Design and radiosynthesis of class-IIa HDAC inhibitor with high molar activity via repositioning the 18F-radiolabel.

The design and radiosynthesis of [18F]NT376, a high potency inhibitor of class-IIa histone deacetylases (HDAC) is reported. We utilized a three-step radiochemical approach that led to the radiosynthesis of [18F]NT376 in a good radiochemical yield, (17.0 ± 3%, decay corrected), high radiochemical purity (> 97%) and relatively high molar activity of 185.0 GBq/µmol (> 5.0 Ci/µmol). The repositioning of the 18F-radiolabel into a phenyl ring (18F-Fluoro-aryl) of the class-IIa HDAC inhibitor avoided the shortcomings of the direct radiolabeling of the 5-trifluoromethyl-1,2,4-oxadiazole moiety that was reported by us previously and was associated with low molar activity (0.74-1.51 GBq/µmol, 20-41 mCi/µmol). This radiochemical approach could find a wider application for radiolabeling similar molecules with good radiochemical yield and high molar activity.

An improved synthesis of 1'-[18F]fluoroethyl-ß-D-lactose ([18F]-FEL) for positron emission tomography imaging of pancreatic cancer.

INTRODUCTION: Earlier, we reported syntheses of ethyl-ß-D-galactopyranosyl-(1,4')-2'-deoxy-2'-[(18)F]fluoro-ß-D-glucopyranoside (Et-[(18)F]FDL) and 1'-[(18)F]fluoroethyl-ß-D-lactose ([(18)F]-FEL) for positron emission tomography (PET) of pancreatic carcinoma. Et-[(18)F]FDL requires a precursor, which involves 11 steps to synthesize and produces overall low yields. Synthesis of precursors for [(18)F]-FEL requires four steps, but those precursors produced low radiochemical yields. Here, we report new precursors and an improved synthesis of [(18)F]-FEL. METHOD: Two precursors, 1'-(methanesulfonyl)ethyl-2',3',6',2,3,4,6-hepta-O-acetyl-ß-D-lactose 2a and 1'-(p-nitrophenyl-sulfonyl)ethyl-2',3',6',2,3,4,6-hepta-O-acetyl-ß-D-lactose 2b, were synthesized from lactose in four steps. Radiofluorination reactions were performed using K(18)F/kryptofix and the crude product [(18)F]-3 was purified by HPLC. Basic hydrolysis of [(18)F]-3 produced 1'-[(18)F]fluoroethyl-ß-D-lactose [(18)F]-4, which was neutralized, diluted with saline, filtered on a 0.22-µm filter, and analyzed by radio-TLC. RESULTS: The average radiochemical yields of [(18)F]-4 (d. c.) from 2a and 2b were 21% (n = 6) and 65% (n = 6), respectively, with >99% radiochemical purity and specific activity of 55.5 GBq/µmol. Synthesis time was 90-95 min from the end of bombardment. CONCLUSION: An improved synthesis of [(18)F]FEL has been achieved in high yields, with high purity and specific activity. Precursor 2b with this method should be applicable for high yield automated production in a commercial synthesis module for clinical application.

High-Contrast PET Imaging with [18F]NT160, a Class-IIa Histone Deacetylase Probe for In Vivo Imaging of Epigenetic Machinery in the Central Nervous System.

We utilized positron emission tomography (PET) imaging in vivo to map the spatiotemporal biodistribution/expression of class-IIa histone deacetylases (class-IIa HDACs) in the central nervous system (CNS). Herein we report an improved radiosynthesis of [18F]NT160 using 4-hydroxy-TEMPO which led to a significant improvement in radiochemical yield and molar activity. PET imaging with [18F]NT160, a highly potent class-IIa HDAC inhibitor, led to high-quality and high-contrast images of the brain. [18F]NT160 displayed excellent pharmacokinetic and imaging characteristics: brain uptake is high in gray matter regions, tissue kinetics are appropriate for a 18F-tracer, and specific binding for class-IIa HDACs is demonstrated by self-blockade. Higher uptake with [18F]NT160 was observed in the hippocampus, thalamus, and cortex while the uptake in the cerebellum was relatively low. Overall, our current studies with [18F]NT160 will likely facilitate the development and clinical translation of PET tracers for imaging of class-IIa HDACs biodistribution/expression in cancer and the CNS.

