Di-bromo-Based Small-Molecule Inhibitors of the PD-1/PD-L1 Immune Checkpoint

Article abstract of DOI:10.1021/acs.jmedchem.0c01260

Immune checkpoint blockade is one of the most promising strategies of cancer immunotherapy. However, unlike classical targeted therapies, it is currently solely based on expensive monoclonal antibodies, which often inflict immune-related adverse events. H

Full text of DOI:10.1021/acs.jmedchem.0c01260

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Article
Di-Bromo-based small-molecule inhibitors
of the PD-1/PD-L1 immune checkpoint
Magdalena Konieczny, Bogdan Musielak, Justyna Kocik, Lukasz Skalniak,
Dominik Sala, Miros#awa Czub, Katarzyna Magiera-Mularz, Ismael Rodríguez,
Maja Myrcha, Malgorzata Stec, Maciej Siedlar, Tad A Holak, and Jacek Plewka
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01260 • Publication Date (Web): 16 Sep 2020
Downloaded from pubs.acs.org on September 16, 2020
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Figure 1 The comparison of selected short biphenyl-based initial scaffolds. BA stands for binding affinity.
1
32x61mm (300 x 300 DPI)
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Figure 2 The w-AIDA-NMR HSQC (A-D) and 1D NMR spectra E. A) N66A-PD-1 w-AIDA – five groups of
characteristic peaks are boxed in green; B) N66A-PD-1/PD-L1 complex – part of PD-1 peaks disappear –
boxed in red; C) N66A-PD-1/PD-L1 complex + 1a – after addition of compound 1a the spectrum of N66A-
PD-1/PD-L1 complex resembles the spectrum A – all PD-1 peaks are restored indicating the displace-ment of
PD-L1 from the complex (peaks boxed in green); D) N66A-PD-1/PD-L1 complex + 1c spectrum resembles
the spectrum A – all PD-1 peaks are restored indicating the displacement of PD-L1 from the complex; E)
aliphatic part of 1D NMR spectrum: Blue- a characteristic spectrum of N66A-PD-1 are highlighted with gray
bars, Red - PD-L1spectrum – peaks in highlighter regions differ, Green – spectrum of N66A-PD-1/PD-L1
complex – spectrum becomes flatten due to complex formation, Purple - N66A-PD-1/PD-L1 complex + 1a –
restoration of characteristic PD-1 peaks due to addition of 1a inhibitor, Orange - N66A-PD-1/PD-L1 complex
+
1c – restoration of characteristic PD-1 peaks due to addition of 1c inhibitor.
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Figure 3 The activity of modified bromo compounds in PD-1/PD-L1 immune checkpoint blockade cell-based
assay. Activities of the reference anti-PD-L1 antibody durvalumab and tested compounds in alleviating the
effect of PD-1/PD-L1 checkpoint on TCR-mediated effector Jurkat T cells are expressed as fold induction of
the luciferase activity, which expression is TCR-inducible (for details please see Materials and Methods). The
graphs present relative luminescence normalized to DMSO- treated controls and are mean +/- SEM values
from 4-7 independent experiments.
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Figure 4 The compound 2k blocks the PD-L1/PD-1 immune checkpoint in primary human T cells, as
evidenced by the increased expression of the late activation and exhaustion marker, PD-1. Peripheral blood
mononuclear cells (PBMCs) were seeded on the pre-seeded hPD-L1 aAPCs and cultured for 48 hours alone or
in the presence of 2k, DMSO (as a solvent for 2k), or atezolizumab (atezo., an anti-PD-L1 therapeutic anti-
body). The expression of PD-1 was tested with flow cytometry. (A) Exemplary histograms, presenting the
increased expression of PD-1 on either the CD4+, or CD8+ T cells. (B) The analysis of the expression of PD-
1 on CD4+, or CD8+ T cells under control conditions and in the presence of PD-L1-blocking molecules, the
antibody atezolizumab, and the compound 2k. Graphs present mean ± SD values calculated for three
independent experiments performed with the engagement of different donors. Statistical significance was
analyzed with the Student’s t-test: * p<0.05, ** p<0.01.
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Figure 5 Interactions between PD-L1 and 3b (top) and 2k (bottom) from modeled complexes (compounds
were docked on PD-L1 taken from PDB ID: 5NIX). Both compounds are involved in a number of hydrophobic
interactions (gray dashed lines) with residues Ile54A Tyr56A and B, Gln66B, Met115B, Ala121A for 3b and
Ala121B for 2h and Tyr123A. There are strong conserved stabiliz-ing π-stacking (green dashed line)
between distal phenyl ring from biphenyl moiety and Tyr56A and phenyl form phenoxymetyl with Tyr56B.
The major differences are in hydrogen bonds (blue solid line) which for 2k are between Tyr56B hydroxyl
group and tertiary amine in piperidine solubilizer and Asn63B amide nitrogen and oxygen from carboxylic
group of solubilizer. For 3b, hydrogen bonds are between cyanopyridine nitrogen in with Arg125A nitro-gen
and nitrile group nitrogen with Asn63B nitrogen.
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Di-Bromo-based small-molecule inhibitors of the PD-1/PD-L1 immune checkpoint
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Magdalena Konieczny , Bogdan Musielak , Justyna Kocik , Lukasz Skalniak , Dominik Sala , Miroslawa Czub , Katarzyna Magiera-
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Mularz , Ismael Rodriguez , Maja Myrcha , Malgorzata Stec , Maciej Siedlar , Tad A. Holak* , Jacek Plewka*
1Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland
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Department of Clinical Immunology, Institute of Pediatrics, Jagiellonian University Medical College, Wielicka 265, 30-663 Krakow,
Poland
*
Correspondence
tadholak@uj.edu.pl (T.A.H.)
jacek.plewka@uj.edu.pl (J.P)
KEYWORDS: immunotherapy, PD-L1 inhibitor, small-molecule inhibitor, cell-based assays, di-bromine
ABSTRACT: Immune checkpoint blockade is one of the most promising strategies of cancer immunotherapy. However, unlike classical
targeted therapies, it is currently solely based on expensive monoclonal antibodies, which often inflict immune-related adverse events.
Herein, we propose a novel small-molecule inhibitor targeted at the most clinically-relevant immune checkpoint - PD-1/PD-L1. The
compound is capable of disrupting the PD-1/PD-L1 complex by antagonizing PD‐L1 and, therefore, restores activation of T cells similarly
to the antibodies, while being cheap in production and possibly non-immunogenic. The final compound is significantly smaller than others
reported in the literature while being non-toxic to cells even at high concentrations. The scaffold was designed using a Structure-Activity-
Relationship screening cascade based on a new antagonist induced dissociation NMR assay, called the weak-AIDA-NMR. Weak-AIDA-
NMR finds true inhibitors, as opposed to only binders to the target protein, in early steps of lead compound development, making it less time
and cost consuming.
Introduction
Programmed cell death protein 1 (PD-1, known also as CD279) and its ligand (PD-L1, known also as CD274 or B7-H1) are
transmembrane receptors involved in the negative regulation of the activated T cells. The interaction of PD-1 with PD-L1 induces T cell
apoptosis, anergy, and functional exhaustion.1,2 Some aggressive cancers, including breast, pancreatic cancers, and non-small-cell lung
carcinomas, are known to overexpress PD-L1, which allows cancer to evade the immune response by suppressing the adaptive immune
system. The constitutive overexpression of PD-L1 on cancer cells reduces activation and proliferation of cancer-reactive T cells and induces
T cells apoptosis. Consequently, disrupting the PD-1/PD-L1 complex at the cancer cell-T cell interface has become an attractive strategy of
3
cancer immunotherapy and was awarded a Nobel Prize in Physiology or Medicine in 2018. Immune checkpoint blockade-based therapies
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using monoclonal antibodies were shown potent in numerous clinical trials for patients with a broad spectrum of cancers, delivering anti-
tumor responses and long‐term remissions.4–9
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The global Checkpoint Inhibitors Market in 2018 was estimated to be almost $ 15 billion and predicted to grow to $ 26 billion by 2023.10
It is solely based, though, on monoclonal antibodies (mAbs) with three PD-1 inhibitors (pembrolizumab, nivolumab, and cemiplimab) and
three PD-L1 inhibitors (avelumab, atezolizumab, and durvalumab) approved by the U.S. Food and Drug Administration and The European
Medicines Agency for various types of cancers, such as bladder, head and neck cancers, and kidney cancer, etc.11,12 Additionally, there are
over 1 500 different clinical studies on PD-1/PD-L1 agents as of 2017, comprising mostly combination therapies with other targeted therapies
and/or chemotherapy aiming at the discovery of -synergistic effects. This clearly indicates how rapidly growing the PD-1/PD-L1
immunotherapy market is.13
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Despite their proven efficacy, mAbs-based therapies are struggling with limitations including high treatment price, immune-related
adverse events (irAEs) and poor tumor penetration related to their large size (150 kDa).14,15 A way to overcome these shortcomings is
presented with small-molecule-based therapeutics, which due to their size (usually below 0.5 kDa) would be orally bioavailable and cheaper
in manufacturing while presenting improved pharmacokinetics and diffusion rates.16 Even though there are plenty of patents regarding potent
small-molecules targeted at PD-L1,17–20 the only small molecule intended to target at PD-L1 currently in clinical trials is CA-170 from Curis
and Aurigene, which is tested for the treatment of advanced solid tumors and lymphomas (NCT02812875, clinicaltrials.gov) and phase II
clinical trials for lung cancer, head and neck/oral cavity cancer, MSI-H positive cancers, and Hodgkin lymphoma in India
(CTRI/2017/12/011026, ctri.nic.in). Three groups, including ours, have recently shown, however, that CA-170 is not a direct PD-L1-binder,
which was evidenced in several independent biophysical and cell-based assays.21–23
PD-1/PD-L1 interface is a challenging target due to its large, flat and hydrophobic interface with a poorly defined binding pocket.24
A
popular way to approach such problematic targets is to use in-silico screening further verified with a biophysical assay to eliminate false-
positive “hits”. However, to analyze the impact of chemical modifications on the inhibitor potency is non-trivial. One can predict how single
changes will modify the chemical properties of the analyzed molecule, such as its solubility, hydrophobicity, etc., but we can only speculate
how it will affect its interactions with the target protein. Therefore, an array of different modifications is usually applied to compare their
influence on the final potency of the drug.
In the present study, we developed novel small-molecule inhibitors targeted at human PD-L1 that is potent in disrupting the PD-1/PD-
L1 complex in biophysical and cell-based assays. To identify these inhibitors, we established a Structure-Activity-Relationship (SAR)
screening cascade that is based on a new antagonist induced dissociation assay nuclear magnetic resonance (NMR) screen, called weak-
AIDA-NMR (w-AIDA-NMR).25 w-AIDA-NMR facilitated the hit-to-lead design of the compounds capable of dissociating the preformed
PD-1/PD-L1 complex. Compared to regular AIDA-NMR,24,26,27 herein we used an N66A mutant of PD-1 that increases the K
of the complex
d
from 8 µM to ca. 100 µM, hence allowing small molecules, which are typically low-affinity binders, competition in the complex formation.
This was further followed by selectivity assays and structural biology directed medicinal chemistry design. The final compound 2k was
intended to be as small as possible and water-soluble, despite bearing two halogen atoms, while fulfilling other requirements from Lipinski’s
rule of five. In parallel, we perform in-silico screening to guide the selection of the intermediate compounds.
2
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Results and Discussion
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Identification of Fragments that Bind to PD-L1
The only small-molecule, non-peptidic inhibitors, for which co-crystal structures with PD-L1 were reported, are based on a
biphenyl core.17,18,27 Because in silico design requires a high-resolution structure of the target, we used this common feature as a starting
point for our SAR analysis. In order to find a seed fragment that could lead to the PD-L1 inhibitory compound, we conducted a w-AIDA-
NMR screen on the available in-house library of decorated biphenyl compounds (data not shown). In parallel, we used a dimeric structure
of PD-L1 with BMS-1166 bound (PDB ID:5NIX) for in silico screening with AutoDock Vina28 integrated into PyRx software to identify
plausible beneficial chemical modification.29 From our in silico screening, we learned that a bromine, fluorine, or chlorine substitutions
decreased the binding affinity from -9.3 kcal/mol for 3-(bromomethyl)-1-1’-biphenyl to -9.8 kcal/mol, whereas hydroxyl and nitriles reduce
the potency of the scaffold indicating that the patch has to be hydrophobic. To validate the in silico predictions, we characterized selected
compounds using the Homogeneous Time-Resolved Fluorescence (HTRF) assay (Figure 1). The IC50 of 1a and 1c were determined to be
around 51 µM and 56 µM respectively, which is impressive for such small fragments with acceptable water solubility (Table S1). Moreover,
the results of HTRF come in hand with our in silico screening where compounds 1a and 1c were the most prominent examples.
