Patent Application
20240327361
Throughout this application, certain publications are referenced in parentheses. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention relates.
The dysregulation of class-lla histone deacetylases (HDACs) is implicated in various cancers and in the neurodegenerative diseases of the central nervous system (CNS). Therefore, molecular imaging of class-lla HDACs with positron emission tomography (PET) enable early diagnosis and select patients for therapeutic intervention with targeted class-lla HDAC inhibitors to improve overall outcomes. Recently, 5-trifluoromethyl-1,2,4-oxadiazole (TFMO) containing small molecules were reported to exhibit potent and specific class-lla HDACs inhibition. Herein, TFMO moiety was successfully radiolabeled with F-18 and validated the utility of the new radiochemical design by the radiosynthesis of 18F-NT57, a radioactive homolog of TMP195, a known TFMO containing potent inhibitor of class-lla HDACs. PET imaging was performed in vivo with the new probe in rats thus mapping the biodistribution of class-lla HDACs in healthy tissues in the periphery and the CNS.
The Human histone deacetylases (HDACs) form a large family of 18 members classified into four classes (I to IV) (Aramsangtienchai, P., et al. 2006; Younes, A., et al. 2016). The class-lla HDACs is a sub family comprised of four enzymes: HDAC-4, HDAC-5, HDAC-7 and HDAC-9 (Li and Yang 2016; Kikuchi, S., et al. 2015; Choi, S. Y., et al. 2018). There is mounting evidence on a key role for the class-Ila HDACs in various cancers and in the disorders of the central nervous system (CNS) including memory and cognitive impairment, dementia and behavioral changes among others (Linares, A., et al. 2019).
Class-lla HDAC-4 and 5 are expressed throughout the brain silencing or truncation led to defect in spatial learning and memory, cognitive impairment and dementia (Bolger T A Y T 2005; Gräff J T L 2013; Sando R G N. 2012). However, despite intense studies, relatively little is known regarding the expression level and distribution of class-lla HDACs in the human brain in its entirety. Although postmortem studies established a link between expression of class-lla HDAC and neurodegenerative diseases such as Alzheimer's and Huntington's (Anderson 2015; Yeh H H Y D, et al. 2013), these studies are limited to one or very few regions of the brain, leaving the rest of the brain unexplored. Therefore, there is critical need to accurately quantify the level of class-lla HDACs expression in vivo in live humans to enable side-by-side comparison of the healthy brain to the disease state in the entire brain.
Despite being initially dismissed as histone deacetylases, recent studies in cancer and neurodegenerative diseases showed that overexpression of class-lla HDACs resulted in relative abundance of deacetylated histone (Lobera M M K, 2013; Yeh H H Y D, et al. 2013; Guerriero J L S A, 2017). Moreover, inhibition of class-lla HDACs restores histone acetylation status and improved outcome, which support class-lla HDACs as functional histone deacetylases. Notably, Guerriero et al. demonstrated that specific inhibition of class-lla HDACs in breast cancer restored the acetylation status of the core histone proteins and resulted in reduced primary tumor size and decreased pulmonary metastases thus highlighting the therapeutic value of class-lla HDACs (Guerriero J L S A, 2017; Cassetta L P J 2017). The deacetylation power of class-lla HDACs is still unclear. It could be explained by their overexpression (higher class-lla protein density increases histone deacetylation). Also, class-lla HDACs have been reported to recruit HDAC3 to form an enzymatically active multiprotein complex (class-lla HDAC-HDAC3-SMRT/N—CoR). In this model, the catalytic pocket of class-lla HDACs was essential for the deacetylase activity of this multiprotein complex (Fischle W D F, 2002). These studies suggest that the class-lla HDACs gain of function is through enzymatically active multiprotein complex and provide evidence for class-lla HDAC inhibition is a viable therapeutic target.
Molecular imaging with positron emission tomography (PET) is a non-invasive powerful tool that can provide quantitative and repeated measurements in real time on the class-lla HDAC protein expression. Currently, reliable class-lla HDAC PET tracers for imaging of cancer and the disorders of the brain are lacking. Therefore, the class-lla HDACs was targeted for PET tracer development to enable non-invasive in vivo visualization, detection and quantitative mapping of the biodistribution of class-lla HDAC protein expression in cancer and brain diseases. This invention developed PET tracer that detect brain disorders at an early point in time when therapeutic intervention is most effective and support and accelerate clinical development of specific class-lla HDACs targeting drugs. Also, PET tracer developed in the current invention further identifies a subset of patient population who may benefit the most from anti-class-lla therapy and monitor their response to treatment.
The expression levels and distribution of class-lla HDACs in human brain is largely unknown. The publicly available human protein atlas (HPA) database shows: (1) High HDAC-4 protein expression in the cerebral cortex and medium expression in the cerebellum, caudate and hippocampus; (2) moderate HDAC-5 protein expression in the cerebral cortex, cerebellum and hippocampus and low expression in the caudate; (3) no data available on the protein expression of HDAC-7 and (4) moderate HDAC-9 protein expression in the cerebral cortex and cerebellum, low expression in the caudate and no expression in the hippocampus. The publicly available database (GTEx portal) reports variable gene expression of class-lla HDACs in the brain (low gene expression of HDAC-4, 7 and 9 and moderate expression of HDAC-5 in the cortex, cerebellar hemisphere and cerebellum). However, these data obtained via semi-quantitative or qualitative measurement and current invention overcomes these limitations.
Despite intensive efforts by many research groups, radiolabeled HDAC inhibitors with either F-18 or C-11 PET radiotracers such as 18F-SAHA, 18F-FESAHA, and 11C-MS-275 and other tracers showed poor blood-brain barrier (BBB) permeability and thus limited their utility for brain imaging (Hendricks, J_A., et al., 2011; Zeglis, B. M., et al., 2011; Hooker, J. M., et al., 2010; Seo, Y. J., et al., 2013).
11C-martinostat was the first successful class-I HDAC (HDAC1, 2 and 3) inhibitor-based tracer (Wang, C., et al., 2014; Wey, H. Y., et al. 2015; Reid, A. E., et al., 2009). Also, a semi-selective HDAC6 PET imaging probe was reported (Wey H Y, G. T., Zurcher N R, et al. 2016). However, these probes do not provide information on the expression or distribution of class-lla HDACs or their involvement in cancer or CNS diseases. Moreover, radiolabeled class-lla HDACs substrates that undergo metabolism by class-lla enzymes to release 18F-fluorinated acetate in vitro and in vivo were previously reported (Bonomi, R., et al., 2015; Laws, M. T., et al., 2013; Guerriero, J. L., et al., 2017). However, these substrates exhibited poor in vivo pharmacokinetics that were detrimental to their clinical utility as PET radiotracers. An independent study that used 18F-FAHA (a substrate-type radiotracer) indicated that fluoroacetate produced in the brain does not remain localized to the point of catabolism of 18F-FAHA by class lla HDACs, thus confounding the PET signal and limits its practical utilization for quantitative imaging (Reid, A. E., et al., 2009). Therefore, a metabolically stable F-inhibitor-type radiotracer was pursued to overcome the inherent limitations of the labelled substrates, thus providing accurate quantification of class-lla HDACs protein expression by PET imaging.
TMP195 and CHDI-390576 were among the very few potent and highly specific class-lla HDAC inhibitors that were recently reported in the literature (
CHDI-390576 is a second generation benzhydryl hydroxamic acid with improved CNS properties and selectivity to class-lla HDACs over the previously reported cyclopropane hydroxamic acids by the same group (Burli, R. W. et al., 2013). Although CHDI-390576 is fluorinated, the peculiar positioning of the fluorine atoms rendered this molecule likely inaccessible for facile F-18 incorporation. In contrast, TMP195 contains the 5-trifluoromethyl-1,2,4-oxadiazole (TFMO) moiety that in theory is accessible for late-stage radiolabeling with F-18. Therefore, a late stage radiochemical synthesis was designed in Scheme 1 (
This invention provides a compound having the structure:
wherein:
wherein when R2 is H, R3 is F, X is N, and R and R2 are in ortho position, R is other than
wherein when R2 is F, R3 is F, X is C, and R and R2 are in ortho position, R is other than
wherein when R2 is H, R3 is F, X is C, and R and R2 are in ortho position, R is other than OH or
This invention provides a composition comprising compound A having the structure:
and
This invention provides a process for preparing compound III having the structure:
each of which is optionally substituted by one or more substituents selected from —C0-6alkyl, —C1-6alkylOC1-6alkyl, halogen, or —C(O)OtButyl;
This invention provides a process for preparing compound IV having the structure:
This invention provides a method of detecting a disease of the central nervous system (CNS) in a subject, the method comprising:
wherein:
The following detailed description of embodiments of the invention will be made in reference to the accompanying drawings. In describing the invention, explanation about related functions or constructions known in the art are omitted for the sake of clearness in understanding the concept of the invention to avoid obscuring the invention with unnecessary detail.