Synthesis and Preclinical Evaluation of a Novel Fluorine-18-Labeled Tracer for Positron Emission Tomography Imaging of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a target for treating B-cell malignancies and autoimmune diseases. To aid in the discovery and development of BTK inhibitors and improve clinical diagnoses, we have developed a positron emission tomography (PET) radiotracer based on a selective BTK inhibitor, remibrutinib. [18F]PTBTK3 is an aromatic, 18F-labeled tracer that was synthesized in 3 steps with a 14.8 ± 2.4% decay-corrected radiochemical yield and ≥99% radiochemical purity. The cellular uptake of [18F]PTBTK3 was blocked up to 97% in JeKo-1 cells using remibrutinib or non-radioactive PTBTK3. [18F]PTBTK3 exhibited renal and hepatobiliary clearance in NOD SCID (non-obese diabetic/severe combined immunodeficiency) mice, and the tumor uptake of [18F]PTBTK3 in BTK-positive JeKo-1 xenografts (1.23 ± 0.30% ID/cc) was significantly greater at 60 min post injection compared to the tumor uptake in BTK-negative U87MG xenografts (0.41 ± 0.11% ID/cc). In the JeKo-1 xenografts, tumor uptake was blocked up to 62% by remibrutinib, indicating the BTK-dependent uptake of [18F]PTBTK3 in tumors.

An investigation on stereospecific fluorination at the 2'-arabino-position of a pyrimidine nucleoside: radiosynthesis of 2'-deoxy-2'-[(18)F]fluoro-5-methyl-1-ß-D-arabinofuranosyluracil.

Direct fluorination at the 2'-arabino-position of a pyrimidine nucleoside has been a long-standing challenge, yet we recently reported such a stereospecific fluorination for the first time in the synthesis of [(18)F]FMAU, albeit in low yields. Herein we report the results of an investigation on stereospecific fluorination on a variety of precursors for synthesis of [(18)F]FMAU. Several precursors were synthesized in multiple steps and fluorination was performed at the 2'-arabino position using K[(18)F]/kryptofix 2.2.2. All precursors produced [(18)F]FMAU in low yields.

PEG2000-DBCO surface coating increases intracellular uptake of liposomes by breast cancer xenografts.

Given our interest in the utility of liposomes for molecular imaging and theranostics, we investigated how coating the outer layer of the liposome affects internalization by breast cancer cell lines in vitro and in breast tumor tissues in vivo. Indeed, we discovered that a remarkably high liposomal uptake can be achieved by DBCO (dibenzocyclooctyne) soft coating. Our data demonstrates that decorating the terminal lipid with a DBCO moiety at a specific density induces increased tumor uptake in vivo (tumor uptake ~ 50%) compared to conventional undecorated liposome (tumor uptake ~ 20%). In this study, we report improved visualization of breast cancer cells in vivo using a 4T1 orthotopic breast cancer model and primary breast tumor xenograft models MDA-MB-231 and MDA-MB-436. L-PEG2000-DBCO coated liposomes demonstrate increased accumulation in breast cancer cells independent of tumor size, type, position, receptor expression, as well as the condition of the host mice. We expect these findings to have a major positive impact on the practical utility of liposomes in image-guided applications and precision medicine theranostics.

Design, synthesis, biochemical evaluation, radiolabeling and in vivo imaging with high affinity class-IIa histone deacetylase inhibitor for molecular imaging and targeted therapy.

