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Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A2a and A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5'-(α,β-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.

Extracellular adenosine (ADO), present in high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T cell and NK cell activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP → AMP) and CD73 (AMP → ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Extensive interrogation of structure-activity relationships (SARs), structure-based drug design, and optimization of pharmacokinetic properties culminated in the discovery of AB680, a highly potent (Ki = 5 pM), reversible, and selective inhibitor of CD73. AB680 is further characterized by very low clearance and long half-lives across preclinical species, resulting in a PK profile suitable for long-acting parenteral administration. AB680 is currently being evaluated in phase 1 clinical trials. Initial data show AB680 is well tolerated and exhibits a pharmacokinetic profile suitable for biweekly (Q2W) iv-administration in human.

AB928 is a potent and selective dual antagonist of the A2a and A2b receptors, which is currently in clinical trials. Here, we report the development of two scalable and practical syntheses of AB928...

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme's natural substrate, adenosine 5'-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (73, IC50 = 12 nM) and 4-({5-[4-chloro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (74, IC50 = 19 nM). Cocrystallization of 74 with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.

Diorganocuprate(I) reagents derived from lithiated heterocycles and CuCN react with enantioenriched secondary propargyl bromides to give the corresponding propargylated heterocycles. While propargyl electrophiles typically undergo SN2′ displacement, this transformation represents the first example of the reaction of hard carbanions with propargyl electrophiles in an SN2 fashion and occurs with excellent levels of stereoinversion. The new method was applied to the formal synthesis of (+)-frondosin B.

Lithium alkynyl(disilyl)zincates obtained from metalated ethynyloxiranes, as well as from N - tert -butanesulfinyl(ethynyl)aziridines or N - tert -butanesulfonyl(ethynyl)aziridines, undergo 1,2-migration of the organosilyl group with ring opening of the oxirane or aziridine ring by S N i displacement. A developed protocol that involves n BuLi for the metalation step offers a straightforward approach to the corresponding δ-oxy- and δ-amino α-silyl allenylzinc intermediates. The reagents derived from the epoxides are amenable to subsequent in situ condensation with aldehydes or ketones to provide 1,3-diols but not those derived from aziridines that only react sluggishly in similar condensations.

International audience; 2-Fluoropyridinyl-6-oxy- precursors derived from phenyl vinyl sulfide react with radicals generated from xanthates via an addition-elimination process to furnish the corresponding vinyl sulfides in good yields. This convergent method is operationally simple and enables a straightforward synthesis of the difficult to access tetrasubstituted vinyl sulfides. Vinyl sulfides were used as more robust enol ether surrogates in highly stereoselective reactions with N-acylium cations leading to nitrogen-containing polycyclic structures.

Diorganocuprate(I) reagents derived from lithiated heterocycles and CuCN react with enantioenriched secondary propagryl bromides to give the corresponding propargylated heterocycles. While propargyl electrophiles typically undergo SN2′ displacement, this transformation represents the first example of the reaction of hard carbanions with propargyl eletrophiles in an SN2 fashion and occurs with excellent levels of stereoinversion. The new method was applied to the formal synthesis of (+)-frondosin B.

α-Trifluoromethyl-styrenes, trifluoromethyl-enynes, and dienes undergo palladium-catalyzed trimethylenemethane cycloadditions under mild reaction conditions. The trifluoromethyl group serves as a unique σ-electron-withdrawing group for the activation of the olefin toward the cycloaddition. This method allows for the formation of exomethylene cyclopentanes bearing a quaternary center substituted by the trifluoromethyl group, compounds of interest for the pharmaceutical, agrochemical, and materials industries. In the diene series, the cycloaddition operates in a [3 + 4] and/or [3 + 2] manner to give rise to seven- and/or five-membered rings. This transformation greatly improves the scope of the TMM cycloaddition technology and provides invaluable insights into the reaction mechanism.

