Full text of DOI:10.1039/d0md00353k

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RESEARCH ARTICLE
Collaborative virtual screening to elaborate an
imidazo[1,2-a]pyridine hit series for visceral
leishmaniasis†
Cite this: RSC Med. Chem., 2021, 12,
384
d
Yuichiro Akao,a Stacie Canan,‡b Yafeng Cao,c Kevin Condroski,§b Ola Engkvist,
Sachiko Itono,¶a Rina Kaki,e Chiaki Kimura,e Thierry Kogej,d Kazuya Nagaoka,f
Akira Naito,e Hiromi Nakai,e Garry Pairaudeau,g Constantin Radu,‖h Ieuan Roberts,g
Mitsuyuki Shimada,**a David Shum,h Nao-aki Watanabe,f Huanxu Xie,c
Shuji Yonezawa,e Osamu Yoshida,e Ryu Yoshida,e
i
Charles Mowbrayi and Benjamin Perry
*
An innovative pre-competitive virtual screening collaboration was engaged to validate and subsequently
explore an imidazo[1,2-a]pyridine screening hit for visceral leishmaniasis. In silico probing of five proprietary
pharmaceutical company libraries enabled rapid expansion of the hit chemotype, alleviating initial concerns
about the core chemical structure while simultaneously improving antiparasitic activity and selectivity index
relative to the background cell line. Subsequent hit optimization informed by the structure–activity
relationship enabled by this virtual screening allowed thorough investigation of the pharmacophore,
opening avenues for further improvement and optimization of the chemical series.
Received 16th October 2020,
Accepted 18th November 2020
DOI: 10.1039/d0md00353k
rsc.li/medchem
visceral leishmaniasis (VL) represents the greatest burden to
global health, with data from 2015 suggesting it is responsible
Introduction
Leishmaniasis is generally considered to be one of the most
neglected tropical diseases and has strong links with
poverty.1–3 It is a complex vector-borne disease caused by
more than 20 species of the protozoan genus Leishmania with
symptoms ranging from localized skin ulcers to a lethal
systemic disease. Of the four leishmaniasis syndromes,
for almost 1.4 million DALYs (disability adjusted life-years)
lost, representing almost 6% of all DALYs caused by diseases
on the World Health Organization′s list of neglected tropical
diseases (NTDs).4 Caused by infection with L. donovani and L.
infantum parasites transmitted by phlebotomine sand flies,
visceral leishmaniasis manifests in patients as swelling of
spleen and liver, renal dysfunction and, if left untreated,
death. Current estimates suggest an annual new infection rate
for VL of 50 000 to 90 000 cases, with most cases found in
eastern Africa, Brazil and India.2,5–8
Although treatments for VL exist, these medications are
rarely fit for purpose, with major issues linked to resistance,
geographic variability in efficacy, toxicity, availability and
affordability, and often long and painful treatment periods
requiring many weeks of hospitalization.9–13 As a result, the
past decade has seen increased efforts to develop new
treatments for Leishmania related syndromes such as VL, with
a primary focus on delivering pre-clinical candidates with
improved profiles (broader geographic utility, reduced or no
toxicity, oral dosing, up to 10 day treatment period).5 Target
product profiles for these next generation VL treatments have
been published and discussed widely.14,15
a Takeda Pharmaceutical Company Limited, 26-1 Muraoka-Higashi 2-chrome,
Fujisawa, Kanagawa 251-8555, Japan
b Celgene Corporation, Celgene Global Health, 10300 Campus Point Drive, San
Diego, California 92121, USA
c WuXi AppTec Company Ltd., 666 Gaoxin Road, East Lake High-Tech Development
Zone, Wuhan 430075, People's Republic of China
d AstraZeneca Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden
e Shionogi & Co., Ltd, 3-1-1, Futaba-cho, Toyonaka-shi, Osaka, Japan
f Eisai Co., Ltd, 1-3,Tokodai 5-chome, Tsukuba, Ibaraki 300-2635, Japan
g AstraZeneca, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK
h Institut Pasteur Korea, 16, Daewangpangyo-ro 712 beon-gil, Bundang-gu,
Seongnam-si, Gyeonggi-do, 13488 Republic of Korea
i Drugs for Neglected Diseases initiative, 15 Chemin Louis Dunant, Geneva 1202,
Switzerland. E-mail: bperry@dndi.org
† Electronic supplementary information (ESI) available: Synthetic chemistry, in
silico screening, and assay experimental details, including analytical data for all
compounds. See DOI: 10.1039/d0md00353k
‡ Current address: Elgia Therapeutics, Inc., San Diego, CA, USA.
