ecto-5'-Nucleotidase (eN, CD73) catalyzes the hydrolysis of extracellular AMP to adenosine. eN inhibitors have potential for use as cancer therapeutics. The eN inhibitor α,β-methylene-ADP (AOPCP, adenosine-5'-O-[(phosphonomethyl)phosphonic acid]) was used as a lead structure, and derivatives modified in various positions were prepared. Products were tested at rat recombinant eN. 6-(Ar)alkylamino substitution led to the largest improvement in potency. N(6)-Monosubstitution was superior to symmetrical N(6),N(6)-disubstitution. The most potent inhibitors were N(6)-(4-chlorobenzyl)- (10l, PSB-12441, Ki 7.23 nM), N(6)-phenylethyl- (10h, PSB-12425, Ki 8.04 nM), and N(6)-benzyl-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (10g, PSB-12379, Ki 9.03 nM). Replacement of the 6-NH group in 10g by O (10q, PSB-12431) or S (10r, PSB-12553) yielded equally potent inhibitors (10q, 9.20 nM; 10r, 9.50 nM). Selected compounds investigated at the human enzyme did not show species differences; they displayed high selectivity versus other ecto-nucleotidases and ADP-activated P2Y receptors. Moreover, high metabolic stability was observed. These compounds represent the most potent eN inhibitors described to date.

Ecto‐5′‐nucleotidase (CD73, EC 3.1.3.5) catalyzes the extracellular hydrolysis of AMP yielding adenosine, which induces immunosuppression, angiogenesis, metastasis, and proliferation of cancer cells. CD73 inhibition is therefore proposed as a novel strategy for cancer (immuno)therapy, and CD73 antibodies are currently undergoing clinical trials. Despite considerable efforts, the development of small molecule CD73 inhibitors has met with limited success. To develop a suitable drug candidate, a high resolution (2.05 Å) co‐crystal structure of the CD73 inhibitor PSB‐12379, a nucleotide analogue, in complex with human CD73 is determined. This allows the rational design and development of a novel inhibitor (PSB‐12489) with subnanomolar inhibitory potency toward human and rat CD73, high selectivity, as well as high metabolic stability. A co‐crystal structure of PSB‐12489 with CD73 (1.85 Å) reveals the interactions responsible for increased potency. PSB‐12489 is the most potent CD73 inhibitor to date representing a powerful tool compound and novel lead structure.

The potent and selective GPR35 agonist 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (12) was obtained in tritium-labeled form, designated [(3)H]PSB-13253, with a specific activity of 36 Ci (1.33 TBq)/mmol. Radiolabeling was achieved by methylation of ethyl 6-bromo-8-(4-((tert-butyldimethylsilyl)oxy)benzamido)-4-oxo-4H-chromene-2-carboxylate (19) with [(3)H]methyl tosylate followed by ester hydrolysis. The radioligand was characterized by kinetic, saturation, and competition assays at membrane preparations of Chinese hamster ovary cells recombinantly expressing the human GPR35. [(3)H]12 labeled the receptor with high affinity (KD = 5.27 nM). Binding was saturable (Bmax = 12.6 pmol/mg of protein) and reversible. Affinities of selected standard ligands and a library of amidochromen-4-one-2-carboxylates were determined. Binding data mostly correlated with potencies determined in β-arrestin assays. On the basis of the test results, several new fluorine-substituted 6-bromo-8-benzamidochromen-4-one-2-carboxylic acids were obtained, which represent the most potent GPR35 agonists known to date. 6-Bromo-8-(2,6-difluoro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (83; Ki = 0.589 nM, EC50 = 5.54 nM) showed the highest affinity with a Ki value in the subnanomolar range.

The lipid-activated G protein-coupled receptor (GPCR) GPR55 has been proposed as a drug target for the treatment of chronic diseases including inflammation, neurodegeneration, neuropathic pain, metabolic diseases, and cancer. A series of chromen-4-one-2-carboxylic acid derivatives was synthesized with the aim to obtain potent and selective ligands for GPR55 by (i) attachment of a variety of substituted 8-benzamido residues, (ii) substitution in position 6 by halogen atoms, and (iii) thioation of the 4-oxo function. The compounds were investigated in β-arrestin recruitment assays using enzyme complementation. Depending on the substitution pattern, a spectrum of efficacies was obtained ranging from (partial) agonists to antagonists. 6-Chloro-8-(3-((5-cyclohexylpentyl)­oxy)­benzamido)-4-oxo-4H-chromene-2-carboxylic acid (74, PSB-18251) displayed the highest efficacy of the series combined with high potency (EC50 0.196 μM). 6-Chloro-8-(3-(heptyloxy)­benzamido)-4-oxo-4H-chromene-2-carboxylic acid (76, PSB-18337) exhibited higher potency (EC50 0.0400 μM) but lower efficacy (39%). Several GPR55 antagonists were discovered including 8-(3-(cyclohexylmethoxy)­benzamido)-4-oxo-4H-chromene-2-carboxylic acid (57, PSB-18263) (IC50 8.23 μM) and 4-oxo-8-(3-phenethoxybenzamido)-4H-chromene-2-carboxylic acid (65, PSB-18270) (IC50 3.96 μM). These potent GPR55 agonists and antagonists showed high selectivity versus the related GPCRs GPR18 and GPR35 tested in the same assay system, while 8-(4-(4-cyclohexylbutoxy)­benzamido)-6-fluoro-4-oxo-4H-chromene-2-carboxylic acid (84, PSB-18177) represents a dual GPR35/GPR55 antagonist (IC50 GPR55: 3.26 μM, GPR35: 2.57 μM). Binding studies of selected compounds at CB1 and CB2 receptors indicated GPR55 selectivity also versus CB receptors. The newly developed GPR55 (partial) agonists and antagonists will be useful tools for evaluating the suitability of GPR55 as a drug target.

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