Terphenyl-Based Small-Molecule Inhibitors of Programmed Cell Death-1/Programmed Death-Ligand 1 Protein-Protein Interaction

Article abstract of DOI:10.1021/acs.jmedchem.1c00957

We describe a new class of potent PD-L1/PD-1 inhibitors based on a terphenyl scaffold that is derived from the rigidified biphenyl-inspired structure. Using in silico docking, we designed and then experimentally demonstrated the effectiveness of the terphenyl-based scaffolds in inhibiting PD-1/PD-L1 complex formation using various biophysical and biochemical techniques. We also present a high-resolution structure of the complex of PD-L1 with one of our most potent inhibitors to identify key PD-L1/inhibitor interactions at the molecular level. In addition, we show the efficacy of our most potent inhibitors in activating the antitumor response using primary human immune cells from healthy donors.

Full text of DOI:10.1021/acs.jmedchem.1c00957

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Article
Terphenyl-Based Small-Molecule Inhibitors of Programmed Cell
Death-1/Programmed Death-Ligand 1 Protein−Protein Interaction
Damian Muszak, Ewa Surmiak,* Jacek Plewka, Katarzyna Magiera-Mularz, Justyna Kocik-Krol,
Bogdan Musielak, Dominik Sala, Radoslaw Kitel, Malgorzata Stec, Kazimierz Weglarczyk, Maciej Siedlar,
Alexander Dömling, Lukasz Skalniak, and Tad A. Holak*
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ABSTRACT: We describe a new class of potent PD-L1/PD-1 inhibitors
based on a terphenyl scaffold that is derived from the rigidified biphenyl-
inspired structure. Using in silico docking, we designed and then
experimentally demonstrated the effectiveness of the terphenyl-based
scaffolds in inhibiting PD-1/PD-L1 complex formation using various
biophysical and biochemical techniques. We also present a high-resolution
structure of the complex of PD-L1 with one of our most potent inhibitors to
identify key PD-L1/inhibitor interactions at the molecular level. In addition,
we show the efficacy of our most potent inhibitors in activating the antitumor
response using primary human immune cells from healthy donors.
INTRODUCTION
additional benefits such as oral bioavailability and lower
production cost.
■
15,16
Cancer immunotherapy aims to stimulate the immune system’s
ability to fight cancer as opposed to directly killing cancer cells
with more traditional methods such as chemotherapy and
Although the PD-1/PD-L1 interface is considered difficult to
treat with SMIs due to its large, flat surface of interaction with
no visible binding pockets, the number of patents and
1
−6
radiation therapy.
Inhibition of negative immune check-
17,18
publications on anti-PD-L1 SMIs is constantly growing.
point regulators is now used in clinics around the world and
was recognized with the 2018 Nobel Prize in Physiology or
A common feature of virtually all reported PD-1/PD-L1
SMI is that they target PD-L1 through a biphenyl-based
scaffold originally developed by Bristol Myers Squibb
7
Medicine. One of the most important immune checkpoints
(
ICs) in cancer immunotherapy consists of programmed cell
death protein 1 (PD-1, also known as CD279) with its ligand
PD-L1, known also as CD274 or B7-H1).
The PD-1/PD-L1 axis is responsible for inhibiting excessive
19−21
(BMS).
However, none of these molecules has progressed
4
(
into clinical trials so far. As of now, only the peptide-derived
compound CA-170 from Curis and Aurigene is undergoing
clinical trials as a small molecule that directly targets the PD-
L1, PD-L2, and the V-domain Ig suppressor of T cell activation
(VISTA) IC. However, it turned out that CA-170 is not a
classic PD-L1-blocker and alternative modes of its action have
stimulation and normally aims at maintaining immune
tolerance to self-antigens by negatively regulating the immune
8
−10
response.
By blocking the PD-1/PD-L1 interaction,
activation of T cells can be achieved for an improved
antitumor response. Therefore, the immune checkpoint
blockade (ICB) of PD-1 or PD-L1 at the tumor cell−T cell
interface has become an attractive strategy for cancer
22,23
been postulated.
Here, we present a new class of potent PD-L1 inhibitors
based on a rigidified biphenyl structure. These compounds,
which comprise the terphenyl scaffold, are effective in blocking
PD-1/PD-L1 interactions in a variety of biophysical and
1
1,12
immunotherapy.
Traditionally, the inhibition of PD-1/PD-L1 interactions is
achieved with monoclonal antibodies (mAbs) that target PD-1
(
(
e.g., pembrolizumab, nivolumab, cemiplimab, etc.) or PD-L1
e.g., atezolizumab, avelumab, and durvalumab). Despite their
3
Received: May 27, 2021
medical and commercial success, mAb-based immunotherapies
suffer from several disadvantages, including high treatment
costs, immune-related adverse events (irAE), and poor
13,14
penetration of solid tumors.
Small-molecule inhibitors
(
SMIs) are expected to overcome these limitations, with
©
XXXX The Authors. Published by
American Chemical Society
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Figure 1. In silico comparison of the prototype m-terphenyl fragment and corresponding BMS core performed with AutoDock Vina integrated into
PyRx software. (A) Structures used in molecular modeling: known PD-L1 inhibitors BMS-200, BMS-202, and BMS-1166 molecules; BMS core
fragment extracted by comparison of the structures of several BMS molecules; m-terphenyl derivativerigidified motif obtained by the ether
linker removal. (B) Imposition of the modeled complexes of found m-terphenyl derivative (blue), BMS core (green), and whole BMS-1166
structure (yellow) in complex with PD-L1 (subunit Agray, subunit Borange, PDB ID: 6R3K).
1
a
Scheme 1. Synthesis Pathway Used in R and Hydroxymethyl Group Position Optimization of m-Terphenyls 4a−4h
a
Reagents and conditions: (a) 1a−c, 4-benzodioxane-6-boronic acid, Pd(dppf)Cl ·DCM, K CO , dioxane/water 2:1, 80 °C, 3 h, 47−61%; (b) 2a−
2
2
3
1
c, (hydroxymethyl)phenylboronic acid, Pd(dppf)Cl ·DCM, K CO , dioxane/water 2:1, 80 °C, 3 h, 32−56%; (c) for 4f: 4d (R = NH ), HBr,
2
2
3
2
1
1
NaNO , CuBr , 70 °C, 2 h, 9%; for 4g: 4d (R = NH ), HCl, NaNO , KI, RT, 16 h, 28%; for 4h: 4d (R = NH ), HCl, NaNO , CuCN, KCN, RT,
2
2
2
2
2
2
4
h, 21%.
cellular assays. We also provide a high-resolution structure of
PD-L1 bound to the terphenyl small molecule to identify key
PD-L1/ligand interactions on the PD-L1 interface.
strategy for enhancing the selectivity and potency of SMIs that
24−27
bind to protein targets.
In our study, we focused on the
effect of rigidifying the known PD-L1 inhibitors from the BMS
19,21
family
(Figure 1A). The core scaffold was extracted by
RESULTS AND DISCUSSION
comparison with several BMS molecules disclosed in
■
19,21
Identification of Potent Terphenyl-Based PD-L1
Inhibitors. Rigidification of the scaffold is a well-known
patents
(Figure 1A, BMS core). Rigidification was then
implemented by removing the ether linker (highlighted in red),
B
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Table 1. Inhibitory Activities of the Optimized Terphenyls Obtained in the HTRF Assay at the First Stage of Optimization of
1
the R Substituent
name
R1
hydroxymethyl position
HTRF % of the undissociated complex at 5 μM
IC50 [μM]
a
4
4
4
4
4
4
4
4
a (m-terphenyl)
Me
Me
Me
NH2
Cl
Br
I
CN
para
meta
ortho
para
para
para
para
para
42
88
98
73
1
15
56
21
5.52 ± 0.04
b
c
d
e
f
0.51 ± 0.01
0.95 ± 0.02
g
h
1.17 ± 0.12
a
4a molecule is modeled terphenyl core fragment presented in Figure 1 and labeled there as m-terphenyl derivative.
a
Scheme 2. Synthesis Routes Leading to Elongated m-Terphenyl Derivatives 5a−5r and 6a−6c
a
Reagents and conditions: (a) (i) 4a−4c, SOCl , DCM, cat. DMF, RT, 2H; (ii) appropriate phenol, K CO /Cs CO , KI, DMF, 80 °C, 16 h, 42−
2
2
3
2
3
8
5%; (iii) 5a/5e/5i/5m/5o/5q, LiOH, dioxane/H O, 2:1, RT, 16 h, 45−89% or 5c/5k, 3-pyridinyl boronic acid, Pd(dppf)Cl ·DCM, K CO ,
2
2
2
3
dioxane/water 2:1, 80 °C, 5 h, 80−92%; (b) (i) 4a, MsCl, DCM, Et N, RT, 2H (ii) NaN , DMF, RT, 6 days; (iii) methyl propiolate, sodium
ascorbate, CuSO , RT, 24 h, 72%; (iv) 5g, LiOH, dioxane/H O, 2:1, RT, 16 h, 61%; (c) (i): 4a or 4b, SOCl , DCM, cat. DMF, RT, 2H (ii)
3
3
4
2
2
appropriate amine, THF, RT, 48 h, 29−70%.
leading to the m-terphenyl derivative (Figure 1A). The
rigidified terphenyl structure was tested in silico against the
aromatic core of BMS molecules (Figure 1B) using AutoDock
mainly by a stronger π−π stacking with Tyr56 (Figure 1B).
B
These promising results prompted us to structurally optimize
the found m-terphenyl motif. All designed molecules were
tested in a homogeneous time-resolved fluorescence (HTRF)
assay to assess their activity as inhibitors of the PD-1/PD-L1
complex (Table S1).
2
8
29
Vina integrated into PyRx software, with the dimeric
structure of PD-L1 bound to BMS-1166 (PDB ID: 6R3K).
Molecular docking shows the high potency of the designed,
rigidified core, with a stronger PD-L1 binding affinity of 1.0
kcal/mol (−11.7 kcal/mol) than the corresponding fragment
of BMS core (Figure 1A, −10.7 kcal/mol). Moreover, the
calculations suggest that the deletion of the ether linker
fragment allows for a better stabilization of the complex,
In the first step of the process, the impact of the type of the
1
C-2′-attached substituent (R ), as well as the position of the
hydroxymethyl group, for the binding affinity was tested
(Scheme 1, Table 1).
C
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To obtain the designed molecules, we applied a synthetic
pathway based mainly on the Suzuki cross-coupling reaction.
Appropriate 1,3-dibromoaryls 1a−c were coupled into their 3-
benzodioxane derivatives 2a−c. Subsequently, biphenyls were
taken into a second cross-coupling reaction with 2, 3, or 4-
Table 2. Inhibitory Activities of the Optimized Terphenyls
Obtained in HTRF Assay in the First Stage of Optimization
of the XR Substituent and Position of Molecule Extension
2
(
hydroxymethyl)phenylboronic acid, leading to the corre-
sponding m-terphenyl derivatives 4a−e. C-2′ nitrile and
brominated and iodinated molecules (compounds 4f−h) were
synthesized by the Sandmeyer reaction, followed by amine-
based diazonium salt formation. All compounds generated in
this step had their potency for the PD-1/PD-L1 complex
dissociation tested using the HTRF assay in the scouting mode
at 5 μM since IC50 determination would not be possible for
some compounds due to their low solubility at this stage. In
this assay, a lower percentage of the undissociated complex
reported in Table 1 indicates higher potency of the tested
compound. Full IC s of several compounds were determined
50
(Table 1) and compared with the corresponding percentages
of the undissociated complex, indicating a correlation between
the two measurements. This approach was used for the activity
estimation of all molecules presented in this article. The HTRF
assay was also validated on the BMS-1166 compound, yielding
IC of 3.89 nM (Table 4) as compared to 1.40 nM reported in
5
0
1
9,30
the literature.
The results of the HTRF assay show clearly
that the optimal substituent attached to C-2′ of the middle ring
of terphenyl is chlorine with IC50 of approximately 500 nM
(
compound 4e); however, comparable results have been
obtained for brominated and cyano derivatives (compounds
f and 4h, respectively). Introduction of a polar fragment
compound 4d) in this region has a negative impact on the
4
(
molecule affinity. Generally, the insertion of the halogen atom
1
at the C-2′ position (the R substituent) is preferred and the
activity of the tested fragments decreases with the growth of
the halogen van der Waals radius (compounds 4e−g).
In parallel, we assessed the influence of further molecular
elongation on both the aromatic and aliphatic fragments with
different hydrogen bond donor−acceptor properties (Scheme
a
Percentage of the undissociated protein complex was determined at
0 μM concentration of the inhibitor.
2
, Table 2). Molecules 5a−r were obtained following a
5
nucleophilic substitution reaction of the previously prepared
benzyl chloride derivatives. The corresponding alkyl chlorides
were synthesized by the transformation of the 4-hydroxyme-
To prepare the desired compounds, we carried out
Williamson alkylation of the 4-bromo-2-hydroxybenzyl alcohol,
leading to the appropriate alkoxy derivative. Then, the
intermediates were transformed into the corresponding
phenylboronic acid pinacol esters (compounds 3a−i, SI) and
coupled with 3-bromobiphenyls 2a or 2c. Hydroxymethyl
groups of terphenyls were then transformed into the
corresponding chlorides, which were used as an amine
alkylating agent. Initially, we tested the piperazine-extended
derivatives of the ethers 6d−e, but we have observed
precipitation during the HTRF assay. This forced us to change
the amine to N-(2-aminoethyl)acetamide, significantly increas-
ing the solubility in the aqueous buffer for compounds 7a−h.
Almost all molecules obtained in this stage of the optimization
were characterized by high affinity to the PD-L1 protein and
caused complete dissociation of the complex at 5 μM.
Therefore, to differentiate potencies of the tested molecules,
the inhibitor concentration was set to 5 nM in further assays.
Introduction of the methoxy fragment in 7a has the greatest
impact on binding to PD-L1, resulting in complete complex
dissociation at 5 nM ligand concentration and a measured IC50
value of 0.82 nM. Further elongation of the ether substituent
decreases the affinity to the target (compounds 7b−g).
thylterphenyl derivatives 4a−c with SOCl and a catalytic
2
amount of anhydrous dimethylformamide (DMF). Then, they
were treated with an appropriate nucleophile, yielding ethers or
tertiary amines (compounds 5a−r and 6a−c). The double
substitution of the amines was prevented by using an excess of
the nucleophile. Furthermore, the bulkiness of the terphenyl
fragment additionally precluded double substitution.
The results obtained from these steps clearly indicated that
only the para elongation, using amine as a building block is
beneficial for the interaction (compound 6b, Table 2).
Attempted meta extension of the motif causes a significant
drop of the affinity from 5% of the undissociated complex for
6
b to about 56% for 6c, tested at 5 μM concentrations.
Furthermore, the introduction of an aromatic fragment in each
position destabilizes the interaction.
In the next stage of the structure optimization, the type of
the C3-attached ether substituent in the proximal phenyl ring
of the terphenyl was considered. We synthesized a series of
different alkoxy derivatives (Scheme 3, Table 3) implementing
different-length alkyl and cycloalkyl substituents, as well as an
acetonitrile fragment (compounds 6d−e and 7a−h).
D
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3
2
Scheme 3. Synthetic Pathway Used during R Alkoxy Fragment and R Hydrophilic Tail Optimization of m-Terphenyls 6d−6e,
a
7a−7h, and 8a−8j
a
Reagents and conditions: (a) 2a or 2c, borane 3a−3i, Pd(dppf)Cl ·DCM, K CO , dioxane/water 2:1, 80 °C, 3 h, 29−89%; (b) (i) 4i or 4j,
2
2
3
SOCl , DCM, cat. DMF, RT, 2H; (ii) appropriate amine, THF, RT, 48 h, 20−38%; (c) (i) 4k−4r, SOCl , DCM, cat. DMF, RT, 2H; (ii)
2
2
appropriate amine, DIPEA, DMF, 80 °C, 16 h, 10−78% (d) (i) 4k, SOCl , DCM, cat. DMF, RT, 2H (ii) appropriate amine, DIPEA, DMF, 80 °C,
2
1
6 h, 33−89%.
Addition of an extra biphenyl motif in compound 7h caused a
drop in the inhibitory potency of the molecule. The
acetonitrile derivative in molecule 7e shows the activity
comparable with the O-methylated 7a molecule, causing 99%
of the complex dissociation at 5 nM.
The last step of the SAR analysis was focused on the
optimization of the hydrophilic tail (Scheme 3, Table 4).
Maintaining all optimized molecule fragments, we synthesized
and evaluated the inhibitory activities of the piperazine-
inhibitory concentration was successful only for 7a and 8g.
The values of 0.82 and 2.07 nM, respectively, were obtained.
For the rest of the tested molecules, the limit of the assay was
exceeded. It has to be noted that HTRF results served only the
purpose of the initial selection of “leads” for further cell-based
assays and as such should not be considered as a sole
determinant of the compound potency in the PD1-/PD-L1
complex formation inhibition.
Terphenyl-Based Inhibitors Induce Dimerization of
PD-L1. Representative molecules from the optimized group
(
compounds 8a−e), ethylenediamine- (compounds 7a, 8f−h),
and 2-aminoethanol-containing (compounds 8i−j) terphenyl
derivatives.
1
were tested for the interaction with PD-L1 using a H NMR
titration experiment and showed protein oligomerization upon
addition of the ligand (Figure 2).
After adding compounds 4a, 7a, 7e, and 8j to human PD-L1
The results showed that the type of solubilizer used has a
great impact on the affinity to the targeted protein. The
piperazine derivative family (compounds 8a−8e) was charac-
terized by the lowest potencies in disrupting the PD-1/PD-L1
interaction, characterized by 88−5% undissociated complex at
(
hPD-L1), we observed the broadening of the hPD-L1 proton
signals in 1D NMR spectra, characteristic of oligomerization of
hPD-L1, previously observed by us with the BMS com-
5
nM ligand concentration. The best PD-L1 binder from this
30
pounds. We additionally compared the spectra of the
interaction of 8j with hPD-L1 (Figure 2B) and murine PD-
L1 (mPD-L1) (Figure 2C). Only for human PD-L1, changes
in NMR were observed upon addition of 8j to the protein. For
murine PD-L1, no interaction was observed between mPD-L1
and the compound, even with the over-titration of mPD-L1
with 8j (1:10, molar ratio, respectively) as there was no change
in the corresponding NMR spectra fingerprints (changes in the
intensity of the peaks or their position compared to each
other). The results are, therefore, consistent with our previous
studies, in which we showed that the biphenyl moiety from
group is the glycine-containing 8d molecule with 5% of the
remaining complex. For 8b−d, we could not precisely
determine the values of the complex dissociation as they are
beyond the quantification limits of the assay. Therefore, we
assigned them the values below 1 nM, the level at which we
can still confidently determine the assay results. The
ethylenediamine derivatives (compounds 7a and 8f−h) were
characterized by moderate to excellent potencies in the
inhibition of the PD-1/PD-L1 interactions, with the percent-
age of the undissociated complex in 32−0% range at 5 nM
inhibitor concentration. Determination of the half-maximal
E
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Table 3. Inhibitory Activities of the Optimized Terphenyls Obtained in HTRF Assay in the Second Step of Optimization of the
3
R Alkoxyl Substituent
anti-PD-L1 inhibitors binds to human PD-L1 and not to the
murine analogue.
Lead Compounds Activate Jurkat Effector Cells in the
ICB Assay. After a successful optimization of the terphenyl
compounds, the activity of the optimized molecules 7a and
provides evidence that the activity of the compounds is PD-
L1-dependent and is not related to the unspecific activation of
T cells. Compounds 8j, 8h, and 8i (Figure 3G) showed the
highest selectivity of the desired PD-1/PD-L1-blocking activity
versus cytotoxicity (Figure 3F). Compounds 8h and 8j, which,
apart from their bioactivity, showed dissociation of the protein
complex in HTRF at 5 nM, were directed for further study.
Lead Compound 8j Activates Primary T Cells in the T-
Cell Activation Assay. A standard ICB assay relies on
artificial Jurkat-ECs, which are surrogates of T-cells. To verify
the potential of the compounds to reactivate the PD-L1-
blocked primary T cells, a modified assay termed T-cell
3
1
8
a−j in a cellular context was assessed. To this end, we first
performed the PD-1/PD-L1 ICB assay. In this assay, T-cell
receptor (TCR)-mediated activation of Jurkat effector cells
(
Jurkat-ECs) is reduced by the presence of PD-L1, exposed at
the surface of CHO/TCRAct/PD-L1 cells (Figure 3A). Upon
the addition of PD-1/PD-L1 blockers, full activation of Jurkat-
ECs is restored, as presented for a therapeutic anti-PD-L1
antibody, atezolizumab (Figure 3B).
32
activation (TCA) was carried out. In this assay, human
primary peripheral blood mononuclear cells (PBMCs) are
allowed to make contact with CHO/TCRAct/PD-L1 cells
alone or in the presence of PD-1/PD-L1 blockers, and the
Most of the compounds selected for the assay (molecules 7a,
and 8f−8j) were able to increase the activation of Jurkat-ECs
(Figure 3B). Only the piperazine-containing molecules do not
+
+
show any changes after the treatment. The best activity,
reflected by the lowest EC50 values (Figure 3E), was observed
for 8j, followed by 8h, 8i, 7a, and 8f. These compounds
provided higher activation of the Jurkat-ECs than the
previously published BMS-1166 compound (Figure 3B). All
active compounds were comparable in terms of toxicity toward
Jurkat-ECs, with compounds 7a, 8f, and 8j slightly less
cytotoxic than 8g, 8h, and 8i (Figure 3C,D). Importantly, none
of the tested compounds was able to increase the activity of
Jurkat-ECs where PD-L1 was absent (Figure S1). This
activation of CD4 and CD8 T cell is monitored by flow
cytometry.
Following the addition of PBMCs to the CHO/TCRAct/
+
+
PD-L1 cells, the activation of both CD4 and CD8 T cells was
observed, as monitored by the expression of early (CD69),
intermediate (CD25 and HLA-DR), and late (PD-1)
activation/exhaustion markers (Figure 4A). This is due to
the TCR-dependent activation of T-cells by CHO/TCRAct/
PD-L1 cells. As reported before, the addition of PD-1/PD-L1-
blockers in such a setup increases the expression of PD-1 on T-
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Table 4. Inhibitory Activities of the Optimized Terphenyls
Obtained in HTRF Assay in the Second Step of
Optimization of Hydrophilic Tail NR2
cells but rarely other markers. In the current experiment, this
was confirmed for three therapeutic anti-PD-L1 antibodies,
durvalumab, atezolizumab, and avelumab (Figure 4A). Also,
for the tested compounds 8h and 8j, a significant increase in
the numbers of the PD-1-positive cells was observed (Figure
4
A). Surprisingly, for the antibodies, a decrease of CD69 and
+
CD25 expression was observed for CD4 cells, which has not
been observed before in our studies. The addition of
compound 8j, but neither 8h nor the therapeutic antibodies,
increased the numbers of activated, CD25-positive, and HLA-
DR-positive T-cells (Figure 4A). Altogether, the presented
data clearly show the biological activity of 8j in a cellular
context.
