Design, Synthesis, and Biological Evaluation of Imidazopyridines as PD-1/PD-L1 Antagonists

Article abstract of DOI:10.1021/acsmedchemlett.1c00033

The PD-1/PD-L1 axis has proven to be a highly efficacious target for cancer immune checkpoint therapy with several approved antibodies. Also, small molecules based on a biphenyl core can antagonize PD-1/PD-L1, leading to the in vitro formation of PD-L1 dimers. However, their development remains challenging, as we do not yet fully understand their mode of action. In this work, we designed a new scaffold based on our previously solved high-resolution structures of low-molecular-weight inhibitors bound to PD-L1. A small compound library was synthesized using the Groebke-Blackburn-Bienaymé multicomponent reaction (GBB-3CR), resulting in the structure-activity relationship of imidazo[1,2-a]pyridine-based inhibitors. These inhibitors were tested for their biological activity using various biophysical assays giving potent candidates with low-micromolar PD-L1 affinities. An obtained PD-L1 cocrystal structure reveals the binding to PD-L1. Our results open the door to an interesting bioactive scaffold that could lead to a new class of PD-L1 antagonists.

Full text of DOI:10.1021/acsmedchemlett.1c00033

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Letter
Design, Synthesis, and Biological Evaluation of Imidazopyridines as
PD-1/PD-L1 Antagonists
⊥
⊥
́
̇
Roberto Butera, Marta Wazynska, Katarzyna Magiera-Mularz, Jacek Plewka, Bogdan Musielak,
Ewa Surmiak, Dominik Sala, Radoslaw Kitel, Marco de Bruyn, Hans W. Nijman, Philip H. Elsinga,
Tad A. Holak, and Alexander Dömling*
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ABSTRACT: The PD-1/PD-L1 axis has proven to be a highly
efficacious target for cancer immune checkpoint therapy with several
approved antibodies. Also, small molecules based on a biphenyl core can
antagonize PD-1/PD-L1, leading to the in vitro formation of PD-L1
dimers. However, their development remains challenging, as we do not
yet fully understand their mode of action. In this work, we designed a
new scaffold based on our previously solved high-resolution structures
of low-molecular-weight inhibitors bound to PD-L1. A small compound library was synthesized using the Groebke−Blackburn−
́
Bienayme multicomponent reaction (GBB-3CR), resulting in the structure−activity relationship of imidazo[1,2-a]pyridine-based
inhibitors. These inhibitors were tested for their biological activity using various biophysical assays giving potent candidates with
low-micromolar PD-L1 affinities. An obtained PD-L1 cocrystal structure reveals the binding to PD-L1. Our results open the door to
an interesting bioactive scaffold that could lead to a new class of PD-L1 antagonists.
́
KEYWORDS: PD-L1 inhibitor, multicomponent reaction, Groebke−Blackburn−Bienayme, imidazo[1,2-a]pyridine
n recent decades, oncology has been revolutionized by
understand and develop small molecules that antagonize PD-
1
Iimmunotherapy. In particular, immune checkpoint block-
1/PD-L1, we present here the design, synthesis, biological
activity, and structural basis of imidazopyridines as PD-1/PD-
L1 antagonists.8,11−14
ade (ICB) targeting the PD-1/PD-L1 axis has shown
impressive clinical benefit, with durable regression and even
cure in a subset of hard-to-treat cancers.2,3 In general, ICB-
responsive cancers are characterized by high levels of
mutations and corresponding neoantigens (“hot” tumors).
