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. 2020 Mar 23;11(4):582–588. doi: 10.1021/acsmedchemlett.0c00058

Discovery and Optimization of Rationally Designed Bicyclic Inhibitors of Human Arginase to Enhance Cancer Immunotherapy

Matthew J Mitcheltree †,*, Derun Li †,*, Abdelghani Achab †, Adam Beard †, Kalyan Chakravarthy §, Mangeng Cheng §, Hyelim Cho §, Padmanabhan Eangoor Λ, Peter Fan Λ, Symon Gathiaka #, Hai-Young Kim °, Charles A Lesburg #, Thomas W Lyons □, Theodore A Martinot □, J Richard Miller §, Spencer McMinn †, Jennifer O’Neil ◊, Anandan Palani ∥, Rachel L Palte #, Josep Saurí °, David L Sloman †, Hongjun Zhang †, Jared N Cumming †, Christian Fischer †
PMCID: PMC7153280  PMID: 32292567

Abstract

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The action of arginase, a metalloenzyme responsible for the hydrolysis of arginine to urea and ornithine, is hypothesized to suppress immune-cell activity within the tumor microenvironment, and thus its inhibition may constitute a means by which to potentiate the efficacy of immunotherapeutics such as anti-PD-1 checkpoint inhibitors. Taking inspiration from reported enzyme–inhibitor cocrystal structures, we designed and synthesized novel inhibitors of human arginase possessing a fused 5,5-bicyclic ring system. The prototypical member of this class, 3, when dosed orally, successfully demonstrated serum arginase inhibition and concomitant arginine elevation in a syngeneic mouse carcinoma model, despite modest oral bioavailability. Structure-based design strategies to improve the bioavailability of this class, including scaffold modification, fluorination, and installation of active-transport recognition motifs were explored.

Keywords: Arginase inhibitor, cancer immunotherapy, structure-based drug design, pharmacokinetic optimization

Recruitment of the immune system to combat cancer, an approach now well-established by clinically approved therapies such as anti-PD-1 checkpoint inhibitors, has revolutionized the field of oncology in ways that impact both clinical and basic-research practice. Targeting and activating lymphocytes has resulted in unprecedented rates of durable response among cancer patients.1 As a result, there is increasing interest in identifying new targets to enhance lymphocyte activation and tumor-cell killing.2 Whereas early chemotherapeutics largely targeted functions critical to cell proliferation, new attention has centered increasingly on nodes that render tumors more susceptible toward clearance by immune cells.

Arginase is one such target whose role in immuno-oncology is supported by emerging genetics and immunopathology.3 Inhibitors of this enzyme, which functions to hydrolyze l-arginine to l-ornithine and urea, were sought historically for their potential to treat inflammatory diseases by enabling nitric oxide biosynthesis.4 However, recent evidence suggests a direct role for arginase inhibition to enhance cancer immunotherapy.5,6 Arginase activity suppresses T-cell activation and proliferation in myeloid-cell coculture experiments in vitro(7) and supports tumor growth in vivo.8,9 Accordingly, levels of arginase protein, enzyme activity, and reaction product (l-ornithine) are significantly elevated in the peripheral blood and tumor tissue of cancer patients.9−12 Myeloid cells expressing hArg1, one of the two major arginase isoforms in humans, are present in a variety of human tumors and are associated with poor prognosis.9 Together, these observations suggest that pharmacologic inhibition of arginase, either as monotherapy or in combination with other immunotherapies, may render the tumor microenvironment more hospitable to active T-cell proliferation and thus to tumor clearance.13,14

As part of an effort to discover novel small-molecule inhibitors of arginase to enhance cancer immunotherapy,15 we took as starting points the reported human hArg1 cocrystal structures of nonhydrolyzable substrate mimetics (S)-2-amino-6-boronohexanoic acid (ABH), its α-substituted derivative 1, and the cyclic analog 2 (Chart 1).16−21 In particular, we were inspired by Van Zandt and co-workers’ observation that, when bound to hArg1, compounds 1 and 2 engage in water-mediated hydrogen bonds with acidic residue Asp181 (Figure 1), leading to increased potency over ABH in a thioornithine generation assay22 (TOGA IC50, Chart 1). We speculated that an appropriately designed binder might engage this residue directly, displacing the intervening water molecule and further stabilizing the resulting enzyme–inhibitor complex. Toward this end, we designed 3, an ABH derivative possessing a rigid bicyclic appendage, as ideally disposed to interact with Asp181 through electrostatic interaction with its secondary amino group (Figure 1C).23

Chart 1. Previously Reported Substrate-Mimetic Arginase Inhibitors.

