Next Article in Journal
Water-Soluble Vitamins: Hypo- and Hypervitaminosis in Pediatric Population
Previous Article in Journal
Amphotericin B Encapsulation in Polymeric Nanoparticles: Toxicity Insights via Cells and Zebrafish Embryo Testing
Previous Article in Special Issue
Pharmacokinetic Evaluation of Neutral Sphinghomyelinase2 (nSMase2) Inhibitor Prodrugs in Mice and Dogs
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Synthesis of Arginase Inhibitors: An Overview

by
Maria Cristina Molaro
1,
Chiara Battisegola
1,
Marica Erminia Schiano
1,
Mariacristina Failla
2,
Maria Grazia Rimoli
1,
Loretta Lazzarato
2,
Konstantin Chegaev
2 and
Federica Sodano
1,*
1
Department of Pharmacy, “Federico II” University of Naples, 80131 Naples, Italy
2
Department of Drug Science and Technology, University of Turin, 10125 Turin, Italy
*
Author to whom correspondence should be addressed.
Pharmaceutics 2025, 17(1), 117; https://doi.org/10.3390/pharmaceutics17010117
Submission received: 17 December 2024 / Revised: 12 January 2025 / Accepted: 14 January 2025 / Published: 16 January 2025

Abstract

:
Arginase (ARG) is a binuclear manganese-containing metalloenzyme that can convert L-arginine to L-ornithine and urea and plays a key role in the urea cycle. It also mediates different cellular functions and processes such as proliferation, senescence, apoptosis, autophagy, and inflammatory responses in various cell types. In mammals, there are two isoenzymes, ARG-1 and ARG-2; they are functionally similar, but their coding genes, tissue distribution, subcellular localization, and molecular regulation are distinct. In recent decades, the abnormal expression of ARG-1 or ARG-2 has been reported to be increasingly linked to a variety of diseases, including cardiovascular disease, inflammatory bowel disease, Alzheimer’s disease, and cancer. Therefore, considering the current relevance of this topic and the need to address the growing demand for new and more potent ARG inhibitors in the context of various diseases, this review was conceived. We will provide an overview of all classes of ARG inhibitors developed so far including compounds of synthetic, natural, and semisynthetic origin. For the first time, the synthesis protocol and optimized reaction conditions of each molecule, including those reported in patent applications, will be described. For each molecule, its inhibitory activity in terms of IC50 towards ARG-1 and ARG-2 will be reported specifying the type of assay conducted.

1. Introduction

Arginase (ARG) has roots in early life forms. It is a manganese-containing binuclear metalloenzyme capable of converting L-arginine to urea and L-ornithine. Urea provides protection against ammonia (NH3), while L-ornithine serves to stimulate cell growth and other physiological functions. This interconnection with various metabolic pathways, such as polyamine synthesis and energy metabolism regulation, underscores the importance of ARG in maintaining metabolic homeostasis [1]. L-arginine is one of the most versatile amino acids in animal cells, serving as a precursor not only for the protein synthesis but also for the production of nitric oxide, urea, polyamines, proline, glutamate, creatine, and agmatine [2]. In mammals, there are two isoenzymes: arginase-1 (ARG-1) and arginase-2 (ARG-2). They are functionally similar, but the coding genes, tissue distribution, subcellular localization, and molecular regulation are distinctive. ARG-1 is localized in the cytoplasm and mainly expressed in the liver, where it is responsible for the detoxification of ammonia in the urea cycle [1]. In contrast, ARG-2 is predominantly found in the mitochondria and is primarily involved in polyamine generation [3]. Both enzymes metabolize L-arginine, which is severely depleted in the immunosuppressive tumour microenvironment (TME), where it is crucial for proliferating cells. This positions them at the forefront of immune escape through various mechanisms.
Within the TME, ARG-1 plays a crucial role in immune evasion by activating immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs), a heterogeneous population of bone marrow-derived cells that suppress immune responses and facilitate tumour progression [4]. In addition to high expression in MDSCs, ARG-1 is also upregulated in tumour-associated macrophages (TAMs) and activated neutrophils within the TME [5,6]. In these cells, it depletes L-arginine, impairing T-cell activation and proliferation, ultimately enabling cancer cells to evade immune surveillance.
Recently, ARG-2 has received special attention. It has been reported that the ARG-2 pathway is a means by which regulatory T cells (Tregs), also called CD4+ cells, regulate inflammation in tissues. Compared to psoriatic Tregs, healthy Tregs express almost 4-fold more ARG-2; similarly, Tregs in metastatic melanoma lesions express high levels of ARG-2 protein, with these levels being higher in tumour-infiltrating Tregs than in Tregs isolated from healthy skin [7]. ARG-2 reduces the survival and proliferation of effector T cells (CD8+), thereby impacting antitumour immune responses. Thus, L-arginine metabolism via ARG-2 is an immunoregulatory pathway used by Tregs in human tissues, with significant implications for both autoimmunity and cancer.
The disturbance in the expression of ARG can lead to a range of vascular, neurological, immunological, and inflammatory disorders, as the isoforms ARG-1 and ARG-2 are involved in the regulation of nitric oxide (NO), polyamines, proline, and, particularly in the immune system, endothelial cells, and neuronal cells [8,9,10].
Regarding the involvement of ARG in the immune system, in the presence of an inflammatory stimulus, the enzyme nitric oxide synthase (NOS) produces NO. This NO interacts with reactive oxygen species (ROS), creating a cytotoxic nitrosative stress environment that inhibits both cellular replication and pathogenic activity [11]. On the other hand, the enzyme ARG metabolizes L-arginine, regulating defence mechanisms and downregulating NO production. This helps prevent uncontrolled cellular apoptosis triggered by ONOO species generated from excess NO reacting with superoxide radicals (O2) [12].
An altered balance in the expression of these two enzymes can cause serious issues for the immune system. Additionally, the dysregulated release of ARG from cells and tissues into extracellular fluids can further compromise the defence mechanisms of macrophages against pathogens by limiting the bioavailability of L-arginine, reducing NO levels, and disrupting cytokine production pathways. It has been shown that ARG is implicated in disorders such as multiple sclerosis, as the increased regulation of the enzyme ARG-2 stimulates the production of cytokines that differentiate T helper 17 cells, thus inducing inflammation [13]. In studies on obesity-induced vasculopathy, it has been observed that high levels of fats and sucrose activate Rho-associated kinases, increasing the expression of ARG-1. The increased synthesis of polyamines mediated by ARG-1 promotes cellular proliferation and fibrosis; additionally, the rise in ROS levels contributes to dysfunction [14].
The enzyme ARG, particularly its isoform ARG-2, plays a crucial role in maintaining the balance of the cardiovascular system by regulating the levels of NO [15]. This helps reduce oxidative damage to the endothelium, promotes blood vessel dilation, and prevents the adhesion and aggregation of leukocytes and platelets. An imbalance between the enzymes that degrade L-arginine (ARG and NOS) can contribute to many age-related cardiovascular complications, such as vascular stiffness, ventricular hypertrophy, hypertension, inflammation, and disorders caused by oxidative stress [16]. Studies have shown that a high-fat, high-cholesterol diet causes liver damage in mice, leading to the overexpression of ARG-1, a consequent reduction in circulating L-arginine levels, and cardioprotective effects mediated by NO [17]. In contrast, a glucose-free diet in mice results in increased expression of ARG-2, triggering signalling pathways that protect hepatocytes from fat accumulation, inflammatory responses, insulin resistance, and glucose intolerance [18].
ARG and NOS are present in both the peripheral and central nervous systems, but their interaction is complicated by the complexity of the brain. ARG is essential for the detoxification of ammonia and the synthesis of polyamines, which are necessary for neuronal development and regeneration. NO, on the other hand, is a neurotransmitter that contributes to synaptic plasticity and cerebral blood flow. During development, high levels of cAMP stimulate ARG-1 to promote neuronal survival. However, with ageing, an imbalance between ARG and NOS can reduce NO production, contributing to neurodegenerative diseases [19].
One neurodegenerative disease associated with the alteration of ARG enzyme expression is Alzheimer’s disease. It has been observed that microglial activation leads to the production of cytokines, which induce increased expression of ARG-1 and ARG-2 in the brain. However, the accumulation of ARG-2 at sites of β-amyloid deposition causes the uncoupling of NOS, generating O2 and neurodegenerative oxidative stress [20]. This may be an attractive molecular imaging target for the evaluation of Alzheimer’s disease progression [21]. In certain contexts, an excess of NO can cause neuronal damage and brain trauma due to excitotoxicity. Notably, the role of ARG-2 is completely reversed in neurodegenerative diseases compared to cardiovascular disorders, highlighting the importance of understanding the distinct contributions of each isoform in different disease contexts. This underscores the need for selective molecules tailored to each isoform.
In line with the significant role of ARG-1 and ARG-2 in numerous diseases, there has been growing interest in developing inhibitors for these enzymes. In particular, identifying the ARG enzymes as critical metabolic checkpoints in the TME has spurred the design of novel ARG inhibitors. Both natural and synthetic compounds have been evaluated in various in vitro, ex vivo, and in vivo models [22,23]. Natural compounds like chlorogenic acid and picetannol, as well as synthetic compounds such as L-arginine-like derivatives and boronic acid derivatives, have been studied. These boronic acids are α-amino acids with a lateral boronic group capable of chelating the two manganese ions required for enzymatic activity. However, their inhibitory activity (IC50) was in the micromolar range, indicating the need for further improvement. The initial approach taken by Van Zandt et al., later upgraded by others, focused on expanding 2-(S)-amino-6-boronohexanoic acid (ABH) with moieties capable of additional interactions with residues Asp181, Asp183, and Asp202 [24,25,26]. Further improvements in the pharmacological profile were achieved by limiting the conformational flexibility of ABH. Notable examples include CB-1158 (numidargistat) and OATD-02 [27,28]. In a recent study, OATD-02 demonstrated superior in vivo antitumor capacity compared to CB-1158, as it inhibited both extracellular ARG-1 within the TME and cellular ARG-2 overexpressed in the TME [29]. By inhibiting ARG-2, OATD-02 was found to regulate the activity of CD8+ cells and Tregs, thus controlling a key player responsible for the metabolic adaptation typical of hypoxic tumours.
This review aims to provide a comprehensive overview of ARG inhibitors, focusing on the ongoing search for these molecules involved in multiple key pathophysiological processes. We categorize these inhibitors into generations and classes, as illustrated in Figure 1. For each molecule, its inhibitory activity (IC50) against ARG-1 and ARG-2 will be reported, with colorimetric assays used for all compounds unless otherwise specified.
Three reviews have previously addressed this topic, each from a different perspective. Borek et al. focused on the SAR and pharmacokinetic properties of each inhibitor [22]; Niu et al. discussed the underlying mechanism of ARG in tumour cell growth and summarized recent clinical research on ARG targeting for cancer therapy [30]. Clemente et al. explored the potential of ARG as a molecular imaging biomarker, stimulating the development of high-affinity, specific ARG imaging probes [16]. Failla et al. analysed the structural characteristics and plasticity of ARG-1 and ARG-2 binding sites, aiming to design inhibitors with new binding patterns [31]. However, none of these reviews have addressed the synthetic procedures of ARG inhibitors, a critical aspect from a medicinal chemistry perspective.
The chemical synthesis of these inhibitors presents numerous challenges, primarily due to their inherent chemical complexity. For instance, replacing the guanidine group with a boronic acid residue has proven effective. This modification preserves the geometry and electrophilicity of the original group while improving the compound’s physicochemical properties, such as reduced polarity and non-basicity. However, synthesizing a boronic acid derivative is challenging due to the reactivity of the functional group, necessitating the use of a protected ester form. Although using a protecting group can simplify the synthetic process, this is not always the case with amino acid derivatives, such as ARG inhibitors. These derivatives must mimic the amino acid L-arginine, requiring both amino and carboxylic groups. Given that these functional groups are as reactive as boronic acid, selecting the optimal protecting group and synthetic strategy is challenging. The inherent chemical complexity of these procedures presents scalability issues and challenges with reproducibility, further complicated by the presence of chiral centres and the need for stereoselective syntheses.
In this review, we provide a complete overview of all classes of ARG inhibitors, including molecules of synthetic and natural origins. For the first time, we also present the synthetic protocols and the optimized reaction conditions for each molecule. Many structures and syntheses of ARG inhibitors have been reported in patents that are often difficult to access and interpret. This review aims to be a comprehensive guide to the synthesis of ARG inhibitors, addressing critical issues from a synthetic chemical perspective and suggesting how these can be overcome through medicinal chemistry strategies.

2. First-Generation Inhibitors

The first ARG inhibitors were developed by analysing the chemical structure of the enzyme’s natural substrates, such as L-arginine and other structurally similar amino acids. The enzyme’s small and highly polar active site favours the accommodation of amino acids with a natural L-configuration (such as L-arginine analogues), which is also the configuration responsible for the enzyme’s activity.
The subsequent inclusion of a boronic group into the inhibitor structures was crucial for two main reasons. First, during the binding mechanism with the ARG enzyme, the boronic group forms a tetrahedral boronate anion by incorporating an OH group, effectively mimicking the transition state of the hydrolysis of the trigonal planar guanidine group in L-arginine by ARG. Second, the boron atom’s electron deficiency facilitates nucleophilic attack by the hydroxide ion, enhancing the activity of the inhibitor. Boronic acid also serves as an excellent guanidine substitute due to its similar geometry and electrophilicity, while offering better physicochemical properties—it is less polar, non-basic, and has fewer hydrogen bond donors [32]. In addition to incorporating the boronic moiety, other structural modifications included varying the length of the alkyl side chain, introducing a sulphur atom along the carbon chain, and adding a phenyl ring to the side chain to create a conformationally restricted analogues. These modifications led to the development of ARG inhibitors, which, divided into α-amino acid derivatives and boronic acid-containing compounds, constitute the class of first-generation inhibitors [22].

2.1. α-Amino Acid Derivatives

ARG inhibitors derived from amino acids include L-homoarginine (1), L-ornithine (2), and L-citrulline (3) categorized as “natural amino acids compounds” (Figure 2). L-homoarginine interacts slowly with ARG, as the enzyme’s hydrolytic efficiency depends on the side chain of the α-amino acid substrate. It inhibits human hARG-1 with IC50 and Ki values of 8.14 ± 0.52 mM and 6.1 ± 0.50 mM, respectively, and hARG-2 with IC50 and Ki values of 2.52 ± 0.01 mM and 1.73 ± 0.10 mM, respectively. ARG activity was assessed by measuring L-ornithine formation in HEK293T cell lysates [33]. L-ornithine shows a significant inhibition of rat ARG, achieving 85.9% inhibition at 10 mM. Similarly, L-citrulline exhibits inhibition levels comparable to L-ornithine, with a bovine liver ARG inhibition of 60% at 20 mM for L-ornithine and 53% at 20 mM for L-Citrulline, as measured by a [14C] urea assay [34,35]. The IC50 values, in the millimolar range, obtained with the aforementioned α-amino acid derivatives in in vitro models indicated that they were weak ARG inhibitors, predicting an even weaker therapeutic potential in vivo.
Nω-hydroxyarginine (L-NOHA, 4), is a hydroxylated derivative of L-arginine and a potent ARG inhibitor. It demonstrated the following activity against hARG with the following results: hARG-1 with a Kd of 3.6 μM (pH 8.5, surface plasmon resonance, SPR) and hARG-2 with Ki values of 1.6 μM, (pH 7.5) and 2 μM (pH 9.5, radioactive assay) [36,37]. For the synthesis of L-NOHA, Wallace et al. adopted Bodanszky’s method (Scheme 1) [38,39]. Starting with Nδ-(benzyloxycarbonyl)-L-ornithine (4a), the carboxylic acid group was protected using tert-butylacetate and HClO4, yielding the corresponding tert-butyl ester (4b). Compound 4b was treated with tert-butyl pyrocarbonate in dichloromethane (DCM) at 0 °C and then at room temperature (r.t.) to protect the amine group, producing compound 4c. Next, hydrogenation with H2 and Pd/C removed the benzyloxycarbonyl group, generating the Nα-(tert-butyroxycarbonyl)-L-ornithine tert-butyl ester (4d) [39]. To form the cyanamide derivative 4e, the reaction of 4d with BrCN in MeOH using NaOAc as a base (Bailey’s method) was performed [40]. The subsequent reaction of 4e with NH2OH in dry dioxane under reflux produced the Nα-(tert-butyloxycarbonyl)-Nω-hydroxy-L-arginine tert-butyl ester (4f). Finally, deprotection of 4f with trifluoroacetic acid (TFA) yielded Nω-hydroxyarginine (4) [39].
Nor-NOHA (5), a hydroxyguanidine derivative with a shorter alkyl chain, is another compound that effectively inhibits the enzyme ARG [41]. It was tested on hARG with the following results: on hARG-1 (pH 8.5) it showed a Kd of 0.517 μM (SPR) and 0.047 μM (isothermal titration colorimetry determination, ITC), while on hARG-2 it exhibited a Ki of 51 nM (pH 7.5). As shown by the data from the ARG inhibition tests, the smaller derivative nor-NOHA demonstrated higher affinity compared to L-NOHA and also exhibited a better pharmacokinetic profile, including improved bioavailability and faster elimination. The synthesis of nor-NOHA, which is very similar to that of L-NOHA, is outlined in Scheme 2 [36,37].
The synthesis of nor-NOHA (5), began with Nα-tert(butyloxycarbonyl)-L-glutamine (5a) as the starting material [42]. After the Hofmann degradation of the carboxamide, the amine group was protected using benzyl chloroformate (CbzCl), followed by the protection of the carboxyl group with a tert-butyl ester, yielding Nα-tert(butyloxycarbonyl)-Nγ-benzyloxycarbonyl-L-2,4-tert-butyl-diaminobutyrate (5b). This intermediate 5b underwent further reactions, including catalytic hydrogenation to selectively deprotect the amine group, followed by treatment with BrCN and NH2OH-HCl to introduce the hydroxyguanidine group, forming Nα-tert(butyloxycarbonyl)-Nω-hydroxy-nor-L-tert-butyl arginine (5c). These steps were streamlined into two stages. Finally, dry HCl removed all protecting groups, yielding the target compound, nor-NOHA.2HCl (5) [43].
Two additional first-generation inhibitors, Nω-hydroxy-indospicin (6) and 4-hydroxy-amino-D,L-phenylalanine (7), are analogues of L-NOHA in which the hydroxyguanidine group is replaced by hydroxyamidine. Both have shown micromolar activity against bovine liver ARG ([14C] urea assay) with IC50 values of 50 ± 10 μM for compound 6 and 230 ± 5 μM for compound 7 [41].
The synthesis of compounds 6 and 7 (see Scheme 3) has been described by Vadon et al. Briefly, N-BOC-glycine was alkylated using 5-bromopentanenitrile (for compound 6) or 4-(bromomethyl)benzonitrile (for compound 7) in dry tetrahydrofuran (THF) using lithium diisopropylamide (LDA). The resulting products, Nα-tert-butoxycarbonyl-6-cyano-D,L-norleucine (6b) and Nα-tert-butoxycarbonyl-p-cyano-D,L-phenylalanine (7b) had their carboxyl groups protected as a tert-butyl ester using benzyltriethylammonium chloride, K2CO3, and tert-butyl bromide in dimethylacetamide (DMAc). The intermediates Nα-tert-butoxycarbonyl-6-cyano-D,L-norleucine-tert-butylester (6c) and Nα-tert-butoxycarbonyl-p-cyano-D,L-phenylalanin-tert-butylester (7c) were refluxed with ethanolic hydroxylamine for 6 h to form the hydroxiimidine derivatives Nα-tert-butoxycarbonyl-Nω-hydroxy-D,L-indospicine-tert-butylester (6d) and Nα-tert-butoxycarbonyl-p-hydroxyamidino-D,L-phenylalanine-tert-butylester (7d). In the final synthetic step, treatment with HCl in dry dioxane at r.t. for 24 h removed the protecting groups, producing the final compounds Nω-hydroxy-D,L-indospicine (6) and 4-hydroxy-amino-D,L-phenylalanine (7), respectively [44].
α-Difluoromethylornithine (DFMO, 8) is an irreversible inhibitor of ornithine decarboxylase (ODC), the enzyme involved in polyamines biosynthesis [34,45]. It also exhibited weak inhibitory activity against intestinal ARG, with a Ki of 3.9 ± 1.0 mM in intact HT-29 cells and 80 ± 3% inhibition at 10 mM on bovine liver arginase [46]. The synthesis of DFMO is shown in Scheme 4 [47]. The process began with glycine ethyl ester hydrochloride salt (8a) reacting with benzaldehyde in the presence of magnesium sulphate, acetonitrile (ACN), and triethylamine (TEA) to form the 2-benzylideneamino glycine ethyl ester (8b). This intermediate was then treated with acrylonitrile in the presence of K2CO3 and triethylbenzylammonium chloride, yielding ethyl 2-benzylideneamino-4-cyanobutyrate (8c). Next, 8c reacted with chlorodifluoromethane and lithium tert-butoxide in THF at 40 °C producing ethyl-2-benzylideneamino-2-difluoromethyl-4-cyanobutyrate (8d). Compound 8d was then deprotected with 4 M HCl in methyl-tert-butyl ether (MTBE) to give ethyl-2-amino-2-difluoromethyl-4-cyanobutyrate (8e), which underwent catalytic hydrogenation in MTBE and 12 M HCl to form ethyl 2,5-diamino-2-difluoromethylpentanoate dihydrochloride (8f). In the final step, 8f was deprotected with 12 M HCl, yielding DFMO monohydrochloride monohydrate (8).