These neoantigens can be recognized by immune effector T-
cells, which under homeostatic conditions would result in
cancer cell elimination.4 In cancer, this elimination is
restrained by the immune checkpoint PD-1 expressed on T-
cells. PD-1 binds to PD-L1 expressed on cancer cells, resulting
in an inhibitory intracellular signaling cascade that prevents
proper T-cell activation.5 Consequently, inhibition of the
interaction of PD-L1 and PD-1 receptors promotes T-cell
activation. Several PD-1/PD-L1-directed antibodies are in
clinical use, and numerous experimental ones are under
development. However, current PD-1/PD-L1-directed thera-
pies are useful only for a small subset of patients, are expensive
to produce, have a risk of adverse effects, and show
development of resistance, which limits their utility.6,7
Therefore, novel therapeutic modalities such as small
molecules or peptides exhibit a lot of promise.8,9 The only
small-molecule inhibitor targeted against PD-L1 currently
undergoing clinical trial is CA-170 (Figure 1B).10 However, by
means of various functional cell assays it was recently proved
not to be a direct binder to PD-1 or PD-L1, and its mode of
action remains unclear.11 As part of our ongoing efforts to
Our recently published cocrystal structures of several small
molecules binding to PD-L1 have been used to propose a
generalized pharmacophore model for small-molecule PD-L1
binders (Figure 1A).9,14−17 These structures triggered a wave
of small-molecule designs and subsequent patent applica-
tions.18,19 Accordingly, a twisted biphenyl moiety is linked via a
two-atom linker to a planar (hetero)aromatic ring fragment
that has a methanamine para to the linker moiety. Symmetrical
central biphenyl moieties with two times the linker (hetero)-
aromatic fragment have also been described as highly potent
PD-L1 binders.20 The diversity of PD-L1 small-molecule
scaffolds based on our proposed pharmacophore model and
claimed in patents is great (Figure 1B).18,19 The biphenyl
component allows for fewer variations, but the linker and
(hetero)aromatic moieties can be executed in a variety of
designs. Finally, the water-exposed part of the molecule allows
Received: January 18, 2021
Accepted: April 21, 2021
Published: April 28, 2021
© 2021 The Authors. Published by
American Chemical Society
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a
Scheme 1. Synthetic Route to Compounds 5a−f
a
Reagents and conditions: (a) Pd(dppf)Cl2, toluene/ethanol/
NaHCO3 (sat. aq.) (5:1:5) (0.3 M), 85 °C, 12 h; (b) 5-fluoro-2-
nitropyridine (4a) or 5-fluoro-4-methyl-2-nitropyridine (4b), KOH,
dry DMSO, 0 °C to rt, 5 min to 1 h; (c) Fe, HCl; EtOH/H2O (5:1)
(0.1 M), 2 h, reflux. R1 = H, p-F, [3,4]-(OC2H4O); R2 = H, CH3.
carbonate solution (5:1:5) and heating this for 12 h at 85 °C
generated compounds 3a−c in excellent yields of 90−97%.
Following the Suzuki reaction, we selected the nucleophilic
aromatic substitution involving 3a−c and 5-fluoro-2-nitro-
pyridine (4a) or 5-fluoro-4-methyl-2-nitropyridine (4b, see the
Supporting Information) as the best option to prepare the
precursors to amidines 6a−d.
Figure 1. Design of PD-1/PD-L1 antagonists. (A) Generalized
pharmacophore model of a PD-L1 antagonist. Aromatic (purple),
hydrophobic (green), and basic (positive charged, blue) pharmaco-
phores are included. (B) Examples of potent PD-L1 dimerizers taken
from the patent literature. The pharmacophore is indicated by red and
blue colors. (C) Design of imidazopyridines accessible by GBB-3CR.
The four variable parts of the scaffold are indicated by the different
colored boxes. (D) Modeling of an imidazopyridine into a PD-L1
dimer structure (PDB ID:5NIX).
By adding finely ground potassium hydroxide in dry DMSO
at 0 °C to generate a superbasic medium in the presence of
3a−c and 4a−b, we were able to synthesize the corresponding
products 5a−d with quantitative conversion and with brief
reaction times of 5 min for 5a−c and 1 h for 5d.26 An
extraction step was performed, and the resulting crude oil was
utilized in the subsequent step. Lastly, to generate the desired
amidines 6a−d, reduction of 5a−d was performed by the use
of hydrochloric acid and iron powder in ethanol/water (5:1)
under reflux conditions to avoid possible benzyl ether cleavage
of the biphenylic component, i.e., via a classical Pd/C H2
reduction. Following 2 h of reaction time and chromatographic
purification, the required amidines were obtained in yields of
72−92%.
for many variations useful to tune drug-like properties such as
water solubility.