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O
Br
F
F
Br
OH
O
OH
OH
1
a
1b
1c
1d
IC = 50.7 µM
IC >100 µM
BA= -6.0 kcal/mol
IC = 55.9 µM
BA= -9.3 kcal/mol
IC ~55.5 µM
BA= -6.5 kcal/mol
5
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50
50
50
BA= -9.3 kcal/mol
NC
O
Br
Br
Br
Br
F
Br
O
Br
1
e
1f
1g
IC = 66.3 µM
BA= -9.0 kcal/mol
IC50 ca. 100 µM
BA= -6.8 kcal/mol
IC50= 64.4 µM
BA= -8.5 kcal/mol
5
0
Figure 1 The comparison of selected short biphenyl-based initial scaffolds. BA stands for binding affinity.
Since HTRF is prone to “false positives”, especially aggregators and autofluorophores, we validated the results using the w-AIDA
NMR assay developed by us. In this assay we introduced a mutation in the PD-1 binding pocket that effectively lowered its affinity towards
PD-L1 so that short fragments could dissociate it. The mutation does not affect the PD-L1 binding interface to its inhibitors as described in
detail in our methodology paper . In the w-AIDA NMR 2D experiment, 15N mutated PD-1 gives a characteristic pattern of peaks (Figure
25
2
A). Peak assignment is not necessary as we observe a relative change in the intensity and positions of the reference peaks upon the addition
1
15
of ligand/inhibitors. Upon the formation of the complex with PD-L1, most of the cross-peaks in the H- N HMQC spectrum of PD-1 became
broader and less intense with some of the cross-peaks disappearing (Figure 2B). If the inhibitor is capable of disrupting the complex, the
resulting spectrum should come back to the reference one of PD-1 alone, as tested inhibitors are PD-L1 binders. Out of tested compounds,
only bromine- and fluorine-substituted biphenyls (1a and 1c) were capable of dissociating mutant PD-1/PD-L1 complex at equimolar
3
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concentrations
in
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the w-AIDA-NMR confirming them not only to be binders but also putative seeds for lead inhibitory compounds as well (Figure 2C and D,
respectively). Similar observations can be made using a 1D version of the w-AIDA-NMR, however, with non-labeled, hence cheaper to
produce protein. A characteristic aliphatic spectrum of PD-1 and PD-L1 is presented in Figure 2E blue and red, respectively with the most
important PD-1 peaks highlighted with gray bars. Upon the complex formation (green curve) highlighted peaks are flattened. However, after
the addition of equimolar concentrations of 1a and 1c, those characteristic PD-1 peaks are restored indicating the dissociation of PD-L1 from
the complex (curve purple and orange, respectively). Unambiguous identifications of short fragments potent to dissociate a PD-1/PD-L1
complex would not be possible using wild type proteins, due to too high binding constant between inhibitor and PD-L1 as presented in Figure
S1.
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Figure 2 The w-AIDA-NMR HSQC (A-D) and 1D NMR spectra E. A) N66A-PD-1 w-AIDA – five groups of characteristic peaks are boxed
in green; B) N66A-PD-1/PD-L1 complex – part of PD-1 peaks disappear – boxed in red; C) N66A-PD-1/PD-L1 complex + 1a – after
addition of compound 1a the spectrum of N66A-PD-1/PD-L1 complex resembles the spectrum A – all PD-1 peaks are restored indicating
the displacement of PD-L1 from the complex (peaks boxed in green); D) N66A-PD-1/PD-L1 complex + 1c spectrum resembles the
spectrum A – all PD-1 peaks are restored indicating the displacement of PD-L1 from the complex; E) aliphatic part of 1D NMR spectrum:
Blue- a characteristic spectrum of N66A-PD-1 are highlighted with gray bars, Red - PD-L1spectrum – peaks in highlighter regions differ,
Green – spectrum of N66A-PD-1/PD-L1 complex – spectrum becomes flatten due to complex formation, Purple - N66A-PD-1/PD-L1
complex + 1a – restoration of characteristic PD-1 peaks due to addition of 1a inhibitor, Orange - N66A-PD-1/PD-L1 complex + 1c –
restoration of characteristic PD-1 peaks due to addition of 1c inhibitor.
Having the potential seed compounds identified, we continued with the second SAR step using in silico screening by designing
the modification starting with the 2-bromo-3-(bromomethyl)-1,1'-biphenyl (calculated binding affinity of -10.2 kcal/mol). Since biphenyl
moiety is known to position resulting molecules within the cleft between the two PD-L1 units, we firstly decorated the phenoxymethyl with
halogens and O-ether benzonitrile or corresponding pyridine-cyanide to enhance the specificity of the binding by increasing the number of
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hydrogen donors/acceptors. To our surprise, we found two potent populations of compounds with the phenoxymethyl ring substituted with
chlorine and O-ethers in the para position and bromine without ether substitution. As the last step, we tested various solubilizers to improve
the water solubility of the scaffold, which would not decrease the potency, resulting in final formulations of binding affinity between -10.5
to -11.5 kcal/mol.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
After in silico identification of potential beneficial extension, we synthesized a group of compounds based on 2-bromo-3-
(bromomethyl)-1,1'-biphenyl moiety (2a-k) collected in Table 1. Firstly, 2-bromo-3-(bromomethyl)-1,1'-biphenyl (1a) O-alkylation of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
various aldehydes in the presence of potassium carbonate was performed. Next, isolated aldehydes (2a-d) were subjected to the reductive
amination mediated by sodium cyanoborohydride resulting in final structures (2g-k). In the case of the compound 2f, we performed a
Williamson reaction between the compound 1a and methyl 3-bromo-4-hydroxybenzoate to isolate 2e as intermediate. Then, we introduced
the amine component in the presence of DBU obtaining the final compound 2f. All described compounds were tested in the HTRF assay to
assess their biological activities as PD-1/PD-L1 complex inhibitors. Not surprisingly, the solubilities of the compounds heavily influence the
resulting biological activities with aldehydes (2a-d) being substantially weaker than corresponding scaffolds equipped with solubilizing
1
groups (Table S1). The best results were obtained for compounds with bromine at R position called from herein “di-bromo” with
N‐[2‐(methylamino)ethyl]acetamide (2g) and pipecolic acid (2k) both exhibiting favourable low nM IC50 values.
Table 1. General Pathway for the Synthesis of Bromo mono-substituted compounds
R1
O
R2
R1
O
R3
Br
Br
Br
A
B
Br
1a
2a-e
2f-k
A
B
Name
R
1
R
2
IC50 [nM]
~50000
Name
R
1
R
3
IC50
[
nM]
13200
±150
O
2
a
-H
H
H
2fa
2g
-Br
H
N
N
H
O
O
O
Cl
2
b
-Br
-Br
-H
H
N
12.2
±0.2
~
50000
50000
N
H
O
2c
-Me
-OMe
-Br
H
H
2h
2i
COOH
COOH
COOH
5
±
22.0
0.5
~
N
N
N
O
O
2d
-Me
175.1
13.3
>
100000
±
2
ea
O
2j
-OMe
2
8 600
500
203.9
6.4
O
±
±
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Journal of Medicinal Chemistry
2
k
-Br
COOH
15.0
0.2
±
1
2
3
N
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Reagents and conditions: (A) substituted 4-hydroxybenzaldehde, K
2d) (B) amine component, NaBH CN, AcOH, DMF, rt, overnight (53% for 2g, 65% for 2h, 56% for 2i, 72% for 2j, 51% for 2k). (A)
methyl 3-bromo-4-hydroxybenzoate, K CO , DMF, rt, overnight (45% for 2e); (B) amine component R , DBU (25% for 2f).
2 3
CO , DMF, rt, overnight (43% for 2a, 39% for 2b, 38% for 2c, 52% for
a
3
2
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To extend the pool of functional groups, we tested a second generation of compounds based on 2-bromo-3-(bromomethyl)-1,1'-
biphenyl (1a), [1,1':2',1''-terphenyl]-3'-ylmethanol (1b) resulting in compounds 3b-d, 3f (Table 2) and based on 1d and 1e-g fragments (Table
3
). We included 1b, 1d and 1e-g fragments to further validate if initial screening using w-AIDA-NMR did not omit a potential hit. The
synthesis started with the Mitsunobu reaction or the Williamson etherification of short fragment 1a or 1b with 5-chloro-2,4-
dihydroxybenzaldehyde resulting in 3a and 3e intermediates. To prepare compounds 3b,c,f, O-alkylation of phenolic aldehydes (3a, 3e) with
4
3
-(bromomethyl)picolinonitrile or 3-(bromomethyl)benzonitrile was carried out. The NaBH CN-mediated reductive amination was used to
convert aldehyde 3b into compound 3d, but unexpectedly the product resulted to be unstable, without the possibility to isolate the pure
compound (purity 64%). Similar reactions on other aldehyde substrates (3a,c,e, and f) were unsuccessful, limiting the possibility to compare
the compounds’ bioactivities. Clearly, the addition of bulky 4‐(hydroxymethyl)pyridine‐2‐carbonitrile or 3‐(hydroxymethyl)benzonitrile at
2
the position R is favourable as they increase the number of hydrogen donors, however at the cost of the significantly higher molecular
weight. Moreover, even though that structure of compound 3d has already been disclosed in the patent WO2017202275A1 (example 3), the
modifications that we did around it are not. Interestingly, our in-silico screening routine failed to find more potent compounds reported in
this patent proving that computational screening is still far from perfect and requires a thorough validation of the results with biophysical
methods.
Table 2. General Pathway for the Synthesis of extended 2-bromo-3-(bromomethyl)-1,1'-biphenyl (1a) and
[1,1':2',1''-terphenyl]-3'-ylmethanol (1b) based compounds 3b-d, 3f.
R2
R2
R2
Cl
R3
R3
R3
R1
R1
R1
C
R1
A
B
X
O
O
O
Cl
Cl
3a
3e
3b-c
3d
1
1b
a
3f
IC50
Name
R
1
R
2
R
3
[nM]
H
3a
-Br
-OH
>100000
O
O
N
CN
H
2
9400
3
b
-Br
-Br
±450
O
O
CN
H
5
±
51.9
13.2
3c
O
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N
CN
COOH
OH
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
3.9
3
d
-Br
-Ph
-Ph
±
0.1
O
H
H
3e
-OH
>100000
O
O
N
CN
2
3800
130
3
f
±
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
Reagents and conditions: (A) for compound 1a where X is -Br: 5-chloro-2,4-dihydroxybenzaldehyde, NaHCO
54% for 3a) for compound 1b where X is -OH: 5-chloro-2,4-dihydroxybenzaldehyde, PPh , DIAD, THF, rt, overnight (20% for 3e) (B)
, DMF, rt, overnight (63% for 3b, 87% for 3c, 90% for 3f) (C) amine component, NaBH CN,
3
, ACN/DMF, rt, overnight
(
3
picolinonitrile/benzonitrile component, K
AcOH, DMF, rt, overnight (for 3d).
2
CO
3
3
The preparation of inhibitors based on 1e-g fragments comprised a similar synthetic pathway to previously described. After the Williamson
ether synthesis, a series of aldehydes (4a,g,i,m) or alcohol (4m) was isolated. The performance of sodium cyanoborohydride-mediated
reductive amination with various amine components led to the conversion of aldehydes (4a,g,i,m) into final compounds (4b,h,j,k,l). To
prepare the compound 4n, the conversion of (3-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)phenyl)methanol (4m) to the corresponding
chloride and a nucleophilic substitution with a thiomorpholine 1,1-dioxide was followed. Two other 1,1':2',1''-terphenyl- and 6-(2-
fluorophenyl)-2,3-dihydrobenzo[b][1,4]dioxine-based aldehydes (4c,e) were obtained by the potassium carbonated promoted O-alkylation
of 3-bromo-4-hydroxybenzaldehyde with 1b and 1d chlorides. The last step involved the NaBH
in potential inhibitors 4d and 4f.
3
CN-mediated reductive amination resulting
Table 3. General Pathway for the Synthesis of 6-(2-bromo-3-(bromomethyl)phenyl)-2,3-dihydrobenzo[b][1,4]dioxine (1g), 1,1':2',1''-
terphenyl (1b), 6-(3-(bromomethyl)-2-fluorophenyl)-2,3-dihydrobenzo[b][1,4]dioxine (1d), [1,1':2',1''-terphenyl]-3'-ylmethanol, 2-bromo-
3
-(bromomethyl)-3'-fluoro-1,1'-biphenyl (1f) and 2'-bromo-3'-(bromomethyl)-[1,1'-biphenyl]-4-carbonitrile (1e) based compounds 4a-n.
