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The identification of a green, versatile, user‐friendly, and efficient methodology is necessary to facilitate the use of Heck‐Cassar‐Sonogashira (HCS) cross‐coupling reaction in drug discovery and industrial production in the pharmaceutical segment. The Heck‐Cassar and Sonogashira protocols, using N‐hydroxyethylpyrrolidone (HEP)/water/N,N,N′,N′‐tetramethyl guanidine (TMG) as green solvent/base mixture and sulfonated phosphine ligands, allowed to recycle the catalyst, always guaranteeing high yields and fast conversion under mild conditions, with aryl iodides, bromides, and triflates. No catalyst leakage or metal contamination of the final product were observed during the HCS recycling. To our knowledge, a turnover number (TON) up to 2375, a turnover frequency (TOF) of 158 h−1, and a process mass intensity (PMI) around 7 that decreased around 3 after solvent, base, and palladium recovery, represent one of the best results to date using a sustainable protocol. The Heck‐Cassar protocol using sSPhos was successfully applied to the telescoped synthesis of Erlotinib (TON: 1380; TOF: 46 h−1)., Recycle and reuse: Heck‐Cassar‐Sonogashira cross‐coupling using the green N‐hydroxyethylpyrrolidone (HEP)/H2O/N,N,N′,N′‐tetramethyl guanidine (TMG) blend, sulfonate phosphines, and aryl iodides, bromides, and triflates affords the corresponding products in high yield free from metal contamination. The catalyst/HEP/H2O solution can be recycled, increasing turnover number and frequency up to 2375 and 158 h−1, respectively, with a low process mass intensity close to 3 after solvent, base, and palladium recovery. Additionally, Erlotinib synthesis is described.

The MT2‐selective melatonin receptor ligand UCM765 (N‐(2‐((3‐methoxyphenyl)(phenyl)amino)ethyl)acetamide), showed interesting sleep inducing, analgesic and anxiolytic properties in rodents, but suffers from low water solubility and modest metabolic stability. To overcome these limitations, different strategies were investigated, including modification of metabolically liable sites, introduction of hydrophilic substituents and design of more basic derivatives. Thermodynamic solubility, microsomal stability and lipophilicity of new compounds were experimentally evaluated, together with their MT1 and MT2 binding affinities. Introduction of a m‐hydroxymethyl substituent on the phenyl ring of UCM765 and replacement of the replacement of the N,N‐diphenyl‐amino scaffold with a N‐methyl‐N‐phenyl‐amino one led to highly soluble compounds with good microsomal stability and receptor binding affinity. Docking studies into the receptor crystal structure provided a rationale for their binding affinity. Pharmacokinetic characterization in rats highlighted higher plasma concentrations for the N‐methyl‐N‐phenyl‐amino derivative, consistent with its improved microsomal stability and makes this compound worthy of consideration for further pharmacological investigation., A taste for stability: Compound 19 is a potent melatonin receptor agonist obtained from the structural optimization of UCM765, an MT2‐selective partial agonist active in vivo, but characterized by poor physicochemical and pharmacokinetic properties. Insertion of a bromine atom on the phenyl‐methylamino scaffold led to a more soluble derivative endowed with higher microsomal stability.

Tuberculosis is one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extensively drug-resistant strains is a reason for concern. We have previously reported a series of substituted 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides with growth inhibitory activity against Mycobacterium tuberculosis strains and low propensity to be substrate of efflux pumps. Encouraged by these preliminary results, we have undertaken a medicinal chemistry campaign to determine the metabolic fate of these compounds and to delineate a reliable body of Structure-Activity Relationships. Keeping intact the (thiazol-4-yl)isoxazole-3-carboxamide core, as it is deemed to be the pharmacophore of the molecule, we have extensively explored the structural modifications able to confer good activity and avoid rapid clearance. Also, a small set of analogues based on isostere manipulation of the 2-aminothiazole were prepared and tested, with the aim to disclose novel antitubercular chemotypes. These studies, combined, were instrumental in designing improved compounds such as 42g and 42l, escaping metabolic degradation by human liver microsomes and, at the same time, maintaining good antitubercular activity against both drug-susceptible and drug-resistant strains.

