Modulators of Caspase-6

Abstract

Modulators of caspase-6 activity are provided for use in the treatment of neurodegenerative diseases.

Claims

1. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with caspase-6 inhibitory activity and one or more pharmaceutically acceptable excipients, wherein the active agent has one of the following structures I or II, or a pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof: ##STR00003## wherein Ra and Rb are independently linear or branched C.sub.1 to C.sub.6 alkyl, aryl, or alkenyl, C.sub.1 to C.sub.9 alkylaryl, C.sub.1 to C.sub.9 substituted alkylaryl, or C.sub.1 to C.sub.9 alkylheteroaryl; and wherein the phenyl group is substituted with 0, 1 or 2 halogens and further wherein the halogens are chloride, fluoride or bromide.

2. The pharmaceutical composition of claim 1, wherein the composition is formulated for oral or topical administration, subcutaneous, intravenous, or intramuscular injection, infusion, inhalation, or intrathecal injection directly into the central nervous system.

3. The pharmaceutical composition of claim 1, wherein the neurological disease is Huntington's disease, Alzheimer's disease, dementia, mild-cognitive impairment, or memory loss.

4. A method of treating a neurological disease comprising administering to a subject in need of such treatment an effective dose of a pharmaceutical composition of claim 1 wherein said disease is Huntington's disease, Alzheimer's disease, dementia, mild-cognitive impairment, or memory loss.

5. The method of claim 4 wherein the composition of claim 1 is administered in combination with one or more additional drugs useful in the treatment of neurological disease.

6. The method of claim 5 wherein the one or more additional drugs is selected from L-DOPA, rasagiline, memantine hydrochloride, donepezil hydrochloride, rivastigmine, galantamine and tetrabenzine.

7. The method of claim 4, wherein administration of an effective dose of a pharmaceutical composition of claim 1 is commenced prior to the appearance of symptoms of said neurological disease in said subject.

8. The method of claim 7, wherein cells of the subject express a mutant htt gene.

9. The method of claim 7, wherein neural cells of the subject overexpress caspase-6 mRNA.

10. A method according to claim 4 wherein the subject is human.

11. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with the structure designated as PG-3a in FIG. 3A, or pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof.

12. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with the structure designated as PG-3b in FIG. 3B, or pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof.

13. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with the structure designated as PG-3c in FIG. 3C, or pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof.

14. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with the structure designated as PG-3d in FIG. 3D, or pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof.

15. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with the structure designated as PG-3e in FIG. 3E, or pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof.

16. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with the structure designated as PG-3f in FIG. 3F, or pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof.

17. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with the structure designated as PG-3g in FIG. 3G, or pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof.

18. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with the structure designated as PG-3h in FIG. 3H, or pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof.

19. A pharmaceutical composition for the treatment or amelioration of a neurological disease wherein the composition comprises a therapeutically effective amount of an active agent with the structure designated as PG-3 in FIG. 3, or pharmaceutically acceptable salt, stereoscopic isomer, derivative or prodrug thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present inventions. The disclosure can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0071] FIG. 1A shows the synthesis scheme for compounds 8a-f FIG. 1B shows possible tautomeric forms of compounds 8a-f.

[0072] FIG. 2 shows the hydrolytic ring opening of compound 8 to compound 9 and decarboxylation of compound 9e to compound 10.

[0073] FIG. 3 and FIGS. 3a-h show the structure of the PG3 compounds and its analogs.

[0074] FIG. 4 shows that the PG3 compounds inhibit the caspase 6 enzyme in a dose dependent fashion. Different concentrations of the PG3 compounds were incubated with the Htt protein and the caspase-6 enzyme. The Htt substrate from COS-7 cell lysates is cleaved by caspase 6 and fragments are detected by FRET between the N-terminal BKP1 antibody and the neo-epitope antibody against amino acid 586. (A) shows the dose response curves for compound PG3, PG3a and PG3b (B) shows the dose response curves for compound PG3c, PG3d and PG3e (C) shows the dose response curves for compound PG3f, PG3g and PG3h.