Chart 1

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Figure 1.

Figure 1

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X-ray cocrystal structures of boronic acid inhibitors bound to hArg1, where the manganese ions comprising the enzyme active site are depicted as plum-colored spheres. (A) The structure of 1 (cyan) bound to hArg1 (PDB 4HWW)21 reveals a water-mediated interaction between the piperidine ring and Asp181 (gold) which stabilizes the inhibitor–enzyme complex and contributes to the biochemical potency of 1. (B) Cyclic analog 2 (green) likewise features a water-bridged interaction with Asp181 (PDB 6Q37).20 (C) Inhibitor 3 (rose) mimics this binding mode, engaging Asp181 directly (PDB 6 V7C).

Synthesis of 3 was achieved through elaboration of its bicyclic core, which in its N-Boc protected form 4 was known and available through commercial sources (Scheme 1). Unexpectedly, allylation of this compound proved challenging, as typical conditions for ketone α-alkylation (LiHMDS, allyl bromide) produced complex product mixtures containing overalkylated and unreacted starting material, leading to low isolated yields of 5 (ca. 20%). Ultimately, we found that monoallylation of 4 could be performed by palladium and enamine dual catalysis, using allyl alcohol as the allylation reagent.24,25 The resulting product (5) was isolated in 46% yield as a 9:1 ratio of epimers and was subjected to Ugi-coupling conditions (t-BuNC, NH4OAc, CF3CH2OH) to install the α-amino acid pharmacophore,26 providing a mixture of diastereomeric products. Chiral separation by supercritical fluid chromatography was carried out at this stage, providing 7 in diastereomerically and enantiomerically pure form before hydroboration and deprotection in hot hydrochloric acid provided the desired compound. It bears mentioning that structural characterization and purity assessment of compound 3 were initially complicated by the observation that, in aqueous solutions of pH 2–10, chromatographically pure samples featured 1H NMR signals corresponding to a minor related species. That this apparent impurity interconverts with 3 was first established by ROESY-NMR, while a combination of 1H–15N HMBC, 11B, and 2D DOSY NMR experiments ultimately cemented its identity as the tricyclic aminoboronate cyclo-3 (Figure 2).27 As a consequence of these findings, NMR characterization of similar compounds was typically performed at pH < 2, under which conditions the equilibrium fraction of cyclic isomer was virtually zero.

Scheme 1. Synthesis of Compound 3.

Scheme 1

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Reagents and conditions: (a) allyl alcohol, [Pd(allyl)Cl]2, DPPF, pyrrolidine, CH3OH, 20 °C, 14 h; (b) t-BuNC, NH4OAc, CF3CH2OH, 35 °C, 16 h; (c) SFC, ChiralPak IC-3; (d) pinacolborane, [Ir(cod)Cl]2, DPPE, 4 Å MS, CH2Cl2, 0 → 23 °C, 1 h; (e) 12 N HCl, 100 °C, 16 h.

Figure 2.

Figure 2

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1H NMR spectra of compound 3 in D2O reveal pH-dependent equilibration with a cyclic aminoboronate (cyclo-3, signals from methylene protons alpha to boronate anion highlighted in blue), whose formation is suppressed at pH ≤ 1 (deuterium chloride).

Consistent with our design hypothesis, compound 3 exhibited potent arginase inhibitory activity (hArg1 TOGA IC50 = 29 nM); and X-ray crystallography revealed that 3 indeed binds hArg1 in the intended fashion, with the pyrrolidine ring nitrogen atom engaging Asp181 in direct electrostatic interaction (Figure 1; N–O distance = 3.3 Å). Comparable potency was observed in tandem liquid chromatography–mass spectrometry analysis of arginase activity in pooled human cancer patient serum (HCS IC50 = 148 nM) and EMT6 tumor-bearing mouse serum (IC50 = 361 nM), while the cellular activity of compound 3 in human HEK 293 cells and mouse bone-marrow derived macrophage (BMDM) cells was substantially reduced (IC50 values of 956 and 1,658 nM, respectively). Based on this promising in vitro profile, 3 was selected for pharmacokinetic profiling in male CL57BL/6 mice dosed intravenously (i.v., 1 mg/kg, n = 2) or orally (p.o., 10 mg/kg, n = 2). In these studies, the results of which are listed in Table 1, 3 displayed an i.v. mean residence time (MRT) of 1.4 h, an apparent volume of distribution (VD) of 0.84 L/kg, and oral bioavailability of 7%.

