Synthesis and biological activity of conformationally restricted indole-based inhibitors of neurotropic alphavirus replication: Generation of a three-dimensional pharmacophore

Article abstract of DOI:10.1016/j.bmcl.2021.128171

We have previously reported the development of indole-based CNS-active antivirals for the treatment of neurotropic alphavirus infection, but further optimization is impeded by a lack of knowledge of the molecular target and binding site. Herein we describe the design, synthesis and evaluation of a series of conformationally restricted analogues with the dual objectives of improving potency/selectivity and identifying the most bioactive conformation. Although this campaign was only modestly successful at improving potency, the sharply defined SAR of the rigid analogs enabled the definition of a three-dimensional pharmacophore, which we believe will be of value in further analog design and virtual screening for alternative antiviral leads.

Full text of DOI:10.1016/j.bmcl.2021.128171

Bioorg. Med. Chem. Lett. 46 (2021) 128171
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of conformationally restricted
indole-based inhibitors of neurotropic alphavirus replication: Generation of
a three-dimensional pharmacophore
,
b
Scott J. Barrazab, Janice A. Sindacb, Craig J. Dobryc, Philip C. Delektac, Pil H. Leea
,
,b,*
David J. Millerc, Scott D. Larsena
a Vahlteich Medicinal Chemistry Core, University of Michigan, Ann Arbor, MI 48109, United States
b Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, United States
c Departments of Internal Medicine and Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
We have previously reported the development of indole-based CNS-active antivirals for the treatment of
neurotropic alphavirus infection, but further optimization is impeded by a lack of knowledge of the molecular
target and binding site. Herein we describe the design, synthesis and evaluation of a series of conformationally
restricted analogues with the dual objectives of improving potency/selectivity and identifying the most bioactive
conformation. Although this campaign was only modestly successful at improving potency, the sharply defined
SAR of the rigid analogs enabled the definition of a three-dimensional pharmacophore, which we believe will be
of value in further analog design and virtual screening for alternative antiviral leads.
Neurotropic alphavirus
Antiviral
Pharmacophore
Conformationally restricted
Western equine encephalitis virus
Neurotropic alphaviruses are NIAID Category B Priority pathogens
that infect the central nervous system (CNS) and have the potential to
cause severe and debilitating disease in humans and animals.1 Among
these, the mosquito-borne equine encephalitis viruses (Eastern, West-
ern, and Venezuelan) are endemic to the Americas and constitute an
emergent public health risk due to the potential for human mortality2
and neurological damage,3–5 in addition to disruption of agriculture.6–7
Despite this risk, prophylactic therapies are inadequate; current vaccines
lack wide availability and exhibit limited efficacy in humans,8–9 and
recent vaccine candidates have demonstrated protection in animal
models only.10–13 Options for treating infection with small molecule
drugs are similarly minimal and are unable to compensate for vaccine
deficiencies. For example, the efficacy of the current standard of treat-
ment, ribavirin, is constrained by poor CNS exposure.14–16 Remarkably,
equine encephalitis viruses have received scant historical attention to
address these shortcomings.17 Only a handful of reports have been
published in recent years detailing the discovery of drug-like small
molecules18–20 and natural products,21–22 so there remains an urgent
need to identify and develop effective therapies that can intercede with
infection.
of indole-based alphavirus RNA replication inhibitors, e.g. 1 and 2
(Figure 1), and, more recently, anthranilamide-based inhibitors pos-
sessing improved pharmacokinetic properties.26 Unfortunately, the
molecular target remains elusive – a consequence of the phenotypic
nature of our primary antiviral assays – and thus the architecture of the
binding site that could guide design of more potent inhibitors is un-
known. An approach that is often effective in such a scenario is the
synthesis and evaluation of rigid analogues.27–30 Of the multiple con-
formations that a drug can adopt, only a small subset are expected to
have maximal activity if the binding site is well defined.31 It can
therefore be informative to restrict lead molecules into specific confor-
mations in an effort to pinpoint the most active ones.32 In addition to the
obvious benefit of potentially defining a 3-dimensional (3D) pharma-
cophore, this strategy can also improve both potency and selectivity
versus off-target effects.33–35
In the present work, we thus pursued two central objectives: (1) to
conformationally restrict rotatable bonds in our leads 1 and 2, and (2) to
develop a 3D pharmacophore model based on the resulting SAR that
could expedite future drug design. Toward this end, we describe three
different approaches to conformational restriction: 1) replacement of
small functionality with sterically demanding functionality; 2) bridging
We have previously reported the discovery23 and optimization24–25
* Corresponding author.
