Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Death-Ligand 1 (PD-1/PD-L1) Interaction via Transiently Induced Protein States and Dimerization of PD-L1

Article abstract of DOI:10.1021/acs.jmedchem.7b00293

Blockade of the PD-1/PD-L1 immune checkpoint pathway with monoclonal antibodies has provided significant advances in cancer treatment. The antibody-based immunotherapies carry a number of disadvantages such as the high cost of the antibodies, their limited half-life, and immunogenicity. Development of small-molecule PD-1/PD-L1 inhibitors that could overcome these drawbacks is slow because of the incomplete structural information for this pathway. The first chemical PD-1/PD-L1 inhibitors have been recently disclosed by Bristol-Myers Squibb. Here we present NMR and X-ray characterization for the two classes of these inhibitors. The X-ray structures of the PD-L1/inhibitor complexes reveal one inhibitor molecule located at the center of the PD-L1 homodimer, filling a deep hydrophobic channel-like pocket between two PD-L1 molecules. Derivatives of (2-methyl-3-biphenylyl)methanol exhibit the structures capped on one side of the channel, whereas the compounds based on [3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol induce an enlarged interaction interface that results in the open "face-back" tunnel through the PD-L1 dimer.

Full text of DOI:10.1021/acs.jmedchem.7b00293

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Article
Small-molecule inhibitors of the Programmed Cell Death-1/
Programmed Death-ligand 1 (PD-1/PD-L1) interaction via
transiently-induced protein states and dimerization of PD-L1
Katarzyna Guzik, Krzysztof M. Zak, Przemyslaw Grudnik, Katarzyna Magiera, Bogdan Musielak,
Ricarda Törner, Lukasz Skalniak, Alexander Dömling, Grzegorz Dubin, and Tad A Holak
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017
Downloaded from http://pubs.acs.org on June 14, 2017
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Smallꢀmolecule inhibitors of the Programmed Cell
Deathꢀ1/Programmed Deathꢀligand 1 (PDꢀ1/PDꢀ
L1) interaction via transientlyꢀinduced protein
states and dimerization of PDꢀL1
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,
,
Katarzyna Guzik,† Krzysztof M. Zak,‡ § Przemyslaw Grudnik,‡ § Katarzyna Magiera,†,§ Bogdan
†
†
†
,
‡ §
∥
Musielak, Ricarda Törner, Lukasz Skalniak, Alexander Dömling, Grzegorz Dubin and
Tad A. Holak*,†,
§
†Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena
3, 30ꢀ060 Krakow, Poland.
‡Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Gronostajowa 7, 30ꢀ387 Krakow, Poland.
§Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30ꢀ387
Krakow, Poland.
∥
Department for Drug Design, University of Groningen, A. Deusinglaan 9, AV 9713
Groningen, the Netherlands.
Keywords: PDꢀL1, dimerization, checkpoint inhibitor, Xꢀray structure, small molecule
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ABSTRACT
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Blockade of the PDꢀ1/PDꢀL1 immune checkpoint pathway with monoclonal antibodies has
provided significant advances in cancer treatment. The antibodyꢀbased immunotherapies
carry a number of disadvantages such as the high cost of the antibodies, their limited halfꢀlife
and immunogenicity. Development of smallꢀmolecule PDꢀ1/PDꢀL1 inhibitors that could
overcome these drawbacks is slow because of the incomplete structural information for this
pathway. The first chemical PDꢀ1/PDꢀL1 inhibitors have been recently disclosed by Bristolꢀ
Myers Squibb. Here we present NMR and Xꢀray characterization for the two classes of these
inhibitors. The Xꢀray structures of the PDꢀL1/inhibitor complexes reveal one inhibitor
molecule located at the center of the PDꢀL1 homodimer, filling a deep hydrophobic channelꢀ
like pocket between two PDꢀL1 molecules. Derivatives of (2ꢀmethylꢀ3ꢀbiphenylyl)methanol
exhibit the structures capped on one side of the channel, whereas the compounds based on [3ꢀ
(2,3ꢀdihydroꢀ1,4ꢀbenzodioxinꢀ6ꢀyl)ꢀ2ꢀmethylphenyl]methanol induce an enlarged interaction
interface that results in the open “faceꢀback” tunnel through the PDꢀL1 dimer.
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INTRODUCTION
Immune checkpoint blocking (ICB) antibodies that target cytotoxicꢀTꢀlymphocyteꢀ
associated protein 4 (CTLA4) or programmed death protein 1 (PDꢀ1)/ programmed cell death
ligand 1 (PDꢀL1) constitute direct clinical proof that cancer can be treated through the
modulation of immunity. ICBꢀbased immunotherapy elicits durable antitumor responses and
longꢀterm remissions in a subset of patients with a broad spectrum of cancers.1ꢀ6
Until now, the antiꢀCTLA4 antibody (ipilimumab) and three therapeutic antibodies
targeting both PDꢀ1 and PDꢀL1 immune checkpoint proteins (nivolumab, pembrolizumab and
atezolizumab) have gained the US Food and Drug Administration (FDA) acceptance and
several others are currently undergoing clinical trials.4ꢀ11 The antibodies approved are
directed against metastatic melanoma, nonꢀsmall cell lung cancer (NSCLC) and renal cell
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carcinoma. In addition encouraging preclinical and clinical results showed that antiꢀPDꢀ1 and
antiꢀCTLAꢀ4 antibodies combined with each other or with other therapeutics can
significantly enhance efficacy and percentage of responders.4ꢀ11
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Development of small molecules that interfere with the PDꢀ1/PDꢀL1 pathway is lagging
in comparison to the development of the antibodies. This partially can be associated with
incomplete structural information about these proteins. The structures of the murine PDꢀ1 in
complexes with the human PDꢀL1 (hPDꢀL1) and murine PDꢀL2 have established the
molecular principles of their interaction.12,13 Structures of human apoꢀPDꢀ1 (PDB 3RRQ) and
apoꢀPDꢀL1 (3BIS, 3FN3, 4Z18, 5C3T) have also been determined. The structure of the fully
human PDꢀ1/PDꢀL1 complex, recently solved by us, indicated that the PDꢀL1 ligand binding
to the human PDꢀ1 (hPDꢀ1) is associated with significant plasticity within the receptor.14
Smallꢀmolecule ICBs of the PDꢀ1/PDꢀL1 interaction would offer complementary and
potentially synergistic therapeutics in comparison to the mAbs. A few series of small
molecules, macrocyclic peptides, peptides and peptidomimetics targeting the PDꢀ1/PDꢀL1
interaction were reported.15ꢀ17 BristolꢀMyersꢀSquibb (BMS) disclosed recently the first nonꢀ
peptidic smallꢀmolecule inhibitors against the PDꢀ1/PDꢀL1 pathway that showed the activity
in a homogenous timeꢀresolved fluorescence (HTRF) binding assay, however no further data
supporting their activity was provided.18ꢀ19 Recently, we reported the first coꢀcrystal
structures of hPDꢀL1 complexed with small molecular weight inhibitors disclosed in the
BMS patent (PDB: 5J89, 5J8O).20 The Xꢀray and biochemical data revealed that protein
dimerization is induced upon ligand binding. Here, we present structural characterization of
two further groups of the BMS compounds: the derivatives based on the scaffold of (2ꢀ
methylꢀ3ꢀbiphenylyl)methanol (compounds 1, Figure 1) and those based on the [3ꢀ(2,3ꢀ
dihydroꢀ1,4ꢀbenzodioxinꢀ6ꢀyl)ꢀ2ꢀmethylphenyl]methanol (compounds 2, Figure 1).18ꢀ19 Both
bind to the dimeric PDꢀL1. Recently, we reported the first coꢀcrystal structures of PDꢀL1
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complexed with small molecular weight inhibitors (PDB: 5J89, 5J8O) disclosed in BMS
patent (Zak et al., 2016). Furthermore, our biochemical data revealed that protein
dimerization is induced upon ligand binding. Here, we present the two novel crystal
structures of disclosed compounds (1a, 2a) both bound to dimeric PDꢀL1. Our structural
studies revealed that upon complex formation one of investigated compound (2a) induces
conformational changes in the PDꢀL1 binding site. Thus, this flexible binding site opens new
possibilities for the design of improved PDꢀ1/PDꢀL1 pathway antagonists.
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RESULTS
Synthesis of the PD-1/PD-L1 inhibitors 1a-d and 2a-c. Figure 1 shows the two groups
of representative BMS compounds synthesized by us. The first group comprises derivatives
of (2ꢀmethylꢀ3ꢀbiphenylyl)methanol, 1a-d. The second one incorporates replacing the distal
phenyl ring of 1 by the 2,3ꢀdihydrobenzo[b][1,4]dioxinyl moiety, compounds 2a-c. Both
groups of the compounds have their IC50 of binding to hPDꢀL1 in the range of 1.4ꢀ146 nM.18ꢀ
20
We also partitioned the large 1 and 2 ligands and screen the resulting fragments for
binding to PDꢀL1 and PDꢀ1. The fragments that were synthesized contain the biphenyl
central scaffold of the BMS compounds (Supporting Information Tables S1 and S2).