Primary hyperoxaluria type I (PH1) is a rare kidney disease due to the deficit of alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5'-phosphate-dependent enzyme responsible for liver glyoxylate detoxification, which in turn prevents oxalate formation and precipitation as kidney stones. Many PH1-associated missense mutations cause AGT misfolding. Therefore, the use of pharmacological chaperones (PCs), small molecules that promote correct folding, represents a useful therapeutic option. To identify ligands acting as PCs for AGT, we first performed a small screening of commercially available compounds. We tested each molecule by a dual approach aimed at defining the inhibition potency on purified proteins and the chaperone activity in cells expressing a misfolded variant associated with PH1. We then performed a chemical optimization campaign and tested the resulting synthetic molecules using the same approach. Overall, the results allowed us to identify a promising hit compound for AGT and draw conclusions about the requirements for optimal PC activity.

UniPR129, an L-β-homotryptophan conjugate of the secondary bile acid lithocholic acid (LCA), acts as an effective protein-protein interaction (PPI) inhibitor of the Eph–ephrin system but suffers from a poor oral bioavailability in mice. To improve UniPR129 bioavailability, a metabolic soft spot, i.e., the 3α-hydroxyl group on the LCA steroidal ring, was functionalized to 3-hydroxyimine. In vitro metabolism of UniPR129 and 3-hydroxyimine derivative UniPR500 was compared in mouse liver subcellular fractions, and main metabolites were profiled by high resolution (HR-MS) and tandem (MS/MS) mass spectrometry. In mouse liver microsomes (MLM), UniPR129 was converted into several metabolites: M1 derived from the oxidation of the 3-hydroxy group to 3-oxo, M2–M7, mono-hydroxylated metabolites, M8–M10, di-hydroxylated metabolites, and M11, a mono-hydroxylated metabolite of M1. Phase II reactions were only minor routes of in vitro biotransformation. UniPR500 shared several metabolic pathways with parent UniPR129, but it showed higher stability in MLM, with a half-life (t1/2) of 60.4 min, if compared to a t1/2 = 16.8 min for UniPR129. When orally administered to mice at the same dose, UniPR500 showed an increased systemic exposure, maintaining an in vitro valuable pharmacological profile as an EphA2 receptor antagonist and an overall improvement in its physico-chemical profile (solubility, lipophilicity), if compared to UniPR129. The present work highlights an effective strategy for the pharmacokinetic optimization of aminoacid conjugates of bile acids as small molecule Eph–ephrin antagonists.

Devising novel strategies to therapeutically favour inflammation resolution and provide neuroprotection is an unmet clinical need. Enhancing endocannabinoid tone by inhibiting the catabolic enzyme fatty acid amide hydrolase (FAAH), or stimulating melatonin receptors has therapeutic potential to treat neuropathological states in which neuroinflammation plays a central role.A rodent hippocampal explant model of inflammatory injury was used to assess the effects of UCM1341, a dual-acting compound with FAAH inhibitory action and agonist activity at melatonin receptors, against neuroinflammatory damage. FAAH activity was measured by a radiometric assay, and N-acylethanolamine levels were assessed by HPLC-MS/MS methods. FAAH distribution, evolution of inflammation and the contribution of UCM1341 to the expression of proteins controlling macrophage behaviour were investigated by biochemical and confocal analyses.UCM1341 exhibited greater neuroprotection against neuroinflammatory degeneration, compared with the reference compounds URB597 (FAAH inhibitor) and melatonin. During neuroinflammation, UCM1341 augmented the levels of anandamide and N-oleoylethanolamine, but not N-palmitoylethanolamine, up-regulated PPAR-α levels, attenuated demyelination and prevented the release of TNF-α. UCM1341 modulated inflammatory responses by contributing to microglia/macrophage polarization, stimulating formation of lipid-laden macrophages and regulating expression of proteins controlling cholesterol metabolism and efflux. The neuroprotective effects of UCM1341 were prevented by PPARα, TRPV1 and melatonin receptor antagonists.UCM1341, by enhancing endocannabinoid and melatoninergic signalling, benefits neuroprotection and stimulates inflammation resolution pathways. Our findings provide an encouraging prospect of therapeutically targeting endocannabinoid and melatoninergic systems in inflammatory demyelinating states in the CNS.

The role of the Eph-ephrin system in the etiology of pathological conditions has been consolidated throughout the years. In this context, approaches directed against this signaling system, intended to modulate its activity, can be strategic therapeutic opportunities. Currently, the most promising class of compounds able to interfere with the Eph receptor-ephrin protein interaction is composed of synthetic derivatives of bile acids. In the present review, we summarize the progresses achieved, in terms of chemical expansions and structure-activity relationships, both in the steroidal core and the terminal carboxylic acid group, along with the pharmacological characterization for the most promising Eph-ephrin antagonists in in vivo settings.