2.2. Boronic Acid Derivatives

Another class of first-generation ARG inhibitors consists of boronic acid derivatives. The compound 2(S)-amino-6-boronohexanoic acid (ABH, 9) is the first boronic acid-based arginine isostere [48]. ABH exhibited strong activity, with a Kd of 5 nM for hARG-1, a Ki of 8.5 nM for hARG-2, and an IC50 of 0.8 μM for rat liver ARG-1 [22]. The synthesis of ABH is outlined in Scheme 5 [48,49]. First, (R)-5-(tert-butoxy)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoic acid (9a) was reduced using sodium borohydride and ethyl chloroformate to produce a primary alcohol derivative (9b). In the next step, 9b underwent Swern oxidation to form an aldehyde 9c, which was used directly in a Wittig reaction with triphenylphosphonium methylilide, yielding olefin (9d). The hydroboration of 9d, followed by treatment with MeOH and protection with (1S,2S,3R,5S)-(+)-pinanediol, yields intermediate 9e. The final step involved complete deprotection using BCl3, yielding ABH (9) as a white semi-crystalline solid.
2-Boronoethyl-L-cysteine (BEC, 10) is an analogue of ABH, where a sulphur atom replaces carbon in the main chain. BEC exhibited activity with a Kd of 270 nM for hARG-1 (ITC) and a Ki of 30 nM for hARG-2 (radioactive assay) [22]. The synthesis of BEC (Scheme 6) involved a single-step reaction [50]. A solution of cysteine (10b) and dibutylenboronate (10a) in MeOH and H2O was refluxed at 80 °C under N2 for 14 h. Afterward, azobisisobutyronitrile was added, yielding the desired compound 10.
ABH and BEC exhibited higher affinity for ARG than α-amino acid derivatives. However, their bioavailability was relatively low, with undetectable plasma levels, likely due to their limited ability to cross biological membranes. Additionally, their unacceptable toxicity to normal cells, stemming from their chemical reactivity and instability, rendered them unsuitable for clinical use in cancer treatment. As a result, a structural optimization study, along with the exploration of medicinal chemistry strategies—such as drug delivery systems—became crucial. Approaches aimed at enhancing the permeability and stability of these ARG inhibitors, potentially through carrier or bioprecursor prodrug strategies, are urgently needed.

3. Second-Generation Inhibitors

Second-generation inhibitors include α-substituted ABH analogues, which have shown promising and potent ARG inhibitors. These inhibitors are further divided into two subclasses: basic side chain α-substituted ABH analogues (see Section 3.1) and non-basic side chain α-substituted ABH analogues (see Section 3.2) [16].

3.1. Basic Side Chain α-Substituted ABH Analogues

The first second-generation molecule discussed in this review is (R)-2-amino-6-borono-2-(2-piperidin-1-yl) ethyl) hexanoic acid (11), which demonstrated inhibition activity against hARG-1 and hARG-2 with IC50 values of 223 nM and 509 nM, respectively [24]. Its cellular activity against hARG-1 in CHO cells was also confirmed (IC50 = 509 nM) [22]. The patented synthesis of compound 11 (Scheme 7) began with (4S,5S)-tert-butyl-6-oxo-4,5-diphenyl-1,3-oxazinane-3-carboxylate (11a), which was alkylated with 2-(4-iodobutyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (11b) using NaHMDS and HMPA in THF at −78 °C, forming boronate (11c). A second alkylation with allyl iodide, using potassium bis(trimethylsilyl)amide (KHMDS) as the base and DME (dimethoxyethane) as the solvent, yielded oxazinone 11d, which followed two possible synthetic routes a or b. In route a, 11d underwent ozonolysis in DCM at −78 °C (11e) followed by reductive amination with piperidine, NaBH(OAc)3, and AcOH in DCM to produce amine 11f in a 95% yield. The auxiliary oxazinone was then removed by treatment with 6 M HCl in a microwave reactor at 170 °C for 40 min, yielding compound 11 in a 87% yield. For route b, the steps were reversed. First 11d was treated with Li in liquid ammonia to produce (R)-methyl 2-allyl-2-(tert-butoxycarbonylamino)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hexanoate (11g) as a colourless oil. This intermediate was then subjected to ozonolysis in DCM at −78 °C (11h), followed by reductive amination with piperidine, NaBH(OAc)3, and AcOH forming amine 11i in 67% yield. The deprotection of 11i with 6 M HCl under reflux for 16 h produced the final compound 11 as a dihydrochloride salt [51]. The two synthetic procedures for compound 11 (route a and route b) were developed to facilitate large-scale synthesis. Route a required fewer synthetic steps and provided a higher overall yield compared to route b. The need to optimize the synthesis of compound 11, addressed by the same authors who designed this inhibitor, was driven by the need for a precise configuration of the target inhibitor and the requirement to start with a flexible intermediate, such as the aldehyde, which could be prepared on a large scale and with high enantioselectivity.
Among α, α-disubstituted amino acid-based ARG inhibitors, compound 2-amino-6-borono-2-(1-(3,4-dichlorobenzyl) piperidin-4-yl) hexanoic acid (12), stands out. This derivative, featuring a piperidine ring linked to a quaternary amino acid centre, was synthesized by Golebiowski et al. (Scheme 8) [26]. It demonstrated potent inhibitory activity with IC50 values of 200 nM for hARG-1 and 290 nM for hARG-2 [16]. The synthesis of compound 12 began with the reaction of 1-tert-butyl-4-methyl piperidine-1,4-dicarboxylate (12a) with N,O-dimethylhydroxylamine and i-PrMgBr in THF, forming Weinreb amide 12b. Next, 12b underwent substitution with but-3-en-1-yl-magnesiumbromide in THF at 0 °C under N2 yielding tert-butyl 4-(pent-4-enoyl) piperidine-1-carboxylate (12c) in a 91% yield. In the third step, an Ugi reaction was performed by treating ketone 12c with tert-butyl isocyanide and ammonium acetate (NH4OAc) in 2,2,2-trifluoroethanol, producing intermediate tert-butyl-4-(2-acetamido-1-(tert-butylamino)-1-oxohex-5-en-2-yl) piperidine-1-carboxylate (12d). This was followed by a hydroboration reaction using pinacolborane, chloro-1,5-cycloctadiene iridium (I) dimer ([Ir(cod)Cl]2), and 1,2-bis(diphenylphosphino)ethane (dppe) in THF, to yield derivative 12e. A subsequent step included the deprotection of the piperidine ring with 2 M HCl in dioxane at r.t. for 2 h. The deprotected piperidine was then N-alkylated with 3,4-dichlorobenzaldehyde and NaBH(OAc)3 in 1,2 dichloroethane (DCE) at r.t. for 16 h. Final acid deprotection with 6 M HCl yielded the target compound 12 [26,51]. This synthetic procedure was optimized by the authors and involved seven steps, each achieving notably high yields. The optimization process incorporated a multi-component reaction strategy, specifically the Ugi reaction, using a ketone derivative (12c) as a model substrate and tert-butyl isocyanide. Optimal conditions were achieved by using ammonium acetate as both the amino and acid component, along with replacing methanol with trifluoroethanol as the solvent. This adjustment effectively suppressed the competitive Passerini reaction.
Two additional N-alkylated piperidine derivatives are 2-amino-6-borono-2-(1-(4-chlorobenzyl) piperidin-4-yl)-hexanoic acid (MARS, 13) and 2-amino-6-borono-2-(1-(4-fluorobenzyl)piperidin-4-yl)hexanoic acid (FMARS, 14), which differ in the halogen substituent. MARS exhibited IC50 values of 0.9 μM for hARG-1 and 0.7 μM for hARG-2, while FMARS showed IC50 values of 1.1 μM for hARG-1 and 0.4 μM for hARG-2 [52]. The synthesis of both compounds (Scheme 9), reported by Clemente et al., followed the patented protocol established by Adam Golebiowski et al. [51,52]. The strategy was similar to that used for compound 12, with the key difference being the use of a reductive amination step (4-chloro and 4-fluoro benzadelhydes for MARS and FMARS, respectively).
Another class of ARG inhibitors includes tropane derivatives (1519), characterized by a two-carbon bridge within the piperidine ring, which enhances their activity. These compounds demonstrated significant inhibition of hARG-1 and hARG-2, with IC50 values ranging from nanomolar to micromolar levels [22,24,26,52,53,54]. The increased potency of tropane derivatives 1519 compared to the simpler piperidine derivatives 1214 has been thoroughly investigated. For derivatives 1214, the piperidine ring adopts a chair conformation, with the nitrogen atom interacting with two aspartate residues in the binding pocket of ARG-1 and ARG-2 through a water molecule. In contrast, for derivatives 1519, the two-carbon bridge of the tropane forces the piperidine ring into a boat conformation, enabling the nitrogen atom to establish direct contact with one of the aspartate residues in the pocket of interest. The other aspartate residue is still contacted via the water molecule. Thus, the tropane derivatives benefit from two key advantages that explain their enhanced potency: the fixed ring geometry positions the nitrogen atom optimally (resulting in an entropy gain) and the nitrogen establishes direct contact with one aspartate residue, bypassing the need for water-mediated interaction. Furthermore, this significant improvement in in vitro activity was accompanied by reduced polarity and an improved pharmacokinetic profile.
The synthesis of the derivatives 1519 involved a seven-step protocol (Scheme 10) [55]. The initial four steps were consistent across all compounds. For the synthesis of compound 2-amino-2-(8-azabicyclo [3.2.1]octan-3-yl)-6-boronohexanoic acid (16), the fifth and sixth steps were omitted, proceeding directly to the final stage. For compounds 2-amino-6-borono-2-((1R,5S)-8-(3,4-dichlorobenzyl)-8-azabicyclo[3.2.1]octan-3-yl)hexanoic acid (ABHtrop, 15), 2-amino-2-(8-benzyl-8-azabicyclo[3.2.1]octan-3-yl)-6-boronohexanoic acid (17), 2-amino-6-borono-2-(8-(3-chlorobenzyl)-8-azabicyclo[3.2.1]octan-3-yl) hexanoic acid (18), and 2-amino-6-borono-2-(8-(3,4-difluorobenzyl)-8-azabicyclo [3.2.1]octan-3-yl) hexanoic acid (19), the sixth step varied depending on the aldehyde reagent used.
The synthetic strategy for these tropane ring analogues closely resembles that developed by the same authors for compound 12. In this case, the intermediate 15b was used as the model substrate in the Ugi reaction. Both this intermediate and the subsequent Ugi product, 15c, were thermodynamically favoured. Notably, the use of the trans isomer of derivative 15b as a substrate for the Ugi reaction resulted in the same relative stereochemistry for 15c. Starting with (1R,5S)-8-(tert-butoxycarbonyl)-8-azabicyclo[3.2.1]octane-3-carboxylic acid (15a), the first step was the coupling with N-methyl-N-methoxyamide using MeNH(OMe), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP) in DCM at r.t. The intermediate underwent substitution with but-3-en-1-yl-magnesium bromide in THF at 0 °C, forming (1R,5S)-tert-butyl 3-(pent-4-enoyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (15b). In the next step, 15b participated in an Ugi reaction with NH4OAc, tert-butyl-isocyanide, and 2,2,2-trifluoroethanol at r.t. for 7 days, yielding (1R,3s,5S)-tert-butyl 3-(2-acetamido-1-(tert-butylamino)-1-oxohex-5-en-2-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (15c). Notably, both ketone 15b and Ugi product 15c were thermodynamically favoured, with the trans isomer of 15b yielding the same stereochemistry in 15c. Next, 15c underwent hydroboration reaction with pinacolborane, [Ir(cod)Cl]2, and dppe in THF, producing a hydroborate intermediate. This was treated with 4 M HCl in dioxane at r.t. for 3 h to selectively deprotect the tropane nitrogen. N-alkylation for compounds 15 and 1719 was carried out using NaBH(OAc)3 and various aldehydes (3,4-dichlorobenzaldehyde for compound 15, benzaldehyde for 17, 3-chlorobenzaldehyde for 18, and 3,4-difluorobenzaldehyde for 19) in DCE. The intermediates were finally deprotected with 6 M HCl at 95 °C for 16 h, producing the desired compounds.
The same strategy was applied to synthesize FBMARS (20, Scheme 11). Starting from amino acid 20a, a coupling reaction with N,O-dimethylhydroxyalamine hydrochloride in the presence of EDC, HOBt, and trimethylamine (TMA) in DCM yielded the Weinreb amide 20b. This intermediate was substituted with but-3-en-1-yl-magnesiumbromide in dry THF, forming ketone 20c. Ketone (20c) underwent an Ugi reaction with NH4OAc, 2,2,2-trifluoroethanol, and tert-butyl-isocyanide, yielding 20d after 3 weeks. Hydroboration with dppe, [Ir(cod)Cl]2, DCM and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane produced 20e. Subsequent treatment with 4 M HCl in dioxane for 1 h removed the tert-butyloxycarbonyl group, yielding 20f as a salt. This salt was N-alkylated with 4-fluorobenzaldehyde in DCE using TMA and sodium triacetoxyborohydride, forming intermediate 20g. Finally, deprotection with 6 M HCl in DCM gave FBMARS (20) [52].
The same research group detailed the synthesis of the 18F-fluoroanalogues of FMARS (14) and FBMARS (20) using copper-mediated late-stage radiofluorination. These radioactive compounds are highly valuable for ARG imaging, which has proven effective in detecting and monitoring ARG-related diseases. The structures of 18F-FMARS and 18F-FBMARS are shown in Figure 3.
Another class of ARG inhibitors includes N-alkylcyclobutylamine derivatives, featuring a nitrogen atom substituted with diverse functional groups such as phenyl and biphenyl moieties, which can be further modified. As summarized in Table 1, these compounds (2146) exhibited activity against hARG-1 and hARG-2, with potency ranging from 0.1 nM to 100 nM.
The synthesis of compounds 2146, patented by Van Zandt et al. and illustrated in Scheme 12, closely resembles previously described methods. The process involved seven steps differing only in the reagent used during the sixth step [55]. The first step was a coupling reaction involving (1S,3S)-3-(tert-butoxycarbonylamino)cyclobutanecarboxylic acid (21a), N,O-dimethylhydroxylamine hydrochloride, N-methyl-morpholine, and EDC in DCM. This reaction, carried out at r.t. over 18 h, produced tert-butyl(1S,3S)3-(methoxy(methyl)carbamoyl)cyclobutylcarbamate (21b). The second step involved the substitution of 21b with 3-butenylmagnesium bromide in THF for 2 h at r.t., yielding tert-butyl-(1S,3S)-3-pent-4-enoylcyclobutylcarbamate (21c). In the third step, an Ugi reaction with 21c in 2,2,2-trifluoroethanol, NH4OAc and tert-butyl isocyanide over 4 days produced tert-butyl(1S,3S)-3-(R-2-acetamido-1-(tert-butylamino)-1-oxohex-5-en-2-yl)cyclobutylcarbamate (21d). Next, 21d underwent hydroboration with pinacolborane in the presence of [Ir(cod)Cl]2 and dppe in DCM, forming tert-butyl-(1S,3S)-3-((R)-2-acetamido-1-(tert-butylamino)-1-oxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hexan-2-yl)cyclobutylcarbamate (21e). In the fifth step, 21e was treated with 4 M HCl in dioxane for 3 h to remove the tert-butyloxycarbonyl group, yielding (S)-2-acetamido-2-((1S, 3R)-3-aminocyclobutyl)-N-tert-butyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hexanamide (21f) as a hydrochloride salt. For the sixth step, 21f was reacted overnight with acetic acid, NaBH(OAc)3, and various aldehydes (depending on the desired derivative) in DCE, producing the respective N-alkylated intermediates (21g). In the final step, these intermediates were treated with 6 M HCl and heated to 100 °C overnight, yielding the final products 2146, whose names are provided in Table 1.
Further investigations into the N-alkylcyclobutylamine scaffold led to the development of tertiary amine derivatives (compounds 4749). However, these derivatives displayed lower potency compared to the parent compounds 2146. Their syntheses and structures are shown in Scheme 13.
The synthesis of compounds 4749 followed an eight-step protocol starting with the reaction of 3-oxocyclobutanecarboxylic acid (47a) in MeOH and p-toluensulfonic acid at 55 °C for 3 days to yield methyl-3,3-dimethoxycyclobutanecarboxylate (47b). This intermediate underwent coupling with N,O-dimethylhydroxylaminehydrochloride and isopropylmagnesium chloride in THF to produce 47c. Next, substitution with 3-butenylmagnesium bromide yielded 1-(3,3-dimethoxycyclobutyl)pent-4-en-1-one (47d), which was subjected to an Ugi reaction with ammonium acetate, tert-butyl-isocyanide, and 2,2,2-trifluoroethanol, producing 2-acetamido-N-tert-butyl-2-(3,3-dimethoxycyclobutyl)hex-5-enamide (47e). The hydroboration of 47e with pinacolborane, [Ir(cod)Cl]2 and dppe generated 2-acetamido-N-tert-butyl-2-(3,3-dimethoxycyclobutyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hexanamide (47f), which was then treated with p-toluensulfonic acid overnight in acetone to form 2-acetamido-N-tert-butyl-2-(3-oxocyclobutyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl hexanamide (47g), containing an oxidized cyclobutyl ring. In the penultimate step, 47g was reacted with either dibenzylamine (for compound 47), its trans isomer (for 48), or isoindoline (for 49), followed by NaBH(OAc)3 reduction to form intermediates 47h. Finally, global deprotection using 6 M HCl at 100 °C yielded the desired compounds 4749 [47].
The basic side chain α-substituted ABH analogues (compounds 5051) also emerged as potent ARG inhibitors. Compound 50, i.e., 2-amino-6-borono-2-(3-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)propyl)hexanoic acid, with a cyclic amine side chain, showed strong inhibitory activity (Ki < 10 nM) against both hARG-1 and 2 [22]. Compound 51, featuring a linear amine side chain, exhibited comparable potency.
Their synthesis involved a nine-step protocol (Scheme 14) [56]. The initial steps consisted of sequential alkylations starting from tert-butyl-2-((diphenylmethylene)amino)acetate (50a) generating tert-butyl-5-(tert-butyldimethylsilyoxy)-2-(diphenylmethyleneamino)pentanoate (50b) and then tert-butyl-2-(3-tert-butylmethylsilyloxy)propyl)-2-(diphenylmethylenamino)hex-4-enoate (50c). After amine deprotection with hydroxylamine hydrochloride, the amino group was re-protected with benzyl chloroformate to yield tert-butyl-2-(benzyloxycarbonylamino)-2-(3-(tert-butidimethylsilyloxy)propyl)hex-4-enoate (50e). The hydroboration of 50e with pinacolborane in the presence of [Ir(cod)Cl]2, and dppe in dry DCM gave tert-butyl-2-(benzyloxycarbonylamino)-2-(3-(tert-butidimethylsilyloxy)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hexanoate (50f), followed by the removal of the tert-butyldimethylsilyl (TBDMS) group under acid conditions to form 50g. The alcohol group in 50g was iodinated to produce tert-butyl-2-(benzyloxycarbonylamino)-2-(3-iodopropyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hexanoate (50h). In the eighth step, N nucleophilic substitution was carried out using 4-(4-chlorophenyl)piperidin-4-ol hydrochloride (for compound 50) or 2-phenylethanamine (for compound 51), yielding tert-butyl-2-(benzyloxycarbonylamino)-2-(3-(3-phenylpiperidin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hexanoate (50i). Finally, global deprotection using 1 M HCl, Pd/C, TFA and DCM under argon, followed by the addition of phenyl boronic acid, afforded final products 5051.
OncoArendi Therapeutics (now Molecure) has developed (R)-2-amino-6-borono-2-(guanidinomethyl)hexanoic acid (52), a compound featuring a methylguanidine side chain at the α position relative to the amino acid group. The pure (R)-enantiomer demonstrated potent ARG inhibition, with an IC50 of 32 nM against hARG-1. The synthesis protocol of this compound is shown in Scheme 15 and is patented. The starting compound was ethyl-2-oxohex-5-enoate (52a), which was treated with (S)-(-)-2-methyl-2-propanesulfinamide, Ti(OEt)4 in DCM to form imine (52b), which was reacted in turn with TBAF and MeNO2 undergoing a nitro methylene addition on the α-carbon atom. The asymmetric, diastereoselective aza-Henry reaction led to the formation of the nitro amine ester (52c), which was subjected to hydroboration using pinacolborane, [Ir(cod)Cl]2, dppe, DCM, and the subsequent reduction of the nitro group with NaBH4 and NiCl2-6H2O in MeOH to obtain boronate (52e). The amine (52e) was then guanylated using tert-butyl(((tert-butoxycarbonyl)amino)(1H-pyrazol-1-yl)methylene) carbamate, TEA, and ACN and was deprotected with a solution of 6 M HCl to obtain the desired compound (52) [25,57].

