen bonds are displayed as black dotted lines.
Figure 8. Ld InMac vs FaSSIF/Ld InMac fold wrt cpd 30. Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani
amastigotes.14 Data are the result of at least three replicates. FaSSIF/EC50 fold wrt cpd 30 is the FaSSIF and Ld InMac ratio of a new compound
compared to 30.
the layers in the crystal structure and therefore influence the
melting point. Thus, a bulkier sulphonamide, as exemplified by
enantiomers 34 and 35, were designed to further exploit this
hypothesis (Table 9). Encouragingly, both enantiomers
demonstrated an increase in FaSSIF solubility, albeit with a
loss of intracellular potency compared to 32 and 33.
Excited by this result, the alternative LHS that could break
planarity and affect the stacking of the crystal structure were
prioritized (i.e., 36, 37, 1, and 38). As Table 9 shows, all these
compounds demonstrated increased FaSSIF solubility alongside
similar or increased in vitro potency compared to 21. It was
postulated that adding OMe to the core and iso-butyl
sulphonamide to these examples could further increase in vitro
potency while maintaining FaSSIF solubility, as had been seen
for 33. Indeed, both examples 39 and 40 showed a similar
increase in potency (in vitro), although 39 had a lower FaSSIF
solubility than expected compared to 40. This observation
between enantiomers was also mirrored for 1/38 and 36/37,
although the rationale for this is still unclear.
Interestingly, 1 crystallized in the noncentrosymmetric space
group (P1) and has four independent molecules in the
asymmetric unit (Figure 7). Although a different space group
L
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 9. Rat PK dose escalation studies for 30, 31, 33, 1, and 39.
Figure 10. Human kinase selectivity profile of 1, 31, and 39. The figure shows all human protein kinases affected by any of the three compounds (no
bar, protein was not identified; maximum compound concentration analyzed was 20 μM).
to 15, analogous intermolecular hydrogen bonds were found to
exist in 1 and 15, giving rise to columns in the same way. In
cross-section, the columns are wider for 1 as they must
accommodate the substituted morpholinyl groups and the
removal of the π−π stacking. Although the structure of 1 has no
centers of symmetry owing to the chiral ring, a pseudocen-
trosymmetric arrangement has been adopted. In effect, the four
independent molecules in 1 increase the flexibility of the system,
allowing the same hydrogen bonds to exist as in 15 despite the
much bulkier, chiral ring being present. Overall, however, the
packing in 1 is clearly less efficient than 15, leading to a density
of 1.405 gcm−3 (versus 1.511 gcm−3) at the temperature of the
experiments (150 K) and a packing coefficient of 0.676 (versus
0.699). In this respect, the deliberate attempt to disrupt the
M
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 11. (a) In vivo efficacy of 31, 1, and 39 compared to miltefosine; (b) mouse liver smears from in vivo efficacy model for untreated, miltefosine,
31, 1, and 39. The mean LDU values from individual animal livers were compared to the control to give the percentage (%) suppression of parasite load
in Balb/c mice (that were infected with L. donovani). Vehicle used for 31, 1, and 39 was 0.5% HPMC, 0.4% Tween 80, and 0.5% benzyl alcohol, and
deionized water for miltefosine. This experiment was carried out using five mice per dose level. Error bars represent the standard deviation (SD). Lead
optimization criteria was 70% suppression in liver parasite burden, while the preclinical candidate was >95%.
packing found in 15 was a success and helped to improve FaSSIF
solubility while maintaining in vitro potency (Table 8 and 9).
Upon analyzing the data shown in Table 9, it became clear
that there was no correlation between FaSSIF solubility and in
vitro potency. Consequently, multiparameter optimization was
pursued (eq 2), whereby the FaSSIF and Ld InMac ratio of a
new compound was compared to a reference compound (i.e.
30), coupled with a potency filter of Ld InMac pEC50 ≥ 5.8, and
this allowed more informed compound progression decisions.
Figure 8 highlights the compounds of interest based on the
selection criteria discussed above (i.e. 31, 33, 1, and 39), which
were subsequently progressed to rat PK dose escalation studies
(Figure 9). Compounds 5 and 7 were rejected due to metabolic
instability when incubated with mouse liver microsomes (Table
2), whereas 32 and 40 showed no advantage in terms of
“FaSSIF/Ld InMac fold wrt cpd 30” profile over their
counterparts 33 and 39, respectively (Table 9).
33 was progressed into rat PK on the basis of high potency
compensating for low FaSSIF solubility. However, it gave very
similar exposures to 30 upon dose escalation (Figure 9),
demonstrating that it was not suitable for further progression.
Pleasingly, however, 3 out of the 4 compounds (31, 1, and 39)
demonstrated notably superior exposure to 30 as measured by
the AUC (Figure 9), in line with the multiparameter hypothesis.
Furthermore, these compounds all showed a dose proportional
exposure increase, which would be important in further
development of the compound series, particularly when
determining potential therapeutic indices.
FaSSIF/Ld InMac fold wrt cpd 30
i
y
i
y
new cpd
cpd 30
cpd 30
new cpd
j
j
j
j
j
z
z
z
z
z
j
j
j
j
j
z
z
z
z
z
= FaSSIF
× Ld InMac EC
50
(2)
k
{
k
{
Equation 2: FaSSIF/Ld InMac fold wrt cpd 30.
The reasoning behind the additional in vitro potency filter was
that compounds from this series with Ld InMac pEC50 < 5.8
were never seen to demonstrate desirable in vivo efficacy at a
further reduced dose (i.e., ≤25 mg/kg b.i.d.), even when having
progressable FaSSIF solubility (for example, compound 37
demonstrated 65% parasite suppression at 25 mg/kg b.i.d. for 10
days).
Chemoproteomic profiling on Kinobeads was performed in a
mixed human cell/tissue extract, to identify potential human off-
targets for 31, 1, and 39 (Figure 10). The compounds were
N
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
tested for their activity against >260 human protein kinases, and
IC50 values were generated. Figure 10 shows only kinases
affected by any of the three compounds, indicating that the three
compounds had different human kinase profiles. Cdk4 was only
identified in the experiment for 39 (IC50 > 20 μM). Additionally,
31 and 39 did show potency against Cdk2, albeit 9- or 35-fold
less potent respectively than the closest analogue from Ding and
co-workers,19 where it was unaffected by 1. However, the extent
of inhibition of these human kinases was not sufficient to
preclude progression for any one compound. Interestingly,
performing similar dose−response experiments using leishma-
nia parasite lysate for 8 identified CRK12 (IC50 3 nM), MPK9
(IC50 105−181 nM), and CRK6 (IC50 194−363 nM).17
On this basis, 31, 1, and 39 were progressed into in vivo
efficacy studies, where all three gave sufficient parasite
suppression to be considered as preclinical development
candidates at 25 mg/kg b.i.d. for 10 days (Figure 11). As such,
all three compounds were profiled in key in vitro and ex vivo
safety assays (Table 10) in order to select a lead compound to
prioritize for a 7 day toxicology study.
Table 10. Comparison of three Key Compounds (31, 1, and
39)
31
1
39
a
Ld InMac (pEC50)
6.4
1.1 (mouse)
5.9
<0.5 (mouse), 0.6 (mouse),
0.7 (human) 2.5 (human)
6.6
b
Cli (mL·min−1·g−1
)
mouse in vivo efficacy 25 mg/kg b.i.d. 25 mg/kg b.i.d. 25 mg/kg b.i.d.
(>95% parasite
@ 10 d
@ 10 d
@ 10 d
suppression)
c
PFI
8.0
25
5.2
5.5
6.1
33
d
e
FaSSIF (μg/mL)
CYP3A4 (pIC50)
Ames38
17
<4.4
−ve @ 1500
μg/plate
−ve @ 120
μg/mL
>30
<4.4
−ve @ 1500
μg/plate
equivocal @
350 μg/mL
−ve @ 1500
μg/plate
−ve @ 5
μg/mL
f
MLA
hERG (IC50, μM)39,40 17.5
>30
g
RVW
QT shortening QT shortening QT shortening
(11.3% @
10 μM)
(8.9% @
30 μM)
(15.2% @
100 μM)
a
Ld InMac is the intramacrophage assay carried out in THP-1 cells
with L. donovani amastigotes.14 Data are the result of at least six
b
c
replicates. Cli is liver microsomal intrinsic clearance. PFI is the
property forecast index;31 −ve is negative. FaSSIF is fasted state
d
simulated intestinal fluid solubility.32 Melting point for this batch was
e
262 °C. MLA is mouse lymphoma assay.41 RVW is rabbit ventricular
f
g
wedge.42
Following a form screen, the most stable form was identified
and the corresponding FaSSIF solubility is reported in Table 10.
Apparent inhibition by 31 in an in vitro hERG assay39,40
prompted progression of all three compounds to the ex vivo
rabbit ventricular wedge (RVW) assay.42 Interestingly, they all
showed a similar effect of QT shortening, which appears to be
independent of hERG in vitro potency. The clinical relevance of
this QT shortening finding and its translation in vivo was
unclear.43 There were no changes to the remaining QT
parameters tested, and the risk of QT prolongation and Torsade
de pointes (TdP) arrhythmia in humans was assessed as low
based on these results. Because there were only minor
differences, all three compounds were progressed into 7 day
rat toxicology and rat cardiovascular studies (Table 11).
O
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
470.5 MHz). Chemical shifts (δ) are expressed in ppm recorded using
the residual solvent as the internal reference in all cases. Signal splitting
patterns are described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), broad (b), or a combination thereof. Coupling constants
(J) are quoted to the nearest 0.1 Hz. LC-MS analyses were performed
with either an Agilent HPLC 1100 series connected to a Bruker
Daltonics MicrOTOF or an Agilent Technologies 1200 series HPLC
connected to an Agilent Technologies 6130 quadrupole LC/MS, where
both instruments were connected to an Agilent diode array detector.
Mobile phase was water/acetonitrile + 0.1% HCOOH, or water/
acetonitrile + 0.1% NH3; linear gradient 80:20 to 5:95 over 3.5 min, and
then held for 1.5 min; flow rate 0.5 mL min−1. All intermediates had a
measured purity of >90% as determined using this analytical LC-MS
system unless otherwise noted. All assay compounds had a measured
purity of ≥95% as determined using this analytical LC-MS system (TIC
and UV). High resolution electrospray measurements were performed
on a Bruker Daltonics MicrOTOF mass spectrometer. Microwave-
assisted chemistry was performed using a Biotage Initiator microwave
synthesizer.