Embodiments of the present invention described herein provide radiolabeled compositions and methods of use thereof. The compositions can be used for imaging of disorders of the central nervous system for the use in early detection of diseases including memory and cognitive impairment, dementia and behavioral changes, Alzheimer's and Huntington diseases and others.
In some embodiments, the present invention provides a compound having the structure:
wherein:
wherein when R2 is H, R3 is F, X is N, and R and R2 are in ortho position, R is other than
wherein when R2 is F, R3 is F, X is C, and R and R2 are in ortho position, R is other than
wherein when R2 is H, R3 is F, X is C, and R and R2 are in ortho position, R is other than OH or
In some embodiments, R is NH—R1 wherein R1 is C1-6alkylC(O)NC1-6alkyl aryl, C1-6alkyl-N—C1-6alkyl aryl, C1-6alkyl-N-heteroaryl-aryl, C1-6alkyl-heteroaryl-aryl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl aryl, C1-6alkylC(O)—NH—C1-6alkyl aryl, cyclic amine, cyclic amine aryl, cyclic amine-C1-6alkyl-aryl, C1-6alkyl-cyclic amine, C1-6alkyl-cyclic amine aryl, C1-6alkylC(O)-cyclic amine or C1-6alkylC(O)-cyclic amine aryl.
In some embodiments, R is —NH—R1 wherein R1 is C1-6alkylC(O)NC1-6alkyl phenyl, C1-6alkyl-N—C1-6alkyl phenyl, C1-6alkyl-N-oxazole-phenyl, C1-6alkyl-oxazole-phenyl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl phenyl, pyrrolidine, pyrrolidine phenyl, C1-6alkyl-pyrrolidine, C1-6alkyl-pyrrolidine-phenyl, C1-6alkylC(O)-pyrrolidine, C1-6alkylC(O)-pyrrolidine-phenyl, piperidine, piperidine phenyl, C1-6alkyl-piperidine, piperidine-C1-6alkyl-phenyl, C1-6alkylC(O)-piperidine, C1-6alkylC(O)-piperidine-phenyl.
In some embodiments, R is substituted aryl, heteroaryl, cycloalkyl, heterocyloalkyl, wherein the substitution is OH, —O—C1-6 alkyl, formyl, carbonyl, —C1-6alkyl, —C1-6alkenyl, —C1-6alkynyl, —C0-6alkyl aryl, —C0-6alkyl heteroaryl, —C0-6alkyl cycloalkyl, —C0-6alkyl heterocyloalkyl, —C0-6alkylC(O)NHC0-6alkyl, —C0-6alkylC(O)NHC0-6alkyl aryl, —C0-6alkylC(O)NHC0-6alkyl heteroaryl, —C0-6alkylC(O)NHC0-6alkyl cycloalkyl, —C0-6alkylC(O)NHC0-6alkyl heterocyloalkyl, —C0-6alkylNHC0-6alkyl, —C0-6alkylNHC0-6alkyl aryl, —C0-6alkylNHC0-6alkyl heteroaryl, —C0-6alkylNHC0-6alkyl cycloalkyl, and —C0-6alkylNHC0-6alkyl heterocyloalkyl, each of which is optionally substituted by one or more substituents selected from —C0-6alkyl, —C1-6alkylOC1-6alkyl, halogen, or —C(O)OtButyl.
In some embodiments, when R2 is H, R3 is F, X is C, and R and R2 are in meta position, R is other than
In some embodiments, when R2 is H, R3 is F, X is N, and R and R2 are in ortho position, R is other than
In some embodiments, when R2 is F, R3 is F, X is C, and R and R2 are in ortho position, R is other than
In some embodiments, wherein when R2 is H, R3 is F, X is C, and R and R2 are in ortho position, R is other than OH or
In some embodiments, R3 is F, Cl, Br or I.
In some embodiments, R3 is radioactive.
In some embodiments, the present invention provides a compound having the structure:
wherein when R2 is H, R3 is F, X is C, and R and R2 are in meta position, R is other than
wherein when R2 is H, R3 is F, X is N, and R and R2 are in ortho position, R is other than
wherein when R2 is F, R3 is F, X is C, and R and R2 are in ortho position, R is other than
wherein when R2 is H, R3 is F, X is C, and R and R2 are in ortho position, R is other than OH or
wherein when R1 is H, R3 is F, X is C, and R and R2 are in meta position, R is other than
wherein when R1 is H, R3 is F, X is N, and R and R2 are in ortho position, R is other than
wherein when R1 is F, R3 is F, X is C, and R and R2 are in ortho position, R is other than
In some embodiments, when R1 is H, R3 is F, X is C, and R and R2 are in ortho position, R is other than OH or
In some embodiments, when R1 is H, R3 is F, X is C, and R and R2 are in meta position, R is other than
In some embodiments, when R1 is H, R3 is F, X is N, and R and R2 are in ortho position, R is other than
In some embodiments, when R1 is F, R3 is F, X is C, and R and R2 are in ortho position, R is other than
In some embodiments, when R1 is H, R3 is F, X is C, and R and R2 are in ortho position, R is other than OH or
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, R or R1 is C1-6alkylC(O)NC1-6alkyl aryl, C1-6alkyl-N—C1-6alkyl aryl, C1-6alkyl-N-heteroaryl-aryl, C1-6alkyl-heteroaryl-aryl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl aryl, C1-6alkylC(O)—NH—C1-6alkyl aryl, cyclic amine, cyclic amine aryl, cyclic amine-C1-6alkyl-aryl, C1-6alkyl-cyclic amine, C1-6alkyl-cyclic amine aryl, C1-6alkylC(O)-cyclic amine or C1-6alkylC(O)-cyclic amine aryl.
In some embodiments, R or R1 is C1-6alkylC(O)NC1-6alkyl phenyl, C1-6alkyl-N—C1-6alkyl phenyl, C1-6alkyl-N-oxazole-phenyl, C1-6alkyl-oxazole-phenyl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl phenyl, pyrrolidine, pyrrolidine phenyl, C1-6alkyl-pyrrolidine, C1-6alkyl-pyrrolidine-phenyl, C1-6alkylC(O)-pyrrolidine, C1-6alkylC(O)-pyrrolidine-phenyl, piperidine, piperidine phenyl, C1-6alkyl-piperidine, piperidine-C1-6alkyl-phenyl, C1-6alkylC(O)-piperidine, C1-6alkylC(O)-piperidine-phenyl.
In some embodiments, oxazole, phenyl, pyrrolidine and piperidine is further substituted with halogen, C1-6alkyl, aryl, or C1-6alkyl aryl.
In some embodiments, halogen is F, Cl, Br, I.
In some embodiments, R or R1 is further substituted with OH, —O—C1-6 alkyl, formyl, carbonyl, —C1-6alkyl, —C1-6alkenyl, —C1-6alkynyl, —C0-6alkyl aryl, —C0-6alkyl heteroaryl, —C0-6alkyl cycloalkyl, —C0-6alkyl heterocyloalkyl, —C0-6alkylC(O)NHC0-6alkyl, —C0-6alkylC(O)NHC0-6alkyl aryl, —C0-6alkylC(O)NHC0-6alkyl heteroaryl, —C0-6alkylC(O)NHC0-6alkyl cycloalkyl, —C0-6alkylC(O)NHC0-6alkyl heterocyloalkyl, —C0-6alkylNHC0-6alkyl, —C0-6alkylNHC0-6alkyl aryl, —C0-6alkylNHC0-6alkyl heteroaryl, —C0-6alkylNHC0-6alkyl cycloalkyl, and —C0-6alkylNHC0-6alkyl heterocyloalkyl, each of which is optionally substituted by one or more substituents selected from —C0-6alkyl, —C1-6alkylOC1-6alkyl, halogen, or —C(O)OtButyl.
In some embodiments, R or R1 is H, OH, methyl, formyl, or carbonyl.
In some embodiments, R or R1 is OH.
In some embodiments, R or R1 is methyl.
In some embodiments, R or R1 is formyl, or carbonyl.
In some embodiments, R or R1 is —O—C1-6 alkyl.
In some embodiments, R or R1 is —O-methyl, —O-ethyl or —O-tert-butyl.