In addition to the expression of surface markers, the
accumulation of immune-related cytokines in the culture
media collected in the TCA assay was monitored with the
cytometric bead array (CBA) human Th1/Th2/Th17 kit. In
the presence of atezolizumab, significantly increased levels of
IL-17A, TNF-α, and IL-10 were observed (Figure 4B). Also,
the levels of IL-6, IFN-γ, and IL-2 increased, but these
increases were not statistically significant. Similarly, the
treatment of PBMCs cocultured with the CHO/TCR-Act/
PD-L1 cells with 8j also increased the accumulation of all six
cytokines, with significant increases observed for IL-6, TNF-α,
IFN-γ, IL-10, and IL-2 (Figure 4B).
Distal Terphenyl Ring Provides Rigidification That
Fits into the PD-L1 Cleft. We were able to crystalize and
solve the structure of the 8g compound in complex with PD-
L1 at the resolution of 2.3 Å that allowed us to decipher the
molecular basis of the interactions between them (Figure 5 and
Table S2). The asymmetric unit of the hPD-L1/8g complex
contains six protein molecules organized into three dimers,
with one inhibitor located at the center of the interface of each
dimer. The electron density map describes each inhibitor
molecule allowing the position of all moieties in the structure
with the fully detailed density for the molecule located at the
interface of the AB dimer. 8g is mostly buried in the deep and
elongated tunnel, which is composed of two PD-L1 monomers.
The 1,4-benzodioxane group creates the standard interaction
with ATyr56 (the monomer molecules are annotated by
a
Limit of quantification of the used assay was achieved.
1
1
Figure 2. H NMR spectra of PD-L1 with terphenyl-based inhibitors. (A) H NMR spectra of the aliphatic part of human PD-L1 protein (blue)
with compound 4a (red), 7a (green), 7e (purple), and 8j (orange) in the molar ratio 1:1. (B,C) Aliphatic region of PD-L1 (blue) with 8j (red):
human PD-L1 (panel B) and mouse PD-L1 (panel C) in the molar ratio 1:1 and 1:10, respectively.
G
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Figure 3. Terphenyl compound bioactivity and toxicity in the ICB assay, (A) schematic representation of the ICB assay. (B) Dose−response curves
of the reactivation of Jurkat-ECs with PD-L1-blocking agents: atezolizumab (left panel) and terphenyl compounds (right panel). Graphs show fold
induction of the luminescence signal relative to either untreated (for atezolizumab) or dimethyl sulfoxide (DMSO)-treated (for compounds) cells.
Data points represent mean ± SD values from 3 to 5 independent experiments. Gray data points were removed from curve-fitting analysis due to
the toxicity of the compounds observed at the 10 μM concentration. (C) Long-term (48 h) cytotoxicity of the tested compounds toward Jurkat-
ECs. The graph shows Jurkat-EC survival relative to DMSO-treated control cells. Data points represent mean ± SD values from three independent
experiments. (D) Half-maximal effective concentrations (EC ) of the cytotoxicity of the compounds, calculated from (C). (E) EC values of the
5
0
50
bioactivity of the compounds in the ICB assay, calculated from (B). (F) Selectivity of the bioactivity of the compounds in the ICB assay over the
cytotoxicity effect toward Jurkat-ECs. Error bars are combined errors of EC50 (tox.) and EC50 (bio.). (G) Chemical structures of the three most potent
terphenyl compounds.
subscripts A and B according to their chain arrangement in the
crystal structure of the dimer), previously observed by us for
the BMS-202, BMS-1001, and BMS-1166 compounds.
against the PD-1/PD-L1 complex both in common biophysical
assays, such as HTRF and NMR, as well as cell-based assays.
Our most potent compounds activated Jurkat-ECs to levels
comparable to those of the control antibodies, despite being
considerably smaller. Similar to other anti-PD-L1 inhibitors
reported in the literature, our compounds dimerize human PD-
L1, as presented here by NMR and X-ray crystallography
studies. They are specific for human PD-L1 and do not cross-
react with the murine PD-L1 analogue. Our most potent
compound, 8j, is characterized by an exceptionally high affinity
to the molecular target (IC50 < 1 nM), as well as a proven
potency in cell-based assays with an EC50 of ca. 1 μM. Most
importantly, adding compound 8j to the coculture of PBMCs
and CHO/TCRAct/PD-L1 cells not only induced PD-1
expression but also increased the numbers of CD25-positive
and HLA-DR-positive T cells, which was not observed even in
PD-L1 therapeutic antibodies. Also, the accumulation of
immune-related cytokines was increased in the presence of
either 8j or atezolizumab. This shows the potency of this
compound toward primary human T cells and designates 8j as
an outstanding candidate for further optimization in the anti-
PD-L1 treatments of cancer. The presented crystal structure
provides an explanation for the enhanced inhibitory effect of
terphenyl-based inhibitors. The introduced phenyl ring
replaces the benzyl linker of BMS-1166 and thus provides
the rigidification of the structures in the conformation that fits
perfectly into the PD-L1 cleft.
30,33
The central terphenyl moiety is stabilized by plenty of
hydrophobic contacts with BTyr56, AMet115, BMet115,
Ala121, and Ala121. The chlorine substituent attached to
A
B
this moiety forms an additional halogen bond with the
backbone carbonyl of Asp122, providing the rationale for the
A
enhanced inhibitory effect of these halogenated inhibitors at
1
the R position. The methoxy substituent at the meta position
of the distal ring of the terphenyl is also additionally stabilized
by hydrophobic interaction with Ile54 and Val68, pointing
B
B
out the importance of this substituent. At the other side of the
tunnel, the sulfonamide polar extension forms a hydrogen
bond with the carbonyl of the Asp122 side chain and is largely
A
water-exposed.
Our structural data confirm that the terphenyl compounds
with the rigidified biphenyl unit bind to PD-L1 at the
interaction site that has been seen in the compounds with the
biphenyl scaffold. Comparison of the PD-L1/8g, PD-L1/BMS-
1
2
166, PD-L1/the C2-symmetric inhibitor, and PD-L1/ARB-
72572 complexes shows that the crystallographic arrange-
ment of the terphenyl core is close to the docking results
3
3−35
(Figure S2).
CONCLUSIONS
■
In this paper, we have demonstrated a new group of small-
molecule anti-PD-L1 inhibitors based on the concept of
rigidifying the biphenyl unit by removing the ether linker from
the known PD-L1 inhibitors of the BMS family. Such
terphenyl-based scaffolds exhibit high inhibitory activity
EXPERIMENTAL PROCEDURES
■
Homogeneous Time-Resolved Fluorescence. HTRF was
performed using the certified CisBio assay according to the
H
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Figure 4. Effect of 8j and 8h on the reactivation of primary T-cells preblocked by PD-L1-overexpressing cells. (A) Expression of early (CD69),
intermediate (CD25, HLA-DR), and late (PD-1) T-cell activation/exhaustion markers was determined by flow cytometry. PBMCs from healthy
donors were exposed to CHO/TCRAct/PD-L1 cells for 2 days in the presence of durvalumab (durva), atezolizumab (atezo), avelumab (avelu), or
increasing concentrations of 8j and 8h (concentrations: 0.1, 0.5, or 2.5 μM). ø indicates untreated cells. The graphs show fractions of positive cells
and represents cumulative data from four donors. Statistical significance was analyzed with one-way ANOVA, followed by Fisher’s posthoc test: *p
<
0.05, **p < 0.01, ***p < 0.01. (B) Accumulation of the indicated cytokines in culture media from the TCA assay. The graphs show mean ±
standard error of the mean values from four independent donors. For each donor, the data were normalized to untreated coculture of PBMCs and
CHO/TCRAct cells as a determinant of donor-dependent cytokine level independent of the PD-L1/PD-1 axis. Statistical significance was analyzed
with one-way ANOVA, followed by the Fisher posthoc test: *p < 0.05, **p < 0.01, ***p < 0.001.
manufacturer’s guidelines. The experiments were performed at 5 nM
h-PD-L1 and 50 nM hPD-1 in the final formulation at 20 μL final
volume in the well. To determine the half-maximal inhibitory
concentration (IC ) of tested compounds, measurements were
performed on two individual dilution series, unless states otherwise.
After mixing all components according to the CisBio protocol, the
plate was left for 2 h incubation at room temperature, followed by
time-resolved fluorescence energy transfer (TR-FRET) measurement
on Tecan Spark 20M. Collected data were 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.
5
0
I
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Article
41−43
or PDB-REDO server.
Water molecules were added automati-
cally and inspected manually. The coordinates and structure factors
were deposited in the Protein Data Bank with accession number
7
NLD.
Cell Culture. CHO-K1 cells, CHO-K1 cells overexpressing TCR
activator (CHO/TCRAct), PD-L1-expressing CHO/TCRAct cells
CHO/TCRAct/PD-L1), and Jurkat-EC T-cells overexpressing PD-1
(
and carrying a luciferase reporter gene under the control of the
nuclear factor of activated T-cell response element (NFAT-RE) were
obtained from Promega and cultured in RPMI-1640 medium
(Biowest) supplemented with 10% fetal bovine serum (FBS, Biowest)
and 200 mM L-glutamine (Biowest). G418 (250 μg/mL, InvivoGen)
and Hygromycin B Gold (50 μg/mL, InvivoGen) were also added to
the culture medium as selection antibiotics. The overexpression of
PD-L1 and TCR ligand in CHO/TCRAct/PD-L1 cells and
overexpression of PD-1 in Jurkat-ECs were confirmed by flow
cytometry and western blot analysis. PCR tests for Mycoplasma sp.
Figure 5. Cocrystal X-ray structure of 8g bound to the Ig-like V-type
domain of human PD-L1 (PDB: 7NLD). (A) Arrangement of the
molecules in the crystal structuretwo PD-L1 molecules (gray and
orange) form a single pocket accommodating 8g compound (blues).
(
B) Detailed interactions of 8g at the binding cleft of PD-L1. The
44
contamination were routinely performed, which indicated negative
results for both cell lines.
inhibitor binds at a hydrophobic cavity formed upon PD-L1
dimerization. Color coding as in (A).
Cytotoxicity Assay. The cytotoxicity of compounds was verified
toward Jurkat-ECs since these cells provide the readout in the ICB
assay and present a much higher degree of susceptibility to the
compounds’ toxicity than CHO-K1 cells (not shown). Jurkat-ECs
were seeded on 96-well transparent plates in the presence of
increasing concentrations of the compounds with DMSO-treated cells
as a control. The concentration of DMSO was kept constant (0.1%).
After 48 h of incubation, a tetrazolium reagent, Biolog redox dye MIX
MB (Biolog), was added according to the manufacturer’s protocol,
and the culture plates were incubated for an additional 2 h (37 °C, 5%
Protein Nuclear Magnetic Resonance. NMR spectra were
recorded in PBS pH 7.4 containing 10% (v/v) of D O added to the
2
samples to provide the lock signal. Water suppression was carried out
using the WATERGATE sequence. All the spectra were recorded at
3
00 K using a Bruker AVANCE 600 MHz spectrometer with the cryo-
platform. The spectra were recorded at the ligand/protein ratio of 1:1,
unless stated otherwise. The samples were prepared by adding small
amounts of a 50 mM ligand stock solution in DMSO to the protein
solution (0.20 mL) of PD-L1 fragment at a concentration of 0.2 mM.
Spectra were visualized using TopSpin 4.0.2.
CO ). The absorbance was measured using a Spark microplate reader
2
(Tecan) at 590 nm with 750 nm as a reference. The data are
presented as Jurkat-EC survival relative to DMSO-treated control
cells. Data points represent mean ± SD values from three
independent experiments. Half-maximal effective concentrations
(EC50 values) were calculated from the dose−response curve using
OriginPro 2020 (OriginLab) software.
In Silico Molecular Docking. For molecular docking, we
generated structures of BMS core and m-terphenyl derivative in
the PDB format using Chem3D 17.1 and used AutoDock Vina1.1.2
integrated into PyRx with the dimeric structure of PD-L1 bound to
BMS-1166 (PDB ID: 6R3K).
Protein Expression and Purification. Escherichia coli strain
BL21 was transformed with a pET-21b plasmid carrying the PD-L1
gene (amino acids 18−134). The bacteria were cultured in LB at 37
PD-1/PD-L1 ICB Assay. The functional assay of the blockade of
PD-1/PD-L1 interaction in vitro (PD-1/PD-L1 Blockade Bioassay,
Promega) was performed according to the manufacturer’s protocol.
CHO/TCRAct/PD-L1 cells or CHO/TCRAct or CHO-K1 cells were
seeded on 96-well white plates at the density of 10,000 cells/well and
the next day overlaid with Jurkat T cells (20,000 cells/well) in the
presence of increasing concentrations of the compounds with DMSO
only as a control (the concentration of DMSO was kept constant at
0.1%) or atezolizumab, as a positive control, anti-PD-L1 monoclonal
antibody (Selleckchem). The 2.5-fold dilutions of atezolizumab were
prepared in the assay buffer (RPMI 1640 + 1% FBS) on the day of the
assay. Activation of the Jurkat T cells, reflected by luciferase activity,
was monitored by luminescence measurements after 6 h of incubation
°C until OD6
of 0.6 when the recombinant protein production
00nm
was induced with 1 mM IPTG. The protein production was
continued overnight. Inclusion bodies were collected by centrifuga-
tion, washed twice with 50 mM Tris-HCl pH 8.0 containing 200 mM
NaCl, 10 mM ethylenediaminetetraacetic acid, 10 mM 2-mercaptoe-
thanol, and 0.5% Triton X-100, followed by a single wash with the
same buffer but with no Triton X-100. The washed inclusion bodies
were resuspended overnight in 50 mM Tris-HCl pH 8.0, 6 M GuHCl,
200 mM NaCl, and 10 mM 2-mercaptoethanol and clarified with
centrifugation. Refolding of PD-L1 was performed by dropwise
dilution into 0.1 M Tris-HCl pH 8.0 containing 1 M L-arginine
hydrochloride, 0.25 mM oxidized glutathione, and 0.25 mM reduced
glutathione. The refolded protein was dialyzed 3 times against 10 mM
Tris-HCl pH 8.0 containing 20 mM NaCl and purified by size
exclusion chromatography using Superdex 75 and dialysis buffer. The
quality of the refolded protein was evaluated by sodium dodecyl
sulphate−polyacrylamide gel electrophoresis and NMR.
(37 °C, 5% CO ), and 20 min of additional incubation with the Bio-
2
Glo assay reagent (Promega) at room temperature. The luminescence
was read on a Spark microplate reader (Tecan). The data are
presented as fold induction of the luminescence signal relative to
either untreated (for atezolizumab) or DMSO-treated (for com-
pound) cells. Data points represent mean ± SD values from three to
five independent experiments. Half-maximal effective concentrations
(EC50 values) were calculated from the Hill curve fitting to the
experimental data using Origin Pro 2020 software (OriginLab).
Isolation of PBMCs and TCA Assay. Anticoagulant citrate
dextrose-A-treated blood from healthy donors was purchased from the
Regional Center of Blood Donation and Blood Therapy in Krakow,
Poland. PBMCs were isolated from whole blood by density gradient
centrifugation using Pancoll human separating solution (PAN-Biotech
GmbH, Aidenbach, Germany). The separated cells were washed and
resuspended in RPMI 1640 medium (Biowest) containing 10% FBS
PD-L1 Cocrystallization, Determination, and Refinement.
Purified PD-L1 in 10 mM Tris-HCl pH 8.0, 20 mM NaCl buffer was
concentrated to 5 mg/mL, mixed with the 8g inhibitor in 1:3 molar
ratio (protein/compound) and clarified. The supernatant was used for
screening using commercially available buffer sets. Diffraction-quality
crystals were obtained at room temperature from the condition
containing 0.1 M sodium cocodylate pH 6.5, 25% PEG 4000. The
crystal was flash-cooled in liquid nitrogen. The X-ray diffraction data
were collected at the BL14.1 beamline operated by the Helmholtz-
Zentrum Berlin (HZB) at the BESSY II (Berlin Adlershof,
5
(Biowest). PBMCs were added at an amount of 3 × 10 to CHO,
36
Germany). The data were indexed, integrated, and scaled using
CHO/TCRAct, or CHO/TCRAct/PD-L1 cells preseeded on 24-well
plates in the presence of either anti-PD-L1 mAbs (at the final
concentration 5 μg/mL): durvalumab (Selleckchem), atezolizumab
(Selleckchem), or avelumab (MedChemExpress) or tested com-
3
7−39
XDS, XSCALE, and Aimless.
The initial phases were obtained by
40
molecular replacement calculated in Phaser. The model building
was performed in Coot and refinement was performed using Phenix
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Article
pounds at the concentrations 0.1, 0.5, and 2.5 μM. Untreated cells
were used as controls for antibody treatment, while DMSO-treated
cells [0.1% (v/v)] were used as controls for compound treatment.
acid, K CO , and dioxane/water mixture (2:1, 7/14 mL for 1 mmol)
2 3
under an argon atmosphere. The mixture was deoxygenated by rinsing
with argon for half an hour; then, the Pd(dppf)Cl dichloromethane
2
The cells were incubated for 48 h (37 °C, 5% CO ) and then
complex was added. The reaction mixture was heated at 80 °C, using
preheated bath, for 3 h and after this time, the progression of the
2
detached from the plates with TrypLe Select Enzyme (Thermo
Fisher). PBMCs were stained for 20 min at room temperature with
the antibodies: anti-CD4-FITC, anti-CD8-BV510, anti-CD69-APC,
anti-CD25-PE, anti-HLA-DR-PerCP, and anti-PD1-PECy7 (Becton
Dickinson Biosciences, BD). Following two washing steps, the cells
were analyzed with a FACSCanto II cytometer. Data analysis was
carried out with FlowJo software, followed by statistical significance
calculations done with Origin Pro 2020 software (OriginLab).
Statistical significance was analyzed with one-way analysis of variance
reaction was controlled using TLC analysis (SiO , hexane/ethyl
2
acetate, 4:1). When the reaction was complete, water was added and
the extraction with ethyl acetate followed. The organic phases were
combined, dried over anhydrous MgSO , and evaporated. The crude
4
product was purified by flash chromatography (SiO , hexane/ethyl
2
acetate), giving the final products 2a−2c with 47−61% yield.
6-(3-Bromo-2-methylphenyl)-1,4-benzodioxane (2a). 2,6-Dibro-
motoluene (1a) (1.93 mL, 14.0 mmol, 1.4 equiv), 1,4-benzodioxane-
6-boronic acid (1.79 g, 10.0 mmol, 1.0 equiv), K CO (5.53 g, 40.0
(
0
ANOVA), followed by the Fisher’s posthoc test: *p < 0.05, **p <
2
3
.01, ***p < 0.01.
mmol, 4.0 equiv), Pd(dppf)Cl complex with dichloromethane (0.41
2
Analysis of Cytokine Accumulation. The concentration of the
g, 0.5 mmol, 0.05 equiv). Product 2a was obtained as a colorless oil
with 52.7% (1.61 g) yield. Compound 2a was previously described in
cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNFα) in
supernatants was determined using the CBA Human Th1/Th2/Th17
kit (BD Biosciences), with data acquisition using the FACSCanto flow
cytometer. The CBA beads were discriminated in FL-4 and FL-5
channels, while the concentration of specified cytokines was
determined by the intensity of FL-2 fluorescence, using the respective
standard reference curve and FCAP Array software (BD Biosciences).
IL-4 was below the detection level and is not presented in the
manuscript. Statistical significance was analyzed with one-way
ANOVA using the Origin Pro 2020 software, followed by the Fisher’s
posthoc test: *p < 0.05, **p < 0.01, ***p < 0.01.
45
patent application.
R = 0.51 (SiO , hexane/ethyl acetate 2:1); H NMR (600 MHz,
1
f
2
CDCl ): δ [ppm] 7.52 (dd, J = 8.0, 1.2 Hz, 1H), 7.14 (dd, J = 7.6, 1.1
3
Hz, 1H), 7.05 (t, J = 7.7 Hz, 1H), 6.90 (d, J = 8.2 Hz; 1H), 6.79 (d, J
= 2.1 Hz, 1H), 6.74 (dd, J = 8.2, 2.2 Hz, 1H), 4.31 (s, 4H), 2.33 (s,
3H); 13C NMR (151 MHz, CDCl ): δ [ppm] 143.6, 143.2, 143.0,
3
135.7, 135.2, 131.5, 129.2, 126.8, 126.3, 122.5, 118.2, 117.1, 64.6,
−
1
21.2; IR (ATR) [cm ]: 2927, 2874, 1582, 1505, 1457, 1303, 1276,
1240, 1066, 993, 897, 878, 820, 789, 780; LC−MS (DAD/ESI): t =
R
9.03 compound did not ionize.
Syntheses. All syntheses were performed according to the
procedures summarized in Schemes 1-3 and as described in the
Supporting Information. Reagents were obtained from commercial
suppliers (Sigma-Aldrich, ABCR, Acros Organics, Appollo Scientific,
AK Scientific) and used without further purification unless otherwise
noted. The anhydrous solvents were purchased from Sigma-Aldrich or
6-(2-Amino-3-bromophenyl)-1,4-benzodioxane (2b). 2,6-Dibro-
moaniline (1b) (5.27 g, 21.0 mmol, 1.4 equiv), 1,4-benzodioxane-6-
boronic acid (2.70 g, 15.0 mmol, 1.0 equiv), K CO (8.29 g, 60.0
2
3
mmol, 4.0 equiv), Pd(PPh3)4 (0.87 g, 0.75 mmol, 0.05 equiv).
Product 2b was obtained as a colorless solid with 61.0% (2.80 g)
yield. Product 2b was crystallized from cyclohexane.
1
Alfa Aesar. Nuclear magnetic resonance spectra were recorded at
R = 0.36 (SiO , hexane/ethyl acetate 4:1); H NMR (600 MHz,
f
2
1
3
00K on a Bruker AVANCE 600 spectrometer { H NMR (600 MHz)
CDCl ): δ [ppm] 7.39 (dd, J = 8.0, 1.5 Hz, 1H), 7.02 (dd, J = 7.5, 1.4
3
13
1
13
and C NMR (151 MHz)}. Chemical shifts for H NMR and
C
Hz, 1H), 6.95−6.93 (m, 2H), 6.89 (dd, J = 8.3, 2.0 Hz, 1H), 6.65 (t, J
NMR were reported in parts per million (δ) referenced to the
appropriate deuterated solvent. Coupling constants were reported in
hertz (Hz). The following abbreviations were used for spin
multiplicity: s = singlet, sbroad = broad singlet, d = doublet, t =
triplet, q = quartet, quin = quintet, dd = double of doublets, ddd =
double doublet of doublets, and m = multiplet. IR absorption spectra
were recorded on a Nicolet IR200 spectrometer using the attenuated
total reflection (ATR) technique. Thin-layer chromatography (TLC)
was performed on Sigma-Aldrich precoated silica gel plates (0.20 mm
thick, particle size 25 mm). Spots were visualized by UV light at 254
and 365 nm. Column chromatography was performed using silica gel
= 7.3 Hz, 1H), 4.30 (s, 4H); 13C NMR (151 MHz, CDCl ): δ [ppm]
3
143.9, 143.3, 141.6, 132.4, 131.7, 129.6, 122.2, 119.0, 118.0, 117.9,
−
1
109.9, 64.6, 64.6; IR (ATR) [cm ]: 3481,3389, 2932, 2876, 1603,
1504, 1462, 1308, 1239, 1055, 898, 881, 821, 780, 737; LC−MS
+
(DAD/ESI): t = 7.77 min, calcd for C H BrNO (m/z) [M + H]
r
14 12
2
+
306.01; found, [M + H] 306.10; HRMS (ESI): calcd for
C H BrNO (m/z) [M + H] 306.0124; found, [M + H]+
+
1
4
12
2
306.0123.