§ Current address: Loxo Oncology at Lilly, Boulder CO, USA.
¶ Current address: Axcelead Drug Discovery Partners, Inc., Kanagawa, Japan.
‖ Current address: Molecular Devices, NYC, NY, USA.
A combination of target-based and phenotypic-based drug
discovery approaches have been applied extensively to
identify new chemical matter for anti-leishmanial discovery
projects, as well as the kinetoplastid field in general.9,16–18
** Current address: The University of Tokyo, Tokyo, Japan.
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Despite some excellent progress in target-based approaches,
these often suffer from poor translation into efficacy in
in vitro infection models,19 demonstrating the importance
not only of a relevant mechanism of action but also the
correct physicochemical properties to access the parasites
once intracellular infection has taken hold. The phenotypic
approach bypasses this issue by engaging cell-infection
assays as the primary screen, however, these approaches
often suffer from a combination of a low hit rate and
identification of a high percentage of unsuitable chemical
matter, with many hit lists comprising compounds rich in
an alternative to this, we engaged an innovative in silico
approach, termed the NTD drug discovery booster (“Booster”).
The Booster process engages collaborative rounds of virtual
ligand-based screening across multiple large, proprietary
pharmaceutical company databases to identify and test both
close analogues to the hit, as well as structurally
differentiated
compounds
fitting
the
same
base
pharmacophore. The hit structure (“Seed”) is shared with
scientists at each partner pharmaceutical company, in silico
similarity searches are performed probing the proprietary
collections of each partner, and up to 100 prospective
“similar” molecules per partner are screened against the
parasite(s) of interest. The definition of “similar” is
independently determined by each partner, and could be
focused on both SAR annotation as well as more general
pharmacophore-led exploration, and could involve the use of
both commercial and proprietary computational approaches.
Sharing the results of these experimental screens with the
providing partners, as well as anonymized sharing of the
structure of the most improved molecule from the entire
collaborative screening endeavor (“Improved Hit”), allows a
second, more refined iteration of in silico screening to be
performed, again providing approximately 100 molecules per
partner. Repetition of this iterative screening/sharing process
continues until improvements cease to be made, or until SAR
around a chemotype emerges.
The use of both orthogonal in silico techniques as well as
orthogonally differentiated libraries, previously demonstrated
to have minimal overlap,35 maximizes both the richness of
the SAR explored around the initial seed phenotype, and
increases the chances of identifying an interesting change of
chemotype with similar or improved anti-parasitic activity,
i.e. a scaffold hop. Furthermore, these newly identified
scaffold hops can be subsequently mined using the same
Booster approach, thus maximizing the value of the hits
coming from HTS.
PAINS,20
toxicophores
and
intractable
chemistries.
Nevertheless, these approaches have yielded an interesting
pipeline of novel potential treatments for VL, some of which
have progressed to the early stages of clinical or preclinical
evaluation.21 These include CRK12 inhibitor GSK3186899,22
the Leishmania proteasome inhibitors GSK3494245 and LXE-
408,23,24 as well as DNDI-6174, DNDI-6148 and DNDI-0690,
compounds identified via phenotypic drug discovery
efforts.25
Despite this promising situation, the reality of clinical-
level attrition in drug discovery R&D dictates that if the
global health community are to ensure delivery of new,
improved treatments to VL patients, further new chemical
entities (NCEs) capable of tackling Leishmania infections still
need to be identified and developed.26,27 Alongside the
traditional target and phenotypic approaches mentioned
above, recent reports have highlighted a number of efforts to
identify novel anti-leishmanial NCEs via repurposing of
existing drugs and clinical stage compounds,28 combined
scaffold and parasite hopping,29 the screening of natural
products,30 as well as investigation into host-mediated
treatments for Leishmania infections.31
We report herein on early discovery efforts around an anti-
leishmanial chemotype derived from the output of
a
kinetoplastid high throughput screening (HTS) campaign.