The outcome of the 7 day studies was the deselection of 39, as
the therapeutic index (TI) was not sufficient for progression
(Table 11). 1 was selected as the preclinical candidate for VL (as
opposed to 31) due to the fact the TI was limited by exposure
and not by an adverse event, coupled with no direct inhibition of
CYP3A4 and no flags within liver gene expression profiling panel
(which can lead to problematic drug−drug interactions that are
particularly relevant due to the frequency of VL/HIV
coinfections).44
SUMMARY AND CONCLUSION
■
Initial hit 4 demonstrated a promising profile (Table 1) but
highlighted the need to optimize in vitro potency and mouse
metabolic stability in order to test this series within an in vivo VL
animal study. With those properties improved and selectivity
against a panel of human kinases, 15 was progressed to such an
in vivo model, where it demonstrated an 85% suppression in
parasite load within the liver (Figure 4). However, due to low
CLND solubility, 15 was not progressed further because it
required the use of a vehicle that was not acceptable in
downstream toxicological studies. Successfully using PFI to
improve solubility of this series led to compound 30, which
demonstrated candidate quality efficacy using a formulation that
could be used for toxicological studies (Table 6). Dose
escalation in rats demonstrated that 30 exhibited variable
nonlinear exposure, limiting the ability to assess its therapeutic
index in toxicological studies (Table 7). It was speculated that
the low FaSSIF solubility was driving this variable nonlinear
exposure. After investigating the small molecule crystal structure
of 15 (Figure 6), disrupting certain hydrogen bonds and
breaking planarity by introducing specific substituents became
the primary medicinal chemistry focus. Multiple compounds
demonstrated improved FaSSIF solubility, however, the use of a
mathematical comparison to 30 and a potency filter (eq 2 and
Figure 8) aided the selection of 31, 33, 1, and 39. All except 33
(where high potency compensated for low FaSSIF solubility)
demonstrated superior exposure compared to 30 (Figure 9).
This demonstrated that FaSSIF solubility gave a better
correlation to rat PK exposure than CLND for this series, likely
due to being more relevant to biological systems and the fact that
it is a thermodynamic measurement of solubility. To further
differentiate between 31, 1, and 39 (Table 10), all were
progressed into 7 day rat toxicological studies (Table 11). 1 was
finally selected as a preclinical candidate for VL, and definitive
safety studies are being conducted to support clinical
progression. Compound 1 is one of only a handful of
compounds worldwide that have reached preclinical candidate
status for VL, highlighting the need and also the achievement.
3,3,3-Trifluoro-N-(trans-4-((3-((S)-2-methylmorpholino)-1H-
pyrazolo[3,4-d]pyrimidin-6-yl)amino)cyclohexyl)propane-1-sulfo-
namide 1. 29a (91 g, 173 mmol) was dissolved in dioxane (1.2 L), then
hydrazine hydrate 85% (173.2 g, 3.5 mol) was added and the reaction
stirred at 90 °C for 18 h. The mixture was cooled to 25 °C, where H2O
(800 mL) and EtOAc (800 mL) was added and the mixture was stirred
at room temperature for 20 min. The organic phase was separated, and
the aqueous phase was extracted with EtOAc (3 × 1.5 L). The
combined organic phases were washed with brine (2 × 2 L) and dried
over Na2SO4. The solvent was removed under vacuum, and EtOAc
(400 mL) was added to the residue and stirred at room temperature for
1 h. The resulting mixture was filtered, the cake was washed with EtOAc
(100 mL), and the filtrate was dried in vacuo to afford a crude solid.
This solid was then purified by recrystallization from methanol (400
1
mL) to give 1 (40 g, 81 mmol, 47% yield) as a light-green solid. H
NMR (DMSO-d6): δ 11.93 (s, 1H), 8.82 (s, 1H), 7.33 (d, J = 7.6 Hz,
1H), 7.14 (s,1H), 3.86 (dd, J = 10.9 and 2.9 Hz, 1H), 3.73−3.55 (m,
5H), 3.31−3.09 (m, 3H), 2.90−2.81 (m, 1H), 2.66−2.50 (m, 3H), 1.92
(br s, 4H), 1.39−1.29 (m, 4H), 1.15 (d, J = 6.2 Hz, 3H). 13C NMR
(DMSO-d6): δ 160.5, 157.3, 154.0, 151.3, 127.9 (q, JCF = 128 Hz), 71.3,
65.9, 54.6, 52.0, 49.9, 47.9, 45.4, 33.1 (2C), 31.2 (2C), 28.9 (q, JCF
=
29.9 Hz), 19.2. 19F NMR (DMSO-d6) δ −64.4. HRMS (ES+): m/z [M
+ H]+ calcd for C19H29N7O3F3S 492.1999, found 492.1998.
Enantiopurity determined using a chiral column: Chiralpak AD-3, 3
mm, 0.46 cm × 10 cm, acetonitrile/MeOH = 50/50 + 0.2% isopropyl
amine, 30 mL/min, 298 K, 254 nm. First enantiomer (compound 38,
minor) Rt = 6.6 min, second enantiomer (1, major) Rt = 9.7 min. ee
measured: 99.2%.
N-Cyclohexyl-3-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-6-amine
3. A mixture of 11a (100 mg, 0.44 mmol) and cyclohexylamine (48 mg,
0.48 mmol) in n-butanol (4 mL) was stirred at 100 °C for 15 min then
cooled to room temperature. Hydrazine hydrate (360 μL, 1.76 mmol)
was added and the mixture stirred in a sealed tube at 150 °C for 45 min
(microwave). The solution was cooled, solvent evaporated, and the
crude mixture chromatographed (0−10% methanol/DCM) to give 3
(64 mg, 0.23 mmol, 53% yield). 1H NMR (DMSO-d6): δ 12.57 (s, 1H),
8.74 (s, 1H), 7.12 (bs, 1H), 3.72 (bs, 1H), 2.64 (d, J = 7.2 Hz, 2H), 2.05
(sept., J = 6.9 Hz, 1H), 1.93−1.89 (m, 2H), 1.77−1.71 (m, 2H), 1.64−
1.58 (m, 1H), 1.35−1.13 (m, 5H), 0.91 (d, J = 6.6 Hz, 6H). 13C NMR
(DMSO-d6): δ 160.6, 157.5, 153.6, 145.7, 106.7, 49.6, 36.5, 32.7, 28.6
(2C), 25.9 (2C), 25.4 (2C), 22.9. HRMS (ES+): m/z [M + H]+ calcd
for C15H24N5 274.2032, found 274.2018.
N-(trans-4-((3-Isobutyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-
amino)cyclohexyl)-2-methylpropane-1-sulfonamide 6. 6 was ob-
tained by an analogous method to 3 (28 mg, 0.07 mmol, 44% yield). 1H
NMR (DMSO-d6): δ 12.50 (bs, 1H), 8.65 (s, 1H), 7.14 (s, 1H), 6.94
(d, J = 7.6 Hz, 1H), 3.03−2.88 (m, 2H), 2.81 (d, J = 6.3 Hz, 2H), 2.55
(d, J = 7.1 Hz, 2H), 2.03−1.91 (m, 2H), 1.87−1.79 (m, 4H), 1.27−1.21
(m, 4H), 0.93 (d, J = 6.8 Hz, 6H), 0.81 (d, J = 6.6 Hz, 6H). HRMS
(ES+): m/z [M + H]+ calcd for C19H33N6O2S 409.2386, found
409.2400.
EXPERIMENTAL SECTION
■
General Experimental Information. Chemicals and solvents
were purchased from the Aldrich Chemical Co., Fluka, Fluorochem,
ABCR, VWR, Acros, Fisher Chemicals, and Alfa Aesar and were used as
received unless otherwise stated. Air- and moisture-sensitive reactions
were carried out under an inert atmosphere of argon in oven-dried
glassware. Analytical thin-layer chromatography (TLC) was performed
on precoated TLC plates (layer 0.20 mm silica gel 60 with fluorescent
indicator UV254, from Merck). Developed plates were air-dried and
analyzed under a UV lamp (UV254/365 nm). Flash column
chromatography was performed using prepacked silica gel cartridges
(230−400 mesh, 40−63 μm, from SiliCycle) using a Teledyne ISCO
Combiflash Companion, or Combiflash Retrieve. 1H NMR, 13C NMR,
19F NMR, and 2D-NMR spectra were recorded on a Bruker Avance
DPX 500 spectrometer (1H at 500.1 MHz, 13C at 125.8 MHz, 19F at
P
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3,3,3-Trifluoro-N-(trans-4-((3-isobutyl-1H-pyrazolo[3,4-d]-
pyrimidin-6-yl)amino)cyclohexyl)propane-1-sulfonamide 7. 7 was
obtained by an analogous method to 3 (18 mg, 0.04 mmol, 25% yield).
1H NMR (DMSO-d6): δ 12.67 (s, 1H), 8.82 (s, 1H), 7.37 (d, J = 7.6 Hz,
1H), 7.25 (s, 1H), 3.63 (s, 1H), 3.32−3.27 (m, 2H), 3.16 (s, 1H),
2.68−2.61 (m, 4H), 2.08−2.00 (m, 1H), 2.08−2.00 (m, 4H), 1.38−
1.28 (m, 4H), 0.91 (d, J = 6.5 Hz, 6H). HRMS (ES+): m/z [M + H]+
calcd for C18H28F3N6O2S 449.1947, found 449.1952.
N-(trans-4-((3-(2-Methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)cyclohexyl)-2-methylpropane-1-sulfonamide 8. 8 was
obtained by an analogous method to 3 (270 mg, 0.59 mmol, 72% yield).
1H NMR (DMSO-d6): δ 13.08 (s, 1H), 8.76 (s, 1H), 7.66 (dd, J = 1.7,
7.6 Hz, 1H), 7.47−7.42 (m, 1H), 7.27−7.17 (m, 2H), 7.09−7.00 (m,
2H), 3.87 (s, 3H), 3.75−3.65 (m, 1H), 3.15−3.08 (m, 1H), 2.91 (d, J =
6.6 Hz, 2H), 2.17−2.07 (m, 1H), 2.02−1.91 (m, 4H), 1.44−1.35 (m,
4H), 1.04 (d, J = 6.6 Hz, 6H). 13C NMR (DMSO-d6): 160.5, 157.6,
155.7, 142.3, 130.5, 130.3, 122.1, 121.1, 112.1, 60.4, 55.8, 51.9, 49.1,
33.3 (2C), 31.2 (2C), 25.0, 22.8 (2C). HRMS (ES+): m/z [M + H]+
calcd for C22H31N6O3S 459.2178, found 459.2183.
2-Methyl-N-(trans-4-((3-(pyridin-2-yl)-1H-pyrazolo[3,4-d]-
pyrimidin-6-yl)amino)cyclohexyl)propane-1-sulfonamide 9. 9 was
obtained by an analogous method to 3 (630 mg, 1.5 mmol, 59% yield).