Synthesis of the (2-methyl-3-biphenylyl)methanol derivatives (1a-d) and other
between (3ꢀbromoꢀ2ꢀmethylphenyl)methanol and phenyl boronic acid. A large number of
biphenyl fragments were obtained using the same methodology (Supporting Information
Tables S1 and S2). The Mistunobu reaction or the palladiumꢀmediated BuchwaldꢀHartwig
cross coupling between compound 3 and an appropriately substituted aldehyde provided
aldehydes 4aꢀ4c.21, 22 In the last step the NaBH3CNꢀmediated reductive amination was used
to convert aldehydes 4aꢀc into final compounds 1aꢀ1d (Table 1).18, 19
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Synthesis of the [3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol
derivatives (2a-c). To prepare inhibitors 2aꢀc, a multistep synthetic route similar to that for
1aꢀd was applied. Compound 5 was obtained by the SuzukiꢀMiyaura coupling between (3ꢀ
bromoꢀ2ꢀmethylphenyl)methanol and (2,3ꢀdihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)boronic acid. A
subsequent Mistunobu etherification reaction between alcohol 5 and an appropiate phenolic
aldehyde afforded aldehydes 6aꢀc.21 The sodium cyanoborohydrideꢀmediated reductive
amination was used to convert aldehyde 6a into compound 2a. Compounds 2b-c were
obtained by the cesium carbonateꢀpromoted Oꢀalkylation of aldehydes 7aꢀb, followed by
reductive amination using NaBH3CN and appropriate amines (Table 2). 18, 19
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NMR indicates that 1 and 2 bind to PD-L1 and induce its oligomerization in
solution. Determination of binding of the lead BMS compounds to PDꢀL1 was carried out
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with the Hꢀ15N 2D HMQC NMR spectroscopy on the 15N isotopically enriched domain of
human PDꢀL1 (hPDꢀL1), which encompassed residues 18ꢀ134 of hPDꢀL1. The 15Nꢀlabeled
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PDꢀL1 was titrated with increasing concentration of an evaluated compound and Hꢀ15N 2D
HMQC spectra were recorded after addition of each new portion of the compound.23.24
Ligandꢀinduced chemical shift perturbations in the 2D HMQC spectra confirmed the
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interaction of 1 and 2’s with PDꢀL1. Additionally, NMR signal doubling in the 2D H−15N
HMQC spectra indicating the KD’s of less than 1 ꢀM (and a slow chemical exchange).25
Moreover, the linewidth broadening in the 1D proton NMR of PDꢀL1 suggests that the
compounds induce protein oligomerization (Supporting Information Figures S1 and S2),
already observed in our recent studies for the (2ꢀmethylꢀ3ꢀbiphenylyl)methanol derivative
1c.20 The approximate size of the oligomers of all of the small molecule/PDꢀL1 complexes
determined from the NMR analysis is that of a tetramer (Supporting Information Figures
S1B). Size exclusion chromatography on PDꢀL1 in the presence and absence of the inhibitor
(1 and 2) indicates the 1,2ꢀinduced dimerization of PDꢀL1 (Supporting Information Figure
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S3). A single peak corresponding to a protein of molecular weight of 17 kDa was observed
for apoꢀPDꢀL1, indicating a monomeric form in solution. In the presence of the compound
the movement of the peak to shorter retention time confirmed the dimeric state of the
complexed PDꢀL1 (34 kDa) (Supporting Information Figure S3). The Xꢀray structural data
below indicates that the principal oligomerization component of the 1,2/PDꢀL1 complexes is
dimeric (although the hPDꢀL1/1a complex contains four protein molecules organized into
two dimers in the asymmetric unit, the molar ratio of the protein to ligand being 4:2, see
below). We have repeated the NMR experiments at lower concentrations to those standard
used above (data not shown). At the concentrations of PDꢀL1 of 0.5 µM, there is a clear cut
large increase of the proton NMR linewidths at the molar concentration ratio protein to ligand
4:1, which is identical to that seen in the Supporting Information Figures S1B. We conclude
that, at least, at high µM concentrations of PDꢀL1, NMR monitors the loosely formed
tetrameric states of the 1,2/PDꢀL1 complexes. However, size exclusion chromatography and
Xꢀray indicates that the most stable unit of the 1,2 induced oligomerization of PDꢀL1 is that
of a dimer.
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We have in addition synthesized and tested a large number of fragments and
intermediates of 1 and 2 that possess the biphenyl core building element. All these structures
induced oligomerization of PDꢀL1 (Supporting Information Table S2).
NMR detects two groups of the compounds that differ in their modes of binding to
PD-L1. The 1Hꢀ15N 2D HMQC NMR spectrum of the 2ꢀbound 15N uniformly labeled PDꢀL1
shows increase of number or crossꢀpeaks and extensive peak overlap compared to the
spectrum of the monomeric apoꢀPDꢀL1 (Supporting Information Figure S4). This is because,
as our recent structure of PDꢀL1 with a similar inhibitor indicates, one inhibitor molecule is
located at the interface of the asymmetric dimer (i.e. the stoichiometry, for example, of 1c:
PDꢀL1 in the complex is 1: 2, respectively).20 The tetrameric state seen in NMR and in the Xꢀ
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ray comprises a symmetric dimer of the 1c/PDꢀL1 dimer. Both the binding site of the
inhibitor and the intermolecular interactions within the dimer involve the PDꢀ1 interaction
surface of PDꢀL1.14, 20 To simplify the 15N NMR spectra, we prepared the amino acidꢀtype
selectiveꢀlabeling PDꢀL1 (Supporting Information Figure S4). The amino acids chosen for
labeling were those involved directly in the binding of PDꢀL1 to PDꢀ1 and/or 1c (i.e.
tyrosines) and those close to the binding interfaces (valines). We therefore prepared two PDꢀ
L1’s labeled with 15NꢀVal and 15NꢀTyr (Supporting Information Figure S4).
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In all NMR spectra recorded for the [15NꢀVal]ꢀPDꢀL1 and [15NꢀTyr]ꢀPDꢀL1 we observed
broadening and/or shifts of the HMQC signals of hPDꢀL1 during the titration. Upon addition
of the compound, compared to the apoꢀPDꢀL1, the largest perturbation in the positions of the
15
NMR signals was observed for the signal at δH=7.13 ppm and δN=109.9 ppm in the NꢀVal
PDꢀL1 spectrum (Figure 2). Several of the 1 and 2 compounds were checked. Two clearly
distinctive patterns of the compoundꢀinduced changes in the spectra have been observed. In
the first group of the compounds, the differences in chemical shifts between the two 15N
valine signals in the dimer complex, compared to the starting signal of the apoꢀPDꢀL1, are
ꢁδH= 0.8 and ꢁδN=2.4 ppm (Figure 2A). This group comprises exclusively of compounds 1.
The second group, which contains only 2, showed these differences to be smaller and equal
ꢁδH= 0.10/ꢁδN=0.4 and ꢁδH= 0.25/ꢁδN=0.8 ppm for 2a and 2b, respectively (Figure 2B).
A similar pattern of grouping was observed for one of the resonances in the spectra of
the 15NꢀTyr labeled hPDꢀL1 (the signal at δH=8.12 and δN=116.5) upon titration with tested
compounds, but the changes in chemical shift were smaller, ca. ꢁδH= 0.50 and ꢁδN=1.7 ppm
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(date not shown). Based on the NMR results for the N ValꢀPDꢀL1, we concluded that these
two groups of the compounds might differently interact with PDꢀL1. The representatives of
these two groups have been therefore subjected to crystallization, which indeed showed their
different extend of binding to hPDꢀL1.
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Structural basis of the interaction 1a and 2a with PD-L1. The crystal structures of 1a
and 2a in complex with hPDꢀL1 have been determined to the resolution of 2.35 Å and 1.7 Å,
respectively (Supporting Information Table S3). The structures follow the stoichiometry and
molecule arrangement presented lately by us (PDB: 5J89, 5J8O)20, nonetheless with novel
details leading to the discovery of the hPDꢀL1s interaction surface subꢀsites highly vulnerable
for ligandꢀinduced changes (Figure S5). The asymmetric unit of the hPDꢀL1/2a complex
contained one dimer exhibiting a pseudo 2ꢀfold rotational symmetry around an axis parallel
to the long axis of the protein molecule. The hPDꢀL1/1a complex crystallized in different
configuration with an asymmetric unit containing four protein molecules organized into two
dimers rotated a 180o around a horizontal axis, with the Cꢀtermini of each dimer molecule
close to another. In both structures each dimer is harboring one inhibitor in the center of
interface of each dimer. Each of the 1a and 2a molecules are arranged perpendicular to the
long axis of protein and its location is explicitly occluding the hPDꢀ1 binding site in both
hPDꢀL1 protein molecules thereby providing a rationale for disrupting the PDꢀ1/PDꢀL1
interaction.