Since 2012 we (DNDi) have tested over 2.2 million
compounds in high throughput high content screening
against kinetoplastid parasites including Leishmania donovani
and Trypanosoma cruzi, the causative agent of Chagas
disease,32 searching for novel and attractive small molecules
as starting points for drug discovery campaigns.†† Seeking to
improve the efficiency and throughput of the triaging of
results from these phenotypic screening efforts led to the
creation of the NTD drug discovery booster, a pre-competitive
virtual screening model comprising key players from
pharmaceutical R&D.33
Results and discussion
Hit identification
Compound 1 was identified as a hit with anti-trypanosomal
activity from a high throughout screen against Trypanosoma
cruzi. Follow up confirmation of activity validated both the anti-
trypanosomal activity of 1 in a T. cruzi intracellular infection
assay (U2OS as host cell) as well as demonstrating the
compound's efficacy against Leishmania donovani in an
intracellular infection assay (THP1 as host cell) (Fig. 1). As
mentioned previously, we are wary of the high hit rate of
intractable, non-drug-like and/or PAINS-like compounds in the
kinetoplastid literature, and our initial analysis of the 1
NTD drug discovery booster
In the traditional approach to initial exploration of chemical
chemotype flagged up the 2-pyridyl
3 amino substituted
space around
a new HTS hit, commercial compound
imidazo[1,2-a]pyridine core as
a
PAINS motif.20 Further
catalogues are probed for similar molecules and small sets of
closely designed analogues are synthesized for testing.34 As
investigation of the literature around 1 identified earlier reports
about a related structure with moderate potency against the
related leishmania parasite L. major, one of the causative
parasitic agents of cutaneous leishmaniasis.36 In this report the
†† Compound libraries for HTS efforts at DNDi were sourced from
a
combination of commercial and proprietary collections.
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Fig. 1 Overview of Booster in silico screening process; first (A) and second (B) round of virtual screening result in data set of compounds with
annotated SAR around 1; data set is further annotated via expansive analoguing (C).
Table 1 Overview of the in silico approach taken by each partner company, along with the number of compounds furnished from each partner
Number of compounds (actives)a
Company and computational approach
On scaffoldb
Scaffold hopc
15 (3)
A- ECFP4 (ref. 40) similarity (initial Tanimoto cut-off 0.7
descending incrementally until sufficient compounds had been identified)
B- Series of substructure-based queries prioritized with
Tanimoto similarity caluculated by use of Morgan fingerprint
approach. Final selection cherry picked by eye
45 (12)
69 (10)
85 (11)
121 (18)
16 (0)
C- ECFP4 (ref. 40) similarity (Tanimoto cut-off 0.6) and in-house
fingerprint search (Tanimoto cut-off 0.7). Subsequent in-house
“quality”, commercial availability, and IP filters
D- Daylight and ChemAxon fingerprint similarity ranking,41,42
followed by Openeye pharmacophore alignment43,44
E- The top scoring 150 compounds were selected by Tanimoto
similarity calculation using the FCFP4 fingerprint, followed by
refinement to 96 compounds based on maximized diversity
All companies
58 (12)
56 (4)
86 (9)
106 (11)
363 (56)
294 (34)
a
b
Actives defined as compounds with SI > 5 against one or both parasites. Compounds with a 2-substituted imidazo[1,2-a]pyridine core.
Compounds without a 2-substituted imidazo[1,2-a]pyridine core.