R3
O
R4
R5
R3
O
R4
R5
R2
R2
R2
A
B
R1
R1
X
1
b,d-g
4a,c,e,g,i,m
4b,d,f,h,j,k,l,n
IC50
[nM]
R
1
R
2
R
3
R
4
R
5
O
O
H
35400
±300
4
a
-Br
-Br
-Br
-H
-H
O
COOH
O
O
4
±
01.0
2.1
4
b
-Br
N
O
O
H
4c
-F
-F
-Br
-Br
-H
-H
>100000
O
O
O
H
N
22.7
±1.0
4
d
N
H
O
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Journal of Medicinal Chemistry
H
4
e
-Ph
-Ph
-Ph
-Ph
-Br
-Br
-H
-H
>100000
1
2
3
4
5
6
7
8
9
O
COOH
2
9400
450
N
4f
±
OH
O
CN
H
4
g
-Br
-Br
-Br
-Br
-Br
-Br
-H
-H
-H
>100000
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CN
H
N
5114
±58
4
h
N
H
O
H
4
i
~55000
O
F
F
COOH
65.3
±0.7
4
j
-Br
-Br
N
-H
H
N
106.8
±20.8
4
k
-Br
-Br
-Br
-Br
-Br
-Br
-H
N
H
-H
-H
F
F
F
F
O
O
4
238
S
O
4
l
N
±86
4m
-H
OH
>100000
22300
O
S
O
4
n
-H
-H
N
±
230
Reagents and conditions: (A) for compounds 1e-g where X is -Br: substituted 4-hydroxybenzaldehde, K
a, 61% for 4g, 57% for 4i, 52% for 4m) for compounds 1b, 1d where X is -OH: 1. SOCl , DCM, 40 °C 2. substituted 4-hydroxybenzaldehde,
CO , DMF, rt, overnight (30% for 4c, 60% for 4e) (B) for compounds 4a,c,e,g,i: amine component, NaBH CN, AcOH, DMF, rt, overnight
43% for 4b, 61% for 4d, 37% for 4f, 26% for 4h, 44% for 4j, 67% for 4k, 33% for 4l) for compounds 4m 1. SOCl , DCM, 40 °C 2. amine
component, K CO , DMF, rt, overnight (29% for 4n).
2
CO
3
, DMF, rt, overnight (88% for
4
K
(
2
2
3
3
2
2
3
Because of the presence of two halogens and the solubilizer group, none of our best compounds passed Lipinski’s rule of five, mainly due
to the violation of mass and/or hydrophobicity requirements as calculated with the software Chemicalize (Table S1). However, the final
3
compound 2k showed the most promising overall parameters being 90 Da smaller and 100 Å Van der Waals volume difference as compared
to the second-best 3d. Compounds 2k, 2j and 2h were additionally subjected to the competitive enzyme-linked immunosorbent assay
(competitive ELISA) to compare their ability to dissociate human PD-1/PD-L1 complex compared to BMS-1166 (a potent PD-1/PD-L1
inhibitor from Bristol-Myers Squibb company) (Figure S2). According to the results, compound 2k is a stronger inhibitor compared to BMS-
1
166 (1.47 ±0.05 nM and 28.04 ±0.36 nM, respectively) further proving the potency of our final product.
NMR-binding assay
1
The direct binding of most prominent compounds 2k and 3d to the human PD-L1 was also confirmed with a H NMR method
(
Figure S3). In the assay, the addition of the tested molecules 2k and 3d caused a characteristic dimerization of hPD-L1 that we observed
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2
7
1
for the biphenyl compounds previously , evidenced by the broadening of the NMR peaks in the aliphatic region of the H NMR spectrum
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compared to the NMR spectrum of the apo hPD-L1.
Cell-based assay
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3 The activity of modified bromo compounds in PD-1/PD-L1 immune checkpoint blockade cell-based assay. Activities of the
reference anti-PD-L1 antibody durvalumab and tested compounds in alleviating the effect of PD-1/PD-L1 checkpoint on TCR-mediated
effector Jurkat T cells are expressed as fold induction of the luciferase activity, which expression is TCR-inducible (for details please see
Materials and Methods). The graphs present relative luminescence normalized to DMSO- treated controls and are mean +/- SEM values
from 4-7 independent experiments.
The most prominent compounds from HTRF assay were tested in the PD-1/PD-L1 immune checkpoint blockade cell-based assay. In the
assay, the PD-1/PD-L1 interaction is provided by the co-cultured artificial antigen-presenting cells (aAPCs) and Jurkat reporter T cells. The
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Journal of Medicinal Chemistry
compounds that disrupt this interaction increase a TCR-mediated activation of the Jurkat cells, evidenced by the increased activity of
luciferase. The luciferase activity thus reflects the activation status of the Jurkat T cells.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Durvalumab, a clinically relevant PD-L1-blocking antibody, released the TCR signaling with the EC50 value of 0.2±0.06 nM, as
revealed by the fitting of the Hill’s equation to the experimental dataset (Figure 3). All the tested compounds were also able to increase the
activation of the effector cells, but at considerably higher concentration ranges than durvalumab. For Hill’s equation fitting, the maximal
response to durvalumab was set as a maximal allowed activation of the Jurkat effector cells, since for the other compounds the activation
plateau could not be reached. Out of the tested compounds, the compound 2k provided the highest level of the Jurkat T cell activation (2.1-
fold at 50 µM concentration of a compound in the assay) and the lowest EC50 value of 6.6±0.8µM (Figure 3). This activation was much
better than the activation observed for the patented compound 3d (EC50 value of 12.2±1.4µM), for which solubility problems in DMSO/water
conditions were observed, disallowing data collection for the two highest concentrations. The compound 2g showed similar activity to 2k
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(EC50 value of 6.8±1.1µM). However, due to the toxicity of the compound 2g, observed above 10 µM concentration, the value had to be
estimated based on partial data. Other compounds exhibit EC50 > 40 µM and low activation levels.
Final compound 2k was also found not to be toxic up to 100 µM final concentration in cell viability assay (Figure S4). Compared
to the BMS compounds tested in Skalniak et. al, (2017) Figure 2a 27 this is a significantly better result, as the potent BMS-1166 decreased
the viability of the cells by 50% at 40 µM concentration, while other BMS compounds at even lower inhibitor concentrations.
The biological activity of the compound 2k was further verified on the primary T cells. For this, peripheral blood mononuclear
cells (PBMCs) isolated from the blood of healthy donors were contacted with hPD-L1 aAPCs to provide TCR-mediated activation of T cells
and the engagement of PD-1/PD-L1 immune checkpoint. The cells were then analyzed with flow cytometry following the gating for CD4+
+
and CD8 T cells. The blockade of PD-1/PD-L1 immune checkpoint was expected to increase the activation of T cells. In the experiment,
atezolizumab and compound 2k were used. Neither of the two molecules was able to increase the expression of an early T-cell activation
marker, CD69 (not shown). Since the prolonged activation of T cells leads to the increase of the expression of immune checkpoint
molecules,30 we have monitored the expression of PD-1 protein itself as an indicator of the late T cell activation and exhaustion. In fact, the
treatment with both the control antibody atezolizumab and the compound 2k led to a significant increase of the expression of PD-1 receptor
+
+
on the surface of both, CD4 and CD8 T cells (Figure 4). This confirms the bioactivity of the compound 2k in the context of primary T
cells.
1
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4 The compound 2k blocks the PD-L1/PD-1 immune checkpoint in primary human T cells, as evidenced by the increased
expression of the late activation and exhaustion marker, PD-1. Peripheral blood mononuclear cells (PBMCs) were seeded on the pre-
seeded hPD-L1 aAPCs and cultured for 48 hours alone or in the presence of 2k, DMSO (as a solvent for 2k), or atezolizumab (atezo., an
anti-PD-L1 therapeutic antibody). The expression of PD-1 was tested with flow cytometry. (A) Exemplary histograms, presenting the
increased expression of PD-1 on either the CD4+, or CD8+ T cells. (B) The analysis of the expression of PD-1 on CD4+, or CD8+ T cells
under control conditions and in the presence of PD-L1-blocking molecules, the antibody atezolizumab, and the compound 2k. Graphs
present mean ± SD values calculated for three independent experiments performed with the engagement of different donors. Statistical
significance was analyzed with the Student’s t-test: * p<0.05, ** p<0.01.
Cross-reactivity of the PD-L1/PD-1-blocking small-molecules for human and murine PD-L1
Several compounds based on the biphenyl core were subjected to the MiscroScale Thermophoresis (MST) experiment to determine their
cross-reactivity towards murine and human PD-L1’s. We used both the human (for the positive control) and the murine PD-L1 for 2k, 2i,
2
g, and BMS-1166, and the murine anti-PD-L1 antibody MIH-5 as the positive control for murine PD-L1 (Figure S5 panels A, B, C, D, E,
respectively). The experiment showed that the compounds based on the biphenyl moiety were not active against the murine PD-L1, as they
did not cause any changes in the fluorescence spectrum upon addition to the protein solution, whereas they have a clear pattern when tested
D
against the human PD-L1, with 2k and 2g being the tighter binders (the dissociation constants K of 4.0 and 3.2 nM, respectively) than
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BMS-1166 (the K
D
of 30.3 nM)(Table S2). This MST result disqualifies the biphenyl-based compounds for the experiments on the syngeneic
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mice as there is no cross-reactivity between human and murine targets.
Modeled interactions with dimeric PD-L1
To explore the molecular interactions of most potent
compounds 2k and 3d with PD-L1, we docked the compounds into
a dimeric PD-L1 using the PyRx software. To refine the docking
results, residues interacting with those compounds were allowed
flexibility in their side chains. Then, the interactions were assessed
using Protein-Ligand Interaction Profiler (Figure 5).31 The core of
the scaffold for both molecules is the same, with the biphenyl
structure positioning the molecules with its strong and conserved
stabilizing π-π stacking with Tyr56A and an aromatic phenyl ring
with Tyr56B. The hydrogen bonding of 2k comes from the
interactions between the Tyr56B hydroxyl group and nitrogen atom
from pipecolic acid moiety and Asn63B amide nitrogen and oxygen
from the carboxylic group of a solubilizer. For 3d, hydrogen bonds
are predicted between cyanopyridine nitrogen with Arg125A
nitrogen and nitrile group nitrogen with Asn63B nitrogen. Clever
usage of solubilizer in 2k allows for a great reduction in molecule
size by 90 Da, down to 557 Da (close to the Lipinski’s size rule
requirement <500 Da). Moreover, the tertiary amine in solubilizers
of both 3d and 2k is predicted to be involved in salt bridges with
Asp122A negatively charged carboxyl group stabilizing the whole
complex. Both compounds are also involved in several hydrophobic
interactions with residues Ile54A Tyr56A and B, Gln66B, Met115B,
and either Ala121A for 2k or Ala121B for 3d, as well as and
Tyr123A that comes from the main scaffold (2a).
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Figure 5 Interactions between PD-L1 and 3b (top) and 2k (bottom)
from modeled complexes (compounds were docked on PD-L1 taken
from PDB ID: 5NIX). Both compounds are involved in a number of
hydrophobic interactions (gray dashed lines) with residues Ile54A
Tyr56A and B, Gln66B, Met115B, Ala121A for 3b and Ala121B
for 2h and Tyr123A. There are strong conserved stabilizing π-
stacking (green dashed line) between distal phenyl ring from
biphenyl moiety and Tyr56A and phenyl form phenoxymetyl with
Tyr56B. The major differences are in hydrogen bonds (blue solid
line) which for 2k are between Tyr56B hydroxyl group and tertiary
amine in piperidine solubilizer and Asn63B amide nitrogen and
oxygen from carboxylic group of solubilizer. For 3b, hydrogen
bonds are between cyanopyridine nitrogen in with Arg125A
nitrogen and nitrile group nitrogen with Asn63B nitrogen.
Conclusions
Herein we demonstrate a new, potent compound that can dissociate the PD-1/PD-L1 complex, with the potency of inciting adaptive immune
system and activating cancer-reactive T cells. Our final compound 2k is significantly smaller than other small molecule inhibitors reported
in the literature. The design process was based on the novel methodology for the identification of potent small-molecular fragments – w-
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AIDA-NMR as the starting block for further extensions. Tested fragments were not able to dissociate native PD-1/PD-L1 complex in AIDA-
NMR (Figure S1) proving the usefulness of w-AIDA-NMR in the identification of small potent fragments for inhibitors.
w-AIDA-NMR offers unambiguity of the results, due to the simplest system possible – protein and compound in the buffer of your choice,
and ensures that the core fragment is capable of dissociating the complex. Moreover, using this technique one can instantly spot compounds
that are precipitating or aggregating target protein as well as control the solubility of the tested fragment – a common issue in other
biophysical assays. Since it is not based on the modification of light, it will not generate “false-positives”, significantly reducing time and
cost commitment in developing “falsified hits”. Each intermediate step was validated with the HTRF affinity determination and later with
the cell-based assay. Final compound 2k was shown to be more potent than other molecules tested here and able to activate Jurkat cells to a
degree close to the control antibody. In fact, 2k is the strongest small-molecule inhibitor of PD-L1 reported in the literature including potent
BMS-1166 and BMS-1001.27 Thanks to its enhanced solubility and small size, 2k is neutral to cell viability even at a 100 µM concentration.