6-(3-Bromo-2-chlorophenyl)-1,4-benzodioxane (2c). 1,3-Dibro-
mo-2-chlorobenzene (1c) (5.16 g, 19.09 mmol, 1.1 equiv), 1,4-
benzodioxane-6-boronic acid (3.12 g, 17.4 mmol, 1.0 equiv), K CO
2
3
6
0 (40−63 mm, Sigma-Aldrich). Flash chromatography was
(9.62 g, 69.6 mmol, 4.0 equiv), Pd(dppf)Cl2 complex with
dichloromethane (0.71 g, 0.87 mmol, 0.05 equiv). Product 2c was
obtained as a salmon solid with 46.8% (2.65 g) yield.
performed on a Reveleris X2 flash chromatography, using Grace
Reveleris silica flash cartridges. The liquid chromatography−mass
spectrometry (LC−MS) measurements were performed on an
ultraperformance liquid chromatography (UPLC)−tandem mass
spectrometry system consisting of a Waters ACQUITY UPLC
1
R = 0.38 (SiO , hexane/ethyl acetate 4:1); H NMR (600 MHz,
f
2
CDCl ): δ [ppm] 7.59 (dd, J = 8.0, 1.6 Hz, 1H), 7.24 (dd, J = 7.6, 1.6
3
Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.93−6.91 (m, 2H), 6.87 (dd, J =
(Waters Corporation, Milford, MA, USA) coupled to a Waters
8.3, 2.1 Hz, 1H), 4.30 (s, 4H); 13C NMR (151 MHz, CDCl ): δ
3
TQD mass spectrometer (electrospray ionization mode ESI-tandem
quadrupole). Chromatographic separations were carried out using the
Acquity UPLC BEH (bridged ethyl hybrid) C18 column, 2.1 × 100
mm and 1.7 μm particle size, equipped with an Acquity UPLC BEH
C18 VanGuard precolumn; 2.1 × 5 mm and 1.7 μm particle size. The
column was maintained at 40 °C and eluted under gradient conditions
using 95−0% of eluent A over 10 min at a flow rate of 0.3 mL min .
Eluent A: water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic
acid (0.1%, v/v). The purity of all final compounds determined using
chromatographic LC−MS was >95%. HRMS was carried out by the
Laboratory for Forensic Chemistry Faculty of Chemistry, Jagiellonian
University, with a microOTOF-QII (Bruker) spectrometer using the
ESI ionization technique.
[ppm] 143.5, 143.1, 142.3, 132.9, 132.9, 132.6, 130.2, 127.4, 123.8,
−1
122.5, 118.3, 117.0, 64.5, 64.4; IR (ATR) [cm ]: 2978, 2931, 2875,
+
1506, 1447, 1312; LC−MS (DAD/ESI ): t = 8.68 compound did
R
not ionize.
General Procedure for the Suzuki Reaction (4a−4e and 4i−
4r). A three-necked round-bottom flask was charged with appropriate
halide 2a−2c, borane 3a−3i (for synthesis of compounds 3a−3i, see
−
1
Supporting Information), K CO , and dioxane/water mixture (2:1, 5/
2 3
10 mL for 1 mmol) under an argon atmosphere. The mixture was
deoxygenated by rinsing with argon for half an hour, and then, the
Pd(dppf)Cl dichloromethane complex was added. The reaction
2
mixture was heated at 80 °C, using preheated bath, for 5 h. After this
time, the progression of the reaction was controlled by TLC. When
the reaction was complete, water was added and the extraction with
ethyl acetate followed. The organic phases were combined, dried over
General Procedure for Preparation of 3-Bromobiphenyl
Intermediates (2a−2c). A three-necked round-bottom flask was
charged with appropriate halide 1a−1c, 1,4-benzodioxane-6-boronic
anhydrous MgSO , and evaporated. The crude product was purified
4
K
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by flash chromatography (SiO , hexane/ethyl acetate), giving the final
(d, J = 8.2 Hz, 1H), 6.88 (t, J = 7.5 Hz, 1H), 4.75 (s, 2H), 4.31 (s,
4H), 3.90 (sbroad, 2H), 2.23 (sbroad, 1H); 13C NMR (151 MHz,
2
products 4a−e and 4i−r with 29−89% yield.
(
3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)-
CDCl ): δ [ppm] 143.7, 142.9, 141.0, 139.9, 139.2, 133.0, 129.8,
3
methanol (4a). 6-(3-Bromo-2-methylphenyl)-1,4-benzodioxane (2a)
129.6, 129.5, 127.5, 127.5, 127.5, 122.4, 118.1, 117.6, 65.1, 64.5, 64.4;
−
1
(
(
2.98 g, 9.78 mmol, 1.0 equiv), 4-(hydroxymethyl)phenylboronic acid
2.03 g, 13.30 mmol, 1.2 equiv), K CO (4.90 g, 35.56 mmol, 3.2
IR (ATR) [cm ]: 3466, 3429, 3372, 3350, 3247, 1507, 1317, 1284;
LC−MS (DAD/ESI): t = 6.64 min, calcd for C H NO [m/z] [M
2
3
R
21 19
3
+
+
equiv), Pd(dppf)Cl complex with dichloromethane (0.36 g, 0.45
+ H] 334.14; found, [M + H] 334.22; HRMS (ESI): calcd for
C H NO [m/z] [M + Na] 356.1257; found, [M + Na] 356.1259.
2
+
+
mmol, 0.04 equiv). Product 4a was obtained as a colorless solid with
21 19 3
5
6.4% (1.62 g) yield.
(3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-[1,1′-biphenyl]-4-yl)-
1
R = 0.23 (SiO , hexane/ethyl acetate 2:1); H NMR (600 MHz,
methanol (4e). 6-(3-Bromo-2-chlorophenyl)-1,4-benzodioxane (2c)
(0.19 g, 0.59 mmol, 1.0 equiv), 4-(hydroxymethyl)phenylboronic acid
(0.11 g, 0.71 mmol, 1.2 equiv), K CO (0.24 g, 1.77 mmol, 4.2 equiv),
f
2
DMSO-d ): δ [ppm] 7.39 (d, J = 8.3 Hz, 2H), 7.33 (dt, J = 8.1, 1.8
6
Hz, 2H), 7.27 (t, J = 7.6 Hz, 1H), 7.17−7.15 (m, 2H), 6.92 (d, J = 8.2
Hz, 1H), 6.86 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.2, 2.1 Hz, 1H), 5.22
2
3
Pd(dppf)Cl complex with dichloromethane (0.048 g, 0.06 mmol, 0.1
2
t, J = 5.8 Hz, 1H), 4.65 (d, J = 5.7 Hz, 2H), 4.28 (s, 4H), 2.07 (s,
equiv). Product 4e was obtained as a colorless solid with 32.6%
H); 13C NMR (151 MHz, DMSO-d ): δ [ppm] 143.4, 143.0, 142.8,
(0.068 g) yield.
6
1
42.3, 141.7, 140.7, 135.4, 132.8, 129.4, 129.1, 129.0, 126.8, 126.0,
R = 0.50 (SiO , hexane/ethyl acetate 1:2); H NMR (600 MHz,
f
2
−
1
22.6, 118.2, 117.2, 64.6, 64.5, 63.2, 19.1; IR (ATR) [cm ]: 3315,
CDCl ): δ [ppm] 7.48 (m, 4H), 7.36−7.30 (m, 3H), 7.03 (d, J = 1.9
3
930, 1578, 1508, 1318; LC−MS (DAD/ESI): t = 7.36 min, calcd
Hz, 1H), 6.98−6.95 (m, 2H), 4.79 (s, 2H), 4.33 (s, 4H), 1.75 (s,
R
+
+
13
1H); C NMR (151 MHz, CDCl ): δ [ppm] 143.2, 143.0, 141.3,
2
2
20
3
2
3
+
141.1, 140.1, 139.6, 133.4, 131.0, 130.5, 130.2, 129.8, 126.7, 126.4,
2
22 20
3
+
+
−1
122.8, 118.6, 116.9, 65.2, 64.5, 64.4; IR (ATR) [cm ]: 3317, 1507,
(
1455, 1247, 795; LC−MS (DAD/ESI): t = 7.29 min, calcd for
R
+
+
C H ClO [m/z] [M + H] 353.09, [M-H O + H] 335.08; found,
2
1
17
3
2
+
2.98 g, 9.78 mmol, 1.0 equiv), 3-(hydroxymethyl)phenylboronic acid
2.03 g, 13.30 mmol, 1.2 equiv), K CO (4.90 g, 35.56 mmol, 3.2
[M-H O + H] 335.15; HRMS (ESI): calcd for C H ClO [m/z]
2 21 17 3
+
+
2
3
[M + Na] 375.0758; found, [M + Na] 375.0761.
2
(3′-(Benzo-1,4-dioxan-6-yl)-3-methoxy-2′-methyl-[1,1′-biphen-
yl]-4-yl)methanol (4i). 6-(3-Bromo-2-methylphenyl)-1,4-benzodiox-
ane (2a) (1.50 g, 4.92 mmol, 1.0 equiv), (2-methoxy-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol (3a) (1.30 g,
4.92 mmol, 1.0 equiv), K CO (2.72 g, 19.66 mmol, 4.0 equiv),
0.1% (1.48 g) yield.
1
R = 0.24 (SiO , hexane/ethyl acetate 2:1); H NMR (600 MHz,
f
2
3
2
3
Pd(dppf)Cl complex with dichloromethane (0.20 g, 0.25 mmol, 0.05
2
equiv). Product 4i was obtained as a colorless solid with 68.7% (1.22
g) yield.
1
R = 0.38 (SiO , hexane/ethyl acetate 1:1); H NMR (600 MHz,
f
3
2
1
1
1
35.8, 133.1, 129.1, 128.8, 128.7, 128.3, 128.0, 125.4, 125.3, 122.6,
18.2, 116.9, 65.4, 64.4, 18.8; IR (ATR) [cm ]: 3360, 2875, 1578,
CDCl ): δ [ppm] 7.34 (d, J = 7.5 Hz, 1H), 7.30−7.28 (m, 1H), 7.26−
3
−
1
7.23 (m, 2H), 6.96 (dd, J = 7.5, 1.4 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H),
506, 1069; LC−MS (DAD/ESI): t = 7.48 min, calcd for C H O
6.91 (m, 2H), 6.86 (dd, J = 8.2, 2.1 Hz, 1H), 4.77 (s, 2H), 4.33 (s,
R
22 20
3
+
+
13
[m/z] [M + H] 333.15, [M-H O + H] 315.14; found, [M-H O +
4H), 3.91 (s, 3H), 2.20 (s, 3H); C NMR (151 MHz, CDCl ): δ
2
2
3
+
+
H] 315.21; HRMS (ESI): calcd for C H O [m/z] [M + Na]
[ppm] 157.1, 143.5, 143.1, 142.7, 142.6, 142.4, 135.7, 133.1, 129.2,
2
2
20
3
+
3
55.1305; found, [M + Na] 355.1304.
3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-2-yl)-
methanol (4c). 6-(3-Bromo-2-methylphenyl)-1,4-benzodioxane (2a)
128.7, 128.5, 127.5, 125.3, 122.5, 121.6, 118.2, 116.9, 111.6, 64.5,
−
1
(
64.4, 62.0, 55.4, 18.8; IR (ATR) [cm ]: 3547, 2936, 1505, 1302,
1229; LC−MS (DAD/ESI): t = 7.58 min, calcd for C H O [m/z]
R
23 22
4
+
+
(
(
2.98 g, 9.78 mmol, 1.0 equiv), 2-(hydroxymethyl)phenylboronic acid
2.03 g, 13.30 mmol, 1.2 equiv), K CO (4.90 g, 35.56 mmol, 3.2
[M + H] 363.15; found, [M + H] 363.26; HRMS (ESI): calcd for
C H O [m/z] [M + Na] 385.1410; found, [M + Na] 385.1411.
+
+
2
3
23 22
4
equiv), Pd(dppf)Cl complex with dichloromethane (0.36 g, 0.45
mmol, 0.04 equiv). Product 4c was obtained as a colorless solid with
3-(((3′-(Benzo-1,4-dioxan-6-yl)-4-(hydroxymethyl)-2′-methyl-
[1,1′-biphenyl]-3-yl)oxy)methyl)benzonitrile (4j). 6-(3-Bromo-2-
methylphenyl)-1,4-benzodioxane (2a) (0.60 g, 2.0 mmol, 1.0
equiv), 3-((2-(hydroxymethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)phenoxy)methyl)benzonitrile (3i) (0.86 g, 2.4 mmol, 1.2
equiv), K CO (1.09 g, 7.9 mmol, 4.0 equiv), Pd(PPh ) (0.11 g, 0.1
2
41.9% (1.24 g) yield.
1
R = 0.25 (SiO , hexane/ethyl acetate 2:1); H NMR (600 MHz,
f
2
CDCl ): δ [ppm] 7.59 (dd, J = 7.6, 0.7 Hz, 1H), 7.43 (dt, J = 7.5, 1.4
3
Hz, 1H), 7.38 (dt, J = 7.5, 1.4 Hz, 1H), 7.28−7.25 (m, 2H), 7.22 (dd,
J = 7.5, 1.3 Hz, 1H), 7.14 (dd, J = 6.6, 2.4 Hz, 1H), 6.93 (d, J = 8.2
Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.84 (dd, J = 8.2, 2.1 Hz, 1H), 4.49
2
3
3 4
mmol, 0.05 equiv). Product 4j was obtained as a bluish oil with 29.1%
(0.266 g) yield.
1
(dd, J = 6.0, 1.2 Hz, 2H), 4.33 (s, 4H), 1.97 (s, 3H), 1.57 (t, J = 5.9
R = 0.45 (SiO , hexane/ethyl acetate 1:1); H NMR (600 MHz,
f
2
13
Hz, 1H); C NMR (151 MHz, CDCl ): δ [ppm] 143.1, 142.6, 142.2,
DMSO-d ): δ [ppm] 7.93 (s, 1H), 7.81 (t, J = 7.9 Hz, 2H), 7.62 (t, J
3
6
1
1
3
7
40.9, 140.8, 138.5, 135.6, 133.5, 129.5, 129.3, 128.3, 127.7, 127.6,
27.5, 125.3, 122.5, 118.2, 116.9, 64.4, 63.2, 18.2; IR (ATR) [cm ]:
= 7.8 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.15
(dd, J = 10.7, 3.5 Hz, 2H), 6.99 (d, J = 1.3 Hz, 1H), 6.95 (dd, J = 7.6,
1.3 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.84 (d, J = 2.1 Hz, 1H), 6.80
(dd, J = 8.2, 2.1 Hz, 1H), 5.24 (s, 2H), 5.07 (t, J = 5.6 Hz, 1H), 4.62
−1
559, 3533, 2933, 1581, 1505, 1317, 1066; LC−MS (DAD/ESI): t =
R
+
.58 min, calcd for C H O [m/z] [M-H O + H] 315.14; found,
2
2
20
3
2
+
13
[
M-H O + H] 315.21; HRMS (ESI): calcd for C H O : [M +
(d, J = 5.6 Hz, 1H), 4.28 (s, 4H), 1.99 (s, 3H); C NMR (151 MHz,
2
22 20
3
+
+
Na] 355.1305; found, [M + Na] 355.1305.
2′-Amino-3′-(benzo-1,4-dioxan-6-yl)-[1,1′-biphenyl]-4-yl)-
methanol (4d). 6-(2-Amino-3-bromophenyl)-1,4-benzodioxane (2b)
6.12 g, 20.00 mmol, 1.0 equiv), 4-(hydroxymethyl)phenylboronic
acid (3.04 g, 20.00 mmol, 1.2 equiv), K CO (9.22 g, 66.68 mmol, 4.2
DMSO-d ): δ [ppm] 154.3, 143.0, 142.5, 142.3, 141.8, 141.4, 139.2,
6
(
134.9, 132.4, 132.1, 131.5, 130.7, 129.7, 129.4, 128.7, 128.4, 127.2,
125.5, 122.1, 121.3, 118.8, 117.7, 116.8, 112.9, 111.4, 67.9, 64.1, 57.9,
−
1
(
18.6; IR (ATR) [cm ]: 3390, 2924, 2873, 2230, 1574, 1505, 1459,
2
3
1402, 1301, 1278, 1221, 1066, 894, 791; LC−MS (DAD/ESI): t =
R
+
equiv), Pd(dppf)Cl complex with dichloromethane (0.68 g, 0.83
mmol, 0.04 equiv). Product 4d was obtained as a colorless solid with
8.16 min, calcd for C H NO (m/z) [M-H O + H] 446.18; found,
[M-H O + H] 446.34; HRMS (ESI): calcd for C H NO (m/z)
2
30 25
4
2
+
2 30 25 4
+
+
32.0% (1.78 g) yield.
[M + Na] 486.1676; found, [M + Na] 486.1676.
1
R = 0.19 (SiO , hexane/ethyl acetate 1:1); H NMR (600 MHz,
(3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphen-
yl]-4-yl)methanol (4k). 6-(3-Bromo-2-chlorophenyl)-1,4-benzodiox-
ane (2c) (0.46 g, 1.41 mmol, 1.0 equiv), (2-methoxy-4-(4,4,5,5-
f
2
CDCl ): δ [ppm] 7.53 (m, 2H), 7.47 (d, J = 8.2 Hz, 2H), 7.12 (m,
3
2H), 7.05 (d, J = 2.00 Hz, 1H), 7.00 (dd, J = 8.3, 2.0 Hz, 1H), 6.97
L
https://doi.org/10.1021/acs.jmedchem.1c00957
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
141.6, 141.2, 141.1, 1335., 131.1, 130.7, 130.2, 128.5, 128.4, 126.5,
122.9, 121.8, 118.7, 117.0, 112.8, 66.7, 64.6, 64.6, 62.4, 38.2, 25.4,
227; IR (ATR) [cm ]: 3376, 3055, 2955, 2930, 2871, 1583, 1507,
−
1
equiv). Product 4k was obtained as a colorless solid with 70.0%
1456, 1303, 1280, 1248, 1225, 1068, 797; LC−MS (DAD/ESI): t =
R
+
(
0.377 g) yield.
9.16 min, calcd for C H ClO (m/z) [M-H O + H] 421.16; found,
2
6
27
4
2
1
+
R = 0.38 (SiO , hexane/ethyl acetate 1:1); H NMR (600 MHz,
f
[M-H O + H] 421.16; HRMS (ESI): calcd for C H ClO (m/z)
[M + Na] 461.1490; found, [M + Na] 461.1490.
2
2
26 27
4
+
+
CDCl ): δ [ppm] 7.35 (d, J = 7.4 Hz, 1H), 7.33−7.28 (m, 3H), 7.02
3
(
dd, J = 7.5, 1.5 Hz, 1H), 7.00 (m, 2H), 6.99−6.92 (m, 2H), 4.74 (d,
2-((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-4-(hydroxymethyl)-
[1,1′-biphenyl]-3-yl)oxy)acetonitrile (4o). 6-(3-Bromo-2-chlorophen-
yl)-1,4-benzodioxane (2c) (0.40 g, 1.24 mmol, 1.0 equiv), 2-(2-
hydroxymethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
J = 6.4 Hz, 2H), 4.31 (s, 4H), 3.89 (s, 3H), 2.30 (t, J = 6.5 Hz, 1H);
1
3
C NMR (151 MHz, CDCl ): δ [ppm] 156.9, 143.2, 143.0, 141.4,
3
1
1
3
41.1, 141.1, 133.3, 131.0, 130.6, 130.1, 128.4, 128.3, 126.4, 122.8,
21.8, 118.6, 116.9, 111.9, 64.5, 64.4, 62.0, 55.4; IR (ATR) [cm ]:
−
1
phenoxy)acetonitrile (3e) (0.50 g, 1.72 mmol, 1.4 equiv), K
CO
2 3
530, 2936, 1506, 1460, 1303, 1227; LC−MS (DAD/ESI): t = 7.43
(0.51 g, 3.72 mmol, 3.0 equiv), Pd(dppf)Cl complex with
2
R
+
+
min, calcd for C H ClO [m/z] [M + H] 383.10, [M-H O + H]
dichloromethane (0.10 g, 0.12 mmol, 0.1 equiv). Product 4o was
22
19
4
2
+
3
65.09; found, [M-H O + H] 365.19; HRMS (ESI): calcd for
obtained as a colorless solid with 56.0% (0.283 g) yield.
2
+
+
1
C H ClO [m/z] [M + Na] 405.0864; found, [M + Na]
R
f
= 0.24 (SiO
2
, hexane/ethyl acetate 1:1); H NMR (600 MHz,
2
2
19
4
4
05.0863.
3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-ethoxy-[1,1′-biphenyl]-
-yl)methanol (4l). 6-(3-Bromo-2-chlorophenyl)-1,4-benzodioxane
CDCl
3
): δ [ppm] 7.49 (d, J = 7.7 Hz, 1H), 7.36−7.32 (m, 2H), 7.29
(
(dd, J = 6.5, 2.8 Hz, 1H), 7.19 (dd, J = 7.7, 1.4 Hz, 1H), 7.06 (d, J =
1.3 Hz, 1H), 7.00−6.99 (m, 1H), 6.96−6.91 (m, 2H), 4.86 (s, 2H),
4
(
2c) (0.50 g, 1.53 mmol, 1.2 equiv), (2-ethoxy-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol (3b) (0.35 g,
.27 mmol, 1.0 equiv), K CO (0.70 g, 5.10 mmol, 4.0 equiv),
4.78 (s, 2H), 4.31 (s, 4H), 1.90 (s , 1H); 13C NMR (151 MHz,
broad
CDCl ): δ [ppm] 153.8, 143.4, 143.2, 141.4, 141.3, 133.2, 131.0,
3
1
130.2, 129.5, 129.2, 126.7, 124.5, 122.9, 118.7, 117.0, 115.0, 113.4,
2
3
−
1
Pd(dppf)Cl complex with dichloromethane (0.05 g, 0.06 mmol, 0.05
64.6, 64.5, 60.8, 54.0; IR (ATR) [cm ]: 3524, 3053, 2985, 2944,
2886, 1586, 1510, 1400, 1321, 1283, 1244, 1069, 1048, 890, 787;
LC−MS (DAD/ESI): t = 6.99 min, calcd for C H ClNO (m/z)
2
equiv). Product 4l was obtained as a colorless solid with 37.9% (0.191
g) yield.
R
23 18
4
1
+
+
R = 0.40 (SiO , hexane/ethyl acetate 1:1); H NMR (600 MHz,
f
[M-H O + H] 390.09; found, [M-H O + H] 390.18; HRMS (ESI):
2
2
2
+
CDCl ): δ [ppm] 7.38 (m, 1H), 7.35−7.31 (m, 3H), 7.05−7.03 (m,
calcd for C H ClNO (m/z) [M + Na] 430.0816; found, [M +
23 28 4
3
+
2
4
H), 7.02 (m, 1H), 6.99−6.95 (m, 2H), 4.79 (s, 2H), 4.33 (s, 4H),
Na] 430.0817.