3.2. Non-Basic Side Chain α-Substituted ABH Analogues

To this subclass belong sulphamide derivatives such as 2-amino-6-borono-2-((sulfamoylamino)methyl)hexanoic acid (53), which exhibited an inhibition activity against hARG-1 with an IC50 of 330 nM [25]. The synthesis of 53 (see Scheme 16) consisted of an alkylation reaction of compound 53a with pinacol-4-bromobutylboronate and sodium hydride (NaH) in N,N-dimethylformamide (DMF) to obtain a quaternary boronic cyanoaminoester (53b), which was then reduced with sodium borohydride (NaBH4) to form the intermediate 53c. Compound 53c was subsequently subjected to sulphamoylation with chlorosulfonyl isocyanate (ClSO2NCO) in the presence of tert-butanol in TEA and DCM at 0 °C. The final step involved acid hydrolysis with HCl/dioxane, yielding the desired boronic acid (53).
Other compounds designed as ARG inhibitors with a non-basic side chain included derivatives 5455, which contained urea and thiourea moieties, respectively. Both 54 (2-amino-6-borono-2-(1-((4-chlorophenyl)carbamoyl)piperidin-4-yl)hexanoic acid) and 55 (2-amino-6-borono-2-(1-((4-chlorophenyl)carbamothioyl)pyrrolidin-3-yl)hexanoic acid) were capable of inhibiting hARG-1 and -2 in the range of 251 to 1000 nM. Despite their similar activities, the synthesis pathways for these compounds differed [22,51]. For compound 54 (Scheme 17), the first four-steps of its synthesis were identical to those for compound 12, as shown in Scheme 8. Starting from intermediate 12e, the fifth step involved an acid deprotection reaction using 6 M HCl solution at 95 °C for 16 h, giving intermediate 54a. This intermediate was then selectively carbamylated at the piperidine nitrogen using 4-chlorophenyl isocyanate (p-Cl–PhNCO) in the presence of TEA and DMF, producing the target compound 54 [26].
For the synthesis of compound 55, as shown in Scheme 18, Van Zandt et al. used 55a as the starting material [51]. This compound underwent a coupling reaction with N,O-dimethylhydroxylamine hydrochloride and EDC, yielding intermediate 55b, which in turn was then subjected to a substitution with 3-butenylmagnesium bromide in THF producing compound 55c. Next, an Ugi reaction was performed with NH4OAc and tert-butyl isocyanide in 2,2,2-trifluoroethanol, generating derivative 55d. This was followed by a hydroboration reaction using pinacolborane, [Ir(cod)Cl]2 and dppe to give tert-butyl-(3R)-3-[1-acetamido-1-(tert-butyl-carbamoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pentyl]pyrrolidine-1-carboxylate (55e). In the fifth step, a solution of 6 M HCl in 1,4-dioxane was added to 55e, yielding intermediate 55f, which was then treated with 4-chlorophenyl-isothiocyanate in TEA and DMF to obtain the desired compound 55. In addition to the inhibitors mentioned, several other compounds featuring different substituents in their side chains were synthesized, through their activities were found to be comparatively lower [58].
Comparing the two subclasses of second-generation ARG inhibitors, it was observed that the presence of a basic side chain in the α-substituted ABH derivatives led to a notable improvement not only in activity but also in selectivity against both ARG-1 and ARG-2, compared to their non-basic counterparts. This enhancement was attributed to the formation of an additional hydrogen bond, mediated by a water molecule, between the basic centre in the side chain of the ARG inhibitor and an aspartate residue in the pocket of both ARG-1 and ARG-2—a feature absent in non-basic side chain α-substituted ABH analogues. However, despite the improved selectivity and activity, the presence of a basic side chain did not result in significant improvements in bioavailability compared to non-basic analogues, and the pharmacokinetic profile still required optimization.