Due to the lack of cross-reactivity between human and murine PD-L1 for biphenyl-based compounds, we were not able to perform
experiments on mice. However, we were able to prove the bioactivity of the compound 2k in the experiments with primary T cells isolated
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from the blood of healthy donors by monitoring the expression of PD-1 receptor on the surface of both, CD4 and CD8 T cells.
Experimental Section
Protein Expression and Purification
All chemicals used for protein expression and purification were purchased from BioShop (Bioshop Canada Inc., Burlington, ON, Canada)
and Sigma Aldrich (Sigma Aldrich Corporation, St. Louis, MO, USA) and were used without any additional purification. The IgV domains
of human and mouse PD-L1 protein (hPD-L1 residues: 18-134, C-terminal His-tag; mPD-L1 residues: 19-134) and the extracellular domain
2
4
of human PD-1 (hPD-1 residues 34-150, C93S) were expressed and purified as described previously . Proteins were expressed in the
Escherichia coli BL21 (DE3). Bacterial cells were cultured at 37 °C in LB or M9 minimal medium containing 15NH
Cl as the sole nitrogen
4
source to achieve 15N isotope labeling. Proteins expression was induced with 1 mM Isopropyl β-d-1-thiogalactopyranosid (IPTG) at OD600
of 0.8 and the cells were cultured overnight. For hPD-1, temperature was lowered to 28 °C. The inclusion bodies purification was carried
out as described previously.32 Afterwards proteins were refolded by drop-wise dilution into solution containing 0.1 M Tris pH 8.0, 0.4 M l-
Arginine hydrochloride, 2 mM EDTA, 5 mM cystamine and 0.5 mM cysteamine for hPD-1 and 0.1 M Tris pH 8.0, 1 M l-Arginine
hydrochloride, 0.25 mM oxidized glutathione and 0.25 mM reduced glutathione for hPD-L1. After refolding, proteins were dialyzed 3 times
against solution containing 10 mM Tris pH 8.0 and 20 mM NaCl. Finally, proteins were purified by SEC (size-exclusion chromatography)
on HiLoad 26/600 Superdex 75 column (GE Healthcare, Chicago, IL, USA) in 25mM sodium phosphate pH 6.4 with 100 mM NaCl for
hPD-1, N66A hPD-1 mutant or in PBS pH 7.4 for hPD-L1, hPD-L1(18-239).
AIDA-NMR
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Uniform 15N labelling was obtained by expressing proteins in the M9 minimal medium containing 15NH
v/v) of D O was added to the samples to provide the lock signal. All spectra were recorded at 300K using a Bruker Avance III 600 MHz
Cl as the sole nitrogen source. 10%
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(
2
spectrometer equipped with the nitrogen cryoprobe. The ability of tested compounds to dissociate PD-L1/(wild type - wt or N66A)PD-1 was
15
evaluated using the (Weak) Antagonist Induced Dissociation Assay (w-AIDA-NMR). In brief, N-labeled wt- and N66A PD-1 (0.15 mM)
1
was slightly overtitrated with the unlabeled PD-L1. The compounds were aliquoted into the resulting mixture. During the experiment the H-
15N signals were monitored by the SOFAST HMQC.
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Homogenous Time-Resolved Fluorescence
The HTRF assay was performed using the certified Cis-Bio assay kit at 20 µL final volume using their standard protocol (5 nM of h
PD-L1 and 50 nM of hPD-1 in the final formulation). To determine the half maximal inhibitory concentration (IC50) of tested compounds,
measurements were performed on individual dilution series. After mixing all components according to Cis-Bio protocol, the plate was left
for 2h incubation at room temperature followed by TR-FRET measurement on Tecan Spark 20M. Collected data was background subtracted
on the negative control, normalized on the positive control, averaged and fitted with normalized Hill’s equation to determine the IC50 value
using Mathematica 12.
Cell Culture
CHO K-1 cells overexpressing hPD-L1 and the recombinant TCR ligand (hPD-L1 Antigen Presenting Cells, hPD-L1 aAPCs) (Promega,
Madison, WI, USA) and Jurkat T cells overexpressing hPD-1 and carrying a luciferase reporter gene under the control of Nuclear Factor of
Activated T-cells Response Element (NFAT-RE) (hPD-1 Effector Cells, hPD-1 ECs, Promega) were cultured in RPMI-1640 medium
(Biowest, Billerica, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Biowest) and 200 mM L- Glutamine (Biowest) in the
presence of G418 (250 µg/mL, InvivoGen, San Diego, CA, USA) and Hygromycin B Gold (50 µg/mL, InvivoGen) as selection antibiotics.
The overexpression of hPD-L1 and TCR ligand in aAPCs and PD-1 in ECs were confirmed by flow cytometry and western blot analysis,
respectively. PCR tests for Mycoplasma sp. contamination 33 were routinely performed and indicated negative results for both cell lines.
hPD-1/hPD-L1 Immune Checkpoint Blockade Assay
The activity of the inhibitors of hPD-1/hPD-L1 immune checkpoint was examined using the hPD-1/hPD-L1 Blockade Bioassay (Promega),
according to the manufacturer’s instructions. hPD-L1 aAPCs were seeded on 96-well (white) plates at the density 10 000 cells/well 17h prior
to the experiment. The 2.5-fold dilutions of the small molecules were first prepared in DMSO. On the day of the assay the compounds were
diluted 1000-fold in the assay buffer (99% RPMI 1640, 1% FBS) to maintain the constant concentration of DMSO (0.1% of total volume).
The 2.5-fold dilutions of durvalumab, a positive control anti-hPD-L1 monoclonal antibody (Imfinzi, Medimmune/AstraZeneca), were
prepared in the assay buffer on the day of the assay. The culture medium was discarded from the wells and serial dilutions of either the small-
molecule or antibody was added. Afterwards, Jurkat hPD-1 cells were seeded at the density of 20,000 cells per well in the assay’s plates.
After 6h of the incubation in standard culture conditions, assay plates were equilibrated at ambient temperature for 10min, followed by a
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0min incubation with the Bio-GloTM Assay reagent (Promega). The luminescence was detected using the Spark microplate reader (Tecan).
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Half maximal effective concentrations (EC50 values) were calculated from the Hill’s curve fitting to the experimental data
The isolation of peripheral blood mononuclear cells (PBMCs)
Blood samples from healthy donors were purchased from the Regional Center of Blood Donation and Blood Therapy in Krakow. PBMCs
were isolated by a density gradient centrifugation using Pancoll human separating solutions (PAN-Biotech) and then resuspended in RPMI
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640 medium (Biowest) containing 10% FBS (Biowest).
Flow cytometry analysis of activation markers
PBMCs were added to the pre-seeded aAPCs at the density 400 000 cells/well and incubated in the presence of: the compound 2k, DMSO,
and with or without atezolizumab (5 µg/ml) as an anti-PD-L1 monoclonal antibody. After 48 hours, cells were detached using TrypLe Select
Enzyme (Thermo Fisher Scientific), collected into 5 mL round bottom tubes and washed with flow cytometry staining buffer (Thermo Fisher
Scientific). For flow cytometry analysis, cells were stained using monoclonal antibodies: anti-CD69-APC, anti-CD4-FITC, anti-CD8-BV510
and anti-PD1-PECy7 (Becton Dickinson, BD). Following by 20 min incubation at room temperature, cells were washed, resuspended in PBS
and analyzed using FACSCanto II flow cytometer (BD) and FACSDiva software (BD). The data analysis was carried out with FACSuite
software (BD).
Molecular docking and in silico screening
The compounds were generated as a permutation of all interesting to us functional groups on biphenyl scaffold (over 1500 structures), which
were then minimized using OpenBabel software. For molecular docking of putative inhibitors we used AutoDock Vina28 integrated into
PyRx with dimeric structure of PD-L1 with BMS-1166 bound (PDB ID:5NIX). Based on the scoring results and feasibility of chemical
synthesis we selected the most interesting compounds.
Chemical Synthesis of Compounds
General Information
Starting materials and solvents were received from Sigma Aldrich, Cool Pharm, Idalia and Alfa Aesar and they were used without additional
purification. H and 13CNMR spectra were recorded on Bruker Avance 600 MHz spectrometer with report of chemical shifts (δ) in ppm and
1
coupling constants (J) in Hz. TMS was used as reference and an internal standard with singlet at δ 0 ppm. Moreover chemical shifts were
6 3
analyzed in correspondence to the solvent peaks (DMSO-d , CDCl ).
Infrared spectra were recorded for the solid samples on a Nicolet IR200 spectrometer due to ATR technique. High-resolution mass
spectrometry (HRMS) analysis were performed on a microTOF-QII apparatus using the ESI ionization mode. Purification of compounds
was conducted thanks to flash chromatography on the Grace Reveleris X2 Flash Chromatography System with the Grace Resolv Silica
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Cartridges. The UPLC-MS’s were recorded on the TQD Waters H-Class spectrometer (column: ACQUITY UPLC BEH C18 1.7um, 2.1 x
0 mm, method: 6 min, gradient: 0-3 min 80% H O - 20% MeCN, 3-3.5 min 100% MeCN, 3.5-6 min 80% H O- 20% MeCN). All final
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compounds were determined to have at least 95 % purity. Thin-layer chromatography (TLC) was performed thanks to use of aluminum
sheets pre-coated Silica Gel 60 F254 (Merck). Techniques of visualization of TLC plates included using an UV lamp with radiation at 254
nm wavelength. Melting points were determined with an Ascon-M5 apparatus.
Synthesis of substrates and preparation of short fragments 1a-1g are described in the Supporting Information.
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4
-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzaldehyde (2a)
1
a (200 mg, 0.62 mmol), 4-hydroxybenzaldehyde (75 mg, 0.62 mmol), potassium carbonate (170 mg, 1.23 mmol) were stirred in anhydrous
DMF (3 mL) at room temperature overnight. The solvent was removed under reduced pressure. Water was added (30 mL) and mixture was
extracted with AcOEt (2x30 mL). Organic layers were combined and concentrated. Crude product was purified by column chromatography
1
on silica gel (0-100% AcOEt in hexane) and crystalized from cyclohexane to yield 2a (97 mg, 43%) as white solid. H NMR (600 MHz,
CDCl
.30 (s, 2H). 13C NMR (151 MHz, CDCl
) δ: 190.8, 163.4, 143.7, 141.1, 136.1, 132.1, 130.9, 130.4, 129.4, 128.1, 127.8, 127.5, 127.4, 122.6,
3
3
) δ: 9.91 (s, 1H), 7.90 – 7.86 (m, 2H), 7.54 – 7.51 (m, 1H), 7.47 – 7.37 (m, 6H), 7.31 (dd, J = 7.5, 1.7 Hz, 1H), 7.15 – 7.12 (m, 2H),
5
-1
115.2, 70.4. IR (ATR): 2916, 2849, 2835, 1684, 1598, 1591 1508, 1422, 1260, 1164, 1046 cm . HRMS ESI-MS-q-TOF for C20
H
15BrO
2
+
+
[
M + Na] found: 389.0148 m/z; Calc. Mass: 389.0153. Mp: 87.7 °C. UPLC-MS (DAD/ESI): t
R
= 8.78 min, for C20
H
15BrO
2
[M + H] found:
3
67.18 m/z; Calc Mass: 367.03.
3-bromo-4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzaldehyde (2b)
Compound 2b was prepared following the procedure for 2a. Preparation involved the use of 1a (220 mg, 0.68 mmol), 3-bromo-4-
hydroxybenzaldehyde (136 mg, 0.68 mmol) and potassium carbonate (187 mg, 1.36 mmol), which were stirred in anhydrous DMF (3 mL)
at room temperature overnight. Crude product was purified by column chromatography on silica gel (0-100% AcOEt in hexane) and yielded
1
2
b (119 mg, 39%) as white solid. H NMR (600 MHz, CDCl
3
) δ: 9.87 (s, 1H), 8.15 (d, J = 2.0 Hz, 1H), 7.83 (dd, J = 8.4, 2.0 Hz, 1H), 7.69
(
d, J = 7.7 Hz, 1H), 7.49 – 7.37 (m, 6H), 7.32 (dd, J = 7.5, 1.5 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H), 5.36 (s, 2H). 13C NMR (151 MHz, CDCl
δ: 189.7, 159.5, 143.6, 141.1, 135.7, 134.8, 131.3, 131.3, 130.9, 129.6, 128.2, 127.9, 127.7, 127.2, 121.9, 113.4, 113.1, 71.1. IR (ATR): 3082,
3
)
-1
+
2923, 2852, 1683, 1593, 1492, 1276, 1256, 1185, 1050 cm . HRMS ESI-MS-q-TOF for C20
2 2
H14Br O [M + Na] found: 466.9249 m/z; Calc.