.16 (q, J = 7.0 Hz, 2H), 2.58 (s , 1H), 1.48 (t, J = 7.0 Hz, 3H);
(3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-cyclobutylmethoxy-
broad
1
3
C NMR (151 MHz, CDCl ): δ [ppm] 156.3, 143.2, 143.0, 141.4,
[1,1′-biphenyl]-4-yl)methanol (4p). 6-(3-Bromo-2-chlorophenyl)-
3
1,4-benzodioxane (2c) (0.49 g, 1.50 mmol, 1.0 equiv), (2-(cyclo-
1
1
41.1, 140.9, 133.3, 131.0, 130.5, 130.1, 128.4, 128.4, 126.4, 122.8,
butylmethoxy)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl)methanol (3f) (0.67 g, 2.10 mmol, 1.4 equiv), K CO (0.62 g,
4.48 mmol, 3.0 equiv), Pd(dppf)Cl complex with dichloromethane
21.7, 118.6, 116.9, 112.8, 64.5, 64.4, 63.7, 62.1, 14.9; IR (ATR)
−1
[
(
3
cm ]: 3219, 2972, 1509, 1399, 1279, 1070, 1052, 1044; LC−MS
2
3
+
DAD/ESI): t = 7.92 min, calcd for C H ClO [m/z] [M + H]
2
R
23 21
4
+
+
97.12 [M-H O + H] 379.11; found, [M-H O + H] 379.21; HRMS
(0.12 g, 0.15 mmol, 0.1 equiv). Product 4p was obtained as a colorless
2
2
+
(
ESI): calcd for C H ClO [m/z] [M + Na] 419.1021; found, [M
solid with 64.8% (0.425 g) yield.
23
21
4
+
−1
+
Na] 419.1021.
R
f
= 0.16 (SiO
2
, hexane/ethyl acetate 4:1); IR (ATR) [cm ]:
(
3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-isopropoxy-[1,1′-biphen-
3414, 2976, 2935, 2869, 1583, 1507, 1456, 1303, 1280, 1249, 1226,
1
yl]-4-yl)methanol (4m). 6-(3-Bromo-2-chlorophenyl)-1,4-benzodiox-
ane (2c) (0.33 g, 1.03 mmol, 1.0 equiv), (2-izopropoxy-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol (3c) (0.36 g,
1069, 1046, 797, 746; H NMR (600 MHz, CDCl ): δ [ppm] 7.34−
3
7.27 (m, 4H), 7.02−6.98 (m, 3H), 6.96−6.92 (m, 2H), 4.75 (s, 2H),
4.31 (s, 4H), 4.02 (d, J = 6.6. Hz, 2H), 2.8702.78 (m, 1H), 2.19−2.12
13
1
.23 mmol, 1.2 equiv), K CO (0.42 g, 3.08 mmol, 3.0 equiv),
(m, 2H), 2.02−1.85 (m, 4H); C NMR (151 MHz, CDCl ): δ
2
3
3
Pd(dppf)Cl complex with dichloromethane (0.08 g, 0.10 mmol, 0.1
[ppm] 156.7, 143.3, 143.1, 141.5, 141.2, 141.1, 133.5, 131.1, 130.7,
130.2, 128.6, 128.4, 126.5, 1229., 121.8, 118.7, 117.0, 113.0, 72.2,
2
equiv). Product 4m was obtained as a colorless oil with 60.8% (0.256
g) yield.
64.6, 64.6, 62.5, 34.7, 25.0, 18.8; LC−MS (DAD/ESI): t = 9.00 min,
R
1
+
R = 0.4 (SiO , hexane/ethyl acetate 1:1); H NMR (600 MHz,
calcd for C H ClNO (m/z) [M-H O + H] 419.14; found, [M-
f
2
26 25
4
2
+
CDCl ): δ [ppm] 7.34−7.32 (m, 4H), 7.01−7.00 (m, 2H), 6.98 (dd, J
H O + H] 419.24; HRMS (ESI): calcd for C H ClO (m/z) [M +
3
2 26 25 4
+
+
=
6
7.6, 1.5 Hz, 1H), 6.96−6.92 (m, 2H), 4.72 (s, 2H), 4.66 (hept, J =
.0 Hz, 1H), 4.31 (s, 4H), 1.39 (d, J = 6.0 Hz, 6H); 13C NMR (151
MHz, CDCl ): δ [ppm] 155.3, 143.3, 143.1, 141.5, 141.2, 140.9,
Na] 459.1334; found, [M + Na] 459.1333.
(3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-cyclopentylmethoxy-
[1,1′-biphenyl]-4-yl)methanol (4q). 6-(3-Bromo-2-chlorophenyl)-
1,4-benzodioxane (2c) (0.29 g, 0.89 mmol, 1.0 equiv), (2-(cyclo-
butylmethoxy)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
3
1
1
3
1
33.4, 131.1, 130.6, 130.2, 129.3, 128.7, 126.5, 122.9, 121.6, 118.7,
17.0, 114.3, 70.4, 64.6, 64.5, 62.6, 22.4; IR (ATR) [cm ]: 3419,
053, 2976, 2931, 2874, 1583, 1507, 1456, 1302, 1280, 1248, 1225,
068, 797; LC−MS (DAD/ESI): t = 8.23 min, calcd for
−
1
phenyl)methanol (3g) (0.42 g, 1.26 mmol, 1.4 equiv), K
CO (0.37 g,
2 3
2.68 mmol, 3.0 equiv), Pd(dppf)Cl complex with dichloromethane
R
2
+
C H ClNO (m/z) [M-H O + H] 393.13; found, [M-H O +
H] 393.23; HRMS (ESI): calcd for C H ClO (m/z) [M + Na]
(0.07 g, 0.09 mmol, 0.1 equiv). Product 4q was obtained as a colorless
2
4
23
4
2
2
+
+
solid with 62.4% (0.251 g) yield.
2
4
23
4
+
1
4
33.1177; found, [M + Na] 433.1177.
R
f
= 0.16 (SiO
, hexane/ethyl acetate 4:1); H NMR (600 MHz,
2
(
3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-isopentyloxy-[1,1′-bi-
CDCl
3
): δ [ppm] 7.35 (m, 4H), 7.02−6.97 (m, 3H), 6.96−6.91 (m,
phenyl]-4-yl)methanol (4n). 6-(3-Bromo-2-chlorophenyl)-1,4-benzo-
dioxane (2c) (0.48 g, 1.47 mmol, 1.0 equiv), (2-izopentyloxy-4-
2H), 4.75 (s, 2H), 4.31 (s, 4H), 3.93 (d, J = 6.9 Hz, 1H), 2.45−2.38
(m, 1H), 1.90−1.83 (m, 2H), 1.69−1.57 (m, 4H), 1.41−1.33 (m,
2H); 13C NMR (151 MHz, CDCl ): δ [ppm] 156.7, 143.3, 143.1,
(
(
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol (3d)
0.66 g, 2.06 mmol, 1.4 equiv), K CO (0.61 g, 4.41 mmol, 3.0
3
2
3
141.6, 141.2, 141.1, 133.5, 131.1, 130.6, 128.5, 128.4, 126.5, 122.9,
equiv), Pd(dppf)Cl complex with dichloromethane (0.12 g, 0.15
mmol, 0.1 equiv). Product 4n was obtained as a colorless oil with
121.7, 118.7, 117.0, 112.8, 72.4, 64.6, 64.6, 62.5, 39.2, 29.7, 25.6; IR
2
−
1
(ATR) [cm ]: 3390, 3054, 2952, 2868, 1583, 1507, 1456, 1303,
5
5.8% (0.361) yield.
R = 0.16 (SiO , hexane/ethyl acetate 4:1); H NMR (600 MHz,
f
1280, 1249, 1225, 1069, 1040, 797, 747; LC−MS (DAD/ESI): t =
R
1
+
2
9.38 min, calcd for C H ClO (m/z) [M-H O + H] 433.16; found,
[M-H O + H] 433.25; HRMS (ESI): calcd for C H ClO (m/z)
27 27
4
2
+
CDCl ): δ [ppm] 7.35 (m, 4H), 7.02−6.98 (m, 3H), 6.96−6.92 (m,
3
2 27 27 4
+
+
2
1
H), 4.75 (s, 2H), 4.31 (s, 4H), 4.08 (t, J = 6.6 Hz, 2 H), 2.39 (s
H), 1.89−1.79 (m, 1H), 1.73 (q, J = 6.6 Hz, 2H), 0.97 (d, J = 6.6
,
[M + Na] 473.1490; found, [M + Na] 473.1488.
broad
3′-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-4-(hydroxymethyl)-
13
Hz, 6H); C NMR (151 MHz, CDCl ): δ [ppm] 156.6,143.3, 143.1,
[1,1′-biphenyl]-3-yl)oxy)methyl)-[1,1′-biphenyl]-3-carbonitrile (4r).
3
M
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
6
-(3-Bromo-2-chlorophenyl)-1,4-benzodioxane (2c) (0.28 g, 0.86
mmol, 1.0 equiv), 3′-((2-(hydroxymethyl)-5-(4,4,5,5-tetramethyl-
,3,2-dioxaborolan-2-yl)phenoxy)methyl)-[1,1′-biphenyl]-3-carboni-
trile (3h) (0.38 g, 0.86 mmol, 1.0 equiv), K CO (0.48 g, 3.45 mmol,
R = 0.47 (SiO , hexane/ethyl acetate 1:2); H NMR (600 MHz,
f 2
CDCl ): δ [ppm] 7.44 (d, J = 8.2 Hz, 2H), 7.37−7.35 (m, 3H), 7.23
(dd, J = 7.6, 1.7 Hz, 1H), 7.21 (dd, J = 7.5, 1.7 Hz, 1H), 6.91 (d, J =
8.3 Hz, 1H), 6.89 (d, J = 2.1 Hz, 1H), 6.84 (dd, J = 8.3, 2.1 Hz, 1H),
4.77 (d, J = 2.6 Hz, 2H), 4.31 (s, 4H), 1.72 (sbroad, 1H); C NMR
(151 MHz, CDCl ): δ [ppm] 147.7, 147.5, 145.1, 143.1, 142.8, 140.2,
139.0, 129.7, 128.8, 128.5, 127.6, 126.5, 122.7, 118.4, 116.7, 104.0,
65.2, 64.5, 64.4; IR (ATR) [cm ]: 3309, 2955, 1505, 1451, 1252,
= 7.47 min, calcd for C21
H O + H] 427.02; found, [M-H O + H] 427.13; HRMS (ESI ):
3
1
2
3
1
3
4
.0 equiv), Pd(dppf)Cl complex with dichloromethane (0.03 g, 0.04
2
mmol, 0.05 equiv). Product 4r was obtained as a colorless oil with
8.8% (0.427 g) yield.
R = 0.37 (SiO , hexane/ethyl acetate 1:1); H NMR (600 MHz,
3
8
1
−1
f
2
+
CDCl ): δ [ppm] 7.86 (t, J = 1.6 Hz, 1H), 7.80 (m, 1H), 7.64 (dt, J =
1071; LC−MS (DAD/ESI ): t
R
H
17IO
3
[M-
3
+
+
+
7
.7, 1.3 Hz, 1H), 7.62 (s, 1H), 7.56−7.48 (m, 4H), 7.41 (d, J = 7.6
2
2
+
+
Hz, 1H), 7.33−7.26 (m, 3H), 7.10 (d, J = 1.4 Hz, 1H), 7.06 (dd, J =
calcd for C21
H17IO [m/z] [M + Na] 467.0115; found, [M + Na]
3
7
4
.6, 1.5 Hz, 1H), 6.98 (t, J = 1.2 Hz, 1H), 6.92 (m, 2H), 5.22 (s, 2H),
467.0115.
3
.81 (s, 2H), 4.30 (s, 4H), 2.27 (sbroad, 1H); 13C NMR (151 MHz,
-(3′-(Benzo-1,4-dioxan-6-yl)-4′-(hydroxymethyl)-[1,1′-biphen-
yl]-2-carbonitrile (4h). In a three-necked round-bottom flask
equipped with a thermometer, (2′-amino-3′-(benzo-1,4-dioxan-6-yl)-
CDCl ): δ [ppm] 155.8, 143.2, 143.0, 142.1, 141.2, 141.1, 141.0,
3
1
1
1
3
39.4, 137.7, 133.2, 131.6, 130.9, 130.8, 130.6, 130.1, 129.7, 129.6,
28.8, 128.6, 127.4, 126.9, 126.4, 126.2, 122.8, 122.4, 118.8, 118.6,
[
1,1′-biphenyl]-4-yl)methanol (4d) (0.21 g, 0.62 mmol, 1 equiv) was
−
1
added and dissolved in 6 mL of dioxane/water (2/1, v/v) mixture.
After dissolution, 0.2 mL of conc. HCl was added and the mixture was
16.9, 113.4, 113.1, 70.0, 64.5, 64.4, 61.8; IR (ATR) [cm ]: 3334,
059, 2931, 2229, 1585, 1507, 1397, 1246, 781; LC−MS (DAD/
+
cooled in an ice bath; then, a solution of NaNO (0.07 g, 0.96 mmol,
2
ESI): t = 8.83 min, calcd for C H ClNO [m/z] [M + H] 560.16
R
35 26
4
+
+
1.5 equiv) in 2 mL of water was added dropwise, maintaining the
temperature below 5 °C. The mixture was stirred for 5 min and then
added to a previously prepared solution of CuCN (0.08 g, 0.86 mmol,
[
M-H O + H] 542.16; found, [M-H O + H] 542.32; HRMS (ESI):
2
2
+
calcd for C H ClNO (m/z) [M + Na] 582.1443; found, [M +
Na] 582.1440.
35
26
4
+
1
.4 equiv) and KCN (0.16 g, 2.37 mmol, 3.8 equiv) in 4 mL of water.
Synthesis of C2′ Halogenated and C2′-Cyano Derivatives
The solution was stirred for 4 h. After this time, 3 mL of 2 M NaOH
was added and the mixture was transferred into a separatory funnel.
The aqueous phase was extracted with ethyl acetate (3 × 15 mL). The
organic phases were collected, dried over anhydrous MgSO , and the
solvent was evaporated. The product was purified by flash
by the Sandmeyer Reaction (4f−h). (3′-(Benzo-1,4-dioxan-6-yl)-
2
′-bromo-[1,1′-biphenyl]-4-yl)methanol (4f). In a three-necked
round-bottom flask equipped with a thermometer, (2′-amino-3′-
benzo-1,4-dioxan-6-yl)-[1,1′-biphenyl]-4-yl)methanol (4d) (0.22 g,
.63 mmol, 1 equiv) was added and dissolved in 1.2 mL of 48% HBr.
(
0
4
chromatography (SiO , hexane/ethyl acetate, 1:2), giving the final
The mixture was cooled in an ice bath and a solution of NaNO (0.09
g, 1.35 mmol, 2.0 equiv) in 2 mL of water was added dropwise,
maintaining the temperature below 5 °C, and the mixture was stirred
for 5 min and a solution of CuBr (0.37 g, 2.59 mmol, 4 equiv) in 1.8
mL of 48% HBr was added dropwise, maintaining the temperature
below 5 °C. The mixture was heated in 70 °C for 2 h. After this time,
the solution was transferred into a separatory funnel, 5 mL of water
was added, and the aqueous phase was extracted with ethyl acetate (3
2
2
product 4h with 20.6% (0.04 g) yield.
1
R = 0.44 (SiO , hexane/ethyl acetate 1:2); H NMR (600 MHz,
f
2
CDCl ): δ [ppm] 7.63 (t, J = 7.8 Hz, 1H), 7.58 (m, 2H), 7.50 (d, J =
3
2
8
=
4
1
1
[
R
3
.2 Hz, 2H), 7.43−7.41 (m, 2H), 7.10 (d, J = 2.1 Hz, 1H), 7.08 (dd, J
8.3, 2.2 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 4.77 (s, 2H), 4,31 (s,
H), 1.82 (sbroad, 1H); 13C NMR (151 MHz, CDCl ): δ [ppm] 146.6,
3
46.4, 144.2, 143.5, 141.4, 138.1, 132.3, 131.9, 129.3, 128.8, 128.5,
27.1, 122.3, 118.1, 118.0, 117.5, 110.3, 65.0, 64.5, 64.4; IR (ATR)
×
15 mL). The organic phases were collected, dried over anhydrous
−
1
+
cm ]: 3418, 2931, 2224, 1513, 1461, 1288; LC−MS (DAD/ESI ):
MgSO , and the solvent was evaporated. The product was purified by
4
+
+
t = 6.60 min, calcd for C H NO [m/z] [M] 343.12; found, [M]
22 17
3
flash chromatography (SiO , hexane/ethyl acetate, 1:2), giving the
final product 4f with 9.1% (0.02 g) yield.
2
43.25; HRMS (ESI ): calcd for C H NO [m/z] [M + Na]+
+
22 17 3
+
1
366.1101; found, [M + Na] 366.1100.
R = 0.60 (SiO , hexane/ethyl acetate 1:2); H NMR (600 MHz,
f
2
General Procedure for Williamson Ether Synthesis (5a, 5c,
CDCl ): δ [ppm] 7.37−2.28 (m, 5H), 7.21−7.16 (m, 2H), 6.88 (m,
3
5
e, 5i, 5k, 5m, 5o, and 5q). Appropriate alcohol 4a−c (1 equiv)
1
H), 6.86−6.80 (m, 2H), 4.69 (s, 2H), 4.23 (s, 4H), 1.55 (s , 1H);
broad
was added in a round-bottom flask with anhydrous dichloromethane
under an argon atmosphere and cooled in an ice bath; thionyl
chloride (3 equiv) was added dropwise, and then, anhydrous DMF
1
3
C NMR (151 MHz, CDCl ): δ [ppm] 143.5, 143.2, 143.1, 142.9,
3
1
1
1
41.6, 140.1, 135.4, 130.3, 123.0, 129.8, 126.9, 126.6, 123.3, 122.8,
−
1
18.5, 116.8, 65.2, 64.5, 64.4; IR (ATR) [cm ]: 3291, 2927, 1505,
(
0.2 mL per 2 mmol alcohol) was added. The reaction mixture was
stirred approximately 2 h at room temperature and controlled by TLC
(SiO , hexane/ethyl acetate, 4/1). When all alcohol disappeared, the
reaction mixture was poured onto saturated NaHCO3 and the
aqueous phase extracted three times with dichloromethane. The
organic phases were collected, dried over anhydrous MgSO , and
evaporated. The crude product was dissolved in anhydrous DMF;
then, K CO or Cs CO (3 equiv), appropriate nucleophile (1.0−1.5
equiv), and KI (0.1 equiv) were added. The reaction mixture was
heated at 80 °C overnight. After this time, the mixture was transferred
into a separatory funnel with water and extracted three times with
ethyl acetate. The organic phases were collected, dried over
+
454, 1251, 1070; LC−MS (DAD/ESI ): t = 7.35 min, calcd for
R
+
+
C H BrO [M-H O + H] 379.03; found, [M-H O + H] 379.14;
21
17
3
2
2
2
+
+
HRMS (ESI ): calcd for C H BrO [m/z] [M + Na] 419.0253, [M
+
4
2
1
17
3
+
+
+
2 + Na] 421.0233; found, [M + Na] 419.0251, [M + 2 + Na]
21.0233.
4
(
3′-(Benzo-1,4-dioxan-6-yl)-2′-iodo-[1,1′-biphenyl]-4-yl)-
methanol (4g). In a three-necked round-bottom flask equipped with
a thermometer, (2′-amino-3′-(benzo-1,4-dioxan-6-yl)-[1,1′-biphen-
yl]-4-yl)methanol (4d) (0.21 g, 0.62 mmol, 1 equiv) was added
and dissolved in 2 mL of dioxane/water (3/1, v/v) mixture. After
dissolution, 2 mL of 10% HCl was added and the mixture was cooled
2
3
2
3
in an ice bath; then, a solution of NaNO (0.05 g, 0.62 mmol, 1.0
2
anhydrous MgSO , and evaporated. The products were separated
4
equiv) in 2 mL of water was added dropwise, maintaining the
temperature below 5 °C. The mixture was stirred for 1 h, and a
solution of KI (0.42 g, 2.47 mmol, 4 equiv) in 4 mL of water was
added dropwise, maintaining the temperature below 5 °C; then, the
solution was stirred overnight at room temperature. After this time,
the solution was transferred into a separatory funnel, 5 mL of water
was added, and the aqueous phase was extracted with ethyl acetate (3
by column chromatography (SiO , hexane/ethyl acetate, or CH Cl ).
2
2
2
Methyl 3-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-
4-yl)methoxy)benzoate (5a). (3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-
[1,1′-biphenyl]-4-yl)methanol (4a) (0.30 g, 0.90 mmol, 1.0 equiv),
SOCl (0.20 mL, 2.70 mmol, 3.0 equiv), K CO (0.37 g, 2.70 mmol,
2
2
3
3.0 equiv), methyl 3-hydroxybenzoate (0.20 g, 1.35 mmol, 1.5 equiv),
KI (0.015 g, 0.09 mmol, 0.1 equiv). Product 5a was obtained as a
colorless solid with 77.7% (0.326 g) yield.
×
15 mL). The organic phases were collected, dried over anhydrous
MgSO , and the solvent was evaporated. The product was purified by
1
R = 0.50 (SiO , hexane/ethyl acetate 2:1); H NMR (600 MHz,
4
f
2
flash chromatography (SiO , hexane/ethyl acetate, 1:2), giving the
CDCl ): δ [ppm] 7.62 (m, 1H), 7.58 (dt, J = 7.7, 1.0 Hz, 1H), 7.41
2
3
final product 4g with 27.6% (0.08 g) yield.
(d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.28 (t, J = 7.9 Hz, 1H),
N
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Article
7
.18−7.11 (m, 4H), 6.82 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 2.0 Hz,
H), 6.74 (dd, J = 8.2, 2.1 Hz, 1H), 5.06 (s, 2H), 4.20 (s, 4H), 3.83
methyl-[1,1′-biphenyl]-3-yl)methanol (4b) (0.60 g, 1.81 mmol, 1.0
1
(
equiv), SOCl (0.40 mL, 5.43 mmol, 3.0 equiv), K CO (0.75 g, 5.43
2
2
3
s, 3H), 2.06 (s, 3H); 13C NMR (151 MHz, CDCl ): δ [ppm] 167.0,
mmol, 3.0 equiv), 2-bromo-5-hydroxybenzaldehyde (0.55 g, 2.72
mmol, 1.5 equiv), KI (0.03 g, 0.18 mmol, 0.1 equiv). Product 5k was
obtained as a colorless solid with 78.2% (0.73 g) yield.
3
1
1
1
1
58.8, 143.1, 142.6, 142.4, 142.4, 135.8, 135.0, 133.1, 131.6, 129.7,
29.5, 129.2, 128.9, 127.5, 125.4, 122.6, 122.4, 120.3, 118.2, 116.9,
−1
1
15.1, 70.1, 64.5, 64.5, 52.2, 18.8; IR (ATR) [cm ]: 2952, 1712,
R = 0.65 (SiO , CH Cl ); H NMR (600 MHz, CDCl ): δ [ppm]
f
2
2
2
3
301, 1278, 1234; LC−MS (DAD/ESI): t = 9.80 min, calcd for
10.34 (s, 1H), 7.57−7.55 (m, 2H), 7.49−7.45 (m, 2H), 7.43 (m, 1H),
7.37 (dt, J = 7.5, 1.4 Hz, 1H), 7.31−7.28 (m, 1H), 7.26−7.23 (m,
2H), 7.14 (dd, J = 8.8, 3.2 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.91 (d,
J = 2.0 Hz, 1H), 6.86 (dd, J = 8.2, 2.1 Hz, 1H), 5.17 (s, 2H), 4.33 (s,
R
+
+
C H O [m/z] [M + H] 467.19; found, [M + H] 467.41; HRMS
30
26
5
+
(
ESI): calcd for C H O [m/z] [M + Na] 489.1672; found, [M +
30 26 5
+
Na] 489.1672.