International audience; 2-Fluoro-6-pyridinyloxy derivatives of 2-ethoxyvinyl carbinols react with radicals derived from xanthates by an addition-fragmentation pathway to give highly functionalized ketones after acid hydrolysis. 1,4-Diketones are readily accessible by this approach. a,ß-Unsaturated ketones can be obtained by starting with geminal acetoxy xanthates prepared by addition of a simpler xanthate to vinyl acetate. © 2011 American Chemical Society.

α-Trifluoromethyl-styrenes, trifluoromethyl-enynes, and dienes undergo palladium-catalyzed trimethylenemethane cycloadditions under mild reaction conditions. The trifluoromethyl group serves as a unique σ-electron-withdrawing group for the activation of the olefin toward the cycloaddition. This method allows for the formation of exomethylene cyclopentanes bearing a quaternary center substituted by the trifluoromethyl group, compounds of interest for the pharmaceutical, agrochemical, and materials industries. In the diene series, the cycloaddition operates in a [3 + 4] and/or [3 + 2] manner to give rise to seven- and/or five-membered rings. This transformation greatly improves the scope of the TMM cycloaddition technology and provides invaluable insights into the reaction mechanism.

ConspectusRadical allylations represent effective routes to various alkenes, but to date they have relied chiefly on organostannane derivatives and still suffer from significant limitations with respect to the substitution pattern of the starting allylating agent. Indeed, while substituents at the β-position relative to the radical leaving group are well-tolerated, introduction of α-substituents induces a major complication due to the rapid and usually irreversible isomerization of the starting allylating agents. Although a number of research groups have made substantial efforts to develop heavy-metal-free radical allylations, methods compatible with α-substitution of the allylating agent are still scarce. Furthermore, quite a few systems are limited by the relative inaccessibility of the substrates. This Account summarizes our sustained efforts regarding the development of allylic alcohols into “ideal” radical allylating agents and presents published as well as some unpublished results. The systems we ha...

Introduction: In the tumor micro-environment, extracellular ATP is sequentially hydrolyzed to adenosine by the ecto-nucleotidases CD39 (ATP→AMP) and CD73 (AMP→adenosine). Adenosine, through activation of the A2a receptor (A2aR), is a potent inhibitor of T-cell activation, resulting in an immunosuppressed phenotype. Thus, inhibition of A2aR has recently generated great interest in immuno-oncology. We present the characterization of a novel, selective, and highly potent small molecule antagonist of A2aR which is slated to enter the clinic in 2017. Methods: The cellular potency of A2aR antagonists was assessed as a function of decreased cAMP levels in CHO cells stably over-expressing hA2aR, a Gs coupled receptor, following stimulation with the agonist NECA. Experiments were conducted in the presence and absence of human serum. Selectivity against the Gi-coupled receptor A1R was assessed similarly as a function of cAMP elevation in CHO cells stably expressing hA1R, following pretreatment with forskolin and stimulation with NECA. The ability of AB928 to reverse adenosine-mediated immune suppression (25 μM) of human or mouse CD8+ T-cells was determined using standard CD3/CD28 activation conditions. CD25 expression and cytokine release were measured by flow cytometry and ELISA, respectively. The pharmacokinetic characteristics of AB928 were assessed in rodent and non-rodent species to facilitate calculation of a projected human dose. Results: AB928 represents a novel series of potent and selective compounds designed to inhibit adenosine-mediated A2aR activation. This molecule is different from most known A2aR antagonists in that it does not cross the blood brain barrier. AB928 inhibited NECA-mediated A2aR activation with a potency of Conclusions: AB928 is a potent, selective and peripherally restricted antagonist of the A2aR receptor which is slated to enter clinical development in 2017. Citation Format: Matt J. Walters, Joanne B. Tan, Annette Becker, Fangfang Yi, Tim Park, Manmohan R. Leleti, Brandon Rosen, Ehesan Sharif, Laurent Debien, Steve Young, Wan Hsin Lim, Stefan Garrido-Shaqfeh, Juan C. Jaen, Jay P. Powers. Characterization of the potent and selective A2aR antagonist AB928 for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4572. doi:10.1158/1538-7445.AM2017-4572

2-Fluoropyridinyl-6-oxy-substituted vinyl sulfides react with xanthate-derived radicals to yield functionalized vinyl sulfides via an addition/elimination process.