+
Mass: 466.9258. Mp: 151.2 °C. UPLC-MS (DAD/ESI): t = 9.37 min, for C20 [M + H] found: 447.07 m/z; Calc Mass: 446.94.
R
2 2
H14Br O
4
-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)-3-methylbenzaldehyde (2c)
Compound 2c was obtained according to the procedure of 2a, using 1a (200 mg, 0.62 mmol), 4-hydroxy-3-methylbenzaldehyde (84 mg,
0
.62 mmol), potassium carbonate (170 mg, 1.23 mmol) and anhydrous DMF (3 mL). Crude product was purified by column chromatography
1
on silica gel (0-100% AcOEt in hexane) and was crystalized from cyclohexane to yield 2c (90 mg, 38%) as white solid. H NMR (600 MHz,
CDCl
.7 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 5.30 (s, 2H), 2.41 (s, 3H). 13C NMR (151 MHz, CDCl
) δ: 191.3, 161.7, 143.7, 141.3, 136.6, 131.8,
3
3
) δ: 9.88 (s, 1H), 7.76 (d, J = 1.1 Hz, 1H), 7.73 (dd, J = 8.3, 2.0 Hz, 1H), 7.57 – 7.53 (m, 1H), 7.47 – 7.38 (m, 6H), 7.32 (dd, J = 7.5,
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30.8, 130.1, 129.6, 128.2, 128.1, 127.9, 127.5, 127.3, 122.5, 111.2, 70.4, 16.7. IR (ATR): 2821, 1679, 1599, 1260, 1240, 1125, cm . HRMS
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+
ESI-MS-q-TOF for C21
min, for C21 17BrO
H
17BrO
2
[M + Na] found: 403.0304 m/z; Calc. Mass: 403.0309. Mp: 144.5 °C. UPLC-MS (DAD/ESI): t
R
= 9.31
+
H
2
[M + H] found: 381.20 m/z; Calc Mass: 381.05.
4
-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)-3-methoxybenzaldehyde (2d)
Compound 2d was prepared following the procedure for 2a. Preparation involved the use of 1a (300 mg, 0.93 mmol), 4-hydroxy-3-
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methoxybenzaldehyde (141 mg, 0.93 mmol) and potassium carbonate (255 mg, 1.85 mmol), which were stirred in anhydrous DMF (3 mL)
at room temperature overnight. Crude product was purified by column chromatography on silica gel (0-100% AcOEt in hexane) and yielded
1
2d (192 mg, 52%) as white solid. H NMR (600 MHz, CDCl
3
) δ: 9.87 (s, 1H), 7.53 (ddd, J = 10.8, 5.8, 5.0 Hz, 1H), 7.49 – 7.35 (m, 8H),
) δ: 191.1, 153.4, 150.2, 143.6,
41.3, 136.2, 130.8, 130.7, 129.6, 128.2, 127.8, 127.6, 127.3, 126.8, 122.2, 112.6, 109.6, 71.0, 56.3. IR (ATR): 2916, 2850, 2834, 1683,
7
1
.29 (dd, J = 7.5, 1.6 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 5.37 (s, 2H), 4.00 (s, 3H). 13C NMR (151 MHz, CDCl
3
-1
[M + Na]+ found: 419.0253 m/z; Calc.
1588, 1507, 1421, 1265, 1235, 1134, 1031 cm . HRMS ESI-MS-q-TOF for C21
Mass: 419.0259. Mp: 109.0 °C. UPLC-MS (DAD/ESI): t = 8.50 min, for C21
H
17BrO
3
+
R
H
17BrO
3
[M + H] found: 397.22 m/z; Calc Mass: 397.04.
methyl 3-bromo-4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzoate (2e)
Compound 2e was prepared according to the protocol for 2a. Preparation involved the use of 1a (200 mg, 0.61 mmol), methyl 3-bromo-4-
hydroxybenzoate (142 mg, 0.61 mmol) and potassium carbonate (169 mg, 1.22 mmol), which were stirred in anhydrous DMF (3 mL) at
1
room temperature overnight. Crude product was purified by precipitation from AcOEt to give 2e (131 mg, 45%) as white solid. H NMR
(600 MHz, CDCl
3
) δ: 8.30 (d, J = 2.1 Hz, 1H), 7.99 (dd, J = 8.6, 2.1 Hz, 1H), 7.71 – 7.68 (m, 1H), 7.47 – 7.38 (m, 6H), 7.30 (dd, J = 7.5,
1
1
1
.7 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 5.32 (s, 2H), 3.90 (s, 3H). 13C NMR (151 MHz, CDCl
3
) δ: 165.7, 158.3, 143.4, 141.1, 135.9, 135.0,
30.6, 129.5, 128.0, 127.7, 127.5, 127.1, 124.3, 121.8, 112.5, 112.1, 70.8, 52.2. IR (ATR): 2948, 2163, 1981, 1781, 1599, 1498, 1431, 1363,
-1
+
311, 1269, 1240, 1108, 1054 cm . HRMS ESI-MS-q-TOF for C21
2 3
H16Br O [M + Na] found: 496.9447 m/z; Calc. Mass: 496.9364. Mp:
+
160.3 °C. UPLC-MS (DAD/ESI): t
R
= 9.99 min, for C21 [M + H] found: 475.12 m/z; Calc. Mass: 474.95.
2 3
H16Br O
4
-(3-(3-bromo-4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzamido)propyl)morpholin-4-ium hydrochloride (2f)
2e (100 mg, 0.21 mmol) and DBU (350 µL) were stirred in 3-morpholinopropan-1-amine (1.5 mL) at room temperature for 2 days.
Afterwards, reaction mixture was solubilized with AcOEt (30 mL). Followingly, water was added (30x2 mL) to wash out the excess of amine
from organic layers. Organic layers were dried, combined and concentrated. Purification involved column chromatography on silica gel (0-
1
0% CHCl
3
in MeOH). After purification, compound was converted into the corresponding hydrochloride salt and product was precipitated
1
from AcOEt to give 2f (30 mg, 25%) as white solid. H NMR (600 MHz, CDCl
3
) δ: 8.14 (t, J = 4.0 Hz, 1H), 8.06 (d, J = 2.2 Hz, 1H), 7.82
(
dd, J = 8.6, 2.2 Hz, 1H), 7.73 – 7.69 (m, 1H), 7.48 – 7.37 (m, 5H), 7.30 (dd, J = 7.5, 1.7 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 5.31 (s, 2H),
3
.78 (t, J = 4.6 Hz, 4H), 3.57 (dd, J = 10.9, 5.8 Hz, 2H), 2.58 (t, J = 5.7 Hz, 2H), 2.53 (s, 4H), 1.84 – 1.74 (m, 2H). 13C NMR (151 MHz,
) δ: 165.6, 157.1, 143.5, 141.2, 136.2, 132.1, 130.7, 129.6, 129.0, 128.2, 128.2, 127.8, 127.6, 127.2, 121.9, 113.0, 112.3, 70.9, 67.1,
CDCl
3
-1
59.2, 54.1, 41.1, 23.9. IR (ATR): 3460, 3299, 2951, 2856, 2822, 1626, 1604, 1553, 1497, 1285, 1267, 1118, 1056 cm . HRMS ESI-MS-q-
1
7
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Journal of Medicinal Chemistry
TOF for [C27
H
29Br
2
N
2
O
3
]+ [M] found: 587.0556 m/z; Calc. Mass: 587.0545. Mp: 128.9 °C. UPLC-MS (DAD/ESI): t
= 6.75 min, for
R
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[
C
27
H
29Br N O
2 2 3
]+ [M] found: 587.12 m/z; Calc Mass: 587.05.
N-(2-((3-bromo-4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)ethyl)acetamide (2g)
A solution of 2b (200 mg, 0.45 mmol), N-(2-aminoethyl)acetamide (209 mg, 2.05 mmol) and AcOH (3 droplets) was stirred in anhydrous
DMF (4 mL) at 25 °C for 2 h. Then NaBH CN (132 mg, 2.24 mmol) was added and resulted mixture was stirred additionally for 20 h. The
residue was concentrated under reduced pressure. Water was added (30 mL) and mixture was extracted with AcOEt (2x30 mL). Organic
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
layers were combined, dried over MgSO
0-30% MeOH in CHCl
4
, filtered and concentrated. The crude product was purified by column chromatography (silica gel,
1
3
) to give off-white solid (126 mg, yield: 53%) as a product. H NMR (600 MHz, DMSO) δ: 7.84 (s, 1H), 7.66 (dd, J
=
7.6, 1.6 Hz, 1H), 7.62 (d, J = 1.9 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.49 – 7.45 (m, 2H), 7.44 – 7.30 (m, 5H), 7.17 (d, J = 8.5 Hz, 1H), 5.26
(
s, 2H), 3.68 (s, 2H), 3.14 (q, J = 6.3 Hz, 2H), 2.55 (t, J = 6.5 Hz, 2H), 1.79 (s, 3H). 13C NMR (151 MHz, DMSO) δ: 169.7, 153.6, 143.4,
141.2, 137.0, 133.2, 131.3, 129.7, 129.3, 128.8, 128.6, 128.2, 122.9, 114.2, 111.4, 71.0, 51.5, 48.2, 38.7, 23.1. IR (ATR): 3280, 3051, 2937,
-
1
+
2
829, 2659, 1654, 1556, 1499, 1445, 1369, 1282, 1259, 1056 cm . HRMS ESI-MS-q-TOF for C24
m/z; Calc. Mass: 531.0283. Mp: 142.9 °C. UPLC-MS (DAD/ESI): t = 6.13 min, for C24 [M + H] found: 531.22 m/z; Calc Mass:
31.03.
2 2 2
H24Br N O [M + H] found: 531.0274
+
R
2 2 2
H24Br N O
5
(S)-1-(4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxylic acid (2h)
3
Compound 2h was prepared following the protocol of 2g, using 2a (90 mg, 0.25 mmol), L-pipecolinic acid (145 mg, 1.22 mmol), NaBH CN
(72 mg, 1.23 mmol), AcOH (3 droplets) and DMF (4 mL). The crude product was purified by flash chromatography (silica gel, 0-50% MeOH
1
in CHCl
3
) to give white solid (77 mg, yield: 65%) as a product. H NMR (600 MHz, DMSO) δ: 7.59 (d, J = 7.1 Hz, 1H), 7.54 – 7.25 (m,
9
H), 7.04 (d, J = 7.9 Hz, 2H), 5.19 (s, 2H), 4.01 (d, J = 11.9 Hz, 1H), 3.11 (s, 1H), 2.98 (s, 1H), 2.40 (s, 1H), 1.87 (s, 1H), 1.69 (s, 1H), 1.61
1.43 (m, 3H), 1.35 (s, 1H). 13C NMR (151 MHz, DMSO) δ: 158.4, 143.5, 141.3, 137.2, 131.8, 131.4, 129.7, 129.5, 128.6, 128.2, 123.4,
–
115.0 , 79.6, 70.3, 58.3, 49.6, 28.5, 23.9, 22.2. IR (ATR): 3055, 2940, 2862, 2352, 2251, 1610, 1513, 1447, 1413, 1386, 1242, 1180, 1028
-
1
+
cm . HRMS ESI-MS-q-TOF for C26
H
3
26BrNO [M + H] found: 480.1169 m/z; Calc. Mass: 480.1174. Mp: 182.5 °C. UPLC-MS (DAD/ESI):
+
t
R
= 6.18 min, for C26 26BrNO
H
3
[M + H] found: 480.24 m/z; Calc Mass: 480.12.
(S)-1-(4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)-3-methylbenzyl)piperidine-2-carboxylic acid (2i)
Compound 2i was obtained according to the procedure of 2g, using 2c (90 mg, 0.24 mmol), L-pipecolinic acid (140 mg, 1.08 mmol),
NaBH
3
CN (69 mg, 1.18 mmol), AcOH (3 droplets) and DMF (4 mL). The crude product was purified by flash chromatography (silica gel,
1
0-50% MeOH in CHCl
3
) to give yellow solid (65 mg, yield: 56%) as a product. H NMR (600 MHz, CDCl
3
) δ: 7.50 (d, J = 7.5 Hz, 1H),
7
1
1
.44 – 7.21 (m, 9H), 6.83 (d, J = 7.0 Hz, 1H), 5.06 (s, 2H), 4.39 (d, J = 55.8 Hz, 2H), 3.42 (d, J = 55.1 Hz, 2H), 2.62 (s, 1H), 2.28 (s, 3H),
.96 (d, J = 9.0 Hz, 2H), 1.85 – 1.69 (m, 2H), 1.37 – 1.20 (m, 2H). 13C NMR (151 MHz, CDCl
3
) δ: 157.8, 143.5, 141.3, 137.0, 134.3, 130.9,
30.6, 129.6, 128.1, 127.9, 127.8, 127.3, 127.2, 122.4, 111.6, 70.1, 58.5, 50.9, 29.8, 28.2, 22.8, 22.1, 16.7. IR (ATR): 2943, 2862, 2390,
1
8
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Journal of Medicinal Chemistry
Page 24 of 35
-1
+
2
250, 1613, 1505, 1447, 1410, 1288, 1258, 1137, 1003 cm . HRMS ESI-MS-q-TOF for C27
H
3
28BrNO [M + H] found: 494.1325 m/z; Calc.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
Mass: 494.1330. Mp: 106.3 °C. UPLC-MS (DAD/ESI): t = 6.54 min, for C27 28BrNO
R
H
3
[M + H] found: 494.26 m/z; Calc Mass: 494.1330.