[0075] FIG. 5 shows that the presence of the PG3d compound inhibits the cleavage of Htt by caspase-6 in a Western blot assay. COS-7 cells co-transfected with the htt-4C construct and the human caspase-6 lacking the pro-domain that were either left untreated, treated with 10 uM of the PG3d compound or treated with 3 uM of the pan-caspase inhibitor (Q-VD-Oph). (A-upper panel) The cleavage of the Htt protein to generate the 586 amino acid fragment was analyzed by Western blot using the pan-HTT BKP1 antibody. The presence of the PG3d inhibitor or the pan caspase inhibitor resulted in a reduced cleavage of the Htt protein. (A lower panel) The presence of the PG3d compound or the pan-caspase inhibitor (Q-VD-Oph) also resulted in a reduced level of active caspase-6 enzyme. (B) shows a graphical representation of the level of the 586 amino acid fragment compared to actin levels from the western blot analysis. Protein levels were quantified using Licor Odyssey imaging software. The expression of the Htt-586 fragment relative to actin expression in shown on the y-axis. The presence of 10 uM of the PG3d compound resulted in a significant reduction in the expression of the Htt cleavage fragment as compared to untreated cells. Student's t test **: p<0.01

[0076] FIG. 6 shows that the PG3 compounds inhibit the cleavage of lamin A in HEK293 cells by caspase-6 as quantified by the Mesoscale ELISA method. (A) shows the dose response curves for compounds PG3a, PG3b, PG3d and PG3d. (B) shows the dose response curves for compounds PG3e, PG3g and PG3h.

[0077] FIG. 7 shows that PG3d inhibit the intraneuronal activation of Caspase-6. Primary cortical neurons from FVB/N mice were treated with 10 uM camptothecin for 30 h in the presence or absence of 10 uM PG3d. Camptothecin treatment leads to the activation of caspase-6, which can be quantified by measuring the cleavage of lamin A (Ehrnhoefer et al. PLoS One, 2011. 6(11):e27680). The presence of PG3d in the neuronal medium significantly reduces caspase-6 activity. Student's t-test, **: p<0.01.

[0078] FIG. 8 shows that PG3d improves the neuronal viability during excitotoxic stress. Primary cortical neurons from FVB/N mice were treated with different amounts of NMDA in the medium for 20 h, which leads to a loss of intracellular ATP levels, a measure of viability. The presence of 10 uM PG3d significantly improves neuronal viability in this paradigm. Two-way ANOVA: NMDA treatment p<0.0001, PG3d treatment p=0.0001. Post-hoc Bonferroni test: **=p<0.01.

[0079] FIG. 9 is the results of cells studies demonstrating the interaction of caspase-6 with Htt fragment in COS-7 cells. Following transfection, the COS-7 cells were exposed to either DMSO, the pan-caspase inhibitor Q-VD-Oph (Q-VD-OPh) or the PG3d compound (PG3d). After a 24 hr incubation period, cell lysates were either processed by Western blot or were immunoprecipitated with Caspase-6 antibody followed by Western blot analysis. (A) Western blot analysis of the lysates from co-transfected COS-7 cells that were either treated with DMSO (n.t), exposed to 3 uM of the pan-caspase inhibitor Q-VD-Oph (Q-VD-OPh) or were treated with 10 uM of the PG3d compound (PG3d). The upper panel shows Western blot analysis with the Htt antibody 2166 (Millipore). The lower panel shows Western blot analysis with Caspase-6 antibody HD91 to detect the full length and active forms of caspase 6. (B) Western blot analysis of the cell lysates from FIG. 9A following immunoprecipitation with a caspase-6 antibody to identify the binding partners of the caspase-6 enzyme. The upper panel shows Western blot analysis with the Htt antibody 2166 (Millipore) and the lower panel shows Western blot analysis with the HD91 caspase-6 antibody.

[0080] FIG. 10 is a bar graph quantitizing co-precipitation data shown in FIG. 9B by densitometric analysis. 1way ANOVA p=0.031, post-hoc Tukey's test *: p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

Sequences:

[0081] SEQ ID NO: 1 (htt-4C). SEQ ID NO: 1 has an additional 10 amino acids at the N-terminus (relative to wild-type huntingtin), comprising the His-tag to enable processing of the expressed polypeptide. Htt-4C is truncated at amino acid 1212 (numbering according to the wild-type huntingtin sequence, and has four D to A amino acid substitutions at amino acids 513, 530, 552 and 589 (numbering according to the wild-type huntingtin sequence) marked by bold, underlined text. The IVLD Caspase-6 cleavage site is marked with a double-underline.