In some embodiments, R2 is H, F, Cl, Br or I.
In some embodiments, R2 is H.
In some embodiments, R2 is F.
In some embodiments, X is C.
In some embodiments, X is N.
In some embodiments, R or R1 contains a radioactive label.
In some embodiments, R or R1 contains a radioactive halogen.
In some embodiments, R or R1 contains a 18F.
In some embodiments, R or R1 contains a radioactive carbon.
In some embodiments, R or R1 contains a 11C.
In some embodiments, R is NH—R1.
In some embodiments, R1 is selected form the group consisting of:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides compounds that are human histone deacetylases (HDACs) inhibitors.
In some embodiments, the HDACs are HDAC-4, HDAC-5, HDAC-7 and HDAC-9.
In some embodiments, the HDACs inhibitors have the following structures:
In some embodiments, the precursor used for radiolabeling has the following structure:
In some embodiments, the radioactive tracer has the following structure:
In some embodiments, the present invention provides a composition comprising compound A having the structure
and
In some embodiments, R or R1 is C1-6alkylC(O)NC1-6alkyl aryl, C1-6alkyl-N—C1-6alkyl aryl, C1-6alkyl-N-heteroaryl-aryl, C1-6alkyl-heteroaryl-aryl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl aryl, C1-6alkylC(O)—NH—C1-6alkyl aryl, cyclic amine, cyclic amine aryl, cyclic amine-C1-6alkyl-aryl, C1-6alkyl-cyclic amine, C1-6alkyl-cyclic amine aryl, C1-6alkylC(O)-cyclic amine or C1-6alkylC(O)-cyclic amine aryl.
In some embodiments, R or R1 is C1-6alkylC(O)NC1-6alkyl phenyl, C1-6alkyl-N—C1-6alkyl phenyl, C1-6alkyl-N-oxazole-phenyl, C1-6alkyl-oxazole-phenyl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl phenyl, pyrrolidine, pyrrolidine phenyl, C1-6alkyl-pyrrolidine, C1-6alkyl-pyrrolidine-phenyl, C1-6alkylC(O)-pyrrolidine, C1-6alkylC(O)-pyrrolidine-phenyl, piperidine, piperidine phenyl, C1-6alkyl-piperidine, piperidine-C1-6alkyl-phenyl, C1-6alkylC(O)-piperidine, C1-6alkylC(O)-piperidine-phenyl.
In some embodiments, R or R1 is further substituted with OH, —O—C1-6 alkyl, formyl, carbonyl, —C1-6alkyl, —C1-6alkenyl, —C1-6alkynyl, —C0-6alkyl aryl, —C0-6alkyl heteroaryl, —C0-6alkyl cycloalkyl, —C0-6alkyl heterocyloalkyl, —C0-6alkylC(O)NHC0-6alkyl, —C0-6alkylC(O)NHC0-6alkyl aryl, —C0-6alkylC(O)NHC0-6alkyl heteroaryl, —C0-6alkylC(O)NHC0-6alkyl cycloalkyl, —C0-6alkylC(O)NHC0-6alkyl heterocyloalkyl, —C0-6alkylNHC0-6alkyl, —C0-6alkylNHC0-6alkyl aryl, —C0-6alkylNHC0-6alkyl heteroaryl, —C0-6alkylNHC0-6alkyl cycloalkyl, and —C0-6alkylNHC0-6alkyl heterocyloalkyl, each of which is optionally substituted by one or more substituents selected from —C0-6alkyl, —C1-6alkylOC1-6alkyl, halogen, or —C(O)OtButyl.
In some embodiments, the composition contains 1-10% compound B by weight.
In some embodiments, the composition contains 2-5% compound B by weight.
In some embodiments, R3 is F, Cl, Br or I.
In some embodiments, R3 is radioactive.
In some embodiments, the composition comprises the precursors used for radiolabeling and the radioactive tracers.
In some emblements, R is NH—R1
In some embodiments, the present invention provides a composition comprising compound having the structure:
In some embodiments, the present invention provides a composition comprising compound having the structure:
In some embodiments, R is NH—R1.
In some embodiments, R1 is C1-6alkylC(O)NC1-6alkyl aryl, C1-6alkyl-N—C1-6alkyl aryl, C1-6alkyl-N-heteroaryl-aryl, C1-6alkyl-heteroaryl-aryl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl aryl, C1-6alkylC(O)—NH—C1-6alkyl aryl, cyclic amine, cyclic amine aryl, cyclic amine-C1-6alkyl-aryl, C1-6alkyl-cyclic amine, C1-6alkyl-cyclic amine aryl, C1-6alkylC(O)-cyclic amine or C1-6alkylC(O)-cyclic amine aryl.
In some embodiments, R1 is C1-6alkylC(O)NC1-6alkyl phenyl, C1-6alkyl-N—C1-6alkyl phenyl, C1-6alkyl-N-oxazole-phenyl, C1-6alkyl-oxazole-phenyl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl phenyl, pyrrolidine, pyrrolidine phenyl, C1-6alkyl-pyrrolidine, C1-6alkyl-pyrrolidine-phenyl, C1-6alkylC(O)-pyrrolidine, C1-6alkylC(O)-pyrrolidine-phenyl, piperidine, piperidine phenyl, C1-6alkyl-piperidine, piperidine-C1-6alkyl-phenyl, C1-6alkylC(O)-piperidine, C1-6alkylC(O)-piperidine-phenyl.
In some embodiments, R or R1 is further substituted with OH, —O—C1-6 alkyl, formyl, carbonyl, —C1-6alkyl, —C1-6alkenyl, —C1-6alkynyl, —C0-6alkyl aryl, —C0-6alkyl heteroaryl, —C0-6alkyl cycloalkyl, —C0-6alkyl heterocyloalkyl, —C0-6alkylC(O)NHC0-6alkyl, —C0-6alkylC(O)NHC0-6alkyl aryl, —C0-6alkylC(O)NHC0-6alkyl heteroaryl, —C0-6alkylC(O)NHC0-6alkyl cycloalkyl, —C0-6alkylC(O)NHC0-6alkyl heterocyloalkyl, —C0-6alkylNHC0-6alkyl, —C0-6alkylNHC0-6alkyl aryl, —C0-6alkylNHC0-6alkyl heteroaryl, —C0-6alkylNHC0-6alkyl cycloalkyl, and —C0-6alkylNHC0-6alkyl heterocyloalkyl, each of which is optionally substituted by one or more substituents selected from —C0-6alkyl, —C1-6alkylOC1-6alkyl, halogen, or —C(O)OtButyl.
In some embodiments, R or R1 is H, OH, methyl, formyl, or carbonyl.
In some embodiments, R or R1 is OH.
In some embodiments, R or R1 is methyl.
In some embodiments, R or R1 is formyl, or carbonyl.
In some embodiments, R or R1 is —O—C1-6 alkyl.
In some embodiments, R or R1 is —O-methyl, —O-ethyl or —O-tert-butyl.
In some embodiments, R2 is H, F, Cl, Br or I.
In some embodiments, R2 is H.
In some embodiments, R2 is F.
In some embodiments, X is C.
In some embodiments, X is N.
In some embodiments, R1 is selected form the group consisting of:
In some embodiments, the present invention provides a composition comprising compound having the structure:
In some embodiments, the present invention provides a composition comprising human histone deacetylases (HDACs) inhibitors.
In some embodiments, the HIDACs are HDAC-4, HDAC-5, HDAC-7 and HDAC-9.
In some embodiments, the present invention provides a process for preparing compound IV(a) having the structure:
In some embodiments, the current invention provides a process for preparing compound III having the structure:
In some embodiments, R is C1-6alkylC(O)NC1-6alkyl aryl, C1-6alkyl-N—C1-6alkyl aryl, C1-6alkyl-N-heteroaryl-aryl, C1-6alkyl-heteroaryl-aryl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl aryl, C1-6alkylC(O)—NH—C1-6alkyl aryl, cyclic amine, cyclic amine aryl, cyclic amine-C1-6alkyl-aryl, C1-6alkyl-cyclic amine, C1-6alkyl-cyclic amine aryl, C1-6alkylC(O)-cyclic amine or C1-6alkylC(O)-cyclic amine aryl.