An operationally simple reaction sequence for the synthesis of α-keto vinyl carbinols is developed.

International audience; Conjugate addition of lithium enolates onto terminal alkynyl- and allenyl-sulfoxides furnishes the corresponding allylic sulfoxides. The latter readily undergo a Mislow-Braverman-Evans rearrangement to yield the targeted α-keto vinyl carbinols. This two-step procedure does not require purification of the intermediates and constitutes the shortest approach to α-keto vinyl carbinols.

Unprotected vinyl carbinols react with the radicals generated from xanthanes involving a rare cleavage of a C—C bond in a non-strained open chain structure.

Title reaction gives zincates that undergo a 1,2-metallate rearrangement along with the transfer of an alkyl or a silyl group affording allenylzinc intermediates.

International audience; A new radical addition/C-C bond fragmentation process is reported. Vinyl carbinols derived from 2-methyl-2-phenylpropanal react with radicals generated from xanthates to give the corresponding ketones. The radical cleavage reaction proceeds under mild conditions, in good to high yield, and in the presence of the unprotected carbinol. Highly functionalized 1,5-diketones and pyridines are readily available using this approach.

A wide range of biologically important title compounds is available under these conditions.

Introduction: Intra-tumoral generation of adenosine (ADO), a potent inhibitor of T-cell activation, requires the coordinated and sequential cleavage of extracellular adenosine triphosphate (ATP) by the ecto-nucleotidases CD39 (which produces adenosine monophosphate, AMP) and CD73 (which hydrolyzes AMP to ADO). For this reason, various anti-CD73 antibodies are being advanced into clinical trials; however, there are few reports of potent, selective, small-molecule CD73 inhibitors, such as those described here. Methods: Enzymatic assays: Ecto-nucleotidase activity was calculated based on the amount of inorganic phosphate (malachite green assay; absorbance at 620 nm) produced after 50-min incubation with 25μM AMP or ATP, in the presence of varying concentrations of test compound(s). The following systems were used. Endogenous expression: hCD73/SKOV-3 cells; mCD73/E771 cells. Stable over-expression: hCD73/CHO; hCD39/CHO. Transient expression: mCD73/CHO; NTPDase2/CHO; NTPDase3/CHO; NTPDase8/CHO. CD8+ T cell functional assays: Human (enriched from buffy coats or leukopaks) and mouse (isolated from spleen of C57BL/6 mice) CD8+ T cells were pretreated with CD73 inhibitor or buffer control. One hour later, cells were stimulated (anti-CD3/CD28; +/- 50 μM AMP) in the presence of ADO deaminase inhibitor (10 μM). 3 days after stimulation, cell activation (CD25), proliferation (CFSE) and effector function (IFN-γ and granzyme B) were quantified by flow cytometry. Tumor model: CT26 cells were subcutaneously implanted into Balb/c mice. When tumor volumes were ∼100 mm3, mice were enrolled into cohorts (1: vehicle control; 2: A000830; 3: PD-1 Ab; 4: A000830 + PD-1 Ab) of ≥13 mice each. Interim immune phenotyping was performed when Group 1 tumor volumes were 800-1,000 mm3. Single-cell suspensions were generated from tumors and spleens (gentleMACS). 106 cells were blocked using purified CD16/32 antibodies (clone 2.4G2) and stained with the appropriate antibody cocktails. Results: We have designed a series of potent and specific small-molecule inhibitors of human and mouse CD73, represented by A000830 (IC50 against human and mouse CD73 of 1.0 nM and 3 nM, respectively). A000830 blocked ADO generation from AMP by human ovarian cancer cells (SKOV-3) with IC50: 0.2 nM and >10,000-fold selectivity relative to other ecto-nucleotidases (CD39, NTPDase2, NTPDase3, NTPDase8) and a large panel of unrelated enzymes, receptors and ion channels. In in vitro models of ADO-driven inhibition of human and mouse CD8+ T-cell activation, A000830 showed robust rescue of proliferation, CD25 expression, and IFNγ and granzyme B production. A000830 was easily administered (and well tolerated) to mice, resulting in sustained plasma concentrations at least 1,000 times higher than its potency against mouse CD73. In the spleen of CT26 tumor-bearing Balb/c mice, CD39 expression was found mainly on natural killer (NK) cells and myeloid-lineage cells, while in the tumor CD39 was highly expressed on myeloid cells (myeloid-derived suppressor cells (MDSC) and dendritic cells (DC)) as well as CD4+ and CD8+ T cells. CD73 expression was limited (spleen and tumor) to NK cells and CD4+ and CD8+ T cells. Therapeutic dosing of A000830 to these mice (tumors ∼100 mm3 in size) in combination with anti-PD1 antibody produced robust tumor growth inhibition, greater than either treatment alone. This effect was associated with increased CD8:Treg ratios in tumors (but not spleens). Conclusions: We describe a novel class of potent and selective small-molecule CD73 inhibitors that effectively block the generation of ADO from extracellular ATP, reverse the ADO-driven inhibition of human and mouse T-cell activation, and display promising anti-tumor activity when dosed in combination with PD-1 blockade. Citation Format: Juan C. Jaen, Jay P. Powers, Ada Chen, Manmohan R. Leleti, Jarek Kalisiak, Ulrike Schindler, Joanne Bl Tan, Steve Young, Shin-Heng Chiou, Laurent Debien. Small-molecule inhibitors of CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR10.