TABLE-US-00001 (SEQIDNO:1) MHHHHHHEFPMATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQPP PPPPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPLH RPKKELSATKKDRVNHCLTICENIVAQSVRNSPEFQKLLGIAMELF LLCSDDAESDVRMVADECLNKVIKALMDSNLPRLQLELYKEIKKNG APRSLRAALWRFAELAHLVRPQKCRPYLVNLLPCLTRTSKRPEESV QETLAAAVPKIMASFGNFANDNEIKVLLKAFIANLKSSSPTIRRTA AGSAVSICQHSRRTQYFYSWLLNVLLGLLVPVEDEHSTLLILGVLL TLRYLVPLLQQQVKDTSLKGSFGVTRKEMEVSPSAEQLVQVYELTL HHTQHQDHNVVTGALELLQQLFRTPPPELLQTLTAVGGIGQLTAAK EESGGRSRSGSIVELIAGGGSSCSPVLSRKQKGKVLLGEEEALEDD SESRSDVSSSALTASVKDEISGELAASSGVSTPGSAGHDIITEQPR SQHTLQADSVALASCDLTSSATDGDEEAILSHSSSQVSAVPSDPAM DLNAGTQASSPISDSSQTTTEGPDSAVTPSDSSEIVLDGTANQYLG LQIGQPQDEDEEATGILPDEASEAFRNSSMALQQAHLLKNMSHCRQ PSDSSVDKFVLRDEATEPGDQENKPCRIKGDIGQSTDDDSAPLVHC VRLLSASFLLTGGKNVLVPDRDVRVSVKALALSCVGAAVALHPESF FSKLYKVPLDTTEYPEEQYVSDILNYIDHGDPQVRGATAILCGTLI CSILSRSRFHVGDWMGTIRTLTGNTFSLADCIPLLRKTLKDESSVT CKLACTAVRNCVMSLCSSSYSELGLQLIIDVLTLRNSSYWLVRTEL LETLAEIDFRLVSFLEAKAENLHRGAHHYTGLLKLQERVLNNVVIH LLGDEDPRVRHVAAASLIRLVPKLFYKCDQGQADPVVAVARDQSSV YLKLLMHETQPPSHFSVSTITRIYRGYNLLPSITDVTMENNLSRVI AAVSHELITSTTRALTFGCCEALCLLSTAFPVCIWSLGWHCGVPPL SASDESRKSCTVGMATMILTLLSSAWFPLDLSAHQDALILAGNLLA ASAPKSLRSSWASEEEANPAATKQEEVWPALGDRALVPMVEQLFSH LLKVINICAHVLDDVAPGPAIKAALPSLTNPPSLSPIRRKGKEKEP GEQASVPLSPKKGSEASAASRVEGYPYDVPDYA

[0082] SEQ ID NO: 2 (caspase-6 delta prodomain). SEQ ID NO: 2 comprises amino acids 24-293 of human caspase-6, with the prodomain (aa 1-23) deleted. This deletion leads to faster intracellular auto-activation of the enzyme after transfection (Klaiman et al, BBA, 2009. 1793(3): 592-601). The protein has additional 31 amino acids at the C-terminus (relative to wild-type caspase-6), comprising the DDK-tag to enable detection of the expressed polypeptide.

TABLE-US-00002 (SEQIDNO:2) MAFYKREMFDPAEKYKMDHRRRGIALIFNHERFFWHLTLPERRGTC ADRDNLTRRFSDLGFEVKCFNDLKAEELLLKIHEVSTVSHADADCF VCVFLSHGEGNHIYAYDAKIEIQTLTGLFKGDKCHSLVGKPKIFII QACRGNQHDVPVIPLDVVDNQTEKLDTNITEVDAASVYTLPAGADF LMCYSVAEGYYSHRETVNGSWYIQDLCEMLGKYGSSLEFTELLTLV NRKVSQRRVDFCKDPSAIGKKQVPCFASMLTKKLHFFPKSNTRTPL EQKLISEEDLAAMISWITRDDDDKV

[0083] SEQ ID NO: 3 (wt htt). SEQ ID NO: 3 has an additional 10 amino acids at the N-terminus (relative to wild-type huntingtin), comprising the His-tag to enable processing of the expressed polypeptide. Wt Htt is truncated at amino acid 1212 (numbering according to the wild-type huntingtin sequence.