1H NMR (DMSO-d6): δ 13.32 (bs, 1H), 9.34 (s, 1H), 8.71 (d, J = 4.2
Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.93−7.89 (m, 1H), 7.45 (bs, 1H),
7.49−7.39 (m, 1H), 7.04 (d, J = 6.0 Hz, 1H), 3.81−3.61 (m, 1H),
3.12−3.06 (m, 1H), 2.92 (d, J = 6.4 Hz, 2H), 2.13−2.05 (m, 1H),
1.97−1.94 (m, 4H), 1.41−1.31 (m, 4H), 1.04 (d, J = 6.7 Hz, 6H). 13C
NMR (DMSO-d6): δ 160.8, 158.2, 156.0, 159.9, 152.7, 150.0, 144.3,
137.4, 123.7, 120.1, 60.5, 51.9, 49.1, 33.3 (2C), 31.2 (2C), 25.0, 22.8.
HRMS (ES+): m/z [M + H]+ calcd for C20H28N7O2S 430.2020, found
430.2024.
N-(trans-4-Aminocyclohexyl)-2-methylpropane-1-sulfonamide
10a. To a solution of tert-butyl N-(4-aminocyclohexyl)carbamate (24
g, 112 mmol) in THF (800 mL) at −78 °C was added n-butyl lithium
(2.5 M in hexanes, 49.3 mL, 123 mmol) dropwise and stirred for 20 min
at −78 °C. The solution was warmed to −20 °C and stirred for 15 min,
then cooled to −78 °C. 3,3,3-Trifluoropropane-1-sulfonyl chloride
(15.4 mL, 118 mmol) was added dropwise, stirred for a further 5 min at
−78 °C, and then warmed to room temperature where it was stirred for
1 h. The reaction mixture was quenched with 1 M HCl (50 mL) and
EtOAc (500 mL), and the aqueous layer separated and extracted with
EtOAc (2 × 500 mL). The combined organic layers were dried with
Na2SO4 and filtered, and the remaining solvent was evaporated to give a
white solid (33.5 g). This crude material (33.5 g) was dissolved in DCM
(400 mL), and TFA (38.3 mL) added at room temperature and stirred
overnight. The solvent was removed under vacuum to give an oil, and
Et2O (70 mL) was added to precipitate 10a as a white solid (35 g, 100
mmol, 89% yield over two steps). 1H NMR (DMSO-d6): δ 7.97 (br s,
2H), 7.15 (d, J = 7.3 Hz, 1H), 3.02−2.96 (m, 1H), 2.86 (d, J = 6.5 Hz,
2H), 2.46 (ddd, J = 3.7, 7.2, 14.5 Hz, 1H), 2.09−2.02 (m, 1H), 1.81 (t, J
= 5.4 Hz, 2H), 1.75−1.69 (m, 2H), 1.27−1.16 (m, 2H), 1.11−1.03 (m,
2H), 1.00 (d, J = 6.8 Hz, 6H).
N-(trans-4-Aminocyclohexyl)-3,3,3-trifluoropropane-1-sulfona-
mide 10b. This reaction was performed in two batches. To a
suspension of tert-butyl ((1,4-trans)-4-aminocyclohexyl)carbamate (30
g, 140 mmol) in THF (1.33 L), cooled at −78 °C, n-butyl lithium (56
mL, 140 mmol) was added dropwise. The resulting mixture was stirred
at −78 °C for 20 min and at −10 °C for 10 min. After cooling to −78
°C, 3,3,3-trifluoropropane-1-sulfonyl chloride (17.64 mL, 140 mmol,)
was added. After stirring for 1.5 h, it was allowed to warm to RT and
stirred for 20 min. The reaction mixture was diluted with H2O (500
mL), followed by addition of 2 M HCl (20 mL) and was extracted with
EtOAc (400 mL). The organic layer was dried over Na2SO4, filtered,
and concentrated to give tert-butyl ((1,4-trans)-4-(3,3,3-
trifluoropropylsulfonamido)cyclohexyl)carbamate as a white solid
(43.5 g, 116 mmol, 83% yield). 1H NMR (DMSO-d6): δ 7.33 (d, J =
6.4 Hz, 1H), 6.77−6.70 (d, J = 6.7 Hz, 1H), 3.30−3.23 (m, 2H), 3.17−
3.01 (m, 2H), 2.70−2.56 (m, 2H), 1.87−1.67 (m, 4H), 1.36 (s, 9H),
1.31−1.13 (m, 4H). TFA (182 mL, 2377 mmol) was added to a
solution of tert-butyl ((1,4-trans)-4-(3,3,3-trifluoropropylsulfon-
amido)cyclohexyl)carbamate (89 g, 238 mmol) in DCM (732 mL),
cooled to 0 °C. The reaction mixture was stirred at RT overnight. The
mixture was concentrated to dryness and coevaporated with Et2O (100
mL) to give 10b as a white solid (93.5 g, 238 mmol, quant yield). 1H
NMR (DMSO-d6): δ 7.89 (br s, 2H), 7.43 (d, J = 7.5 Hz, 1H), 3.34−
3.24 (m, 2H), 3.16−3.05 (m, 1H), 2.99−2.86 (m, 1H), 2.72−2.56 (m,
2H), 1.95−1.84 (m, 4H), 1.43−1.21 (m, 4H).
1-(2-Chloro-4-methoxypyrimidin-5-yl)-3-methylbutan-1-one
11a. To a solution of 5-bromo-2-chloro-4-methoxypyrimidine (1.11 g,
5 mmol) in THF (20 mL) at −40 °C was added a solution of iso-propyl
magnesium chloride (3 mL, 6.5 mmol, 2 M in ether) dropwise over 10
min and the resulting suspension stirred for 15 min. A solution of iso-
valeraldehyde (700 μL, 6.5 mmol) in THF (2 mL) was added dropwise
over 10 min, stirring continued for 30 min and the reaction quenched
with methanol (1 mL), allowed to warm to room temperature, and the
solvent evaporated. Crude material was taken up in DCM (20 mL),
washed with 2 M HCl (20 mL), dried, and evaporated. Crude material
was dissolved in DCM (20 mL), Dess−Martin periodinane (2.97 g, 7
mmol) added, and the reaction mixture stirred for 1 h. A mixture of satd
sodium bicarbonate (20 mL)/satd sodium thiosulfate (20 mL) was
added and stirring continued for 30 min. The organic layer was
separated, dried, evaporated, and crude material chromatographed (0−
40% EtOAc/heptane) to yield 11a (0.65 g, 2.9 mmol, 57% yield over 2
steps). 1H NMR (DMSO-d6): δ 8.84 (s, 1H), 4.24 (s, 3H), 2.90 (d, J =
6.8 Hz, 2H), 2.34−2.27 (m, 1H), 1.06 (d, J = 6.6 Hz, 6H). MS (ES+) m/
z 229.1, 231.1 [M + H]+.
3,3,3-Trifluoro-N-(trans-4-((3-(pyridin-2-yl)-1H-pyrazolo[3,4-d]-
pyrimidin-6-yl)amino)cyclohexyl)propane-1-sulfonamide 15. 11c
(1.25 g, 5 mmol), 10b (1.50 g, 5.5 mmol), and DIPEA (1.05 mL, 6
mmol) in ethanol (20 mL) were stirred at 120 °C for 30 min
(microwave). After cooling to room temperature, hydrazine hydrate
(1.1 mL, 20 mmol) was added and the mixture stirred at 150 °C for 30
min (microwave). Upon cooling, a precipitate formed, which was
collected, washed with ethanol, and dried in vacuo to give 15 (1.67 g,
3.6 mmol, 71% yield). 1H NMR (DMSO-d6): δ 13.29 (s, 1H), 9.34 (s,
1H), 8.71 (d, J = 4.1 Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.91 (ddd, J =
7.8, 7.8, 1.8 Hz, 1H), 7.51 (s, 1H), 7.43−7.37 (m, 2H), 3.71−3.67 (m,
1H), 3.34−3.29 (m, 2H), 3.18 (d, J = 2.7 Hz, 1H), 2.73−2.64 (m, 2H),
1.98−1.91 (m, 4H), 1.42−1.35 (m, 4H). 13C NMR (DMSO-d6): δ
160.8, 158.2, 156.0, 152.7, 150.0, 144.3, 137.4, 125.4 (q, JCF = 129 Hz),
123.8, 120.1, 52.0, 49.0, 45.4, 33.1 (2C), 31.1 (2C), 29.0 (q, J = 30.0
Hz). MS (ES+) m/z 470.0 [M + H]+.
3,3,3-Trifluoro-N-(trans-4-((3-morpholino-1H-pyrazolo[3,4-d]-
pyrimidin-6-yl)amino)cyclohexyl)propane-1-sulfonamide 21. 21
was obtained by an analogous method to 1 (5.27 g, 11 mmol, 81%
yield). 1H NMR (DMSO-d6): δ 11.98 (bs, 1H), 8.82 (s, 1H), 7.35 (d, J
= 7.5 Hz, 1H), 7.19 (s, 1H), 3.76 (m, 4H), 3.62 (bs, 1H), 3.33−3.30 (m,
6H), 3.16 (bs, 1H), 2.72−2.62 (m, 2H), 1.93 (m, 4H), 1.40−1.28 (m,
4H). HRMS (ES+): m/z [M + H]+ calcd for C18H27F3N7O3S 478.1843,
found 478.1821.
(S)-(2,4-Dichloropyrimidin-5-yl)(2-methylmorpholino)-
methanone 26a. A solution of (S)-2-methylmorpholine (141 g, 1.39
mol) and TEA (141.1 g, 1.4 mol) in DCM (1 L) was added dropwise to
a stirred solution of 2,4-dichloropyrimidine-5-carbonyl chloride (268 g,
1.3 mol) in dry DCM (3000 mL) at 0 °C under N2. The reaction
mixture was stirred for 3 h at 0 °C and then quenched with H2O (500
mL). The phases were separated, and the aqueous phase was extracted
with DCM (2 × 300 mL). The combined organic phases were washed
with 0.5 M HCl (1 L) and brine (2 L), dried over Na2SO4, and then
evaporated under reduced pressure. The residue was purified by flash
column chromatography (petroleum/EtOAc 20/1 to 1/1) to give 26a
as a yellow oil (188 g, 684 mmol, 53% yield). 1H NMR (DMSO-d6): δ
8.86 (s, 1H), 4.33−4.25 (m, 1H), 3.95−3.68 (m, 1H), 3.58−3.37 (m,
3H), 3.17−2.64 (m, 2H), 1.20−0.98 (m, 3H). MS (ES+) m/z 276.0 [M
+ H]+.