The investigation of new green biogenic pyrrolidinones as alternative solvents to N,N-dimethylformamide (DMF) for solid phase peptide synthesis (SPPS) led to the identification of N-octyl pyrrolidone (NOP) as the best candidate. NOP showed good performances in terms of swelling, coupling efficiency and low isomerization generating peptides with very high purity. A mixture of NOP with 20% dimethyl carbonate (DMC) allowed a decrease in solvent viscosity, making the mixture suitable for the automated solid-phase protocol. Aib-enkephalin and linear octreotide were successfully used to test the methodologies. It is worth noting that NOP, DMC and the piperidine used in the deprotection step could be easily recovered by direct distillation from the process waste mixture. The process mass intensity (PMI), being reduced by 63–66%, achieved an outstanding value representing a clear step forward in achieving green SPPS.

The emergence of the C797S mutation in EGFR is a frequent mechanism of resistance to osimertinib in the treatment of non-small cell lung cancer (NSCLC). In the present work, we report the design, synthesis and biochemical characterization of UPR1444 (compound 11), a new sulfonyl fluoride derivative which potently and irreversibly inhibits EGFR

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.

Second- and third-generation inhibitors of EGFR possess an acrylamide group which alkylates Cys797, allowing to overcome resistance due to insurgence of T790M mutation. Less reactive warheads, yet capable to bind the target cysteine, may be useful to design newer and safer inhibitors. In the present work, we synthesized a 2-chloro-N-(4-(phenylamino)quinazolin-6-yl)acetamide (8) derivative as a prototype of EGFR inhibitor potentially able to react with Cys797 by nucleophilic substitution. We then tuned the reactivity of the acetamide fragment by replacing the chlorine leaving group with (hetero)-aromatic thiols or carboxylate esters. Among the synthesized derivatives, the 2-((1H-imidazol-2-yl)thio)acetamide 16, while showing negligible reactivity with cysteine in solution, caused long-lasting inhibition of wild-type EGFR autophosphorylation in A549 cells, resulted able to bind recombinant EGFR L858R/T790M in a time-dependent manner, and inhibited both EGFR autophosphorylation and proliferation in gefitinib-resistant H1975 lung cancer cells (expressing EGFR L858R/T790M mutant) at low micromolar concentration.

Inhibition of FGF/FGFR signaling is a promising strategy for the treatment of malignances dependent from FGF stimulation, including multiple myeloma (MM). The steroidal derivative NSC12 (compound 1) is a pan-FGF trap endowed with antitumor activity in vivo. Chemical modifications of compound 1 were explored to investigate structure-activity relationships, focusing on the role of the bis(trifluoromethyl)1,3-propanediol chain, the stereochemistry at C20 and functionalization of C3 position. Our studies unveiled compound 25b, the pregnane 3-keto 20R derivative of compound 1 as an effective agent, blocking the proliferation of MM cells in vitro by inhibiting FGF-dependent receptor activation and slowing MM growth in vivo. Importantly, the absence of the hydroxyl group at C3 prevents binding to estrogen receptors, which might concur to the antitumor activity observed for compound 1, leading to a specific FGF/FGFR system inhibitor, and further supporting the role of FGFR in anticancer therapy in MM.

The hallmark of joint diseases, such as osteoarthritis (OA), is pain, originating from both inflammatory and neuropathic components, and compounds able to modulate the signal transduction pathways of the cannabinoid type-2 receptor (CB2R) can represent a helpful option in the treatment of OA. In this perspective, a set of 18 cannabinoid type-2 receptor (CB2R) ligands was developed based on an unprecedented structure. With the aim of improving the physicochemical properties of previously reported 4-hydroxy-2-quinolone-3-carboxamides, a structural optimization program led to the discovery of isosteric 7-hydroxy-5-oxopyrazolo[4,3

We report an in vitro phase I metabolism study on COR659 (1), a 2-acylaminothiophene derivative able to suppress alcohol and chocolate self-administration in rats, likely via positive allosteric modulation of the GABA