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The 1a/PDꢀL1 complex represents the inhibitorꢀbinding mode reported by us in the
previous publication.20 The compound is located in a deep, hydrophobic cleft with roughly
cylindrical shape in the cross section. The binding cleft is closed from one side by sidechain
ATyr56 which creates the Tꢀstacking interaction with a distal phenyl moiety of the inhibitor
and an additional πꢀsigma and πꢀalkyl interactions with Ala121 and Met115, respectively
B
A
(the protein molecules are annotated by subscripts A, B, C and D according to the chain
arrangement in the crystal structure; the inhibitor disposition is described here based on the
AB dimer and is similar in the CD dimer unless noted otherwise, Figure 3C, Supporting
Information Figure S6). The methyl phenyl ring, rotated around 45o with respect to the distal
phenyl ring, located in the center of the cleft is stabilized by hydrophobic interactions with
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both A (methyl group) and Met115 (ring and methyl group) with additional contacts with
B
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AAla121 sidechain. The 1,3ꢀdimetoxyphenyl group, connected with the methylꢀphenyl ring
through the ether bond, which is close to the solvent accessible part of the cleft, interacts with
BTyr56 through the πꢀπ stacking thereby providing strong stabilization for this part of the
inhibitor with minor contribution from hydrophobic interactions with the sidechain of
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AAsp122. The Nꢀ(2ꢀaminoehtyl)acetamide tail creates a hydrogen bond with Gln66 and
B
AAsp122 (Figure 3AꢀC, Supporting Information Figures S7 and S8).
The 2a inhibitor is located perpendicularly to the C, F and G strands of PDꢀL1, creating
the interface similar to that of the PDꢀ1 interacting surface (canonical Igꢀstrand designations
are used; Figure S9). The linear arrangement of the compound accommodates it into a 16 Å
long, cylindrical, hydrophobic tunnel between two hPDꢀL1 monomers. The 2,3ꢀdihydroꢀ1,4ꢀ
benzodioxinyl group of 2a provokes movement of the Tyr56 C(β)ꢀC(γ) by 77° around the
A
C(α)ꢀC(β) axis making this part of the compound accessible for the solvent thereby changing
deep hydrophobic cleft into deep, hydrophobic tunnel (Figures 3ꢀ4, Supporting Information
Figures S7, S8 and S10). The most distal ring of 2a interacts with AAla121 through a 2.1 Åꢀ
long hydrogen bond and πꢀsigma interaction with Tyr56, while its second ring (hidden
A
deeper in the tunnel) provides additional hydrophobic contacts with Met115 and Ala121
A
B
(πꢀalkyl and πꢀsigma, respectively). The central methyl phenyl ring is rotated roughly around
45° to the plane of the 2,3ꢀdihydroꢀ1,4ꢀbenzodioxinyl moiety with the methyl group pointing
towards chain A with major interactions involving the hydrophobic contacts with Met115 of
the chain A and B and Ala121. Alkyl interactions with Tyr123 are also present providing
A
A
more rigid arrangement of the compound between PDꢀL1 chains. The substituted 2,5ꢀ
difluorophenyl ring of 2a is stabilized mostly through πꢀπ stacking interaction with Tyr56
B
ring with minor contribution from the halogen bonding between fluorine atom and AAsp122.
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Additionally, (S)ꢀ4ꢀaminoꢀ3ꢀhydroxybutyric acid moiety of 2a creates two hydrogen bonds
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with AThr20 and BGln66 (Figure 3D, Supporting Information Figure S8 and S11).
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DISSCUSSION
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Several crystal structures of the antibodies complexed with both PDꢀ1 and PDꢀL1 have
been determined, revealing the structural basis of the antibodyꢀbased immune checkpoint
blockade.26 Crystal structures of the antiꢀPDꢀ1 pembrolizumab Fab fragment complexed with
hPDꢀ1 (PDB 5GGS) and PDꢀ1 in complex with nivolumab Fab (PDB 5GGR) have showed
that the mechanisms of their interaction are highly similar because of partially shared
epitopes. Structures of the fully human IgG4 antibody (the Fab protein BMS-936559 from
BristolꢀMyers Squibb) (PDB 5GGT) and the avelumab single chain Fv fragment (scFv) (PDB
5GRJ) complexed with PDꢀL1 were also determined.27ꢀ31 Additionally, an ultraꢀhighꢀaffinity
mutants of PDꢀ1 were described.31, 32 The mutant by Maute et al.29, termed the highꢀaffinity
consensus (HAC) PDꢀ1, exhibits a 35,000ꢀfold enhanced affinity for PDꢀL1 in comparison to
the wildꢀtype protein. Structure of the complex of an (HAC) PDꢀ1 mutant bound to hPDꢀL1
was also determined, providing the structural and biophysical basis for enhanced binding of
these proteins.33
Compared to the antibodies, conspicuously missing from this class of therapies are
traditional smallꢀmolecule drugs. Although smallꢀmolecule pharmacological modulators of
the PDꢀ1/PDꢀL1 axis are highly awaited, to date only a few series of small molecules,
macrocyclic peptides, peptides or peptidomimetics targeting this interaction were reported.15ꢀ
17
Among them the group of entirely smallꢀmolecule compounds based on the hydrophobic
biphenyl core scaffold has been described recently by BristolꢀMyers Squibb.18,19 We provided
structural basis of interactions between two examples of compounds 1b-c with PDꢀL1,
evidencing that PDꢀ1/PDꢀL1 axis may be targeted not only by mAbs, but also by small
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molecules.20 In the present study we describe two new crystal structures of compounds 1a
and 2a, both bound to the dimeric PDꢀL1. Surprisingly, the binding mode of inhibitor 2a,
which contains the 2,3ꢀdihydroꢀ1,4ꢀbenzodioxinyl group, significantly differs from that of 1a
and those reported by us previously (1bꢀc) (PDB: 5J89, 5J8O) (Supporting Information
Figure S5). Our structural data shows that inhibitor 2a upon binding to PDꢀL1 induces
conformational changes in PDꢀL1, showing the ligand binding site of PDꢀL1 to be more
flexible than it was previously seen. This quite unexpected formation of the hydrophobic
tunnel, induced by the 2,3ꢀdihydroꢀ1,4ꢀbenzdioxinyl group of 2a, redefines the described
pharmacophore model and creates new possibilities for further design of PDꢀL1 inhibitors
(Figures 3 and 4).20 The formation of the tunnel is associated with the movement of the
aromatic ring of ATyr56.
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We also partition 1,2ꢀlike large ligands (MW ca. 600) into smaller fragments and screen
the resulting fragments for binding to PDꢀL1. All these structures have shown binding to PDꢀ
l1, albeit weaker. They induce dimerization of PDꢀL1, indicating that this structural part of
the compounds is most likely involved in dimerization (Supporting Information Table S2).
These initial small derivative fragments, with indication of binding properties at the site of
interest determined by the NMR 1Hꢀ15N HMQC titration, could be used to construct libraries
1
of larger structures, which can be submitted to the same HꢀI5N HMQC titration experiments
in the future.
EXPERIMENTAL SECTION
Gel filtration experiment. A Superdex S75 10/30 HR column was equilibrated with
buffer containing 10 mM Tris and 20 mM NaCl. Protein was concentrated to 3 mg/ml and
200 µl of sample was loaded onto the column (apoꢀhPDꢀL1 separation). In case of the hPDꢀ
L1/inhibitor complexes, the protein at the 3 mg/ml concentration was mixed with the
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inhibitor in 3 : 1 (inhibitor : protein) molar ratio, centrifuged for 10 min at 15 000 x g and 200
µl of resulting supernatant was loaded onto the column.
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Protein Expression and Purification. The proteins was expressed and purified as
described earlier.20 Briefly, hPDꢀL1 (residues 18ꢀ134) was expressed in E. coli BL21 (DE3)
as inclusion bodies. After bacteria collection, the pellet was washed and the obtained
inclusion bodies were dissolved in the GuHCl buffer. Refolding was performed by a dropwise
dilution in buffer containing 1 M Lꢀarginine, 100 mM Tris pH 8.0, 2 mM EDTA, 0.25mM
reduced glutathione and 0.25 mM oxidized glutathione. After refolding, protein was dialyzed
against 10 mM Tris pH 8.0 containing 20 mM NaCl three times, concentrated and purified
using a Superdex S75 gel filtration column equilibrated with 10 mM Tris pH 8.0 containing
20 mM NaCl.