(S)-(2-Chloro-4-methoxypyrimidin-5-yl)(2-methylmorpholino)-
methanone 27a. Sodium methoxide (40.5 g, 748.9 mmol) was added
in portions to a stirred solution of 26a (188 g, 680.9 mmol) in dry THF
(2 L) at −40 °C under N2. The reaction mixture was stirred for 1 h at
−40 °C, and further sodium methoxide (9.2 g, 170.2 mmol) was added
Q
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
in one portion. The reaction mixture was stirred at −40 °C for 1 h and
gradually warmed to −10 °C for 1 h. The reaction mixture was
quenched with satd aq soln ammonium chloride (500 mL) at 5−10 °C
and extracted with EtOAc (3 × 500 mL). The combined organic phases
were washed with brine (1 L), dried over Na2SO4, and evaporated
under reduced pressure. The resulting crude material was purified by
flash column chromatography (petroleum ether/EtOAc from 20/1 to
1/1) to give two fractions of 27a (75 g, 277 mmol, 38% yield and 30 g,
111 mmol, 14% yield) as a yellow oil. This material was used in the next
step without further purification.
3,3,3-Trifluoro-N-(trans-4-((4-methoxy-5-((S)-2-methylmorpho-
line-4-carbonyl)pyrimidin-2-yl)amino)cyclohexyl)propane-1-sulfo-
namide 28a. To a solution of 27a (75 g, 276 mmol) in dry dioxane
(1500 mL) was added DIPEA (178.4 g, 1.4 mol) and 10b (139.4 g,
358.8 mmol) in one portion at 27 °C. The resulting mixture was stirred
at 120 °C for 16 h. The mixture was cooled to room temperature and
water (800 mL) and EtOAc (500 mL) added. The reaction mixture was
stirred for 10 min, then the organic phase was separated and the
aqueous layer was extracted with more EtOAc (2 × 500 mL). The
combined organic phases were washed with brine (2 L), dried over
Na2SO4, and filtered. The solvent was removed under vacuum to afford
crude material which was purified by flash column chromatography
(DCM/MeOH 50/1 to 20/1) to afford 28a (100 g, 196 mmol, 71%
yield) as a pale solid. 1H NMR (DMSO-d6): δ 8.03 (s, 1H), 7.49−7.31
(m, 2H), 3.91−3.60 (m, 5H), 3.48−3.11 (m, 9H), 2.68−2.62 (m, 2H),
1.98−1.96 (m, 4H), 1.43−1.09 (m, 7H).
HRMS (ES+): m/z [M + H]+ calcd for C22H28F3N6O4S 529.1840,
found 529.1822.
N-(trans-4-((4-Methoxy-3-morpholino-1H-pyrazolo[3,4-d]-
pyrimidin-6-yl)amino)cyclohexyl)-2-methylpropane-1-sulfonamide
33. 44c (2.63 g, 4.71 mmol) was dissolved in methanolic HCl solution
(218 mL, 1.25 M) and stirred at 55 °C for 1 h. The mixture was
concentrated under vacuum and EtOAC added and washed with
saturated NaHCO3 solution. The organic phase was washed with brine,
dried over Na2SO4, and evaporated under vacuum to give crude
product which was purified by flash chromatography (DCM/EtOAc/
MeOH 60/30/10) to give 33 (1.82 g, 3.92 mmol, 83% yield). 1H NMR
(DMSO-d6): δ 11.98 (bs, 1H), 6.98 (d, J = 7.6 Hz, 1H), 6.91 (d, J = 7.4
Hz, 1H), 3.93 (s, 3H), 3.72−3.70 (m, 4H), 3.67−3.57 (m, 1H), 3.22−
3.20 (m, 4H), 3.10−3.02 (m, 1H), 2.90 (d, J = 6.4 Hz, 2H), 2.11−2.04
(m, 1H), 1.95−1.87 (m, 4H), 1.38−1.26 (m, 4H), 1.04−1.00 (d, J = 6.7
Hz, 6H). 13C NMR (DMSO-d6): δ 162.9, 160.6, 160.2, 151.7, 87.3, 66.3
(2C), 60.5, 53.8, 51.9, 49.9 (2C), 33.4 (2C), 31.1 (2C), 25.0, 22.8
(2C). HRMS (ES+): m/z [M + H]+ calcd for C20H34F3N7O4S
468.2387, found 468.2367.
N-(trans-4-((4-Methoxy-3-((R)-3-methylmorpholino)-1H-
pyrazolo[3,4-d]pyrimidin-6-yl)amino)cyclohexyl)-2-methylpro-
pane-1-sulfonamide 39. 39 was obtained by an analogous method to
1
33 (33g, 68.8 mmol, 78% yield). H NMR (DMSO-d6): δ 11.96 (bs,
1H), 6.98 (d, J = 7.4 Hz, 1H), 6.90 (d, J = 7.4 Hz,1H), 3.99−3.93 (m,
1H), 3.91 (s, 3H), 3.79−3.70 (m, 2H), 3.64−3.51 (m, 3H), 3.27−3.09
(m, 2H), 3.08−3.01 (m, 1H), 2.88 (d, J = 6.4 Hz, 2H), 2.06 (sept, J =
6.6 Hz, 1H), 1.93−1.85 (m, 4H), 1.36−1.26 (m, 4H), 1.04−0.99 (m,
9H). 13C NMR (DMSO-d6): δ 163.0, 160.4, 160.0, 150.8, 87.5, 71.0,
66.6, 60.5, 53.8, 51.9, 51.1, 49.0, 44.3, 33.3 (2C), 31.4 (2C), 26.8, 25.0,
22.8 (2C), 12.9. HRMS (ES+): m/z [M + H]+ calcd for C21H36N7O4S
482.2544, found 482.2523. Enantiopurity determined using a chiral
column: 250 mm Chiralpak IA, 150 mm × 4.6 mm 5 μm, heptane/
EtOH = 65/35 + 0.2% iso-propyl amine, 1.0 mL/min, 298 K, 254 nm.
First enantiomer (39, only peak detected) Rt = 8.5 min, second
enantiomer (compound 40, not detected) Rt = 15.5 min. ee measured:
100%.
3-Bromo-4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-
pyrazolo[3,4-d]pyrimidine 41a. A solution of 3-bromo-4,6-dichloro-
1H-pyrazolo[3,4-d]pyrimidine (40 g, 149 mmol), 3,4-dihydro-2H-
pyran (41 mL, 447 mmol), and p-toluenesulfonic acid monohydrate
(5.7 g, 30 mmol) in THF (650 mL) was stirred at 70 °C for 2 h. After
cooling, the solvent was evaporated, ether (200 mL) added, and the
resulting suspension stirred for 2 h at 40 °C, slowly cooled, and the
resulting solid collected, washed with ether, and dried to give 41a (41.5
g, 118 mmol, 79% yield). 1H NMR (CDCl3-d6): δ 6.00 (dd, J = 2.6 and
6.0 Hz, 1H), 4.18−4.10 (m, 1H), 3.84−3.76 (m, 1H), 2.62−2.49 (m,
1H), 2.20−2.13 (m, 1H), 1.86−1.63 (m, 4H). MS (ES+) m/z 349.0,
351.0, 353.0 [M + H]+.
3-Bromo-4,6-dichloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
pyrazolo[3,4-d]pyrimidine 41b. A solution of 3-bromo-4,6-dichloro-
1H-pyrazolo[3,4-d]pyrimidine (65.5 g, 244 mmol) in DCM (1.5 L) at 0
°C was added DIPEA (125 mL, 733 mmol) dropwise (30 min) and the
resulting solution stirred at 0 °C for 5 min. After this time, 2-
(trimethylsilyl)ethoxymethyl chloride (51.5 mL, 293 mmol) was added
dropwise (40 min) and stirred at 3−4 °C for 1.5 h. Water (1 L) was
added to the reaction and the phases separated. The organic layer was
washed with H2O (1 L), dried over Na2SO4, filtered, and concentrated
to give 41b as a brown solid which was used without purification (101 g,
245 mmol, quant yield). 1H NMR (DMSO-d6): δ 5.7 (s, 2H), 3.6 (dd, J
= 8.1, 8.1 Hz, 2H), 0.85 (dd, J = 8.1, 8.1 Hz, 2H), 0.95 (s, 9H). MS
(ES+) m/z 395.0, 397.0 [M + H]+.
3-Bromo-6-chloro-4-methoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-
pyrazolo[3,4-d]pyrimidine 42a. To 41a (35.8 g, 102 mmol) in MeOH
(160 mL) was added a solution of sodium methoxide (5.5 g, 102 mmol)
in MeOH (500 mL) dropwise over 1 h and the resulting suspension
stirred for 30 min. Solvent was evaporated, EtOAc added and washed
with satd aq NaHCO3 then dried over sodium sulfate and solvent
evaporated to give 42a which was used without purification (35.5 g). 1H
NMR (DMSO-d6): δ 5.84 (dd, J = 2.5 and 5.8 Hz, 1H), 4.14 (s, 3H),
3.99−3.93 (m, 1H), 3.73−3.68 (m, 1H), 2.35−2.26 (m, 1H), 2.03−
3,3,3-Trifluoro-N-(trans-4-((4-methoxy-5-((S)-2-methylmorpho-
line-4-carbonothioyl)pyrimidin-2-yl)amino)cyclohexyl)propane-1-
sulfonamide 29a. 28a (117 g, 230 mmol) was dissolved in dry THF
(1700 mL). Lawesson’s reagent (162.5 g, 401.8 mmol) was added in
portions, and the resulting mixture was stirred for 2 h at 45 °C. The
reaction mixture was cooled to room temperature and quenched by
addition of satd aq NaHCO3 (800 mL). The reaction mixture was
stirred for 10 min at RT and then extracted with EtOAc (3 × 500 mL).
The combined organic phases were washed with brine (1 L) and dried
over Na2SO4. The solvent was removed under vacuum to afford a crude
product which was purified by flash column chromatography (DCM/
MeOH 100/1 to 20/1) to afford 29a (91 g, 173 mmol, 74% yield) as a
1
green solid. H NMR (DMSO-d6): δ 8.11−8.04 (m, 1H), 7.48−7.31
(m, 2H), 5.34−5.11 m, 1H), 3.96−2.84 (m, 13H), 2.73−2.59 (m, 2H),
2.00−1.78 (m, 4H), 1.44−1.27 (s, 4H), 1.21−1.02 (m, 3H).
2-Methyl-N-(trans-4-((3-morpholino-1H-pyrazolo[3,4-d]-
pyrimidin-6-yl)amino)cyclohexyl)propane-1-sulfonamide 30. 30
was obtained by an analogous method to 1(4g, 9.1 mmol, 34%
1
yield). H NMR (DMSO-d6): δ 12.04 (s, 1H), 8.86 (s, 1H), 7.22 (s,
1H), 7.07−7.00 (m, 1H), 3.85−3.74 (m, 4H), 3.73−3.62 (m, 1H),
3.41−3.31 (m, 4H), 3.18−3.09 (m, 1H), 2.99−2.90 (m, 2H), 2.19−
2.10 (m, 1H), 2.03−1.92 (m, 4H), 1.43−1.33 (m, 4H), 1.08 (d, J = 5.8
Hz, 6H). 13C NMR (DMSO-d6): δ 160.5, 157.3, 154.0, 151.5, 66.2
(2C), 60.5, 51.9, 48.9 (2C), 48.7, 33.3 (2C), 31.3 (2C), 25.0, 22.8
(2C). HRMS (ES+): m/z [M + H]+ calcd for C19H32N7O3S 438.2282,
found 438.2262.