4. Third-Generation Inhibitors

The third generation of inhibitors features ring-constrained cyclic ABH analogues, including compounds based on modified cyclopentyl and pyrrolidine structures. These compounds have an alkyl linker with a boronic acid group inserted at ring position 2. One example is (1S,2S,4R)-1-amino-4-(aminomethyl)-2-(3-boronopropyl)cyclopentanecarboxylic acid (56), which showed an IC50 of 0.1–0.250 nM for both hARG-1 and hARG-2 in a colorimetric assay. While the cited patent describes many analogues of 56, the lack of its further development is likely due to its unsatisfactory pharmacokinetic properties. These issues could potentially be addressed through strategies such as the prodrug approach or the design of nanodelivery systems, which, as demonstrated in thousands of studies, have been shown to overcome various pharmacokinetic and related limitations. Compound 56 was synthesized by Van Zandt et al. according to the Scheme 19 [59].
The synthesis began with commercially available 5-(propene-3-yl)cyclopent-2-enone (56a), which was treated in nitromethane with the DOWEX® 550A-OH resin at 60 °C to obtain the nitroderivative (56b). This was then dissolved in 2,2,2-trifluoroethanol and reacted with NH4OAc and tert-butylisonitrile under N2 for 2 days, affording two isomers, 56c1 and 56c2, with acetamino and allyl substituents in the syn-relative position. Isomer 56c1 was stirred in DCM and treated with pinacolborane, [Ir(cod)Cl]2 and Diphos® at –25 °C, yielding 56d (isomers 56d1 and 56d2). For the final step, a solution of (1S,2S,4R)-1 -acetamido-N-tert-butyl-4-(nitromethyl)-2-(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)propyl)cyclopentanecarboxamide (isomer 56d1) in ethanol and THF under N2 was treated with Raney nickel. The mixture was purged with H2 and stirred for 20 h. After purging with nitrogen, the mixture was filtered through Celite®, and the filtrate concentrated under reduced pressure. The crude product was dissolved in HCl/acetic acid/H2O in a pressure bottle and stirred for 2 h at 60 °C, then capped and heated for 18 h at 130 °C. After cooling to r.t. and uncapping, the desired compound 56 was obtained after chromatography purification as a white foam.
A wide range of pyrrolidine-derived compounds has been developed through further optimization. The ring-constrained pyrrolidines help reduce entropy by positioning the quaternary amino acid moiety in an optimal orientation for binding. Notably, the presence of a cyclopentane derivative forces the amino group and boronic acid side chain into an anti orientation, preventing van der Waals interactions with active site residues. Furthermore, the atoms within the ring act as scaffolds for incorporating additional substituents that can form hydrogen bonds with the aspartate residues in the ARG-1 and ARG-2 pockets. These structural features collectively contribute to the enhanced potency of these compounds. The most effective compound in this series was NED-3228 (57, Figure 4), which showed IC50 values for hARG-1 and hARG-2 of 1.3 and 8.1 nM, respectively. Another notable compound, 58 (see Figure 4), features an N-(piperidin-2-yl-methyl) substituent in place of the N-(2-amino-3-phenylpropyl) group in 57. This compound exhibited IC50 values for hARG-1 and hARG-2 of 2.6 and 14 nM, respectively.
Compound 57 was synthesized by Van Zandt et al. through a nine-step process, as outlined in Scheme 20 [60]. The synthesis began by reacting a solution of tert-butyl-6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate (57a) in dry Et2O with allyl magnesium bromide at 0 °C. After stirring for 15 min, the reaction produced tert-butyl-trans-3-allyl-4-hydroxypyrrolidine-1-carboxylate (57b). This was then treated with sulphur trioxide pyridine complex in DMSO under nitrogen, yielding ketone intermediate 57c. Compound 57c was reacted with tert-butylisocyanide and ammonium acetate, resulting in a mixture of anti- and syn-isomers of 57d. Next, the BOC group was removed using DCM and TFA giving racemic 57e. To obtain the pure enantiomer 57e1, racemic 57e was treated with (2S,3S)-2,3-bis(benzoyloxy)-4-(isopropylamino)-4-oxobutanoic acid in warm MeOH/i-PrOH. After cooling, the desired salt crystallized out, and the crystals were filtered, washed, and dried to give 57e1 with 99.7% ee. In the following step, 57e1 was reacted with pinacolborane in the presence of [Ir(cod)Cl]2 and dppe in DCM to afford 57f as a white solid. A BOC deprotection with an excess of TFA in dry DCM under N2 yielded 57g. This was then reacted with tert-butyl-(S)-4-phenyl-1,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide in ACN at r.t. for 2 days, producing 57h, which was used directly without further purification. Finally, 57h was deprotected in glacial acetic acid, water, and concentrated hydrochloric acid in a pressure bottle. After stirring at 60 °C for 2 h, the mixture was capped and heated to 130 °C for 18 h to yield NED-3228 (compound 57) as a white solid.
Compound (3R,4S)-3-amino-1-(N-(2-aminoethyl)sulfamoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid (59) also demonstrated strong activity against hARG-1 and hARG-2, with IC50 values ranging from 0.1 to 100 nM for both enzymes. Compound 59 was synthesized by Wan et al. in 2018, following the procedure outlined in Scheme 21 [61]. The synthesis began with the reaction between sulfurisocyanatidic chloride and 2-bromoethan-1-ol in DCM, followed by the addition of tert-butyl-(2-aminoethyl)carbamate and TEA in DCM. The intermediate (tert-butyl (2-((2-oxooxazolidine)-3-sulfonamido)ethyl)carbamate) 59a was reacted with a solution of (rac)benzyl trans-4-allyl-3-azidopyrrolidine-3-carboxylate in ACN and TEA, resulting in 59b ((rac)(benzyl trans-(4-allyl-3-azido-1-(N-(2-((tert-butoxycarbonyl)amino)ethyl)sulfamoyl)pyrrolidine-3-carboxylate). Next, 59b was hydroborated with pinacolborane, [Ir(cod)Cl]2 and dppe in DCM producing 59c ((rac)(benzyl trans-3-azido-1-(N-(2-((tert-butoxycarbonyl)amino)ethyl)sulfamoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate)). Compound 59c was catalytically hydrogenated in a mixture of EtOAc/EtOH giving rise to 59d ((rac)-trans-3-amino-1-(N-(2-((tert-butoxycarbonyl)amino)ethyl)sulfamoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylic acid). Finally, 59d was deprotected in HCl at 50° C. This reaction yielded the desired compound, (rac)-3-amino-1-(N-(2-aminoethyl)sulfamoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid (59) that was purified by HPLC and isolated as a 1:2 trifluoroacetic acid salt.
The bicyclic inhibitors (3aR,4S,5S,6aR)-5-amino-4-(3-boronopropyl)-2-(1-chloro-3-hydroxypropan-2-yl)octahydrocyclopenta[c]pyrrole-5-carboxylic acid (60), (1S,2S,3aR,4S,5S,6aS)-2-amino-1-(3-boronopropyl)-4-fluoro-5-(methylamino)octahydropentalene-2-carboxylic acid (61), and (5S,7S,8S)-7-amino-8-(3-boronopropyl)-1-azaspiro[4.4]nonane-7-carboxylic acid (62) represent a distinct class of inhibitors, independently developed by Merck Sharp & Dohme Corp. and Arcus Biosciences Inc. These compounds are derivatives of octahydrocyclopenta[c]pyrrole with various nitrogen atom substituents. Foley et al. synthesized a range of bicyclic boronic acid derivatives, with compound 60 proving to be the most promising, showing an IC50 < 100 nm against hARG-1. Compound 61 demonstrated an IC50 of 6.9 nM against hARG-1 performed by Thioornithine Generating Assay (TOGA). The general synthesis scheme for compound 60 is shown in Scheme 22 [62].
In the first step, tert-butyl-5-oxohexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (60a) was alkylated under the action of allyl bromide and LiHMDS in THF. This produced the allylated compound tert-butyl 4-allyl-5-oxohexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (60b). Subsequently, a reaction of ketone 60b with CHCl3 and chlorotrimethylsilane in strongly basic conditions, followed by the deprotection of trimethylsilyl group under the action of acetic acid and tetrabutylammonium fluoride in THF gave rise to the racemic trichloromethyl derivative 60c. Chiral column chromatography was used to isolate the desired enantiomer. The treatment of 60c with NaN3 and NaOH produced the crude carboxilic acid 60d ((3aR,4S,5S,6aR)-4-allyl-5-azido-2-(tert-butoxycarbonyl)octahydrocyclopenta[c]pyrrole-5-carboxylic acid). The carboxylic group was protected as benzylic ester using benzyl bromide and K2CO3 in dry ACN. Next, 60e was subjected to a hydroboration reaction with [Ir(cod)Cl]2, dppe, and pinacolborane in DCM. After purification, the product 5-benzyl 2-(tert-butyl) (3aR,4S,5S,6aR)-5-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)hexahydrocyclopenta[c]pyrrole-2,5(1H)-dicarboxylate (60f) was obtained. The BOC group was removed by treating 60f with DCM and TFA, giving the free amine (60g) in a 99% yield. Amine (60g) in DCM was reacted with 1-chloro-3-hydroxy acetone and Na(OAc)3BH, yielding benzyl (3aR,4S,5S,6aR)-5-azido-2-(1-chloro-3-hydroxypropan-2-yl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)octahydrocyclopenta[c]pyrrole-5-carboxylate (60h), which was used without purification. Finally, 60 was obtained by the hydrogenation of 60h with 10% Pd/C in MeOH and deprotection with 3M HCl at r.t. for 1 h. Purification by reverse phase C18 chromatography yielded the target compound 60 as a white solid.
The synthesis of 61 (Scheme 23) began by the introduction of allyl group into the 4-position of (3αR,6αS)-tetrahydro-1H-spiro[pentalene-2,2′-[1,3]dioxolan]-5(3H)-one (61a) under the action of allyl alcohol, 1,1′-ferrocenediyl-bis(diphenylphosphine) (dppf), and allylpalladium(II) chloride dimer in MeOH, producing 61b. To obtain 61c, an Ugi reaction with (3aR,4S,6aS)-4-allyltetrahydro-1H-spiro[pentalene-2,2′-[1,3]dioxolan]-5(3H)-one (61b), NH4OAc, and tert-butyl isocyanide in 2,2,2-trifluoroethanol was performed. (3αR,4S,6αS)-5-acetamido-4-allyl-N-(tert-butyl)hexahydro-1H-spiro[pentalene-2,2′-[1,3]dioxolane]-5-carboxamide (61c) was used without further purification. The deprotection of the dioxalan moiety with p-toluenesulfonic acid in acetone yielded a racemic mixture of epimers (~1:1) resolved using chiral supercritical-fluid chromatography (SFC) to isolate 61d. For the next step, (1S,2S,3aS,6aR)-2-acetamido-1-allyl-N-(tert-butyl)-5-oxooctahydropentalene-2-carboxamide (61d), was reacted with 1-fluoro-4-hydroxy-1,4-diazoniabicyclo[2,2,2]octane bis(tetrafluoroborate) in MeOH at 65 °C for 40 min. This reaction gave two products: (1S,2S,3aS,6R,6aR)-2-acetamido-1-allyl-N-(tert-butyl)-6-fluoro-5,5-dimethoxyoctahydropent alene-2-carboxamide (61e1) and (1S,2S,3aR,4S,6aS)-2-acetamido-1-allyl-N-(tert-butyl)-4-fluoro-5,5-dimethoxyoctahydropentalene-2-carboxamide (61e2). Treating 61e2 with H2O and TFA in DCM at 23 °C and stirring for 1 h provided 1S,2S,3aS,6R,6aR)-2-acetamido-1-allyl-N-(tert-butyl)-6-fluoro-5-oxooctahydropentalene-2-carboxamide (61f) as a white solid. The hydroboration of 61f was performed by adding it in DCM to a solution of (+)-pinanediolborane, [Ir(cod)Cl]2, and dppe under N2 at 23 °C. Stirring for 20 min yielded crude (1S,2S,3aS,6R,6aR)-2-acetamido-N-(tert-butyl)-6-fluoro-5-oxo-1-(3-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2yl)propyl)octahydropentalene-2-carboxamide (61g), used in the next step without further purification. The reaction of 61g with methylamine in absolute EtOH at 0 °C, followed by sodium cyanoborohydride, produced 61h. Finally, the deprotection of 61h using 6 M HCl provided the final product 61 as a white solid [63].
Bicyclic systems, like 62, demonstrated strong activity against both hARG-1 and hARG-2. Compound 62 inhibited hARG-1 with an IC50 of 2.1 nM. However, comparing its activity to other compounds is challenging because Merck utilized the (TOGA), while other groups used a colorimetric assay based on ARG-induced urea production.
The synthesis of 62 (Scheme 24), as reported by Mitcheltree et al., started with the oxidation of 1-azaspiro[4.4]non-7-ene (62a) to produce crude 62b, which was used without purification. Reacting 6-oxaspiro[bicyclo[3.1.0]hexane-3,2′-pyrrolidine (62b) with allylmagnesium bromide in Et2O at 0 °C under N2 gas for 15 h yielded (7R,8R)-8-allyl-1-azaspiro[4.4]nonan-7-ol (62c) as a pale-yellow oil. This intermediate was also used directly. Next, TEA and benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (Cbz-OSu) were added to 62c in DCM, stirred for 2 h at r.t. and yielded (7R,8R)-7-allyl-8-hydroxy-1-azaspiro[4.4]nonane-1-carboxylate (62d) as a colourless oil. Oxidizing 62d with Dess–Martin periodinane provided benzyl 7-allyl-8-oxo-1-azaspiro[4.4]nonane-1-carboxylate (62e), also a colourless oil. The subsequent reaction of 62e with NH4OAc and tert-butyl isocyanide in 2,2,2-trifluoroethanol at 35 °C for 15 h produced benzyl (5S,7S,8S)-7-acetamido-8-allyl-7-(tert-butylcarbamoyl)-1-azaspiro[4.4] nonane-1-carboxylate (62f), a racemic mixture. Chiral separation using SFC gave the enantiomer (5S,7S,8S)-7-acetamido-8-allyl-7-(tert-butylcarbamoyl)-1-azaspiro[4.4]nonane-1-carboxylate (62g) as a viscous oil. For further modification, 62g was reacted with dppe, [Ir(cod)Cl]2, and pinacolborane in DCM under inert conditions, yielding (5S,7S,8S)-7-acetamido-7-(tert-butylcarbamoyl)-8-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)-1-azaspiro[4.4]nonane-1-carboxylate (62h) as a viscous semisolid. Finally, the deprotection of 62h in 12 M HCl at 110 °C for 12 h produced the target compound (5S,7S,8S)-7-amino-8-(3-boronopropyl)-1-azaspiro[4.4]nonane-7-carboxylic acid (62) as a viscous oil [63].
Highly effective inhibitors of ARG activity include 2-substituted alkylamines, such as the compound (1R,2S,5R)-1-acetamido-5-(2-boronoethyl)-2-(piperidin-1-ylmethyl)cyclohexanecarboxylic acid (63). This compound exhibited potent inhibitory activity, with IC50 values for hARG-1 and hARG-2, ranging from 1 to 100 nM and 100 to 1000 nM, respectively. In cellular assays, compound 63 demonstrated notable activity with an IC50 of up to 100 nM. Other derivatives, like compound 64 (featuring a hydroxy group at position 2), were less potent than the piperidine analogue 63. Compound 64 showed intracellular activity between 10 and 100 μM and IC50 < 1000 nM for both ARGs.
The synthesis of 63 is outlined in Scheme 25 and began with the preparation of ethyl 2-oxocyclohex-3-enecarboxylate (63b) via the acylation of 2-cyclohexen-1-one (63a) with ethyl chloroformate under strongly basic conditions (LDA in THF). Next, ethyl 2-hydroxy-4-vinylcyclohex-1-ene-1-carboxylate (63c) was synthesized by reacting 63b with vinylmagnesium bromide and CuBr x Me2S in the presence of TMSCl under argon at −78 °C. The treatment of 63c with NH4OAc and tert-butyl isocyanide in 2,2,2 trifluoroethanol produced 63d in a 56% yield. The subsequent reduction of ethyl rac-(1R,2R,4R)-2-acetamido-2-(tert- butylcarbamoyl)-4-vinylcyclohexane-1-carboxylate (63d) with diisobutylaluminium hydride (DIBAL-H) at −78 °C, followed by treatment with piperidine and sodium triacetoxyborohydride produced rac-(1R,2S,5R)-1-acetamido-N-tert-butyl)-2-(piperidin-1-yl-methyl)-5-vinylcyclohexanecarboxamide (63e) as a single diastereoisomer. The boronation of 63e using pinacolborane, dppe, and [Ir(cod)Cl]2 in DCM at r.t. formed (1R,2S,5R)-1-acetamido-N-(tert-butyl)-2-(piperidin-1-ylmethyl)-5-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ethyl)cyclohexanecarboxamide (63f). Finally, refluxing 63f with 6 M HCl for 6h produced compound 63 as a white solid [64].
Compound 64 was synthesized similarly to 63, as shown in Scheme 26. Refluxing a mixture of 6-methyl-2-cyclohexenone (64a), benzene, acetic acid, and manganese(III) acetate dihydrate for 6 h produced 64b. Subsequently, 1-methyl-2-oxo-4-vinylcyclohexyl acetate (64c) was synthesized via the 1,4-addition of vinylmagnesium bromide, catalysed by CuBr x Me2S in THF. Next, the Ugi reaction of 64c with NH4OAc and tert-butyl isocyanide in 2,2,2 trifluoroethanol produced 2-acetamido-2-(tert-butylcarbamoyl)-1-methyl-4-vinylcyclohexyl acetate (64d). The hydroboronation of 64d using pinacolborane, dppe, and [Ir(cod)Cl]2 in DCM at r.t. gave 64e as a single diastereoisomer. Finally, refluxing 64e with 6M HCl yielded 1-amino-5-(2-boronoethyl)-2-hydroxy-2-methylcyclohexanecarboxylic acid hydrochloride (64) as a single diastereoisomer (white solid). A by-product of 64f was also obtained as a single diastereosomer [65].
Compound 65 was synthesized similarly to compound 64, but with a different starting material, as illustrated in Scheme 27. The process is not described in detail. After purification via flash chromatography on DOWEX® ion exchange resin, the target product, rac-(3R,5R)-3-aminno-5-(2-boronoethyl)tetrahydro-2H-pyran-3-carboxylic acid hydrochloride (65), was obtained as a single diastereoisomer in a 56% yield (white solid). Compound 65 demonstrated activity against both hARG-1 (IC50 = 100–1000 nM) and hARG-2 (IC50 = 1–10 mM).
A novel class of ARG inhibitors with a proline scaffold was developed by Sichuan Kelun-Biotech Biopharmaceutical, AstraZeneca, and Merck Sharp & Dohme. These inhibitors include proline derivatives with a boronic acid group either in position 1 (alongside a carboxyl group) or at position 2 (as in Merck’s compounds). Merck introduced compounds with various substitutions, such as hydroxyl, alkyl, piperidine, pyrrolidine, and aliphatic amino groups. Among these, a standout was the compound (2S,3R,4R)-4-amino-3-(3-boronopropyl)pyrrolidine-2-carboxylic acid (66), which had an amino group at position 4. This compound demonstrated significant inhibitory activity with an IC50 of 3.2 nM (TOGA) for hARG-1. Another notable compound, (2S,3S,4R)-3-(3-boronopropyl)-4-hydroxypyrrolidine-2-carboxylic acid (67), featured a hydroxyl group at position 4, arranged trans to carboxylic acid and the propylboronic acid linker. Its activity against hARG-1 was slightly lower, with an IC50 of 6 nM. An azetidine-based derivative, (2S,3S)-3-(aminomethyl)-3-(3-boronopropyl)azetidine-2-carboxylic acid (68), was the most effective homolog, showing an IC50 of 8 nM for hARG-1. AstraZeneca and Kelun also contributed to this class of inhibitors. AstraZeneca reported derivatives like (2R,4R)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxilic acid (69), which showed IC50 values of 10 nM for hARG-1 and 20 nM for hARG-2. Its methyl analogue, (2R,4R)-2-(4-boronobutyl)-4-(methylamino)pyrrolidine-2-carboxylic acid (70), displayed even greater potency, with IC50 values of 3 nM for hARG-1 and 10 nM for hARG-2. Kelun’s derivatives included proline analogues substituted with hydroxyl or amino groups. Among these, compound 69 showed moderate activity in a standard assay, with an IC50 activity of 4.3 μM for hARG-1. The synthesis of compounds 6670 follows detailed procedures outlined in Scheme 28, Scheme 29, Scheme 30, Scheme 31 and Scheme 32.
Compound 66 was synthesized as described by Achab et al. (Scheme 28) [66]. The process began with the addition of KHMDS to a solution of 1-(tertbutyl)-2-methyl (2S)-3-allyl-4-oxopyrrolidine-1,2-dicarboxylate (66a) in THF at –78 °C under N2. After stirring and warming to –20 °C, CSA was added, followed by pre-cooled MeOH and NaBH4 at –78 °C. Purification through silica gel chromatography yielded 1-(tert-butyl)2-methyl (2S,3S,4S)-3-allyl-4-hydroxypyrrolidine-1,2-dicarboxylate (66b) as a black oil. In the next step, 66b was reacted with 2,6-dimethylpyridine and chloromethanesulfonyl chloride in DCM at 0 °C, producing crude 1-(tert-butyl) 2-methyl(2S,3S,4S)-3-allyl-4-(((chloromethyl)sulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (66c). Without further purification, 66c was treated with sodium azide in DMSO at 80 °C, yielding crude 1-(tert-butyl) 2-methyl (2S,3S,4R)-3-allyl-4-azidopyrrolidine-1,2-dicarboxylate (66d). Next, pinacolborane, [Ir(cod)Cl]2 and dppe were used in DCM under argon to convert 66d into 1-(tert-butyl) 2-methyl (2S,3S,4R)-4-azido-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-1,2-dicarboxylate (66e) as a colourless oil. This intermediate underwent azide reduction using Pd/C in EtOAc, forming crude 1-(tert-butyl)-2-methyl (2S,3R,4R)-4-amino-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-1,2-dicarboxylate (66f). Finally, 66f was heated with 6 M HCl in a microwave reactor at 120 °C for 1 h to produce (2S,3R,4R)-4-amino-3-(3-boronopropyl)pyrrolidine-2-carboxylic acid (66) as a light brown solid. The same study described the synthesis of 67 (Scheme 29) using the intermediate 66c (Scheme 28).
Caesium acetate and 18-crown-6 were added to a solution of intermediate 66c in toluene stirred under N2 at r.t. This reaction yielded 1-(tert-butyl)-2-methyl (2S,3S,4R)-4-acetoxy-3-allylpyrrolidine-1,2-dicarboxylate (67a) as a colourless oil. In the next step, a mixture of pinacolborane, [Ir(cod)Cl]2, and dppe in DCM was prepared under N2 and stirred at r.t. The solution of 67a was then added, producing 1-(tert-butyl)-2-methyl-(2S,3S,4R)-4-acetoxy-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-1,2-dicarboxylate (67b). For the final step, 67b was deprotected by treatment with 12 M HCl in water, resulting in the formation of (2S,3S,4R)-3-(3-boronopropyl)-4-hydroxypyrrolidine-2-carboxylic acid (67) as a yellowish solid.
The synthesis of 68, outlined in Scheme 30, began with the intermediate 68a, prepared following established literature methods. Intermediate (2S,3S)-tert-butyl-3((benzyloxy)methyl)-1-(9-phenyl-9H-fluoren-9-yl)-3-(3-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-2-yl)prpyl)azetidine-2-carboxylate (68a), used in the synthesis of various other compounds, was subjected to catalytic hydrogenation using 10% Pd/C in MeOH, yielding crude (2S,3S)-tert-butyl 3-(hydroxymethyl)-3-(3-(4,4,5,5-tetramethyl-1,3,3-di-oxaborolan-2-yl)propyl)azetidine-2-carboxylate (68b). This intermediate was used directly in the next step without further purification. To synthesize 68c, TEA and BOC-anhydride were added to 68b at 0 °C, resulting in (2S,3S)-di-tert-butyl 3-((benzyloxy)methyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propryl)azetidine-1,2-di-carboxylate (68c). Subsequent catalytic hydrogenation with 10% Pd/C in MeOH produced (2S,3S)-di-tert-butyl 3-(hydroxymethyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propryl)azetidine-1,2-dicarboxylate (68d), the corresponding hydroxymethyl derivative. Methanesulfonyl chloride and TEA were added to 68d in DCM at 0 °C to form crude (2S,3S)-di-tert-butyl 3-(((methylsulfonyl)oxy)methyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propryl)azetidine-1,2-dicarboxylate (68e), a methylsulfonyl ester derivative. Without purification, 68e was reacted with sodium azide in DMF at 80 °C for 15 h yielding (2S,3S)-di-tert-butyl 3-(azidomethyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propryl)azetidine-1,2-dicarboxylate (68f), an azide derivative. The oxidation of 68f with NH4OAc and sodium periodate in a THF-H2O mixture at r.t. for 15 h yielded crude (3-((2S,3S)-3-(azidomethyl)-1,2-bis(tert-butoxycarbonyl)azetidine-3-l)prpyl)boronic acid (68g), a boronic acid derivative. This intermediate underwent reduction with PPh3 in THF—H2O at 60 °C under N2, forming (3-((2S,3S)-3-(aminomethyl)-1,2-bis(tert-butoxycarbonyl)azetidine-3-l)proyl)boronic acid (68h), an aminomethyl derivative. Finally, TFA was added to 68h in DCM at 20 °C, deprotecting the BOC groups and yielding (2S,3S)-3-(aminomethyl)-3-(3-boronopropyl)azetidine-2-carboxylic acid (68) as the free base [67].
Mlynarski et al. reported the synthesis of many proline derivatives, including 69 (Scheme 31) and 70 (Scheme 32) [68].
The synthesis of 69 began with (2S, 4S)-1-tert-butyl-2-methyl-4-hydroxypyrrolidine-1,2-dicarboxylate (69a), which was treated with methanesulfonyl chloride and TEA in DCM at 0 °C. This yielded 1-(tert-butyl)-2-methyl(2S,4S)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate, an intermediate used directly in the next step. Reaction with sodium azide in DMF produced 69b as a mixture of rotamers. A solution of NaOH in H2O was added to 69b dissolved in THF/MeOH, generating (2S, 4R)-2-benzyl 1-tert-butyl-4-azidopyrrolidine-1,2-dicarboxylate (69c). This compound was treated with crotyl bromide in THF at –78 °C under N2, followed by KHMDS in toluene. The reaction mixture was warmed to r.t. and stirred for 3 h, yielding 69d as a mixture of rotamers and E/Z olefins in a 78% yield. Next, 69d was subjected to hydroboration using pinacolborane, [Ir(cod)Cl]2 and dppe in DCM under N2. Stirring overnight yielded 69e, which was purified by SFC to separate the diastereoisomers. The major diastereoisomer 69f1 was identified as the anti-addition product, while the minor diastereomer 69f2 was the syn-addition product. (2R,4R)-2-benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyk-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (69f2) was subjected to catalytic hydrogenation using 10% Pd/C in a mixture of EtOAc and MeOH. This step produced 69g, which was used without further purification. In the final step, 69g was first treated with TFA in DCM at r.t. and then with 1 M HCl and phenylboronic acid in Et2O, obtaining (2R,4R)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxilic acid (69) as a white solid.
Compound 70, an analogue of 69 with a methyl amino group at position 4 of the proline ring, was synthesized starting from 1-(tert-butyl)2-methyl (2S,4R)-4-aminopyrrolidine-1,2-dicarboxylate (70a). The primary amino group in 70a was protected using BOC-anhydride, and then methylated with NaH and CH3I in DMF to form intermediate 70c. The synthesis from 70c followed the same procedures as those for compound 69, starting from the second step onward. The final product, (2R,4R)-2-(4-boronobutyl)-4-(methylamino)pyrrolidine-2-carboxylic acid (70), was obtained as a white solid.

5. Fourth-Generation Inhibitors

In this section of the review, we introduce a new class of compounds: the fourth-generation ARG inhibitors. These inhibitors are cyclic dipeptides composed of natural and non-natural amino acids, with the amino acids linked to a pyrrolidine or piperidine nitrogen, or an exocyclic amine group in proline residues [22]. The development of this new generation of ARG inhibitors was driven by the limitations of earlier compounds, which exhibited poor pharmacokinetic profiles, including very low oral bioavailability and a highly polar zwitterionic nature.