E-mail address: sdlarsen@med.umich.edu (S.D. Larsen).
https://doi.org/10.1016/j.bmcl.2021.128171
Received 15 February 2021; Received in revised form 26 May 2021; Accepted 30 May 2021
Available online 15 June 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S.J. Barraza et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128171
amine 44.
Distinct from the other intermediates, alkenes 47 and 48 were syn-
thesized via a traditional Wittig reaction and the Schlosser modification
thereof,44 respectively, from N-boc-4-formylpiperidine 45 and phos-
phonium bromide 4645 (Scheme 2). Although a large number of highly
E- and Z-selective olefinations exist,46 this divergent approach allowed
for convenient and rapid access to both alkenes from the same reactants.
All new analogues were evaluated for antiviral activity in a phenotypic
cell-based assay against the WEEV genome replicon as previously
described.25 Analogues were also tested for cytotoxicity under similar
conditions using a colorimetric MTT assay. The data appear in Tables 1-
4.
Fig. 1. Indole-based alphavirus replication inhibitor leads.
As noted earlier, definition of a 3D pharmacophore was our primary
goal through the evaluation of conformationally-restricted analogs. Our
initial efforts focused on the restriction of the indole substituents of the
lead 125 (Table 1). Utilizing a non-covalent steric approach, two com-
pounds were designed on the notion that strategically placed methyl
groups would impede bond rotations of adjacent sidechains with mini-
mal impact on molecular weight. Both the 7-methylindole 3 and the 3-
methylindole 4 possessed substantially reduced potency, indicating that
the methyl groups may be preventing rotatable access of the N-benzyl
group and the 2-carboxamide to active conformations.
of nonadjacent atoms; and 3) freezing of ring conformations. We also
strove to minimize the number of additional atoms used in locking
conformations because higher molecular weight could impede CNS
permeability.
Analogs were synthesized according to one of two general proced-
ures (Scheme 1). In all cases where chiral centers were present, the
analogs were prepared and tested as racemates. General Procedure A
was used to incorporate methyl groups onto the indole and to modify the
benzyl group of lead 1. N-(4-Chlorobenzyl)indole-2-carboxylic acids
30a–c36 were condensed with ethyl isonipecotate or ethyl nipecotate
and the respective esters were subsequently hydrolyzed to afford car-
boxylic acid intermediates 31a–c. These acids were condensed with a
variety of amines (see Supporting Information) to produce analogs 3 –
16 (Tables 1 and 2).
We next turned to restricting the flexibility of the N-benzyl amide on
Table 1
Modification of the indole of 1.
Alternatively, General Procedure B was employed to explore changes
to the piperidine and amide moieties of lead 2 (Scheme 1). Piperidines,
piperazines, and bicyclic amines were functionalized through a variety
of reactions to afford intermediates with the general structure 34
(Scheme 2 and Supp. Info). Following deprotection, these intermediates
were condensed with N-(4-chlorobenzyl)indole-2-carboxylic acid 30a to
afford analogs 17– 29 (Table 3).
No
125
R1
R2
R3
IC50 (µM)a
15.6 ± 1.8
CC50 (µM)b
>100
H
H
The majority of intermediates of general structure 34 were amides
and as such were prepared by condensation of an amine with a car-
boxylic acid followed by acid-promoted Boc-deprotection (e.g. 37 and
42, Scheme 2 ; for others, see SI). Urea 40 was prepared by condensation
of two different amines with phosgene and was likewise deprotected. N-
Tosyl-protected spirocyclic amino acid 4337–39 was similarly condensed
with 4-(2-aminoethyl)-pyridine 39 but proved challenging to deprotect.
Samarium (II) iodide either failed to remove the tosyl under neutral
conditions or afforded reductive cleavage of the amide in the presence of
tertiary amines,40,41 and other methods42,43 were similarly unsuccessful.