To circumvent the lengthy and linear sequential syntheses of
many small-molecule PD-1/PD-L1 antagonists, we decided to
explore multicomponent reactions for the one-pot assembly of
the central (hetero)aromatic part of the pharmacophore
model. For this, we chose the Groebke−Blackburn−Bienayme
reaction (GBB-3CR) which is a versatile three-component
reaction of heterocyclic amidines, aldehydes, and isocyanides
́
that gives access to drug-like molecules (Figure 1C).21−23
A
Having the desired amidines in hand, we were able to
execute the GBB reaction (Scheme 2). On the basis of the
key fragment of the scaffold is a bicyclic imidazo ring.
In our design, the bicyclic heteroaromatic moiety (Figure 1
C, gray) is the central element of the scaffold, to which three
suitable substituents are attached, namely, the biphenyl (Figure
1 C, blue), methanamine (Figure 1 C, yellow), and amino
(Figure 1 C, green) moieties. To decide on the position of the
substituents, we used molecular modeling performed via
Moloc and Scorpion software (Figure 1D).24,25 The best fit
into the receptor was to introduce the methanamine moiety by
the GBB-3CR aldehyde component and the biphenyl moiety
through a C−O coupling to the aminopyridine linker.
a
Scheme 2. GBB Reaction
a
Reagents and conditions: (A) Sc(OTf)3 (10 mol %), DCM/MeOH
(2:1) (0.3 M), 1 h, 120 °C, microwave-assisted heating; (B) 7 N HCl
in 2-propanol, rt, 20 h.
Following the design and docking studies (Figure 1D), we
opted for the imidazopyridine-containing scaffold (Figure 1C)
and started on the development of a corresponding synthesis
route that would yield the established scaffold.
We envisioned a synthetic route in which the step with the
highest introduction of variation should be carried out last.
Therefore, the GBB reaction was chosen as one of the last
stages. Consequently, the preparation of the biphenyl-
substituted amidines was approached first (Scheme 1). We
started synthesizing the twisted biphenyl moiety via the Suzuki
cross-coupling reaction between (3-bromo-2-methylphenyl)-
methanol (2) and unprotected boronic acids 1a−c. Using
Pd(dppf)Cl2 as the catalyst in a solvent system consisting of
toluene, ethanol, and saturated aqueous sodium hydrogen
docking studies, we selected phenyl-containing isocyanides for
the possibility of generating π stacking with Tyr-123 in the
pocket of the PD-L1 dimer. As the aldehyde component, we
chose polar substrates to increase the polarity, solubility, and
possibility of hydrogen bonding. Therefore, we used tert-butyl
(2-oxoethyl)carbamate and both enantiomers of the Garner
aldehyde, (S)- and (R)-(−)-3-Boc-2,2-dimethyloxazolidine-4-
carboxaldehyde.
We found the optimal reaction conditions for the used
substrates to be scandium triflate (10 mol %) as the catalyst,
2:1 DCM/MeOH as the solvent system, a concentration of 0.3
M with regard to the amidine, and 1.7 equiv. of the isocyanide
and aldehyde components. Microwave-assisted heating for 1 h
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generated the corresponding GBB products in good to
excellent yields (48−86%). Subsequently, a chromatographic
purification over silica was performed. This purification step
yielded generally still slightly impure compounds. Therefore,
we continued with the deprotection of the tert-butyl carbamate
group and, in the case of compounds 9d and 9e, the additional
aminal protecting group. Performing the deprotection with 7 N
HCl in 2-propanol at room temperature for 20 h resulted in
full conversion. Following an additional chromatographic
purification over silica, the final compounds 9a−k (Chart 1)
were obtained and were analyzed via high-resolution mass
1
spectrometry and H and 13C NMR spectroscopy (see the
Supporting Information).
Chart 1. Compound Library of Imidazopyridine PD-L1
Antagonists
Figure 2. Binding studies. (A) Aliphatic part of the 1H NMR
spectrum of PD-L1 (blue) and PD-L1 with compounds 9a (red), 9b
(green), 9c (purple), and 9f (orange) in a 1:1 molar ratio. (B) HTRF
scouting based on a concentration of 50 μM. (C) HTRF inhibition
assay and calculated IC50 values for the best compounds from HTRF
scouting.