c
chemotype did not contain the 2-pyridyl motif nor the amino
Combining the above findings with the nature of the assay
used to identify the anti-leishmanial effect of the compound
(high content imaging with parasite count) and the fact that
the compound demonstrated anti-parasitic activity in more
than one assay and against multiple related kinetoplastid
parasites, we decided to cautiously investigate further. We
were aware from the literature of the potential for a metal-
chelation-driven pharmacological effect due to the bidentate
heteroaryl-heteroaryl system in the core, indeed it could be
hypothesized that this is the source of PAINS-like activity in
other assays.39 Therefore, a key step for further investigation
of 1 was to gain confidence via SAR and orthogonal assays
that the observed anti-parasitic activity was unlikely to be
driven by metal chelation, and that the PAINS flag could be
substituent at the 3-position of the imidazo[1,2-a]pyridine, but
rather a 2,3 biaryl substitution pattern. In this instance the
structure is, therefore, not classified as a PAINS motif, however
the reported SAR demonstrated potencies around an order of
magnitude lower than those observed for 1, despite the authors
using an axenic promastigote assay which circumvents the
additional challenge for compounds to access intracellular
parasites. In addition, we found examples of launched drugs
which contain the imidazo[1,2-a]pyridine core from 1, including
the so-called Z-drugs zolpidem and alpidem, as well as reports
of structures similar to 1 confirmed as hits in other infectious
disease screens such as Mycobacterium tuberculosis and vaccina
virus.37,38
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the background host cell, and b) clear evidence that altering
the core away from the PAINS-like chemotype maintained
anti-parasitic activity, as suggested by the earlier paper from
Marhadour et al.36
Hit elaboration and validation via collaborative in silico
screening
Compound 1 was submitted as a “seed” to five partners of
the NTD Booster consortium: AstraZeneca plc, Celgene
Corporation, Eisai Co., Ltd, Shionogi & Co., Ltd, and Takeda
Pharmaceutical Company Ltd. Since the pre-competitive
mechanism of the Booster project requires anonymity and a
high degree of confidentiality on in silico methods and library
composition between partners, identities of the partners are
presented as blinded in this study as “Companies A–E”.‡‡
The partners performed two successive iterations of
ligand-based virtual screening against their proprietary
compound collections: cycle A and cycle B (see Fig. 1). Each
distinct library was probed using a partner-specific in silico
similarity search approach as described in Table 1 (see ESI†
for detailed description). The compounds identified via these
virtual screening efforts were investigated in a high content
cell-based L. donovani infection assay using THP1 host cells.
Assay read-out from this high content approach
demonstrated the compound's efficacy at total parasite
clearance as well as host cell cytotoxicity, yielding an anti-
parasitic IC50, a host cell CC50 and a selectivity index (SI)
between the two read-outs (SI = host cell IC50/parasite IC50;
see ESI† for more details).
Fig.
2 A) Diverse compounds identified from the booster giving
confidence in the viability of the chemical series; B) dose response
curves for 4; C) high content images of L. donovani infected THP1
cells treated with various concentrations of 4.
Compounds with
a
SI
>
5
were considered as
demonstrating legitimate anti-parasitic activity. Results of
this process are shown in Fig. 1. The first sequence of virtual
screening (Fig. 1A) identified a total of 367 compounds from
4 partners, of which 25 demonstrated at least some degree of
potency against L. donovani and shared the core imidazo[1,2-
a]pyridine motif. Interestingly a new batch of compound 1,
which displayed similar potency – cytotoxicity profile to the
batch from the original HTS, appeared within this set
(Fig. 2A). Compound 2 also appeared within this set, which
demonstrated the potential of a new vector for exploration at
the 8-position of the imidazo[1,2-a]pyridine core, as well as
demonstrating efficacy with no background cell cytotoxicity.
Parasitology data on each company's selection of compounds
was fed back to the respective partner organizations.
Parasitology data and the structure of compound 2 was
shared with all partners and a second round of virtual
screening was then completed using this elaborated
parasitology data (Fig. 1B). The second round of in silico
Scheme 1 i) ArCOCH3, I2, NaOH, H2O, 110 °C; ii) NBS, MeCN, 20 °C;
iii) R3-NC, TsOH, ArCHO, MeOH, 70 °C; iv) R3R4NH, t-BuONa,
Brettphos-Pd-G3, t-amylalcohol, 90 °C; v) HCl, MeOH, 20 °C; vi)
R7COOH, EDCI, pyridine, 25 °C; vii) BH3SMe2, THF, 60 °C; viii) ArX,
t-BuONa, Pd2(dba)3, XantPhos, PhMe, 110 °C; ix) ix) Ti(i-PrO)4, NaBH3-
CN, dioxane, 60 °C
screening yielded
a
further 290 compounds from
5
participating organizations, of which 16 contained the
imidazo[1,2-a]pyridine core and demonstrated some level of
efficacy against L. donovani. Retrospective analysis of these
two rounds of in silico screening demonstrated the power
legitimately discounted. The two key read-outs we targeted to
achieve this were a) the ability of the chemotype to achieve a
broader selectivity index for Leishmania donovani relative to
‡‡ Assignment of blinded names A–E is random. Specific contributions of each
partner were, and remain, anonymous within the collaboration.