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Crystallization of the hPD-L1 in complex with small molecule inhibitor. The purified
protein was mixed with 1a or 2a in 3 : 1 (inhibitor : protein) molar ratio, concentrated to 5
mg/ml and the crystallization screening was carried out. Diffractionꢀquality crystals were
obtained at room temperature from the conditions containing 0.1 M Tris pH 8.5, 0.2 M
magnesium chloride, 30% (w/v) PEG 4000 and 0.01 M Tris pH 8.4 , 0.28 M sodium chloride,
27% (w/v) PEG 4000 for 1a and 2a, respectively.
Crystal Structure Determination. The crystals were flashꢀcooled in liquid nitrogen
without cryoprotection. The diffraction data were collected at the Helmholtz Centrum 14.1
beamline at BESSY (Berlin, Germany)34. The data were indexed and integrated using XDS33,
36, scaled and merged using Aimless.37 The initial phases were obtained by molecular
replacement using Phaser and PDB 5C3T as a search model.38 The models were manually
built in the resulting electron density maps using Coot.39 Restrained refinement was
performed using Phenix.40
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NMR Experiments. Uniform 15N labeling was obtained by expressing the proteins in the
M9 minimal medium containing 15NH4Cl as the sole nitrogen source. For NMR
measurements the buffer was exchanged by gel filtration to PBS pH 7.4 (hPDꢀL1) or 25 mM
sodium phosphate containing 100 mM NaCl pH 6.4 (hPDꢀ1). 10% (v/v) of D2O was added to
the samples to provide lock signal. All spectra were recorded at 300K using a Bruker Avance
600 MHz spectrometer. Binding of the compounds was analyzed by titrating the 15Nꢀlabeled
PDꢀL1 (0.11 mM) and recording the 1H and 1Hꢀ15N HMQC spectra prior and after addition of
the compound. The approximate molecular weights of protein populations present in the
sample were determined by analyzing the linewidth (relaxation time) of well separated NMR
signals.
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General chemistry. The compounds were synthesized as shown in Tables 1ꢀ2 according
to the procedures similar to those described in the disclosures.18, 19 All reagents were obtained
from Sigma Aldrich and used without additional purification unless noted otherwise. NMR
spectra were recorded on Bruker Avance 600 MHz spectrometer. All chemical shifts (δ) are
reported in ppm and coupling constants (J) in Hz. All chemical shifts are related to the
solvent peaks (DMSOꢀd6, CDCl3). Infrared spectra were recorded for the solid
samples on a Nicolet IR200 spectrometer using ATR technique. Highꢀresolution mass
spectrometry (HRMS) spectra were recorded on a microTOFꢀQII apparatus using the ESI
ionisation mode. Flash chromatography was performed on the Grace Reveleris X2 Flash
Chromatography System with the Grace Resolv Silica Cartridges. Elemental analyses were
performed on a Vario Micro Cube apparatus. The UPLCꢀMS’s were carried out on the TQD
Waters HꢀClass spectrometer (column: ACQUITY UPLC BEH C18 1.7um, 2.1 x 50 mm,
method: 6 min, gradient: 0ꢀ3 min 80% H2O ꢀ 20% MeCN, 3ꢀ3.5 min 100% MeCN, 3.5ꢀ6 min
80% H2Oꢀ 20% MeCN). Purities of all final compounds ꢀ determined using chromatographic
UPLC or elemental analysis ꢀ were 95% or higher. Thinꢀlayer chromatography (TLC) was
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performed on TLC silica plates (60 F254) and visualized with ultraviolet light at 254ꢁnm.
Melting points were determined with an AsconꢀM5 apparatus. Details are given in Supporting
Information.
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N-{2-[({2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)methoxy]phenyl}methyl)amino]ethyl}ac-
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etamide hydrochloride (1a).
A solution of 4a (90 mg, 0.25 mmol), Nꢀ(2ꢀ
aminoethyl)acetamide (104 mg, 1.02 mmol), sodium cyanoborohydride (83.5 mg, 1.3 mmol)
and AcOH (2 drops) in DMF (5 mL) was stirred at 80 °C for 3 h. The mixture was
concentrated under reduced pressure. Purification by flash chromatography (0ꢀ60% MeOH in
1
CHCl3) provided the product as colorless solid. (41 mg, yield: 36%). H NMR (600 MHz,
DMSOꢀd6) δ: 7.75 (t, J = 5.4 Hz, 1H), 7.47 (dd, J = 7.6, 1.05 Hz, 1H), 7.48–7.45 (m, 2H),
7.39–7.37 (m, 1H), 7.33–7.30 (m, 2H), 7.29 (t, J = 7.6 Hz, 1H), 7.21 (dd, J = 7.6, 1.2 Hz,
1H), 6.36 (s, 2H), 5.15 (s, 2H), 3.76 (s, 6H), 3.59 (s, 2H), 3.08 (q, J = 6.3 Hz, 2H), 2.43 (t, J
= 6.4 Hz, 2H), 2.22 (s, 3H), 1.77 (s, 3H) ppm. 13C NMR (151 MHz, DMSOꢀd6) δ: 169.0,
159.2, 158.9, 142.2, 141.4, 135.6, 134.0, 129.7, 129.2, 128.2, 127.0, 125.5, 108.7, 91.5, 68.6,
55.7, 47.9, 40.5, 40.1, 38.8, 22.6, 15.9 ppm. IR (ATR) 3317, 2934, 2836, 1647, 16155, 1597,
1498, 1200, 1147, 1033 cmꢀ1. HRMS ESIꢀMSꢀqꢀTOF for C27H32N2O4 [M+H]+ found:
449.2440 m/z Calc. Mass: 449.2440. Mp: 112–113 °C. UPLCꢀMS (DAD/ESI): tR=6.11 min,
for C27H32N2O4 [M+H]+ found: 449.20 m/z; Calc Mass: 449.24.
1-({3-bromo-4-[(2-methyl-3-phenylphenyl)methoxy]phenyl}methyl)piperidine-2-carboxy-
lic acid (1b). Compound 1b was prepared following the protocol for 1a, using of 4b (150 mg,
0.39 mmol), piperidineꢀ2ꢀcarboxylic acid (148 mg, 1.17 mmol), NaBH3CN (74 mg, 1.17
mmol) and AcOH (2 drops) dissolved in DMF (4 mL). Purification by flash chromatography
1
(0ꢀ60% MeOH in EtOAc) provided the product as colorless solid (50 mg, yield: 26%). H
NMR (300 MHz, DMSOꢀd6) δ: 17.45 (br.s, 1H), 7.73 (d, J = 2.0 Hz, 1H), 7.55 (dd, J = 7.5,
1.2 Hz, 1H), 7.49–7.35 (m, 8H), 7.22 (dd, J = 7.5, 1.2 Hz, 1H), 5.29 (s, 2H), 4.62 (s, 2H),
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3.78 (t, J = 7.0 Hz, 1H), 3.26–3.17 (m, 2H), 2.23 (s, 3H), 2.03–1.93 (m, 2H), 1.86–1.56 (m,
3H), 1.50–1.34 (m, 1H) ppm. 13C NMR (75 MHz, DMSOꢀd6) δ: 170.2, 155.7, 137.1, 134.9,
133.8, 133.7, 129.7, 129.2, 128.3, 127.6, 127.0, 125.6, 122.0, 113.7, 110.8, 69.9, 69.4, 68.6,
59.9, 25.0, 20.1, 19.6, 15.8 ppm. IR (ATR) 3329, 2946, 2520, 1728, 1605, 1500, 1452, 1290,
1265, 1056 cmꢀ1. HRMS ESIꢀMSꢀqꢀTOF for C27H28BrNO3 [M+Na]+ found: 516.1137 m/z;
Calc. Mass: 516.1150. Mp: 119–121 °C. UPLCꢀMS (DAD/ESI): tR=6.49 min, for
C27H28BrNO3 [M+H]+ found: 494.06 m/z; Calc Mass: 494.13.
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Nꢀ{2ꢀ[({2ꢀmethoxyꢀ6ꢀ[(2-methyl-3-phenylphenyl)methoxy]pyridin-3-yl}methyl)amino]et-
hyl}acetamide hydrochloride (1c). Combined sodium cyanoborohydride (200 mg, 3.18
mmol), Nꢀ(2ꢀaminoethyl)acetamide (250 mg, 2.45 mmol), and of 4c (200 mg, 0.60 mmol) in
DMF (20 mL) and AcOH (5 drops) were stirred at room temperature for 16 h. The mixture
was concentrated under reduced pressure. The product was purified by flash chromatography
on silica gel (0ꢀ60% MeOH in EtOAc) to give the product as a yellow oil. The product was
converted into the corresponding hydrochloride salt and recrystallized from acetone (76 mg,
yield: 27%). 1H NMR (600MHz, DMSOꢀd6) δ: 8.97 (br. s., 2H), 8.20 (t, J = 5.6 Hz, 1H), 7.82
(d, J = 8.0 Hz, 1H), 7.47–7.44 (m, 3H), 7.39–7.36 (m, 1H), 7.31–7.30 (m, 2H), 7.21 (t, J =
7.6, 1H), 7.19 (dd, J = 7.7, 1.2 Hz, 1H), 6.53 (d, J = 8.0 Hz, 1H), 5.45 (s, 2H), 4.04 (s, 2H),
3.95 (s, 3H), 3.36 (q, J = 6.3 Hz, 2H), 2.95 (t, J = 6.3 Hz, 2H), 2.22 (s, 3H), 1.83 (s, 3H) ppm.