In some embodiments, R is C1-6alkylC(O)NC1-6alkyl phenyl, C1-6alkyl-N—C1-6alkyl phenyl, C1-6alkyl-N-oxazole-phenyl, C1-6alkyl-oxazole-phenyl, C1-6alkylC(O)—C1-6alkyl-NH—C1-6alkyl phenyl, pyrrolidine, pyrrolidine phenyl, C1-6alkyl-pyrrolidine, C1-6alkyl-pyrrolidine-phenyl, C1-6alkylC(O)-pyrrolidine, C1-6alkylC(O)-pyrrolidine-phenyl, piperidine, piperidine phenyl, C1-6alkyl-piperidine, piperidine-C1-6alkyl-phenyl, C1-6alkylC(O)-piperidine, C1-6alkylC(O)-piperidine-phenyl.
In some embodiments, R is further substituted with OH, —O—C1-6 alkyl, formyl, carbonyl, —C1-6alkyl, —C1-6alkenyl, —C1-6alkynyl, —C0-6alkyl aryl, —C0-6alkyl heteroaryl, —C0-6alkyl cycloalkyl, —C0-6alkyl heterocyloalkyl, —C0-6alkylC(O)NHC0-6alkyl, —C0-6alkylC(O)NHC0-6alkyl aryl, —C0-6alkylC(O)NHC0-6alkyl heteroaryl, —C0-6alkylC(O)NHC0-6alkyl cycloalkyl, —C0-6alkylC(O)NHC0-6alkyl heterocyloalkyl, —C0-6alkylNHC0-6alkyl, —C0-6alkylNHC0-6alkyl aryl, —C0-6alkylNHC0-6alkyl heteroaryl, —C0-6alkylNHC0-6alkyl cycloalkyl, and —C0-6alkylNHC0-6alkyl heterocyloalkyl, each of which is optionally substituted by one or more substituents selected from —C0-6alkyl, —C1-6alkylOC1-6alkyl, halogen, or —C(O)OtButyl.
In some embodiments, the [18F] fluorinating agent in a solvent is Cs 18F and K222 in N, N-dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO).
In some embodiments, R3 is Cl, Br or I.
In some embodiments, compound III has the structure:
In some embodiments, compound III has the structure:
In some embodiments, R or R1 is H.
In some embodiments, R or R1 is OH.
In some embodiments, R or R1 is methyl.
In some embodiments, R or R1 is formyl, or carbonyl.
In some embodiments, R or R1 is —O—C1-6 alkyl.
In some embodiments, R or R1 is —O-methyl, —O-ethyl or —O-tert-butyl.
In some embodiments, R2 is F, Cl, Br or I.
In some embodiments, R2 is H.
In some embodiments, R2 is F.
In some embodiments, X is C.
In some embodiments, X is N.
In some embodiments, R1 is selected form the group consisting of:
In some embodiments, compound III has the structure:
In some embodiments, compound III is a human histone deacetylases (HDACs) inhibitor.
In some embodiments, HDACs are HDAC-4, HDAC-5, HDAC-7 and HDAC-9.
In some embodiments, the current invention provides a process for preparing compound IV having the structure:
In some embodiments, the [18F] fluorinating agent in a solvent is potassium 18F-fluoride (K18F) in dimethyl sulfoxide (DMSO).
In some embodiments, R or R1 is H.
In some embodiments, R or R1 is OH.
In some embodiments, R or R1 is methyl.
In some embodiments, R or R1 is formyl, or carbonyl.
In some embodiments, R or R1 is —O—C1-6 alkyl.
In some embodiments, R or R1 is —O-methyl, —O-ethyl or —O-tert-butyl.
In some embodiments, R2 is F, Cl, Br or I.
In some embodiments, R2 is H.
In some embodiments, R2 is F.
In some embodiments, X is C.
In some embodiments, X is N.
In some embodiments, means a racemic mixture.
In some embodiments, compound IV has the structure:
In some embodiments, compound IV has the structure:
In some embodiments, R is selected form the group consisting of:
In some embodiments, compound IV has the structure:
In some embodiments, compound IV is a human histone deacetylases (HDACs) inhibitor.
In some embodiments, HDACs are HDAC-4, HDAC-5, HDAC-7 and HDAC-9.
In some embodiments, the process for preparing compound IV is a late stage radiochemical synthesis,
In some embodiments, compound IIIb is reacted with Cs 18F and K222 in DMSO to obtain compound II having the structure:
In some embodiments, compound IIIb is reacted with benzyl amine.
In some embodiments, the process for preparing compound V is a late stage radiochemical synthesis, In some embodiments, compound IIIb is
In some embodiments, wherein compound V is
In some embodiments, compound IVb is
In some embodiments, compound IV is
In some embodiments, the DMSO is added at a temperature range from 145° C. to 160° C.
In some embodiments, the DMSO is added at 150° C.
In some embodiments, the current invention provides a method of detecting a disease of the central nervous system (CNS) in a subject, the method comprising:
In some embodiments, R is H, OH, —O—C1-6 alkyl, carbonyl, C1-6alkyl, —C1-6alkenyl, —C1-6alkynyl, —C0-6alkyl aryl, —C0-6alkyl heteroaryl, —C0-6alkyl cycloalkyl, —C0-6alkyl heterocyloalkyl, —C0-6alkylC(O)NHC0-6alkyl, —C0-6alkylC(O)NHC0-6alkyl aryl, —C0-6alkylC(O)NHC0-6alkyl heteroaryl, —C0-6alkylC(O)NHC0-6alkyl cycloalkyl, —C0-6alkylC(O)NHC0-6alkyl heterocyloalkyl, —C0-6alkylNHC0-6alkyl, —C0-6alkylNHC0-6alkyl aryl, —C0-6alkylNHC0-6alkyl heteroaryl, —C0-6alkylNHC0-6alkyl cycloalkyl, or —C0-6alkylNHC0-6alkyl heterocyloalkyl.
In some embodiments, R is H.
In some embodiments, R is OH.
In some embodiments, R is methyl.
In some embodiments, R is formyl, or carbonyl.
In some embodiments, R is —O—C1-6 alkyl.
In some embodiments, R is —O-methyl, —O-ethyl or —O-tert-butyl.
In some embodiments, R2 is F, Cl, Br or I.
In some embodiments, R2 is H.
In some embodiments, R2 is F.
In some embodiments, X is C.
In some embodiments, X is N.
In some embodiments, 18F source used in radiofluorination reaction is Cs18F
18F—F2, or any molecule containing 18F which is commonly used in PET imaging.
In some embodiments, R1 is selected form the group consisting of:
In some embodiments, HDACs inhibitor has the structure:
In some embodiments, the mammal is a rodent.
In some embodiments, the rodent is a rat.
In some embodiments, the disease is cancer.
In some embodiments, the HDAC inhibitors has blood-brain barrier (BBB) permeability.
In some embodiments, the HDAC inhibitors are used for brain imaging.
In some embodiments, the disease is cancer, disorders of the central nervous system, memory and cognitive impairment, dementia, rheumatoid arthritis or behavioral changes.
In some embodiments, the HDAC inhibitors accumulated in the liver of a mammal.
In some embodiments, the HDAC inhibitors accumulated in the brown fat adipose tissues (BATs).
In some embodiments, the present invention provides a method of identifying a suitable HDAC inhibitor which comprises creating the library of HDAC inhibitors containing a 18F radiolabeled group, using the HDAC inhibitors to PET image a subject and identifying biodistribution of DAC inhibitors in the subject.
As used herein, a “symptom” associated with a disease or disorder includes any clinical or laboratory manifestation associated with the disease or disorder and is not limited to what the subject can feel or observe.
As used herein, 11C-source is carbon dioxide (11CO2), carbon monoxide (11CO), 11CH4, 11CH3 or any molecule containing 11C that is commonly used in the PET imaging process.
As used herein,
indicates the functional group R can be attached to either of 1, 2, 3, 4, 5 or 6 position.
As used herein,
indicates the functional group R and R2 can be in ortho, meta, or para position.
As used herein, “precursors” is a chemical compound preceding another in a metabolic pathway. The precursors used in the current invention were radiolabeled and produced compounds in Table 3.
As used herein, “radioactive tracer” is a chemical compound in which one or more atoms have been replaced by a radionuclide so by virtue of its radioactive decay.
As used herein, “treating”, e.g. of an infection, encompasses inducing prevention, inhibition, regression, or stasis of the disease or a symptom or condition associated with the infection.
As used herein, “radiochemical purity” or “RCP” is defined as the ratio of the activity of a radionuclide in a stated chemical species in a material over the total activity of all species containing that radionuclide in this material.
As used herein; “Biodistribution” is defined as a method of tracking where compounds of interest travel in an experimental animal or human subject.
As used herein, “about” mean±5%.