-Bromo-5-((3′-(benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphen-
yl]-4-yl)methoxy)benzaldehydem (5c). (3′-(Benzo-1,4-dioxan-6-yl)-
′-methyl-[1,1′-biphenyl]-4-yl)methanol (4a) (1.20 g, 3.62 mmol, 1.0
equiv), SOCl (0.79 mL, 10.86 mmol, 3.0 equiv), K CO (1.50 g,
2
4H), 2.15 (s, 3H); 13C NMR (151 MHz, CDCl ): δ [ppm] 191.7,
3
158.4, 143.1, 143.0, 142.6, 142.4, 142.3, 135.8, 135.7, 134.7, 134.0,
133.1, 129.4, 129.3, 128.8, 128.5, 128.5, 125.9, 125.4, 123.8, 122.6,
2
−1
2
2
3
118.3, 118.2, 116.9, 113.9, 70.5, 64.5, 18.8; IR (ATR) [cm ]: 2976,
2928, 2873, 1692, 1507, 1467, 1278, 1229, 1069; LC−MS (DAD/
1
5
0.86 mmol, 3.0 equiv), 2-bromo-5-hydroxybenzaldehyde (1.09 g,
.43 mmol, 1.5 equiv), KI (0.06 g, 0.36 mmol, 0.1 equiv). Product 5c
+
ESI): t = 10.13 min, calcd for C H BrO [m/z] [M + H] 515.08;
R
29 23
4
+
was obtained as a colorless solid with 84.9% (1.58 g) yield.
found, [M + H] 515.22; HRMS (ESI): calcd for C H BrO [m/z]
29 23 4
1
+
+
R = 0.55 (SiO , hexane/ethyl acetate 2:1); H NMR (600 MHz,
[M + Na] 537.0672; found, [M + Na] 537.0672.
f
2
CDCl ): δ [ppm] 10.33 (s, 1H), 7.55 (m, 2H), 7.48 (d, J = 8.1 Hz,
Methyl 3-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-
3-yl)methoxy)isoxazole-5-carboxylate (5m). (3′-(Benzo-1,4-dioxan-
6-yl)-2′-methyl-[1,1′-biphenyl]-3-yl)methanol (4b) (0.30 g, 0.90
mmol, 1.0 equiv), SOCl (0.20 mL, 2.70 mmol, 3.0 equiv), K CO
3
2
2
H), 7.40 (d, J = 8.1 Hz, 2H), 7.27−7.25 (m, 1H), 7.23−7.17 (m,
H), 7.15 (dd, J = 8.8, 3.2 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.88 (d,
J = 2.0 Hz, 1H), 6.84 (dd, J = 8.2, 2.1 Hz, 1H), 5.14 (s, 2H), 4.31 (s,
2
2
3
4
1
1
1
2
1
+
H), 2.13 (s, 3H); 13C NMR (151 MHz, CDCl ): δ [ppm] 191.8,
(0.37 g, 2.70 mmol, 3.0 equiv), methyl 3-hydroxyisoxazole-5-
carboxylate (0.13 g, 0.90 mmol, 1.0 equiv), KI (0.015 g, 0.09
mmol, 0.1 equiv). Product 5m was obtained as a colorless solid with
47.8% (0.197 g) yield.
3
58.4, 143.0, 142.6, 142.6, 142.4, 142.2, 137.5, 134.7, 134.3, 134.0,
33.1, 129.7, 129.2, 128.8, 127.4, 125.3, 123.8, 122.5, 118.3, 118.2,
−
1
16.9, 113.8, 70.4, 64.4, 64.4, 18.8; IR (ATR) [cm ]: 3052, 2953,
1
874, 2754, 1688, 1508, 1305, 1167, 993; LC−MS (DAD/ESI): t =
R = 0.42 (SiO , hexane/ethyl acetate 2:1); H NMR (600 MHz,
R
f
2
+
0.14 min, calcd for C H BrO [m/z] [M + H] 515.08; found, [M
H] 515.18; HRMS (ESI): calcd for C H BrO [m/z] [M + Na]
CDCl ): δ [ppm] 7.47−7.44 (m, 2H), 7.42 (m, 1H), 7.37 (dt, J = 7.4,
2
9
23
4
3
+
+
1.6 Hz, 1H), 7.28−7.25 (m, 1H), 7.23−7.20 (m, 2H), 6.91 (d, J = 8.2
Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.2, 2.1 Hz, 1H), 6.58
2
9
23
4
+
5
37.0672; found, [M + Na] 537.0679.
1
3
Methyl 3-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-
-yl)methoxy)isoxazole-5-carboxylate (5e). (3′-(Benzo-1,4-dioxan-
-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)methanol (4a) (0.30 g, 0.90
(s, 1H), 5.37 (s, 2H), 4.30 (s, 4H), 3.95 (s, 3H), 2.12 (s, 3H);
C
4
NMR (151 MHz, CDCl ): δ [ppm] 171.3, 160.4, 157.1, 143.1, 143.0,
3
6
142.6, 142.4, 142.2, 135.7, 135.1, 133.1, 129.7, 129.3, 129.3, 128.8,
128.4, 126.7, 125.4, 122.6, 118.2, 116.9, 101.0, 72.1, 64.5, 52.9, 18.8;
mmol, 1.0 equiv), SOCl (0.20 mL, 2.70 mmol, 3.0 equiv), K CO
2
2
3
−
1
(
0.37 g, 2.70 mmol, 3.0 equiv), methyl 3-hydroxyisoxazole-5-
IR (ATR) [cm ]: 3140, 1743, 1506, 1303; LC−MS (DAD/ESI): t
R
+
carboxylate (0.13 g, 0.90 mmol, 1.0 equiv), KI (0.015 g, 0.09
= 9.07 min, calcd for C H NO [m/z] [M + H] 458.16; found, [M
+ H] 458.37; HRMS (ESI): calcd for C H NO [m/z] [M + Na]
27 23 6
480.1418; found, [M + Na] 480.1418.
27 23 6
+
+
mmol, 0.1 equiv). Product 5e was obtained as a colorless solid with
6
+
9.2% (0.285 g) yield.
1
R = 0.34 (SiO , hexane/ethyl acetate 2:1); H NMR (600 MHz,
Methyl 3-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-
2-yl)methoxy)benzoate (5o). (3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-
[1,1′-biphenyl]-2-yl)methanol (4c) (0.30 g, 0.90 mmol, 1.0 equiv),
SOCl (0.20 mL, 2.70 mmol, 3.0 equiv), K CO (0.37 g, 2.70 mmol,
f
2
CDCl ): δ [ppm] 7.54 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H),
3
7
.31−7.22 (m, 3H), 6.94 (d, J = 8.2 Hz, 1H), 6.92 (d, J = 2.0 Hz,
1
(
[
H), 6.86 (dd, J = 8.2, 2.1 Hz, 1H), 6.63 (s, 1H), 5.41 (s, 2H), 4.33
2
2
3
s, 4H), 3.99 (s, 3H), 2.17 (s, 3H); 13C NMR (151 MHz, CDCl ): δ
3.0 equiv), methyl 2-hydroxybenzoate (0.20 g, 1.35 mmol, 1.5 equiv),
KI (0.015 g, 0.09 mmol, 0.1 equiv). Product 5o was obtained as a
colorless solid with 49.0% (0.206 g) yield.
3
ppm] 171.4, 160.4, 157.1, 143.1, 142.6, 142.4, 142.2, 135.7, 133.7,
1
7
1
33.1, 129.7, 129.3, 128.8, 128.2, 125.4, 122.6, 118.2, 116.9, 101.0,
−
1
1
2.1, 64.5, 64.5, 52.9, 18.8; IR (ATR) [cm ]: 3144, 1747, 1607,
504, 1313; LC−MS (DAD/ESI): tR = 8.98 min, decomposition
R = 0.58 (SiO , CH Cl ); H NMR (600 MHz, CDCl ): δ [ppm]
f
2
2
2
3
7.65 (m, 2H), 7.53 (m, 1H), 7.46−7.42 (m, 2H), 7.34−7.30 (m, 2H),
7.28−7.24 (m, 2H), 7.21 (dd, J = 7.0, 1.8 Hz, 1H), 7.04 (dd, J = 8.2,
2.6 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.81
(dd, J = 8.2, 2.0 Hz, 1H), 4.92 (s, 2H), 4.32 (s, 4H), 3.93 (s, 3H),
during ionization; HRMS (ESI): calcd for C H NO [m/z] [M +
2
7
23
6
+
+
Na] 480.1418; found, [M + Na] 480.1418.
Methyl 3-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-
2.05 (s, 3H); 13C NMR (151 MHz, CDCl ): δ [ppm] 166.9, 158.7,
3
-yl)methoxy)benzoate (5i). (3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-
3
[
1,1′-biphenyl]-3-yl)methanol (4b) (0.30 g, 0.90 mmol, 1.0 equiv),
143.1, 142.6, 142.2, 141.7, 140.5, 135.6, 134.3, 133.6, 131.4, 123.0,
129.4, 129.4, 128.5, 128.3, 128.0, 127.6, 125.2, 122.5, 122.2, 120.1,
SOCl (0.20 mL, 2.70 mmol, 3.0 equiv), K CO (0.37 g, 2.70 mmol,
2
2
3
−
1
3
.0 equiv), methyl 3-hydroxybenzoate (0.20 g, 1.35 mmol, 1.5 equiv),
118.2, 116.9, 115.3, 68.3, 64.4, 52.2, 18.3; IR (ATR) [cm ]: 2949,
KI (0.015 g, 0.09 mmol, 0.1 equiv). Product 5i was obtained as
1722, 1507, 1278; LC−MS (DAD/ESI): t = 9.75 min, calcd for
R
+
+
colorless solid with 58.8% (0.247 g) yield.
C H O [m/z] [M + H] 467.19, [M-OCH ] 435.16; found, [M-
30 26 5 3
1
+
+
R = 0.47 (SiO , CH Cl ); H NMR (600 MHz, CDCl ): δ [ppm]
OCH ] 435.31; HRMS (ESI): calcd for C H O [m/z] [M + Na]
3 30 26 5
f
2
2
2
3
+
7
7
1
2
.22 (m, 1H), 7.70 (dt, J = 7.7, 1.0 Hz, 1H), 7.49−7.46 (m, 3H),
.40−7.37 (m, 2H), 7.32−7.25 (m, 3H), 7.23 (ddd, J = 8.2, 2.6, 0.9,
H), 6.95 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 2.0, 1H), 6.87 (dd, J = 8.2,
.1 Hz, 1H), 5.20 (s, 2H), 4.33 (s, 4H), 3.95 (s, 3H), 2.17 (s, 3H);
489.1672; found, [M + Na] 489.1672.
Methyl 3-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-
2-yl)methoxy)isoxazole-5-carboxylate (5q). (3′-(Benzo-1,4-dioxan-
6-yl)-2′-methyl-[1,1′-biphenyl]-2-yl)methanol (4c) (0.30 g, 0.90
mmol, 1.0 equiv), SOCl (0.20 mL, 2.70 mmol, 3.0 equiv), K CO
1
3
C NMR (151 MHz, CDCl ): δ [ppm] 166.9, 158.8, 143.1, 142.9,
3
2
2
3
1
1
1
1
42.6, 142.5, 142.4, 136.5, 135.8, 133.1, 131.5, 129.5, 129.2, 129.2,
28.8, 128.6, 128.4, 126.0, 125.4, 122.6, 122.3, 120.3, 118.2, 116.9,
15.2, 70.2, 64.5, 52.2, 18.8; IR (ATR) [cm ]: 2950, 1722, 1507,
(0.37 g, 2.70 mmol, 3.0 equiv), methyl 3-hydroxyisoxazole-5-
carboxylate (0.13 g, 0.90 mmol, 1.0 equiv), KI (0.015 g, 0.09
mmol, 0.1 equiv). Product 5q was obtained as a colorless solid with
−1
279; LC−MS (DAD/ESI): t = 9.81 min, calcd for C H O [m/z]
42.1% (0.173 g) yield.
R
30 26
5
+
+
1
[
M + H] 467.19; found, [M + H] 467.28; HRMS (ESI): calcd for
R
f
= 0.35 (SiO
, hexane/ethyl acetate 2:1); H NMR (600 MHz,
2
+
+
C H O [m/z] [M + Na] 489.1672; found, [M + Na] 489.1671.
CDCl
3
): δ [ppm] 7.50 (m, 1H), 7.35−7.31 (m, 2H), 7.19−7.17 (m,
30
26
5
2
-Bromo-5-((3′-(benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphen-
1H), 7.13−7.10 (m, 2H), 7.02 (dd, J = 6.4, 2.6 Hz, 1H), 6.81 (d, J =
8.2 Hz, 1H), 6.75 (d, J = 2.0 Hz, 1H), 6.69 (dd, J = 8.2, 2.1 Hz, 1H),
yl]-3-yl)methoxy)benzaldehyde (5k). (3′-(Benzo-1,4-dioxan-6-yl)-2′-
O
https://doi.org/10.1021/acs.jmedchem.1c00957
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
.38 (s, 1H), 5.05 (d, J = 11.7 Hz, 1H), 5.00 (d, J = 11.7 Hz, 1H),
.20 (s, 4H), 3.84 (s, 3H), 1.86 (s, 3H); 13C NMR (151 MHz,
Product 5b was obtained as a colorless solid with 88.8% (0.080 g)
yield.
R = 0.44 (SiO , DCM/MeOH, 9:1); 1H NMR (600 MHz,
3
f
2
1
1
40.2, 135.5, 133.5, 133.1, 130.1, 129.5, 129.2, 128.7, 128.4, 127.6,
DMSO-d ): δ [ppm] 7.59−7.57 (m, 2H), 7.53 (d, J = 8.0 Hz, 2H),
6
25.2, 122.5, 118.2, 116.8, 100.8, 70.3, 64.4, 52.8, 18.2; IR (ATR)
7.43 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.30 (dd, J = 8.1,
2.4 Hz, 1H), 7.28−7.26 (m, 1H), 7.17 (d, J = 7.6 Hz, 2H), 6.91 (d, J
= 8,2 Hz, 1H), 6.86 (d, J = 2.0, 1H), 6.81 (dd, J = 8.2, 2.0 Hz, 1H),
−1
[
9
cm ]: 3140, 2953, 1743, 1505, 1288; LC−MS (DAD/ESI): t =
R
+
.00 min, calcd for C H NO [m/z] [M + H] 458.16; found, [M +
27 23 6
+
+
13
H] 458.30; HRMS (ESI): calcd for C H NO [m/z] [M + Na]
5.21 (s, 2H), 4.27 (s, 4H), 2.07 (s, 3H); C NMR (151 MHz,
2
7
23
6
+
4
80.1418; found, [M + Na] 480.1418.
DMSO-d ): δ [ppm] 167.6, 158.9, 143.4, 143.0, 142.4, 142.3, 141.9,
6
Methyl 1-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-
135.9, 135.3, 132.8, 132.7, 130.3, 129.7, 129.3, 129.0, 128.1, 126.0,
4
-yl)methyl)-1H-1,2,3-triazole-4-carboxylate (5g). (3′-(Benzo-1,4-
1
22.6, 122.3, 120.2, 118.2, 117.2, 115.3, 69.7, 64.6, 64.6, 19.1; IR
dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)methanol (4a) (0.33 g,
−1
(
(
4
ATR) [cm ]: 2981, 2572, 1683, 1506, 1454, 1314, 1245; LC−MS
1
.0, 1.0 equiv) was added in a round-bottom flask with anhydrous
−
DAD/ESI): t = 8.67 min, calcd for C H O [m/z] [M − H]
R
29 24
5
dichloromethane and triethylamine (0.21 mL, 1.5 mmol, 1.5 equiv)
and cooled in an ice bath; then, mesyl chloride (0.12 mL, 1.5 mmol,
−
51.16; found, [M − H] 451.19; HRMS (ESI): calcd for C H O
2
9
24
5
+
+
[
m/z] [M + Na] 475.1516; found, [M + Na] 475.1517.
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-
yl)methoxy)isoxazole-5-carboxylic Acid (5f). Methyl 3-((3′-
1
.5 equiv) was added dropwise. The reaction mixture was stirred
3
approximately 2 h at room temperature and controlled by TLC. When
all substrate disappeared, the reaction mixture was poured onto water
and extracted with dichloromethane. The organic phases were
(
benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)methoxy)-
isoxazole-5-carboxylate (5e) (0.090 g, 0.20 mmol, 1.0 equiv), LiOH·
collected, dried over anhydrous MgSO , and evaporated. The crude
product was dissolved in anhydrous DMF, and NaN (0.097 g, 1.5
mmol, 1.5 equiv) was added. The reaction mixture was stirred at
room temperature for 6 days. After this time, water was added and the
reaction mixture was extracted with ethyl acetate. The organic layers
were collected, washed with brine, dried over anhydrous MgSO , and
evaporated. Crude azide was roughly purified with flash chromatog-
raphy on a silica pad (SiO , hexane/ethyl acetate 4:1). The obtained
azide (0.25 g, 0.69 mmol, 1.0 equiv) was added to a round-bottom
flask, and then, methyl propiolate (0.041 mL, 0.69 mmol, 1.0 equiv)
in a THF/water mixture (1:2), sodium ascorbate (0.068 g, 0.32
mmol, 0.5 equiv), and copper(II) sulfate pentahydrate (0.034 g, 0.14
mmol, 0.2 equiv) were added. The reaction mixture was stirred at
room temperature for 24 h. After this time, water was added, and the
reaction mixture was extracted with ethyl acetate. The organic layers
were collected, washed with brine, dried over anhydrous MgSO , and
evaporated. The crude product was crystallized from the cyclohexane/
ethyl acetate (4:1), giving the final product 5g with 72.1% (0.220 g)
4
H O (0.016 g, 0.40 mmol, 2.0 equiv). Product 5f was obtained as a
2
3
colorless solid with 65.2% (0.058 g) yield.
1
R = 0.14 (SiO , ethyl acetate/MeOH, 3:1); H NMR (600 MHz,
f
2
DMSO-d ): δ [ppm] 7.56 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz,
6
2
6
2
H), 7.29 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 7.7 Hz, 2H), 7.00 (s, 1H),
4
.92 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.2,
13
.0 Hz, 1H), 2.17 (s, 2H), 4.28 (s, 4H), 2.07 (s, 3H) C NMR (151
2
MHz, DMSO-d ): δ [ppm] 171.8, 162.2, 158.0, 143.4, 143.0, 142.5,
6
1
1
2
42.4, 142.3, 135.3, 134.6, 132.8, 129.8, 129.4, 129.0, 128.9, 126.1,
22.6, 118.2, 117.3, 100.7, 71.9, 64.6, 64.6, 19.1; IR (ATR) [cm ]:
−1
929, 2606, 1727, 1497, 1274; LC−MS (DAD/ESI): t = 7.89 min,
R
−
−
calcd for C H NO [m/z] [M − H] 442.13; found, [M − H]
2
6
21
6
42.15; HRMS (ESI): calcd for C H NO [m/z] [M + Na]+
4
4
2
6
21
6
+
66.1261; found, [M + Na] 466.1263.
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)-
methyl)-1H-1,2,3-triazole-4-carboxylic Acid (5h). Methyl 1-((3′-
benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)methyl)-1H-
,2,3-triazole-4-carboxylate (5g) (0.150 g, 0.34 mmol, 1.0 equiv),
1
4
(
1
1
R = 0.88 (SiO , ethyl acetate/methanol 1:1); H NMR (600 MHz,
f
2
LiOH·H O (0.042 g, 1.00 mmol, 3.0 equiv). Product 5h was obtained
2
CDCl ): δ [ppm] 8.00 (s, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.27 (d, J =
3
as a colorless solid with 61.4% (0.089 g) yield.
8
7
2
4
.2 Hz, 2H), 7.19 (t, J = 7.5 Hz, 1H), 7.15 (dd, J = 7.6, 1.6 Hz, 1H),
.09 (dd, J = 7.3, 1.6 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.79 (d, J =
.0 Hz, 1H), 6.74 (dd, J = 8.2, 2.1 Hz, 1H), 5.56 (s, 2H), 4.23 (s,
1
R = 0.39 (SiO , ethyl acetate/methanol 1:1); H NMR (600 MHz,
f
2
CDCl ): δ [ppm] 8.85 (s, 1H), 7.42−7.37 (m, 4H), 7.26 (t, J = 7.6
3
Hz, 1H), 7.17−7.13 (m, 2H), 6.91 (d, J = 8.2 Hz, 1H), 6.84 (d, J =
H), 3.87 (s, 3H), 2.03 (s, 3H); 13C NMR (151 MHz, CDCl ): δ
3
2
4
.0 Hz, 1H), 6.80 (dd, J = 8.2, 2.0 Hz, 1H), 5.71 (s, 2H), 4.27 (s,
[
ppm] 161.3, 143.7, 143.2, 142.8, 142.6, 141.8, 140.5, 135.7, 133.1,
H), 2.04 (s, 3H); 13C NMR (151 MHz, CDCl ): δ [ppm] 162.1,
3
1
6
1
32.2, 130.4, 129.6, 128.8, 128.2, 127.6, 125.6, 122.6, 118.3, 117.0,
4.6, 64.6, 54.4, 52.4, 18.9; IR (ATR) [cm ]: 3142, 2999, 1726,
509, 1456, 1509, 1438, 1360, 1318, 1244, 1068, 1047, 901, 813, 780;
−
1
143.4, 134.0, 142.3, 142.2, 140.4, 135.2, 134.7, 132.8, 130.1, 129.6,
29.4, 129.0, 128.3, 126.1, 122.6, 118.2, 117.2, 64.6, 53.2, 19.1; IR
1
−
1
(
ATR) [cm ]: 3115, 2977, 2874, 2559, 1687, 1506, 1439, 1229,
LC−MS (DAD/ESI): t = 7.78 min, calcd for C H N O [m/z] [M
R
26 23
3
4
+
+
1071, 1053, 899, 783; LC−MS (DAD/ESI): t = 6.99 min, calcd for
R
+
H] 442.18; found, [M + H] 442.30; HRMS (ESI): calcd for
+
+
C H N O (m/z) [M + Na]+ 464.1581; found, [M + Na]
+
C H N O [m/z] [M + H] 428.16; found, [M + H] 428.28;
25 21 3 4
HRMS (ESI): calcd for C H N O (m/z) [M + Na] 450.1424;
2
6
23
3
4
+
2
5
21
3
4
4
64.1581.
+
found, [M + Na] 450.1422.