3,3,3-Trifluoro-N-(trans-4-((4-methoxy-3-(2-methoxyphenyl)-1H-
pyrazolo[3,4-d]pyrimidin-6-yl)amino)cyclohexyl)propane-1-sulfo-
namide 31. To 44a (5.95 g, 9.7 mmol) was added 1.25 M HCl in
MeOH (450 mL) and the resulting mixture stirred at 60 °C for 30 min.
The reaction mixture was concentrated under vacuum to a final volume
of 100 mL, satd aq NaHCO3 (300 mL) added, and extracted with
EtOAc (400 mL). The aqueous layer was extracted twice with EtOAc,
and the combined organic layers dried over Na2SO4 and evaporated
under vacuum to obtain crude material which was purified by flash
column chromatography (10−90% EtOAc in cyclohexane) to give 31
(3.5 g, 9.7 mmol, 68% yield). 1H NMR (DMSO-d6): δ 12.88 (s, 1H),
7.29−7.43 (m, 3H), 7.09 (d, 1H), 6.94−7.03 (m, 2H), 3.83 (s, 3H),
3.62−3.75 (m, 4H), 3.26−3.31 (m, 2H), 3.17 (s, 1H), 2.59−2.73 (m,
2H), 1.87−2.01 (m, 4H), 1.27−1.44 (m, 4H). 13C NMR (DMSO-d6):
δ 164.1, 160.5, 159.7, 158.1, 142.0, 131.1, 130.2, 126.2 (q, JCF = 127
Hz), 123.5, 120.4, 111.8, 95.8, 55.7, 53.5, 52.0, 49.0, 45.5, 33.2 (2C),
31.3 (2C), 28.9 (q, JCF = 29.9 Hz). 19F NMR (DMSO-d6):δ −64.3.
R
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1.96 (m, 1H), 1.93−1.87 (m, 1H), 1.80−1.72 (m, 1H), 1.60−1.53 (m,
2H). MS (ES+) m/z 347, 349 [M + H]+.
(DMSO-d6): δ 7.18−7.09 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 5.55−5.49
(m, 1H), 4.00−3.91 (m, 4H), 3.78−3.64 (m, 5H), 3.58−3.50 (m, 1H),
3.30−3.22 (m, 4H), 3.11−3.05 (m, 1H), 2.91 (d, J = 6.4 Hz, 2H),
2.42−2.31 (m, 1H), 2.09 (sept, J = 6.7 Hz, 1H), 2.03−1.89 (m, 5H),
1.80−1.62 (m, 2H), 1.55−1.50 (m, 2H), 1.41−1.30 (m, 4H), 1.04 (d, J
= 6.7 Hz, 6H). MS (ES+) m/z 552.5 [M + H]+.
3-Bromo-6-chloro-4-methoxy-1-((2-(trimethylsilyl)ethoxy)-
methyl)-1H-pyrazolo[3,4-d]pyrimidine 42b. To 41b (772 g, 1.94
mol) in MeOH (20 L) was added sodium methoxide (115.2 g, 2.13
mol) in MeOH (230 mL) dropwise and the resulting solution stirred at
room temperature for 30 min. The reaction mixture was partitioned
between EtOAc (10 L) and brine (2 L) and the organic layer separated,
washed with brine (2 L), dried over Na2SO4, filtered, and concentrated.
This residue was purified by column chromatography on silica gel
(100−200 mesh silica gel) eluted with petroleum ether:EtOAc
(100:1−100:2) to give a crude product. This crude product was
purified by recrystallization from petroleum ether (1 L) to give 42b as a
(S)-N-trans-(4-((4-Methoxy-3-(3-methylmorpholino)-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-
amino)cyclohexyl)-2-methylpropane-1-sulfonamide 44e. A mixture
of 43d (35 g, 59.2 mmol) in 1,4-dioxane (1000 mL) was purged with
argon, (R)-3-methylmorpholine (59.8 g, 592 mmol), Tris-
(dibenzylideneacetone)dipalladium(0) (5.42 g, 5.92 mmol), and
dicyclohexyl(2′,6′-diisopropoxy-[1,1′-biphenyl]-2-yl)phosphine (5.52
g, 11.83 mmol) added and purged with argon. Potassium bis
(trimethylsilyl)amide (0.5 M in toluene, 237 mL, 118 mmol) was
added and purged and the mixture stirred at 110 °C for 2 h. The crude
mixture was cooled, filtered through Celite, partitioned between ethyl
acetate (500 mL) and brine (1 L), the phases separated, and the organic
phase washed with further brine (1 L). The combined aqueous layers
were extracted with ethyl acetate (1 L) and the organic layers
combined, dried over Na2SO4, filtered, and concentrated. Crude
material was purified by flash chromatography (cyclohexane/EtOAc
gradient 0−30%) to give 44e as a brown solid (16.91 g, 27.6 mmol,
1
white solid (450 g, 1.14 mol, 58.8% yield). H NMR (DMSO-d6): δ
5.62 (s, 2H), 4.14 (s, 3H), 3.59 (t, J = 8.2 Hz, 2H), 0.84 (t, J = 8.1 Hz,
2H), 0.07 (s, 9H). MS (ES+) m/z 392.9. 395.0 [M + H]+.
N-(trans-4-((3-Bromo-4-methoxy-1-(tetrahydro-2H-pyran-2-yl)-
1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)cyclohexyl)-2-methylpro-
pane-1-sulfonamide 43a. To a stirred solution of 42a (7.82 g, 22.44
mmol) and 10a (6 g, 17.26 mmol) in ethanol (172 mL) was added
DIPEA (12 mL, 69.04 mmol) and the mixture heated to 70 °C and
stirred for 2 days. Solvent was evaporated and the crude material
purified by flash chromatography (30−50% ethyl acetate/cyclohexane)
to afford 43a (5.5 g, 10.1 mmol, 58% yield). 1H NMR (DMSO-d6): δ
7.52 (dd, J = 9.5, 17.0 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 5.65−5.55 (m,
1H), 4.05−3.96 (m, 2H), 3.67 (s, 2H), 3.33 (s, 3H), 2.92−2.89 (m,
2H), 2.36−2.27 (m, 1H), 2.13−2.06 (m, 1H), 1.94 (s, 3H), 1.83 (dd, J
= 13.2, 13.2 Hz, 1H), 1.54 (s, 2H), 1.36 (d, J = 18.6 Hz, 6H), 1.05−1.01
(m, 6H). MS (ES+) m/z 547.3, 545.3 [M + H]+.
3,3,3-Trifluoro-N-(trans-4-((4-methoxy-3-(2-methoxyphenyl)-1-
((2-(trimethylsilyl) ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)amino)cyclohexyl)propane-1-sulfonamide 44a. To 43b (3.9 g,
6.6 mmol) and 2-methoxyphenylboronic acid (1.37 g, 8.9 mmol) was
added [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichlor-
ide (483 mg, 0.66 mmol), sodium carbonate (2.1 g, 39.6 mmol), DME
(160 mL), and H2O (80 mL) under argon. The resulting mixture was
stirred at 110 °C for 30 min. The reaction mixture was filtered then
concentrated under vacuum. The residue was taken up in EtOAc and
washed with a satd aq soln NH4CI. The layers were separated and the
organic phase washed with brine, dried over Na2SO4, and concentrated.
The crude material was purified by flash column chromatography (6:4
cyclohexane in EtOAc) to afford 44a which was used without further
purification (4.04 g, 6.6 mmol, quant yield). MS (ES+) m/z 613.3 [M +
H]+.
3,3,3-Trifluoro-N-(trans-4-((4-methoxy-3-morpholino-1-(tetrahy-
dro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-
cyclohexyl)propane-1-sulfonamide 44b. A mixture of 43b (0.25 g,
0.43 mmol), morpholine (0.186 mL, 2.14 mmol), cesium carbonate
(0.39 g, 1.20 mmol), 4.5-bis(diphenylphospino)-9.9-dimethylxanthene
(0.03 g, 0.05 mmol), and palladium acetate (9.6 mg, 0.043 mmol) in
1,4-dioxane (4.08 mL) was purged with argon and heated at 110 °C
overnight. The resulting mixture was partitioned between ethyl acetate
(75 mL) and water (150 mL). The phases were separated and the
aqueous phase extracted with ethyl acetate (1 × 75 mL). The organic
phases were combined with two further additional experiments, dried
with Na2SO4, filtered, and concentrated to give 44b as a brown oil (250
mg, 0.42 mmol, 45% overall yield from all three experiments) which was
used without purification. MS (ES+) m/z 592.2 [M + H]+.
1
47%). H NMR (DMSO-d6): δ 7.20−7.10 (m, 1H), 7.04−6.96 (m,
1H), 5.35−5.25 (m, 2H), 4.05−3.90 (m, 4H), 3.82−3.63 (m, 3H),
3.61−3.49 (m, 3H), 3.26−3.15 (m, 2H), 3.09−3.03 (m, 1H), 2.89 (d, J
= 6.4 Hz, 2H), 2.07 (sept, J = 6.7 Hz, 1H), 1.95−1.86 (m, 3H), 1.38 (s,
3H), 1.36−1.28 (m, 3H), 1.04 (d, J = 6.6 Hz, 3H), 1.00 (d, J = 6.7 Hz,
6H), 0.84−0.76 (m, 2H), −0.10 (s, 9H). MS (ES+) m/z 612.3 [M +
H]+.
Intramacrophage Leishmania donovani Assay. This assay was
conducted as previously described.14
Kinetic Aqueous Solubility Assessment.23 The aqueous
solubility of test compounds were measured using an in-house method
utilizing quantification via chemiluminescent nitrogen detection
(CLND): a 5 μL of 10 mM DMSO stock solution was diluted to 100
μL with pH 7.4 phosphate buffered saline and equilibrated for 1 h at RT,
filtered through Millipore Multiscreen HTS-PCF filter plates (MSSL
BPC). The eluent is quantified by suitably calibrated flow injection
CLND. This assay has a dynamic range between the lower detection
limit of 1 and 500 μM, governed by the protocol’s 1:20 dilution into pH
7.4 phosphate buffer solution from nominal 10 mM DMSO stock.