As used herein, “alkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and may be unsubstituted or substituted. Thus, C1-Cn as in “C1-Cn alkyl” is defined to include groups having 1, 2, . . . , n−1 or n carbons in a linear or branched arrangement. For example, C1-C6, as in “C1-C6 alkyl” is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl, hexyl, and octyl.
As used herein, “alkenyl” refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon-carbon double bonds may be present, and may be unsubstituted or substituted. For example, “C2-C6 alkenyl” means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and up to 1, 2, 3, 4, or 5 carbon-carbon double bonds respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl.
The term “alkynyl” refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present, and may be unsubstituted or substituted. Thus, “C2-C6 alkynyl” means an alkynyl radical having 2 or 3 carbon atoms and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl.
“Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, a divalent alkane, alkene and alkyne radical, respectively. It is understood that an alkylene, alkenylene, and alkynylene may be straight or branched. An alkylene, alkenylene, and alkynylene may be unsubstituted or substituted.
As used herein, “aryl” is intended to mean any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.
As used herein, the term “polycyclic” refers to unsaturated or partially unsaturated multiple fused ring structures, which may be unsubstituted or substituted.
The term “alkylaryl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aryl group as described above. It is understood that an “alkylaryl” group is connected to a core molecule through a bond from the alkyl group and that the aryl group acts as a substituent on the alkyl group. Examples of arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1-phenylethyl, 2-phenylethyl, 3-phenylpropyl, 2-phenylpropyl and the like.
The term “heteroaryl”, as used herein, represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Heteroaryl groups within the scope of this definition include but are not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.
The term “alkylheteroaryl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an heteroaryl group as described above. It is understood that an “alkylheteroaryl” group is connected to a core molecule through a bond from the alkyl group and that the heteroaryl group acts as a substituent on the alkyl group. Examples of alkylheteroaryl moieties include, but are not limited to, —CH2—(C5H4N), —CH2—CH2—(C5H4N) and the like.
The term “heterocycle”, “heterocyclyl” or “heterocyclic” refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms. Preferred heteroatoms include N, O, and/or S, including N-oxides, sulfur oxides, and dioxides. Preferably the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation. The heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed. Such rings may be optionally fused to one or more of another “heterocyclic” ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s). Examples of heterocycles include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.
The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise.
In the compounds of the present invention, alkyl, alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
As used herein, the term “halogen” refers to F, Cl, Br, and I.
As used herein, “heteroalkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch.
As used herein, “heterocycle” or “heterocyclyl” as used herein is intended to mean a 5- to 10-membered nonaromatic ring containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. “Heterocyclyl” therefore includes, but is not limited to the following: imidazolyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, dihydropiperidinyl, tetrahydrothiophenyl and the like. If the heterocycle contains a nitrogen, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.
As used herein, “cycloalkyl” shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).
As used herein, “monocycle” includes any stable polyatomic carbon ring of up to 10 atoms and may be unsubstituted or substituted. Examples of such non-aromatic monocycle elements include but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of such aromatic monocycle elements include but are not limited to: phenyl.
As used herein, “bicycle” includes any stable polyatomic carbon ring of up to 10 atoms that is fused to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted. Examples of such non-aromatic bicycle elements include but are not limited to: decahydronaphthalene. Examples of such aromatic bicycle elements include but are not limited to: naphthalene.
The term “ester” is intended to a mean an organic compound containing the R—O—CO—R′ group.
The term “amide” is intended to a mean an organic compound containing the R—CO—NH—R′ or R—CO—N—R′R″ group.
The term “phenyl” is intended to mean an aromatic six membered ring containing six carbons and five hydrogens.
The term “benzyl” is intended to mean a —CH2R1 group wherein the R1 is a phenyl group.
The term “substitution”, “substituted” and “substituent” refers to a functional group as described above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; amino groups, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different.
It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.
In choosing the compounds of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
The compounds of the present invention include all hydrates, solvates, and complexes of the compounds used by this invention. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. The compounds described in the present invention are in racemic form or as individual enantiomers. The enantiomers can be separated using known techniques, such as those described in Pure and Applied Chemistry 69, 1469-1474, (1997) IUPAC. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention.
The compounds of the subject invention may have spontaneous tautomeric forms. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
In the compound structures depicted herein, hydrogen atoms are not shown for carbon atoms having less than four bonds to non-hydrogen atoms. However, it is understood that enough hydrogen atoms exist on said carbon atoms to satisfy the octet rule.
This invention also provides isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is 2H and/or wherein the isotopic atom 13C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic forms.
It is understood that the structures described in the embodiments of the methods hereinabove can be the same as the structures of the compounds described hereinabove.
It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.
Except where otherwise specified, if the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in “Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, N Y, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.
The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.
It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as 12C, 13C, or 14C. Furthermore, any compounds containing 13C or 14C may specifically have the structure of any of the compounds disclosed herein.
It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as 1H, 2H, or 3H. Furthermore, any compounds containing 2H or 3H may specifically have the structure of any of the compounds disclosed herein.
Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.
In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.
It is understood that substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.
In choosing the compounds used in the method of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.
In choosing the compounds of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
The various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.
The compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.
The compounds used in the method of the present invention may be prepared by techniques described in Vogel's Textbook of Practical Organic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Furnis, A. J. Hannaford, P. W. G. Smith, (Prentice Hall) 5th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.
The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat an infection or disease caused by a pathogen, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkali earth metal salts, sodium, potassium or lithium. The term “pharmaceutically acceptable salt” in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).
The compounds of the present invention may also form salts with basic amino acids such a lysine, arginine, etc. and with basic sugars such as N-methylglucamine, 2-amino-2-deoxyglucose, etc. and any other physiologically non-toxic basic substance.
As used herein, “administering” an agent may be performed using any of the various methods or delivery systems well known to those skilled in the art. The administering can be performed, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery, subcutaneously, intraadiposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventicularly, intratumorally, into cerebral parenchyma or intraparenchchymally.
The compounds used in the method of the present invention may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.
The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.
A dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional antitumor agents. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or topically onto a site of disease or lesion, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
The compounds used in the method of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or in carriers such as the novel programmable sustained-release multi-compartmental nanospheres (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, nasal, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.
Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
The compounds used in the method of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids such as lecithin, sphingomyelin, proteolipids, protein-encapsulated vesicles or from cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions.
The compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
The compounds used in the method of the present invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.
Parenteral and intravenous forms may also include minerals and other materials such as solutol and/or ethanol to make them compatible with the type of injection or delivery system chosen.
The compounds and compositions of the present invention can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by topical administration, injection or other methods, to the afflicted area, such as a wound, including ulcers of the skin, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
Specific examples of pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903,297 to Robert, issued Sep. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.
The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, powders, and chewing gum; or in liquid dosage forms, such as elixirs, syrups, and suspensions, including, but not limited to, mouthwash and toothpaste. It can also be administered parentally, in sterile liquid dosage forms.
Solid dosage forms, such as capsules and tablets, may be enteric-coated to prevent release of the active ingredient compounds before they reach the small intestine. Materials that may be used as enteric coatings include, but are not limited to, sugars, fatty acids, proteinaceous substances such as gelatin, waxes, shellac, cellulose acetate phthalate (CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), and methyl methacrylate-methacrylic acid copolymers.
The compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.
Variations on those general synthetic methods will be readily apparent to those of ordinary skill in the art and are deemed to be within the scope of the present invention.
Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.
This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.
While the invention has been shown and described with reference to certain embodiments of the present invention thereof, it will be understood by those skilled in the art that various changes in from and details may be made therein without departing from the spirit and scope of the present invention and equivalents thereof.
Solvents and starting material were obtained from commercial sources and were used as received. High-Performance Liquid Chromatography (HPLC) was performed with a 1260 series pump, (Agilent Technologies, Stuttgart, Germany), with a built-in UV detector operated at 254 nm and a radioactivity detector with a single-channel analyzer (lab logic) using a semipreparative C18 reverse-phase column (10×250 mm, Phenomenex) and an analytical C18 column (4.6×250 mm, ASCENTIS RP-AMIDE, Sigma). An acetonitrile/ammonium acetate buffer (MeCN/NH4QAc: 20 mM)) or acetonitrile/water (MeCN/water) solvent systems with various composition (solvent systems were developed specific to each compound), was used for quality control analyses at a flow of 1 mL/min. High resolution mass spectroscopy (HRMS) was performed using Agilent 1260HPLC/G6224A-TOF MS. NMR spectroscopy was performed using 400 MHz Bruker instrument.