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Table 2 SAR exploration of the 3-position for L. donovani
a
c
Compound
R3
R6
R7
IC50 μM
CC50b μM
Clint
4
5
9
N-Cyclopentylamino-
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
Me
H
H
H
H
H
1.80
1.16
10.2
>50
30.4
3.57
>50
29.4
>50
>50
9.34
1.11
2.71
3.9
3.70
1.73
1.91
3.91
1.71
4.53
3.49
7.76
2.31
2.53
2.61
42.5
25.6
>50
>50
>50
19.4
>50
>50
>50
>50
>50
6.69
9.61
>50
>50
5.75
4.3
8.93
16.0
>50
>50
>50
10.7
13.2
>50
N-(2,3-Dihydro-1,4-benzodioxin-6-yl)-amino
N-2,2,2-Trifluoroethylamino-
N-2-(N′,N'Dimethyl)-ethyl-1,2 diamino-
N-2-Methyl-2-hydroxy-propylamino-
N-Cyclopropylmethylamino-
N-Oxetan-2-ylmethylamino-
N-Tetrahydrofuran-3-ylamino-
N-Pyrolidin-3-ylamino-
N-Morpholino-
N-Benzylamino-
N-Phenylamino-
N-4-Fluorophenylamino-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
171
N-(N-Methyl)-4-fluorphenylamino-
N-3-Fluorophenylamino-
N-2-Fluorophenylamino-
>1000
242
133
N-3,5-Difluorophenylamino-
N-2,5-Dichlorophenylamino-
N-5-Fluoropyridin-2-ylamino-
N-Pyridin-2-ylaminoamino-
4-Trifluoromethoxyphenylamino-
N-4-Cyananophenylamino-
N-4-Methoxyphenylamino-
N-3-Methoxyphenylamino-
N-2-Methoxyphenylamino-
62
81
130
223
326
a
L. donovani parasite clearance in infected THP1 cells. b THP1 cytotoxicity. c Mouse microsomes, μL min−1 (mg.pr)−1
.
and versatility of this approach to hit to lead elaboration. Of
the 657 compounds identified via in silico screening, almost
45% of the compounds represented a scaffold hop, defined
number of initial findings: firstly, there appeared to be at
least 5 vectors around the core which tolerated changes
whilst retaining anti-leishmanial activity (positions 2, 3, 6, 7
and 8). Secondly, although certain structural changes in the
chemotype induced significant cytotoxicity against the host
THP1 cell line, we identified key examples which
demonstrate acceptable differentiation between anti-parasitic
activity and cell based cytotoxicity, with the most selective
compounds achieving a selectivity index (SI) of >20 (4, 5, 6,
7). Analysis of the anti-parasitic activity and cell line
cytotoxicity curves and high content images clearly
demonstrated concentration points for most compounds
where a full antiparasitic effect was observed with no
cytotoxic observation on the background cell line (see
Fig. 2B and C). Thirdly, we were able to identify compounds
which achieved an antiparasitic effect with no background
cell cytotoxicity whilst removing the 2-pyridyl functionality at
the 2 position of the core (8), removing some suspicions
in this instance as
a
compound not containing
a
2-substituted imidazo[1,2-a]pyridine as the core. The hit rate
of actives within this set of scaffold hops was only marginally
lower than for those “on-scaffold” compounds which
contained the original chemotype (11.5% compared to
15.4%) – yet these more structurally ambitious scaffold hop
compounds would arguably not be included in a traditional
hit to lead campaign due to the inherent increase in
synthetic complexity of scoping multiple chemotypes.