5.1. Peptide Boronic Acid Derivatives

5.1.1. Peptide Cyclic Inhibitors

The fourth generation of inhibitors includes ABH derivatives, which feature a ring-constrained pyrrolidine that reduces entropy by positioning the quaternary amino acid in an optimal orientation for binding [48,54]. A notable example is numidargistat ((3R,4S)-3-amino-1-((S)-2-aminopropanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid (71), a potent ARG inhibitor with IC50 values of 86 nM for hARG-1 and 296 nM for hARG-2 [22]. In 2016, it was approved by the Food Drug Administration (FDA) for clinical trials to treat patients with metastatic solid tumours, both as a monotherapy and in combination with chemotherapy and immunotherapy [69]. Compound 71 and its analogues (7273) were synthesized from Sjogren et al., with their synthetic protocols being patent-protected, as shown in Scheme 33 [70]. Specifically, compounds (3R,4S)-3-amino-1-((S)-2-amino-3-methylbutanoyl)-4-(3-boronopropylpyrrolidine)-3-carboxylic acid (72) and (3R,4S)-3-amino-1-((S)-2-amino-3-hydroxypropanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid (73) were prepared similarly to numidargistat (71), with the only difference being the reagents used with intermediate 71g. For 72, (tert-butoxycarbonyl)-L-valine was used, while for 73, (S)-3-(tert-butoxycarbonyl)-2,2-dimethyloxazolidine-4-carboxylic acid was employed.
The synthesis started with tert-butyl 6-oxa-3-azabicyclo [3.1.0]hexane-3-carboxylate (71a), which was reacted with allyl-MgBr in a Grignard reaction to form compound 71b. This was then oxidized using a sulphur trioxide pyridine complex (Py-SO3) to generate the ketone 71c. Next, tert-butyl 3-allyl-4-oxopyrrolidine-1-carboxylate (71c) was treated with CHCl3 and LiHMDS, followed by NaN3 and NaOH to yield compound 71d. The next step involved the protection of compound 71d with BnBr and K2CO3, resulting in ((3R,4S)-3-benzyl 1-tert-butyl-4-allyl-3-azidopyrrolidine-1,3-dicarboxylate (71e), which was then subjected to hydroboration with pinacolborane and [Ir(cod)Cl]2 in DCM to produce compound 71f. Compound 71f underwent BOC deprotection with TFA in DCM, yielding 71g. This was then alkylated with BOC-L-Alanine and EDC in DCM to form 71h, which was deprotected again with TFA in DCM. In the final steps, compound 71i was reacted with isobutylboronic acid, hexane, methanol, a solution of HCl, and K2CO3 to obtain the boronic derivative 71l. Finally, hydrogenation with Pd/C resulted in the desired product, (3R,4S)-3-amino-1-((S)-2-aminopropanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid (71).
An analogue of (2S,3R,5S)-3-amino-1-((S)-2-aminopropanoyl)-5-(2-boronoethyl)-2-methylpiperidine-3-carboxylic acid (74), which has a methyl group in position 2 of the piperidine ring, also exhibits activity against hARG-1 and hARG-2 with an IC50 of 249 nM or lower [22]. The full synthesis of this compound is detailed in Scheme 34 and is described briefly here.
The patented synthesis of compound 74 began with a substitution reaction between ethyl 2–bromopropionate (74a) and allylamine hydrochloride in the presence of TEA in ACN, yielding compound 74b. This was followed by amine protection using BOC-anhydride, TEA, and DCM, stirred overnight at r.t., producing compound 74c. Basic hydrolysis with NaOH and EtOH converted 74c into N-allyl-N-(tert-butoxycarbonyl)alanine (74d). To a solution of 74d in DCM, N,N-diisopropylethylamine (DIPEA), N,O–dimethylhydroxyamine, and HATU were added, forming the amide 74e after overnight stirring at r.t. This intermediate underwent alkylation reaction with vinyl magnesium bromide in THF at –20 °C, followed by ring-closing metathesis using Grubbs catalyst 2nd generation in DCM, yielding cyclic compound 74g. A second Grignard reaction with vinylmagnesium bromide and CuBr x Me2S in THF was performed, followed by the addition of tert-butyl-2-methyl -3-oxo-3,6-dihydropyridin–1(2H)–carboxylate (74g) and chlorotrimethyl silane at –78° C, which was then stirred overnight at r.t. to yield tert-butyl-2-methyl-3-oxo-5–vi nylpiperidine-1–carboxylate (74h). To a solution of 74h in 2,2,2–trifluoroethanol, NH4OAc and tert- butylisocyanide were added dropwise, and the mixture was stirred at r. t. overnight. After chiral HPLC resolution, compound 74l underwent a N-deprotection with HCl /AcOEt, yielding 74m. This intermediate was protected with benzaldehyde, and sodium triacetoxyborohydride in DCE to obtain 74n as a white foam. A hydroboration reaction was performed on 74n using dppe, [Ir(cod)Cl]2, and pinacolborane in DCM, followed by treatment with 12 M HCl, resulting in the boronic acid derivative 74p. This was protected using BOC anhydride, a solution 1M NaOH and acetone, producing compound 74q as a white solid. The next step involved hydrogenation over 20% Pd (OH)2/C in MeOH, followed by N-alkylation with BOC-L-Ala-Osu in DMF, resulting in 74s. In the final step, treatment of 74s with a solution of 4 M HCl in EtOAc yielded the desired compound (2S,3R,5S)-3-amino-1-((S)-2-aminopropanoyl)-5-(2-boronoethyl)-2-methylpiperidine-3-carboxylic acid (74) [71].
The compound (3R,5S)-3-amino-1-(2-aminoacetyl)-5-(2-boronoethyl)piperidine-3-carboxylic acid (75) is a glycine derivative with IC50 values ranging from 1 to 249 nM against hARG-1 and 500–999 nM against hARG-2. Its synthesis, outlined in Scheme 35, involves a patented two-step procedure. Starting from (3R, 5S)-5-(2-boronoethyl)-3-((tert-butoxycarbonyl)amino)piperidine-3-carboxylic acid (75a), Blaszczyk et al. performed an amidation reaction using BOC-Gly-OSu in DMF, followed by deprotection with 4 M HCI in EtOAC, yielding the final product 75 [22,71].
Another ARG inhibitor, (3R,5S)-1-(l-histidyl)-3-amino-5-(2-boronoethyl)piperidine-3-carboxylic acid trihydrochloride (76) is a histidine derivative with the same IC50 profile as 75 (1–249 nM for hARG-1 and 500–999 nM for hARG-2) [64]. Its synthesis follows the same procedure as 75 but uses BOC-L-His-(1-BOC)-Osu in the first step (Scheme 35).
Among peptide inhibitors, notable examples include (3aR,4S,5S,6aR)-5-amino-2-((S)-2-aminopropanoyl)-4-(3 boronopropyl) octahydrocyclopenta[c] pyrrole-5-carboxylic acid (77) with an IC50 of 108 nM against hARG-1 (Merck,TOGA assay), and (3aR,4S,5S,6aR)-5-amino-2-((S)-2-amino-3-methylbutanoyl)-4-(3-boronopropyl)octahydrocyclopenta[c]pyrrole-5-carboxylic acid (78), containing a valine moiety and exhibiting an improved IC50 of 52 nM, (TOGA assay) [22]. The synthesis of both compounds follows a patented 10-step protocol outlined in Scheme 36 [62,63]. The processes are identical except for the coupling reagent used in the seventh step: BOC-Ala-OH for 77 and BOC-Val-OH for 78.
The synthesis of compound 77 began with the resolution of 77a via SFC, yielding tert-butyl-(3aR,4S,6aR)-4-allyl-5-oxohexahydrocyclopenta[c]pyrrolo-2(1H)-carboxylate (77b). In the second step, 77b was treated with CHCl3, chlorotrimethylsilane, and LiHMDS (1 M in THF) under N2 at –78 °C, then warmed to –30 °C and stirred for 1.5 h. Following the addition of tetrabutylammonium acetate in DMF, the mixture was heated to 0 °C and stirred for 12 h, forming tert-butyl-(3aR,4S,6aR)-4-allyl-5-(trichloromethyl)-5-((trimethylsilyl)oxy)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (77c). In the third step, the product was treated with acetic acid and tetra-n-butylammonium fluoride in THF, to produce 77d, which was then reacted with sodium azide and sodium hydroxide in water to give (3aR,4S,6aR)-4-allyl-5-azido-2-(tert-butoxycarbonyl)octahydrocyclopenta[c]pyrrole-5-carboxylic acid (77e). The benzylation of 77e with K2CO3 and benzyl bromide yielded 5-benzyl 2-(tert-butyl)(3aR,4S,5S,6aR)-4-allyl-5-azidohexahydrocyclopenta[c]pyrrole-2,5(1H)-dicarboxylate (77f). A hydroboration reaction with [Ir(cod)Cl]2 and pinacolborane in DMC then produced 5-benzyl 2-(tert-butyl) (3aR,4S,5S,6aR)-5-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)hexahydro cyclopenta[c]pyrrole-2,5(1H)-dicarboxylate (77g), which was deprotected with 4 M HCl in EtOAc, yielding benzyl(3aR,4S,5S,6aR)-5-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)propyl)octahydrocyclopenta[c]pyrrole-5-carboxylate (77h). Compound 77h was N-alkylated with BOC-Ala-OH or BOC-Val-OH in DMF and in the presence of propanephosphonic acid anydride (T3P) and TEA to form (3aR,4S,5S,6aR)-5-azido-2-((tert-butoxycarbonyl)-L-alanyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)octahydrocyclopenta[c]pyrrole-5-carboxylate or (3aR,4S,5S,6aR)-5-azido-2-((tert-butoxycarbonyl)-L-valin)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)octahydrocyclopenta[c]pyrrole-5-carboxylate (77i). Subsequent deprotection with H2 over Pd-C gave 77l, which was used directly in the final step. The treatment of 77l with 6 M HCl at 20 °C for 13 h yielded the final product (3αR,4S,5S,6αR)-2-(L-alanyl)-5-amino-4-(3-boronopropyl)octahydrocyclopenta[c]pyrrole-5-carboxylic acid (77) and (3αR,4S,5S,6αR)-2-(L-vanil)-5-amino-4-(3-boronopropyl)octahydrocyclopenta[c]pyrrole-5-carboxylic acid (78) as white solids (TFA salt).

5.1.2. Proline Cyclic Inhibitors

Other peptide inhibitors of ARG are based on a proline scaffold, where the amino group of the proline core is coupled with various natural amino acids such as valine, leucine, and proline, or unnatural amino acids like cyclopentylglycine, tert-leucine, and indanylglycine. Examples include (2S,3R,4R)-4-((S)-2-amino-3-methylbutanamido)-3-(3-boronopropyl)pyrrolidine-2-carboxylic acid (79), (2S,3R,4R)-4-((S)-2-amino-2-cyclopentylacetamido)-3-(3-boronopropyl)pyrrolidine-2-carboxylic acid (80), and (2S,3R,4R)-4-((S)-2-amino-3,3-dimethylbutanamido)-3-(3-boronopropyl)pyrrolidine-2-carboxylic acid (81). These compounds, reported in Figure 5, exhibit improved inhibitory properties.
Compound 79, featuring an isopropyl group as a substituent, inhibits hARG-1 with an IC50 = 320 nM and hARG-2 with IC50 = 330 nM (TOGA assay). In contrast, compounds 80 and 81, containing cyclopentylglycine and tert-leucine as unnatural amino acids linked to the proline scaffold, are significantly more potent, inhibiting hARG-1 with an IC50 = 0.8 nM (TOGA assay) [22]. Compounds 80 and 81 were synthesized following the same method as compound 79 (Scheme 37) but using appropriate starting materials [72].
The patented synthesis of compound 79, described by Achab AA et al., involves three steps [72]. Starting with (2S,3R,4R)-1-tert-butyl 2-methyl 3-allyl-4-aminopyrrolidine-1,2-dicarboxylate (79a), the compound undergoes N-alkylation using BOC-L-Val-OH, TEA, and HATU, yielding intermediate 79b. This is followed by a hydroboration reaction with pinacolborane, [Ir(cod)Cl]2, and dppe to produce compound 79c. In the final step, the intermediate is treated with potassium trimethylsilanolate, then with 6 M HCl, to obtain the target compound (2S,3R,4R)-4-((S)-2-amino-3-methylbutanamido)-3-(3-boronopropyl)pyrrolidine-2-carboxylic acid (79).
Another ARG inhibitor is (2S,3R,4R)-4-((S)-2-amino-4-methylpentanamido)-3-(3-boronopropyl)pyrrolidine-2-carboxylic acid (82), which incorporates leucine and the natural amino acid attached to the amine group of the proline scaffold. This compound inhibits hARG-1 with an IC50 = 1.6 nM (TOGA) and is synthesized in three steps, as outlined in Scheme 38 [22,72]. Starting with 1-(tert-butyl)-2-methyl(2S,3R,4R)-3-allyl-4-aminopyrrolidine-1,2-dicarboxylate (82a), a hydroboration reaction with a pinanediolborane derivative, (3aR,4R,6R,7aS)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborole, [Ir(cod)Cl]2, and dppe yields compound 82b. This intermediate is then N-alkylated using BOC-L-Leu-OH, TEA, and HATU to produce compound 82c. In the final step, compound 82c reacts with potassium trimethylsilanolate, followed by treatment with 6 M HCl, resulting in the desired compound, (2S,3R,4R)-4-((S)-2-amino-4-methylpentanamido)-3-(3-boronopropyl)pyrrolidine-2-carboxylic acid (82).
The compound (2R,4R)-4-((S)-2-amino-3-methylbutanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (AZD0011, 83), a valine analogue, exhibited IC50 values of 320 nM against hARG-1 and 330 nM against hARG-2, as determined by the TOGA assay. Furthermore, pharmacological tests using the mouse xenograft model revealed that 83 acted as prodrug, releasing the parent compound 69 in vivo [73]. The synthesis of 83, outlined in Scheme 39 [62], involved two steps starting from (2R,4R)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (69g). In the first step, 69g underwent N-alkylation with BOC-Val-OH, TEA, and HATU yielding intermediate 83a. The second step involved deprotection using TFA, Et2O, a solution 1 M HCl, and phenylboronic acid, producing the final compound (2R,4R)-4-((S)-2-amino-3-methylbutanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (83).
The compound (2R,4R)-4-((S)-2-amino-3-hydroxy-3-methylbutanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (84), a hydroxyvaline analogue, displayed IC50 values of 340 nM and 520 nM against hARG-1 and hARG-2, respectively, as determined by the TOGA assay [22]. Its synthesys outlined in Scheme 40, involved two steps starting with (2R,4R)-2-benzyl-1-tert-butyl 4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (84a). The first step was N-alkylation with (S)-N-α-tert-butoxycarbonyl-3,3-dimethyl-serine, DIPEA, and HATU, yielding intermediate 84b. In the second step, deprotection was achieved using Pd/C in EtOAc, followed by treatment with TFA, Et2O, a solution 1 M HCl, and phenylboronic acid, producing the final compound (2R,4R)-4-((S)-2-amino-3-hydroxy-3-methylbutanamido)-2-(4-boronobutyl) pyrrolidine-2-carboxylic acid (84) [68].
As observed in the structures of the fourth-generation ARG inhibitors presented thus far, the nitrogen atom is linked to an amino acid of a varying nature through a peptide bond. In some cases, the design of these compounds has resulted in improvements in both activity and pharmacokinetic profiles, fulfilling the objectives for which this generation was developed. However, in other instances, these inhibitors exhibited lower inhibitory activity compared to the non-peptide derivatives of the third generation and could only be considered prodrugs in specific cases. For example, it has been demonstrated that peptide derivatives, such as compound 83, were rapidly metabolized in vivo through the hydrolysis of the peptide bond, releasing the non-peptide derivative 69, which exhibited superior activity.
OATD-02 (85), developed by OncoArendi Therapeutics (now Molecure), is a boronic acid derivative and an intracellular dual inhibitor of ARG-1 and ARG-2, with potent IC50 values of 20 nM and 48 nM for hARG-1 and -2, respectively [22]. Currently in preclinical development [74], its synthesis involves five steps, as detailed in Scheme 41 [28,75]. Starting from 2-cyclohexen-1-one (85a), the reaction with triethoxyvinylsilane under the catalysis of Rh(cod)(MeCN)2BF4 and R-BINAP in dioxane produced intermediate 85b. Further processing with sodium hydride and diethyl carbonate yielded 85c, which was converted to 85d by reaction with NH4OAc in 2,2,2-trifluoroethanol and tert-butyl isocyanide. The subsequent reduction of (1R,2R,4R)-2-acetamido-2-(tert-butylcarbamoyl)-4-vinylcyclohexane-1-carboxylate (85d) with a solution 1 M DIBAL-H in DCM at –78 °C, followed by the addition of dimethylamine in THF and sodium triacetoxyborohydride, led to the formation of (1R,2S,5R)-1-acetamido-N-(tert-butyl)-2-((dimethylamino)methyl)-5-vinylcyclohexanecarboxamide (85e). Reacting 85e with pinacolborane in the presence of dppe and [Ir(cod)Cl]2, followed by reflux in 6 M HCl, yielded the crude product. Purification through chromatography on the silica gel C-18 (isocratic elution, H2O), and, next, flash chromatography on the DOWEX® 50WX8 ion exchange resin (eluent 0.1 M ammonia in water) produced the final compound (OATD-02) 85.

6. Side Chain β-Substituted ABH Analogues

Recently, Shields and colleagues developed novel ARG inhibitors by exploring the previously little-studied β-position of the compound ABH (9) [76]. Specifically, they synthesized promising inhibitors by alkylating the β-position of ABH with various functionalized alkyl chains to increase potency. Until now, companies and research groups had focused primarly on substitution at the α-position or the simultaneous substitution of both the α- and β-positions through cyclization. Through their chemical modifications, Shields et al. have extensively occupied the channel leading to the active site by targeting the β-position, without relying on cyclization. Among the twenty compounds synthesized, the most potent inhibitors able to inhibit ARG-1 with an IC50 value lower than that ABH (i.e., lower than the 470 nM obtained from the biochemical assays performed by the same authors) were compounds 86–88, shown in Figure 6. Notably, compounds 87 and 88 shared the same chemical structure but differed in spatial arrangement: 87 was the anti derivative, and 88 was the syn derivative. The anti arrangement (87) exhibited higher potency. The X-ray crystallography of the 87 and 88-ARG-2 complexes prompted the authors to add an amino acid residue to 87 and 88, forming a peptide bond to further improve potency. Various amino acid residues were tested, and the most promising inhibitors were derivatives 89 and 90 (Figure 6). For the syn compound, alanine was added while valine was added to the anti compound. Interestingly, the syn compound (88) became more potent after alanine was added (89), surpassing both 87 and its corresponding derivative, 90. Moreover, pharmacological tests confirmed that 89 and 90 acted as prodrugs, releasing the respective parent compounds in vivo [76].
The synthetic procedure of compounds 87 and 88 is shown in Scheme 42.
The synthesis began with the mesylation of tert-butyl (2S,3S)-2-(((benzyloxy)carbonyl)amino)-3-(hydroxymethyl)hex-5-enoate (87a) and tert-butyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-(hydroxymethyl)hex-5-enoate (88a) and the subsequent protection of the amino function using potassium phthalimide to give the corresponding protected amines tert-butyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((1,3-dioxoisoindolin-2-yl)methyl)hex-5-enoate (87c) and tert-butyl (2S,3S)-2-(((benzyloxy)carbonyl)amino)-3-((1,3-dioxoisoindolin-2-yl)methyl)hex-5-enoate (88c). Subsequently, the hydroboration reaction of 87c and 88c was performed in the presence of [Ir(cod)Cl]2, dppm and pinacolborane yielding 87d and 88d, respectively. In the last step, a global deprotection in acidic medium was performed to remove the benzyl carbamate, tert-butyl ester, and the pinacol-protecting groups, revealing the final compounds (2S,3R)-2-amino-3-(aminomethyl)-6-boronohexanoic acid dihydrochloride (87) and (2S,3S)-2-amino-3-(aminomethyl)-6-boronohexanoic acid dihydrochloride (88) as hydrochloride salts. These salts were freebased by passage through an ion exchange column and then freeze-dried.

7. Natural Compounds as ARG Inhibitors

Plant-derived compounds, often inspired by traditional medicine, offer a promising avenue for discovery ARG inhibitors. Natural and semi-synthetic compounds not only enhance molecular diversity but also hold the potential to reduce toxicity [34,77].
Among natural ARG inhibitors, polyphenols stand out for their bioactivity and safety. This class includes flavonoids, phenolic acids, and tannins, which have demonstrated significant ARG inhibitory activity and could serve as valuable lead compounds. On the semi-synthetic side, cinnamide derivatives, derived from cinnamic acid, show great potential. These compounds can be chemically modified to improve their potency and selectivity. By combining natural and semi-synthetic approaches, researchers can create a robust platform for developing new ARG inhibitors, benefiting from molecular diversity, reduced toxicity, and structure–activity relationship (SAR) insights.