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-3-yl)-
methoxy)benzoic Acid (5j). Methyl 3-((3′-(benzo-1,4-dioxan-6-yl)-
General Procedure of Ester Saponification (5b, 5f, 5h, 5j,
n, 5p, and 5r). The appropriate ester was added in a one-necked
3
5
round-bottom flask and dissolved in a dioxane/water (2:1, 10:5 mL
per 1 mmol of ester) mixture. After complete dissolution, LiOH
monohydrate was added and the mixture was stirred at room
temperature overnight. The progression of the reaction was controlled
2
′-methyl-[1,1′-biphenyl]-3-yl)methoxy)benzoate (5i) (0.093 g, 0.20
mmol, 1.0 equiv), LiOH·H O (0.016 g, 0.40 mmol, 2.0 equiv).
2
Product 5j was obtained as a colorless solid with 58.8% (0.053 g)
yield.
using TLC analysis (SiO , ethyl acetate/methanol, or dichloro-
2
R = 0.58 (SiO , DCM/MeOH, 9:1); 1H NMR (600 MHz,
methane (DCM)/methanol). After completion of the reaction, the
mixture was transferred into a separatory funnel, 7 mL of water was
added, and then extracted once with a small amount of ethyl acetate.
The aqueous phase was acidified to pH ≈ 4 and extracted three times
with ethyl acetate. The organic phases obtained from extraction after
acidification were collected, washed with brine, dried over anhydrous
f
2
CDCl ): δ [ppm] 7.66−7.64 (m, 2H), 7.38−7.34 (m, 3H), 7.30 (t, J
3
=
8.2 Hz, 1H), 7.25 (dt, J = 6.9, 1.7 Hz, 1H), 7.19−7.13 (m, 4H),
6
.82 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 8.2,
3
2
.0 Hz, 1H), 5.09 (s, 2H), 4.21 (s, 4H), 2.04 (s, 3H); 1 C NMR (151
MHz, CDCl ): δ [ppm] 171.7, 158.8, 143.1, 142.9, 142.6, 142.5,
3
1
1
42.4, 136.4, 135.8, 133.1, 130.7, 129.6, 129.2, 129.2, 128.8, 128.6,
28.5, 126.0, 125.4, 123.0, 122.6, 121.3, 118.2, 116.9, 115.6, 70.2,
MgSO , and the solvent was evaporated. Further purification of the
product was not required.
4
−
1
3
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)-
methoxy)benzoic Acid (5b). Methyl 3-((3′-(benzo-1,4-dioxan-6-yl)-
′-methyl-[1,1′-biphenyl]-4-yl)methoxy)benzoate (5a) (0.093 g, 0.20
mmol, 1.0 equiv), LiOH·H O (0.016 g, 0.40 mmol, 2.0 equiv).
64.5, 18.8; IR (ATR) [cm ]: 2922, 2565, 1679, 1506, 1455, 1302,
1247; LC−MS (DAD/ESI): t = 8.68 min, calcd for C29 [m/z]
[M − H] 451.16; found, [M − H] 451.12; HRMS (ESI): calcd for
H O
24 5
R
−
−
2
+
+
C H O [m/z] [M + Na] 475.1516; found, [M + Na] 475.1516.
2
29 24
5
P
https://doi.org/10.1021/acs.jmedchem.1c00957
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
3
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-3-yl)-
5-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)-
methoxy)-2-(pyridin-3-yl)benzaldehyde (5d). 2-Bromo-5-((3′-
(benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)methoxy)-
benzaldehyde (5c) (0.20 g, 0.40 mmol, 1.0 equiv), 3-pyridinylboronic
acid (0.072 g, 0.60 mmol, 1.5 equiv), K CO (0.16 g, 1.20 mmol, 3.0
methoxy)isoxazole-5-carboxylic Acid (5n). Methyl 3-((3′-(benzo-
1
,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-3-yl)methoxy)isoxazole-5-
carboxylate (5m) (0.090 g, 0.20 mmol, 1.0 equiv), LiOH·H O (0.016
2
g, 0.40 mmol, 2.0 equiv). Product 5n was obtained as a colorless solid
2
3
with 45.1% (0.040 g) yield.
equiv), Pd(dppf)Cl complex with dichloromethane (0.016 g, 0.02
mmol, 0.05 equiv). Product 5d was obtained as a colorless solid with
2
1
R = 0.31 (SiO , ethyl acetate/MeOH, 3:1); H NMR (600 MHz,
f
2
CDCl ): δ [ppm] 7.47−7.45 (m, 2H), 7.43 (m, 1H), 7.37 (dt, J = 7.4,
92.6% (0.190 g) yield.
3
1
1
.4 Hz, 1H), 7.28−7.25 (m, 1H), 7.23−7.20 (m, 2H), 6.91 (d, J = 8.2
R = 0.37 (SiO , ethyl acetate); H NMR (600 MHz, CDCl ): δ
f
2
3
Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.2, 2.1 Hz, 1H), 6.68
[ppm] 9.95 (s, 1H), 8.68 (dd, J = 4.9, 1.5 Hz, 1H), 8.66 (m, 1H),
7.71−7.69 (m, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.42−7.38 (m, 4H),
7.35 (dd, J = 8.4, 2.7 Hz, 1H), 7.27 (m, 1H), 7.24−7.21 (m, 2H), 6.91
(d, J = 8.2 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.2, 2.0 Hz,
1H), 5.23 (s, 2H), 4.31 (s, 4H), 2.15 (s, 3H); 13C NMR (151 MHz,
13
(
s, 1H), 5.38 (s, 2H), 4.31 (s, 4H), 2.12 (s, 3H); C NMR (151
MHz, CDCl ): δ [ppm] 171.4, 159.7, 159.3, 143.1, 143.0, 142.6,
3
1
1
42.4, 142.2, 135.7, 135.0, 133.1, 129.8, 129.3, 129.3, 128.8, 128.5,
26.7, 125.4, 122.6, 118.2, 116.9, 102.2, 72.3, 64.5, 64.5, 18.8; IR
−
1
(
ATR) [cm ]: 3147, 2954, 2614, 360, 1736, 1505; LC−MS (DAD/
CDCl ): δ [ppm] 191.1, 159.1, 150.3, 149.1, 143.0, 142.6, 142.4,
3
−
ESI): t = 7.79 min, calcd for C H NO [m/z] [M − H] 442.13;
found, [M − H] 442.15; HRMS (ESI): calcd for C H NO [m/z]
142.3, 137.4, 135.7, 135.1, 134.8, 134.6, 133.4, 133.1, 132.4, 129.7,
129.2, 128.8, 127.4, 125.3, 123.2, 122.5, 122.2, 118.2, 116.9, 112.0,
70.3, 64.4, 64.4, 18.8; IR (ATR) [cm ]: 3049, 2997, 2876, 1684,
R
26 21
6
−
2
6
21
6
+
+
−1
[
M + Na] 466.1261; found, [M + Na] 466.1260.
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-2-yl)-
methoxy)benzoic Acid (5p). Methyl 3-((3′-(benzo-1,4-dioxan-6-yl)-
3
1504, 1320, 1169; LC−MS (DAD/ESI): t = 8.56 min, calcd for
R
+
+
C H NO [m/z] [M + H] 514.19; found, [M + H] 514.33;
3
4
27
4
+
2
′-methyl-[1,1′-biphenyl]-2-yl)methoxy)benzoate (5o) (0.093 g, 0.20
HRMS (ESI): calcd for C H NO : [M + Na] 536.1832; found, [M
34 27 4
+
mmol, 1.0 equiv), LiOH·H O (0.016 g, 0.40 mmol, 2.0 equiv).
+ Na] 536.1830.
2
Product 5p was obtained as a colorless solid with 58.8% (0.053 g)
yield.
5-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-3-yl)-
methoxy)-2-(yridine-3-yl)benzaldehyde (5l). 2-Bromo-5-((3′-
(benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-3-yl)methoxy)-
benzaldehyde (5k) (0.20 g, 0.40 mmol, 1.0 equiv), 3-pyridinylboronic
R = 0.70 (SiO , DCM/MeOH, 9:1); 1H NMR (600 MHz,
f
2
DMSO-d ): δ [ppm] 12.96 (s, 1H), 7.61 (m, 1H), 7.50 (d, J = 7.7 Hz,
6
1
H), 7.43 (m, 2H), 7.35 (t, J = 7.9 Hz, 1H), 7.31 (m, 1H), 7.27−7.21
acid (0.072 g, 0.60 mmol, 1.5 equiv), K
2 3
CO (0.160 g, 1.20 mmol, 3.0
(m, 2H), 7.14 (t, J = 7.5 Hz, 2H), 7.06 (dd, J = 8.2, 2.1 Hz, 1H), 6.88
d, J = 8.2 Hz, 1H), 6.77 (d, J = 2.0 Hz, 1H), 6.72 (dd, J = 8.3, 2.0 Hz,
equiv), Pd(dppf)Cl complex with dichloromethane (0.016 g, 0.02
2
(
mmol, 0.05 equiv). Product 5l was obtained as a colorless solid with
1
4
1
1
1
2
H), 4.89 (d, J = 11.5 Hz, 1H), 4.85 (d, J = 11.5 Hz, 1H), 4.27 (s,
H), 1.92 (s, 3H); 13C NMR (151 MHz, DMSO-d ): δ [ppm] 167.5,
1
): δ
6
3
58.7, 143.4, 142.9, 142.0, 142.0, 140.8, 135.2, 134.5, 133.4, 132.6,
30.2, 130.1, 129.5, 129.4, 128.6, 128.5, 128,00, 125.7, 122.5, 122.2,
−
1
19.9, 118.1, 117.2, 115.2, 68.3, 66.8, 64.6, 18.6; IR (ATR) [cm ]:
876, 2647, 1690, 1507, 1279, 1244; LC−MS (DAD/ESI): t = 8.61
R
−
−
min, calcd for C H O [m/z] [M − H] 451.16; found, [M − H]
2
9
24
5
+
13
4
4
51.12 HRMS (ESI): calcd for C H O [m/z] [M + Na]
2
9
24
5
+
75.1516; found, [M + Na] 475.1516.
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-2-yl)-
methoxy)isoxazole-5-carboxylic Acid (5r). Methyl 3-((3′-(benzo-
3
−
1
1
,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-2-yl)methoxy)isoxazole-5-
carboxylate (5q) (0.090 g, 0.20 mmol, 1.0 equiv), LiOH·H O (0.016
g, 0.40 mmol, 2.0 equiv). Product 5r was obtained as a colorless solid
with 55.3% (0.049 g) yield.
2
R
+
+
+
1
R = 0.45 (SiO , ethyl acetate/MeOH, 1:3); H NMR (600 MHz,
f
2
CDCl ): δ [ppm] 7.21 (m, 1H), 7.46−7.41 (m, 2H), 7.26−7.23 (m,
General Procedure for the Amine Formation (6a−e, 7a−h,
and 8a−j). Appropriate alcohol 4a−h was added in a round-bottom
flask with anhydrous dichloromethane and few drops of anhydrous
DMF (0.2 mL for 2 mmol alcohol) and cooled in an ice bath; then,
thionyl chloride (5 equiv) was added dropwise. The reaction mixture
was stirred approximately 2 h at room temperature and controlled by
TLC. When all substrates disappeared, the reaction mixture was
3
2
6
2
H), 7.16 (dd, J = 7.6, 0.8 Hz, 1H), 7.09 (dd, J = 7.4, 0.9 Hz, 1H),
.90 (d, J = 8.2 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.76 (dd, J = 8.3,
.0 Hz, 1H), 6.17 (s, 1H), 4.97 (d, J = 11.8 Hz, 1H), 4.93 (d, J = 11.8
13
Hz, 1H), 4.27 (s, 4H), 1.88 (s, 3H); C NMR (151 MHz, CDCl ): δ
3
[
ppm] 171.2, 170.6, 159.5, 143.4, 143.0, 142.0, 140.6, 135.1, 133.9,
1
9
1
33.3, 130.1, 129.5, 128.9, 128.6, 128.0, 125.7, 122.6, 118.1, 117.3,
−
1
5.6, 69.4, 64.6, 18.5; IR (ATR) [cm ]: 3396, 2931, 1595, 1505,
359; LC−MS (DAD/ESI): t = 7.78 min, calcd for C H NO [m/
poured onto saturated NaHCO
The organic phases were collected, dried over anhydrous MgSO
3
and extracted with dichloromethane.
, and
4
R
26 21
6
−
−
z] [M − H] 442.13; found, [M − H] 442.15; HRMS (ESI): calcd
evaporated. The crude product was used without further purification
in the next step.
+
+
for C H NO [M + Na] 466.1261; found, [M + Na] 466.1262.
26
21
6
General Procedure for 2-Pyridinebenzaldehyde Prepara-
tion (5d and 5l). A three-neck round-bottom flask was charged with
appropriate halide 5c or 5k, borane 3-pyridinylboronic acid, K CO ,
and a dioxane/water mixture (2:1, 5/10 mL for 1 mmol) under an
argon atmosphere. The mixture was deoxygenated by rinsing with
argon for half an hour; then, the Pd(dppf)Cl2 dichloromethane
complex was added. The reaction mixture was heated at 80 °C, using
preheated bath, for 5 h. After this time, the progression of the reaction
was controlled by TLC. When the reaction was complete, water was
added and the extraction with ethyl acetate followed. The organic
Method A. Crude alkyl chloride was dissolved in anhydrous
acetonitrile or THF, and excess of appropriate amine dissolved in
anhydrous acetonitrile or THF was added (if the hydrochloride of
amine was used, an equimolar amount of triethylamine was added to
the reaction mixture). The reaction mixture was stirred at room
temperature for 48 h and controlled by TLC. After completion of the
reaction, the mixture was poured into saturated NaHCO3 and
extracted with ethyl acetate. The organic phases were collected, dried
2
3
over anhydrous MgSO , and evaporated. The crude products were
4
purified by flash column chromatography (SiO , hexane/ethyl
2
phases were combined, dried over anhydrous MgSO , and evaporated.
The crude product was purified by flash chromatography (SiO2,
hexane/ethyl acetate), giving the final products 5d or 5l with 93 and
acetate).
4
Method B. Crude alkyl chloride was dissolved in anhydrous THF,
and excess of Boc-protected piperazine dissolved in anhydrous THF
was added. The reaction mixture was stirred at room temperature 48
80% yields, respectively.
Q
https://doi.org/10.1021/acs.jmedchem.1c00957
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
h and controlled by TLC. After the reaction was complete, the solvent
was evaporated and the crude Boc-protected product was purified by
equiv), thionyl chloride (0.26 mL, 3.6 mmol, 5.0 equiv), 1-Boc-
piperazine (0.67 g, 3.6 mmol, 5.0 equiv). Product 6b was obtained as
a light-yellow oil with 66.3% (0.191 g) yield.
flash chromatography (SiO , hexane/ethyl acetate 1:1). The purified
2
−
1
Boc-protected product was dissolved in anhydrous dioxane, and HCl
in dioxane was added (10 equiv, 4 M). The reaction mixture was
stirred at room temperature overnight and then poured into saturated
R = 0.09 (SiO , methanol); IR (ATR) [cm ]: 3298, 2933, 2811,
f
2
1
1578, 1507, 1463, 1414, 1317, 1279, 1245, 1123, 1069, 899, 790; H
NMR (600 MHz, DMSO-d ): δ [ppm] 7.36 (d, J = 8.0 Hz, 2H), 7.31
6
NaHCO and extracted with ethyl acetate. The organic phases were
(d, J = 8.2 Hz, 2H), 7.27 (t, J = 7.4 Hz, 1H), 7.16 (dd, J = 7.6, 2.0 Hz,
2H), 6.92 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 2.1 Hz, 1H), 6.82 (dd, J =
8.2, 2.1 Hz, 1H), 4.28 (s, 4H), 3.47 (s, 2H), 3.17 (s, 1H), 2.71−2.66
3
collected, dried over anhydrous MgSO , and evaporated. The
4
products were purified by column chromatography (SiO , pure
2
13
methanol, or methanol/7 M NH in methanol).
(m, 4H), 2.35−2.27 (m, 4H), 2.07 (s, 3H); C NMR (151 MHz,
3
Method C. Crude alkyl chloride was dissolved in anhydrous THF
and an appropriate amine dissolved in anhydrous THF was added (if
the hydrochloride of amine was used, an equimolar amount of
triethylamine was added to the reaction mixture). The reaction
mixture was stirred at 50 °C overnight. After completion of the
DMSO-d ): δ [ppm] 142.9, 142.5, 142.2, 141.8, 140.3, 137.0, 134.9,
6
132.3, 128.9, 128.7, 128.6, 125.5, 122.1, 117.7, 116.8, 64.1, 62.6, 54.2,
45.6, 18.7; LC−MS (DAD/ESI): t = 5.01 min, calcd for
R
+
+
C H N O [m/z] [M + H] 401.22; found, [M + H] 401.30;
HRMS (ESI): calcd for C H N O (m/z) [M + H] 401.2223;
2
6
28
2
2
+
2
6
28
2
2
+
reaction, it was poured into saturated NaHCO and extracted with
found, [M + H] 401.2226.
3
ethyl acetate. The organic phases were collected, dried over
1-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-3-yl)-
methyl)piperazine (6c). Method B. (3′-(Benzo-1,4-dioxan-6-yl)-2′-
methyl-[1,1′-biphenyl]-3-yl)methanol (4b) (0.20 g, 0.60 mmol, 1.0
anhydrous MgSO , and evaporated. The crude products were purified
4
by column chromatography (SiO , DCM/methanol, methanol, or
2
methanol/7 M NH in methanol).
equiv), SOCl (0.22 mL, 3.0 mmol, 5.0 equiv), 1-Boc-piperazine (0.56
2
3
Method D. Crude alkyl chloride was dissolved in anhydrous DMF,
and an appropriate amine dissolved in anhydrous DMF was added (if
the hydrochloride of the amine was used, equimolar amount of
triethyl amine was added to the reaction mixture) together with
diisopropylethylamine (DIPEA). The reaction mixture was stirred at
g, 3.0 mmol, 5.0 equiv). Product 6c was obtained as a colorless solid
with 29.1% (0.070 g) yield.
1
R
f
= 0.11 (SiO , MeOH); H NMR (600 MHz, CDCl
2
): δ [ppm]
3
7.25 (t, J = 7.5 Hz, 1H), 7.23 (s, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.16−
7.13 (m, 2H), 7.10 (m, 2H), 6.80 (d, J = 8.2 Hz, 1H), 6.79 (d, J = 2.0
Hz, 1H), 6.73 (dd, J = 8.2, 2.0 Hz, 1H), 4.17 (s, 4H), 3.45 (s, 2H),
2.80 (m, 5H), 2.36 (sbroad, 4H), 2.03 (s, 3H); 13C NMR (151 MHz,
8
0 °C overnight. After completion of the reaction, it was poured into
saturated NaHCO and extracted with ethyl acetate. The organic
3
phases were collected, dried over anhydrous MgSO , and evaporated.
CDCl
3
): δ [ppm] 143.1, 142.8, 142.6, 142.3, 137.8, 135.9, 133.1,
4
The crude products were purified by column chromatography (SiO2,
130.3, 129.0, 128.8, 128.1, 128.0, 127.7, 125.3, 122.6, 118.2, 116.9,
−
1
ethyl acetate, DCM/methanol, or CHCl /7 M NH in methanol or
64.5, 64.4, 63.6, 54.1, 45.8, 18.9; IR (ATR) [cm ]: 3319, 2934, 1507,
1279, 1069; LC−MS (DAD/ESI): t = 5.11 min, calcd for
3
3
methanol/7 M NH in methanol).
R
3
+
+
Method E. Crude alkyl chloride was dissolved in anhydrous DMF,
and an appropriate amine dissolved in anhydrous DMF was added (if
the hydrochloride of the amine was used, an equimolar amount of
triethylamine was added to the reaction mixture) together with
DIPEA. The reaction mixture was stirred at 80 °C overnight. After
completion of the reaction, it was poured into saturated NaHCO3,
extracted three times with ethyl acetate, and the crude Boc-protected
C H N O [m/z] [M + H] 401.22; found, [M + H] 401.34;
26 28 2 2
HRMS (ESI): calcd for C26
found, [M + H] 401.2222.
+
H
N
O
2
[m/z] [M + H] 401.2223;
28
2
+
1-((3′-(Benzo-1,4-dioxan-6-yl)-3-methoxy-2′-methyl-[1,1′-bi-
phenyl]-4-yl)methyl)piperazine (6d). Method B. (3′-(Benzo-1,4-
dioxan-6-yl)-3-methoxy-2′-methyl-[1,1′-biphenyl]-4-yl)methanol (4i)
(0.20 g, 0.5 mmol, 1.0 equiv), thionyl chloride (0.19 mL, 2.7 mmol,
5.0 equiv), 1-Boc-piperazine (0.51 g, 2.7 mmol, 5.0 equiv). Product
product was purified by flash chromatography (SiO , hexane/ethyl
2
acetate 1:1). The purified Boc-protected product was dissolved in
anhydrous dioxane, and HCl in dioxane was added (10 equiv, 4 M).
The reaction mixture was stirred at room temperature overnight, then
6d was obtained as a light-yellow oil with 19.9% (0.047 g) yield.
1
R
f
= 0.08 (SiO
, MeOH); H NMR (600 MHz, DMSO-d
): δ
2
6
[ppm] 7.35 (d, J = 7.6 Hz, 1H), 7.27 (t, J = 7.5 Hz, 1H), 7.19 (d, J =
6.6 Hz, 1H), 7.16 (dd, J = 7.5, 1.2 Hz, 1H), 6.95−6.89 (m, 3H), 6.88
(d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.2, 2.1 Hz, 1H), 4.28 (s, 4H), 3.80
(s, 3H), 3.45 (s, 2H), 3.17 (s, 1H), 2.74−2.60 (sbroad, 4H), 2.40−2.27
poured into saturated NaHCO , and extracted three times with ethyl
3
acetate. The organic phases were collected, dried over anhydrous
MgSO , and evaporated. The products were purified by column
4
(sbroad, 4H), 2.09 (s, 3H); 13C NMR (151 MHz, DMSO-d
): δ [ppm]
chromatography (SiO , DCM/methanol, or methanol/7 M NH in
6
2
3
methanol).
157.4, 143.4, 143.0, 142.3, 142.0, 135.4, 132.9, 129.9, 129.1, 129.0,
125.9, 125.0, 122.6, 121.3, 118.2, 117.2, 112.3, 64.6, 56.6, 55.9, 54.9,
4
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)-
−
1
methyl)morpholine (6a). Method A. (3′-(Benzo-1,4-dioxan-6-yl)-2′-
methyl-[1,1′-biphenyl]-4-yl)methanol (4a) (0.33 g, 1.0 mmol, 1.0
equiv), thionyl chloride (0.36 mL, 5.0 mmol, 5.0 equiv), morpholine
46.2, 19.2; IR (ATR) [cm ]: 3322, 2934, 2829, 1577, 1507, 1463,
1399, 1301, 1280, 1227, 1068, 872, 792, 742; LC−MS (DAD/ESI):
+
t = 4.72 min, calcd for C H N O [m/z] [M + H] 431.34; found,
R
27 30
2
3
+
(
0.35 mL, 5.0 mmol, 5.0 equiv). The crude product was purified by
[M + H] 431.23; HRMS (ESI): calcd for C H N O (m/z) [M +
27 30 2 3
+
+
flash chromatography (SiO , hexane/ethyl acetate 2:1), giving the
H] 431.2329; found, [M + H] 431.2329.
2
final compound 6a as a colorless oil with 70.1% (0.281 g) yield.