3
4
CH3
H
H
61 ± 39
>100
>100
>100
CH3
aInhibition of luciferase expression in WEEV replicon assay. bCell viability determined
by the MTT reduction assay. Values are mean of at least n = 3 independent
experiments.
–
Only sodium naphthalenide cleanly cleaved the S N bond to afford
Scheme 1. General Procedure A reagents and conditions: (a) ethyl isonipecotate or ethyl nipecotate, EDC, HOBT, TEA, DCM, r.t.; (b) NaOH, H2O, THF, r.t.; (c)
R3R4NH, EDC, HOBT, TEA, DCM, r.t.; General Procedure B reagents and conditions: (d) various reactions – see Scheme 2 and SI; (e) 30a, EDC, TEA, HOBT, DCM, r.t.
PG = protecting group; G = reactive group.
2

S.J. Barraza et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128171
target that is very well defined and discriminating.
Table 2
Modification of the benzyl amide of 1.
Our attention then turned towards the central piperidine ring and the
piperidine-4-carboxamide. Compound 224 was employed as the lead for
this series due to its equipotency with 12 and greater ease of analog
synthesis. Biological results are summarized in Table 3. The carbox-
amide imparts significant conformational bias to the linker between the
piperidine and the pyridine, so we initially explored replacing it with E
or Z olefins 17 and 18, respectively, which are conformationally locked
bioisosteres for the two possible rotamers of the carboxamide.47
Remarkably, both analogs lost all activity, indicating that the cabox-
amide itself is likely making at least one key binding interaction. To
explore this further, inverse amide 19 and shifted amide 20 were pre-
pared, each proving to be over 30-fold less active than 2. This supported
that a carboxamide bound directly to the piperidine 4-carbon through
the carbonyl is a critical part of the pharmacophore. We therefore
retained this motif through the remainder of our investigation.
No
125
R
IC50 (µM)a
15.6 ± 1.8
CC50 (µM)b
>100
5c
30.6 ± 7.3
>100
6
7
>100
>100
>100
>100
Urea analog 21 and unsaturated amide 22 were examined next, each
of which retained all of the atoms of the carboxamide and its position
relative to the piperidine, but would be expected to have some minor
conformational differences. The simple unsaturated amide retained
more activity than the urea, but still lost nearly 10-fold potency. Methyl
analogs 23 and 24 were particularly informative. While the previously
prepared N-methyl analog 2424 only lost 2-fold potency, suggesting that
8
9
>100
>100
>100
>100
–
the N H is not functioning as a hydrogen bond donor, the new
α-methyl
10
11
>100
>100
>100
>100
analog 23 lost all activity, indicating that the carbonyl likely plays a
much more significant role. The α-methyl group is either occluding the
amide carbonyl from binding (sterically or conformationally) or is
impeding favorable chair conformations of the piperidine. Finally, since
–
we had shown with 24 that the N H was not critical for activity, we
12
13
14
0.53 ± 0.04
1.7 ± 0.1
7.1 ± 0.9
>100
>100
>100
prepared the highly rigid bicyclic analog 25. This analog lost most po-
tency, obviously indicating that it is not locked in the optimal confor-
mation. Collectively, the results for 17 – 25 highlight the importance of a
sterically unhindered and flexible carboxamide to the pharmacophore.
For the next three analogs 26 – 28, we retained the “flexible” car-
boxamide moiety in the linker, and focused on freezing the conforma-
tional mobility of the piperidine ring. While the exo-bicycle 26 and the
spirocycle 28 lost over 20-fold potency, the tricyclic analog 27 retained
almost all of the potency of lead 2, suggesting that the locked equatorial-
equatorial relationship between the 1- and 4-substituents may reflect the
unknown bioactive conformation, Unfortunately, no activity improve-
ment was realized by this conformational restraint.