7.1. Polyphenols

Polyphenols are among the most prominent natural compounds identified as ARG inhibitors. These secondary plant metabolites, abundant in fruits, vegetables, and medicinal plants, offer a wide array of health benefits. Key classes of polyphenols include flavonoids, phenolic acids, stilbenes, and lignans, many of which have demonstrated ARG-inhibitory activity, partly linked to their influence on NO production.
Notable examples (Table 2) include chlorogenic acid (91) (a phenolic acid), picetannol (92) and resveratrol (93) (stilbens), and taxifolin (94) (flavanoid). These compounds showed IC50 values of 10.6 μM, 12.1 μM, 18.2 μM, and 23.2 μM, respectively, against ARG-1 in assays using mammalian bovine liver [78]. Piceatannol-3′-O-β-D-glucopyranoside (95), a stilbene glycoside, inhibited both ARG-1 and ARG-2 in a dose-dependent manner with IC50 values of 11.2 μM and 11.0 μM, measured in rat liver and kidney lysates, respectively [79]. Studies by Arraki et al. [80] highlighted several polyphenols, including ellagic acid (96),various luteolin derivatives extracted from the leaves of Morus alba, like luteolin-7-diglucoside (97), luteolin-7-glucoside (98), and luteolin (99), as well as stilbenes like scirpusin B (100), ε-viniferin (101), cyperusphenol B (102), carexinol A (103), and the newly identified virgatanol (104) stilbenes and polyphenols isolated from various species of Cyperus and Carex, from the Cyperaceae family—which showed inhibitory effects on purified bovine liver (Table 2). Additionally, Sauchinone (105), isolated from Saurus chinensis extract, showed an IC50 of 61.4 μM against ARG-2 in murine kidney lysates [81]. Methanolic extracts of Scutellaria indica also showed promise, with compounds (2S)-5,7-dihydroxy-8,20-dimethoxyflavanone (106) and (2S)-5,20,50-trihydroxy-7, 8-dimethoxyflavanone (107) displaying IC50 values of 25.1 μM and 11.6 μM, respectively, against ARG-2 [82]. However, comparing these findings is challenging due to the variability in assay conditions. Despite this limitation, structural analyses suggest that features like the caffeoyl (3,4-dioxycinnamoyl) group and catechol functionality are crucial for ARG inhibition. Although the identified natural compounds mostly exhibit micromolar-range activity, these insights provide a foundation for designing more potent ARG inhibitors and expanding the chemical space of natural product-derived therapeutics.

7.2. Cinnamide Derivatives

Pham et al. simplified the structure of chlorogenic acid—an ester of caffeic and quinic acid—to create compound (108) (Table 2), also known as caffeic acid phenylamide (CAPA). In tests using a micro-assay on purified bovine liver arginase (bARG-1), CAPA showed slightly better activity than chlorogenic acid, with IC50 values of 6.9 µM and 10.6 µM, respectively. However, when tested on recombinant human arginase (hARG-1), CAPA’s activity decreased significantly, with an IC50 of 60.3 µM.
This study highlights the value of using bARG-1, a cost-effective alternative, for initial screening of potential mammalian ARG inhibitors. However, it also emphasizes the importance of testing promising compounds on hARG-1 to ensure their relevance before advancing to further studies. The research identified the cinnamoyl group and catechol moiety as essential structural features for inhibitory activity. Despite CAPA’s relatively high IC50 against hARG-1, its structure points to cinnamide derivatives as promising lead compounds for developing therapeutically effective ARG inhibitors [83].

8. Conclusions and Future Perspectives

In conclusion, ARG, the enzyme responsible for the metabolism of the amino acid L-arginine, plays a key role in numerous pathophysiological processes, making it a significant target for researchers aiming to combat various disorders. Although the enzyme was discovered many years ago, recent advancements have spurred renewed research into ARG and its inhibitors, leading to the development of new molecules designed to target this enzyme. This review traces the evolution of ARG inhibitors, categorizing them into different generations and classes based on structure variations, including recently developed molecules and those derived from natural or semi-synthetic sources. For the first time, in this review the structures and syntheses of ARG inhibitors described in patent applications have been reported.
As emphasized throughout the review, various pathologies are characterized by the uncontrolled expression of one of the two ARG isoforms, underscoring the need for selective inhibitors targeting either ARG-1 or ARG-2. Despite numerous studies and clinical trials demonstrating the potential of ARG as a biomarker and diagnostic tool for cancer progression, there is still a need for standardized clinical definitions of ARG activity and for the consistent measurement of ARG-1 and ARG-2 expression levels in blood or tissues for cancer diagnosis. To address this challenge, an artificial intelligence-based prediction model could be developed, leveraging deep learning of clinical data, including cancer types, ARG activity values, expression levels, and patient information, to assess cancer progression.
Despite significant progress, a fully selective inhibitor remains elusive due to the structural similarities between the two isoforms. Notable examples include numidargistat and OATD-02, although neither shows significant selectivity for one isoform over the other. However, OATD-02 offers a unique advantage as the first potent dual ARG-1/ARG-2 inhibitor in its class. Its enhanced antitumor activity in vivo has been attributed to its complex mechanism of action, targeting both intracellular and extracellular ARGs. Consequently, OATD-02 stands out as the only pharmacological tool capable of effectively harnessing the benefits of inhibiting both isoenzymes, while also regulating also CD8+ and Treg cell activity in contrast to numidargistat.
Both numidargistat and OATD-02 have advanced to the clinical phase, ref. [84] with OATD-02 still in the recruitment phase. Numidargistat has been evaluated in five separate phase I studies for advanced or metastatic solid tumours, primarily in combination with other immunotherapies or conventional chemotherapies. However, the literature suggests that immunochemotherapy has shown superior efficacy compared to immunotherapy alone [85]. While numidargistat has a manageable safety profile, characterized primarily by inhibition of the urea cycle, immune-related adverse events, and a pharmacodynamic increase in plasma arginine levels [86,87], it has not yet advanced beyond phase I due to a response below expectations in terms of overall response rate and disease control rate. Another ARG inhibitor in clinical trials, CB-280 (structure undisclosed), is being tested for cystic fibrosis [88]. This compound has shown good tolerability, with no dose-limiting toxicities or severe grade adverse reactions [89]. In addition to small molecule inhibitors, a peptide vaccine targeting ARG-1 has been tested in clinical studies demonstrating a good safety profile, with no severe adverse reactions and with 90% of patients showing a measurable immune response to the peptide, though the clinical antitumor response has been modest [90]. Further details on the clinical studies have been published in Failla et al. [31].
The challenge of developing selective inhibitors for the ARG isoforms remains highly relevant. Structural modifications, such as incorporating boronic acid in place of the guanidine group and side chain derivatizations, have led to significant improvements, though an ideal compound has yet to be achieved. High-throughput screening offers a promising solution, especially when the target structure is not fully defined, and it can be used alongside computational strategies.
Moreover, improving the pharmacokinetic profile of these molecules presents another challenge. A promising approach involves leveraging medicinal chemistry techniques to achieve the desired selectivity for one of the two isoforms while simultaneously enhancing pharmacokinetic properties, as discussed in our previous perspective [31]. Strategies such as the prodrug approach, utilizing small molecules or polymers as carriers, could improve drug targeting [91]. Alternatively, molecular hybridization techniques hold potential for improving pharmacokinetic profile of ARG inhibitors, thereby boosting their pharmacological activity [92,93,94]. Furthermore, since ARG inhibitors have shown promise as probes for molecular imaging, improving their selectivity and biopharmaceutical properties could pave the way for both therapeutic and diagnostic applications.

Author Contributions

Conceptualization, F.S.; methodology, M.C.M.; validation, M.F.; investigation, C.B.; data curation, M.G.R.; writing—original draft preparation, M.C.M., C.B., M.E.S. and K.C.; writing—review and editing, K.C. and L.L.; supervision, F.S.; project administration, F.S.; funding acquisition, F.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded AIRC SIS 2022 (ID 28705, P.I. Federica Sodano).