Pharmacological inhibition of the enzyme fatty acid amide hydrolase (FAAH), which terminates signaling of the endocannabinoid N-arachidonoylethanolamine (or anandamide, AEA), exerts favourable effects in rodent models of stress-related depression. Yet although depression seems to be more common among women than men and in spite of some evidence of sex differences in treatment efficacy, preclinical development of FAAH inhibitors for the pharmacotherapy of stress-related depression has been predominantly conducted in male animals. Here, adult female rats were exposed to six weeks of social isolation and, starting from the second week, treated with the FAAH inhibitor URB694 (0.3 mg/kg/day, i.p.) or vehicle. Compared to pair-housed females, socially isolated female rats treated with vehicle developed behavioral (mild anhedonia, passive stress coping) and physiological (reduced body weight gain, elevated plasma corticosterone levels) alterations. Moreover, prolonged social isolation provoked a reduction in brain-derived neurotrophic factor (BDNF) and AEA levels within the hippocampus. Together, these changes are indicative of an increased risk of developing a depressive-like state. Conversely, pharmacological inhibition of FAAH activity with URB694 restored both AEA and BDNF levels within the hippocampus of socially isolated rats and prevented the development of behavioral and physiological alterations. These results suggest a potential interplay between AEA-mediated signaling and hippocampal BDNF in the pathogenesis of depression-relevant behaviors and physiological alterations and antidepressant action of FAAH inhibition in socially isolated female rats.

We report an in vitro phase I metabolism study on COR659 (1), a 2-acylaminothiophene derivative able to suppress alcohol and chocolate self-administration in rats, likely via positive allosteric modulation of the GABAB receptor and antagonism/inverse agonism at the cannabinoid CB1 receptor. Given the identification of the methyl ester group at C-3 of the thiophene ring as a metabolic soft spot, we also report the chemical optimization project aimed to balance metabolic stability with in vitro and in vivo potency on a set of 3-substituted COR659 analogues. High performance liquid chromatography coupled to tandem and high resolution mass spectrometry was employed for the characterization of in vitro metabolism and in vivo pharmacokinetics of COR659 in rats. In vitro [35S]GTPγS binding assays on stimulated GABAB and CB1 receptors, in combination with alcohol and chocolate self-administration experiments in rats, were employed to assess the pharmacological profile of this novel set of analogues, using COR659 as reference compound. Eight metabolites of COR659 were discovered in liver microsomal incubates; two of them (M1, M2) were identified by comparison with synthetic reference standards. M2, oxidation product of methyl group at C-5 of the thiophene ring, was a major metabolite in vitro, but showed a low systemic exposure in vivo. M1, cleavage product of the methyl ester group at C-3, revealed in vitro an unusual mechanism of metabolism by a NADPH-dependent route and, in vivo, it maintained high and persistent levels in plasma, which could represent a potential pharmacokinetic and toxicological issue. In the novel set of COR659 analogues, those bearing branched alkyl substituents on the ester group, showed an improved in vitro metabolic stability (2-4), had an in vitro GABAB PAM (2-4) and/or CB1 partial agonist/antagonist profile (2-3) and maintained the ability to reduce alcohol (2-4) and/or chocolate (4) self-administration in rats. Both PK and PD data ruled out any involvement of metabolite M1 in the in vivo potency of COR659 and 4. The present results, therefore, highlight the importance to design and synthesize novel compounds endowed with the dual activity profile and devoid of metabolic liabilities.

The hallmark of joint diseases, such as osteoarthritis (OA), is pain, originating from both inflammatory and neuropathic components, and compounds able to modulate the signal transduction pathways of the cannabinoid type-2 receptor (CB2R) can represent a helpful option in the treatment of OA. In this perspective, a set of 18 cannabinoid type-2 receptor (CB2R) ligands was developed based on an unprecedented structure. With the aim of improving the physicochemical properties of previously reported 4-hydroxy-2-quinolone-3-carboxamides, a structural optimization program led to the discovery of isosteric 7-hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-carboxamide derivatives. These new compounds are endowed with high affinity for the CB2R and moderate to good selectivity over the cannabinoid type-1 receptor (CB1R), associated with good physicochemical characteristics. As to the functional activity at the CB2R, compounds able to act either as agonists or as inverse agonists/antagonists were discovered. Among them, compound 51 emerged as a potent CB2R agonist able to reduce pain in rats carrying OA induced by injection of monoiodoacetic acid (MIA).