Compound 3 is a key example of such a scaffold hop
originating from this approach, demonstrating significant
potencies against both T. cruzi and L. donovani whilst
representing
a
significant deviation away from the
imidazo[1,2-a]pyridine core.§§
Combining the parasitology data for the key active
compounds containing the original imidazo[1,2-a]pyridine
core structure of 1, we collated data to form a hit series and
move forward into detailed investigation and targeted
analoguing as detailed below. An initial survey of the data
generated by these rounds of virtual screening resulted in a
about
a
metal-chelation driven SAR. These findings
convinced us that the cytotoxicity observed for the series of
compounds was not uniquely responsible for the observed
anti-parasitic effect, and that the series merited further
investigation. Finally, we also observed that the anti-
leishmanial SAR around the scaffold was potentially non-
additive, with the impact of certain functionality changes
being influenced by substitution patterns elsewhere in the
structure (Fig. 2A). This appeared particularly true for
§§ A full data set containing the compounds synthesized during this effort can
be found in the supporting information and has been made fully available
online: https://dndi.org/research-development/portfolio/drug-discovery-booster/
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Table 3 SAR exploration of the R2 position
a
c
Compound
R3
R2
R6
R7
IC50 μM
CC50b μM
Clint
932
4
Cyclopentyl
Pyrid-2-yl
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
Me
H
H
H
H
H
Me
H
H
H
H
1.80
5.26
1.76
>50
42.2
16.2
7.37 (3.2)d
7.9
10.2
25.9
22.0
>50
>50
5.93
6.51
9.4
42.5
19.3
27.1
>50
>50
>50
>50 (29.3)d
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
15.4
4.22
>50
>50
40
32
33
34
35
36
37
38
39
40
41
42
43
8
44
45
46
47
48
49
50
51
52
53
5-Methoxypyrid-2-yl
4-Methoxypyrid-2-yl
Pyrid-3-yl
2-Methylpyrid-4-yl
3,4-Dimethoxyphenyl
4-(Dimethylamino)phenyl
>1000
4-Methoxyphenyl
2-Fluorophenyl
2-Fluoro-4-methoxyphenyl
Thiazol-2-yl
N-Methylimidazo2-yl
4-N,N-Dimethylaminophenyl
4-Methoxyphenyl
Cyclohexyl
4-Ethoxyphenyl
4-Tertbutylphenyl
5-Methoxypyrid-2-yl
4-Methoxypyrid-2-yl
5-Chloropyrid-2-yl
N-Methylimidazo-2-yl
N-Methylimidazo-4-yl
Thiazol-4-yl
13.2
2.5
4-Fluorophenyl
3-Fluorophenyl
126
2.85
11.5
>50
4.46
15.7
36.2
>1000
H
H
OMe
OMe
OMe
<12
40.6
>50
N-Methylpyrazol-3-yl
a
b
c
d
L. donovani parasite clearance in infected THP1 cells. THP1 cytotoxicity. Mouse microsomes, μL min−1 (mg.pr)−1
.
Value for library batch
(value for resynthesized batch).
substitutions of the 6- and 7-position of the core imidazo[1,2-
a]pyridine, as demonstrated by compounds 4–6, where the
impact of a 6-methyl group improves potency when the
3-position is aryl substituted, but negatively impacts potency
when the 3-position is alkylated. We therefore decided that
rather than follow a rigid systematic investigation of each
area of the molecule as an SAR follow-up strategy, we would
use an empirical approach combining both single- and multi-
dimensional SAR exploration. We made subtle variations
around the 6- and 7-position substitution at the same time as
exploring the 2- and 3-position. This was intended to give as
broad a coverage of the chemotype as possible in a smaller
number of compounds, and to avoid missing key
improvements masked by the hypothesized non-additive SAR.
converted into representative final products D via palladium
mediated coupling with various primary and secondary
amines. In some instances, representative final compounds
D were obtained directly in one step from aminopyridines A
via tosic acid-mediated three-component cyclization with
isonitriles and aryl aldehydes. One such example of D,
2-(pyridin-2-yl)-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]
pyridin-3-amine (E), was cleaved under acidic conditions to
give 3-aminoimidazo[1,2-a]pyridine F, and subsequently
converted into representative final compounds
G via
palladium mediated coupling with various aryl bromides.
Further exploitation of intermediate F was achieved via
amidation followed by borane mediated reduction of the
amide to give N-alkylated representative final compounds K.
Structure activity relationship (SAR)
Synthesis and analoging
SAR elaboration of the core chemotype was focused on three
distinct regions (Tables 2–4).‖‖ Scanning of the 3-position of
the imidazo[1,2-a]pyridine core revealed some initial SAR of
this position (Table 2), suggesting that compounds with both
amino alkyl and amino aryl moieties at this position could
demonstrate anti-parasitic activity. Although small lipophilic
alkyl groups demonstrated moderate anti-parasitic activities
Analoging was driven using the representative synthetic
pathway shown in Scheme 1.¶¶ Various substituted
2-aminopyridines A were converted into 2-aryl imidazo[1,2-a]
pyridines
B
via iodine-mediated condensation with
arylmethylketones. Subsequent regioselective bromination of
the 3-position of the imidazo[1,2-a]pyridines yielded
3-bromoimidazo[1,2-a]pyridines C, which were then
‖‖ 3 was followed up further by subsequent rounds of in silico screening via this
¶¶ Full synthetic details for all compounds in ESI.†
consortium approach, results to be communicated in future correspondence.