For compound 9f, an adjusted synthesis route was chosen in
which instead of (3-bromo-2-methylphenyl)methanol we used
1-(benzyloxy)-3-bromo-2-methylbenzene (A) for the Suzuki
reaction, yielding 3-(benzyloxy)-2-methyl-1,1′-biphenyl (B)
(see the Supporting Information). Additionally, a hydro-
genation was performed with Pd/C and H2 (1 bar) in
methanol for 2 h at 40 °C, reaching full conversion to 2-
methyl-[1,1′-biphenyl]-3-ol (C). The remainder of the syn-
thesis route was performed analogously to the other
compounds, leading to compound 9f.
Moreover, we also explored a postsynthetic modification of
compound 9h wherein we formed a tetrazole from the cyanide
function on the isocyanide substrate, resulting in compound 9i.
Compound 9h was reacted with sodium azide and zinc
chloride in n-propanol at 95 °C for 20 h. After chromato-
graphic purification and subsequent deprotection, we achieved
a 39% yield of compound 9i. Conceptually, we expected that
this modification would increase the affinity via hydrogen
bonding between the tetrazole (as a carboxylic acid isostere)
and Arg125.
To support our binding hypothesis from protein−ligand
docking, we performed binding studies, which included NMR
studies of ligand−PD-L1 binding and a homogeneous time-
resolved fluorescence (HTRF) assay with PD-1 and PD-L1
proteins (Figure 2). Their binding to PD-L1 was verified using
NMR titrations and HTRF assays (see the Supporting
Information). The results of the NMR titration experiments
demonstrated that our synthesized compounds generally
disrupt the PD-1/PD-L1 complex and bind to PD-L1 as well
as induced imerization of PD-L1.
HTRF experiments were conducted to further prove the
ability of our compounds to disrupt the PD-1/PD-L1 complex
and for IC50 value determination. The results of the HTRF
experiments proved that all of the tested compounds were
capable of disrupting the PD-1/PD-L1 complex, showing the
potential of this scaffold. HTRF scouting revealed that parts of
the GBB-3CR scaffold selected as binding parts in docking and
cocrystallization studies (represented here as compounds 3a, 6,
and B) have some disruption potential but that the full
structure is needed for effective PD-L1 blocking, which we
report in compounds 9a−e, 9j, and 9k. The phenyl ether (9f)
and benzyl ether (9a) analogues show different affinity to the
target protein: the presence of the linking methyl group
increases the binding potential. Fluorine substitution on the
biphenyl core is not tolerated (9g−i), whereas dioxane
addition is accepted (9j and 9k). Additionally, the presence
of a methyl group on the imidazopyridine ring (9j) improves
the inhibitory activity of the GBB-3CR scaffold. IC50 values
were determined for selected compounds and found to be in
the range of 1.8−22.9 μM. The compounds with the lowest
IC50 values were 9b (9.3 μM) and 9j (1.8 μM).
Next, the molecular basis of the interaction between 9c and
the PD-L1 dimer was elucidated by X-ray structure analysis
(Figure 3). The resolution was 2.45 Å, and the electron density
of 9c was shown well, with the exception of the phenyl moiety
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group helps to keep the biphenyl moiety in a twisted shape
close to the receptor conformation. In other compound
designs, the o-methyl group was replaced by a bromide,
chloride, or nitrile at the ortho position.12,27 The oxymethylene
linker between the biphenyl and imidazopyridine fragments is
rather flexible and in other designs can be an alkene, although
structural data are missing.28
Why does imidazopyridine compound 9c show a rather low
PD-L1 affinity? Detailed analysis of the cocrystal structure
reveals that the π-stacking interaction of the pyridine and
Tyr56(A) is suboptimal because of the low overlap of the two
aromatic rings. Also, the electrostatics of this interaction is
poor because the two dipole moments are parallel-aligned
(Figure 3E). The distances between the Asp122(B) oxygens
and the solvent-exposed aminomethyl substituent are rather
long (4.2 and 4.8 Å), resulting in a poor charge−charge
interaction (Figure 3F). Moreover, the largely hydrophobic
pocket formed by Ile54(A), Val68(A), and Gln66(A) is not
filled.
To understand the molecular basis accounting for the
differences in affinity between 9c and 9j, we docked compound
9j to the dimer of PD-L1 (PDB entry 6R3K). The predicted
binding mode of 9j matches perfectly the overall orientation of
9c and reveals the origins of the greater potency of 9j (Figure
4). First, the presence of the extended biphenyl is predicted to
Figure 3. Binding of 9c to the PD-L1 dimer. (A) 2D structure of 9c.