Table 1. Pharmacokinetic Profile of 3 in CL57BL/6 Mice.

Route of delivery i.v. p.o.
Dose (mg/kg) 1 10
AUC (μM·h) 6.4 4.8
C1h (μM) 0.91 1.2
MRT (h) 1.4 3.6
CL (mL/min/kg) 10  
VD (L/kg) 0.84  
%F   7%
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Encouraged by these results, and seeking an optimal interaction distance with Asp181,28 we applied analogous design principles and synthetic routes to test a range of additional arginase inhibitors with secondary amine groups (Chart 2). Fused 5,5-bicyclic analogs 9 and 10 (isomeric with 3) as well as spirocyclic analogs (±)-11,2912, and 13 were prepared, with 5,5-bicycles 12 and 13 demonstrating particularly strong enzymatic potency. Our computational models suggested that these compounds’ differentiated potencies in vitro reflected differences in distance separating their respective secondary-amine nitrogen atoms from Asp181, and subsequent X-ray cocrystal structures of 10, 12, and 13 bound to hArg1 supported this hypothesis by confirming predicted binding modes. Neutral cyclopentane analog 14(20) accordingly showed much lower potency, offering further validation of our strategy to target this key residue within the enzyme.

Chart 2. Other Cyclic Scaffolds Investigated.

Chart 2

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The totality of data emerging from these scaffolds, including encouraging pharmacokinetic behavior of 3 in higher species (vide infra), led us to invest effort in the structural optimization of inhibitor 3 in particular. Molecules possessing the boronic acid–amino acid pharmacophore native to 3 and its relatives—a motif required for potency among these substrate-mimetic arginase inhibitors—reliably demonstrated low permeability (MDCKII Papp < 2 × 10–6 cm/s). Thus, with the understanding that active uptake or paracellular transport mechanisms would likely underpin bioavailability of such polar molecules, our team followed several strategies to improve this pharmacokinetic parameter. We began with installation of aminoacyl groups to the pyrrolidine ring of 3 to potentially improve bioavailability through recruitment of active amino-acid transport mechanisms (Chart 3).30−32 Unfortunately, in this case, glycyl, alanyl, valyl, and seryl analogs 15–18 failed to display improved bioavailability relative to their parent. Remarkably, these analogs maintained activity in spite of their masked pyrrolidine nitrogen atoms, likely by virtue of new electrostatic interactions formed between their aminoacyl groups and Asp181. The corresponding neutral N-acetyl analog of 3 accordingly was much less potent (TOGA IC50 = 1,391 nM, structure not shown).

Chart 3. Aminoacyl Derivatives of 3.

Chart 3

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We also explored simple alkylation of the bicyclic core of 3, aiming to mask its hydrogen-bond donor features through steric occlusion, basicity attenuation, or both.33 While methylation of the primary amine resulted in a substantial loss in activity (data not shown), methylation of the pyrrolidine nitrogen atom afforded a marginal boost in bioavailability at a slight cost to potency (19). Methylation adjacent to the nitrogen atom afforded similarly bioavailable compounds with comparable (20) or substantially reduced (21) activity in the enzymatic assay (Chart 4), corroborating X-ray evidence and docking models which suggested that methylated regioisomer 21 might suffer from steric clash with Thr246 of hArg1 (Figure 1). Notably, N-trifluoroethyl analog 22 displayed improved bioavailability over 3, but the trifluoroethylamine group’s bulk and attenuated basicity (calculated pKa = 4.9 ± 0.4) together led to a >100-fold drop in enzyme inhibition.

Chart 4. Derivatives Bearing Pyrrolidine Ring Alkylation.

Chart 4

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The boost in bioavailability observed upon lipophilic N-trifluoromethylation motivated us to explore deletion of the pyrrolidine nitrogen atom altogether, resulting in pentalane analog 23 (Chart 5). This compound indeed displayed exceptionally improved mouse%F (63%) relative to 3 without substantial changes to its elimination profile due to compensating shifts in volume and clearance (i.v. MRT = 1.2 h; VD = 1.9 L/kg; CL = 25 mL/min/kg). This compound’s modest enzymatic potency was attributed to its lack of a suitably disposed polar group with which to engage Asp181, and so we sought to identify appropriate decoration of this new pentalane scaffold to recover activity. While hydroxylated compounds 24a and 24b displayed improved activity over their aliphatic progenitor, only amino analogs 25 and 26 achieved comparable or superior activity when compared to 3. Unfortunately, all of these monosubstituted pentalanes displayed disappointing mouse bioavailability.