Reaction of dialkylzincs with lithiated acetylenic epoxides is described to give zincates that undergo a 1,2-metallate rearrangement by an anti-S(N)2' pathway. This rearrangement occurs with the transfer of an alkyl or a silyl group affording allenylzinc intermediates. Allenic and/or homopropargylic alcohols are obtained upon hydrolysis. Quenching the reaction mixture with aldehydes or ketones is shown to give access to 2-alkynyl-1,3-diols in a stereoselective manner.

International audience; A modular, convergent, and operationally simple route to trifluoromethyl alkenes and vinyl fluorides involving a unique carbon-oxygen bond homolysis is reported. Highly functionalized trifluoromethyl alkenes and vinyl fluorides were obtained in good yields and good selectivity.

The current treatment for leishmaniasis is based on chemotherapy, which relies on a handful of drugs with serious limitations, such as high cost, toxicity, and a lack of efficacy in regions of endemicity. Therefore, the development of new, effective, and affordable antileishmanial drugs is a global health priority. Leishmania synthesizes a range of mannose-rich glycoconjugates that are essential for parasite virulence and survival. A prerequisite for glycoconjugate biosynthesis is the conversion of monosaccharides to the activated mannose donor, GDP-mannose, the product of a reaction catalyzed by GDP-mannose pyrophosphorylase (GDP-MP). The deletion of the gene encoding GDP-MP in Leishmania led to a total loss of virulence, indicating that the enzyme is an ideal drug target. We developed a phosphate sensor-based high-throughput screening assay to quantify the activity of GDP-MP and screened a library containing ∼80,000 lead-like compounds for GDP-MP inhibitors. On the basis of their GDP-MP inhibitory properties and chemical structures, the activities of 20 compounds which were not toxic to mammalian cells were tested against ex vivo amastigotes and in macrophage amastigote assays. The most potent compound identified in the primary screen (compound 3), a quinoline derivative, demonstrated dose-dependent activity in both assays (50% inhibitory concentration = 21.9 μM in the macrophage assay) and was shown to be nontoxic to human fibroblasts. In order to elucidate signs of an early structure-activity relationship (SAR) for this class of compounds, we obtained and tested analogues of compound 3 and undertook limited medicinal chemistry optimization, which included the use of a number of SAR probes of the piperazinyl aryl substituent of compound 3. We have identified novel candidate compounds for the design and synthesis of antileishmanial therapeutics.