(
S)-1-(4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)-3-methoxybenzyl)piperidine-2-carboxylic acid (2j)
Compound 2j was obtained as described for 2g. Preparation involved the use of 2d (100 mg, 0.25 mmol), L-pipecolinic acid (149 mg, 1.15
mmol), NaBH
3
CN (74 mg, 1.26 mmol), AcOH (3 droplets) and DMF (4 mL). The crude product was purified by flash chromatography
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
(silica gel, 0-50% MeOH in CHCl
3
) to give white solid (93 mg, yield: 72%) as a product. H NMR (600 MHz, DMSO) δ: 7.60 (dd, J = 7.6,
1
.5 Hz, 1H), 7.52 – 7.33 (m, 7H), 7.09 (s, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 7.9 Hz, 1H), 5.17 (s, 2H), 4.08 (d, J = 12.8 Hz, 1H),
3.80 (s, 3H), 3.15 (s, 1H), 3.04 (s, 1H), 1.96 – 1.87 (m, 1H), 1.72 (d, J = 9.7 Hz, 1H), 1.61 – 1.53 (m, 3H), 1.41 – 1.33 (m, 1H). 13C NMR
(
151 MHz, DMSO) δ: 149.4, 147.9, 143.4, 141.3, 137.4, 131.4, 129.7, 129.3, 128.6, 128.2, 123.3, 123.1, 114.5, 113.7, 79.6, 71.0, 58.5, 56.1,
-1
4
9.7, 28.3, 23.6, 22.1. IR (ATR): 3345, 3055, 2939, 2867, 2394, 2251, 1633, 1606, 1517, 1448, 1416, 1268, 1238, 1102, 1029 cm . HRMS
+
ESI-MS-q-TOF for C27
min, for C27 28BrNO
H
28BrNO
4
[M + H] found 510.1274 m/z; Calc. Mass: 510.1280. Mp: 154.6 °C. UPLC-MS (DAD/ESI): t
R
= 6.10
+
H
4
[M + H] found: 510.28 m/z; Calc Mass: 510.13.
(
S)-1-(3-bromo-4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxylic acid (2k)
Compound 2k was prepared following the protocol of 2g, using 2b (119 mg, 0.27 mmol), L-pipecolinic acid (158 mg, 1.22 mmol), NaBH
3
CN
(
79 mg, 1.33 mmol), AcOH (3 droplets), which were stirred in DMF (4 mL). The crude product was purified by flash chromatography (silica
1
3
gel, 0-50% MeOH in CHCl ) to give white solid (77 mg, yield: 51%) as a product. H NMR (600 MHz, DMSO) δ: 7.67 (d, J = 6.5 Hz, 1H),
7.62 (s, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.50 – 7.35 (m, 6H), 7.32 (d, J = 7.9 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 5.26 (s, 2H), 3.84 (d, J = 13.3
Hz, 1H), 3.49 – 3.39 (m, 1H), 3.05 (s, 1H), 2.90 – 2.83 (m, 1H), 2.24 – 2.16 (m, 1H), 1.86 – 1.76 (m, 1H), 1.74 – 1.62 (m, 1H), 1.58 – 1.40
(
m, 3H), 1.39 – 1.29 (m, 1H). 13C NMR (151 MHz, DMSO) δ: 153.4, 142.9, 140.7, 136.5, 133.7, 130.9, 129.8, 129.2, 128.3, 128.2, 127.7,
22.4, 113.6, 110.9, 70.6, 64.4, 57.8, 49.2, 28.7, 24.4, 22.0. IR (ATR): 3649, 3075, 2941, 2859, 2392, 2251, 1621, 1496, 1411, 1288, 1260,
1
-1
+
1102, 1055 cm . HRMS ESI-MS-q-TOF for C26
H
25Br
2
3
NO [M + Na] found 580.0097 m/z; Calc. Mass: 580.0099. Mp: 129.1 °C. UPLC-
+
MS (DAD/ESI): t = 6.51 min, for C26 NO
R
H
25Br
2
3
[M + H] found: 558.20 m/z; Calc Mass: 558.02.
4
-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)-5-chloro-2-hydroxybenzaldehyde (3a)
5
3
-chloro-2,4-dihydroxybenzaldehyde (657 mg, 2.43 mmol) and NaHCO (200 mg, 2.43 mmoL) were dissolved in dry ACN (13 mL) and
were stirred for 20 minutes. To the resulted mixture 1a (528 mg, 1.62 mmol) in dry DMF (18 mL) was added dropwise. Solution was allowed
to stir at room temperature for 20 h. The residue was concentrated under reduced pressure. Water was added (60 mL) and mixture was
extracted with AcOEt (2x60 mL). Organic layers were combined, dried over MgSO
4
, filtered and concentrated. The crude product was
1
purified by flash chromatography (silica gel, 0-100% AcOEt in hexane) and yielded white solid (368 mg, yield: 54%) as a product. H NMR
(
600 MHz, CDCl
H), 6.61 (s, 1H), 5.30 (d, J = 4.3 Hz, 2H). 13C NMR (151 MHz, CDCl
) δ: 193.9, 163.1, 160.7, 143.7, 141.1, 135.4, 134.2, 131.0, 129.6,
3
3
) δ: 11.43 (s, 1H), 9.72 (s, 1H), 7.62 (dd, J = 7.7, 0.7 Hz, 1H), 7.58 (s, 1H), 7.47 – 7.38 (m, 6H), 7.32 (dd, J = 7.5, 1.4 Hz,
1
-1
128.2, 127.9, 127.6, 127.1, 122.1, 115.4, 114.9, 102.0, 71.2 ppm. IR (ATR): 2925, 2850, 1644, 1618, 1490, 1360, 1274, 1200, 1056 cm .
1
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Journal of Medicinal Chemistry
+
HRMS ESI-MS-q-TOF for C20
H
14BrClO
3
[M + Na] found: 438.9691 m/z; Calc. Mass: 438.9713. Mp: 192.8 °C. UPLC-MS (DAD/ESI): t
R
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
=
9.34 min, for C20 14BrClO
H
3
[M + H] found: 417.09 m/z; Calc Mass: 416.99.
4
-((5-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)-4-chloro-2-formylphenoxy)methyl)picolinonitrile (3b)
3a (224 mg, 0.54 mmol), 4-(bromomethyl)picolinonitrile (235 mg, 1.19 mmol), potassium carbonate (507 mg, 3.67 mmol) were stirred in
anhydrous DMF (18 mL) at room temperature overnight. The solvent was removed under reduced pressure. Water was added (30 mL) and
mixture was extracted with AcOEt (2x30 mL). Organic layers were combined and concentrated. Purification involved flash chromatography
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
(
0-100% AcOEt in hexane) and yielded 3b (180 mg, 63%) as white solid. H NMR (600 MHz, CDCl
3
) δ: 10.34 (s, 1H), 8.74 (d, J = 5.1 Hz,
1H), 7.95 (s, 1H), 7.72 (d, J = 0.7 Hz, 1H), 7.61 – 7.55 (m, 2H), 7.51 – 7.29 (m, 7H), 6.51 (s, 1H), 5.35 (s, 2H), 5.21 (s, 2H). 13C NMR (151
3
MHz, CDCl ) δ: 186.4, 159.9, 159.6, 151.7, 146.5, 143.8, 141.0, 135.3, 134.7, 131.3, 131.2, 129.5, 128.3, 128.1, 128.0, 127.4, 125.9, 124.3,
22.1, 119.7, 117.5, 117.0, 98.7, 71.1, 68.4. IR (ATR): 2923, 28853, 2237, 1677, 1598, 1500, 1444, 1380, 123, 1280, 1213, 1038 cm-1.
1
+
HRMS ESI-MS-q-TOF for C27 18BrClN
H
2 3
O [M + Na] found: 555.0107 m/z; Calc. Mass: 555.0087. Mp: 199.7 °C. UPLC-MS (DAD/ESI):
+
t
R
= 9.20 min, for C27 18BrClN O [M + H] found: 533.21 m/z; Calc Mass: 533.03.
H
2 3
3
-((5-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)-4-chloro-2-formylphenoxy)methyl)benzonitrile (3c)
Compound 3c was prepared following the protocol of 3b, using 3a (135 mg, 0.33 mmol), 3-(bromomethyl)benzonitrile (76 mg, 0.39 mmol)
and potassium carbonate (89 mg, 0.65 mmol), which were stirred in anhydrous DMF (4 mL). Purification involved crystallization from
1
AcOEt and gave 2e (150 mg, 87%) as white solid. H NMR (600 MHz, CDCl
3
) δ: 10.32 (s, 1H), 7.93 (s, 1H), 7.70 (s, 1H), 7.67 – 7.60 (m,
3H), 7.52 (t, J = 7.7 Hz, 1H), 7.48 – 7.40 (m, 4H), 7.40 – 7.36 (m, 2H), 7.33 (dd, J = 7.5, 1.6 Hz, 1H), 6.57 (s, 1H), 5.32 (s, 2H), 5.18 (s,
2
1
1
H). 13C NMR (151 MHz, CDCl
3
) δ: 186.8, 160.7, 159.5, 143.7, 141.0, 137.2, 135.5, 132.3, 131.6, 131.2, 130.7, 130.5, 129.9, 129.5, 128.3,
28.0, 127.9, 127.4, 122.1, 119.7, 118.4, 117.0, 113.3, 98.9, 71.0, 70.0. IR (ATR): 2919, 2856, 2225, 1678, 1595, 1441, 1408, 1380, 1320,
-1
+
277, 1199, 1066 cm . HRMS ESI-MS-q-TOF for C28
H
19BrClNO
3
[M + Na] found: 554.0112 m/z; Calc. Mass: 554.0135. Mp: 189.4 °C.
+
UPLC-MS (DAD/ESI): t
R
= 9.64 min, for C28 19BrClNO
H
3
[M + H] found: 532.15 m/z; Calc Mass: 532.03.
4
-([1,1':2',1''-terphenyl]-3'-ylmethoxy)-5-chloro-2-hydroxybenzaldehyde (3e)
3
5-chloro-2,4-dihydroxybenzaldehyde (657 mg, 3.81 mmol), PPh (11.07 g, 42.21 mmol) and 1b (1000 mg, 3.84 mmol) were dissolved in
dry THF (30 mL). To the ice-cooled solution of resulted mixture DIAD (0.83 mL, 4.23 mmol) in THF (30 mL) was added dropwise. Solution
was allowed to warm to room temperature and was stirred for 20 h. The mixture was concentrated under reduced pressure. The crude product
1
was purified by column chromatography on silica gel using 0-60% AcOEt in hexane to give white solid (313 mg, yield: 20%). H NMR (600
MHz, CDCl
3
) δ: 11.36 (s, 1H), 9.66 (s, 1H), 7.67 (d, J = 6.7 Hz, 1H), 7.54 – 7.48 (m, 2H), 7.45 (dd, J = 7.7, 1.2 Hz, 1H), 7.24 – 7.06 (m,
0H), 6.26 (s, 1H), 4.93 (s, 2H). 13C NMR (151 MHz, CDCl
) δ: 193.7, 162.8, 160.8, 142.0, 141.1, 139.9, 138.2, 133.9, 133.4, 130.3, 130.2,
3
1
1
1
29.8, 128.0, 127.9, 127.6, 127.2, 127.1, 126.4, 114.9, 114.7, 101.5, 69.7. IR (ATR): 3055, 2920, 2851, 1638, 1622, 1573, 1482, 1326, 1269,
-1
+
199, 1181, 1047 cm . HRMS ESI-MS-q-TOF for C26
H
3
19ClO [M + Na] found: 437.0915 m/z; Calc. Mass: 437.0920. Mp: 152.4 °C. UPLC-
+
MS (DAD/ESI): t
R
= 9.51 min, for C26
H
19ClO
3
[M + H] found: 415.30 m/z; Calc Mass: 415.11.