3-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-4-(piperazin-1-ylmeth-
yl)-[1,1′-biphenyl]-3-yl)oxy)methyl)benzonitrile (6e). Method B. 3-
(((3′-(Benzo-1,4-dioxan-6-yl)-4-(hydroxymethyl)-2′-methyl-[1,1′-bi-
phenyl]-3-yl)oxy)methyl)benzonitrile (4j) (0.24 g, 0.5 mmol, 1.0
equiv), thionyl chloride (0.14 mL, 2.0 mmol, 5.0 equiv), 1-Boc-
piperazine (0.37 g, 2.0 mmol, 5.0 equiv). Product 6e was obtained as a
1
R = 0.36 (SiO , ethyl acetate/hexane 1:1); H NMR (600 MHz,
f
2
DMSO-d ): δ [ppm] 7.37 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz,
6
2
H), 7.27 (t, J = 7.6 Hz, 1H), 7.16 (d, J = 7.6 Hz, 2H), 6.91 (d, J = 8.2
Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.81 (dd, J = 8.2, 2.1 Hz, 1H), 4.28
(
(
s, 4H), 3.62−3.57 (m, 4H), 3.51 (s, 2H), 2.43−2.32 (m, 4H), 2.06
13
s, 3H); C NMR (151 MHz, DMSO-d ): δ [ppm] 142.9, 142.5,
colorless solid with 38.4% (0.104 g) yield.
6
1
1
1
42.1, 141.8, 140.5, 136.5, 134.9, 132.3, 29.0, 128.7, 128.5, 125.5,
R
f
= 0.12 (SiO
2
, methanol H NMR (600 MHz, DMSO-d
): δ
6
22.1, 117.7, 116.8, 66.2, 64.1, 64.1, 62.2, 53.2, 18.7; IR (ATR)
[ppm] 7.94 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 7.7 Hz,
1H), 7.62 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.26 (t, J = 7.3
Hz, 1H), 7.16 (d, J = 7.7 Hz, 2H), 7.02 (d, J = 1.4 Hz, 1H), 6.94−
6.90 (m, 2H), 6.84 (d, J = 2.1 Hz, 1H), 6.81 (dd, J = 8.2, 2.1 Hz, 1H),
−1
[
cm ]: 2957, 2923, 2842, 1582, 1506, 1455, 1317, 1292, 1242, 1228,
1
114, 1070, 1006, 898, 881, 845, 791; LC−MS (DAD/ESI): t = 5.65
R
+
+
+
min, calcd for C H NO (m/z) [M + H] 402.27; found, [M + H]
26
27
3
4
4
02.21; HRMS (ESI): calcd for C H NO (m/z) [M + H]
02.2064; found, [M + H] 402.2062.
5.25 (s, 2H), 4.28 (s, 4H), 3.51 (s, 2H), 2.73−2.67 (m, 4H), 2.39−
2
6
27
3
+
13
2.32 (m, 4H), 2.00 (s, 3H); C NMR (151 MHz, DMSO-d
): δ
6
4
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-methyl-[1,1′-biphenyl]-4-yl)-
[ppm] 155.6, 143.0, 142.5, 142.2., 141.8, 141.7, 139.3, 134.9, 132.4,
132.0, 131.5, 130.6, 130.2, 129.7, 128.7, 128.4, 125.5, 125.5, 122.1,
121.3, 118.7, 117.7, 116.8, 113.6, 111.4, 68.0, 64.1, 56.7, 54.5, 45.7,
methyl)piperazine (6b). Method B. (3′-(Benzo-1,4-dioxan-6-yl)-2′-
methyl-[1,1′-biphenyl]-4-yl)methanol (4a) (0.24 g, 0.72 mmol, 1.0
R
https://doi.org/10.1021/acs.jmedchem.1c00957
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
−
1
1
1
1
5
+
8.6); IR (ATR) [cm ]: 2932, 2875, 2814, 2230, 1576, 1506, 1461,
R = 0.30 (SiO , ethyl acetate/methanol 1:1); H NMR (600 MHz,
f 2
402, 1301, 1244, 1225, 1068, 1001, 792; LC−MS (DAD/ESI): t =
CDCl ): δ [ppm] 7.34−7.26 (m, 4H), 7.00−6.97 (m, 3H), 6.95−6.91
(m, 2H), 6.23 (s, 1H), 4.31 (s, 1H), 4.04 (7, J = 6.6 Hz, 2H), 3.83 (s,
2H), 3.37 (q, J = 5.5 Hz, 2H), 2.79 (t, J = 5.9 Hz, 2H), 1.99 (s, 3H),
1.82 (sept, J = 6.7 Hz, 1H), 0.97 (d, J = 6.6 Hz, 6H); C NMR (151
MHz, CDCl ): δ [ppm] 170.4, 156.7, 143.3, 143.1, 141.5, 141.2,
140.7, 133.4, 131.1, 130.6, 130.2, 129.6, 126.9, 126.5, 122.9, 121.6,
118.7, 117.0,113.0, 66.6, 64.6, 64.5, 48.9, 47.9, 39.1, 38.2, 25.4, 23.5,
22.8; IR (ATR) [cm ]: 3292, 3062, 2956, 2931, 2872, 1652, 1507,
R
3
+
.93 min, calcd for C H N O (m/z) [M + H] 532.26; found, [M
3
4
33
3
3
+
+
H] 532.41; HRMS (ESI): calcd for C H N O (m/z) [M + H]
3
4
33
3
3
+
13
5
32.2595; found, [M + H] 532.2594.
N-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-bi-
phenyl]-4-yl)methyl)amino)ethyl)acetamide (7a). Method C. (3′-
3
(
Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphenyl]-4-yl)-
−
1
methanol (4k) (0.15 g, 0.39 mmol, 1.0 equiv), SOCl (0.1 mL, 1.95
2
mmol, 5.0 equiv) N-(2-aminoethyl)acetamide (0.19 mL, 1.95 mmol,
1456, 1302, 1280, 1249, 1226, 1069, 796, 733; LC−MS (DAD/ESI):
+
5
(
.0 equiv). Product 7a was obtained as a brownish oil with 28.0%
0.051 g) yield.
R = 0.26 (SiO , MeOH); H NMR (600 MHz, DMSO-d ): δ
t
= 6.62 min, calcd for C H ClN O (m/z) [M + H] 523.24;
R
30 35
2
4
+
found, [M + H] 523.37; HRMS (ESI): calcd for C H ClN O (m/
z) [M + H] 523.2358; found, [M + H] 523.2358.
30 35 2 4
1
+
+
f
2
6
[
(
(
ppm] 7.33 (m, 4H), 7.00−6.98 (m, 3H), 6.95−6.91 (m, 2H), 6.33
s, 1H), 4.29 (s, 4H), 3.86 (s, 3H), 3.81 (s, 2H), 3.36 (m, 2H), 2.78
t, J = 5.8 Hz, 2H), 2.00 (sbroad, 1H), 1.98 (s, 3H); 13C NMR (151
N-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-(cyanomethoxy)-
[1,1′-biphenyl]-4-yl)methyl)amino)ethyl)acetamide (7e). Method
D. 2-((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-4-(hydroxymethyl)-
[1,1′-biphenyl]-3-yl)oxy)acetonitrile (4o) (0.22 g, 0.54 mmol, 1.0
MHz, DMSO-d ): δ [ppm] 170.3, 157.1, 143.2, 143.0, 141.4, 141.1,
6
1
1
40.5, 133.3, 130.9, 130.5, 130.1, 129.5, 127.2, 126.4, 122.8, 121.6,
equiv), SOCl (0.12 mL, 1.62 mmol, 3.0 equiv), N-(2-aminoethyl)-
2
18.6, 116.9, 112.0, 64.5, 64.4, 55.4, 48.7, 47.9, 39.1, 23.3; IR (ATR)
acetamide (0.21 mL, 2.24 mmol, 4.0 equiv), DIPEA (0.20 mL, 1.12
mmol, 2.0 equiv). Product 7e was obtained as a colorless oil with
−1
[
(
4
cm ]: 3294, 2934, 1652, 1507, 1457, 1302, 1068, 732; LC−MS
+
DAD/ESI): t = 5.43 min, calcd for C H ClN O [m/z] [M + H]
9.8% (0.026 g) yield.
R
26 27
2
4
+
1
67.17; found, [M + H] 467.28; HRMS (ESI): calcd for
R
f
= 0.24 (SiO
2
, ethyl acetate/methanol 1:1); H NMR (600 MHz,
C H ClN O [m/z] [M + H]+ 467.1732; found, [M + H]
+
CDCl
): δ [ppm] 7.37 (d, 7.7 Hz, 1H), 7.35−7.31 (m, 2H), 7.28 (dd,
2
6
27
2
4
3
4
67.1732.
N-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-ethoxy-[1,1′-bi-
phenyl]-4-yl)methyl)amino)ethyl)acetamide (7b). Method D. (3′-
J = 6.7, 2.7 Hz, 1H), 7.15 (dd, J = 7.6, 1.5 Hz, 1H), 7.05 (d, J = 1.4
Hz, 1H), 6.99−6.98 (m, 1H), 6.95−6.91 (m, 2H), 6.09 (sbroad, 1H),
4.86 (s, 2H), 4.30 (s, 4H), 3.85 (s, 2H), 3.38 (q, J = 5.5 Hz, 2H), 2.81
(t, J = 5.6 Hz, 2H), 1.99 (s, 3H); 13C NMR (151 MHz, CDCl ): δ
(
Benzo-1,4-dioxan-6-yl)-2′-chloro-3-ethoxy-[1,1′-biphenyl]-4-yl)-
3
methanol (4l0.15 g, 0.27 mmol, 1.0 equiv), SOCl (0.14 mL, 1.95
mmol, 5.0 equiv), N-(2-aminoethyl)acetamide (0.080 g, 0.78 mmol,
[ppm] 170.3, 154.1, 143.3, 143.0, 141.2, 140.8, 140.5, 133.1, 130.9,
130.1, 128.5, 126.5, 124.3, 122.8, 118.5, 116.9, 115.1, 113.6, 64.5,
2
−
1
2
.0 equiv), DIPEA (0.094 mL, 0.54 mmol, 2.0 equiv). Product 7b was
64.4, 53.9, 48.1, 48.0, 39.1, 23.4; IR (ATR) [cm ]: 3292, 3059, 2932,
1652, 1582, 1508, 1457, 1319, 1303, 1281,1251, 1068, 798, 737; LC−
MS (DAD/ESI): t = 5.29 min, calcd for C H ClN O (m/z) [M +
obtained as a brownish oil with 48.7% (0.091 g) yield.
1
R = 0.29 (SiO , MeOH); H NMR (600 MHz, CDCl ): δ [ppm]
f
2
3
R
27 26
3
4
+
+
7
.25−7.10 (m, 4H), 6.92−6.89 (m, 3H), 6.87−6.84 (m, 2H), 6.14
H] 492.17; found, [M + H] 492.27; HRMS (ESI): calcd for
C H ClN O (m/z) [M + H] 492.1685; found, [M + H]+
+
(
(
s
, 1H), 4.22 (s, 4H), 4.02 (q, J = 7.0 Hz, 2H), 3.75 (s, 2H), 3.29
broad
27 26
2
4
13
m, 2H), 2.70 (m, 2H), 1.90 (s, 3H), 1.35 (t, J = 7.0 Hz, 3H);
C
492.1683.
NMR (151 MHz, CDCl ): δ [ppm] 170.2, 156.4, 143.2, 143.0, 141.4,
1
N-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-(cyclobutylme-
thoxy)-[1,1′-biphenyl]-4-yl)methyl)amino)ethyl)acetamide (7f).
Method D. (3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-cyclobutylme-
thoxy-[1,1′-biphenyl]-4-yl)methanol (4p) (0.36 g, 0.83 mmol, 1.0
3
41.1, 140.4, 133.3, 131.0, 130.5, 130.1, 129.5, 126.9, 126.4, 122.8,
1
21.5, 118.6, 116.9, 112.9, 64.5, 64.4, 63.6, 48.8, 47.8, 39.1, 23.4, 15.0;
−
1
IR (ATR) [cm ]: 3293, 3066, 2979, 2878, 1652, 1507, 1456, 1303,
250, 1068; LC−MS (DAD/ESI): t = 5.69 min, calcd for
1
equiv), SOCl (0.30 mL, 4.16 mmol, 5.0 equiv), N-(2-aminoethyl)-
2
R
+
+
C H ClN O [m/z] [M + H] 481.18; found, [M + H] 481.30;
acetamide (0.23 mL, 2.49 mmol, 3.0 equiv), DIPEA (0.29 mL, 1.66
mmol, 2.0 equiv). Product 7f was obtained as a colorless oil with
27
29
2
4
+
HRMS (ESI): calcd for C H ClN O [m/z] [M + H] 481.1889;
found, [M + H] 481.1889.
2
7
29
2
4
+
29.6% (0.128 g) yield.
1
N-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-isopropoxy-[1,1′-
R
f
= 0.30 (SiO
2
, ethyl acetate/methanol 1:1); H NMR (600 MHz,
biphenyl]-4-yl)methyl)amino)ethyl)acetamide (7c). Method D. (3′-
DMSO-d ): δ [ppm] 7.82 (t, J = 5.5 Hz, 1H), 7.43 (t, J = 7.6 Hz,
6
(
Benzo-1,4-dioxan-6-yl)-2′-chloro-3-isopropoxy-[1,1′-biphenyl]-4-yl)-
1H), 7.38−7.36 (m, 2H), 7.35 (dd, J = 7.5, 1.8 Hz, 1H), 7.00 (d, J =
1.5 Hz, 1H), 6.97 (dd, J = 7.6, 1.6 Hz, 1H), 6.95−6.93 (m, 2H), 6.90
(dd, J = 8.3, 2.1 Hz, 1H), 4.29 (s, 4H), 3.99 (d, J = 6.3 Hz, 2H), 3.72
(s, 2H), 3.15 (q, J = 6.3 Hz, 2H), 2.78−2.71 (m, 1H), 2.58 (t, J = 6.5
methanol (4m) (0.21 g, 0.53 mmol, 1.0 equiv), SOCl (0.12 mL, 1.59
2
mmol, 3.0 equiv), N-(2-aminoethyl)acetamide (0.21 mL, 2.24 mmol,
4
.0 equiv), DIPEA (0.20 mL, 1.12 mmol, 2.0 equiv). Product 7c was
1
3
obtained as a colorless oil with 36.6% (0.096 g) yield.
Hz, 2H), 2.11−2.03 (m, 2H), 1.94−1.85 (m, 4H), 1.79)s, 3H);
C
1
R = 0.30 (SiO , ethyl acetate/methanol 1:1; H NMR (600 MHz,
NMR (151 MHz, DMSO-d ): δ [ppm] 169.14, 156.1, 143.1, 142.8,
f
2
6
CDCl ): δ [ppm] 7.34−7.26 (m, 4H), 7.01−6.98 (m, 2H), 6.97−6.91
141.1, 140.4, 139.0, 132.5, 130.4, 130.3, 129.9, 128.5, 128.5, 126.9,
122.4, 121.0, 118.0, 116.7, 112.7, 79.2, 71.3, 64.1, 64.1, 48.5, 47.3,
3
(
m, 3H), 6.15 (s , 1H), 4.65−4.58 (m, 1H), 4.31 (s, 4H), 3.81 (s,
broad
−
1
2
1
1
1
6
3
1
H), 3.37 (q, J = 5.8 Hz, 2H), 2.78 (t, J = 5.8 Hz, 2H), 1.99 (s, 3H),
34.1, 24.2, 22.6, 18.1; IR (ATR) [cm ]: 3292, 3062, 2975, 2934,
2867, 1652, 1507, 1456, 1302, 1280, 1249, 1226, 1068, 796, 747;
LC−MS (DAD/ESI): t = 6.37 min, calcd for C H ClN O (m/z)
.36 (d, J = 6.0 Hz, 6H); 13C NMR (151 MHz, CDCl ): δ [ppm]
3
70.3, 155.5, 143.3, 143.1, 141.6, 141.2, 140.5, 139.5, 131.5, 130.6,
30.3, 129.9, 128.0, 126.5, 122.9, 121.4, 118.7, 117.0, 114.5, 70.2,
R
30 33
2
4
+
+
[M + H] 521.22; found, [M + H] 521.38; HRMS (ESI): calcd for
−
1
C H ClN O (m/z) [M + H] 521.2202; found, [M + H]+
+
4.6, 64.6, 49.1, 47.8, 39.1, 23.5, 22.4); IR (ATR) [cm ]: 3295,
3
0
33
2
4
057, 2976, 2931, 2874, 1652, 1507, 1456, 1302, 1280, 1249, 1226,
521.2202.
113, 1069, 796, 734; LC−MS (DAD/ESI): t = 5.86 min, calcd for
N-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-(cyclopentylme-
thoxy)-[1,1′-biphenyl]-4-yl)methyl)amino)ethyl)acetamide (7g).
Method D. (3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-cyclopentylme-
thoxy-[1,1′-biphenyl]-4-yl)methanol (4q) (0.21 g, 0.51 mmol, 1.0
R
+
+
C H ClN O (m/z) [M + H] 495.32; found, [M + H] 495.20;
HRMS (ESI): calcd for C H ClN O (m/z) [M + H] 495.2045;
found, [M + H] 495.2045.
28
31
2
4
+
2
8
31
2
4
+
N-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-isopentyloxy-
equiv), SOCl (0.18 mL, 2.55 mmol, 5.0 equiv), N-(2-aminoethyl)-
2
[
1,1′-biphenyl]-4-yl)methyl)amino)ethyl)acetamide (7d). Method
acetamide (0.18 mL, 1.02 mmol, 3.0 equiv), DIPEA (0.15 mL, 1.53
mmol, 2.0 equiv). Product 7g was obtained as a colorless oil with
70.9% (0.193 g) yield.
D. (3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-isopentyloxy-[1,1′-biphen-
yl]-4-yl)methanol (4n) (0.15 g, 0.34 mmol, 1.0 equiv), SOCl (0.12
2
1
mL, 1.70 mmol, 5.0 equiv), N-(2-aminoethyl)acetamide (0.097 mL,
R = 0.30 (SiO , ethyl acetate/methanol 1:1); H NMR (600 MHz,
f
2
1
.02 mmol, 3.0 equiv), DIPEA (0.12 mL, 0.68 mmol, 2.0 equiv).
CDCl ): δ [ppm] 7.33−7.27 (m, 4H), 7.00−6.95 (m, 3H), 6.95−6.91
3
Product 7d was obtained as a colorless oil 73.7% (0.131 g) yield.
(m, 2H), 6.18 (s, 1H), 4.31 (s, 4H), 3.89 (d, J = 6.9 Hz, 2H0, 3.84 (s,
S
https://doi.org/10.1021/acs.jmedchem.1c00957
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
2
H), 3.37 (q, J = 5.5 Hz, 2H), 2.79 (t, J = 5.8 Hz, 2H), 2.44−2.35 (m,
H), 1.88−1.81 (m, 2H), 1.68−1.56 (m, 4H), 1.39−1.33 (m, 2H);
494.18; found, [M + H]+ 494.33; HRMS (ESI): calcd for
C H ClN O [m/z] [M + H] 494.1841; found, [M + H]+
494.1841.
+
1
2
7
28
3
4
1
3
C NMR (151 MHz, CDCl ): δ [ppm] 156.7, 143.2, 143.0, 141.4,
3
1
1
40.6, 133.3, 131.0, 130.5, 130.1, 129.6, 122.8, 121.4, 118.6, 116.9,
1-(4-((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-bi-
phenyl]-4-yl)methyl)piperazin-1-yl)ethan-1-one (8c). Method D.
(
12.8, 72.2, 64.5, 64.4, 49.0, 47.8, 39.1, 39.0, 29.5, 25.5, 23.3; IR
−
1
3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphenyl]-4-
(
1
ATR) [cm ]: 3292, 3060, 2947, 2867, 1652, 1507, 1456, 1303,
280, 1249, 1226, 1068, 796, 748; LC−MS (DAD/ESI): t = 6.88
yl)methanol (4k) (0.15 g, 0.39 mmol, 1.0 equiv), SOCl (0.14 mL,
2
R
+
1
.95 mmol, 5.0 equiv), 1-acetylpiperazine (0.10 g, 0.78 mmol, 2.0
equiv), DIPEA (0.14 mL, 0.78 mmol, 2.0 equiv). Product 8c was
min, calcd for C H ClN O (m/z) [M + H] 535.24; found, [M +
31
35
2
4
+
+
H] 535.41; HRMS (ESI): calcd for C H ClN O (m/z) [M + H]
3
1
35
2
4
+
obtained as a brownish oil with 76.3% (0.147 g) yield.
5
35.2358; found, [M + H] 535.2358.
1
R = 0.46 (SiO , DCM/MeOH, 9:1); H NMR (600 MHz,
N-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-((3′-cyano-[1,1′-
f
2
biphenyl]-3-yl)methoxy)-[1,1′-biphenyl]-4-yl)methyl)amino)ethyl)-
acetamide (7h). Method D. 3′-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-
chloro-4-(hydroxymethyl)-[1,1′-biphenyl]-3-yl)oxy)methyl)-[1,1′-bi-
CDCl
3
): δ [ppm] 7.30 (d, J = 7.7 Hz, 1H), 7.23−7.17 (m, 3H), 6.92
(dd, J = 7.6, 1.5 Hz, 1H), 6.91 (d, J = 1.9 Hz, 1H), 6.88 (d, J = 1.5 Hz,
1H), 6.85−6.82 (m, 2H), 4.18 (s, 4H), 3.74 (s, 3H), 3.56 (t, J = 4.9
Hz, 2H), 3.54 (s, 2H), 3.39 (t, J = 4.8 Hz, 2H), 2.44 (m, 4H), 1.99 (s,
phenyl]-3-carbonitrile (4r) (0.15 g, 0.27 mmol, 1.0 equiv), SOCl
2
3H); 13C NMR (151 MHz, CDCl ): δ [ppm] 169.0, 157.3, 143.2,
(
0.10 mL, 1.35 mmol, 5.0 equiv), N-(2-aminoethyl)acetamide (0.077
3
mL, 0.81 mmol, 3.0 equiv), DIPEA (0.094 mL, 0.54 mmol, 2.0 equiv).
143.0, 141.5, 141.1, 140.3, 133.3, 130.9, 130.5, 130.2, 130.1, 126.4,
Product 7h was obtained as a brownish oil with 78.5% (0.136 g) yield.
124.7, 122.8, 121.6, 118.6, 116.9, 112.1, 64.4, 64.4, 55.8, 55.5, 53.1,
52.7, 46.4, 41.5, 21.4; IR (ATR) [cm ]: 3436, 2933, 1644, 1507,
1
−1
R = 0.26 (SiO , DCM/methanol, 9:1 H NMR (600 MHz,
f
2
CDCl ): δ [ppm] 7.77 (t, J = 1.5 Hz, 1H), 7.72 (dt, J = 7.9, 1.2 Hz,
1458, 1247, 1228; LC−MS (DAD/ESI): t = 5.38 min, calcd for
3
R
+
+
1
7
6
H), 7.54 (m, 2H), 7.47−7.39 (m, 4H), 7.29 (d, J = 7.7 Hz, 1H),
.23−7.18 (m, 2H), 7.16−7.13 (m, 1H), 7.02 (d, J = 1.3 Hz, 1H),
.93 (dd, J = 7.6, 1.4 Hz, 1H), 6.88 (m, 1H), 6.83−6.80 (m, 3H), 5.13
C H ClN O [m/z] [M + H] 493.19; found, [M + H] 493.33;
28 29 2 4
+
HRMS (ESI): calcd for C H ClN O [M + H] 493.1888; found,
2
8
29
2
4
+
[M + H] 493.1889.