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Caldwell, R.B.; Toque, H.A.; Narayanan, S.P.; Caldwell, R.W. Arginase: An Old Enzyme with New Tricks. Trends Pharmacol. Sci. 2015, 36, 395–405. [Google Scholar] [CrossRef]
  2. Wu, G.; Morris, S.M. Arginine Metabolism: Nitric Oxide and Beyond. Biochem. J. 1998, 336, 1–17. [Google Scholar] [CrossRef]
  3. Morris, S.M. Recent Advances in Arginine Metabolism: Roles and Regulation of the Arginases. Br. J. Pharmacol. 2009, 157, 922–930. [Google Scholar] [CrossRef]
  4. Ostrand-Rosenberg, S.; Sinha, P. Myeloid-derived suppressor cells: Linking inflammation and cancer. J. Immunol. 2009, 182, 4499–4506. [Google Scholar] [CrossRef]
  5. Kumar, V.; Patel, S.; Tcyganov, E.; Gabrilovich, D.I. The nature of myeloid-derived suppressor cells in the tumor microenvironment. Trends Immunol. 2016, 37, 208–220. [Google Scholar] [CrossRef]
  6. Canè, S.; Geiger, R.; Bronte, V. The roles of arginases and arginine in immunity. Nat. Rev. Immunol. 2024; Epub ahead of print. [Google Scholar] [CrossRef] [PubMed]
  7. Lowe, M.M.; Boothby, I.; Clancy, S.; Ahn, R.S.; Liao, W.; Nguyen, D.N.; Schumann, K.; Marson, A.; Mahuron, K.M.; Kingsbury, G.A.; et al. Regulatory T Cells Use Arginase 2 to Enhance Their Metabolic Fitness in Tissues. JCI Insight 2019, 4, 129756. [Google Scholar] [CrossRef]
  8. Munder, M. Arginase: An Emerging Key Player in the Mammalian Immune System. Br. J. Pharmacol. 2009, 158, 638–651. [Google Scholar] [CrossRef]
  9. Durante, W. Role of Arginase in Vessel Wall Remodeling. Front. Immunol. 2013, 4, 111. [Google Scholar] [CrossRef]
  10. Wiesinger, H. Arginine Metabolism and the Synthesis of Nitric Oxide in the Nervous System. Prog. Neurobiol. 2001, 64, 365–391. [Google Scholar] [CrossRef]
  11. Wink, D.A.; Hines, H.B.; Cheng, R.Y.S.; Switzer, C.H.; Flores-Santana, W.; Vitek, M.P.; Ridnour, L.A.; Colton, C.A. Nitric Oxide and Redox Mechanisms in the Immune Response. J. Leukoc. Biol. 2011, 89, 873–891. [Google Scholar] [CrossRef]
  12. Gotoh, T.; Mori, M. Arginase II Downregulates Nitric Oxide (NO) Production and Prevents NO-Mediated Apoptosis in Murine Macrophage-Derived RAW 264.7 Cells. J. Cell Biol. 1999, 144, 427–434. [Google Scholar] [CrossRef]
  13. Choudry, M.; Tang, X.; Santorian, T.; Wasnik, S.; Xiao, J.; Xing, W.; Lau, K.H.W.; Mohan, S.; Baylink, D.J.; Qin, X. Deficient Arginase II Expression without Alteration in Arginase I Expression Attenuated Experimental Autoimmune Encephalomyelitis in Mice. Immunology 2018, 155, 85–98. [Google Scholar] [CrossRef]
  14. Bhatta, A.; Yao, L.; Xu, Z.; Toque, H.A.; Chen, J.; Atawia, R.T.; Fouda, A.Y.; Bagi, Z.; Lucas, R.; Caldwell, R.B.; et al. Obesity-Induced Vascular Dysfunction and Arterial Stiffening Requires Endothelial Cell Arginase 1. Cardiovasc. Res. 2017, 113, 1664–1676. [Google Scholar] [CrossRef]
  15. Berkowitz, D.E.; White, R.; Li, D.; Minhas, K.M.; Cernetich, A.; Kim, S.; Burke, S.; Shoukas, A.A.; Nyhan, D.; Champion, H.C.; et al. Arginase Reciprocally Regulates Nitric Oxide Synthase Activity and Contributes to Endothelial Dysfunction in Aging Blood Vessels. Circulation 2003, 108, 2000–2006. [Google Scholar] [CrossRef]
  16. Clemente, G.S.; van Waarde, A.; Antunes, I.F.; Dömling, A.; Elsinga, P.H. Arginase as a Potential Biomarker of Disease Progression: A Molecular Imaging Perspective. Int. J. Mol. Sci. 2020, 21, 5291. [Google Scholar] [CrossRef]
  17. Erdely, A.; Kepka-Lenhart, D.; Salmen-Muniz, R.; Chapman, R.; Hulderman, T.; Kashon, M.; Simeonova, P.P.; Morris, S.M., Jr. Arginase activities and global arginine bioavailability in wild-type and ApoE-deficient mice: Responses to high fat and high cholesterol diets. PLoS ONE 2010, 5, e15253. [Google Scholar] [CrossRef]
  18. Zhang, Y.; Higgins, C.B.; Fortune, H.M.; Chen, P.; Stothard, A.I.; Mayer, A.L.; Swarts, B.M.; DeBosch, B.J. Hepatic arginase 2 (Arg2) is sufficient to convey the therapeutic metabolic effects of fasting. Nat. Commun. 2019, 10, 1587. [Google Scholar] [CrossRef]
  19. Lange, P.S.; Langley, B.; Lu, P.; Ratan, R.R. Arginine Metabolism: Enzymology, Nutrition, and Clinical Significance Novel Roles for Arginase in Cell Survival, Regeneration, and Translation in the Central Nervous System. J. Nutr. 2004, 134, 2812S–2817S. [Google Scholar] [CrossRef]
  20. Polis, B.; Srikanth, K.D.; Gurevich, V.; Bloch, N.; Gil-Henn, H.; Samson, A.O. Arginase Inhibition Supports Survival and Differentiation of Neuronal Precursors in Adult Alzheimer’s Disease Mice. Int. J. Mol. Sci. 2020, 21, 1133. [Google Scholar] [CrossRef]
  21. Liu, P.; Fleete, M.S.; Jing, Y.; Collie, N.D.; Curtis, M.A.; Waldvogel, H.J.; Faull, R.L.M.; Abraham, W.C.; Zhang, H. Altered arginine metabolism in Alzheimer’s disease brains. Neurobiol. Aging 2014, 35, 1992. [Google Scholar] [CrossRef]
  22. Borek, B.; Gajda, T.; Golebiowski, A.; Blaszczyk, R. Boronic Acid-Based Arginase Inhibitors in Cancer Immunotherapy. Bioorganic Med. Chem. 2020, 28, 115658. [Google Scholar] [CrossRef]
  23. Muller, J.; Cardey, B.; Zedet, A.; Desingle, C.; Grzybowski, M.; Pomper, P.; Foley, S.; Harakat, D.; Ramseyer, C.; Girard, C.; et al. Synthesis, Evaluation and Molecular Modelling of Piceatannol Analogues as Arginase Inhibitors. RSC Med. Chem. 2020, 11, 559–568. [Google Scholar] [CrossRef]
  24. Van Zandt, M.C.; Whitehouse, D.L.; Golebiowski, A.; Ji, M.K.; Zhang, M.; Beckett, R.P.; Jagdmann, G.E.; Ryder, T.R.; Sheeler, R.; Andreoli, M.; et al. Discovery of (R)-2-Amino-6-Borono-2-(2-(Piperidin-1-Yl)Ethyl)Hexanoic Acid and Congeners as Highly Potent Inhibitors of Human Arginases i and II for Treatment of Myocardial Reperfusion Injury. J. Med. Chem. 2013, 56, 2568–2580. [Google Scholar] [CrossRef]
  25. Blaszczyk, R.; Brzezinska, J.; Dymek, B.; Stanczak, P.S.; Mazurkiewicz, M.; Olczak, J.; Nowicka, J.; Dzwonek, K.; Zagozdzon, A.; Golab, J.; et al. Discovery and Pharmacokinetics of Sulfamides and Guanidines as Potent Human Arginase 1 Inhibitors. ACS Med. Chem. Lett. 2020, 11, 433–438. [Google Scholar] [CrossRef]
  26. Golebiowski, A.; Whitehouse, D.; Beckett, R.P.; Van Zandt, M.; Ji, M.K.; Ryder, T.R.; Jagdmann, E.; Andreoli, M.; Lee, Y.; Sheeler, R.; et al. Synthesis of Quaternary α-Amino Acid-Based Arginase Inhibitors via the Ugi Reaction. Bioorganic Med. Chem. Lett. 2013, 23, 4837–4841. [Google Scholar] [CrossRef]
  27. Steggerda, S.M.; Bennett, M.K.; Chen, J.; Emberley, E.; Huang, T.; Janes, J.R.; Li, W.; MacKinnon, A.L.; Makkouk, A.; Marguier, G.; et al. Inhibition of Arginase by CB-1158 Blocks Myeloid Cell-Mediated Immune Suppression in the Tumor Microenvironment. J. Immunother. Cancer 2017, 5, 101. [Google Scholar] [CrossRef]
  28. Blaszczyk, R.; Nowicka, J.; Borek, B.; Brzezinska, J.; Gzik, A.; Dziegielewski, M.; Golebiowski, A.; Jedrzejczak, K.; Matyszewski, K.; Olczak, J. Arginase Inhibitors and Their Therapeutic Applications. World Intellectual Property Organization. WO2017191130A2, 9 November 2017. [Google Scholar]
  29. Grzybowski, M.M.; Stańczak, P.S.; Pomper, P.; Błaszczyk, R.; Borek, B.; Gzik, A.; Nowicka, J.; Jędrzejczak, K.; Brzezińska, J.; Rejczak, T.; et al. OATD-02 Validates the Benefits of Pharmacological Inhibition of Arginase 1 and 2 in Cancer. Cancers 2022, 14, 3967. [Google Scholar] [CrossRef]
  30. Niu, F.; Yu, Y.; Li, Z.; Ren, Y.; Li, Z.; Ye, Q.; Liu, P.; Ji, C.; Qian, L.; Xiong, Y. Arginase: An Emerging and Promising Therapeutic Target for Cancer Treatment. Biomed. Pharmacother. 2022, 149, 112840. [Google Scholar] [CrossRef]
  31. Failla, M.; Molaro, M.C.; Schiano, M.E.; Serafini, M.; Tiburtini, G.A.; Gianquinto, E.; Scoccia, R.; Battisegola, C.; Rimoli, M.G.; Chegaev, K.; et al. Opportunities and Challenges of Arginase Inhibitors in Cancer: A Medicinal Chemistry Perspective. J. Med. Chem. 2024, 67, 19988–20021. [Google Scholar] [CrossRef]
  32. Christianson, D.W. Arginase: Structure, Mechanism, and Physiological Role in Male and Female Sexual Arousal. Acc. Chem. Res. 2005, 38, 191–201. [Google Scholar] [CrossRef]
  33. Tommasi, S.; Elliot, D.J.; Da Boit, M.; Gray, S.R.; Lewis, B.C.; Mangoni, A.A. Homoarginine and Inhibition of Human Arginase Activity: Kinetic Characterization and Biological Relevance. Sci. Rep. 2018, 8, 3697. [Google Scholar] [CrossRef] [PubMed]
  34. Pudlo, M.; Demougeot, C.; Girard-Thernier, C. Arginase Inhibitors: A Rational Approach Over One Century. Med. Res. Rev. 2017, 37, 475–513. [Google Scholar] [CrossRef] [PubMed]
  35. Boucher, J.L.; Custot, J.; Vadon, S.; Delaforge, M.; Lepoivre, M.; Tenu, J.P.; Yapo, A.; Mansuy, D. N Omega-Hydroxyl-L-Arginine, an Intermediate in the L-Arginine to Nitric Oxide Pathway, Is a Strong Inhibitor of Liver and Macrophage Arginase. Biochem. Biophys. Res. Commun. 1994, 203, 1614–1621. [Google Scholar] [CrossRef]
  36. Di Costanzo, L.; Ilies, M.; Thorn, K.J.; Christianson, D.W. Inhibition of Human Arginase I by Substrate and Product Analogues. Arch. Biochem. Biophys. 2010, 496, 101–108. [Google Scholar] [CrossRef]
  37. Colleluori, D.M.; Ash, D.E. Classical and Slow-Binding Inhibitors of Human Type II Arginase. Biochemistry 2001, 40, 9356–9362. [Google Scholar] [CrossRef]
  38. Wallace, G.C.; Fukuto, J.M. Synthesis and Bioactivity of N Omega-Hydroxyarginine: A Possible Intermediate in the Biosynthesis of Nitric Oxide from Arginine. J. Med. Chem. 1991, 34, 1746–1748. [Google Scholar] [CrossRef]
  39. Bodanszky, M.; Bodanszky, A. In The Practice of Peptide Synthesis, 1st ed.; Springer: New York, NY, USA, 1984. [Google Scholar]
  40. Bailey, D.M.; Degrazia, C.G.; Lape, H.E.; Frering, R.; Fort, D.; Skulan, T. Hydroxyguanidines. A New Class of Antihypertensive Agents. J. Med. Chem. 1973, 16, 151–156. [Google Scholar] [CrossRef]
  41. Moali, C.; Brollo, M.; Custot, J.; Sari, M.A.; Boucher, J.L.; Stuehr, D.J.; Mansuy, D. Recognition of α-Amino Acids Bearing Various C=NOH Functions by Nitric Oxide Synthase and Arginase Involves Very Different Structural Determinants. Biochemistry 2000, 39, 8208–8218. [Google Scholar] [CrossRef]
  42. Teng, H.B. Synthesis of Nω-Hydroxy-nor-L-Arginine. Chin. J. Appl. Chem. 2010, 27, 1111–1113. [Google Scholar]
  43. Custot, J.; Moali, C.; Brollo, M.; Boucher, J.L.; Delaforge, M.; Mansuy, D.; Tenu, J.P.; Zimmermann, J.L. Nω-Hydroxy-nor-L-Arginine: A High-Affinity Inhibitor of Arginase Well Adapted To Bind to Its Manganese Cluster. J. Am. Chem. Soc. 1997, 119, 4086–4087. [Google Scholar] [CrossRef]
  44. Vadon, S.; Custot, J.; Boucher, J.L.; Mansuy, D. Synthesis and Effects on Arginase and Nitric Oxide Synthase of Two Novel Analogues of N"-Hydroxyarginine, Nu-Hydroxyindospicine and p-h y Drox y Amidinophen y Lalanine. J. Chem. Soc. 1996, 1, 645–648. [Google Scholar]
  45. Metcalf, B.W.; Bey, P.; Danzin, C.; Jung, M.J.; Casara, P.; Vevert, J.P. Catalytic Irreversible Inhibition of Mammalian Ornithine Decarboxylase (E.C.4.1.1.17) by Substrate and Product Analogs. J. Am. Chem. Soc. 1978, 100, 2551–2553. [Google Scholar] [CrossRef]
  46. Selamnia, M.; Mayeur, C.; Robert, V.; Blachier, F. Difluoromethylornithine (DFMO) as a Potent Arginase Activity Inhibitor in Human Colon Carcinoma Cells. Biochem. Pharmacol. 1998, 55, 1241–1245. [Google Scholar] [CrossRef]
  47. Zhu, J.; Chadwick, S.T.; Price, B.A.; Zhao, S.X.; Costello, C.A.; Vemishetti, P. Processes for the Production of Alpha-Difluoromethyl Ornithine (DFMO). WO2003020209A2, 4 May 2004. [Google Scholar]
  48. Baggio, R.; Elbaum, D.; Kanyo, Z.F.; Carroll, P.J.; Christopher Cavalli, R.; Ash, D.E.; Christianson, D.W. Inhibition of Mn2+2-Arginase by Borate Leads to the Design of a Transition State Analogue Inhibitor, 2(S)-Amino-6-Boronohexanoic Acid. J. Am. Chem. Soc. 1932, 119, 8107–8108. [Google Scholar] [CrossRef]
  49. Preite, M.D.; Manriquez Mujica, J.M.; Correa Vargas, J.M.; Iturriaga Aguera, R.M.; Casanello Toledo, P.C.; Krause Leyton, B.J. Method for the Enantioselective Synthesis of 2(s)-Amino-6-Boronohexanoic Acid (ABH) and Purification Thereof. WO2016037298A1, 17 May 2016. [Google Scholar]
  50. Matteson, D.S.; Soloway, A.H.; Tomlinson, D.W.; Campbell, J.D.; Nixon, G.A. Synthesis and Biological Evaluation of Water-Soluble 2-Boronoethylthio Compounds. J. Med. Chem. 1964, 7, 640–643. [Google Scholar] [CrossRef]
  51. Van Zandt, M.C.; Golebiowski, A.; Ji, M.K.; Whitehouse, D.; Ryder, T.; Beckett, P. Inhibitor Arginase and Their Therapeutic Application. US20120083469A1, 5 April 2012. [Google Scholar]
  52. Clemente, G.S.; Antunes, I.F.; Kurhade, S.; Van Den Berg, M.P.M.; Sijbesma, J.W.A.; Van Waarde, A.; Buijsman, R.C.; Willemsen-Seegers, N.; Gosens, R.; Meurs, H.; et al. Mapping Arginase Expression with 18F-Fluorinated Late-Generation Arginase Inhibitors Derived from Quaternary a-Amino Acids. J. Nucl. Med. 2021, 62, 1163–1170. [Google Scholar] [CrossRef]
  53. Moretto, J.; Pudlo, M.; Demougeot, C. Human-Based Evidence for the Therapeutic Potential of Arginase Inhibitors in Cardiovascular Diseases. Drug Discov. Today 2021, 26, 138–147. [Google Scholar] [CrossRef]
  54. Ilies, M.; Di Costanzo, L.; Dowling, D.P.; Thorn, K.J.; Christianson, D.W. Binding of α,α-Disubstituted Amino Acids to Arginase Suggests New Avenues for Inhibitor Design. J. Med. Chem. 2011, 54, 5432–5443. [Google Scholar] [CrossRef]
  55. Van Zandt, M.; Golebiowski, A.; Koo Ji, M.; Whitehouse, D.; Ryder, T.; Beckett, R.P. Inhibitor Arginase and Their Therapeutic Application. WO 2013/059437A1, 25 April 2013. [Google Scholar]
  56. Tomczuk, B.E.; Olson, G.L.; Pottorf, R.S.; Wang, J.; Nallaganchu, B.R. Arginase Inhibitors and Methods of Use Thereof. WO2012091757A1, 5 July 2012. [Google Scholar]
  57. Błaszczyk, R.; Brzezinska, J.; Golebiowski, A.; Olczak, J. Arginase Inhibitors and Their Therapeutic Applications. WO2016108707A1, 7 July 2016. [Google Scholar]
  58. Golebiowski, A.; Paul Beckett, R.; Van Zandt, M.; Ji, M.K.; Whitehouse, D.; Ryder, T.R.; Jagdmann, E.; Andreoli, M.; Mazur, A.; Padmanilayam, M.; et al. 2-Substituted-2-Amino-6-Boronohexanoic Acids as Arginase Inhibitors. Bioorganic Med. Chem. Lett. 2013, 23, 2027–2030. [Google Scholar] [CrossRef]
  59. Van Zandt, M.; Jagdmann, G.E., Jr. Boronates as Arginase Inhibitors. WO2012058065A1, 3 March 2012. [Google Scholar]
  60. Van Zandt, M.C.; Jagdmann, G.E.; Whitehouse, D.L.; Ji, M.; Savoy, J.; Potapova, O.; Cousido-Siah, A.; Mitschler, A.; Howard, E.I.; Pyle, A.M.; et al. Discovery of N-Substituted 3-Amino-4-(3-Boronopropyl)Pyrrolidine-3-Carboxylic Acids as Highly Potent Third-Generation Inhibitors of Human Arginase i and II. J. Med. Chem. 2019, 62, 8164–8177. [Google Scholar] [CrossRef]
  61. Wang, Z.; Li, N.; Ma, J.; Shao, Y. Heterocyclic Compounds as Arginase Inhibitors. WO2019120296A1, 27 June 2019. [Google Scholar]
  62. Foley, C.N.; Grange, R.L.; Guney, T.; Kalisiak, J.; Newcomb, E.T.; Tran, A.T. Arginase Inhibitors. WO2019173188A1, 19 September 2019. [Google Scholar]
  63. Mitcheltree, M.J.; Li, D.; Achab, A.; Beard, A.; Chakravarthy, K.; Cheng, M.; Cho, H.; Eangoor, P.; Fan, P.; Gathiaka, S.; et al. Discovery and Optimization of Rationally Designed Bicyclic Inhibitors of Human Arginase to Enhance Cancer Immunotherapy. ACS Med. Chem. Lett. 2020, 11, 582–588. [Google Scholar] [CrossRef] [PubMed]
  64. Blaszczyk, R.; Brzezinska, J.; Gzik, A.; Golebiowski, A.; Nowicka, J.; Borek, B.; Dziegielewski, M.; Jedrzejczak, K.; Matyszewski, K.; Olczak, J. Arginase Inhibitors and Their Therapeutic Applications. US 10391077, 27 August 2019. [Google Scholar]
  65. Marques, F.A.; Lenz, C.A.; Simonelli, F.; Noronha Sales Maia, B.H.L.; Vellasco, A.P.; Eberlin, M.N. Structure Confirmation of a Bioactive Lactone Isolated from Otoba Parvifolia through the Synthesis of a Model Compound. J. Nat. Prod. 2004, 67, 1939–1941. [Google Scholar] [CrossRef] [PubMed]
  66. Lu, M.; Zhang, H.; Li, D.; Childers, M.; Pu, Q.; Palte, R.L.; Gathiaka, S.; Lyons, T.W.; Palani, A.; Fan, P.W.; et al. Structure-Based Discovery of Proline-Derived Arginase Inhibitors with Improved Oral Bioavailability for Immuno-Oncology. ACS Med. Chem. Lett. 2021, 12, 1380–1388. [Google Scholar] [CrossRef] [PubMed]
  67. Achab, A.A.; Childers, M.L.; Cumming, J.N.; Fischer, C.A.; Gathiaka, S.; Gunadyn, H.; Lesburg, C.A.; Li, D.; Lu, M.; Palani, A.; et al. Arginase Inhibitors and Methods of Use. US20210040127A1, 11 February 2021. [Google Scholar]
  68. Mlynarski, S.N.; Grebe, T.; Kawatkar, S.; Verschoyle Finley, M.R.; Simpson, I.; Wang, J.; Cook, S. Arginase Inhibitors and Methods of Use Thereof. WO2019159120A1, 22 August 2019. [Google Scholar]
  69. Gross, M.; Chen, J.; Emberley, E.; Janes, J.; Li, W.; Mackinnon, A.; Pan, A.; Parlati, F.; Rodriguez, M.; Steggerda, S.; et al. Abstract A195: CB-1158 Inhibits the Immuno-Oncology Target Arginase and Causes an Immune Mediated Anti-Tumor Response. Mol. Cancer Ther. 2015, 14, A195. [Google Scholar] [CrossRef]
  70. Sjogren, E.B.; Li, J.; Van Zandt, M.; Whitehouse, D. Compositions and Methods for Inhibiting Arginase Activity. WO2017075363A1, 4 May 2017. [Google Scholar]
  71. Blaszczyk, R.; Gzik, A.; Borek, B.; Dziegielewski, M.; Jedrzejczak, K.; Nowicka, J.; Chrzanowski, J.; Brzezinska, J.; Golebiowski, A.; Olczak, J.; et al. Dipeptide Piperidine Derivatives. US20190300525A1, 3 October 2019. [Google Scholar]
  72. Achab, A.A.; Childers, M.L.; Cumming, J.N.; Fischer, C.; Gathiaka, S.; Gunaydin, H.; Lesburg, C.A.; Li, D.; Lu, M.; Palani, A.; et al. Arginase Inhibitors and Methods of Use. WO2019177873A1, 19 September 2019. [Google Scholar]
  73. Mlynarski, S.N.; Aquila, B.M.; Cantin, S.; Cook, S.; Doshi, A.; Finlay, M.R.V.; Gangl, E.T.; Grebe, T.; Gu, C.; Kawatkar, S.P.; et al. Discovery of (2R,4R)-4-((S)-2-Amino-3-methylbutanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic Acid (AZD0011), an Actively Transported Prodrug of a Potent Arginase Inhibitor to Treat Cancer. J. Med. Chem. 2024, 67, 20827–20841. [Google Scholar] [CrossRef]