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Table 4 SAR exploration of the 6- and 7-position of the core
b
c
Compound
R3
R6
R7
IC50a μM
CC50 μM
Clint
19
54
55
56
57
58
59
60
61
21
67
62
63
64
65
66
1
N-4-Fluorophenylamino-
Me
CN
CF3
MeO
Cl
H
H
H
H
H
H
H
H
CN
CF3
MeO
Me
H
Me
H
2.71
>50
35.4
2.38
>50
>50
11.0
9.61
>50
>50
4.26
>50
>50
23.7
923
146
1.77
4.56
117
355
H
H
H
0.71 (2.45)d
3.7
5.9 (11.8)d
>50
N-3-Fluorophenylamino
2.47
7.19
5.12
2.74
14.24
16.0
>50
7.37
33.3
CH2OMe
CH2NMe2
H
H
H
Me
H
H
CH2OMe
CH2NMe2
OH
H
H
92
4.54
16.87
4.67 (2.9)e
1.80
43.3
N-Cyclopentylamino-
28.0 (24.0)e
42.5
4
68
5
6
H
H
Me
Cl
H
Me
H
H
Me
H
H
H
H
H
H
Me
H
H
1.88
1.16
>50
25.6
>1000
N-(2,3-Dihydro-1,4-benzodioxin-6-yl)-amino-
0.62 (1.98)d
1.91 (6.33)d
9.34
16.0 (28.3)d
>50 (17.4)d
>50
411
>1000
7
17
69
18
70
10
16
71
N-Benzylamino-
3.37
1.11
0.82
>50
H
17.9
>50
6.69
9.59
>50
>50
>50
>1000
N-Phenylamino-
N-2-(N′,N'Dimethyl)-ethyl1,2 diamino-
H
N-Morpholino
>50
137
H
OMe
a
b
c
d
L. donovani parasite clearance in infected THP1 cells. THP1 cytotoxicity. Mouse microsomes, μL min−1 (mg.pr)−1
.
Library batch
(resynthesized batch). e Library batch (initial HTS batch).
and selectivity indices (9, 12), presence of both basic (10, 15)
and non-basic (13, 16) heteroatoms appear detrimental to
anti-parasitic activity. Conversely, presence of additional
polar heteroatoms or halogens in the case of aniline-type
substitution in the 3-position seemed to enhance potency
and in certain cases selectivity over host cell cytotoxicity (5,
21), although, as previously mentioned, this selectivity
seemed to partly depend on the nature of additional
substitution at positions 6 and 7 on the imidazo[1,2-a]
pyridine core (22–25, 29–30). A combination of alkyl and aryl
substituents on the 3-position amine appeared to result in an
interesting combination of both potency and selectivity over
background cytotoxicity (20). It became clear that there were
extensive opportunities to modulate physicochemical
properties of the compound at the 3-position of the
chemotype.
Investigation of the 2-position of the core (Table 3)
revealed that small substitutions such as chloro and methoxy
on the 4- and 5-position 2-pyridyl were tolerated (32, 33,
42–47) and could be exploited to tune microsomal stability.
Moving the position of the pyridyl nitrogen to the 3 or 4
position removed anti-leishmanial activity (34, 35), however
replacement of the 2-pyridyl with 5-membered nitrogen-
Table 5 In vitro ADME properties for select compounds
Clint μL min−1
Kinetic solubility μg
(mg.pr)−1
mL−1
Compound
Mouse
Human
pH 7.4
pH 2.0
6
7
25
37
61
411
>1000
81
>1000
354
95
>100
35
>100
>100
8
72
>76
>76
67
<0.5
>76
<0.5
<0.5
Fig. 3 Pharmacokinetic profiling of 23 in mouse.
73
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containing heteroaromatic rings was tolerated in certain
instances (51), and this change also had a major impact on
the metabolic stability of the compound. Interestingly,
replacement of the pyridyl with electron enriched phenyls
such as 4-anilino (37, 38) and 4-alkoxy substituted (44, 45)
retained moderate activity and generally removed any
evidence of cytotoxicity, suggesting that the anti-parasitic
efficacy of the chemotype is likely not driven by a bidentate
metal chelation effect. This observation of multiple non-2-
pyridyl analogues further supported our earlier hypothesis
regarding the redundancy of the PAINS flag for the initial
hit 1.