1525
16
15.2 ± 4.7
>100
62 ± 13
>50
Conversely, we examined an azapane analog 29 that we expected
would be more flexible than piperidine 2 but about the same overall
length. Interestingly, this compound was equally potent with the lead
despite being a racemate. Depending on the eudismic ratio (not deter-
mined), the active enantiomer could potentially be two-fold more active,
marking this compound as the most active compound to-date in the
WEEV replicon assay. The simplest explanation for this positive result is
that a larger population of azapane conformers are available that are
favorable to binding than are available to the piperidine.
aInhibition of luciferase expression in WEEV replicon assay. bCell viability determined
by the MTT reduction assay. Values are mean of at least n = 3 independent
experiments. cBased on nipecotic acid central ring (racemate).25
the isonipecotic acid frame of 1 (Table 2). Previously prepared analogs25
5 and 15 are included in the table for comparison. Remarkably, the
majority of new analogs (6 – 11) exhibited no activity whatsoever,
suggesting that the binding pocket for the N-benzyl amide is well
defined and intolerant of many rigid conformations. One compound
(12), however, proved to possess markedly better potency than all
others in this series, which we do not believe can be attributed to simple
increased lipophilicity or chain extension because analogs of similar
ClogP (14) and length (1525 and 16) were less potent (Table 2). Inter-
estingly, the racemic methyl analog 13 was also quite active; however its
lower potency relative to 12 is further evidence that the amide binding
site is well defined and not tolerant of even modest additional bulk or
altered conformation. Importantly, the increased potency of 12 has
apparently been achieved without a commensurate increase in cyto-
toxicity vs 1. Finally, the dramatic loss of potency of analogs 7 and 8,
each a single methylene shorter than the corresponding active analogs
12 and 14, respectively, suggests that some degree of flexibility is
required to engage the binding site. Overall, the results in Table 2 are
consistent with an amide binding pocket in the unknown molecular
The replicon activity of azapane 29 was confirmed in an antiviral
assay in FMV-infected BE(2)-C cells. At 5 µM, 29 demonstrated a nearly
two-fold improvement in infected cell viability (36.1 ± 4.0% versus
negative control 19.6 ± 4.0%), similar to the two-fold enhancement
observed for lead 2 at 25 µM. At higher concentration improvement was
more modest (22.9 ± 4.9% cell viability at 25 µM), possibly due to
concentration-dependent cytotoxicity.
The primary objective of this work was to establish a 3D pharma-
cophore model that could facilitate future optimization and enable vir-
tual screening to identify alternative templates. We first noted that a
number of small changes in the structure resulted in large differences in
biological activity, suggesting that an “activity cliff” analysis48 could be
useful for identifying key pharmacophoric elements. Employing Data-
Warrior49, we identified pairs of compounds exhibiting both high 2D
structural similarity and divergent potency and ranked them according
to structure activity landscape index (SALI)50 scores (Table S1 in
3

S.J. Barraza et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128171
Scheme 2. Reagents and conditions: (a) EDC, HOBT, Et3N, DCM, r.t.; (b) HCl, dioxane, r.t.; (c) COCl2, Et3N, toluene, DCM, 0 ◦C; (d) Na0, naphthalene, THF, r.t.; (e)
PhLi, t-BuOH, LiBr, THF, ꢀ 78 ◦C ꢀ r.t.; (f) n-BuLi, THF, r.t.
Supporting Information). Based on the pairs having the greatest SALI
values, there appear to be three pharmacophoric features most sensitive
to structural changes: the distance between the terminal aryl group and
the piperidine (e.g. 16 vs 15, 12 vs 6, 12 vs 14), the flexibility of the
linker to the terminal aryl group (7 vs 12, 8 vs 14) and the piperidine-4-
carboxamide carbonyl on the piperidine (2 vs 23, 2 vs 17/18, 2 vs 20, 2
vs 19).
The overall accuracy of this query is 0.93 out of 1.0; all the actives
except for one (27) match this query, all of the inactives except for one
do not match this query, and the overlap of the actives is 5.22 out of 6.0.
The alignment of the six most active compounds overlaid on top of the
pharmacophoric query is shown in Figure 2. This query contains the
terminal hydrophobic group, which is consistent with the activity cliff
analysis above. It also contains two hydrogen bond acceptors, one of
which is the piperidine-4-carboxamide which was also shown to be
important in the activity cliff analysis. A detailed description of how the
pharmacophore elucidation was performed is provided in the Support-
ing Information.