Eph/ephrin system is an emerging target for cancer therapy but the lack of potent, stable and orally bioavailable compounds is impairing the development of the field. Since 2009 our research group has been devoted to the discovery and development of small molecules targeting Eph/ephrin system and our research culminated with the synthesis of UniPR129, a potent but problematic Eph/ephrin antagonist. Herein, we describe the in vitro pharmacological properties of two derivatives (UniPR139 and UniPR502) stemmed from structure of UniPR129. These two compounds acted as competitive and reversible antagonists of all Eph receptors reducing both ephrin-A1 and -B1 binding to EphAs and EphBs receptors in the low micromolar range. The compounds acted as antagonists inhibiting ephrin-A1-dependent EphA2 activation and UniPR139 exerted an anti-angiogenic effect, inhibiting HUVEC tube formation in vitro and VEGF-induced vessel formation in the chick chorioallantoic membrane assay. Finally, the oral bioavailability of UniPR139 represents a step forward in the search of molecules targeting the Eph/ephrin system and offers a new pharmacological tool useful for future in vivo studies.

The emergence of the C797S mutation in EGFR is a frequent mechanism of resistance to osimertinib in the treatment of non-small cell lung cancer (NSCLC). In the present work, we report the design, synthesis and biochemical characterization of UPR1444 (compound 11), a new sulfonyl fluoride derivative which potently and irreversibly inhibits EGFRL858R/T790M/C797S through the formation of a sulfonamide bond with the catalytic residue Lys745. Enzymatic assays show that compound 11 displayed an inhibitory activity on EGFRWT comparable to that of osimertinib, and it resulted more selective than the sulfonyl fluoride probe XO44, recently reported to inhibit a significant part of the kinome. Neither compound 11 nor XO44 inhibited EGFRdel19/T790M/C797S triple mutant. When tested in Ba/F3 cells expressing EGFRL858R/T790M/C797S, compound 11 resulted significantly more potent than osimertinib at inhibiting both EGFR autophosphorylation and proliferation, even if the inhibition of EGFR autophosphorylation by compound 11 in Ba/F3 cells was not long lasting.

Eph/ephrin interactions and their bidirectional signaling are integral part of the complex communication system between β-cells, essential for glucose homeostasis. Indeed, Eph/ephrin system was shown to be directly involved in the glucose-stimulated insulin secretion (GSIS) process occurring in the pancreatic islets. Here we tested the Eph antagonist UniPR500 as GSIS enhancer. UniPR500 was validated as EphA5-ephrin-A5 inhibitor in vitro and its efficacy as GSIS enhancer was assessed on EndoC-βH1 cells. The selectivity of UniPR500 was evaluated by testing this compound on a panel of well-known molecular targets responsible for the regulation of glucose homeostasis. Plasmatic levels of UniPR500 were measured by HPLC/MS approach after oral administration. Finally, UniPR500 was tested as hypoglycemic agent in healthy mice, in a non-genetic mouse model of insulin resistance (IR) and in a non-genetic mouse model of type 1 diabetes (T1D). The compound is an orally bioavailable and selective Eph antagonist, able to increase GSIS from EndoC-βH1 cells. When tested in vivo UniPR500 showed to improve glucose tolerance in healthy and IR mice. As expected by a GSIS enhancer acting on healthy β-cells, UniPR500 was ineffective when tested on a non-genetic mouse model of type 1 diabetes, where pancreatic function was severely compromised. In conclusion our findings suggest that Eph targeting is a new and valuable pharmacological strategy in the search of new hypoglycemic agents.

The EphA2 receptor has been validated in animal models as new target for treating tumors depending on angiogenesis and vasculogenic mimicry. In the present work, we extended our current knowledge on structure-activity relationship (SAR) data of two related classes of antagonists of the EphA2 receptor, namely 5β-cholan-24-oic acids and 5β-cholan-24-oyl l-β-homotryptophan conjugates, with the aim to develop new antiangiogenic compounds able to efficiently prevent the formation of blood vessels. As a result of our exploration, we identified UniPR505, N-[3α-(Ethylcarbamoyl)oxy-5β-cholan-24-oyl]-l-β-homo-tryptophan (compound 14), as a submicromolar antagonist of the EphA2 receptor capable to block EphA2 phosphorylation and to inhibit neovascularization in a chorioallantoic membrane (CAM) assay.