(
s, 2H), 4.42 (s , 1H), 4.20 (s, 4H), 3.92 (s, 2H), 3.30 (m, 2H),
.82 (t, J = 5.5 Hz, 2H), 1.80 (s, 3H); C NMR (151 MHz, CDCl ):
2-Amino-1-(4-((3′-(benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-
[1,1′-biphenyl]-4-yl)methyl)piperazin-1-yl)ethan-1-one (8d). Meth-
od E. (3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphen-
broad
13
2
3
δ [ppm] 170.9, 156.2, 143.3, 143.0, 142.0, 141.6, 141.2, 140.9, 139.3,
1
1
1
37.7, 133.1, 131.6, 131.0, 130.8, 130.8, 130.7, 130.4, 130.0, 129.8,
29.6, 127.4, 126.9, 126.5, 126.2, 124.1, 122.7, 122.3, 118.8, 118.5,
16.9, 113.7, 113.0, 70.1, 64.5, 64.4, 47.9, 47.9, 38.0, 23.1); IR (ATR)
yl]-4-yl)methanol (4k) (0.15 g, 0.39 mmol, 1.0 equiv), SOCl (0.14
2
mL, 1.95 mmol, 5.0 equiv), tert-butyl(2-oxo-2-(piperazin-1-yl)ethyl)-
carbamate (0.19 g, 0.78 mmol, 2.0 equiv), DIPEA (0.14 mL, 0.78
mmol, 2.0 equiv). Product 8d was obtained as a brownish solid with
−1
[
cm ]: 3293, 3062, 2932, 2229, 1652, 1507, 1456, 1303, 1249, 1068;
LC−MS (DAD/ESI): t = 6.68 min, calcd for C H ClN O (m/z)
57.3% (0.114 g) yield.
R
39 34
3
4
+
+
1
[
M + H] 644.23; found, [M + H] 644.42; HRMS (ESI): calcd for
R
f
= 0.26 (SiO , methanol/7 M NH
2
3
in methanol, 20:1); H NMR
C H ClN O (m/z) [M + H]+ 644.2311; found, [M + H]
+
(600 MHz, CDCl
): δ [ppm] 7.30 (d, J = 7.7 Hz, 1H), 7.25−7.19 (m,
3
9
34
3
4
3
6
44.2311.
3H), 6.94−6.92 (m, 2H), 6.89 (m, 1H), 6.87−6.83 (m, 2H), 4.22 (s,
4H), 3.75 (s, 3H), 3.59 (m, 2H), 3.54 (s, 2H), 3.48 (sbroad, 2H), 3,34
(m, 2H), 3.05 (sbroad, 2H), 2.45 (m, 4H); 13C NMR (151 MHz,
1
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-bi-
phenyl]-4-yl)methyl)piperazine (8a). Method B. (3′-(Benzo-1,4-
dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphenyl]-4-yl)methanol (4k)
CDCl ): δ [ppm] 170.2, 157.2, 143.2, 143.0, 141.5, 141.1, 140.3,
3
(0.15 g, 0.39 mmol, 1.0 equiv), SOCl (0.14 mL, 1.95 mmol, 5.0
133.3, 130.9, 130.5, 130.2, 130.0, 126.3, 124.8, 122.8, 121.6, 118.6,
2
equiv), 1-Boc-piperazine (0.36 g, 1.95 mmol, 5.0 equiv). Product 8a
116.9, 112.1, 64.5, 64.4, 55.9, 55.6, 52.9, 52.8, 44.3, 42.7, 42.2; IR
−
1
was obtained as a colorless solid with 33.3% (0.058 g) yield.
(ATR) [cm ]: 3371, 2935, 1651, 1507, 1459, 1246, 1229, 732; LC−
1
+
R = 0.10 (SiO , MeOH); H NMR (600 MHz, DMSO-d ): δ
MS (DAD/ESI): t = 4.50 min, calcd C H ClN O [M + H]
f
2
6
R
28 30
3
4
+
+
[
7
=
=
ppm] 7.43 (m, 1H), 7.38 (m, 2H), 7.35 (dd, J = 7.5, 1.5 Hz, 1H),
508.20; found, [M + H] 508.35; HRMS (ESI ): calcd for
+
+
.02 (s, 1H), 6.99 (d, J = 7.6 Hz, 1H), 6.95−6.93 (m, 2H), 6.90 (dd, J
C H ClN O [M + H] 508.1998; found, [M + H] 508.1998.
28 30 3 4
8.3, 1.8 Hz, 1H), 4.29 (s, 4H), 3.80 (s, 3H), 3.46 (s, 2H), 2.70 (t, J
1-((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-bi-
4.3 Hz, 4H), 2.35 (m, 5H); 13C NMR (151 MHz, DMSO-d ): δ
phenyl]-4-yl)methyl)-4-(methylsulfonyl)piperazine (8e). Method D.
(3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphenyl]-4-
6
[
ppm] 157.3, 143.6, 143.3, 141.5, 140.9, 139.6, 133.0, 130.9, 130.8,
1
6
1
30.4, 129.8, 127.4, 125.9, 122.9, 121.6, 118.5, 117.2, 112.5, 64.6,
yl)methanol (4k) (0.15 g, 0.39 mmol, 1.0 equiv), SOCl (0.14 mL,
2
−
1
4.6, 56.5, 55.9, 54.9, 46.2; IR (ATR) [cm ]: 3306, 2931, 1507,
1.95 mmol, 5.0 equiv), 1-(methylsulfonyl)piperazine (0.13 g, 0.78
mmol, 2.0 equiv), DIPEA (0.14 mL, 0.78 mmol, 2.0 equiv). Product
457, 796; LC−MS (DAD/ESI): t = 4.71 min, calcd for
R
+
+
C H ClN O [m/z] [M + H] 451.18; found, [M + H] 451.32;
8e was obtained as a brownish solid with 88.9% (0.183 g) yield.
26
27
2
3
+
1
HRMS (ESI): calcd for C H ClN O [M + H] 451.1783; found,
R
f
= 0.27 (SiO
2
, EtOAc); H NMR (600 MHz, CDCl
): δ [ppm]
3
2
6
27
2
3
+
[
M + H] 451.1783.
-((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-bi-
phenyl]-4-yl)methyl)piperazine-1-carboxamide (8b). Method C.
3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphenyl]-4-
yl)methanol (4k) (0.15 g, 0.39 mmol, 1.0 equiv), SOCl (0.14 mL,
7.39 (d, J = 7.7 Hz, 1H), 7.36−7.31 (m, 3H), 7.03 (dd, J = 7.6, 1.5
Hz, 1H), 7.02 (d, J = 1.8 Hz, 1H), 7.00 (d, J = 1.4 Hz, 1H), 6.97−
6.94 (m, 2H), 4.32 (s, 4H), 3.86 (s, 3H), 3.68 (s, 2H), 3.30 (m, 4H),
4
2.80 (s, 3H), 2.69 (t, J = 4.2 Hz, 4H); 13C NMR (151 MHz, CDCl ):
(
3
2
δ [ppm] 157.3, 143.2, 143.0, 141.4, 141.1, 140.4, 133.3, 130.9, 130.5,
1
.95 mmol, 5.0 equiv), piperazine-1-carboxamide hydrochloride (0.13
130.1, 130.0, 126.4, 124.7, 122.8, 121.6, 118.6, 116.9, 112.2, 64.5,
−
1
g, 0.78 mmol, 2.0 equiv), triethylamine (0.11 mL, 0.78 mmol, 2.00
64.4, 55.7, 55.6, 52.3, 46.0, 34.1; IR (ATR) [cm ]: 2929, 1507, 1323,
1160; LC−MS (DAD/ESI): t = 5.81 min, calcd for C H ClN O S
equiv). Product 8b was obtained as a brownish solid with 36.3%
R
27 29
2
5
+
+
(
0.070 g) yield.
[m/z] [M + H] 529.16; found, [M + H] 529.29; HRMS (ESI):
calcd for C H ClN O S [M + Na] 551.1378; found, [M + Na]
R = 0.38 (SiO , DCM/MeOH, 9:1); 1H NMR (600 MHz,
+
+
f
2
27 29
2
5
CDCl ): δ [ppm] 7.31 (d, J = 7.7 Hz, 1H), 7.24−7.17 (m, 3H), 6.93
551.1378.
3
(
1
3
dd, J = 7.7, 1.5 Hz, 1H), 6.91 (d, J = 1.9 Hz, 1H), 6.89 (d, J = 1.5 Hz,
2-Amino-N-(2-(((3′-(benzo-1,4-dioxan-6-yl)-2′-chloro-3-me-
thoxy-[1,1′-biphenyl]-4-yl)methyl)amino)ethyl)acetamide (8f).
Method E. (3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-
biphenyl]-4-yl)methanol (4k) (0.15 g, 0.39 mmol, 1.0 equiv),
SOCl2 (0.14 mL, 1.95 mmol, 5.0 equiv), tert-butyl (2-((2-
aminoethyl)amino)-2-oxoethyl)carbamate (0.17 g, 0.78 mmol, 2.0
equiv), DIPEA (0.14 mL, 0.78 mmol, 2.0 equiv). Product 8f was
obtained as a brownish solid with 43.3% (0.081 g) yield.
H), 6.86−6.83 (m, 2H), 4.71 (s, 2H), 4.20 (s, 4H), 3.74 (s, 3H),
.54 (s, 2H), 3.34 (t, J = 5.0 Hz, 4H), 2.45 (t, J = 5.1 Hz, 4H); 13
C
NMR (151 MHz, CDCl ): δ [ppm] 158.3, 157.2, 143.2, 143.0, 141.5,
3
41.1, 140.2, 133.4, 130.9, 130.5, 130.2, 130.0, 126.4, 125.0, 122.8,
21.6, 118.6, 116.9, 112.1, 64.5, 64.4, 56.0, 55.6, 52.8, 44.1; IR (ATR)
−1
+
R
27 28
3
4
T
https://doi.org/10.1021/acs.jmedchem.1c00957
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
R = 0.29 (SiO , methanol/7M NH in methanol, 20:1); IR (ATR)
cm ]: 3306, 2934, 1650, 1581, 1507, 1459, 1068; H NMR (600
2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-bi-
f
2
3
−
1
1
[
phenyl]-4-yl)methyl)amino)-2-(hydroxymethyl)propane-1,3-diol
(
8j). Method D. (3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-
MHz, CDCl ): δ [ppm] 7.47 (s, 1H), 7.22 (m, 4H), 6.91−6.85 (m,
3
[
1,1′-biphenyl]-4-yl)methanol (4k) (0.15 g, 0.39 mmol, 1.0 equiv),
5
H), 4.22 (s, 4H), 3.77 (s, 5H), 3.38−3.38 (m, 4H), 2.74 (s, 2H);
1
3
SOCl (0.14 mL, 1.95 mmol, 5.0 equiv), tris(hydroxymethyl)-
C NMR (151 MHz, CDCl ): δ [ppm] 172.8, 157.1, 143.2, 143.0,
2
3
aminomethane (0.094 g, 0.78 mmol, 2.0 equiv), DIPEA (0.094 mL,
.54 mmol, 2.0 equiv). Product 8j was obtained as a brownish oil with
1
1
41.4, 141.1, 140.4, 133.3, 130.9, 130.5, 130.1, 129.5, 126.4, 122.8,
0
7
21.6, 118.6, 116.8, 112.0, 64.5, 64.4, 55.4, 48.5, 48.0, 44.9, 38.6; LC−
7.7% (0.147 g) yield.
MS (DAD/ESI): t = 4.25 min, calcd C H ClN O [m/z] [M +
R
26 28
3
4
1
+
+
R
f
= 0.26 (SiO , CHCl
2
3
/methanol, 1:2); H NMR (600 MHz,
H] 482.18; found, [M + H] 482.30; HRMS (ESI): calcd for
C H ClN O [m/z] [M + H]+ 482.1841; found, [M + H]
+
CDCl
3
): δ [ppm] 7.23 (d, J = 7.8 Hz, 1H), 7.21−7.17 (m, 3H), 6.90
2
6
28
3
4
(
3
[
1
6
1
m, 3H), 6.85−6.82 (m, 2H), 4.20 (s, 4H), 3.78 (s, 3H), 3.68 (s, 2H),
.52 (s, 6H), 3.13 (s , 3H); C NMR (151 MHz, CDCl ): δ
4
82.1840.
13
broad 3
N-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-bi-
ppm] 156.9, 143.2, 143.0, 141.3, 141.1, 140.9, 133.3, 130.9, 130.6,
phenyl]-4-yl)methyl)amino)ethyl)methanesulfonamide (8g). Meth-
od C. (3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphen-
30.1, 129.8, 127.1, 126.4, 122.8, 122.0, 118.6, 116.9, 112.2, 64.5,
−1
4.4, 62.9, 60.4, 55.6, 40.9; IR (ATR) [cm ]: 3343, 2935, 1507,
yl]-4-yl)methanol (4k) (0.15 g, 0.39 mmol, 1.0 equiv), SOCl (0.14
2
457, 1068, 1042, 796; LC−MS (DAD/ESI): t = 5.18 min, calcd
R
mL, 1.95 mmol, 5.0 equiv), N-(2-aminoethyl)methanesulfonamide
0.27 g, 1.95 mmol, 5.0 equiv). Product 8g was obtained as a
+
+
C H ClNO [m/z] [M + H] 486.17, found, [M + H] 486.29;
HRMS (ESI): calcd for C H ClNO [m/z] [M + H] 486.1678;
2
6
28
6
(
+
2
6
28
6
brownish oil with 36.2% (0.071 g) yield.
+
found, [M + H] 486.1.
R = 0.28 (SiO , DCM/methanol, 1:1); 1H NMR (600 MHz,
f
2
CDCl ): δ [ppm] 7.26−7.21 (m, 3H), 7.19 (dd, J = 6.6, 2.3 Hz, 1H),
3
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00957.
6
4
2
1
1
4
1
.94−6.91 (m, 3H), 6.87−6.84 (m, 2H), 4.85 (s , 2H), 4.22 (s,
■
broad
H), 3.83 (s, 2H), 3.81 (s, 3H), 3.22 (t, J = 5.7 Hz, 2H), 2.87 (s, 3H),
*
.84 (t, J = 5.6 Hz, 2H); 13C NMR (151 MHz, CDCl ): δ [ppm]
3
57.2, 143.2, 143.0, 141.4, 141.1, 141.1, 133.2, 130.9, 130.7, 130.1,
26.4, 124.5, 122.8, 121.8, 118.6, 116.9, 112.2, 64.5, 64.4, 55.6, 48.1,
7.6, 41.7, 40.0; IR (ATR) [cm ]: 3320, 2932, 1507, 1457, 1318,
303, 1151, 797; LC−MS (DAD/ESI): t = 5.50 min, calcd
−
1
Experimental procedures for substrate preparation (S1,
S2a−i, and 3a−3i); results of the HTRF assay; data
collection and refinement statistics (molecular replace-
ment) for the PD-L1 cocrystal structure with 8j; results
of the ICB assay; comparison of the terphenyl-based
with biphenyl-based inhibitor structures in complex with
PD-L1; and spectral data and purity of target
compounds (PDF)
R
+
+
C H ClN O S [m/z] [M + H] 503.14; found, [M + H] 503.30;
HRMS (ESI): calcd for C H ClN O S [m/z] [M + H] 503.1402;
found, [M + H] 503.1402.
-(2-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-bi-
phenyl]-4-yl)methyl)amino)ethyl)urea (8h). Method D. (3′-(Benzo-
,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphenyl]-4-yl)methanol
4k) (0.15 g, 0.39 mmol, 1.0 equiv), SOCl (0.142 mL, 1.95 mmol,
25
27
2
5
+
2
5
27
2
5
+
1
1
(
2
5
.0 equiv), (2-aminoethyl)urea hydrochloride (0.11 g, 0.78 mmol, 2.0
equiv), triethylamine (0.054 mL, 0.39 mmol, 1.00 equiv), DIPEA
0.094 mL, 0.54 mmol, 2.0 equiv). Product 8h was obtained as a
brownish oil with 54.9% (0.100 g) yield.
R = 0.18 (SiO , CHCl /7M NH in methanol, 10:1); H NMR
Molecular formula strings (CSV)
(
Accession Codes
NLD; authors will release the atomic coordinates and
experimental data upon article publication.
7
1
f
2
3
3
(
1
600 MHz, CDCl ): δ [ppm] 7.33−7.27 (m, 3H), 7.24 (d, J = 7.5 Hz,
3
H), 6.99−6.96 (m, 3H), 6.94−6.91 (m, 2H), 5.70 (s , 1H), 4.93
broad
AUTHOR INFORMATION
(s
, 2H), 4.29 (s, 4H), 3.84 (s, 3H), 3.80 (s, 2H), 3.27 (m, 2H),
■
broad
2
.77 (t, J = 5.5 Hz, 2H), 2.19 (sbroad, 1H); 13C NMR (151 MHz,
Corresponding Authors
CDCl ): δ [ppm] 159.7, 157.1, 143.2, 143.0, 141.4, 141.1, 140.6,
Ewa Surmiak − Department of Organic Chemistry, Faculty of
Chemistry, Jagiellonian University, 30-387 Krakow, Poland;
orcid.org/0000-0002-4103-4675; Email: ewa.surmiak@
uj.edu.pl
Tad A. Holak − Department of Organic Chemistry, Faculty of
Chemistry, Jagiellonian University, 30-387 Krakow, Poland;
Email: tadholak@uj.edu.pl
3
1
1
3
33.3, 130.9, 130.5, 130.1, 129.5, 126.9, 126.4, 122.8, 121.6, 118.6,
−1
16.9, 112.0, 64.5, 64.4, 55.4, 48.9, 48.8, 40.3; IR (ATR) [cm ]:
335, 2933, 1658, 1582, 1507, 1457, 1303, 1168; LC−MS (DAD/
+
ESI): t = 5.23 min, calcd C H ClN O [m/z] [M + H] 468.17;
R
25 26
3
4
+
found, [M + H] 468.27; HRMS (ESI): calcd for C H ClN O [m/
2
5
26
3
4
+
+
z] [M + H] 468.1685; found, [M + H] 468.1687.
-(((3′-(Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-bi-
phenyl]-4-yl)methyl)amino)propane-1,3-diol (8i). Method D. (3′-
2
Authors
(
Benzo-1,4-dioxan-6-yl)-2′-chloro-3-methoxy-[1,1′-biphenyl]-4-yl)-
Damian Muszak − Department of Organic Chemistry, Faculty
of Chemistry, Jagiellonian University, 30-387 Krakow,
Poland; orcid.org/0000-0002-4876-382X
Jacek Plewka − Malopolska Centre of Biotechnology,
Jagiellonian University, 30-387 Kraków, Poland;
orcid.org/0000-0002-0307-0907
methanol (4k) (0.15 g, 0.39 mmol, 1.0 equiv), SOCl (0.14 mL, 1.95
2
mmol, 5.0 equiv), serinol (0.071 g, 0.78 mmol, 2.0 equiv), DIPEA
(
0.094 mL, 0.54 mmol, 2.0 equiv). Product 8i was obtained as a
brownish oil with 68.0% (0.121 g) yield.
R = 0.32 (SiO , CHCl /methanol, 1:2); H NMR (600 MHz,
1
f
2
3
CDCl ): δ [ppm] 7.26 (d, J = 7.6 Hz, 1H), 7.23−7.21 (m, 2H), 7.18
3
(
m, 1H), 6.93−6.90 (m, 3H), 6.86−6.83 (m, 2H), 4.84 (s , 2H),
broad
Katarzyna Magiera-Mularz − Department of Organic
Chemistry, Faculty of Chemistry, Jagiellonian University, 30-
4
2
1
1
6
1
.22 (s, 4H), 3.98 (s, 2H), 3.80 (s, 3H), 3.72 (m, 2H), 3.61 (m, 2H),
.86 (m, 1H), 1.84 (s, 1H); 13C NMR (151 MHz, CDCl ): δ [ppm]
57.2, 143.2, 143.0, 141.8, 141.1, 141.1, 133.2, 130.9, 130.7, 130.3,
30.1, 126.4, 123.3, 122.8, 122.0, 118.6, 116.9, 112.2, 77.1, 64.5, 64.4,
3
3
87 Krakow, Poland; orcid.org/0000-0002-4826-6380
Justyna Kocik-Krol − Department of Organic Chemistry,
Faculty of Chemistry, Jagiellonian University, 30-387
Krakow, Poland; orcid.org/0000-0002-3779-3118
Bogdan Musielak − Department of Organic Chemistry,
Faculty of Chemistry, Jagiellonian University, 30-387
Krakow, Poland; orcid.org/0000-0002-1665-5920
−
1
0.7, 59.6, 55.6, 46.1, 24.0; IR (ATR) [cm ]: 3304, 2935, 2246,
507, 1458, 1303, 1246, 1069, 732; LC−MS (DAD/ESI): t = 5.27
R
+
+
+
min, calcd C H ClNO [m/z] [M + H] 456.16; found, [M + H]
4
4
2
5
26
5
56.24; HRMS (ESI): calcd for C H ClNO [m/z] [M + H]
25 26 5
+
56.1572; found, [M + H] 456.1573.
U
https://doi.org/10.1021/acs.jmedchem.1c00957
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Dominik Sala − Department of Organic Chemistry, Faculty of
Chemistry, Jagiellonian University, 30-387 Krakow, Poland;
orcid.org/0000-0002-9417-9149
Radoslaw Kitel − Department of Organic Chemistry, Faculty
of Chemistry, Jagiellonian University, 30-387 Krakow,
Poland
protein 1; PD-L1, programmed cell death ligand 1 m-murine,
h-human; SMI, small-molecule inhibitors; TCA, T-cell
activation; TCR, T-cell receptor; TCRAct, TCR activator;
TPSA, topological polar surface area; TR-FRET, time-resolved
fluorescence energy transfer
Malgorzata Stec − Department of Clinical Immunology,
Institute of Pediatrics, Jagiellonian University Medical
College, 30-663 Krakow, Poland; orcid.org/0000-0003-
REFERENCES
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(
2
(
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00957
(
Notes
3
(
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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■
This research was funded by Preludium grant no. UMO-2020/
7/N/ST4/02691 from the National Science Centre, Poland
to D.M.), by the project POIR.04.04.00-00-420F/17-00,
2
(
42.
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which is carried out within the TEAM program of the
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Union under the European Regional Development Fund (to
T.A.H.), by Preludium grant no. UMO-2019/35/N/NZ5/
4435 from the National Science Centre, Poland (to J.K.-K.).
Further funding was received to A.D. through an NIH Grant
2R01GM097082-05). 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. J.K.-K. acknowledges the
fellowship with project no. POWR.03.02.00-00-I013/16 from
the National Center for Research and Development. The
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to the financial support of the European Regional Develop-
ment Fund in the framework of the Polish Innovation
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ABBREVIATIONS
■
DMSO, dimethyl sulfoxide; EC , half-maximal effective
5
0
(
19) Chupak, L.; Ding, M.; Martin, S.; Zheng, X.; Hewawasam, P.;
concentration; FBS, fetal bovine serum; GuHCl, guanidinium
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