Herein, we describe the design, synthesis and deciphering of the key characteristics of the structure activity relationship (SAR) of trifluoromethyloxadiazole (TFMO) bearing class-IIa HDAC inhibitors. Our medicinal chemistry campaign of 23 compounds identified compound 1 as a highly potent inhibitor with sub nM affinity to class-IIa HDAC4 isoform. Therefore, We radiolabeled compound 1 (named thereafter as NT160) with [18F]fluoride thus producing the identical [18F]-NT160 as a diagnostic tool for positron emission tomography (PET). [18F]-NT160 was produced in high radiochemical purity (>95%), moderate radiochemical yield (2-5%) and moderate molar activity in the range of 0.30-0.85 GBq/umol (8.0-23.0 mCi/umol). We also established that [18F]-NT160 can cross the blood brain barrier and bind to class-IIa HDACs in vivo. The combination of [18F]-NT160 and 1 represent a novel theranostic pair using the same molecule to enable diagnostic PET imaging with [18F]-NT160 followed by targeted therapy with NT160.

Fluorinated cannabinoid CB2 receptor ligands: synthesis and in vitro binding characteristics of 2-oxoquinoline derivatives.

Cannabinoid receptor 2 (CB2) plays an important role in human physiology and the pathophysiology of different diseases, including neuroinflammation, neurodegeneration, and cancer. Several classes of CB2 receptor ligands, including 2-oxoquinoline derivatives, have been previously reported. We report the synthesis and results of in vitro receptor binding of a focused library of new fluorinated 2-oxoquinoline CB2 ligands. Twelve compounds, 13-1618, 19, 21-24, 27, and 28 were synthesized in good yields in multiple steps. Human U87 glioma cells expressing either hCB1 (control) or hCB2 were generated via lentiviral transduction. In vitro competitive binding assay was performed using [(3)H]CP-55,940 in U87hCB1 and U87hCB2 cells. Inhibition constant (K(i)) values of compounds 13-16, 18, 19, 21-24, 27, and 28 for CB2 were >10,000, 2.8, 5.0, 2.4, 22, 0.8, 1.4, >10,000, 486, 58, 620, and 2400 nM, respectively, and those for CB1 were >10,000 nM. Preliminary in vitro results suggest that six of these compounds may be useful for therapy of neuropathic pain, neuroinflammatory diseases and immune disorders. In addition, compound 19, with its subnanomolar K(i) value, could be radiolabeled with (18)F and explored for PET imaging of CB2 expression.

Novel late-stage radiosynthesis of 5-[18F]-trifluoromethyl-1,2,4-oxadiazole (TFMO) containing molecules for PET imaging.

Small molecules that contain the (TFMO) moiety were reported to specifically inhibit the class-IIa histone deacetylases (HDACs), an important target in cancer and the disorders of the central nervous system (CNS). However, radiolabeling methods to incorporate the [18F]fluoride into the TFMO moiety are lacking. Herein, we report a novel late-stage incorporation of [18F]fluoride into the TFMO moiety in a single radiochemical step. In this approach the bromodifluoromethyl-1,2,4-oxadiazole was converted into [18F]TFMO via no-carrier-added bromine-[18F]fluoride exchange in a single step, thus producing the PET tracers with acceptable radiochemical yield (3-5%), high radiochemical purity (> 98%) and moderate molar activity of 0.33-0.49 GBq/umol (8.9-13.4 mCi/umol). We validated the utility of the novel radiochemical design by the radiosynthesis of [18F]TMP195, which is a known TFMO containing potent inhibitor of class-IIa HDACs.

Positron Emission Tomography Imaging of Staphylococcus aureus Infection Using a Nitro-Prodrug Analogue of 2-[18F]F-p-Aminobenzoic Acid.