Chart 5. Pentalane Analog 23 and Its Monosubstituted Derivatives.

Chart 5

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With this new pentalane scaffold in hand, we again evaluated aminoacylation as a tactic to boost oral absorption (Chart 6). We selected exo-amino compound 25b as a starting point, as docking simulations suggested that, when acylated, the nitrogen atom would be more favorably disposed for hydrogen-bond donation to Asp181 than the corresponding endo diastereoisomer. Once synthesized, however, alanyl and valyl analogs 27 and 28 appeared unstable toward amide-bond hydrolysis in serum, as evidenced by good potency in human cancer patient serum (HCS) despite modest enzyme inhibitory potency (TOGA). This suspicion was borne out in mouse pharmacokinetic experiments, where rapid clearance of intravenously administered 28 was observed, and where hydrolysis product 25b was detected exclusively in the blood of animals receiving an oral dose of 28. Notably, such instability was not observed for aminoacyl analogs 15–18, illustrating the risks inherent with aminoacylation strategies to boost oral absorption, as recruitment of active-transport mechanisms and resilience toward enzymatic degradation are both highly contingent upon the scaffold chosen.30−32

Chart 6. Aminoacylated Derivatives of 25b.

Chart 6

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Ultimately, modest improvements were made to both the potency and the pharmacokinetic properties of 24a through incorporation of fluorine within the pentalane core (Chart 7).34,35 Reasoning that fluorination, through inductive withdrawal, might improve the hydrogen-bond donor abilities of neutral hydroxypentalanes 24, or might attenuate the basicity of methylaminopentalanes 26, we identified chemistry to prepare regioisomerically fluorinated congeners of these compounds by a single divergent route (see Supporting Information for details). Gratifyingly, the resulting analogs featured improved potencies in both enzymatic and serum assays relative to their desfluoro congeners, while showing boosted bioavailability in the case of endo-hydroxypentalanes 29a and 31a (relative to 24a). Induced-fit docking of 31a to hArg1 revealed an antiperiplanar disposition of the β-fluoro hydroxyl motif within the optimal pose (Figure 3), representing a conformation known to increase the hydrogen-bond donor strength of fluorohydrins.36 Thus, we speculate that these improvements in potency and pharmacokinetics are attributable to fluorine’s unique ability to improve the hydrogen-bond donor strength of vicinal alcohols (for stronger interaction with Asp181) while mimicking the nonpolar surface of the unsubstituted—and exceptionally bioavailable—pentalane analog 23.

Chart 7. Fluorinated Pentalanes Bearing Hydroxy and Methylamino Substitution.

Chart 7

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Figure 3.

Figure 3

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Induced fit docking pose of 31a with hArg1, featuring an antiperiplanar fluorohydrin conformation, and a predicted hydroxyl–carboxylate (O–O) distance of 2.9 Å.

Concurrent with these optimization efforts, we collected pharmacokinetic data of 3 in higher-order species, where its greater oral bioavailability (29% F dog; 25% F rhesus) prompted us to select it for mouse pharmacokinetic–pharmacodynamic experiments. In our first study, 3 was administered to EMT6 tumor-implanted BALB/c mice (n = 3) at a dose of 300 mg/kg twice daily by oral gavage. Compound and arginine levels in the serum were measured after the fourth dose by LCMS at 2 and 7 h postadministration (Figure 4A). At both time points, serum levels of 3 surpassed the measured EMT6 mouse serum arginase IC50 of 361 nM by >10-fold in the experimental arm; accordingly, serum arginine levels were significantly elevated among animals receiving 3 relative to the control group (saline, n = 3).

Figure 4.

Figure 4

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Pharmacokinetic–pharmacodynamic assessment of 3 (p.o., 300 mg/kg, BID) in EMT6 tumor-bearing BALB/c mice confirmed exposure and pharmacological inhibition of arginase in both 2-day (A) and 14-day (B) experiments.