In order to generate a hypothetical 3D pharmacophore, we used the
Pharmacophore Elucidation tool in MOE 2019.0102.51 The approach for
Pharmacophore Elucidation is to exhaustively search for all pharmaco-
phore queries (arrangements of molecular features such as aromatic
rinds, hydrophobic groups, hydrogen bond acceptors/donors, etc.) that
induce good overlay of most of the active molecules and separate actives
from inactives. It was assumed that all or most of the active compounds
bind to the same biological target in a similar way and can therefore be
overlaid.
In conclusion, we have explored a variety of conformationally
restricted analogs of our WEEV antiviral leads 1 and 2, with several
objectives in mind: 1) improving antiviral potency; 2) increasing selec-
tivity vs off-targets that could reduce cytotoxicity; and 3) beginning to
define a 3D pharmacophore for the unknown molecular target to facil-
itate future design, including virtual screening to potentially identify
new lead chemotypes. Both covalent and non-covalent approaches to
restricting rotational freedom were examined. As expected, many rigid
analogs completely lost activity, suggesting that the binding site is well
defined and highly discriminating based on molecular shape. Significant
improvements in antiviral potency were realized with terminal amide
analogs of 1 (e.g. 12) without increasing cytotoxicity, suggesting some
improvement in selectivity for the unknown viral target. Modification of
the carboxamide and piperidine of more potent lead 2 were less suc-
cessful at improving potency but helped to identify important pharma-
cophoric elements. The piperidine ring had little tolerance for
conformational rigidity, with only the locked 1,4-diequatorial analog 27
retaining most of the potency of lead 2. Conversely, increasing flexi-
bility, as in azapane analog 29, actually improved potency slightly. We
applied computational modeling to identify the most important SAR
(activity cliffs) and to subsequently define a common pharmacophore
which rationalized most of the SAR. Key elements of this pharmaco-
phore include an optimum linker length between the indole and the
terminal aromatic group of the amide N-substituent, as well as the
requirement for a carboxamide with precise placement on the linker and
an unhindered carbonyl. We believe this pharmacophore could be useful
for designing new compounds and may enable virtual screening to
identify novel viral replication inhibitors.
“Active” compounds were defined as those with IC50 < 5 µM,
affording a set of 7 actives and 22 inactives. These sets were used to
generate a collection of pharmacophore queries such that all or most of
the active compounds satisfy the queries. The generated queries were
then scored by the accuracy of separating actives and inactives. The
overall accuracy = m/N where N is the number of compounds in the
input database and m is the sum of the number of actives that match the
query and the number of inactives that do not match the query. Queries
were also scored by degree of overlap of the actives with the pharma-
cophoric features. The overlap is between 0 and n where n is the number
of actives. The larger the overlap the better the alignment. The overlap is
calculated by the heavy atom root mean square deviation among the
aligned compounds.
The program generated a database of 8850 queries from our
analogue set. The output database contained the conformations of the
high scoring alignment induced by the query, features of each query, the
number of actives that match the query, the score of the alignment, the
accuracy of the query in separating actives and inactives, the accuracies
of the query in matching the actives/inactives, etc. The queries were
sorted by the accuracies, and the top queries were examined for the
alignment of actives (see Table S2 in Supporting Information for the top
10 queries.) One query (H H H a a) was selected as the best on the
aggregate basis of accuracy, overlap, and agreement with the activity
cliff analysis. The selected query has 5 features: 3 hydrophobic groups
and 2 hydrogen bond acceptors (defined as the postulated locations of
potential hydrogen bond donors).
4

S.J. Barraza et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128171
Table 3
Modification of the piperidine amide of 2.
No
224
R
IC50 (µM)a
0.53 ± 0.08
CC50 (µM)b
65.0
17
>50
>50
18
19
>50
>50
Fig. 2. The overlay of the six active analogs with the pharmacophoric query.
Green represents location of hydrophobic groups and cyan indicates hydrogen
bond acceptors (represented by optimal location of potential hydrogen bond
donors in binding site).
17.6c
66.7c
Acknowledgements
20
24.7 ± 3.5
24.4 ± 4.0
3.9 ± 0.4
>50
77.3
78.0
74.2
>50
47.9
This work was supported by an NIH Partnerships for Biodefense Viral
Pathogens grant (R01 AI089417, Principal Investigator DJM) and a
University of Michigan Rackham Merit Scholarship for SJB.
21
Appendix A. Supplementary data
22
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128171.
23
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