TMP195 was prepared similar to the previously described procedures (Lobera, M., et al., 2013)
General procedure for amine/carboxylic acid coupling reactions (3-4, 12-13 and 23). The acid (1.0 eq) and HATU (1.2 eq.) in DMF (1.0-3.0 mL) were stirred for 15 min followed by simultaneous addition of the amine (1.2 eq) and NMM (excess: ˜1.0 mL). The reaction mixture was stirred for 3 hours. The DMF was removed under vacuum (˜70° C. water bath) and the residue was purified by column chromatography followed by trituration in cold pentane to afford the final products in 50-70% yield. The general procedure is described in Scheme A (
General procedure for synthesis of (N′-Hydroxycarbamimidoyl) benzoic acid (7-8 and 16-17). To the nitrile (1.0 g) in 30 ml ethanol was added first hydroxylamine hydrochloric acid (1.0 g) dissolved in water (8.0 ml) followed by sodium carbonate (1.2 g) dissolve in water (12.0 ml). The mixture was heated under reflux for 4 h. Ethanol was removed under reduced pressure and the residue was diluted with water, acidified with 10% HCl to pH ˜3, and filtrated, washed with water and then dried under reduced pressure to afford compound (N′-hydroxycarbamimidoyl) benzoic acid in 50-80% yield.
General synthesis of the bromodifluorooxadiazoles (bromo-Precursors: 10-11 and 20-21). Bromodifluoroacetic anhydride (2) ml neat was added to N′-hydroxycarbamimidoyl benzoic acid (1 g). The reaction mixture heated to 50° C. for 3 h. The volatiles were evaporated, and the crude product was purified by column chromatography using ethyl acetate/hexanes or dichloromethane/methanol to afford the bromodifluoro-analogs. The general scheme is shown in
General synthesis of the trifluorooxadiazoles (19-20). The synthesis of the TFMO moiety was performed similar to the synthesis described above for bromodifluorooxadiazole except that neat trifluoroacetic anhydride was used.
N-benzyl-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl) benzamide (3): H NMR (400 MHZ, CDCb) o 8.80 (s, 1H), 8.30 (d, J=8.6 Hz, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.64 (t, J=8.6 Hz, 1H), 7.34 (m, 5H), 6.56 (s, 1H), 4.70 (d, J=8.6 Hz, 2H). 13C NMR (126 MHZ): o 170.00 (t, J=23.9), 168.42, 166.10, 137.82, 135.48, 131.16, 130.54, 129.69, 128.89, 128.07, 127.82, 125.78, 125.56, 107.52 (t, J=217.60), 44.36. 19F NMR (CDCb), o −51.98. HRMS: Calculated for C11H13F3N302 19F NMR (CDCb), o −64.73. HRMS: Calculated for [M+H] 348.0954, Found: 348.0950. C11H13F3N302 [M+H] 348.0954, Found: 348.0950.
N-benzyl-4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl) benzamide (4): 1H NMR (CDCl3): o 8.20 (d, 2H), 7.95 (d, 2H), 7.35 (m, 5H), 6.5 (s, 1H), 4.69 (d, 2H). 13C NMR (126 MHZ): o 168.46, 166.25, 166.0 (q, J=31.8 Hz), 137.78, 137.75, 128.90, 128.03, 127.99, 127.84, 127.83, 127.69, 116.5 {q, J=196.2 Hz), 44.35. 19F NMR (CDCb), o −65.30. HRMS: Calculated for C11H13F3N302 [M+H] 348.0954, Found: 348.0952.
3-(5-(bromodifluoromethyl)-1,2,4-oxadiazol-3-yl) benzoic acid (10): 1H NMR (400 MHZ, DMSO-d6): o 8.54 (s, 1H), 8.27 (d, J=6, 1H), 8.19 (d, J=6, 1H), 7.75 (s, 1H). 13C NMR (126 MHZ) δ 170 (t, J=24), 168.28, 166.77, 133.45, 132.43, 131.79, 130.57, 128.40, 125.45, 107.52 (t, J=215.5). 19F NMR (CDCl3), o−54.01.
4-(5-(bromodifluoromethyl)-1,2,4-oxadiazol-3-yl) benzoic acid (11): 1H NMR (400 MHZ, DMSO-d6): o 8.14 (m, SH). 13C NMR (126 MHZ): 0170 (t, J=23.8), 168.28, 166.94, 134.60, 130.76, 128.71, 128.12, 107.52 (t, J=215.4). 19F NMR (CDCb), o−54.01.
N-benzyl-3-{5-(bromodifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (12): 1H NMR (400 MHz, CDCb) o 8.49 (s, 1H), 8.28 (d, J=8.6 Hz, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.64 (t, J=8.6 Hz, 1H), 7.34 (m, 5H), 6.56 (s, 1H), 4.70 (d, J=8.6 Hz, 2H). 13C NMR (126 MHZ): o 170.00 (t, J=23.9), 168.42, 166.10, 137.82, 135.48, 131.16, 130.54, 129.69, 128.89, 128.07, 127.82, 125.78, 125.56, 107.52 (t, J=217.60), 44.36. 19F NMR (CDCb), o −51.98. HRMS: Calculated for C11H138rF2N302 [M+H]: 408.0154, Found: 408.0152.
N-benzyl-4-(5-(bromodifluoromethyl)-1,2,4-oxadiazol-3-yl) benzamide (13): H NMR (400 MHZ, CDCb) o 8.20 (d, J=8.6 Hz, 2H}, 7.95 (d, J=8.4 Hz, 2H), 7.35 (m, 5H), 6.5 (s, 1H), 4.69 (d, J=5.6 Hz, 2H). 13C NMR (126 MHZ): o 170.01 (t, J=23.9), 168.33, 166.25, 137.78, 137.65, 128.91, 128.05, 127.99, 127.92, 127.86, 127.77, 107.07 (t, J=217.74), 44.37. 19F NMR (CDCb), −δ −51.97. HRMS: Calculated for C11H138rF2N302[M+H]: 408.0154, Found: 408.0153.
Ethyl 3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzoate (17): 1H NMR (400 MHZ, CDCb) o 8.79 (s, 1H}, 8.31 (d, J=5.9 Hz, 1H), 8.28 (d, J=5.9 Hz, 1H}, 7.64 (t, J=S.9 Hz, 1H), 4.45 (q, J=5.37, J=10.7 Hz, 2H), 1.45 (t, J=5.34, 3,H). 13C NMR (126 MHZ): 5168.60, 166.98 (q, J=31, J=63 Hz}, 165.50, 165.49, 133.18, 131.83, 131.72, 129.33, 128.78, 125.32, 115.51 (q, J=194, J=388 Hz), 61.52, 14.31. 19F NMR (400 MHZ, CDCl3): o 65.34. HRMS: Calculated for C12H9F3N203 [M+H]: 287.0644, Found: 287.0638.
Ethyl 3-(5-(bromodifluoromethyl)-1,2,4-oxadiazol-3-yl)benzoate (18): 1H NMR (400 MHZ, CDCb) o 8.79 (s, 1H), 8.32 (d, J=5.9 Hz, 1H), 8.30 (d, J=5.9 Hz, 1H), 7.63 (t, J=5.9 Hz, 1H), 4.45 (q, J=5.37, J=10.7 Hz, 2H), 1.45 (t, J=5.34, 3H). 13C NMR (126 MHZ): 0169.98 (t, J=56.6 Hz), 168.50, 165.56, 133.11, 131.71, 131.68, 129.31, 128.79, 125.53, 107.11 (t, J=215 Hz), 61.52, 14.35. 19F NMR (400 MHZ, CDCl3): o 51.95. HRMS: Calculated for C12H10BrF2N203 [M+H]: 346.9843, Found: 346.9837.
3-(5-(bromodifluoromethyl)-1,2,4-oxadiazol-3-yl)-N-(2-methyl-2-(2-phenyloxazol-4-yl) propyl)benzamide (23): 1H NMR (400 MHZ, CDCb) o 8.66 (s, 1H) 8.2 (m, 5H}, 7.65 (t, J=6.6 Hz, 1H), 7.52 (s, 1H), 7.45 (m, 3H), 3.66 (d, J=4.0 Hz, 2H), 1.50 (s, 6H). 13C NMR (126 MHz): 0169.92 (t, J=33.4 Hz), 168.62, 166.15, 161.81, 149.14, 136.18, 133.09, 131.16, 131.09, 130.69, 130.45, 129.50, 127 0.09, 126.49, 125.84, 125.57, 107.13 (t, J=300 Hz), 50.62, 34.33, 25.37.19F NMR (400 MHz, CDCl3): o 51.88. HRMS: Calculated for C23H20BrF2N4Q3 [M+H]: 517.0681, Found: 517.0681.