13C NMR (151MHz, DMSOꢀd6) δ: 170.2, 162.5, 160.5, 144.0, 142.2, 141.4, 135.6, 133.9,
129.7, 129.2, 128.3, 128.3, 127.0, 125.5, 105.3, 101.5, 66.5, 53.7, 46.1, 44.1, 35.2, 22.6, 15.9
ppm. IR (ATR) 3253, 3062, 2934, 2702, 1651, 1605, 1587, 1463, 1309, 1003 cmꢀ1. HRMS
ESIꢀMSꢀqꢀTOF for C25H29N3O3 [M+H]+ found: 420.2299 m/z; Calc. Mass: 420.2287. Mp:
139–140 °C. UPLCꢀMS (DAD/ESI): tR= 5.79 min, for C25H29N3O3 [M+H]+ found: 420.22
m/z; Calc Mass: 420.23.
(2R)-2-[({2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)methoxy]phenyl}methyl)amino]-
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3-methylbutan-1-ol hydrochloride (1d). Compound 1d was prepared according to the protocol
of 1a, using of 4a (181 mg, 0.5 mmol), Lꢀvalinol (210 mg, 2.03 mmol), NaBH3CN (167 mg,
2.6 mmol) and AcOH (2 drops) in DMF (4 mL). The residue was purified by silica gel flash
chromatography (0ꢀ60% MeOH in EtOAc). After conversion into the corresponding
hydrochloride salt product was recrystallized from isopropanol and diisopropyl ether (55 mg,
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1
yield: 48%). H NMR (600 MHz, DMSOꢀd6) δ: 8.08 (br. d, J = 54.5 Hz, 2H), 7.5 (dd, J =
6.6, 1.0 Hz, 1H), 7.46–7.43 (m, 2H), 7.38–7.35 (m, 1H), 7.31–7.27(m, 3H), 7.21 (dd, J = 6.6,
1.2 Hz, 1H), 6.46 (s, 2H), 5.34 (t, J = 4.9 Hz, 1H), 5.21 (s, 2H), 4.12 (t, J = 5.6 Hz, 2H), 3.83
(s, 6H), 3.72–3.68 (m, 1H), 2.80–2.75 (m, 1H), 2.22 (s, 3H), 2.09–2.06 (m, 1H), 1.23 (s, 1H),
0.96 (d, J = 6.9 Hz, 3H), 0,92 (d, J = 6.9 Hz, 3H) ppm. 13C NMR (151 MHz, DMSOꢀd6) δ:
161.9, 159.9, 142.7, 141.8, 135.8, 134.6, 130.3, 129.6, 128.8, 128.7, 127.5, 126.0, 100.2,
92.0, 69.3, 67.8, 64.5, 57.7, 56.5, 38.7, 26.8, 23.3, 19.6, 18.1, 16.4 ppm. IR (ATR) 3289 (br),
2965, 1611, 1595, 1463, 1150 cmꢀ1. HRMS ESIꢀMSꢀqꢀTOF for C28H35NO4 [M+H]+ found:
450.2640 m/z Calc. Mass: 450.2644. Mp: 74–76 °C. UPLCꢀMS (DAD/ESI): tR=6.68 min, for
C28H35NO4 [M+H]+ found: 450.20 m/z; Calc Mass: 450.26.
(3S)-4-{[(4-{[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methoxy}-2,5-difluo-
rophenyl)methyl]amino}-3-hydroxybutanoic acid hydrochloride (2a). Compound 2a was
prepared following the protocol for 1a, using of 6a (396 mg, 1.0 mmol), (S)ꢀ4ꢀaminoꢀ3ꢀ
hydroxybutanoic acid (224 mg, 2.0 mmol), NaBH3CN (333 mg, 5.3 mmol), AcOH (3 drops)
in DMF (10 mL). After purification by gel flash chromatography (0ꢀ60% MeOH in CHCl3)
the product was converted into the corresponding hydrochloride salt and recrystallized from
diethyl ether and ethanol (192 mg, yield: 36%). 1H NMR (600 MHz, CDCl3 δ: 7.39–7.37 (m,
1H), 7.24–7.22 (m, 2H), 7.06 (dd, 3,4JHꢀF= 10.9, 6.9 Hz, 1H), 6.90 (d, 8.2 Hz, 1H), 6.82 (d, J
3,4
= 2.1 Hz, 1H), 6,79 (dd,
J
=10.9, 6.8 Hz, 1H), 6.77 (dd, J = 8.2, 2.1 Hz, 1H), 5.11 (s,
HꢀF
2.0, 2H), 4.54–4.50 (m, 1H), 4.48 (d, J = 14.8 Hz, 1H), 4.43 (d, J = 14.8 Hz, 1H), 4.30 (s,
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4H), 3.57 (dd, J = 10.8, 5.5 Hz, 1H), 3.26 (dd, J = 10.8, 1.2 Hz, 1H), 2.73 (dd, J = 17.4, 6.7
Hz, 1H), 2.43 (dd, J = 17.4, 2.2 Hz, 1H), 2.26 (s, 3H), 1.27 (d, J = 5.5 Hz, 1H) ppm. 13C
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NMR (151 MHz, CDCl3) δ: 172.8, 156.7 (d, JCꢀF = 242.9 Hz), 149.3 (d, JCꢀF = 242.4 Hz),
147.3, 147.2, 147.1, 143.2, 142.8, 142.6, 135.3, 134.6, 134.2, 130.6, 127.8, 125.7, 122.7,
118.4, 117.3, 117.1 (dd, 2,3JCꢀF = 20.8, 5.5 Hz), 117.0, 115.2 (dd, 2,3JCꢀF = 17.5, 5.5 Hz), 103.5,
103.4, 71.0, 70.9, 70.9, 64.7, 64.6, 55.9, 41.2, 39.2, 16.3 ppm. HRMS ESIꢀMSꢀqꢀTOF for
C27H27F2NO6 [MꢀH2O+Na]+found: 504.1625 m/z; Calc. Mass: 504.1598. Mp: 82–83°C.
UPLCꢀMS (DAD/ESI): tR=6.87 min, for C27H27F2NO6 [MꢀOH]+ found: 482.18 m/z; Calc
Mass: 482.16.
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(2R)-2-[({2-[(3-cyanophenyl)methoxy]-4-{[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-met-
hylphenyl]methoxy}-5-methylphenyl}methyl)amino]-3-hydroxypropanoic acid hydrochloride
(2b). Compound 2b was prepared according to the protocol of 1a, using of 7a (140 mg, 0.3
mmol), Dꢀserine (116 mg, 1.1 mmol), NaBH3CN (91 mg, 1.4 mmol) and AcOH (2 drops) in
DMF (4 mL). The residue was purified by silica gel flash chromatography (0ꢀ60% MeOH in
EtOAc). The product was converted into the corresponding hydrochloride salt and
1
recrystallized from methanol (62 mg, yield: 40%). H NMR (600 MHz, DMSOꢀd6) 8.03 (s,
1H), 7.91 (d, J = 7.8 Hz, 1H), 7.81 (dt, J = 7.8, 1.2 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7,41
(dd, J = 7.8, 0.8 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H ), 7.19 (s, 1H), 7.16 (dd, J = 7.7, 1.0 Hz,
1H), 6.93 (s, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.79 (d, J = 2.1 Hz, 1H), 6.75 (dd, J = 8.2, 2.1
Hz, 1H), 5.30 (d, J = 12.6 Hz, 1H), 5.26 (d, J = 12.6 Hz, 1H), 5.14 (s, 2H), 4.28 (s, 4H), 4.09
(d, J = 13.0 Hz, 1H), 4.02 (d, J = 13.0 Hz, 1H), 3.77 (dd, J = 11.4, 6.6 Hz, 1H), 3.66 (dd, J
=11.4, 6.6 Hz, 1H), 3.24–3.21 (m, 2H), 2.22 (s, 3H), 2.10 (s, 3H) ppm. 13C NMR (151 MHz,
DMSOꢀd6) δ: 168.5, 157.5, 155.5, 143.0, 142.5, 138.6, 133.9, 132.5, 131.7, 131.1, 129.7,
129.6, 127.4, 125.5, 122.1, 118.7, 118.0, 117.7, 116.8, 111.5, 98.4, 68.9, 68.7, 64.3, 61.8,
60.1, 45.0, 15.9, 15.3 ppm. IR (ATR) 3637, 3080 (br), 2928, 2880, 2230, 1616, 1506, 1448,
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1313, 1279, 1125, 1052, 792 cmꢀ1. HRMS ESIꢀMSꢀqꢀTOF for C35H34N2O7 [M+Na]+ found:
617.2278 m/z; Calc. Mass: 617.2264. Mp: 153.5–154.5 °C. UPLCꢀMS (DAD/ESI): tR=6.47
min, for C35H34N2O7 [M+H]+ found: 595.25 m/z; Calc Mass: 595.24.