Solubility of Solid Compounds in Fasted Simulated
Intestinal Fluid.32 Solubility of test compounds in fasted simulated
intestinal fluid (FaSSIF) were determined at pH 6.5 after 4 h
equilibration at room temperature. One mL of FaSSIF buffer (3 mM
sodium taurocholate, 0.75 mM lecithin in sodium phosphate buffer at
pH 6.5) was added to manually weighed 1 mg of solid compound in a 2
mL HPLC autosampler vial. The resulting suspension is shaken at 900
rpm for 4 h at room temperature and then transferred to a Multiscreen
HTS, 96-well solubility filter plate. The residual solid was removed by
filtration. The supernatant solution was quantified by HPLC-UV using
single-point calibration of a known concentration of the compound in
DMSO. The dynamic range of the assay was 1−1000 μg/mL.
Measurement of ChromLogD.23,31 The chromatographic hydro-
phobicity index (CHI)45 values are measured using reversed phase
HPLC column (50 mm × 2 mm 3 μM Gemini NX C18, Phenomenex,
UK) with fast acetonitrile gradient at starting mobile phase of pHs 2,
7.4, and 10.5. CHI values are derived directly from the gradient
retention times by using a calibration line obtained for standard
compounds. The CHI value approximated the volume% organic
concentration when the compound was eluted. CHI is linearly
transformed into ChromLogD23,31 by least-squares fitting of exper-
imental CHI values to calculated ClogP values for over 20K research
compounds using the following formula: ChromlogD = 0.0857 × CHI-
2.00.
N-(trans-4-((4-Methoxy-3-morpholino-1-(tetrahydro-2H-pyran-
2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)cyclohexyl)-2-meth-
ylpropane-1-sulfonamide 44c. The reaction was run in two batches:
To a mixture of 43a (2.6 g, 4.8 mmol), sodium tert-butoxide (1.4 g, 14.3
mmol), xantphos (0.66 g, 1.14 mmol), and Pd2 dba3 (0.436 g, 0.48
mmol) in dioxane (160 mL) was added morpholine (4.16 mL, 47.6
mmol) and the mixture heated at 110 °C for 30 min. The reaction
mixture was filtered through Celite, then diluted with ethyl acetate and
washed with satd NH4Cl. The aqueous phase was extracted with ethyl
acetate, washed with brine, dried over Na2SO4, filtered, and evaporated.
The crude material from both batches was combined and purified by
flash chromatography (50−80% EtOAc/cyclohexane) to give 44c (2.6
Stability in Microsomes. Test compound (0.5 μM) was incubated
with either female CD1 mouse (Xenotech) or human (IVT) liver
microsomes and their action started with addition of excess NADPH (8
mg/mL 50 mM potassium phosphate buffer, pH 7.4). Immediately, at
1
g, 4.7 mmol, 49% overall yield from both experiments). H NMR
S
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
time zero, then at 3, 6, 9, 15, and 30 min, an aliquot (50 μL) of the
incubation mixture was removed and mixed with acetonitrile (100 μL)
to stop the reaction. Internal standard was added to all samples, the
samples centrifuged to sediment precipitated protein, and the plates
then sealed prior to UPLC-MS/MS analysis using a Quattro Premier
XE (Waters Corporation, USA). XLfit (IDBS, UK) was used to
calculate the exponential decay and consequently the rate constant (k)
from the ratio of the peak area of test compound to internal standard at
each time point. The rate of intrinsic clearance (Cli) of each test
compound was then calculated using the equation Cli (mL/min/g
liver) = k × V × microsomal protein yield, where V (mL/mg protein) is
the incubation volume/mg protein added and microsomal protein yield
is taken as 52.5 mg protein/g liver. Verapamil (0.5 μM) was used as a
positive control to confirm acceptable assay performance.
Plasma Protein Binding Experiments. In brief, a 96-well
equilibrium dialysis apparatus was used to determine the free fraction
in mouse plasma for each compound (HT Dialysis LLC, Gales Ferry,
CT). Membranes (12−14 kDa A cutoff) were conditioned in deionized
water for 60 min, followed by conditioning in 80:20 deionized
water:ethanol for 20 min and then rinsed in isotonic buffer before use.
Female CD1 mouse plasma was removed from the freezer and allowed
to thaw on the day of experiment. Thawed plasma was then centrifuged
(Allegra X12-R, Beckman Coulter, USA), spiked with test compound
(final concentration 10 μg/mL), and 150 μL aliquots (n = 6 replicate
determinations) loaded into the 96-well equilibrium dialysis plate.
Dialysis vs isotonic buffer (150 μL) was carried out for 5 h in a
temperature controlled incubator at ca. 37 °C (Barworld scientific Ltd.,
UK) using an orbital microplate shaker at 100 rpm (Barworld scientific
Ltd., UK). At the end of the incubation period, 50 μL aliquots of plasma
or buffer were transferred to micronic tubes (Micronic BV, The
Netherlands) and the composition in each tube balanced with control
fluid (50 μL), such that the volume of buffer to plasma was the same.
Sample extraction was performed by the addition of 200 μL of
acetonitrile containing an appropriate internal standard. Samples were
allowed to mix for 1 min and then centrifuged at 3000 rpm in 96-well
blocks for 15 min (Allegra X12-R, Beckman Coulter, USA), after which
150 μL of supernatant was removed to 50 μL of water. All samples were
analyzed by UPLC-MS/MS on a Quattro Premier XE mass
spectrometer (Waters Corporation, USA). The unbound fraction was
determined as the ratio of the peak area in buffer to that in plasma.
Melting Point (mtp) Determination. Determination of melting
point of compounds were measured by thermal analysis, performing
differential scanning calorimetry, (DSC) experiments, which measured
temperatures and heat flows associated with thermal transitions in a
material. The equipment utilized was from TA Instruments (Q20, RCS
Refrigerator), using the system Software Q Advantage for Q Series and
Analysis Software TA universal analysis.
measured curve by manipulating the pKa. The pKa that provided the
best fit was taken to be the measured pKa.
Usually 0.5−1 mg of solid material is required for the measurements.
When the compound precipitated at some point during the pH
titration, the cosolvent method using methanol was applied using
various concentration of cosolvent. The pKa in water was calculated
using the Yasuda−Shedlovsky extrapolation method.
X-ray Diffraction Studies of 1 and 15. Data for both studies were
collected with an Oxford Diffraction Gemini A Ultra diffractometer at
150(2) K using Cu Kα X-radiation (λ = 1.54178 Å).
Crystal Data and Refinement Summary for 1. C19H28F3N7O3S; M
= 491.54; colorless tablet from the slow evaporation of a solution of 1 in
DMSO and 1-butanol; 0.32 mm × 0.15 mm × 0.07 mm; triclinic; space
group P1 (#1); a = 8.68517(18) Å, b = 16.0121(3) Å, c = 17.5996(4) Å,
α = 95.8623(16)°, β = 102.6295(18)°, γ = 100.2962(16)°, V =
2324.30(8) Å3; Z = 4; Dcalc = 1.405 Mgm−3; θmax = 67.03°; reflections
collected = 33974; independent reflections = 33974; Rint = 0.0000
(HKLF 5 treatment owing to crystal splitting); coverage = 99.2%;
restraints = 896; parameters = 1395; S = 1.041; R1 [I > 2σ(I)] = 0.0526;
wR2 (all data) = 0.1479; absolute structure parameter = 0.022(9); and
largest difference peak and hole = 0.572 and 0.322 eÅ−3.
Crystal Data and Refinement Summary for 15. C19H22F3N7O2S;
M = 469.50; colorless block from the slow evaporation and seeding of a
solution of 15 in DMSO and DMF; 0.21 mm × 0.14 mm × 0.06 mm;
triclinic; space group P1 (#2); a = 8.5590(2) Å, b = 8.6492(2) Å, c =
14.0372(4) Å, α = 85.196(2)°, β = 89.219(2)°, γ = 85.087(2)°, V =
1031.68(4) Å3; Z = 2; Dcalc = 1.511 Mgm−3; θmax = 66.74°; reflections
collected = 22081; independent reflections = 3644; Rint = 0.0351;
coverage = 99.4%; restraints = 0; parameters = 301; S = 1.048; R1 [I >
2σ(I)] = 0.0346; wR2 (all data) = 0.0956; and largest difference peak
and hole = 0.253 and 0.454 eÅ−3.
A description of the refinements and the full tables associated with
the crystal structures are given in the Supporting Information.
Crystallographic information files have been deposited with the
Cambridge Crystallographic Data Centre. CCDC 1857125 (1) and
1857128 (15) contain the supplementary crystallographic data for this
paper. The data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures.
In Vivo Pharmacokinetics. Mouse. Test compound 15 was dosed
intravenously or orally by gavage as a solution at 3 or 10 mg free base/kg
respectively (dose volume, 5 or 10 mL/kg respectively; dose vehicle,
10% (v/v) dimethyl sulfoxide (DMSO), 60% polyethylene glycol 400,
and 30% deionized water) to female Balb/c mice (n = 3). Blood samples
were taken from each mouse at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post
dose and mixed with two volumes of distilled water. After suitable
sample preparation, the concentration of test compound in blood was
determined by UPLC-MS/MS using a Quattro Premier XE (Waters,
USA). Pharmacokinetic parameters were derived from the mean blood
concentration time curve using PK solutions software v 2.0 (Summit
Research Services, USA).
As general operating conditions, the following were set up: start
temperature 25 °C, end temperature 350 °C, heating rate 10 °C/min.
The sample quantity ranged from 1 to 3 mg, placed in a hermetic pan.
The corresponding analysis with the thermal transitions occurred was
obtained based on a reference blank sample (empty pan) to get the
difference between the sample and the reference in the energy response
when heating equally both pans.
Rat. Male Sprague−Dawley rats (n = 3) were dosed with 1, 30, 31,
33, and 39 either intravenously at a target dose of 1 mg/kg or orally at a
target dose of 10, 100, and 300 mg/kg.
pKa Determination.35 The Sirius T3 (Sirius Analytical Inc., UK)
instrument has been used for pKa determination of the compounds. The
pKa determination is based on acid−base titration, and the
protonation/deprotonation of the molecule is measured either by UV
spectroscopy or potentiometrically. The pKa value is calculated from
the pH, where the 50−50% of the protonated and unprotonated form of
the molecules are present.
Following discrete intravenous tail vein dosing from a solution of 5%
DMSO:20% Encapsin in saline, serial blood samples (∼200 μL) were
taken via lateral tail vein at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 24 h post dose
and placed into individual tubes containing potassium EDTA and
thoroughly mixed. Blood samples were placed on ice immediately after
collection. Within 2 h of collection, 70 μL of blood sample was
transferred to a tube containing 130 μL of 0.1N Hepes buffer. Following
dilution, samples were stored at approximately −20 °C or below until
analysis.
Following discrete oral gavage dosing from a suspension of 1%
methyl cellulose at target doses of 10, 100, and 300 mg/kg, respectively,
serial blood samples (∼200 μL) were taken via lateral tail vein at 0.25,
0.5, 0.75, 1, 2, 4, 6, 8, and 24 h post dose and placed into individual tubes
containing potassium EDTA and thoroughly mixed. Blood samples
were placed on ice immediately after collection. Within 2 h of
collection, 70 μL of blood sample was transferred to a tube containing
The UV-metric method provides pKa results for samples with
chromophores whose UV absorbance changes as a function of pH. It
typically required 5 μL of a 10 mM solution of the samples, and the UV
absorbance was monitored over 54 pH values in a buffered solution in
about 5 min.