Biochemical assay: Tracer candidate were screened against recombinant human HDACs 1-11 using 9 dilutions using the commercially available HDACs enzyme assay kits (BPS Bioscience, Inc.) as described in the manufacturer protocol. The IC50 values are shown in Table. 1. The class lla HDAC fluorogenic assay functions in a two-step process: first, the class lla HDAC removes the trifluoroacetylate (TFA) from TFA-lysine substrate to generate free lysine which is recognized by trypsin and releases a fluorescent fluorophore (intact substrate is not recognized by trypsin) that can be detected using microplate reader (Lobera M 2013). IC50 was determined using nonlinear fit curves (GraphPad Prism) (% HDAC activity inhibition vs. compound concentration).
Cell-based deacetylase assay. The cell-based assay was conducted similar to the previous work (Lobera M 2013; Luckhurst C A 2019) Briefly, compounds were diluted in 5 uL DMSO (11-point concentration curve, 5% final DMSO) and were added to 96 wells with positive (5 μM Trichostatin A (TSA)) and negative control (5 uL DMSO) wells. HT-29 cells (obtained from ATCC) with the addition of either 100 μM Boc-Lys-TFA (class-IIa selective substrate) or 200 μM Boc-Lys-Ac (class-I/IIb selective substrate) were plated into 96 well plates at 200,000 cells/well in 45 uL cellular assay buffer (RPMI without phenol red, 0.1% Fetal Bovine Serum) and were incubated for 3 h at 37° C. The deacetylation achieved by the addition of 50 uL HDAC developer solution (2.5 mg/ml trypsin in DMEM without Fetal Bovine Serum and 10% tween 80) for a further 1 h to sensitize the substrate and to lyse the cells. Fluorescent counts were read with microplate reader at an excitation wavelength of 360 nm and detection of emitted light of 460 nm.
Recipe for the preparation of kryptofix/K2CO3 solution: K2CO3 (25 mg) and kryptofix (100 mg) were dissolved in acetonitrile (15.0 mL) and water (5.0 mL).
Recipe for the preparation of kryptofix/Cs2CO3 solution: Cs2CO3 (40 mg) and kryptofix (100 mg) were dissolved in acetonitrile (15.0 mL) and water (5.0 mL).
The solution of [18F] was purchased from NCM USA (Bronx, NY). The [18F] is trapped on a QMA cartridge and then eluted with 1.0-1.2 mL of a solution that contains kryptofix/K2CO3 or kryptofix/Cs2CO3 to a V-vial (Wheaton) with 92-96% recovery. The solvent was removed under a stream of Argon at 110° C. Water residue was removed aziotropically with the addition of acetonitrile (3×1.0 mL) and repeated drying under a stream of Argon at 110° C.
General example: A solution of the bromo-precursor (6-8 mg) in the appropriate solvent (i.e. DMSO) (0.4 mL) was added to the dried K[18F]/kryptofix or Cs[18F]/kryptofix and the mixture was heated at 150° C. for 20 minutes. The reaction mixture was cooled and passed through a silica gel cartridge (waters, 900 mg) and eluted with 30% methanol in dichloromethane (2.5 mL). After evaporating of the solvent under a stream of argon at 60-80° C., the residue was re-dissolved in the appropriate HPLC solvent and purified by semipreparative HPLC. The solvent was evaporated under reduced pressure. Each radioactive product was co-injected with an authentic non-radiolabeled compound into an analytical column to confirm its purity and identity. The radiochemical purity was >95% for all tracers.
18F-NT57 was isolated with 75% acetonitrile/water solution in 17-19 minutes. The solvent was evaporated under reduced pressure and was re-dissolved in 20% ethanol/saline for animal injection. Each radioactive product was co-injected with an authentic non-radiolabeled compound into an analytical column to confirm its purity and identity. The radiochemical purity was >95% for all tracers.
Radiotracer formulation: The tracer was re-dissolved in 0-20% ethanol/saline for animal injection.
The radioactive peak was detected with radioactivity detector co-injected with the relevant authentic cold compound which was detected with ultraviolet detector (254 nM) using analytical HPLC. The retention times for the radiotracers are:
18F-3 was eluted at 5.93 with 60% acetonitrile/water solution.
18F-4 was eluted at 6.0 with 60% acetonitrile/water solution.
18F-19 was eluted at 6.8 with 70% acetonitrile/water solution.
18F-20i was eluted at 6.2 with 70% acetonitrile/water solution.
18F-1 was eluted at 1.8 with 50% acetonitrile/water solution.
18F-2 was eluted at 1.8 with 50% acetonitrile/water solution.
The specific activity was determined from the area under the curve of the tracer attributed ultraviolet peak in the HPLC chromatogram against a calibration curve pre-prepared with the unlabeled reference standard. Specific activity of tracers ranged from 0.8 to 1.2 Ci/μmol. It is important to note, F-18 was purchased from a commercial source with significant decay prior to the start of radiochemical experiment. significantly higher specific activities for tracers were expected once cyclotron becomes operational. Furthermore, automated synthesis expected to further improve the radiochemical yield and specific activity due to more efficient synthesis and shorter overall production time. Moreover, starting with higher amount of radioactivity may also, further improve the yield and specific activities.
Log P for 18F-NT57 was determined using the method similar to previous work (Turkman N, S. A., Paolillo V, et al. 2012). The log P was determined by partitioning the tracer between octanol and phosphate buffer at pH 7.4 and measuring the concentration of the tracer in each layer. The radioactivity of each layer is counted using gamma-counter. The partition coefficient (P) is calculated as [radioactivity (cpm/ml) in 1-octanol)]/[(radioactivity (cpm/ml) in phosphate buffer pH 7.4].
Precursors prepared by the current invention are shown in Table 2 below.
The radioactive tracers used for PET imaging are showing in the tale below.
In Vivo microPET (uPET) Imaging of Healthy Rats
Sprague Dawley rats (200-250 g, N=3,) were anesthetized with 3% isoflurane in oxygen and maintained at 2% isoflurane in oxygen throughout the imaging studies. Anesthetized rat was placed in the uPET (Siemens, Knoxville, TN) with the skull positioned in the center of the field of view. The tracer (500-700 μCi/animal) was administered intravenously into each Rat (n=3 per group) in a total volume 0.5-1.0 ml solution. Dynamic PET images were obtained over 60 minutes. Images were reconstructed with attenuation correction using an ordered subset expectation maximization (OSEM2D) algorithm with 16 subsets and 4 iterations.
Dynamic PET images were analyzed with Inveon Research Workplace 4.0 (Siemens, Knoxville, TN) and AMIDE software using manual segmentation of regions of interest (ROI), were drawn over ROI on the decay-corrected coronal images. The radioactivity accumulation (tracer uptake) within the ROI were obtained as percentage of the injected dose (ID) per gram of tissue (% ID/g). The time-activity curve (TAC) was generated from the region of interest (ROI) by plotting the radioactivity/cc vs time.
Further studies are warranted to determine the extent of the metabolic profile of 18F-NT57 in the blood and brain tissues. It is important to note that other PET tracer candidates identified from extensive structure activity relationship studies are undergoing preclinical evaluation in the laboratory to optimize the in vivo profile (i.e. increasing BBB permeability and tracer uptake in the brain). The new data will be published in due course.
The TFMO bearing molecules are an attractive target for PET tracer development and SAR studies because they contain the three distinctive HDAC pharmacophore motifs (color coded,
A radiochemical route (Scheme 1) was designed to radiolabel the trifluoromethyl-oxadiazole (TFMO) moiety with F-18 and it was utilized to radiolabel TMP195 with F-18 as shown in Scheme 3 (
The chemical synthesis and radiolabeling reactions of the TFMO containing molecules are shown in Schemes 1 and 2 (
The radiochemistry was performed initially by treating the bromodifluoromethyl-precursors 12 or 13 with potassium 18F-fluoride (K18F) to produce the 18F-TFMO containing tracers: 18F-3 or 18F-4 with very high radiochemical purity (>98%), albeit the radiochemical yield was very low (<0.5%). Remarkably, using cesium 18F-fluoride (Cs18F) led to a significantly improved radiochemical yield of 2-5% (decay corrected).