(B) Extracted 3D structure of 9c. (C) Overall view of 9c at the
interface of two PD-L1 monomers (A and B, shown in green and red,
respectively). (D−F) Scorpion software ligand−receptor interaction
analysis. (D) vdW interactions between 9c and the PD-L1 dimer. (E)
π stacking interactions. (F) Charge−charge interactions between
Asp122 and the solvent-exposed aminomethyl substituent.
of the phenylethyl fragment. 9c is mostly buried in the deep
and elongated receptor site, which is composed of two PD-L1
monomers. In the absence of 9c the protein dimer complex has
C2 symmetry. However, the binding of the small molecule
breaks the symmetry. The biphenyl methylene moiety is
embedded within two and three antiparallel β-sheets of
monomers A and B, respectively. Monomer A (in green)
contributes to the binding pocket with the 11 amino acids
Lys124, Tyr56, Ala121, Ser93, Ser117, Ile54, Met115, Tyr123,
Arg125, Lys124, and Asp122. Monomer B (in red) contributes
to the binding pocket with the 12 amino acids Tyr56, Met115,
Tyr123, Ala121, Ser117, Gln66, Ile54, Asp122, Ala121, Ser117,
Ile54, and Gln66. The imidazopyridine moiety is embedded
among Tyr56 and Gln66 of monomer B and Tyr123, Asp122,
and Ala121 of monomer A. The phenylethylamine substituent
is surrounded by Lys124, Arg125, and Tyr123 of monomer A
and the hydroxy group of Tyr56 of monomer B. Finally, the
aminomethyl group is largely water-exposed.
Figure 4. Comparison of the binding modes of 9c and 9j. (A)
Superimposition of 9c from the cocrystal structure with the
anticipated orientation of 9j obtained from molecular docking. (B)
Structural basis of the better potency of 9j. Additional π−π stacking
interactions are highlighted as red dashes, and transparent amino
acids (R125 and Y56) denote residues anticipated to change their
conformation upon binding of 9j to the PD-L1 dimer.
induce structural rearrangement of Tyr56 from monomer B.
Thus, 9c occupies a larger surface area of the PD-L1 dimer and
forms an open channel between monomers, as shown
previously for compounds bearing an “extended” biphenyl
ring system with the 2,3-dihydrobenzo[b][1,4]dioxine moi-
ety.17 Second, the application of a shorter linker between the
imidazopyridine core and ring C locates the latter one closer to
the surface of monomer B and allows for additional π stacking
with Tyr123, which is not observed in case of 9c (Figure 4).
Inspired by our previously published cocrystal structures of
PD-L1, we designed a novel scaffold, imidazopyridine, as a PD-
L1 dimerizer. By exploitation of the multicomponent reaction
(MCR) principle, imidazopyridines can be conveniently
synthesized by the GBB-3CR with high variations of aldehydes,
aminopyridines, and isocyanides. A small library consisting of
11 compounds was synthesized and tested for their efficacy
through various biological assays, exhibiting IC50 values of
9c undergoes a diversity of hydrophobic, π-stacking, and
charge−charge interactions (Figure 3D). Ring A of the
biphenyl moiety is the deepest-buried part of 9c. The ring
makes a short T-shaped π-stacking contact of 3.8 Å with Tyr56
of monomer A (Figure 3E). Plenty of hydrophobic contacts
between ring A and Tyr123(B), Ile54(A), Ala121(B),
Ser117(A), and Met115(A) can be observed. The twisted
C−C bond connecting rings A and B is approximately at the
position of the C2 axis of the imaginary PD-L1 dimer complex
(A−B).
The dihedral angle defined by the two planes of the biphenyl
is 124° (Figure 3B). This is comparable to other dihedral
angles of similar molecules (e.g., BMS116617). The o-methyl
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16.8−1.8 μM. We were further able to cocrystallize compound
9c with PD-L1, providing an explanation for the rather low
affinities of the compound series. Overall, 9c can be regarded
as a model compound that indicates the need of further
improvements to achieve practical affinities, which are ongoing
in our laboratory.
Tad A. Holak − Department of Crystal Chemistry and Crystal
Physics Faculty of Chemistry, Jagiellonian University, 30-387
Kraków, Poland
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00033
Author Contributions
⊥R.B. and M.W. contributed equally.