When the ionization center was far from the UV, the chromophore
pH-metric method based on potentiometric acid−base titration was
used. The pH of each point in the titration curve was calculated using
equations that contain pKa, and the calculated points were fitted to the
T
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
130 μL of 0.1N Hepes buffer. Following dilution, samples were stored
at approximately −20 °C or below until analysis.
Mouse Lymphoma Assay (MLA). The standard protocol was used
for this assay.41
Human Ether-a-go-go Related Gene (hERG). The standard
Samples were analyzed for parent using a method based on protein
precipitation followed by LCMS/MS with a quantitative range between
2.90 and 2860 ng/mL.
protocol was used for this assay.39,40
Rat 7-Day Toxicology. Test compounds were administered to male
Crl:WI(Han) rats in a 7 day, oral gavage, repeat-dose study at doses of
100, 300, and 1000 mg/kg/day.
Rat Cardiovascular Study. Conscious unrestrained telemetered
male rats (CRL:WI (Han) were given a single oral dose of either vehicle
or test article (up to 1000 mg/kg) cardiovascular and electrocardio-
graphic parameters, and body temperature were monitored for 2 h prior
to and for 24 h after dose.
Ethical Statements. Mouse and Rat Pharmacokinetics. All
animal studies were ethically reviewed and carried out in accordance
with Animals (Scientific Procedures) Act 1986 and the GSK/Dundee
University Policy on the Care, Welfare, and Treatment of Animals
In Vivo Efficacy. All regulated procedures, at the University of
Dundee, on living animals was carried out under the authority of a
project license issued by the Home Office under the Animals (Scientific
Procedures) Act 1986, as amended in 2012 (and in compliance with EU
Directive EU/2010/63). License applications will have been approved
by the University’s Ethical Review Committee (ERC) before
submission to the Home Office. The ERC has a general remit to
develop and oversee policy on all aspects of the use of animals on
University premises and is a subcommittee of the University Court, its
highest governing body.
Once analyzed, if concentrations were above the dynamic range of
the standard curve, prediluted samples were diluted with blank matrix
(1:2.86 whole blood:0.1N Hepes buffer) and processed normally for
analysis.
Data Analysis. Data analysis and calculation of pharmacokinetic
parameters was performed using Phoenix, WinNonLin version 6.3.
Following the intravenous administration, the whole blood clearance
(Cl) was calculated by determining the dose administered to each
animal and dividing by the AUC0−∞. The estimate of the volume of
distribution at steady state (Vss) was calculated as MRT × Cl, where
MRT is the mean residence time, calculated by AUMC0−∞/AUC0−∞
For both intravenous and oral administration, the systemic exposures
were determined by calculating the area under the blood concen-
tration−time curve (AUC) from the start of dosing to the last observed
quantifiable concentration (AUC0‑t) by using the linear up-log down
trapezoidal rule. The slope of the terminal elimination phase was
estimated by linear regression of the terminal data points (minimum 3
points) from a natural log concentration vs time plot of the data. The
half-life (t1/2) of the terminal elimination phase was calculated as t1/2
0.693/k (where k is the elimination rate constant).
Graphs were generated using Microsoft Excel.
=
Rat Toxicology Studies. All animal studies were ethically reviewed
and carried out in accordance with Animals (Scientific Procedures) Act
1986 and the GSK Policy on the Care, Welfare, and Treatment of
Animals
Human Biological Samples. All were sourced ethically and their
research use was in accord with the terms of the informed consents.
In Vivo Efficacy Studies. Groups of female BALB/c mice (5 per
group) were inoculated intravenously with approximately 2 × 107 L.
donovani amastigotes (LV9; WHO designation: MHOM/ET/67/
HU3) harvested from the spleen of an infected hamster.46 From day 7
postinfection, groups of mice were treated with either drug vehicle only
(orally), with pentostam (15 mg/kg subcutaneously) with miltefosine
(12 or 30 mg/kg orally), with compound 15 (50 mg/kg orally), with
compound 30 (10, 25, 50, and 100 mg/kg orally), with compound 37
(25 mg/kg orally), or with compounds 1, 31, and 39 (10 and 25 mg/kg
orally). Miltefosine was dosed once daily for 5 or 10 days, compound 15
was dosed twice daily for 5 days, compound 30 was dosed twice daily for
5 and 10 days, and compounds 1, 31, 37, and 39 were dosed twice daily
for 10 days. Drug dosing solutions were prepared fresh each day and the
vehicle was deionized water for miltefosine and pentostam, 10% (v/v)
dimethyl sulfoxide (DMSO), 60% polyethylene glycol 400, and 30%
deionized water for 15 and 0.5% HPMC, 0.4% Tween 80, and 0.5%
benzyl alcohol for 1, 31, 37, and 39. On day 14 (5 days dosing groups)
or 19 (10 days dosing groups) all animals were humanely euthanized,
liver smears made, and parasite burdens determined by counting the
number of amastigotes/500 liver cells. Parasite burden is expressed in
Leishman−Donovan units (LDU): the number of amastigotes per 500
nucleated cells multiplied by the organ weight in grammes.30
Preclinical Safety Studies. Cytochrome P450 Enzyme Isoform
3A4. CYP3A4 inhibition and metabolism dependent inhibition (MDI)
was assessed using a marker substrate (midazolam), monitoring
production of CYP3A4 specific metabolite in the presence and absence
of test compound at eight concentrations up to 100 μM (0.1 to 100 μM,
final organic content ≤0.5%). Known inhibitors of CYP3A4 were
included as positive control (troleandomycin, ketoconazole, and
erythromycin). Data quoted as IC50 and fold shift in IC50 (MDI).
Incubation mixture containing pooled human liver microsomes
(final concentration 0.1 mg/mL), and either substrate, standard
inhibitor, or test compound was preincubated at 37 °C for 20 min.
Either substrate or cofactor was then added and incubated in plate
shaker for another 5 min at 37 °C. Ice-cold acetonitrile 2:1 (v/v) was
added to terminate reaction. Depletion of substrates was measured by
LC-MS/MS, and pIC50 was measured. If a greater than 1.5-fold change
in pIC50 was observed upon preincubation with cofactor values
compared to preincubation with substrate values, this suggests that the
test compound is a metabolism dependent inhibitor.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b01218.
Chemistry experimental; crystallography; supplementary
data table; synthetic details for all compounds (PDF)
Experimental set up and supplementary tables (XLS)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*For T.J.M.: phone, +34 650 349771; E-mail, tim.j.miles@gsk.
com.
■
*For K.D.R.: phone, +44 1382 388 688; E-mail, k.read@dundee.
ac.uk.
*For P.G.W.: phone, +44 1382 386 231; E-mail, p.g.wyatt@
dundee.ac.uk.
ORCID
Manu De Rycker: 0000-0002-3171-3519
Ian H. Gilbert: 0000-0002-5238-1314
Paul G. Wyatt: 0000-0002-0397-245X
Kevin D. Read: 0000-0002-8536-0130
Timothy J. Miles: 0000-0001-7407-7404
Notes
The authors declare the following competing financial
interest(s): The following authors have shares in GlaxoSmithK-
line: M.Bo., R.C.B.C., S.D.C., A.D., G.D., S.F., S.G.-D., S.G.,
A.P.H., S.J.H., R.L., F.M., V.L.N., I.H.R., T.U., D.G., J.M.F.,
P.G.W., K.D.R., and T.J.M. The other authors declare no
competing interests.
Chemoproteomic Profiling on Kinobeads. This assay was
conducted as previously described.17
Ames. The standard protocol was used for this assay.38
U
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(14) De Rycker, M.; Hallyburton, I.; Thomas, J.; Campbell, L.; Wyllie,
S.; Joshi, D.; Cameron, S.; Gilbert, I. H.; Wyatt, P. G.; Frearson, J. A.;
Fairlamb, A. H.; Gray, D. W. Comparison of a high-throughput high-
content intracellular leishmania donovani assay with an axenic
amastigote assay. Antimicrob. Agents Chemother. 2013, 57 (7), 2913−
2922.
(15) Don, R.; Ioset, J.-R. Screening strategies to identify new chemical
diversity for drug development to treat kinetoplastid infections.
Parasitology 2014, 141 (1), 140−146.
(16) Miles, T. J.; Thomas, M. G. Pyrazolo[3,4-d]pyrimidin Derivative
and its use for the Treatment of Leishmaniasis. WO Patent
WO2016116563A1, 2016.
(17) Wyllie, S.; Thomas, M.; Patterson, S.; Crouch, S.; De Rycker, M.;
Lowe, R.; Gresham, S.; Urbaniak, M. D.; Otto, T. D.; Stojanovski, L.;
Simeons, F. R. C.; Manthri, S.; MacLean, L. M.; Zuccotto, F.; Homeyer,
N.; Pflaumer, H.; Boesche, M.; Sastry, L.; Connolly, P.; Albrecht, S.;
Berriman, M.; Drewes, G.; Gray, D. W.; Ghidelli-Disse, S.; Dixon, S.;
Fiandor, J. M.; Wyatt, P. G.; Ferguson, M. A. J.; Fairlamb, A. H.; Miles,
T. J.; Read, K. D.; Gilbert, I. H. Cyclin-dependent kinase 12 is a drug
target for visceral leishmaniasis. Nature 2018, 560 (7717), 192−197.
(18) Woodland, A.; Grimaldi, R.; Luksch, T.; Cleghorn, L. A. T.; Ojo,
K. K.; Van Voorhis, W. C.; Brenk, R.; Frearson, J. A.; Gilbert, I. H.;
Wyatt, P. G. From on-target to off-target activity: identification and
optimization of trypanosoma brucei GSK3 Inhibitors and their
characterisation as anti-trypanosoma brucei drug discovery lead
molecules. ChemMedChem 2013, 8 (7), 1127−1137.
(19) Ding, Q.; Jiang, N.; Roberts, J. L. Preparation of Pyrazolopyr-
imidines as Antitumor Agents. US Patent US20050277655A1, 2005.
(20) Merritt, C.; Silva, L. E.; Tanner, A. L.; Stuart, K.; Pollastri, M. P.
Kinases as druggable targets in trypanosomatid protozoan parasites.
Chem. Rev. 2014, 114 (22), 11280−11304.
(21) Dichiara, M.; Marrazzo, A.; Prezzavento, O.; Collina, S.;
Rescifina, A.; Amata, E. Repurposing of human kinase Inhibitors in
neglected protozoan diseases. ChemMedChem 2017, 12 (16), 1235−
1253.