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(2R, 4R)-1-({5-chloro-2-[(3-cyanophenyl)methoxy]-4-{[3-(2,3-dihydro-1,4-benzodioxin-
6-yl)-2-methylphenyl]methoxy}phenyl}methyl)-4-hydroxypyrrolidine-2-carboxylic acid hydr-
ochloride (2c). Compound 2c was prepared following the protocol for 1a, using of 7b (230
mg, 0.4 mmol), (2R, 4R)ꢀ4ꢀhydroxypyrrolidineꢀ2ꢀcarboxylic acid (262 mg, 2.0 mmol),
sodium cyanoborohydride (145 mg, 2.3 mmol), AcOH (3 drops) in DMF (4 mL). After
purification by flash chromatography (0ꢀ60% MeOH in EtOAc) the product was converted
into the hydrochloride salt and recrystallized from isopropanol (62 mg, yield: 40%). 1H NMR
(600 MHz, DMSOꢀd6) δ: 7.98 (s, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.83 (dt, J = 7.8, 1.3 Hz,
1H), 7.62 (t, J = 7.8 Hz, 1H), 7.48–7.42 (m, 2H), 7.24 (t, J = 7.6 Hz, 1H), 7.18 (dd, J = 7.6
Hz, 1H), 7.1 (s, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.78 (d, J = 2.11 Hz, 1H), 6.75 (dd, J = 8.2,
2.1 Hz, 1H), 5.31 (d, J = 12.5 Hz, 1H), 5.29 (d, J = 12.5 Hz, 1H), 5.23 (s, 2H), 4.28 (s, 4H),
4.22–4.18 (m, 1H), 3.95 (d, J = 13.2 Hz, 1H), 3.80 (d, J = 13.2 Hz, 1H), 3.41–3.39 (m, 1H),
2.95 (d, J = 10.6 Hz, 1H), 2.71 (dd, J = 10.6, 4.9 Hz, 1H), 2.34 (ddd, J = 13.2, 9.7, 5.8 Hz,
1H), 2.24 (s, 3H), 1.84 (br.d, J = 13.2 Hz, 1H) ppm. 13C NMR (151 MHz, DMSOꢀd6) δ:
172.6, 155.9, 153.4, 143.0, 142.5, 138.4, 135.0, 134.4, 134.0, 132.5, 131.8, 131.5, 131.1,
129.9, 125.6, 122.06, 118.7, 117.7, 116.8, 113.0, 111.5, 100.5, 69.7, 69.0, 68.6, 64.1, 62.0,
61.0, 51.5, 38.2, 15.8 ppm. IR (ATR) 3270, 2966, 2230, 1633, 1607, 1507, 1312, 1166, 1067
cmꢀ1. HRMS ESIꢀMSꢀqꢀTOF for C36H33ClN2O7 [M+Na]+ found: 663.1855 m/z; Calc. Mass:
641.21. Mp: 81–83 °C. UPLCꢀMS (DAD/ESI): tR=6.51 min, for C36H33ClN2O7 [M+H]+
found: 641.16 m/z; Calc Mass: 641.21.
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(2ꢀmethylꢀ3ꢀbiphenylyl)methanol (3). A mixture of (3ꢀbromoꢀ2ꢀmethylphenyl)methanol
(4.6 g, 22.8 mmol), phenylboronic acid (5.7 g, 46.3 mmol) and Pd(dppf)Cl2·DCM (0.2 g, 0.1
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mmol) in toluene (34.5 mL) and ethanol (11.3 mL) was placed under argon. To this solution
sodium bicarbonate, 2M (34.5 mL, 69.0 mmol) was added and the mixture was heated at
80 °C for 30 min. Ethyl acetate (44 mL) and (11 mL) water were added to the reaction
mixture. The organic extract was concentrated by rotatory evaporation. The crude product
was chromatographed on silica gel eluting with 0ꢀ40% EtOAc in hexane to afford 4.6 g
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(Yield: 98%) of an offꢀwhite solid. H NMR (600 MHz, CDCl3) δ: 7.43–7.49 (m, 3H), 7.37–
7.34 (m, 1H), 7.31–7.29 (m, 2H), 7.47–7.44 (m, 3H), 7.26 (t, J = 7.5 Hz, 1H), 7.20 (dd, J =
7.6, 1.3 Hz, 1H), 4.78 (s, 2H), 2.25 (s, 3H) ppm. 13C NMR (151 MHz, CDCl3) δ: 143.0,
142.2, 140.0, 133.8, 129.7, 129.5, 128.2, 127.0, 126.9, 125.7, 64.2, 16.0 ppm. IR (ATR):
3365, 3054, 1601, 1469, 1047, 757 cmꢀ1. Mp: 58–59 °C. UPLCꢀMS (DAD/ESI): tR= 6.35
min, for C14H14O [MꢀOH]+ found: 181.03 m/z; Calc Mass: 181.11
2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)methoxy]benzaldehyde (4a). To the iceꢀ
cooled solution of 2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)methoxy]benzaldehyde (0.92
g, 5.04 mmol), triphenylphosphine (1.45 g, 5.55 mmol) and 3 (1.0 g, 5.04 mmol) in dry THF
(21 mL), DIAD (1.08 mL, 5.55 mmol) in THF (21 mL) was added dropwise. The resulting
yellow solution was allowed to warm to room temperature and stirred for additional 20 h. The
mixture was concentrated under reduced pressure. Purification by flash chromatography (0ꢀ
60% EtOAc in hexane) yielded 4a (1.02 g, 56%). colorless solid.1H NMR (600 MHz,
DMSOꢀd6) δ: 10.37 (s, 1H), 7.44–7.40 (m, 3H), 7.38–7.35 (m, 1H), 7.32–7.28 (m, 4H), 6.20
(s, 2H), 5.16 (s, 2H), 3.89 (s, 6H), 2.27 (s, 3H) ppm. 13C NMR (151 MHz, DMSOꢀd6) δ:
187.9, 165.6, 164.3, 143.4, 141.9, 134.7, 134.3, 130.8, 129.5, 128.5, 128.3, 127.1, 125.9,
109.1, 91.1, 69.7, 56.2, 16.4 ppm. IR (ATR) 3013, 2936, 1668, 1607, 1607, 1582, 1465, 1166
cmꢀ1. HRMS ESIꢀMSꢀqꢀTOF for C23H22O4 [M+Na]+ 385.1420 m/z; Calc. Mass: 385.1416.
Mp: 161.5–162 °C. UPLCꢀMS (DAD/ESI): tR=8.08 min, for C23H22O4 [M+H]+ found: 363.13
m/z; Calc Mass: 363.16.
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3-bromo-4-[(2-methyl-3-phenylphenyl)methoxy]benzaldehyde (4b).Compound 4b was
prepared as described for 4a, using of 3ꢀbromoꢀ4ꢀhydroxybenzaldehyde (0.71 g, 3.51 mmol),
PPh3 (1.02 g, 3.89 mmol), 3 (0.70 g, 0.52 mmol) dissolved in THF (21 mL) and solution of
DIAD (0.74 mL, 3.89 mmol) in THF (21 mL). The residue was purified by flash
chromatography on silica gel using 0ꢀ60% EtOAc in hexane. Recrystallization from MeOH
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yielded colourless solid (0.47 g, yield: 35%). H NMR (600MHz, DMSOꢀd6) δ: 9.87 (s, 1H),
8.14 (d, J = 2.0 Hz, 1H), 7.97 (dd, J = 6.5, 2.0 Hz, 1H), 7.55–7.52 (m, 2H), 7.48–7.45 (m,
2H), 7.40–7.37 (m, 1H), 7.33–7.30 (m, 3H), 7.22 (dd, J = 6.5 Hz, 1H), 5.39 (s, 2H), 2.23 (s,
3H) ppm. 13C NMR (151MHz, DMSOꢀd6) δ: 190.6, 159.2, 142.2, 141.2, 134.6, 134.0, 133.9,
131.2, 130.8, 129.8, 129.2, 128.3, 127.5, 127.0, 125.6, 114.1, 111.9, 69.8, 15.9 ppm. IR
(ATR) 3063, 2852, 1689, 1594, 1278, 1254, 1189, 1048 cmꢀ1. HRMS ESIꢀMSꢀqꢀTOF for
C21H17BrO2 [M+Na]+ found: 403.0302 m/z; Calc. Mass: 403.0310. Mp: 101–102 °C. UPLCꢀ
MS (DAD/ESI): tR=9.17 min, for C21H17BrO2 [M+H]+ found: 381.00 m/z; Calc Mass: 381.05.