2
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4
-((5-([1,1':2',1''-terphenyl]-3'-ylmethoxy)-4-chloro-2-formylphenoxy)methyl)picolinonitrile (3f)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound 3f was prepared following the protocol of 3b, using 3e (278 mg, 0.67 mmol), 4-(bromomethyl)picolinonitrile (159 mg, 0.81
mmol), K CO (185 mg, 1.34 mmol) and anhydrous DMF (4 mL). The product was isolated by crystallization from AcOEt as white solid
175 mg, yield: 90%). H NMR (600 MHz, CDCl
2
3
1
(
(
(
3
) δ: 10.27 (s, 1H), 8.73 (d, J = 5.0 Hz, 1H), 7.88 (s, 1H), 7.66 (d, J = 0.6 Hz, 1H), 7.63
dd, J = 7.5, 1.3 Hz, 1H), 7.52 – 7.44 (m, 3H), 7.23 – 7.19 (m, 3H), 7.18 – 7.12 (m, 3H), 7.10 – 7.02 (m, 4H), 6.14 (s, 1H), 5.04 (s, 2H), 4.95
s, 2H). 13C NMR (151 MHz, CDCl
3
) δ: 186.3, 159.7, 159.6, 151.5, 146.4, 142.1, 140.9, 139.8, 138.3, 134.5, 133.2, 130.9, 130.6, 130.3,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
29.8, 128.1, 128.0, 127.7, 127.4, 127.2, 126.6, 125.8, 124.2, 119.2, 117.3, 116.9, 98.3, 69.8, 68.1. IR (ATR): 3060, 2875, 2242, 1673, 1595,
-1
+
1499, 1449, 1402, 1320, 1278, 1263, 1166, 1034 cm . HRMS ESI-MS-q-TOF for C33
2 3
H23ClN O [M + Na] found: 553.1289 m/z; Calc.
+
Mass: 553.1295. Mp: 196.7 °C. UPLC-MS (DAD/ESI): t = 9.32 min, for C33H23ClN O [M + H] found: 531.35 m/z; Calc Mass: 531.15.
R
2 3
3
-bromo-4-((2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyl)oxy)benzaldehyde (4a)
Compound 4a was prepared as described in the protocol of 2a, using 1g (250 mg, 0.65 mmol), 3-bromo-4-hydroxybenzaldehyde (130 mg,
0
.65 mmol), potassium carbonate (180 mg, 1.31 mmol) and anhydrous DMF (4 mL). Crude product was purified by column chromatography
1
on silica gel (0-100% AcOEt in hexane) and yielded 4a (291 mg, 88%) as white solid. H NMR (600 MHz, DMSO) δ: 9.88 (s, 1H), 8.15 (d,
J = 2.0 Hz, 1H), 7.97 (dd, J = 8.5, 2.0 Hz, 1H), 7.64 (dd, J = 7.6, 1.6 Hz, 1H), 7.52 – 7.44 (m, 2H), 7.35 (dd, J = 7.6, 1.7 Hz, 1H), 6.94 (d, J
=
8.3 Hz, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.84 (dd, J = 8.2, 2.1 Hz, 1H), 5.40 (s, 2H), 4.29 (s, J = 4.6 Hz, 4H). 13C NMR (151 MHz, DMSO)
δ: 190.7, 159.0, 143.2, 142.9, 142.5, 135.8, 134.1, 133.8, 131.3, 131.2, 131.0, 128.5, 127.8, 122.9, 122.3, 118.0, 116.8, 114.1, 111.9, 71.2,
-1
6
4.2. IR (ATR): 2983, 2952, 1685, 1598, 1494, 1314, 1275, 1258, 1243, 1190, 1062 cm . HRMS ESI-MS-q-TOF for C22
H
16Br
2
O
4
[M +
+
+
Na] found: 524.9348 m/z; Calc. Mass: 524.9313. Mp: 196.2 °C. UPLC-MS (DAD/ESI): t
R
= 8.98 min, for C22
H
16Br
2
O
4
[M + H] found:
5
03.04 m/z; Calc Mass: 502.95.
(S)-1-(3-bromo-4-((2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyl)oxy)benzyl)piperidine-2-carboxylic acid (4b)
Compound 4b was prepared according to the procedure of 2g, using 4a (310 mg, 0.61 mmol), L-pipecolinic acid (364 mg, 2.82 mmol),
NaBH
3
CN (181 mg, 3.07 mmol), addition of AcOH (3 droplets) and anhydrous DMF (4 mL) as a solvent. The crude product was purified
1
by flash chromatography (silica gel, 0-50% MeOH in CHCl
3
) to give white solid (164 mg, yield: 43%) as a product. H NMR (600 MHz,
DMSO) δ: 7.66 – 7.59 (m, 2H), 7.48 (t, J = 7.6 Hz, 1H), 7.35 – 7.29 (m, 2H), 7.18 (d, J = 8.5 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.86 (d, J =
.1 Hz, 1H), 6.83 (dd, J = 8.3, 2.1 Hz, 1H), 5.24 (s, 2H), 4.28 (s, 4H), 3.85 (d, J = 13.4 Hz, 1H), 3.06 (dd, J = 8.2, 3.8 Hz, 1H), 2.93 – 2.81
2
(
m, 1H), 2.30 – 2.17 (m, J = 16.9, 8.1 Hz, 1H), 1.86 – 1.77 (m, 1H), 1.74 – 1.61 (m, J = 12.6, 6.6 Hz, 1H), 1.57 – 1.41 (m, 3H), 1.38 – 1.28
m, 1H), 1.23 (s, 1H). 13C NMR (151 MHz, DMSO) δ: 153.6, 143.1, 142.8, 142.4, 136.5, 133.8, 133.7, 131.0, 129.9, 128.2, 127.7, 122.7,
(
122.3, 118.0,116.8, 113.7, 111.0, 70.7, 64.2, 57.9, 49.2, 28.6, 28.5, 24.1, 22.0. IR (ATR): 2932, 2864, 2356, 2323, 2286, 1728, 1634, 1589,
-1
+
1
500, 1454, 1368, 1315, 1278, 1260, 1246, 1197, 1065 cm . HRMS ESI-MS-q-TOF for C28
H
27Br
2
NO
5
[M + H] found: 616.0327 m/z; Calc.
+
Mass: 616.0334. Mp: decomposition at 191.9 °C. UPLC-MS (DAD/ESI): t
R
= 6.25 min, for C28
H
27Br
2
NO
5
[M + H] found: 616.23 m/z;
Calc Mass: 616.03.
2
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Journal of Medicinal Chemistry
3
-bromo-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-fluorobenzyl)oxy)benzaldehyde (4c)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
d (700 mg, 2.69 mmoL) was dissolved in anhydrous DCM (10 mL) under Ar. SOCl (3.2 mL) was added carefully and resulted mixture
was stirred at 45 °C. After 3 h reaction was stopped and mixture was concentrated under reduced pressure. Generated in situ chloride, 3-
bromo-4-hydroxybenzaldehyde (1.6 g, 8.08 mmol) and K CO (1.8 g, 13.46 mmol) were stirred in anhydrous DMF (10 mL) at room
2
3
temperature overnight. The solvent was removed under reduced pressure. Water was added (100 mL) and mixture was extracted with
AcOEt (2x100 mL). Organic layers were combined and concentrated. The crude product was chromatographed on silica gel eluting with 0-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6
0% AcOEt in hexane to give 180 mg of a white solid with yield: 30%. H NMR (600 MHz, CDCl
3
) δ: 9.85 (s, 1H), 8.12 (d, J = 2.0 Hz,
1H), 7.81 (dd, J = 8.5, 2.0 Hz, 1H), 7.55 (t, J = 7.1 Hz, 1H), 7.39 (td, J = 7.6, 1.6 Hz, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H),
7
1
.09 (t, J = 1.7 Hz, 1H), 7.04 (dt, J = 8.3, 1.8 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 5.36 (s, 2H), 4.31 (s, 4H). 13C NMR (151 MHz, CDCl
3
) δ:
89.7, 159.7, 157.0 (d, 1JC-F = 248.2 Hz), 143.6 (d, 2JC-F = 14.0 Hz), 134.8, 131.3, 131.2, 130.7 (d, 3JC-F = 3.1 Hz), 128.8, 128.7, 128.7, 127.8
(
d, 3JC-F = 3.1 Hz), 124.7 (d, 3JC-F = 3.8 Hz), 123.6 (d, 2JC-F = 15.1 Hz), 122.4, 118.1, 117.5, 113.4, 113.0, 65.2 (d, 3JC-F = 6.3 Hz), 64.6, 64.5.
-1
IR (ATR): 2927, 2823, 2738, 1691, 1592, 1493, 1450, 1323, 1278, 1191, 1064, 1047 cm . HRMS ESI-MS-q-TOF for C22
H
4
16BrFO [M +
+
+
Na] found: 465.0109 m/z; Calc. Mass: 465.0114. Mp: 118.0 °C. UPLC-MS (DAD/ESI): t
R
= 8.56 min, for C22
H
16BrFO
4
[M + H] found:
4
43.15 m/z; Calc Mass: 443.03.
N-(2-((3-bromo-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-fluorobenzyl)oxy)benzyl)amino)ethyl)acetamide (4d)
Compound 4d was prepared following to the procedure of 2g, using 4c (120 mg, 0.27 mmol), N-(2-aminoethyl)acetamide (126 mg, 1.24
mmol), NaBH
3
CN (79 mg, 1.35 mmol), addition of AcOH (3 droplets) and anhydrous DMF (4 mL) as a solvent. The crude product was
1
purified by column chromatography (silica gel, 0-30% MeOH in CHCl
3
) to give white solid (88 mg, yield: 61%) as a product. H NMR (600
MHz, CDCl
3
) δ: 7.60 (d, J = 2.0 Hz, 1H), 7.55 (t, J = 6.4 Hz, 1H), 7.36 (td, J = 7.6, 1.6 Hz, 1H), 7.25 (d, J = 2.0 Hz, 1H), 7.21 (t, J = 7.6 Hz,
1
H), 7.08 (t, J = 1.5 Hz, 1H), 7.04 (dt, J = 8.4, 1.8 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.30 (s, 1H), 5.25 (s, 2H), 4.30
(
s, 4H), 3.79 (s, 2H), 3.39 (dd, J = 11.1, 5.5 Hz, 2H), 2.84 (t, J = 5.6 Hz, 2H), 2.00 (s, 3H). 13C NMR (151 MHz, CDCl
3
) δ: 171.0, 157.0 (d,
1JC-F = 248.1 Hz), 154.6, 143.6 (d, 3JC-F = 6.9 Hz), 133.8, 131.5, 130.4 (d, 4JC-F = 2.6 Hz), 129.1, 128.8, 128.6 (d, 2JC-F = 13.3 Hz), 127.9 (d,
3JC-F = 3.2 Hz), 124.5 (d, 3JC-F = 3.7 Hz), 124.4 (d, 2JC-F = 15.2 Hz), 122.4, 118.1, 117.4, 113.9, 112.8, 65.0 (d, 3JC-F = 6.0 Hz), 64.6, 64.5,
5
2.1, 47.9, 38.7, 23.4. IR (ATR): 3283, 2925, 2803, 2163, 1692, 1655, 1578, 1502, 1454, 1417, 1373, 1319, 1283, 1261, 1244, 1192, 1128,
-1
+
1066, 1048 cm . HRMS ESI-MS-q-TOF for C26
H
2 4
26BrFN O [M + H] found: 529.1135 m/z; Calc. Mass: 529.1138. Mp: 121.6 °C. UPLC-
+
MS (DAD/ESI): t = 5.66 min, C26 26BrFN
R
H
2
O
4
[M + H] found: 529.22 m/z; Calc Mass: 529.11.
4
-([1,1':2',1''-terphenyl]-3'-ylmethoxy)-3-bromobenzaldehyde (4e)
Compound 4e was prepared following the procedure of 4c. Preparation of chloride involved the use of 1b (300 mg, 1.15 mmoL), which was
dissolved in anhydrous DCM (4.5 mL) with addition of SOCl (1.4 mL) under Ar. In the second step, generated in situ chloride was stirred
with 3-bromo-4-hydroxybenzaldehyde (231 mg, 1.15 mmol) and K CO (319 mg, 2.31 mmol) in anhydrous DMF (3.3 mL) at room
2
2
3
temperature overnight. The crude product was chromatographed on silica gel, eluting with 0-60% AcOEt in hexane to give 308 mg of an
1
off-white solid with yield: 60%. H NMR (600 MHz, CDCl
3
) δ: 9.8 (s, 1H), 8.1 (d, J = 2.0 Hz, 1H), 7.7 (m, 1H), 7.7 (dd, J = 8.5, 2.0 Hz,
2
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1
H), 7.5 (t, J = 7.7 Hz, 1H), 7.4 (dd, J = 7.7, 1.2 Hz, 1H), 7.2 – 7.0 (m, 10H), 6.7 (d, J = 8.5 Hz, 1H), 5.0 (s, 2H). 13C NMR (151 MHz,
CDCl ) δ: 189.7, 159.8, 142.0, 141.3, 139.8, 138.4, 134.7, 133.9, 131.1, 130.9, 130.4, 130.3, 129.9, 128.1, 128.0, 127.7, 127.3, 127.2, 126.5,
13.2, 112.9, 69.7. IR (ATR): 3029, 2733, 1685, 1592, 1285, 1263, 1194, 1005 cm . HRMS ESI-MS-q-TOF for C26
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
-1
19BrO
2
[M + Na]+
1
H
+
found: 465.0465 m/z; Calc. Mass: 465.0466. Mp: 142.3 °C. UPLC-MS (DAD/ESI): t
R
= 9.51 min, for C26
H
19BrO
2
[M + H] found: 445.34
m/z; Calc Mass: 445.06.