Notes
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
The authors declare no competing financial interest.
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00033.
ACKNOWLEDGMENTS
■
Experimental procedures, analytical data, NMR spectra,
NMR binding assay, PD-L1 cocrystallizations, HRTF
assay, and molecular docking (PDF)
This research was funded by Project POIR.04.04.00-00-420F/
17-00 (to T.A.H.), which is carried out within the TEAM
Program of the Foundation for Polish Science cofinanced by
the European Union under the European Regional Develop-
ment Fund, COFUNDs ALERT (Grant Agreement 665250),
and a KWF Kankerbestrijding grant (Grant Agreement
10504). Furthermore, we acknowledge the MCB Structural
Biology Core Facility (supported by the TEAM TECH CORE
FACILITY/2017-4/6 Grant from the Foundation for Polish
Science) for valuable support.
AUTHOR INFORMATION
■
Corresponding Author
Alexander Dömling − Department of Drug Design, University
of Groningen, 9713 AV Groningen, The Netherlands;
orcid.org/0000-0002-9923-8873; Email: a.s.s.domling@
rug.nl, www.drugdesign.nl
REFERENCES
■
(1) Dömling, A.; Holak, T. A. Programmed Death-1: Therapeutic
Success after More than 100 Years of Cancer Immunotherapy. Angew.
Chem., Int. Ed. 2014, 53 (9), 2286−2288.
(2) Sharma, P.; Allison, J. P. Dissecting the Mechanisms of Immune
Checkpoint Therapy. Nat. Rev. Immunol. 2020, 20 (2), 75−76.
(3) Okazaki, T.; Honjo, T. The PD-1-PD-L Pathway in
Immunological Tolerance. Trends Immunol. 2006, 27 (4), 195−201.
(4) He, X.; Xu, C. Immune Checkpoint Signaling and Cancer
Immunotherapy. Cell Res. 2020, 30, 660.
(5) Iwai, Y.; Ishida, M.; Tanaka, Y.; Okazaki, T.; Honjo, T.; Minato,
N. Involvement of PD-L1 on Tumor Cells in the Escape from Host
Immune System and Tumor Immunotherapy by PD-L1 Blockade.
Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (19), 12293−12297.
(6) Martins, F.; Sofiya, L.; Sykiotis, G. P.; Lamine, F.; Maillard, M.;
Fraga, M.; Shabafrouz, K.; Ribi, C.; Cairoli, A.; Guex-Crosier, Y.;
Kuntzer, T.; Michielin, O.; Peters, S.; Coukos, G.; Spertini, F.;
Thompson, J. A.; Obeid, M. Adverse Effects of Immune-Checkpoint
Inhibitors: Epidemiology, Management and Surveillance. Nat. Rev.
Clin. Oncol. 2019, 16 (9), 563−580.
(7) Kalbasi, A.; Ribas, A. Tumour-Intrinsic Resistance to Immune
Checkpoint Blockade. Nat. Rev. Immunol. 2020, 20 (1), 25−39.
(8) Konstantinidou, M.; Zarganes-Tzitzikas, T.; Magiera-Mularz, K.;
Holak, T. A.; Dömling, A. Immune Checkpoint PD-1/PD-L1: Is
There Life Beyond Antibodies? Angew. Chem., Int. Ed. 2018, 57,
4840−4848.
(9) Magiera-Mularz, K.; Skalniak, L.; Zak, K. M.; Musielak, B.;
Rudzinska-Szostak, E.; Berlicki, Ł.; Kocik, J.; Grudnik, P.; Sala, D.;
Zarganes-Tzitzikas, T.; Shaabani, S.; Dömling, A.; Dubin, G.; Holak,
T. A. Bioactive Macrocyclic Inhibitors of the PD-1/PD-L1 Immune
Checkpoint. Angew. Chem., Int. Ed. 2017, 56 (44), 13732−13735.
(10) Curis, Inc. A Study of CA-170 (Oral PD-L1, PD-L2 and VISTA
Checkpoint Antagonist) in Patients with Advanced Tumors and
Lymphomas (NCT02812875). https://clinicaltrials.gov/ct2/show/
NCT02812875?term=PD-L1++small+molecule&draw=2&rank=3
(accessed 2020-11-07).