2ꢀmethoxyꢀ6ꢀ[(2ꢀmethylꢀ3ꢀphenylphenyl)methoxy]pyridineꢀ3ꢀcarbaldehyde (4c). Palladꢀ
ium (II) acetate (0.08 g, 0.36 mmol), Cs2CO3 (2.23 g, 6.83 mmol), tꢀBuXPhos (0.29 g, 0.68
mmol), 2ꢀchloroꢀ6ꢀmethoxyꢀ3ꢀpyridinecarboxaldehyde (0.59 g, 3.41 mmol), 3 (0.88 g, 4.44
mmol) and toluene (30 mL) were combined and purged by a stream of argon for 3 minutes.
The reaction was sealed and heated at 80 °C for 4h. The mixture was filtered through a pad of
Celite. The filtrate was concentrated under reduced pressure. The product was purified by
flash chromatography on silica gel using 0ꢀ60% EtOAc in hexane. The product was
recrystallized from diethyl ether (488 mg, 35%). 1H NMR (600MHz, CDCl3) δ: 10.22 (d, J =
0.8 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.44–7.41 (m, 3H), 7.37–7.34 (m, 1H), 7.32–7.30 (m,
2H), 7.28 (d, J = 7.5 Hz, 1H), 7.26–7,25 (m, 1H), 6.45 (dd, J = 8.4, 0.8 Hz, 1H), 5.52 (s, 2H),
4.08 (s, 3H), 2.28 (s, 3H) ppm. 13C NMR (151 MHz, CDCl3 δ: 187.8, 166.6, 166.2, 143.2,
142.0, 140.5, 135.0, 134.8, 130.5, 129.5, 128.7, 128.3, 127.1, 125.7, 112.5, 103.9, 67.8, 54.1,
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16.5 ppm. IR (ATR): 3063, 2961, 2870, 1671, 1591, 1460, 1330, 1277 cmꢀ1. HRMS ESIꢀMSꢀ
qꢀTOF for C21H19NO3 [M+Na]+ found: 356.1256 m/z; Calc Mass: 356.1263. Mp: 132–133
°C. UPLCꢀMS (DAD/ESI): tR= 9.18 min, for C21H19NO3 [M+H]+ found: 334.15 m/z; Calc
Mass: 334.14.
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[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol (5). Compound 5 was
prepared as described for 3, using a (3ꢀbromoꢀ2ꢀmethylphenyl)methanol (2.0 g, 10.3 mmol),
(2,3ꢀdihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)boronic acid (2.2 g, 12.2 mmol) , Pd(dppf)Cl2·DCM
(0.11 g, 0.13 mmol) dissolved in toluene (15.5 mL) and ethanol (5.2 mL). Purification by
flash chromatography (0ꢀ40% EtOAc in hexane) yielded 5 (2.0 g, 76%) as an offꢀwhite solid.
1H NMR (600 MHz, CDCl3) δ: 7.35 (d, J = 7.4 Hz, 1H), 7.21 (t, J = 7.5, 1H), 7.17 (dd, J =
7.5,1.2 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 2.1 Hz, 1H), 6.75 (dd, J = 8.3, 2.1 Hz,
1H), 4.73 (d, J = 4.8 Hz, 2H), 4.28 (s, 4H), 2.24 (s, 3H) ppm. 13C NMR (151 MHz, CDCl3):
143.1, 142.7, 142.4, 139.3, 135.6, 133.8, 129.7, 126.7, 125.7, 122.7, 118.3, 116.9, 64.5, 64.2,
16.0 ppm. HRMS ESIꢀMSꢀqꢀTOF for C15H14O3 [M+Na]+ 279.0993 m/z; Calc. Mass:
279.0997. IR (ATR) 3324, 3062, 2980, 2934, 2881, 1507, 1248, 1231 cmꢀ1. Mp: 80.5–81.5
°C. UPLCꢀMS (DAD/ESI): tR=5.92 min, for C15H14O3 [MꢀOH]+ found: 239.12 m/z; Calc
Mass: 239.11.
4-{[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methoxy}-2,5-difluorobenzald-
ehyde (6a). Compound 6a was prepared as described for 4a, using of 2,5ꢀdifluoroꢀ4ꢀ
hydroxybenzaldehyde (0.43 g, 2.7 mmol), PPh3 (0.79 g, 3.0 mmol), 5 (0.70 g, 2.7 mmol)
dissolved in THF (21 mL) and solution of DIAD (0.59 mL, 3.0 mmol) in THF (21 mL).
Purification by flash chromatography (0ꢀ60% EtOAc in hexane) yielded 6a (0.39 g, 36%)
1
3,4
colorless solid. H NMR (600 MHz, CDCl3) δ: 10.20 (d, J = 2.9 Hz, 1H), 7.59 (dd,
J
=
HꢀF
10.5, 6.5 Hz, 1H), 7.40–7.37 (m, 1H), 7.25–7.24 (m, 2H), 6.92 (d, J = 8.2 Hz, 1H), 6.85 (dd,
3,4J = 11.4, 6.4 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.77 (dd, J = 8.3, 2.0 Hz, 1H), 5.21 (s, 2H),
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4.31 (s, 4H), 2.27 (s, 3H) ppm. 13C NMR (151 MHz, CDCl3) δ: 185.2 (dd,
J
= 9.0, 3.0
CꢀF
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Hz), 161.9 (d, JCꢀF = 255.6 Hz), 153.6, 153.7, 149.4 (d, JCꢀF = 246.9 Hz), 143.2, 142.9,
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142.8, 135.1, 134.6, 133,2, 131.0, 127.9, 125.9, 122.7, 118.4, 118.3, 117.1, 114.2 (dt, 2,3JCꢀF
=
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J
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= 26.0, 4.5 Hz), 71.0, 71.1, 64.6, 16.4 ppm. IR (ATR) 3050,
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2924, 1672, 1627, 1508, 1283, 1108, 1063, 985 cmꢀ1. HRMS ESIꢀMSꢀqꢀTOF for C23H18F2O4
[M+Na]+ 419.1075 m/z; Calc. Mass: 419.1071. Mp: 160–161 °C. UPLCꢀMS (DAD/ESI):
tR=8.45 min, for C23H18F2O4 [M+H]+ found: 397.09 m/z; Calc Mass: 397.12.
4-{[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methoxy}-2-hydroxy-5-methyl-
benzaldehyde (6b). Compound 6b was prepared as described for 4a, using of 2,4ꢀdihydroxyꢀ
5ꢀmethylbenzaldehyde (0.59 g, 3.9 mmol), PPh3 (1.01 g, 3.5 mmol) and 5 (0.90 g, 2.7 mmol)
dissolved in THF (21 mL) and solution of DIAD (0.74mL, 3.9 mmol) in THF (21 mL).
Purification by flash chromatography (0ꢀ60% EtOAc in hexane) yielded 6b (0.95 g, 69%)
colorless solid. 1H NMR (600 MHz, CDCl3) δ: 11.45 (s, 1H), 9.70 (s, 1H), 7.42 (dd, J = 6.7,
2.1 Hz, 1H), 7.28–7.23 (m, 4H), 6.92 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 2.1 Hz. 1H), 6.79 (dd,
J = 8.2, 2.1 Hz, 1H), 5.13 (s, 2H), 4.31 (s, 4H), 2.25 (s, 3H), 2.21 (s, 3H) ppm. 13C NMR
(151 MHz, CDCl3) δ: 194.5, 164.3, 163.3, 143.2, 142.8, 142.6, 135.3, 134.7, 134.5, 134.3,
130.4, 127.3, 125.7, 122.7, 119.7, 118.4, 117.0, 114.6, 99.6, 69.6, 64.6, 16.3, 15.7 ppm. IR
(ATR) 2994, 2945, 1635, 1507, 1494, 1304, 1317, 1229, 1131, 1064, 786 cmꢀ1. HRMS ESIꢀ
MSꢀqꢀTOF for C24H22O5 [M+Na]+ 413.1343 m/z; Calc. Mass: 413.1365. Mp: 157–158 °C.
UPLCꢀMS (DAD/ESI): tR=8.91 min, for C27H27F2NO6 [M+H]+ found: 391.11 m/z; Calc
Mass: 391.16.
5-chloro-4-{[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methoxy}-2-hydroxy-
benzaldehyde (6c). Compound 6c was prepared as described for 4a, using solution of 5ꢀ
chloroꢀ2,4ꢀdihydroxybenzaldehyde (0.67 g, 3.9 mmol), PPh3 (1.01 g, 3.9 mmol), 5 (0.90 g,
3.5 mmol) in THF (21 mL) and DIAD (0.74 mL, 3.9 mmol) dissolved in THF (21 mL).