Deep-seated bacterial infections caused by pathogens such as Staphylococcus aureus are difficult to diagnose and treat and are thus a major threat to human health. In previous work we demonstrated that positron emission tomography (PET) imaging with 2-[18F]F-p-aminobenzoic acid (2-[18F]F-PABA) could noninvasively identify, localize, and monitor S. aureus infection with excellent sensitivity and specificity in a rodent soft tissue infection model. However, 2-[18F]F-PABA is rapidly N-acetylated and eliminated, and in an attempt to improve radiotracer accumulation in bacteria we adopted a prodrug strategy in which the acid was protected by an ester and the amine was replaced with a nitro group. Metabolite analysis indicated that the nitro group of ethyl 2-[18F]fluoro-4-nitrobenzoate (2-[18F]F-ENB) is converted to the corresponding amine by bacteria-specific nitroreductases while the ester is hydrolyzed in vivo into the acid. PET/CT imaging of 2-[18F]F-ENB and the corresponding acid 2-[18F]F-NB in a rat soft tissue infection model demonstrated colocalization of the radiotracer with the bioluminescent signal arising from S. aureus Xen29, and demonstrated that the tracer could differentiate S. aureus infection from sterile inflammation. Significantly, the accumulation of both 2-[18F]F-ENB and 2-[18F]F-NB at the site of infection was 17-fold higher than at the site of sterile inflammation compared to 8-fold difference observed for 2-[18F]F-PABA, supporting the proposal that the active radiotracer in vivo is 2-[18F]F-NB. Collectively, these data suggest that 2-[18F]F-ENB and 2-[18F]F-NB have the potential for translation to humans as a rapid, noninvasive diagnostic tool to identify and localize S. aureus infections.

Molecular Imaging of Sirtuin1 Expression-Activity in Rat Brain Using Positron-Emission Tomography-Magnetic-Resonance Imaging with [18F]-2-Fluorobenzoylaminohexanoicanilide.

Sirtuin 1 (SIRT1) is a class III histone deacetylase that plays significant roles in the regulation of lifespan, metabolism, memory, and circadian rhythms and in the mechanisms of many diseases. However, methods of monitoring the pharmacodynamics of SIRT1-targeted drugs are limited to blood sampling because of the invasive nature of biopsies. For the noninvasive monitoring of the spatial and temporal dynamics of SIRT1 expression-activity in vivo by PET-CT-MRI, we developed a novel substrate-type radiotracer, [18F]-2-fluorobenzoylaminohexanoicanilide (2-[18F]BzAHA). PET-CT-MRI studies in rats demonstrated increased accumulation of 2-[18F]BzAHA-derived radioactivity in the hypothalamus, hippocampus, nucleus accumbens, and locus coeruleus, consistent with autoradiographic and immunofluorescent (IMF) analyses of brain-tissue sections. Pretreatment with the SIRT1 specific inhibitor, EX-527 (5 mg/kg, ip), resulted in about a 20% reduction of 2-[18F]BzAHA-derived-radioactivity accumulation in these structures. In vivo imaging of SIRT1 expression-activity should facilitate studies that improve the understanding of SIRT1-mediated regulation in the brain and aid in the development and clinical translation of SIRT1-targeted therapies.

A Novel Substrate Radiotracer for Molecular Imaging of SIRT2 Expression and Activity with Positron Emission Tomography.

PURPOSE: The purpose of this study was to develop a SIRT2-specific substrate-type radiotracer for non-invasive PET imaging of epigenetic regulatory processes mediated by SIRT2 in normal and disease tissues. PROCEDURES: A library of compounds containing tert-butyloxycarbonyl-lysine-aminomethylcoumarin backbone was derivatized with fluoroalkyl chains 3-16 carbons in length. SIRT2 most efficiently cleaved the myristoyl, followed by 12-fluorododecanoic and 10-fluorodecanoic groups (Kcat/Km 716.5 ± 72.8, 615.4 ± 50.5, 269.5 ± 52.1/s mol, respectively). Radiosynthesis of 12- [18F]fluorododecanoic aminohexanoicanilide (12-[18F]DDAHA) was achieved by nucleophilic radiofluorination of 12-iododecanoic-AHA precursor. RESULTS: A significantly higher accumulation of 12-[18F]DDAHA was observed in MCF-7 and MDA-MB-435 cells in vitro as compared to U87, MiaPaCa, and MCF10A, which was consistent with levels of SIRT2 expression. Initial in vivo studies using 12-[18F]DDAHA conducted in a 9L glioma-bearing rats were discouraging, due to rapid defluorination of this radiotracer upon intravenous administration, as evidenced by significant accumulation of F-18 radioactivity in the skull and other bones, which confounded the interpretation of images of radiotracer accumulation within the tumor and other regions of the brain. CONCLUSIONS: The next generation of SIRT2-specific radiotracers resistant to systemic defluorination should be developed using alternative sites of radiofluorination on the aliphatic chain of DDAHA. A SIRT2-selective radiotracer may provide information about SIRT2 expression and activity in tumors and normal organs and tissues, which may help to better understand the roles of SIRT2 in different diseases.

Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography.

PURPOSE: We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. PROCEDURES: We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19+ artificial antigen-presenting cells. RESULTS: After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19+ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2'-deoxy-2'-[18F]fluoro-5-ethyl-1-ß-D-arabinofuranosyl-uracil ([18F]FEAU). CONCLUSION: This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain.

Histone deacetylases (HDAC's) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa-specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([18F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed in vitro, in a panel of recombinant HDACs, and in vivo using PET/CT imaging in rats. [18F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [18F]DFAHA and previously reported [18F]FAHA. PET imaging with [18F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo. Furthermore, PET imaging with [18F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.

Synthesis and preliminary evaluation of [18F]-labeled 2-oxoquinoline derivatives for PET imaging of cannabinoid CB2 receptor.

INTRODUCTION: The cannabinoid receptor type 2 (CB(2)) is an important target for development of drugs and imaging agents for diseases, such as neuroinflammation, neurodegeneration and cancer. Recently, we reported synthesis and results of in vitro receptor binding of a focused library of fluorinated 2-oxoquinoline derivatives as CB(2) receptor ligands. Some of the compounds demonstrated to be good CB(2)-specific ligands with Ki values in the nanomolar to subnanomolar concentrations; therefore, we pursued the development of their (18)F-labeled analogues that should be useful for positron emission tomography (PET) imaging of CB(2) receptor expression. Here, we report the radiosynthesis of two (18)F-labeled 2-oxoquinoline derivatives and the preliminary in vitro and ex vivo evaluation of one compound as a CB(2)-specific radioligand. METHODS: 4-[(18)F]fluorobenzyl amine [(18)F]-3 was prepared by radiofluorination of 4-cyano-N,N,N-trimethylanilinium triflate salt followed by reduction with LiAlH(4) and then coupled with acid chlorides 11 and 12 to afford [(18)F]-13 and [(18)F]-14. In vitro CB(2) receptor binding assay was performed using U87 cells transduced with CB(2) and CB(1) receptor. Ex vivo autoradiography was performed with [(18)F]-14 on spleen and on CB(2)- and CB(1)-expressing and wild-type U87 subcutaneous tumors grown in mice. RESULTS: The radiochemical yields of [(18)F]-13 and [(18)F]-14 were 10%-15.0% with an average of 12% (n=10); radiochemical purity was >99% with specific activity 1200 mCi/µmol. The dissociation constant Kd for [(18)F]-14 was 3.4 nM. Ex vivo autoradiography showed accumulation of [(18)F]-14 in the CB(2)-expressing tumor. CONCLUSION: Two new [(18)F]-labeled CB(2) ligands have been synthesized. Compound [(18)F]-14 appears to be a potential PET imaging agent for the assessment of CB(2) receptor expression; however, poor solubility restrain its use in vivo.

One-pot desulfurative-fluorination-bromination. Synthesis of 2,5-dibromo-3-(1,1-difluoroalkyl)thiophenes.

A unique one-pot, high-yield desulfurative-fluorination-bromination reaction which leads to the synthesis of 2,5-dibromo-3-(1,1-difluoroalkyl)thiophenes is reported. A single dithiolane reactant can be used to produce both 2,5-dibromo-3-(1,1-difluoroalkyl)thiophene and 3-(1,1-difluoroalkyl)thiophene with longer alkyl chains.