(11) Musielak, B.; Kocik, J.; Skalniak, L.; Magiera-Mularz, K.; Sala,
D.; Czub, M.; Stec, M.; Siedlar, M.; Holak, T. A.; Plewka, J. CA-170 −
A Potent Small-Molecule PD-L1 Inhibitor or Not? Molecules 2019,
24, 2804.
Authors
Roberto Butera − Department of Drug Design, University of
Groningen, 9713 AV Groningen, The Netherlands
́
̇
Marta Wazynska − Department of Obstetrics and
Gynaecology, University Medical Center Groningen,
University of Groningen, 9713 GZ Groningen, The
Netherlands
Katarzyna Magiera-Mularz − Department of Crystal
Chemistry and Crystal Physics Faculty of Chemistry,
Jagiellonian University, 30-387 Kraków, Poland;
orcid.org/0000-0002-4826-6380
Jacek Plewka − Department of Crystal Chemistry and Crystal
Physics Faculty of Chemistry, Jagiellonian University, 30-387
Kraków, Poland; orcid.org/0000-0002-0307-0907
Bogdan Musielak − Department of Crystal Chemistry and
Crystal Physics Faculty of Chemistry, Jagiellonian University,
30-387 Kraków, Poland; orcid.org/0000-0002-1665-
5920
Ewa Surmiak − Department of Crystal Chemistry and Crystal
Physics Faculty of Chemistry, Jagiellonian University, 30-387
Kraków, Poland; orcid.org/0000-0002-4103-4675
Dominik Sala − Department of Crystal Chemistry and Crystal
Physics Faculty of Chemistry, Jagiellonian University, 30-387
Kraków, Poland
Radoslaw Kitel − Department of Crystal Chemistry and
Crystal Physics Faculty of Chemistry, Jagiellonian University,
30-387 Kraków, Poland
Marco de Bruyn − Department of Obstetrics and Gynaecology,
University Medical Center Groningen, University of
Groningen, 9713 GZ Groningen, The Netherlands
Hans W. Nijman − Department of Obstetrics and
Gynaecology, University Medical Center Groningen,
University of Groningen, 9713 GZ Groningen, The
Netherlands
Philip H. Elsinga − Department of Nuclear Medicine and
Molecular Imaging, University Medical Center Groningen,
University of Groningen, 9713 GZ Groningen, The
Netherlands
(12) Konieczny, M.; Musielak, B.; Kocik, J.; Skalniak, L.; Sala, D.;
Czub, M.; Magiera-Mularz, K.; Rodriguez, I.; Myrcha, M.; Stec, M.;
Siedlar, M.; Holak, T. A.; Plewka, J. Di-Bromo-Based Small-Molecule
772
https://doi.org/10.1021/acsmedchemlett.1c00033
ACS Med. Chem. Lett. 2021, 12, 768−773

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Inhibitors of the PD-1/PD-L1 Immune Checkpoint. J. Med. Chem.
2020, 63 (19), 11271−11285.
(13) Musielak, B.; Janczyk, W.; Rodriguez, I.; Plewka, J.; Sala, D.;
Magiera-Mularz, K.; Holak, T. Competition NMR for Detection of
Hit/Lead Inhibitors of Protein−Protein Interactions. Molecules 2020,
25, 3017.
(14) Guzik, K.; Zak, K. M.; Grudnik, P.; Magiera, K.; Musielak, B.;
Törner, R.; Skalniak, L.; Dömling, A.; Dubin, G.; Holak, T. A. Small-
Molecule Inhibitors of the Programmed Cell Death-1/Programmed
Death-Ligand 1 (PD-1/PD-L1) Interaction via Transiently Induced
Protein States and Dimerization of PD-L1. J. Med. Chem. 2017, 60
(13), 5857−5867.
(15) Zak, K. M.; Kitel, R.; Przetocka, S.; Golik, P.; Guzik, K.;
Musielak, B.; Dömling, A.; Dubin, G.; Holak, T. A. Structure of the
Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-
L1. Structure 2015, 23 (12), 2341−2348.
(16) Zak, K. M.; Grudnik, P.; Guzik, K.; Zieba, B. J.; Musielak, B.;
Dömling, A.; Dubin, G.; Holak, T. A. Structural Basis for Small
Molecule Targeting of the Programmed Death Ligand 1 (PD-L1).