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Purification by flash chromatography (0ꢀ60% EtOAc in hexane) yielded 6c (0.54 g, 37%)
colorless solid.1H NMR (600 MHz, CDCl3) δ: 11.43 (s, 1H), 9.70 (s, 1H), 7.55 (s, 1H), 7.45
(dd, J = 6.9, 2.2 Hz, 1H), 7.27–7.24 (m, 2H), 6.91 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 2.1 Hz,
1H),6.77 (dd, J = 8.2, 2.1 Hz, 1H), 6.63 (s, 1H), 5.2 (s, 2H), 4.31 (s, 4H), 2.27 (s, 3H) ppm.
13C NMR (151 MHz, CDCl3) δ: 193.9, 163.1, 161.1, 143.2, 142.8, 142.6, 135.2, 134.2, 133.6,
130.6, 127.4, 125.8, 122.7, 118.4, 117.1, 115.2, 101.7, 70.5, 64.6, 16.4 ppm. IR (ATR) 2954,
2923, 2847, 1646, 1624, 1575, 1493, 1462, 1367, 1243, 1203, 1049, 885, 729 cmꢀ1. HRMS
ESIꢀMSꢀqꢀTOF for C23H19ClO5 [M+Na]+ 433.0819 m/z; Calc. Mass: 433.0819. Mp: 219–220
°C. UPLCꢀMS (DAD/ESI): tR=8.72 min, for C23H19ClO5 [M+H]+ found: 411.11 m/z; Calc
Mass: 411.10.
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3-(5-{[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methoxy}-2-formyl-4-meth-
ylphenoxymethyl)benzonitrile (7a). Cs2CO3 (0.25 g, 0,8 mmol), 6b (0.16 g, 0.8 mmol), 3ꢀ
(bromomethyl)benzonitrile (0.15 g, 0.8 mmol) were stirred in DMF (3 mL) at room
temperature overnight. The solvent was removed under reduced pressure, and the residue was
adsorbed on silica gel 60 and purified by flash chromatography (silica gel, 0ꢀ60% EtOAc in
1
hexane). The product was obtained as colorless solid (0.17 g, yield: 85%). H NMR (600
MHz, DMSOꢀd6) δ: 10.26 (s, 1H), 8.02 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.83 (dt, J = 7.7,
1.4 Hz, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.53 (d, J = 0.8 Hz, 1H), 7.45 (dd, J = 7.7, 0.8 Hz),
7.26 (t, J = 7.7 Hz, 1H), 7.2 (dd, J = 7.7, 1.2 Hz, 1H), 7.04 (s, 1H), 6.93 (d, J = 8.2 Hz, 1H),
6.80 (d, J = 2.1 Hz, 1H), 6.76 (dd, J = 8.2, 2.1 Hz), 5.40 (s, 2H), 5.28 (s, 2H), 4.28 (s, 4H),
2.23 (s, 3H), 2.13 (s, 3H) ppm. 13C NMR (151 MHz, DMSOꢀd6) δ: 187.4, 162.8, 160.9,
143.0, 142.5, 141.7, 138.2, 134.9, 134.4, 134.0, 132.4, 131.9, 129.8, 129.3, 127.6, 125.6,
122.2, 119.4, 118.7, 117.8, 116.8, 111.6, 98.1, 69.1, 69.1, 64.1, 15.9, 15.2 ppm. IR (ATR)
2921, 2229, 1679, 1609, 1580, 1506, 1466, 1273, 1246, 1125, 1052, 783 cmꢀ1. HRMS ESIꢀ
MSꢀqꢀTOF for C32H27NO5 [M+Na]+ 528.1775 m/z; Calc. Mass: 528.1787. Mp: 175–176 °C.
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UPLCꢀMS (DAD/ESI): tR=9.09 min, for C32H27NO5 [M+H]+ found: 506.16 m/z; Calc Mass:
506.19.
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3-(4-chloro-5-{[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methoxy}-2-formy-
lphenoxymethyl)benzonitrile (7b). Compound 7b was prepared according to the protocol of
7a, using Cs2CO3 (1.7 g, 5.2 mmol), 6c (0.54 g, 1.3 mmol), 3ꢀ(bromomethyl)benzonitrile (1.0
g, 5.2 mmol) in DMF (3 mL). Purification by flash chromatography (0ꢀ60% EtOAc in
hexane) yielded 7b (0.23 g, yield: 33%) colorless solid. 1H NMR (600 MHz, CDCl3) δ: 10.37
(s, 1H),7.91 (s, 1H), 7.72 (s, 1H), 7.69–7.66 (m, 2H), 7.54 (t, J = 7.8 Hz, 1H), 7.40–7.37 (m,
2H), 7.26–7.25 (m, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 2.1 Hz, 1H), 6.77 (dd, J = 8.2,
2.1 Hz, 1H), 6.60 (s, 1H), 5.20 (s, 2H), 5.18 (s, 2H), 4.31 (s, 4H), 2.28 (s, 3H) ppm. 13C NMR
(151 MHz, CDCl3) δ: 186.8, 160.7, 160.1, 142.8, 137.3, 135.0, 134.4, 133.7, 132.4, 131.5,
130.8, 130.7,130.4, 130.0, 127.5, 125.9, 122.7, 119.5, 118.4, 118.4, 117.2, 113.3, 98.8, 70.7,
70.0, 64.6, 16.5 ppm. IR (ATR) 3048, 2928, 2850, 2231, 1678, 1596, 1581, 1507, 1409, 1275,
1199, 1067, 1045, 789 cmꢀ1. HRMS ESIꢀMSꢀqꢀTOF for C31H24ClNO5 [M+Na]+ 548.1222
m/z; Calc. Mass: 548.1241. Mp: 151.5–152 °C. UPLCꢀMS (DAD/ESI): tR=9.14 min, for
C31H24ClNO5 [M+H]+ found: 525.10 m/z; Calc Mass: 525.14.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI:
Details on chemical synthesis of compounds, Xꢀray, NMR (PDF)
Molecular formula strings (CSV)
Accession Codes
Authors will release the atomic coordinates and experimental data upon publication.
Coordinates and structure factors have been deposited in the Protein Data Bank with
accession numbers 5N2D (1a) and 5N2F (2a).
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AUTHOR CONTRIBUTIONS
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K.G. synthesized the compounds and wrote the draft of the manuscript. K.M.Z. performed
crystallization of the complexes and solved the structures. P.G. collected Xꢀray data and
refined the structures. K.M., R.T. and L.S. provided support with preparation of expression
plasmids and protein purification. B.M. performed and analyzed NMR experiments. G.D.,
A.D. and T.A.H. analyzed data and wrote the final version of the manuscript. All authors
discussed the experiments and commented on the manuscript.
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AUTHOR INFORMATION
Corresponding Author
* T. A. Holak: holak@chemia.uj.edu.pl
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research has been supported in parts by a Marie Curie FP7ꢀReintegrationꢀGrant within
the 7th European Community Framework Programme and by the UMOꢀ2012/06/A/ST5/ꢀ
00224 and UMOꢀ2014/12/W/NZ1/00457 grants (to TAH), and by the UMOꢀ2011/01/D/ꢀ
NZ1/01169 and UMOꢀ2012/07/E/NZ1/01907 grants (to GD) from the National Science
Centre. The research has been carried out with the equipment purchased thanks to the
financial support of the European Union structural funds (grants POIG.02.01.00ꢀ12ꢀ064/08
and POIG.02.01.00ꢀ12ꢀ167/08). The research was carried out with the equipment purchased
thanks to the financial support of the European Regional Development Fund in the
framework of the Polish Innovation Economy Operational Program (contract no.
POIG.02.01.00ꢀ12ꢀ023/08). P.G. was supported the National Centre of Science (grant UMOꢀ
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2015/19/D/NZ1/02009). KG and KMZ have received financial support from the National
Science Centre (grants UMOꢀ2015/19/N/ST5/00347 to KG UMOꢀ2016/20/T/NZ1/00519 to
KZ). KMZ has received the financial support from the Faculty of Biochemistry, Biophysics
and Biotechnology of Jagiellonian University, which is a partner of the Leading National
Research Centre (KNOW) supported by the Ministry of Science and Higher Education.
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ABBREVIATIONS USED
ICB, Immune checkpoint blockage, PDꢀ1, Programmed Cell Deathꢀ1, PDꢀL1, Programmed
Deathꢀligandꢀ1, PDꢀL2, Programmed Deathꢀligandꢀ2, CTLAꢀ4, cytotoxic Tꢀlymphocyte–
associated antigen 4, NSCLC, nonꢀsmall cell lung cancer, mAbs, monoclonal antibodies,
FDA, US Food and Drug Administration, HTRF, homogenous timeꢀresolved fluorescence,
HAC, highꢀaffinity consensus, GuHCl, Guanidine Hydrochloride, DIAD, diisopropyl
azodicarboxylate, Fab, antigenꢀbinding fragment, KD, dissociation constant, tꢀBu XPhos, 2ꢀ
Diꢀtertꢀbutylphosphinoꢀ2′,4′,6′ꢀtriisopropylbiphenyl.
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