Encouraged by these results, we sought to confirm this pharmacodynamic effect in a 14-day tolerability study, in which a larger cohort of EMT6 tumor-bearing mice (n = 10) were dosed as before (300 mg/kg, BID, p.o.). Chronic dosing of 3 was well-tolerated, and 2 h after the final dose of the study was administered, drug and arginine levels in the mice were quantified (Figure 4B). We noted that peak exposure to 3 was substantially greater at the end of the 14-day study than at 2 days (serum [3] = 33 ± 5 μM versus 7 ± 1 μM), consistent with drug accumulation based on one-compartment model pharmacokinetic curve fit (data not shown). Nonetheless, animals receiving 3 in the 14-day study displayed similar elevations in serum arginine to those observed in the 2-day study (5–10-fold over untreated controls). In the tumor tissue, comparable drug exposure and arginine elevation were observed. Together these results established the ability of orally administered 3 to achieve a pharmacodynamic effect within the tumor environment of a widely used syngeneic mouse model, positioning this molecule and its relatives as potentially valuable tools to study arginase cancer biology.

In summary, application of structure-based drug design led us to discover compound 3, a novel boronic-acid arginase inhibitor with promising pharmacodynamic effects in mouse tumor models. Seeking to improve this compound’s pharmacokinetic limitations, we marshalled several distinct tactics. While many of these approaches—aminoacylation, core alkylation, and scaffold homologation—presented trade-offs in potency and bioavailability, simultaneous improvement in both these parameters was achieved to some extent through fluorination of a scaffold derived from 24a. Hence, we hope that the approaches described here might serve as a valuable survey of design options for those seeking to improve in a systematic sense the drug properties of similar bioavailability-limited scaffolds.

Acknowledgments

This report is dedicated to the memory of our colleague, Peter Spacciapoli. The authors gratefully acknowledge Min Lu and Qinglin Pu for contributions to the synthesis and biological profiling of compounds described here. We also recognize Jialiang Wang, Qingxin Li, Xiong Xiao, Peng Zhang, Wenqin Hu, Jiuyin Yi, Minfang Zhang, Zhenxiang Xie, Yaheng Wang, and Yang Wang from WuXi Apptec for their synthetic efforts. We thank Donovon Adpressa for NMR structure-elucidation work and Brian Hall and Angie Sun for providing purified protein for crystallography studies.

Glossary

Abbreviations

ABH

2-(S)-amino-6-boronohexanoic acid

AUC

area under curve

BID

twice daily

BMDM

bone marrow-derived macrophage

cod

1,5-cyclooctadiene

DOSY

diffusion-ordered spectroscopy

DPPE

1,2-bis(diphenylphosphino)ethane

DPPF

1,1′-bis(diphenylphosphino)ferrocene

hArg1

human arginase I

HCS

human cancer patient serum

HMBC

heteronuclear multiple bond correlation

i.v.

intravenous

LiHMDS

lithium hexamethyldisilazide

LCMS

tandem liquid chromatography–mass spectrometry

MDCKII

Madin–Darby canine kidney cell subclone II

MS

molecular sieves

NMR

nuclear magnetic resonance

p.o.

per os

ROESY

rotating-frame nuclear Overhauser effect correlation spectroscopy

SFC

supercritical fluid chromatography

TOGA

thioornithine-generating assay

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00058.

  • Experimental procedures for protein preparation, X-ray crystallography, the synthesis of all new compounds described, and detailed NMR characterization of compound 3 (PDF)

Accession Codes

Coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with accession codes 6V7C, 6V7D, 6V7E, and 6V7F.

Author Present Address

α Takeda Pharmaceuticals, 35 Landsdowne St., Cambridge, Massachusetts 02139, United States (H.C.).

Author Present Address

β Pfizer, Inc., 1 Burtt Rd. Andover, Massachusetts 01810, United States (H.-Y.K.).

Author Present Address

γ Infinity Pharmaceuticals, 1100 Massachusetts Ave., Cambridge, Massachusetts 02138, United States (T.A.M.).

Author Present Address

δ Akrevia Therapeutics, LabCentral 610, 610 Main St., Cambridge, Massachusetts 02139, United States (J.O.).

Author Present Address

ε Cerevel Therapeutics, 131 Dartmouth St., Suite 502, Boston, Massachusetts 02116, United States (H.Z.).

Author Present Address

ζ LifeMine Therapeutics, 30 Acorn Park Dr., Floor 4, Cambridge, Massachusetts, 02140 (J.N.C.).

Author Contributions

‡ M.J.M. and D.L. contributed equally to this work. All authors have given approval to the final version of the manuscript.

This work was funded entirely by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.

The authors declare no competing financial interest.

Supplementary Material

ml0c00058_si_001.pdf (3.4MB, pdf)

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