Drug resistance and off-organ toxicity remain unsolved issues in chemotherapy of advanced-stage melanoma patients. Thus, the creation of new molecular conjugates able to combine a selective accumulation, high ability of internalization and signaling pathway inhibition, are highly requested. Recently, we reported a new class of molecular conjugates, compounds 1-3, where the anti-αVβ3 integrin peptidomimetic c(AmpRGD), which is a selective ligand for αVβ3 integrin, was covalently bound to the tyrosine kinase inhibitor sunitinib. Here, we report that these c(AmpRGD)-sunitinib conjugates and, in particular, compound 3, are selectively internalized by human melanoma cells through αVβ3 receptor-mediated endocytosis. Compound 3 is more effective than sunitinib in reducing in vitro melanoma cells proliferation, cloning efficiency, migration, and invasion. More interestingly, compound 3 is able to significantly reduce the growth of xenografted melanoma tumor developed in immune-compromised mice, more efficiently than an equimolar dose of sunitinib. Indeed, its targeting ability was demonstrated by the selective localization at the tumor level with respect to healthy tissues. Thus, c(AmpRGD)-sunitinib conjugates such as compound 3 could serve as intriguing multiple-target agents to selectively reach melanoma cells and interfere with the progression of the disease.

High glycolitic activity of multiple myeloma (MM) cells is the rationale for the use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both medullary and extramedullary disease. However, FDG-PET has some limitations, since there is a good portion of MM patients who are false-negative. Besides enhanced glycolysis, glutamine (Gln) addiction has been recently described as a metabolic feature of MM by our group. To sustain high Gln demand, MM cells increase the expression of several Gln transporters (ASCT2, SNAT1, LAT1) and are endowed with fast Gln uptake. Yet, at variance with other Gln-addicted cancers, the possible exploitation of Gln as a PET tracer in MM has never been assessed and was investigated in this study. To this purpose, we have firstly synthesized enantiopure (2S,4R)-4-Fluoroglutamine (4-FGln) and validated it as a Gln analogue in human MM cell lines (RPMI8226 and JJN3) comparing its uptake with that of 3H-labelled Gln. The intracellular levels of 4-FGln were determined by HPLC-MS/MS employing a HILIC gradient separation and multiple reaction monitoring (MRM) detection. Both Gln and 4-FGln were actively accumulated by MM cells and exhibited a strong reciprocal competition, pointing to shared transporters. Inhibition analysis revealed that ASCT2 was the major entry route of both compounds, with minor contributions from the other transporters. However, compared with Gln, 4-FGln exhibited higher affinity for both ASCT2 and LAT1 transporters. On the basis of these results, we then tested [18F]4-FGln uptake for MM detection by Positron Emission Tomography (PET) in two different in vivo murine models. Firstly, to investigate sensitivity of human MM to [18F]4-FGln in vivo, JJN3 cells were subcutaneously injected in immunodeficient NSG mice In this xenograft model, [18F]4-FGln- and[18F]FDG-PET scans were performed after plasmacytomas became palpable and repeated after one week. All the tumours were positive for [18F]FDG and displayed [18F]4-FGln uptake with Standard Uptake Values (SUV) of 1.21±1.9 and 0.99±0.07 after 2 weeks, respectively. Thereafter, the effect of bortezomib (BOR) was investigated to evaluate the potential use of [18F]4-FGln to monitor anti-MM treatment. Ten NGS mice were injected with JJN3 cells and, after 14 days, treated twice weekly with BOR, 1mg/kg, or vehicle for two weeks. PET scans were performed before and after 5 and 12 days of BOR treatment. As expected, BOR reduced tumour size as compared to vehicle. At the first post-BOR PET scan, [18F]4-FGln (SUV mean: pre 0.85±0.31; post 0.45±0.10, P Thereafter, to mimic BOR-resistant MM in a syngeneic mouse model, C57BL/6 mice were injected intravenously with Vk12598 cells obtained from transgenic Vk*MYC mice repeatedly treated with sub-optimal doses of BOR. Upon injection into C57BL/6 mice, Vk12598 cells colonize the BM without lytic lesions and extensively colonize the spleen generating an aggressive MM that brings animals to death within five weeks. PET scans were performed with [18F]4-FGln and [18F]FDG before Vk*MYC MM cells injection and after three, four and five weeks. Blood samples for M-spike evaluation were obtained in parallel. Four weeks after MM cells injection a significant increase of both [18F]4-FGln and [18F]FDG uptake was detected in spleens (SUV mean: 1.14±0.23, P=0.018; 0.94±0.24, P= 0.005). In both MM models, the volume of distribution of [18F]4-F-Gln did not overlap that of [18F]FDG. In conclusion, our data indicate that [18F]-(2S,4R)-4-Fluoroglutamine is a new potential PET tracer in pre-clinical MM models especially of extramedullary disease, either in a BOR-sensitive or in a BOR-resistant context, supporting the exploitation of Gln addiction for diagnostic purposes in MM patients. Disclosures Giuliani: Janssen: Research Funding.