ACKNOWLEDGMENTS
■
Funding for this work was provided by the Wellcome (nos.
092340 and 100476). We thank Gina MacKay and Daniel
Fletcher for performing HRMS analyses and for assistance with
performing other NMR and MS analyses, Irene Hallyburton for
performing the axenic assay, Raul Fernandez Velasco and the
rest of the Galchimia chemistry team plus Colin P. Leslie and the
rest of the Aptuit chemistry team for synthesizing key
compounds, Professor W. Clegg and Dr. R. W. Harrington
plus Dr. M. R. Probert for the GSK-funded crystal structures of 1
and 15, Alastair Pate and Francesco Gastaldello for data
management, and finally Dr. Robert J. Young for critical
evaluation of the manuscript.
ABBREVIATIONS USED
■
THP-1, human monocytic cell line derived from an acute
monocytic leukemia patient; HPMC, hydroxypropyl methyl-
cellulose; VL, visceral leishmaniasis; CRK-12, cdc2-related
kinase 12
REFERENCES
■
(1) World Health Organization (WHO) List of Neglected Tropical
Diseases; World Health Organization: Geneva, 2018; http://www.who.
int/neglected_diseases/diseases/en/ (accessed Aug 2, 2018).
(2) Field, M. C.; Horn, D.; Fairlamb, A. H.; Ferguson, M. A. J.; Gray,
D. W.; Read, K. D.; De Rycker, M.; Torrie, L. S.; Wyatt, P. G.; Wyllie, S.;
Gilbert, I. H. Anti-trypanosomatid drug discovery: an ongoing
challenge and a continuing need. Nat. Rev. Microbiol. 2017, 15 (4),
217−231.
(3) World Health Organization (WHO) Leishmaniasis Fact Sheet;
World Health Organization: Geneva, 2018; http://www.who.int/
mediacentre/factsheets/fs375/en/ (accessed Aug 2, 2018).
(4) Moore, E. M.; Lockwood, D. N. Treatment of visceral
leishmaniasis. J. Glob. Infect. Dis. 2010, 2 (2), 151−158.
(5) Sundar, S.; Singh, A.; Rai, M.; Prajapati, V. K.; Singh, A. K.; Ostyn,
B.; Boelaert, M.; Dujardin, J.-C.; Chakravarty, J. Efficacy of Miltefosine
in the Treatment of Visceral Leishmaniasis in India After a Decade of
Use. Clin. Infect. Dis. 2012, 55 (4), 543−550.
(6) den Boer, M. L.; Alvar, J.; Davidson, R. N.; Ritmeijer, K.;
Balasegaram, M. Developments in the treatment of visceral
leishmaniasis. Expert Opin. Emerging Drugs 2009, 14 (3), 395−410.
(7) Mueller, M.; Ritmeijer, K.; Balasegaram, M.; Koummuki, Y.;
Santana, M. R.; Davidson, R. Unresponsiveness to AmBisome in some
sudanese patients with kala-azar. Trans. R. Soc. Trop. Med. Hyg. 2007,
101 (1), 19−24.
(8) Drugs for Neglected Diseases initative (DNDi) Portfolio; Drugs for
Neglected Diseases initative (DNDi), June 2018; https://www.dndi.
org/diseases-projects/portfolio/ (accessed Oct 5, 2016).
(9) Drugs for Neglected Diseases initative (DNDi) Target Product Profile
for Visceral Leishmaniasis; Drugs for Neglected Diseases initative
(DNDi), 2016; https://www.dndi.org/diseases-projects/
leishmaniasis/tpp-vl/ (accessed Oct 5, 2016).
(22) Ferguson, F. M.; Gray, N. S. Kinase inhibitors: the road ahead.
Nat. Rev. Drug Discovery 2018, 17 (5), 353−377.
(23) Hill, A. P.; Young, R. J. Getting physical in drug discovery: a
contemporary perspective on solubility and hydrophobicity. Drug
Discovery Today 2010, 15 (15−16), 648−655.
(24) Gupta, S.; Nishi. Visceral leishmaniasis: experimental models for
drug discovery. Indian J. Med. Res. 2011, 133, 27−39.
(25) Seifert, K.; Croft, S. L. In vitro and in vivo interactions between
miltefosine and other antileishmanial drugs. Antimicrob. Agents
Chemother. 2006, 50 (1), 73−79.
(26) Escobar, P.; Yardley, V.; Croft, S. L. Activities of hexadecylphos-
phocholine (miltefosine), AmBisome, and sodium stibogluconate
(Pentostam) against leishmania donovani in immunodeficient scid
mice. Antimicrob. Agents Chemother. 2001, 45 (6), 1872−1875.
(27) Dorlo, T. P. C.; Huitema, A. D. R.; Beijnen, J. H.; de Vries, P. J.
Optimal dosing of miltefosine in children and adults with visceral
leishmaniasis. Antimicrob. Agents Chemother. 2012, 56 (7), 3864−3872.
(28) Dorlo, T. P. C.; Rijal, S.; Ostyn, B.; de Vries, P. J.; Singh, R.;
Bhattarai, N.; Uranw, S.; Dujardin, J.-C.; Boelaert, M.; Beijnen, J. H.;
Huitema, A. D. R. Failure of miltefosine in visceral leishmaniasis is
associated with low drug exposure. J. Infect. Dis. 2014, 210 (1), 146−
153.
(10) Freitas-Junior, L. H.; Chatelain, E.; Kim, H. A.; Siqueira-Neto, J.
L. Visceral leishmaniasis treatment: what do we have, what do we need
and how to deliver it? Int. J. Parasitol.: Drugs Drug Resist. 2012, 2, 11−
19.
(29) Wyllie, S.; Patterson, S.; Stojanovski, L.; Simeons, F. R.; Norval,
S.; Kime, R.; Read, K. D.; Fairlamb, A. H. The anti-trypanosome drug
fexinidazole shows potential for treating visceral leishmaniasis. Sci.
Transl. Med. 2012, 4 (119), 119re1.
(30) Bradley, D. J.; Kirkley, J. Regulation of leishmania populations
within the host. I. the variable course of leishmania donovani infections
in mice. Clin. Exp. Immunol. 1977, 30 (1), 119−129.
(31) Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. P. Getting
physical in drug discovery II: the impact of chromatographic
hydrophobicity measurements and aromaticity. Drug Discovery Today
2011, 16 (17−18), 822−830.
(11) Gilbert, I. H. Drug discovery for neglected diseases: molecular
target-based and phenotypic approaches. J. Med. Chem. 2013, 56 (20),
7719−7726.
(12) Palmer, M. J.; Wells, T. N. C.; Eds., Neglected Diseases and Drug
Discovery. [In: RSC Drug Discovery Ser., 2012; 14; 438]. Royal Society of
Chemistry: 2012; p 134−202.
(13) Harhay, M. O.; Olliaro, P. L.; Costa, D. L.; Costa, C. H. N. Urban
parasitology: visceral leishmaniasis in Brazil. Trends Parasitol. 2011, 27
(9), 403−409.
V
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(32) Klein, S. The use of biorelevant dissolution media to forecast the
in vivo performance of a drug. AAPS J. 2010, 12 (3), 397−406.
(33) Gilbert, I. H.; Thomas, M. G. Preparation of Antileishmanial
Pyrazolopyrimidines. WO Patent WO2016116752A1, 2016.
(34) Di, L.; Fish, P. V.; Mano, T. Bridging solubility between drug
discovery and development. Drug Discovery Today 2012, 17 (9−10),
486−495.
(35) The Sirius Gold Standard pKa Assays; Pion, 2018; http://www.
sirius-analytical.com/analytical-services/gold-standard-assays (ac-
cessed Aug 2, 2018).
(36) Jain, N.; Yalkowsky, S. H. Estimation of the aqueous solubility I:
application to organic nonelectrolytes. J. Pharm. Sci. 2001, 90 (2), 234−
252.
(37) Young, R. J. Physical properties in drug design. Topics in
Medicinal Chemistry; Tactics in Contemporary Drug Design, Springer:
Berlin, Heidelberg, 2014; Vol. 9, pp 1−68, DOI: 10.1007/
7355_2013_35.
(38) Ames, B. N.; McCann, J.; Yamasaki, E. Methods for detecting
carcinogens and mutagens with the salmonella/mammalian-microsome
mutagenicity test. Mutat. Res. 1975, 31 (6), 347−364.
(39) Tao, H.; Santa Ana, D.; Guia, A.; Huang, M.; Ligutti, J.; Walker,
G.; Sithiphong, K.; Chan, F.; Guoliang, T.; Zozulya, Z.; Saya, S.;
Phimmachack, R.; Sie, C.; Yuan, J.; Wu, L.; Xu, J.; Ghetti, A. Automated
tight seal electrophysiology for assessing the potential hERG liability of
pharmaceutical compounds. Assay Drug Dev. Technol. 2004, 2 (5),
497−506.
(40) Ly, J. Q.; Shyy, G.; Misner, D. L. Assessing hERG channel
inhibition using PatchXpress. JALA 2005, 10 (4), 225−230.
(41) Clive, D.; Johnson, K. O.; Spector, J. F.; Batson, A. G.; Brown, M.
M. Validation and characterization of the L5178Y/TK mouse
lymphoma mutagen assay system. Mutat. Res., Fundam. Mol. Mech.
Mutagen. 1979, 59 (1), 61−108.
(42) Liu, T.; Brown, B. S.; Wu, Y.; Antzelevitch, C.; Kowey, P. R.; Yan,
G.-X. Blinded validation of the isolated arterially perfused rabbit
ventricular wedge in preclinical assessment of drug-induced proar-
rhythmias. Heart Rhythm 2006, 3 (8), 948−956.
(43) Shah, R. R. Drug-induced QT interval shortening: potential
harbinger of proarrhythmia and regulatory perspectives. Br. J.
Pharmacol. 2010, 159 (1), 58−69.
(44) Alvar, J.; Velez, I. D.; Bern, C.; Herrero, M.; Desjeux, P.; Cano, J.;
Jannin, J.; den Boer, M. Leishmaniasis worldwide and global estimates
of its incidence. PLoS One 2012, 7 (5), No. e35671.
(45) Valko, K.; Bevan, C.; Reynolds, D. Chromatographic hydro-
phobicity index by fast-gradient RP-HPLC: a high-throughput
alternative to Log P/log D. Anal. Chem. 1997, 69 (11), 2022−2029.
(46) Wyllie, S.; Fairlamb, A. H. Refinement of techniques for the
propagation of leishmania donovani in hamsters. Acta Trop. 2006, 97
(3), 364−369.
(47) Sacks, D.; Sher, A. Evasion of innate immunity by parasitic
protozoa. Nat. Immunol. 2002, 3 (11), 1041−1047.
W
DOI: 10.1021/acs.jmedchem.8b01218
J. Med. Chem. XXXX, XXX, XXX−XXX