Oncotarget 2016, 7 (21), 30323−30335.
(17) Skalniak, L.; Zak, K. M.; Guzik, K.; Magiera, K.; Musielak, B.;
Pachota, M.; Szelazek, B.; Kocik, J.; Grudnik, P.; Tomala, M.; Krzanik,
S.; Pyrc, K.; Dömling, A.; Dubin, G.; Holak, T. A. Small-Molecule
Inhibitors of PD-1/PD-L1 Immune Checkpoint Alleviate the PD-L1-
Induced Exhaustion of T-Cells. Oncotarget 2017, 8 (42), 72167−
72181.
(18) Zarganes-Tzitzikas, T.; Konstantinidou, M.; Gao, Y.; Krzemien,
D.; Zak, K.; Dubin, G.; Holak, T. A.; Dömling, A. Inhibitors of
Programmed Cell Death 1 (PD-1): A Patent Review (2010−2015).
Expert Opin. Ther. Pat. 2016, 26 (9), 973−977.
(19) Shaabani, S.; Huizinga, H. P. S.; Butera, R.; Kouchi, A.; Guzik,
K.; Magiera-Mularz, K.; Holak, T. A.; Dömling, A. A Patent Review
on PD-1/PD-L1 Antagonists: Small Molecules, Peptides, and
Macrocycles (2015−2018). Expert Opin. Ther. Pat. 2018, 28 (9),
665−678.
(20) Basu, S.; Yang, J.; Xu, B.; Magiera-Mularz, K.; Skalniak, L.;
Musielak, B.; Kholodovych, V.; Holak, T. A.; Hu, L. Design,
Synthesis, Evaluation, and Structural Studies of C2-Symmetric Small
Molecule Inhibitors of Programmed Cell Death-1/Programmed
Death-Ligand 1 Protein−Protein Interaction. J. Med. Chem. 2019,
62 (15), 7250−7263.
(21) Shaaban, S.; Abdel-Wahab, B. F. Groebke−Blackburn−
́
Bienayme Multicomponent Reaction: Emerging Chemistry for Drug
Discovery. Mol. Diversity 2016, 20 (1), 233−254.
(22) Baenziger, M.; Durantie, E.; Mathes, C. Development of an
Industrial Process Based on the Groebke−Blackburn−Bienayme
Multicomponent Reaction: Efficient Preparation of 3-Aminoimidazo-
[1,2-a]Pyrazines. Synthesis 2017, 49 (10), 2266−2274.
́
́
(23) Boltjes, A.; Dömling, A. The Groebke-Blackburn-Bienayme
Reaction. Eur. J. Org. Chem. 2019, 2019 (42), 7007−7049.
(24) Gerber, P. R.; Muller, K. MAB, a Generally Applicable
̈
Molecular Force Field for Structure Modelling in Medicinal
Chemistry. J. Comput.-Aided Mol. Des. 1995, 9 (3), 251−268.
(25) Kuhn, B.; Fuchs, J. E.; Reutlinger, M.; Stahl, M.; Taylor, N. R.
Rationalizing Tight Ligand Binding through Cooperative Interaction
Networks. J. Chem. Inf. Model. 2011, 51 (12), 3180−3198.
(26) Yuan, Y.; Thomé, I.; Kim, S. H.; Chen, D.; Beyer, A.;
Bonnamour, J.; Zuidema, E.; Chang, S.; Bolm, C. Dimethyl Sulfoxide/
Potassium Hydroxide: A Superbase for the Transition Metal-Free
Preparation of Cross-Coupling Products. Adv. Synth. Catal. 2010, 352
(17), 2892−2898.
(27) Li, Z.; Wu, L.; Yao, W. Heterocyclic Compounds as
Immunomodulators. WO 2017205464 A1, 2017.
(28) Wang, Y.; Xu, Z.; Wu, T.; He, M.; Zhang, N. Aromatic
Acetylene or Aromatic Ethylene Compound, Intermediate, Prepara-
tion Method, Pharmaceutical Composition and Use Thereof. WO
2018/006795 A1, 2018.
773
https://doi.org/10.1021/acsmedchemlett.1c00033
ACS Med. Chem. Lett. 2021, 12, 768−773