Broader substitution of the 6- and 7-positions of the core
imidazo[1,2-a]pyridine revealed a clear negative effect of
withdrawing electron density from the core heterocycle, with
cyano (54, 58) and trifluoromethyl (55, 59) substituents
negatively impacting the antiparasitic effect (Table 4).
Attempts to probe further away from the core negatively
impacted potency and/or cytotoxicity (62–65), however
incorporation of heteroatoms in this position could be used
to improve microsomal stability (64). Scanning the impact of
6- and 7-substitution relative to the functionality at position 3
reconfirmed the previous observation that SAR appears non-
additive, with changes having negative or positive impact on
potency and cytotoxicity depending on the nature of the
3-position substitution.
compound observed in the plasma after 24 hours. Such
exposure relative to the anti-parasitic activity of the
compound was considered insufficient to progress the
compound to an in vivo infection model of Leishmania.
Conclusions
Work continues to optimize the imidazo[1,2-a]pyridine
series informed by the SAR generated by the in-silico
Booster process and subsequent informed design. The
primary focus for this future work will be further
improvement of the in vitro ADME parameters in order to
achieve prolonged exposure in vivo in line with a profile
capable of demonstrating efficacy in an infection model of
Leishmania. This may, however, be complicated by the
clearly non-additive anti-parasitic and cytotoxicity SAR,
although certain key findings from the SAR identified so
far should enable improvement of anti-parasitic activity,
microsomal stability and widening of the selectivity index
between anti-parasitic effect and cell cytotoxicity. The
overall approach of using the drug discovery booster as a
multi-party in silico screening approach to complete the
first phase of hit-to-lead and hit validation allowed us to
rapidly confirm and validate the activity and relevance of
the series as well as inform and guide the initial SAR
exploration. Further analysis of the impact and utility of
the Booster approach, which has to date been applied to
over 20 different hit series,33 will be the subject of future
communication.
ADME and pharmacokinetic profiling
To complete the initial scoping of this chemical series, we
profiled a selection of compounds in preliminary in vitro
solubility and metabolic stability assays (Table 5). In general
compounds appeared poorly soluble at pH 7.4, although this
could be optimized through incorporation of additional
polarity in the 3-position of the core (25). Kinetic solubility at
pH 2.0 was consistently favourable, and the compounds
generally demonstrated rapid intrinsic clearance in both
human and mouse microsomes, with the intrinsic clearance
of both species tracking when moderate improvements were
observed in microsomal stability for compounds 25 and 57,
which again suggested that ADME property optimization was
strongly tied to the presence of polar heteroatoms in the
3-position of the imidazo[1,2-a]pyridine. Compiling the
broader SAR around intrinsic microsomal mouse clearance
in vitro (Tables 2–5) shows that improvement was generally
achievable through the introduction of additional polarity,
for example via incorporation of additional nitrogen in the
pendant aryl groups at positions 2- and 3- of the core, or
through ether groups at the 7-position of the core scaffold.
To understand the translation of these in vitro ADME
parameters into in vivo exposure, compound 23 was profiled
in mouse pharmacokinetic studies at 1 mg kg−1 i.v. and 50
mg kg−1 p.o. (Fig. 3). Despite moderate in vitro Clint and
in vivo bioavailability, 23 demonstrated high clearance in the
mouse, resulting in relatively low overall exposure in terms of
CMax and time of exposure after oral dosing, with no
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We would like to acknowledge John Cuff, Leelapavan Tadoori,
Jean Robert Ioset, Tatsuro Kuzuki, Daisuke Imoto, Midori
Morioka, Dominique Junod and Jean Pierre Paccaud for their
help in implementation and logistics of the Booster project,
Chen Min and Jiajia Zhang for ADME-PK studies, Louise
Burrows for help in manuscript preparation. We acknowledge
the generous support of Global Health Innovative Technology
Fund (GHIT) for financial support of this project, and our
collaboration partners at Abbvie Inc, Astellas Pharma Inc and
Merck KGaA for participation in the Booster project outside
of this particular series. The Drugs for Neglected Diseases
initiative (DNDi) is grateful to its donors, public and private,
who have provided funding to DNDi since its inception in
2003. A full list of DNDi's donors can be found at http://www.
dndi.org/donors/donors/.
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