(2S,4S)-1-(4-([1,1':2',1''-terphenyl]-3'-ylmethoxy)-3-bromobenzyl)-4-hydroxypyrrolidine-2-carboxylic acid (4f)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of the compound 4f was performed following to the procedure of 2g. Preparation include the use of 4e (200 mg, 0.45 mmol), (2R,
4R)-4-hydroxypyrrolidine-2-carboxylic acid (272 mg, 2.07 mmol), NaBH CN (142 mg, 2.26 mmol), AcOH (3 droplets) and anhydrous DMF
6.0 mL). The crude product was purified by flash chromatography (silica gel, 0-50% MeOH in AcOEt) to give white solid (93 mg, yield:
3
(
1
3
3
7%) as a product. H NMR (600 MHz, CDCl ) δ: 7.7 (d, J = 7.2 Hz, 1H), 7.6 – 7.5 (m, 2H), 7.4 (dd, J = 7.7, 1.1 Hz, 1H), 7.2 – 7.1 (m, 9H),
7.1 – 7.0 (m, 2H), 6.7 (d, J = 8.4 Hz, 1H), 4.8 (s, 2H), 4.1 (s, 1H), 3.9 (d, J = 13.1 Hz, 1H), 3.5 (d, J = 13.0 Hz, 1H), 3.1 (s, J = 19.4 Hz, 1H),
2
1
5
.8 (d, J = 9.4 Hz, 1H), 2.4 (s, 1H), 2.2 (s, 1H), 1.7 (d, J = 8.8 Hz, 1H), 1.2 (s, 1H). 13C NMR (151 MHz, CDCl
39.8, 138.0, 134.6, 133.6, 130.1, 129.9, 129.7, 129.5, 129.0, 127.8, 127.7, 127.7, 127.7, 126.9, 126.4, 113.2, 110.7, 68.8, 68.6, 65.4, 60.7,
3
) δ:153.4, 141.3, 141.0,
-1
+
5.8, 48.6. IR (ATR): 3297, 3057, 2659, 1600, 1494, 1410, 1253, 1050 cm . HRMS ESI-MS-q-TOF for C31
H
4
28BrNO [M + Na] found:
+
580.1078 m/z; Calc. Mass: 580.1099. Mp: 152.3 °C. UPLC-MS (DAD/ESI): t
R
= 6.55 min, for C31
H
28BrNO
4
[M + H] found: 558.27 m/z;
Calc Mass: 558.13.
2
'-bromo-3'-((2-bromo-4-formylphenoxy)methyl)-[1,1'-biphenyl]-4-carbonitrile (4g)
Synthesis of the compound 4g was performed according to the procedure of 2a, using 1e (559 mg, 1.59 mmol), 3-bromo-4-
hydroxybenzaldehyde (320 mg, 1.59 mmol), potassium carbonate (440 mg, 3.18 mmol) and anhydrous DMF (8 mL). Product was
1
precipitated from AcOEt and yielded 4g (461 mg, 61%) as white solid. H NMR (600 MHz, CDCl
3
) δ: 9.88 (s, 1H), 8.15 (d, J = 2.0 Hz, 1H),
7.84 (dd, J = 8.4, 2.0 Hz, 1H), 7.79 – 7.73 (m, 3H), 7.53 – 7.50 (m, 2H), 7.48 (t, J = 7.7 Hz, 1H), 7.28 (dd, J = 7.6, 1.6 Hz, 1H), 7.11 (d, J =
8
.5 Hz, 1H), 5.34 (s, 2H). 13C NMR (151 MHz, CDCl
3
) δ: 189.6, 159.4, 145.6, 141.7, 136.3, 134.9, 132.1, 131.4, 131.3, 130.5, 128.3, 128.0,
-1
1
21.3, 118.8, 113.4, 113.1, 111.9, 70.9. IR (ATR): 2844, 2227, 1688, 1590, 1566, 1489, 1276, 1256, 1184, 1050 cm . HRMS ESI-MS-q-
+
2 2 R
TOF for C21H13Br NO [M + Na] found: 491.9203 m/z; Calc. Mass: 491.9211. Mp: over 200 °C. UPLC-MS (DAD/ESI): t = 8.71 min, for
+
21 2 2
C H13Br NO [M + H] found: 469.93 m/z; Calc Mass: 469.94.
N-(2-((3-bromo-4-((2-bromo-4'-cyano-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)ethyl)acetamide (4h)
Compound 4h was prepared following to the procedure of 2g, using 4g (100 mg, 0.21 mmol), N-(2-aminoethyl)acetamide (99 mg, 0.97
mmol), NaBH
3
CN (63 mg, 1.06 mmol), addition of AcOH (3 droplets) and anhydrous DMF (5 mL) as a solvent. The crude product was
1
purified by column chromatography (silica gel, 0-30% MeOH in CHCl
600 MHz, CDCl
Hz, 1H), 7.25 (dd, J = 7.6, 1.5 Hz, 1H), 7.21 (dd, J = 8.3, 1.9 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.00 (s, 1H), 5.23 (s, 2H), 3.73 (s, 2H), 3.35
3
) to give off-white solid (30 mg, yield: 26%) as a product. H NMR
(
3
) δ: 7.79 (d, J = 7.7 Hz, 1H), 7.74 (d, J = 8.3 Hz, 2H), 7.58 (d, J = 1.9 Hz, 1H), 7.51 (d, J = 8.3 Hz, 2H), 7.46 (t, J = 7.7
2
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Journal of Medicinal Chemistry
(
dd, J = 11.3, 5.6 Hz, 2H), 2.76 (t, J = 5.8 Hz, 2H), 1.99 (s, 3H). 13C NMR (151 MHz, CDCl
3
) δ: 170.5, 153.9, 145.8, 141.5, 137.3, 134.5,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
5
33.3, 132.1, 130.5, 130.1, 128.4, 128.4, 127.9, 121.2, 118.9, 113.7, 112.5, 111.8, 70.8, 52.5, 48.1, 39.2, 23.5. IR (ATR): 3282, 2925, 2228,
-1
+
688, 1654, 1591, 1554, 1490, 1447, 1362, 1279, 1256, 1185, 1052, 1025 cm . HRMS ESI-MS-q-TOF for C25
H
23Br
2
N
3
O
2
[M + H] found:
+
56.0230 m/z; Calc. Mass: 556.0235. Mp: 152.8 °C. UPLC-MS (DAD/ESI): t = 5.67 min, for C25
R
H23Br N O [M + H] found: 556.21 m/z;
2 3 2
Calc Mass: 556.02.
3
-bromo-4-((2-bromo-3'-fluoro-[1,1'-biphenyl]-3-yl)methoxy)benzaldehyde (4i)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 4i was obtained according to the procedure of 2a, using 1f (700 mg, 2.03 mmol), 4-hydroxy-3-methylbenzaldehyde (409 mg,
2.03 mmol), potassium carbonate (562 mg, 4.07 mmol) and anhydrous DMF (10 mL). Product was purified by precipitation from AcOEt
1
and yielded 4i (534 mg, 57%) as white solid. H NMR (600 MHz, CDCl
3
) δ: 9.87 (s, 1H), 8.15 (d, J = 2.0 Hz, 1H), 7.83 (dd, J = 8.4, 2.0 Hz,
1
H), 7.75 – 7.69 (m, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.42 – 7.38 (m, 1H), 7.30 (dd, J = 7.5, 1.7 Hz, 1H), 7.19 – 7.14 (m, 1H), 7.14 – 7.05 (m,
3H), 5.35 (s, 2H). 13C NMR (151 MHz, CDCl
) δ: 189.6, 162.5 (d, 1JC-F = 246.4 Hz), 159.5, 143.1 (d, 3JC-F = 7.9 Hz), 142.3, 135.9, 134.8,
3
1
31.3, 130.7, 129.8 (d, 3JC-F = 8.3 Hz), 127.8, 127.6, 125.4 (d, 4JC-F = 2.2 Hz), 121.7, 116.8 (d, 2JC-F = 22.0 Hz), 114.8 (d, 2JC-F = 21.0 Hz),
-1
1
13.4, 113.1, 71.0. IR (ATR): 3060, 2922, 2853, 1683, 1594, 1490, 1461, 1418, 1368, 1313, 1278, 1255, 1190, 1049 cm . HRMS ESI-
+
MS-q-TOF for C20
H
13Br
2
2 R
FO [M + Na] found: 484.9150 m/z; Calc. Mass: 484.9164. Mp: 168.1 °C. UPLC-MS (DAD/ESI): t = 9.32 min,
+
for C20
H
13Br
2
FO
2
[M + H] found: 463.02 m/z; Calc Mass: 462.93.
(S)-1-(3-bromo-4-((2-bromo-3'-fluoro-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxylic acid (4j)
Compound 4j was prepared following to the procedure of 2g, using 4i (135 mg, 0.29 mmol), L-pipecolinic acid (172 mg, 1.33 mmol),
NaBH
3
CN (86 mg, 1.45 mmol), addition of AcOH (3 droplets) and anhydrous DMF (5 mL) as a solvent. The crude product was purified by
1
column chromatography (silica gel, 0-40% MeOH in CHCl
3
) to give white solid (73 mg, yield: 44%) as a product. H NMR (600 MHz,
DMF) δ: 7.82 (dd, J = 7.7, 1.5 Hz, 1H), 7.73 (s, 1H), 7.63 – 7.56 (m, 2H), 7.46 – 7.41 (m, 2H), 7.33 – 7.27 (m, 4H), 5.36 (s, 2H), 3.97 (d, J
= 13.3 Hz, 1H), 3.56 (d, J = 12.8 Hz, 1H), 3.29 – 3.23 (m, 1H), 3.03 – 2.97 (m, 1H), 2.39 – 2.30 (m, 1H), 1.95 – 1.89 (m, 1H), 1.85 – 1.77
(
m, 1H), 1.62 – 1.48 (m, 3H), 1.48 – 1.39 (m, 1H). 13C NMR (151 MHz, DMF) δ: 175.0, 167.2 (d, 1JC-F = 63.5 Hz), 155.0, 144.4 (d, 3JC-F
=
=
7
2
1
.9 Hz), 143.1, 138.1, 134.9, 131.9, 131.2 (d, 3JC-F = 8.4 Hz), 130.9, 129.8, 128.9, 126.7, 123.2, 117.4 (d, 2JC-F = 22.2 Hz), 115.6 (d, 2JC-F
1.0 Hz), 114.7, 112.3, 71.9, 65.4, 59.4, 50.5, 25.7, 24.0, 23.2. IR (ATR): 2923, 2853, 1614, 1495, 1461, 1363, 1288, 1259, 1196, 1157,
-1
+
055, 1023 cm . HRMS ESI-MS-q-TOF for C26
2 3
H24Br FNO [M + H] found: 576.0180 m/z; Calc. Mass: 576.0185. Mp: 175.6 °C. UPLC-
+
MS (DAD/ESI): t
R
= 6.65 min, for C26 FNO [M + H] found: 576.15 m/z; Calc Mass: 576.02.
H
24Br
2
3
N-(2-((3-bromo-4-((2-bromo-3'-fluoro-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)ethyl)acetamide (4k)
Compound 4k was prepared according to the procedure of 2g, using 4i (200 mg, 0.43 mmol), N-(2-aminoethyl)acetamide (201 mg, 1.97
mmol), NaBH
purified by column chromatography (silica gel, 0-40% MeOH in CHCl
(600 MHz, DMSO) δ: 7.98 (t, J = 5.2 Hz, 1H), 7.70 (d, J = 1.9 Hz, 1H), 7.68 (dd, J = 7.7, 1.6 Hz, 1H), 7.56 – 7.49 (m, 2H), 7.41 – 7.37 (m,
3
CN (127 mg, 2.16 mmol), addition of AcOH (3 droplets) and anhydrous DMF (4 mL) as a solvent. The crude product was
1
3
) to give white solid (159 mg, yield: 67%) as a product. H NMR
2
4
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