  74. Grzybowski, M.M.; Stańczak, P.S.; Pęczkowicz-Szyszka, J.; Wolska, P.; Zdziarska, A.M.; Mazurkiewicz, M.; Brzezińska, J.; Blaszczyk, R.; Gołębiowski, A.; Dobrzański, P.; et al. Abstract P71: Novel Dual Arginase 1/2 Inhibitor OATD-02 (OAT-1746) Improves the Efficacy of Immune Checkpoint Inhibitors. Ann. Oncol. 2017, 28, xi20–xi21. [Google Scholar] [CrossRef]
  75. Borek, B.; Nowicka, J.; Gzik, A.; Dziegielewski, M.; Jedrzejczak, K.; Brzezinska, J.; Grzybowski, M.; Stanczak, P.; Pomper, P.; Zagozdzon, A.; et al. Arginase 1/2 Inhibitor OATD-02: From Discovery to First-in-Man Setup in Cancer Immunotherapy. Mol. Cancer Ther. 2023, 22, 807–817. [Google Scholar] [CrossRef]
  76. Shields, J.D.; Aquila, B.M.; Emmons, D.; Finlay, M.R.V.; Gangl, E.T.; Gu, C.; Mlynarski, S.N.; Petersen, J.; Pop-Damkov, P.; Sha, L.; et al. Design and Synthesis of Acyclic Boronic Acid Arginase Inhibitors. J. Med. Chem. 2024, 67, 20799–20826. [Google Scholar] [CrossRef]
  77. Girard-Thernier, C.; Pham, T.N.; Demougeot, C. The Promise of Plant-Derived Substances as Inhibitors of Arginase. Mini Rev. Med. Chem. 2015, 15, 798–808. [Google Scholar] [CrossRef]
  78. Bordage, S.; Pham, T.N.; Zedet, A.; Gugglielmetti, A.S.; Nappey, M.; Demougeot, C.; Girard-Thernier, C. Investigation of Mammal Arginase Inhibitory Properties of Natural Ubiquitous Polyphenols by Using an Optimized Colorimetric Microplate Assay. Planta Med. 2017, 83, 647–653. [Google Scholar] [CrossRef]
  79. Woo, A.; Min, B.; Ryoo, S. Piceatannol-3’-O-β-D-Glucopyranoside as an Active Component of Rhubarb Activates Endothelial Nitric Oxide Synthase through Inhibition of Arginase Activity. Exp. Mol. Med. 2010, 42, 524–532. [Google Scholar] [CrossRef] [PubMed]
  80. Arraki, K.; Totoson, P.; Attia, R.; Zedet, A.; Pudlo, M.; Messaoud, C.; Demougeot, C.; Girard, C. Arginase Inhibitory Properties of Flavonoid Compounds from the Leaves of Mulberry (Morus Alba, Moraceae). J. Pharm. Pharmacol. 2020, 72, 1269–1277. [Google Scholar] [CrossRef] [PubMed]
  81. Lim, C.J.; Cuong, T.D.; Hung, T.M.; Ryoo, S.; Lee, J.H.; Kim, E.H.; Woo, M.H.; Choi, J.S.; Min, B.S. Arginase II Inhibitory Activity of Phenolic Compounds from Saururus Chinensis. Bull. Korean Chem. Soc. 2012, 33, 3079–3082. [Google Scholar] [CrossRef]
  82. Kim, S.W.; Cuong, T.D.; Hung, T.M.; Ryoo, S.; Lee, J.H.; Min, B.S. Arginase II Inhibitory Activity of Flavonoid Compounds from Scutellaria Indica. Arch. Pharm. Res. 2013, 36, 922–926. [Google Scholar] [CrossRef]
  83. Pham, T.N.; Bordage, S.; Pudlo, M.; Demougeot, C.; Thai, K.M.; Girard-Thernier, C. Cinnamide Derivatives as Mammalian Arginase Inhibitors: Synthesis, Biological Evaluation and Molecular Docking. Int. J. Mol. Sci. 2016, 17, 1656. [Google Scholar] [CrossRef]
  84. ClinicalTrials.gov. National Library of Medicine. 2024. Available online: https://clinicaltrials.gov (accessed on 7 January 2025).
  85. Gandhi, L.; Rodríguez-Abreu, D.; Gadgeel, S.; Esteban, E.; Felip, E.; De Angelis, F.; Domine, M.; Clingan, P.; Hochmair, M.J.; Powell, S.F.; et al. Pembrolizumab plus Chemotherapy in Metastatic Non-Small-Cell Lung Cancer. N. Engl. J. Med. 2018, 378, 2078–2092. [Google Scholar]
  86. Naing, A.; Bauer, T.; Papadopoulos, K.P.; Rahma, O.; Tsai, F.; Garralda, E.; Naidoo, J.; Pai, S.; Gibson, M.K.; Rybkin, I.; et al. Phase I study of the arginase inhibitor INCB001158 (1158) alone and in combination with pembrolizumab (PEM) in patients (Pts) with advanced/metastatic (adv/met) solid tumours. Ann. Oncol. 2019, 30, v160. [Google Scholar]
  87. Naing, A.; Papadopoulos, K.P.; Pishvaian, M.; Rahma, O.; Hanna, G.J.; Garralda, E.; Saavedra, O.; Gogov, S.; Kallender, H.; Cheng, L.; et al. First-in-human phase 1 study of the arginase inhibitor INCB001158 alone or combined with pembrolizumab in patients with advanced or metastatic solid tumours. BMJ Oncol. 2024, 3, e000249. [Google Scholar] [CrossRef]
  88. National Library of Medicine, Study to Evaluate the Safety of CB-280 in Patients with Cystic Fibrosis-NCT04279769. Available online: https://clinicaltrials.gov/study/NCT04279769 (accessed on 17 June 2024).
  89. Boas, S.; Donaldson, S.; McBennett, K.; Liou, T.; Howrylak, J.; Johnson, L.; Teneback, C.; Dozor, A.; Sawicki, G.; Dumlao, J.; et al. 529: A phase 1b, randomized, double-blind, placebo-controlled, dose-escalation trial of CB-280, an arginase inhibitor, in patients with cystic fibrosis. J. Cyst. Fibros. 2021, 20, S250. [Google Scholar] [CrossRef]
  90. Lorentzen, C.L.; Martinenaite, E.; Kjeldsen, J.W.; Holmstroem, R.B.; Mørk, S.K.; Pedersen, A.W.; Ehrnrooth, E.; An-dersen, M.H.; Svane, I.M. Arginase-1 targeting peptide vaccine in patients with metastatic solid tumors—A phase I trial. Front. Immunol. 2022, 13, 1023023. [Google Scholar] [CrossRef]
  91. Sodano, F.; Cristiano, C.; Rolando, B.; Marini, E.; Lazzarato, L.; Cuozzo, M.; Albrizio, S.; Russo, R.; Rimoli, M.G. Galactosylated Prodrugs: A Strategy to Improve the Profile of Nonsteroidal Anti-Inflammatory Drugs. Pharmaceuticals 2022, 15, 552. [Google Scholar] [CrossRef] [PubMed]
  92. Sodano, F.; Gazzano, E.; Fraix, A.; Rolando, B.; Lazzarato, L.; Russo, M.; Blangetti, M.; Riganti, C.; Fruttero, R.; Gasco, A.; et al. A Molecular Hybrid for Mitochondria-Targeted NO Photodelivery. ChemMedChem 2018, 13, 87–96. [Google Scholar] [CrossRef] [PubMed]
  93. Sodano, F.; Rolando, B.; Spyrakis, F.; Failla, M.; Lazzarato, L.; Gazzano, E.; Riganti, C.; Fruttero, R.; Gasco, A.; Sortino, S. Tuning the Hydrophobicity of a Mitochondria-Targeted NO Photodonor. ChemMedChem 2018, 13, 1238–1245. [Google Scholar] [CrossRef] [PubMed]
  94. Sodano, F.; Gazzano, E.; Rolando, B.; Marini, E.; Lazzarato, L.; Fruttero, R.; Riganti, C.; Gasco, A. Tuning NO release of organelle-targeted furoxan derivatives and their cytotoxicity against lung cancer cells. Bioorg Chem. 2021, 111, 104911. [Google Scholar] [CrossRef] [PubMed]
Figure 1. Schematic representation of all ARG inhibitor classes.
Figure 1. Schematic representation of all ARG inhibitor classes.
Pharmaceutics 17 00117 g001
Figure 2. Chemical structures of compounds 13.
Figure 2. Chemical structures of compounds 13.
Pharmaceutics 17 00117 g002
Scheme 1. Synthesis of L-NOHA (4).
Scheme 1. Synthesis of L-NOHA (4).
Pharmaceutics 17 00117 sch001
Scheme 2. Synthesis of compound 5.
Scheme 2. Synthesis of compound 5.
Pharmaceutics 17 00117 sch002
Scheme 3. Synthesis of compounds 6 and 7.
Scheme 3. Synthesis of compounds 6 and 7.
Pharmaceutics 17 00117 sch003
Scheme 4. Synthesis of compound 8.
Scheme 4. Synthesis of compound 8.
Pharmaceutics 17 00117 sch004
Scheme 5. Synthesis of compound 9.
Scheme 5. Synthesis of compound 9.
Pharmaceutics 17 00117 sch005
Scheme 6. Synthesis of compound 10.
Scheme 6. Synthesis of compound 10.
Pharmaceutics 17 00117 sch006
Scheme 7. Synthesis of compound 11.
Scheme 7. Synthesis of compound 11.
Pharmaceutics 17 00117 sch007
Scheme 8. Synthesis of compound 12.
Scheme 8. Synthesis of compound 12.
Pharmaceutics 17 00117 sch008
Scheme 9. Synthesis of compounds 13 and 14.
Scheme 9. Synthesis of compounds 13 and 14.
Pharmaceutics 17 00117 sch009
Scheme 10. Synthesis of compounds 1519.
Scheme 10. Synthesis of compounds 1519.
Pharmaceutics 17 00117 sch010
Scheme 11. Synthesis of compound 20.
Scheme 11. Synthesis of compound 20.
Pharmaceutics 17 00117 sch011
Figure 3. Chemical structures of compounds 18F-FMARS and 18F-FBMARS.
Figure 3. Chemical structures of compounds 18F-FMARS and 18F-FBMARS.
Pharmaceutics 17 00117 g003
Scheme 12. Synthesis of compounds 2146.
Scheme 12. Synthesis of compounds 2146.
Pharmaceutics 17 00117 sch012
Scheme 13. Synthesis of compounds 4749.
Scheme 13. Synthesis of compounds 4749.
Pharmaceutics 17 00117 sch013
Scheme 14. Synthesis of compounds 5051.
Scheme 14. Synthesis of compounds 5051.
Pharmaceutics 17 00117 sch014
Scheme 15. Synthesis of compound 52.
Scheme 15. Synthesis of compound 52.
Pharmaceutics 17 00117 sch015
Scheme 16. Synthesis of compound 53.
Scheme 16. Synthesis of compound 53.
Pharmaceutics 17 00117 sch016
Scheme 17. Synthesis of compound 54.
Scheme 17. Synthesis of compound 54.
Pharmaceutics 17 00117 sch017
Scheme 18. Synthesis of compound 55.
Scheme 18. Synthesis of compound 55.
Pharmaceutics 17 00117 sch018
Scheme 19. Synthesis of compound 56.
Scheme 19. Synthesis of compound 56.
Pharmaceutics 17 00117 sch019
Figure 4. Chemical structures of compounds 5758.
Figure 4. Chemical structures of compounds 5758.
Pharmaceutics 17 00117 g004
Scheme 20. Synthesis of compound 57.
Scheme 20. Synthesis of compound 57.
Pharmaceutics 17 00117 sch020
Scheme 21. Synthesis of compound 59.
Scheme 21. Synthesis of compound 59.
Pharmaceutics 17 00117 sch021
Scheme 22. Synthesis of compound 60.
Scheme 22. Synthesis of compound 60.
Pharmaceutics 17 00117 sch022
Scheme 23. Synthesis of compound 61.
Scheme 23. Synthesis of compound 61.
Pharmaceutics 17 00117 sch023
Scheme 24. Synthesis of compound 62.
Scheme 24. Synthesis of compound 62.
Pharmaceutics 17 00117 sch024
Scheme 25. Synthesis of compound 63.
Scheme 25. Synthesis of compound 63.
Pharmaceutics 17 00117 sch025
Scheme 26. Synthesis of compound 64.
Scheme 26. Synthesis of compound 64.
Pharmaceutics 17 00117 sch026
Scheme 27. Synthesis of compound 65.
Scheme 27. Synthesis of compound 65.
Pharmaceutics 17 00117 sch027
Scheme 28. Synthesis of compound 66.
Scheme 28. Synthesis of compound 66.
Pharmaceutics 17 00117 sch028
Scheme 29. Synthesis of compound 67.
Scheme 29. Synthesis of compound 67.
Pharmaceutics 17 00117 sch029
Scheme 30. Synthesis of compound 68.
Scheme 30. Synthesis of compound 68.
Pharmaceutics 17 00117 sch030
Scheme 31. Synthesis of compound 69.
Scheme 31. Synthesis of compound 69.
Pharmaceutics 17 00117 sch031
Scheme 32. Synthesis of compound 70.
Scheme 32. Synthesis of compound 70.
Pharmaceutics 17 00117 sch032
Scheme 33. Synthesis of compounds 7173.
Scheme 33. Synthesis of compounds 7173.
Pharmaceutics 17 00117 sch033
Scheme 34. Synthesis of compound 74.
Scheme 34. Synthesis of compound 74.
Pharmaceutics 17 00117 sch034
Scheme 35. Synthesis of compounds 7576.
Scheme 35. Synthesis of compounds 7576.
Pharmaceutics 17 00117 sch035
Scheme 36. Synthesis of compounds 7778.
Scheme 36. Synthesis of compounds 7778.
Pharmaceutics 17 00117 sch036
Figure 5. Chemical structures of compounds 7981.
Figure 5. Chemical structures of compounds 7981.
Pharmaceutics 17 00117 g005
Scheme 37. Synthesis of compound 79.
Scheme 37. Synthesis of compound 79.
Pharmaceutics 17 00117 sch037
Scheme 38. Synthesis of compound 82.
Scheme 38. Synthesis of compound 82.
Pharmaceutics 17 00117 sch038
Scheme 39. Synthesis of compound 83.
Scheme 39. Synthesis of compound 83.
Pharmaceutics 17 00117 sch039
Scheme 40. Synthesis of compound 84.
Scheme 40. Synthesis of compound 84.
Pharmaceutics 17 00117 sch040
Scheme 41. Synthesis of compound 85.
Scheme 41. Synthesis of compound 85.
Pharmaceutics 17 00117 sch041
Figure 6. Chemical structures of compounds 8690.
Figure 6. Chemical structures of compounds 8690.
Pharmaceutics 17 00117 g006
Scheme 42. Synthesis of compounds 87–88.
Scheme 42. Synthesis of compounds 87–88.
Pharmaceutics 17 00117 sch042
Table 1. Chemical structures of compounds 2146.
Table 1. Chemical structures of compounds 2146.
Pharmaceutics 17 00117 i001
CompoundRIC50
21
(S)-2-amino-6-borono-2-(1S,3R)-3-(3-phenylpropylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i0020.1–25 nM (hARG-1)
26–100 nM (hARG-2)
22
(S)-2-amino-6-borono-2-((1S,3R)-3-(3-(3-chloro-5fluorophenyl)propylamino)
cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i00326–100 nM (hARG-1)
0.1–25 nM (hARG-2)
23
(S)-2-amino-6-borono-2-((1s,3R)-3-(3-(3,4-difluorophenyl)propylamino)cyclobutyl) hexanoic acid
Pharmaceutics 17 00117 i00426–100 nM (hARG-1)
26–100 nM (hARG-2)
24
(S)-2-amino-6-borono-2-((1S,3R)-3-(3-(2,4-dichlorophenyl)propylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i00526–100 nM (hARG-1)
26–100 nM (hARG-2)
25
(S)-2-amino-6-borono-2-((1S,3R)-3-(2,3-dihydro-1h-inden-2yl-amino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i0060.1–25 nM (hARG-1)
26–100 nM (hARG-2)
26
(S)-2-amino-6-borono-2-((1S,3R)-3-(4-tert-butylbenzylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i00726–100 nM (hARG-1)
26–100 nM (hARG-2)
27
(S)-2-amino-2-((1S,3R)-3-(biphenyl-3-ylmethylamino)cyclobutyl)-6-boronohexanoic acid
Pharmaceutics 17 00117 i0080.1–25 nM (hARG-1)
26–100 nM (hARG-2)
28
(S)-2-amino-6-borono-2-((1S,3R)-3-((4′-(trifluoromethyl)biphenyl-3-yl) methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i00926–100 nM (hARG-1)
26–100 nM (hARG-2)
29
(S)-2-amino-6-borono-2-((1S,3R)-3-((4′-chlorobiphenyl-3yl)methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i0100.1–25 nM (hARG-1)
0.1–25 nM (hARG-2)
30
(S)-2-amino-6-borono-2-((1S,3R)-3-((4-fluoronaphthalen-1yl)methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i0110.1–25 nM (hARG-1)
0.1–25 nM (hARG-2)
31
(S)-2-amino-6-borono-2-((1S,3R)-3-((5-fluoronaphthalen-1yl)methylaminocyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i0120.1–25 nM (hARG-1)
26–100 nM (hARG-2)
32
(S)-2-amino-2-(1S,3R)-3-(anthracen-9-ylmethylamino)cyclobutyl)-6-borono hexanoic acid
Pharmaceutics 17 00117 i01326–100 nM (hARG-1)
0.1–25 nM (hARG-2)
33
(S)-2-amino-6-borono-2-((1S,3R)-3-(2-morpholinobenzylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i01426–100 nM (hARG-1)
26–100 nM (hARG-2)
34
(S)-2-amino-6-borono-2-((1R,3R)-3-(((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i01526–100 nM (hARG-1)
26–100 nM (hARG-2)
35
(S)-2-amino-6-borono-2-((1S,3R)-3-((2,3-dihydrobenzofuran-5-yl)methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i01626–100 nM (hARG-1)
26–100 nM (hARG-2)
36
(S)-2-amino-6-borono-2-((1S,3R)-3-((3′,4′-dichlorobiphenyl-4yl)methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i0170.1–25 nM (hARG-1)
0.1–25 nM (hARG-2)
37
(S)-2-amino-6-borono-2-((1S,3R)-3-((4′-chlorobiphenyl-4yl)methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i0180.1–25 nM (hARG-1)
0.1–25 nM (hARG-2)
38
(S)-2-amino-6-borono-2-((1S,3R)-3-((4′-(trifluoromethyl)biphenyl-4-yl)methylamino)cyclobutyl) hexanoic acid
Pharmaceutics 17 00117 i0190.1–25 nM (hARG-1)
0.1–25 nM (hARG-2)
39
(S)-2-amino-6-borono-2-((1S,3R)-3-((4′-fluorobiphenyl-4yl)methylamino)cyclobutyl)hecanoic acid
Pharmaceutics 17 00117 i0200.1–25 nM (hARG-1)
26–100 nM (hARG-2)
40
(S)-2-amino-6-borono-2-((1S,3R)-3-(4-hydroxybenzylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i02126–100 nM (hARG-1)
26–100 nM (hARG-2)
41
(S)-2-amino-6-borono-2-((1S,3R)-3-(4-(4-chlorophenoxy)benzylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i0220.1–25 nM (hARG-1)
0.1–25 nM (hARG-2)
42
(S)-2-amino-6-borono-2-(1S,3R)-3-((4′-chlorobiphenyl-2-yl)methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i02326–100 nM (hARG-1)
26–100 nM (hARG-2)
43
(S)-2-amino-6-borono-2-((1S,3R)-3-((6-phenylpyridin-3-yl)methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i02426–100 nM (hARG-1)
0.1–25 nM (hARG-2)
44
(S)-2-((1S,3R)-3-((9h-fluoren-2-yl)methylamino)cyclobutyl)-2-amino-6-borono-hexanoic acid
Pharmaceutics 17 00117 i0250.1–25 nM (hARG-1)
0.1–25 nM (hARG-2)
45
(S)-2-amino-6-borono-2-((1S,3R)-3-((4′-(trifluoromethyl)biphenyl-2 yl)methylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i02626–100 nM (hARG-1)
26–100 nM (hARG-2)
46
(S)-2-amino-6-borono-2-((1S,3R)-3-(4-cyclohexylbenzylamino)cyclobutyl)hexanoic acid
Pharmaceutics 17 00117 i0270.1–25 nM (hARG-1)
26–100 nM (hARG-2)
Table 2. Structures and IC50 values of natural and semi-synthetic compounds.
Table 2. Structures and IC50 values of natural and semi-synthetic compounds.
CompoundStructureARG-1 ActivityARG-2 Activity
91
Chlorogenic acid
Pharmaceutics 17 00117 i028IC50 = 10.6 µM a
92
Picetannol
Pharmaceutics 17 00117 i029IC50 = 12.1 µM a
93
Resveratrol
Pharmaceutics 17 00117 i030IC50 = 18.2 µM a
94
Taxifolin
Pharmaceutics 17 00117 i031IC50 = 23.2 µM a
95
Piceatannol-3′-O-β-D-glucopyranoside
Pharmaceutics 17 00117 i032IC50 = 11.22 µM bIC50 = 11.06 µM c
96
Ellagic acid
Pharmaceutics 17 00117 i033IC50 = 78.9 µM a
97
Luteolin-7-diglucoside
Pharmaceutics 17 00117 i034IC50 = 152.4 µM a
98
Luteolin-7-glucoside
Pharmaceutics 17 00117 i035IC50 = 99.4 µM a
99
Luteolin
Pharmaceutics 17 00117 i036IC50 = 95.3 µM a
100
Scirpusin B
Pharmaceutics 17 00117 i037IC50 = 22.6 µM a
101
ε-Viniferin
Pharmaceutics 17 00117 i038IC50 = 27.8 µM a
102
Cyperusphenol B
Pharmaceutics 17 00117 i039IC50 = 12.2 µM a
103
Carexinol A
Pharmaceutics 17 00117 i040IC50 = 25.3 µM a
104
Virgatanol
Pharmaceutics 17 00117 i041IC50 = 182.1 µM a
105
Sauchinone
Pharmaceutics 17 00117 i042 IC50 = 61.4 µM d
106
(2S)-5,7-dihydroxy-8,20-dimethoxyflavanone
Pharmaceutics 17 00117 i043 IC50 = 25.1 µM c
107
(2S)-5,20,50-trihydroxy-7, 8-dimethoxyflavanone
Pharmaceutics 17 00117 i044 IC50 = 11.6 µM c
108
Caffeic acid phenylamide
(CAPA)
Pharmaceutics 17 00117 i045IC50 = 6.9 µM a
IC50 = 60.3 µM e
a bovine liver ARG; b rat liver ARG; c rat kidney ARG; d murine kidney ARG; e human ARG.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Molaro, M.C.; Battisegola, C.; Schiano, M.E.; Failla, M.; Rimoli, M.G.; Lazzarato, L.; Chegaev, K.; Sodano, F. Synthesis of Arginase Inhibitors: An Overview. Pharmaceutics 2025, 17, 117. https://doi.org/10.3390/pharmaceutics17010117

AMA Style

Molaro MC, Battisegola C, Schiano ME, Failla M, Rimoli MG, Lazzarato L, Chegaev K, Sodano F. Synthesis of Arginase Inhibitors: An Overview. Pharmaceutics. 2025; 17(1):117. https://doi.org/10.3390/pharmaceutics17010117

Chicago/Turabian Style

Molaro, Maria Cristina, Chiara Battisegola, Marica Erminia Schiano, Mariacristina Failla, Maria Grazia Rimoli, Loretta Lazzarato, Konstantin Chegaev, and Federica Sodano. 2025. "Synthesis of Arginase Inhibitors: An Overview" Pharmaceutics 17, no. 1: 117. https://doi.org/10.3390/pharmaceutics17010117

APA Style

Molaro, M. C., Battisegola, C., Schiano, M. E., Failla, M., Rimoli, M. G., Lazzarato, L., Chegaev, K., & Sodano, F. (2025). Synthesis of Arginase Inhibitors: An Overview. Pharmaceutics, 17(1), 117. https://doi.org/10.3390/pharmaceutics17010117

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop