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Design, synthesis, evaluation and structural studies of C2symmetric small molecule inhibitors of the programmed cell death-1/ programmed death-ligand 1 (PD-1/PD-L1) protein-protein interaction Subhadwip Basu, Jeffrey Yang, Bin Xu, Katarzyna Magiera-Mularz, Lukasz Skalniak, Bogdan Musielak, Vladyslav Kholodovych, Tadeuz (Tad) A. Holak, and Longqin Hu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00795 • Publication Date (Web): 12 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019
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Journal of Medicinal Chemistry
Design, synthesis, evaluation and structural studies of C2-symmetric small molecule inhibitors of the programmed cell death-1/programmed death-ligand 1 (PD-1/PD-L1) protein-protein interaction Subhadwip Basu†, Jeffrey Yang†, Bin Xu , Katarzyna Magiera-Mularz‡, Lukasz Skalniak‡, Bogdan Musielak‡, Vladyslav Kholodovych†,§, Tad A. Holak‡, Longqin Hu, † Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854 ‡
Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387, Krakow, Poland Office of Advanced Research Computing, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854 §
-
The Cancer Institute of New Jersey, New Brunswick, New Jersey 08901
KEYWORDS immunomodulator; programmed cell death-1; PD-1/PD-L1 protein-protein interaction; small molecule inhibitor; C2-symmetric inhibitor; PD-L1/PD-L1 homodimer
ABSTRACT: A series of C2-symmetric inhibitors was designed and evaluated for inhibitory activity against the PD-1/PD-L1 protein-protein interaction (PPI) in a homogenous time-resolved fluorescence (HTRF) assay and PD-1 signaling in cellbased co-culture assays. C2-symmetric inhibitors 2a (LH1306) and 2b (LH1307) exhibited IC50’s of 25 and 3.0 nM, respectively, in the HTRF assay. While 2a was ~3.8-fold more potent than previously reported inhibitor 1a, 2b could not be differentiated from 1b due to their high potency and the limit of our HTRF assay conditions. In one cell-based coculture PD-1 signaling assay, 2a and 2b were 8.2- and 2.8-fold more potent in inhibiting PD-1 signaling than 1a and 1b, respectively. NMR and X-ray co-crystal structural studies provided more structural insights into the interaction between 2b and PD-L1; 2b binds to PD-L1 at the PD-1 binding site and induces the formation of a more symmetrically arranged PDL1 homodimer than previously reported for other inhibitors.
INTRODUCTION The clinical success of the immune checkpoint inhibitors has provided incontrovertible evidence that the immune system is critical in containing and eliminating malignantly transformed cells and that tumors evoke immunosuppressive mechanisms to evade immune attack.1 The PD-1/PD-L1 immune checkpoint serves to mediate peripheral immune tolerance and to protect against excessive inflammation and autoimmunity. Through their continual evolution, tumor cells have acquired the ability to co-opt the PD-1/PD-L1 axis in order to dampen the antitumor activities of tumor-specific T lymphocytes. Cancer immunotherapies targeting the PD1/PD-L1 immune checkpoint pathway have reversed this immunosuppression, resulting in remarkable clinical responses—tumor shrinkage, durable responses, and
prolonged survival—in patients with advanced solid and hematologic malignancies.2,3 Although this novel treatment modality has been significantly more effective and safer than traditional chemotherapy, the response rates are still only between 20% and 50% in most solid tumors.4-9 One major reason for the variable response rates stems from the fact that several alternative immune escape mechanisms exist and are activated upon PD1/PD-L1 inhibition.10 As a result, combination therapies are being sought out in order to prolong treatment responses and broaden benefits to a greater number of cancer patients.11 Current FDA-approved immune checkpoint inhibitors (anti-CTLA-4: ipilimumab; anti-PD-1: nivolumab, pembrolizumab, cemiplimab; anti-PD-L1: atezolizumab, avelumab, durvalumab) are all monoclonal antibodies
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(mAbs).1 These antibody-based therapies have demonstrated high target specificity and potent inhibitory activities, but possess several limitations including a poor pharmacokinetic (PK) profile for oral administration, inadequate tumor penetration, difficultto-manage high-grade autoimmune side effects, and high manufacturing and healthcare costs. These drawbacks of mAbs may hinder the development and clinical evaluation of combination therapies. Small molecules would make it more feasible and economical to design combination dosage forms. In addition, they have more appropriate PK properties allowing for higher oral bioavailability, broader distribution and enhanced tumor penetration, and easier reversal of adverse reactions due to their relatively shorter half-lives. For these reasons, the PD-1/PD-L1 protein-protein interaction (PPI) has become an attractive therapeutic target for small molecules.1,12,13 Bristol-Myers Squibb recently disclosed the first small molecule inhibitors of the PD-1/PD-L1 immune checkpoint pathway based on two core structures: (2methyl-3-biphenyl)methanol and [3-(2,3-dihydro-1,4benzodioxin-6-yl)-2-methylphenyl]methanol.14,15 In their disclosure, these small molecule inhibitors of the PD1/PD-L1 PPI exhibited IC50 values ranging from 920 pM to 14.25 L in an europium (Eu)-allophycocyanin (APC) homogeneous time-resolved fluorescence (HTRF) binding assay. Recently, Holak and coworkers revealed that these small molecule inhibitors bind directly to PD-L1 based on 1H-15N HMQC NMR studies.16 They also reported on the ability of these compounds to induce and stabilize the formation of PD-L1/PD-L1 homodimers, resulting in the blockade of the PD-1-binding surfaces on the PD-L1 proteins which may explain how these compounds interfere with the PD-1/PD-L1 PPI.16,17 These inhibitors were shown to restore the activity of T lymphocytes by disrupting the PD-1/PD-L1 interaction, albeit to a lesser degree than that observed for the therapeutic mAbs.18 Several additional crystal structures have been deposited to the Protein Data Bank (PDB) illustrating the binding mode of these compounds to the pocket of the homodimer.1,19 In the present study, we report our structure-based design of a series of C2-symmetric small molecule inhibitors of the PD-1/PD-L1 PPI and a preliminary CH3
R1 O
structure-activity relationship study demonstrating the importance of the 2,2.-dimethyl-1,1.-biphenyl core in binding to PD-L1. These novel C2-symmetric inhibitors were found to induce PD-L1 protein dimerization upon binding to PD-L1 as shown in the X-ray co-crystal structure of PD-L1 in complex with a C2-symmetric inhibitor 2b (LH1307). These inhibitors were also capable of interfering with the PD-1/PD-L1 PPI and blocking PD-1 signaling in a co-culture assay.
RESULTS AND DISCUSSION Design principle Understanding the interaction surface of the PD-1/PDL120 and PD-L1/small molecule16-18 interactions provided a starting point for our design of the C2-symmetric inhibitors (Figure 1). The PD-1 and PD-L1 proteins utilize their single extracellular IgV-like domains, organized into nine parallel O/ (ABCC.C..DEFG) and several interconnecting loops, to make contact with each other.1 Specifically, both proteins use the front GFCC. faces of their V-domain to interact via hydrophobic and polar interactions. The small molecule inhibitor 1a (previously disclosed as BMS-202)14 binds to PD-L1 at its PD-1-binding site and effectively inhibits the binding of PD-1 to PD-L1. Examination of the 1a/PD-L1 interaction showed that the biphenyl moiety binds to and occupies a hydrophobic cleft created by two PD-L1 proteins.16,17 The polar moiety, i.e. the 2-(acetamido)ethylamino group, of 1a extends out of the hydrophobic cleft and occupies the solvent-exposed region sandwiched between the AG and C.C O-strands of the respective PD-L1 proteins, further enhancing binding to the two PD-L1 proteins via several hydrogen bond and electrostatic interactions. Collectively, this network of hydrophobic and polar interactions is responsible for the interesting dimerization phenomenon discovered by Holak and coworkers.17 Upon binding to PD-L1, 1a induces the formation of a near symmetrically arranged PDL1/PD-L1 homodimer with a global asymmetry score (GloA_Sc)21,22 of 0.55, where asymmetry is mostly caused by binding of the asymmetric ligand 1a (Figure 2A). In this orientation, the PD-1-binding surfaces of the PD-L1 proteins are aligned in opposite directions, with the AGFCC. face of one PD-L1 interacting with the C.CFGA face of the second PD-L1. From these observations, we
R3 X
R2
1a-c
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X
R1
R2
R3
a
N
-H
-OCH3
-NHCH2CH2NHCOCH3
b
CH
-CH3
O
CN
-NHCH2CH2NHCOCH3
N
c
CH
-CH3
CN
O
-NHCH2CH3
N
CH3 R2 R3
X
O R1
R1 O
R3 X
R2
CH3 2a-c
d
e
f
Figure 1. Design of C2-symmetric small molecule inhibitors of PD-1/PD-L1 protein-protein interaction.
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Journal of Medicinal Chemistry homodimer with the bound 1a was duplicated and crosssuperimposed onto the original structure (A over B, B over A), we realized that the biphenyl structure of the two 1a molecules overlaid on top of each other and that a C2symmetric analog 2a could be a better inducer of PD-L1 dimerization and thus a better inhibitor of the PD-1/PDL1 PPI (Figure 2B). Accordingly, 1b and 1c were also converted to C2-symmetric analogs 2b and 2c, respectively. 2a and 2b were constructed and then docked into the PD-L1 homodimer pocket of 1a resulting in more favorable docking scores (Table 1). As anticipated, the biphenyl moiety of 2a and 2b positioned these inhibitors in the same relative location in the hydrophobic core of the homodimer as that in 1a. Both 2a and 2b opened up the hydrophobic cleft previously created by 1a, forming a hydrophobic tunnel through the homodimer. The polar moieties of 2a and 2b extended from the biphenyl system and across the AGFCC. faces of the two PD-L1 proteins and made extensive water-mediated, hydrogen bond, and electrostatic interactions with the AG and CC. O-strands at the ends of the homodimer. These polar interactions could strengthen the resulting PD-L1 homodimer structure, making it harder to dissociate as compared to 1a and in turn yielding more potent inhibitors of the PD1/PD-L1 PPI. We also performed preliminary scaffold hopping to explore the possibility of replacing the 2,2.dimethyl-1,1.-biphenyl core with 1,4- or 1,3-phenylene (2de) or 1,4- or 2,6-naphthalenylene (2f-g) scaffolds to establish a preliminary structure-activity relationship and highlight the importance of the 1,1.-biphenyl moiety.
Figure 2. (A) Co-crystal structure of the 1a:PD-L1 homodimer (PDB ID: 5J89). The interaction surface of 1a within the binding pocket indicates hydrophobic regions in red, polar regions in blue, and solvent-exposed regions in green. (B) Cross-superimposition of two sets of 1a:PD-L1 homodimers leading to the design of C2-symmetric inhibitor 2a. The PD-L1 proteins are represented as blue and green ribbon structures. The two 1a molecules are represented as yellow (1st) and magenta (2nd) stick structures.
hypothesized that more potent small molecule inhibitors of PD-1/PD-L1 PPI could be designed by converting the known inhibitors into C2-symmetric inhibitors centered around a 2,2'-dimethyl-1,1'-biphenyl core like 2a and 2b. Such inhibitors have been reported for homodimeric protein targets with C2 symmetry such as HIV-1 protease.23-26
Chemical synthesis Synthesis of the (2-methyl-3-biphenyl)methanol derivatives (1a-c). Compound 1a was synthesized as previously reported17 except the reducing agent used for the reductive amination was sodium triacetoxyborohydride. The synthesis of compounds 1b and 1c employed a selective Mitsunobu reaction27 of 3 with aldehyde 5, a cesium carbonate-promoted Oalkylation17 of the resulting intermediate aldehyde 7 with
Initial docking studies using the crystal structure of PDL1 in complex with 1a (PDB ID: 5J89) in the Molecular Operating Environment (MOE) program were undertaken to design our C2-symmetric inhibitors. When the PD-L1
Scheme 1. Synthesis of the (2-methyl-3-biphenylyl)methanol derivatives (1a-c)a O H N
H CH3
N
Cl
H 2N
O
OCH3
CH3 O
4
CH3
OH I
CH3
H O
N
O
II
OCH3
H N
CH3 O
1a
6
(2-Methyl-[1,1'-biphenyl]3-yl)methanol (3)
N
N H OCH3
O H 3C
H
HO
III
OH 5
CN
Cl O CH3
H 3C
8
N
O CH3
H 3C
H
O O
OH
CH3
H 2N R
H O
O
IV
CN N
a
N H
R
O
V
CN
7 9
H3C
1b: R = 1c: R =
O CH2CH2NHCCH3 CH2CH3
N
Reagents and conditions: (I) Cs2CO3, Pd(OAc)2, t-BuXPhos, toluene, 80°C, overnight; 71% (II) cat. AcOH, DCM, r.t., 3 h; NaBH(OAc)3, r.t., 15 h; 65% (III) PPh3, DIAD, THF, 0°C to r.t. to 40°C, overnight; 37% (IV) Cs2CO3, DMF, r.t., 5 h; 62% (V) Amine (R-NH2), cat. AcOH, DCM, r.t., 5 h; NaBH(OAc)3, r.t., 18 h; 60-70%.
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Journal of Medicinal Chemistry
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Scheme 2. Synthesis of the C2-symmetric (2,2'-dimethyl-[1,1'-biphenyl]-3,3'-diyl)dimethanol derivatives (2a-c)a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
O CH3
CH3 HO
OH CH3
Br
N
O
H
H 2N
N
O
OCH3
CH3
CH3
H 3C
N H
H3CO H N
N
O
O
N
N H OCH3
CN
14
H 2N R
NC
N II
CH3 O
CH3 O
R
O
CH3
CH3
N
H N
H
O
O
II
H 3C
O
H
O
O
O CH3
11
H N
V
8
NC
CH3 O
13
O
H
OH
CH3
CH3
O
O
H
O
N
I
H 3C
O
H
12
CH3 H3CO
HO
IV
CH3
(2,2'-Dimethyl-[1,1'-biphenyl]3,3'-diyl)dimethanol (10) 4
5
Br III
CH3
H 3C
O
H N
O
2b: R = 2c: R =
2a
R
O
CH3
CH3
CH3
N H
CN
O CH2CH2NHCCH3 CH2CH3
N
a Reagents and conditions: (I) Cs CO , Pd(OAc) , t-BuXPhos, toluene, 80°C, overnight; 31% (II) cat. AcOH, DCM, r.t., 2 3 2 overnight; NaBH(OAc)3, r.t., ~24 h; 40-63% (III) PBr3, 1:1 DCM:DMF, 0°C to r.t., 1 h; quantitative (IV) NaHCO3, CH3CN, 60°C, overnight; 76% (V) Cs2CO3, DMF, r.t., 5 h; quantitative.
Scheme 3. Synthesis of the C2-symmetric analogs containing phenylene (2d-e) and naphthalenylene (2f-g) scaffoldsa O H HO X
AR
OH
X
H
HO
O
AR
O
O
N
O
H
NC
8
O
OH
I 16d-g CH3 O
H 3C
N
H N
N H O
N H
O CN
O
AR
17d-g
O
NC O
III
O
H O
H 2N
AR
II H
15d-g H N
O
O
CH3 O
O
H N
CN 2d-g
N
AR
= d
N
e
f
g
a Reagents and conditions: (I) KHCO , acetone, reflux, 3 days; 9-74% (II) Cs CO , DMF, r.t., 5 h; 41-100% (III) cat. AcOH, 3 2 3 DMF, r.t., overnight; NaBH(OAc)3, r.t., ~24 h; 20-41%.
5-(chloromethyl)nicotinonitrile (8), and a sodium triacetoxyborohydride-mediated reductive amination of intermediate aldehyde 9 with either Nacetylethylenediamine or ethylamine (Scheme 1). The overall yield for compounds 1a-c ranged from 14% to 46%. Synthesis of the (2,2'-dimethyl-1,1'-biphenyl-3,3'diyl)dimethanol derivatives (2a-c). The starting material 10 was prepared using Suzuki-Miyaura coupling followed by a lithium aluminum hydride reduction.28,29 Compound 2a was prepared using the same synthetic strategy as described for compound 1a: Buchwald-Hartwig crosscoupling reaction resulting in intermediate aldehyde 11, followed by reductive amination with Nacetylethylenediamine. The dibromo intermediate 12 for the synthesis of compounds 2b and 2c was prepared from the starting material 10 using phosphorus tribromide. For compounds 2b and 2c, owing to the prolonged reaction time and poor yield of the Mitsunobu conditions
previously employed, a regioselective potassium bicarbonate-promoted O-alkylation of aldehyde 5 with the dibromo compound 12 was performed to obtain intermediate aldehyde 13.30 Cesium carbonate-promoted O-alkylation17 of 13 followed by sodium triacetoxyborohydride-mediated reductive amination of intermediate aldehyde 14 with either Nacetylethylenediamine or ethylamine yielded compounds 2b and 2c, respectively (Scheme 2). The overall yield for compounds 2a-c ranged from 12% to 48%. Synthesis of the phenylenedimethanol (2d-e) and naphthalenedimethanol (2f-g) derivatives. Starting from 1,4- or 1,3-bis(chloromethyl)benzene (15d-e) and 1,4- or 2,6- bis(bromomethyl)naphthalene (15f-g), compounds 2d-g were prepared using a multistep synthetic route similar to compounds 2b-c, involving a regioselective potassium bicarbonate-promoted O-alkylation with 2,4dihydroxybenzaldehyde to afford intermediate aldehydes
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Journal of Medicinal Chemistry
Table 1. Structure-activity relationship of small molecule PD-1/PD-L1 inhibitors 1a-c and C2-symmetric analogs 2a-g. EC50 (nM) Ratiof GI50 ( M) PD-1 (SHP-1) PD-1/PD-L1 PD-1 (SHP-1) PD-1/PD-L1 Toxicitye signalingc blockaded signaling blockade 1a U6$=96 ± 10 2,764 ± 603 >10,000 (0.98) 4.5 ± 0.4 1.6