To extend the utility of the new radiochemical design, TFMO containing esters with F-18 was radiolabeled using the same strategy. Also, the radioactive acids 18F-1 and 18F-2 were generated which are expected to be generated in vivo from cleavage of the amide bond of the class-lla HDAC inhibitors (i.e. 18F-NT57) by the metabolizing enzymes. As such, the acid tracers could be useful to determine the metabolic profile of the new tracers in vivo.
The chemical synthesis was performed similar to Scheme 1 above. The commercially available ethyl 3- or 4-cyanobenzoate 14 or 15 was heated under reflux with hydroxylamine to afford (Z)-ethyl 3 or 4-(N′ hydroxycarbamimidoyl) benzoate (16-17) in quantitative yield and was then treated with trifluoroacetic anhydride (18) to afford the ethyl 3 or 4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl) benzoate (19-20) in 70-80% yield. Similarly, the bromo-precursor analogs ethyl 3- or 4-(5-(bromodifluoromethyl)-1,2,4-oxadiazol-3-yl) benzoate (20-21) was synthesized by reacting the oximes 16-17 with the bromodifluoroacetic anhydride (9) as shown in Scheme 2 (
Despite the attempts to improve the radiochemical yield by varying the solvents and temperature as summarized in Table 1, dimethyl sulfoxide (DMSO) at 150° C. gave the best radiochemical yield. N, N-dimethylacetamide (OMA) gave slightly lower radiochemical yield compared to DMSO, however, the reaction seems to be cleaner (less radioactive and non-radioactive impurities). The use of acetonitrile failed to yield any product. As a result, DMSO was selected as the solvent of choice for subsequent radiolabeling reactions of class-lla HDAC inhibitors. Notably, no reaction occurred below 145° C. and temperatures higher than 160° C. produced lower radiochemical yields. The use of microwave technology is explored to further improve the radiochemical yield.
18F-incorporatation into the TFMO moiety.
Next, the utility of novel radiochemical design to produce the 18F-TFMO containing class-lla HDAC inhibitors were examined. The radiosynthesis of 18F-NT57, a radioactive homolog of TMP195 is outlined in Scheme 3 (
Owing to the single step radiosynthesis, a radioactive dose sufficient for multiple human studies (typically ˜5.0-10.0 mCi/subject) can be readily produced starting with ˜1.0 Ci of F-18 which can be easily generated in most cyclotron facilities. In fact, due to the shorter time of the radiosynthesis and dose formulation, it is likely that performing the radiosynthesis using a fully automated module will significantly improve the amount of the final dose. Notably, the yield was sufficient for performing preclinical studies and will also be sufficient to produce a clinical dose for human studies, if a TFMO based tracer from the studies becomes available for clinical translation. Also, other sites for radiolabeling are currently being pursued to improve the radiochemical yield while preserving the high affinity to class-lla HDACs. PET imaging of class-lla HDACs, an important target for cancer and brain imaging obtained with the 18F-TFMO-containing molecules underscores the significance of the radiochemical labeling method reported in this invention. In addition to the radiolabeled class-lla HDAC inhibitors, it is likely that the radiochemical methods reported herein can be extended to other classes of trifluoromethyl containing heterocyclic small molecules that appear in inhibitors of highly pursued imaging targets such as cyclooxygenases (Cox-1 and Cox-2) and estrogen receptors (Kumar, J. S. D., et al., 2018; Long, S., et al. 2009; Zhang, S., et al., 2017).
The new tracer was purified using a semi-preparative HPLC system (C18 column with 4.0 ml/min flow rate using 75% acetonitrile/water). The overall radiochemical yield of 18F-NT57 was within 2-5% (end of radiosynthesis, decay-corrected, n=10) and the radiochemical purity was >98%. The identity of 18F-NT57 was confirmed by co-injection together with the corresponding cold (unlabeled) molecule using analytical HPLC as shown in
Currently, the F-18 is purchased from an outside source, as a result it undergoes significant decay (several cycles) before reaching the laboratory which reduces the overall specific activity. The radiosynthesis, HPLC purification and formulation of the final radioactive dose was accomplished manually in less than 100 min. The partition coefficients (Log D) using the octanol/PBS shake flask method was determined (Turkman, N., et al., 2012). The observed Log D value of 18F-NT57 was >6.0 which is expected for such a highly lipophilic compound.
To further validate the utility of the new radiochemical approach in vivo, PET imaging with 18F-NT57 in healthy Sprague Dawley rats was performed.
Anesthetized Sprague Dawley rats (200-250 g, N=6) were placed in the Inveon uPET (Siemens, Knoxville, TN) in the supine position with the skull positioned in the center of the field of view. [18F]-NT160 was formulated in ethanol:polysorbate 80:saline (10:10:80%) and it was stable in this formulation at 37° C. at least for 4 hours. [18F]-NT160 (18.5-25.9 MBq/animal) was administered via the tail-vein injection in a total volume 0.5-1.0 ml. Dynamic PET images were obtained over 60 minutes followed by CT imaging (15 min scan). Images were reconstructed with attenuation correction using an ordered subset expectation maximization (OSEM2D) algorithm with 16 subsets and 4 iterations. PET image analysis was performed using the AMIDE software by using manual segmentation of regions of interest (ROI), including: whole brain, cortex, hippocampus, thalamus, cerebellum, and brain stem. Rat Brain Atlas was used for alignment and identification of specific anatomical markers of the brain. The time-activity curve (TAC) was generated from the region of interest (ROI) by plotting the radioactivity/cc vs time. Levels of accumulation of the radiotracer in tissues were expressed as standard uptake values (SUV) that were calculated for the regions of interest (ROI) using the AMIDE software. The SUV is defined as the ratio of the tissue radioactivity concentration C (e.g. expressed as Bq/g tissue) at given time point post injection T, and the injected dose (e.g. in Bq, decay-corrected to the same time T), and normalized by the body weight in grams. GraphPad Prism (Graph Pad Software La Jolla, CA) was used for image data analysis. [18F]-NT2-160 was also prepared by the same or similar procedure. Their PET imaging results were shown in
Dynamic PET imaging studies also provided real time information on the biodistribution of 18F-NT57 as a surrogate to TMP195 in rat tissues over 60 min as summarized in
There is also a notable intense and blockable uptake in the wrist (
Given the specific interest in molecular imaging of class-lla HDACs in the CNS, 18F-NT57 exhibited relatively rapid uptake that peaked at ˜1% ID/g in the brain indicating a successful CNS penetration. This finding is highly significant, since it provides a proof of principle that TFMO containing molecules can penetrate the BBB and can map the class-lla HDAC expression and biodistribution in the brain. Class-lla HDACs are known to be highly expressed in various regions of the brain and their overexpression has been indicated in various brain disorders (Kikuchi, S., et al., 2015; Choi, S. Y., et al., 2018). The tracer exhibited heterogeneous biodistribution in brain regions with high uptake in the grey matter. The in vivo findings are consistent with ex-vivo findings of Broide R S, et. al. on the heterogeneous expression of class-lla HDACs in the rat brain (Trazzi, S., et al., 2016). A blocking experiment was performed to further confirm the specificity of 18F-NT57 to class-lla HDAC in vivo in the brain. The blocking study confirmed the specific binding of the tracer to class-lla HDACs in the brain. The brain uptake was reduced when comparing images obtained with the tracer administered alone (baseline) or with a cold dose of TMP195 (2 mg/kg). A time activity curve obtained over 60 min which further confirms the reduction in brain uptake due to self-blocking with TMP195 (
A late-stage radiosynthesis and a successful in vivo imaging with new class-lla HDAC targeting PET tracer 18F-NT57 was developed. The late-stage radiolabeling strategy produced an identical radioactive homolog of the cold class-lla HDAC inhibitor TMP195 ensuring maintenance of the identical inhibition affinity. This strategy is successfully applied to produce a new generation of 18F-TFMO containing class-lla HDAC inhibitor-based PET tracers. The single radiofluorination-step facilitate automated routine production and produces and formulates the tracer in relatively short time. The reported radiochemistry can be extended to other target molecules that contain trifluoromethyl-heterocyclic moiety. In vivo imaging with 18F-NT57 indicates a successful lead tracer for PET imaging and delineation of class-lla HDAC expression and biodistribution in rat tissues. The findings herein allow for extensive SAR studies to identify clinically translatable TEMO containing class-lla HDAC inhibitor-based PET tracer for PET imaging of cancer and the disorders of the CNS.
This application claims priority of U.S. Provisional Application No. 63/149,505, filed Feb. 15, 2021, the contents of which are hereby incorporated by reference.
This invention was made with government support under AG067417-01 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/016464 | 2/15/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63149505 | Feb 2021 | US |