TABLE-US-00003 (SEQIDNO:3) MHHHHHHEFPMATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQPP PPPPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPLH RPKKELSATKKDRVNHCLTICENIVAQSVRNSPEFQKLLGIAMELF LLCSDDAESDVRMVADECLNKVIKALMDSNLPRLQLELYKEIKKNG APRSLRAALWREAELAHLVRPQKCRPYLVNLLPCLTRTSKRPEESV QETLAAAVPKIMASFGNFANDNEIKVLLKAFIANLKSSSPTIRRTA AGSAVSICQHSRRTQYFYSWLLNVLLGLLVPVEDEHSTLLILGVLL TLRYLVPLLQQQVKDTSLKGSFGVTRKEMEVSPSAEQLVQVYELTL HHTQHQDHNVVTGALELLQQLFRTPPPELLQTLTAVGGIGQLTAAK EESGGRSRSGSIVELIAGGGSSCSPVLSRKQKGKVLLGEEEALEDD SESRSDVSSSALTASVKDEISGELAASSGVSTPGSAGHDIITEQPR SQHTLQADSVALASCDLTSSATDGDEEAILSHSSSQVSAVPSDPAM DLNAGTQASSPISDSSQTTTEGPDSAVTPSDSSEIVLDGTANQYLG LQIGQPQDEDEEATGILPDEASEAFRNSSMALQQAHLLKNMSHCRQ PSDSSVDKFVLRDEATEPGDQENKPCRIKGDIGQSTDDDSAPLVHC VRLLSASELLTGGKNVLVPDRDVRVSVKALALSCVGAAVALHPESF FSKLYKVPLDTTEYPEEQYVSDILNYIDHGDPQVRGATAILCGTLI CSILSRSRFHVGDWMGTIRTLTGNTFSLADCIPLLRKTLKDESSVT CKLACTAVRNCVMSLCSSSYSELGLQLIIDVLTLRNSSYWLVRTEL LETLAEIDFRLVSFLEAKAENLHRGAHHYTGLLKLQERVLNNVVIH LLGDEDPRVRHVAAASLIRLVPKLFYKCDQGQADPVVAVARDQSSV YLKLLMHETQPPSHFSVSTITRIYRGYNLLPSITDVTMENNLSRVI AAVSHELITSTTRALTFGCCEALCLLSTAFPVCIWSLGWHCGVPPL SASDESRKSCTVGMATMILTLLSSAWFPLDLSAHQDALILAGNLLA ASAPKSLRSSWASEEEANPAATKQEEVWPALGDRALVPMVEQLFSH LLKVINICAHVLDDVAPGPAIKAALPSLTNPPSLSPIRRKGKEKEP GEQASVPLSPKKGSEASAASRVEGYPYDVPDYA

[0084] SEQ ID NO: 4 (full-length caspase-6). SEQ ID NO: 4 comprises amino acids 1-293 of human caspase-6. The protein has additional 31 amino acids at the C-terminus (relative to wild-type caspase-6), comprising the DDK-tag to enable detection of the expressed polypeptide.

TABLE-US-00004 (SEQIDNO:4) MSSASGLRRGHPAGGEENMTETDAFYKREMFDPAEKYKMDHRRRGIA LIFNHERFFWHLTLPERRGTCADRDNLTRRFSDLGFEVKCFNDLKAE ELLLKIHEVSTVSHADADCFVCVFLSHGEGNHIYAYDAKIEIQTLTG LFKGDKCHSLVGKPKIFIIQACRGNQHDVPVIPLDVVDNQTEKLDTN ITEVDAASVYTLPAGADFLMCYSVAEGYYSHRETVNGSWYIQDLCEM LGKYGSSLEFTELLTLVNRKVSQRRVDFCKDPSAIGKKQVPCFASML TKICLHFFPKSNTRTPLEQKLISEEDLAAMISWITRDDDDKV

Cell Transfection and Cell Lysis

[0085] COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 1%/penicillin/streptomycin and 0.5% glutamine. Transfections were performed using the Fugene reagent (Roche) according to manufacturer's instructions. The transfected DNA encodes amino acids 1-1212 of the human Htt protein with 15 glutamines and a C-terminal HA-tag, under control of a CMV promoter (Warby, 2008, supra; Wellington et al., 2000. J Biol Chem 275:19831-19838). The construct that was transfected is 4c Htt, which contains D.fwdarw.A mutations at amino acids 513, 530, 552 and 589 (SEQ ID NO: 1). 24 h after transfection, cells were harvested by trypsinization, pellets washed in PBS and lysed by suspension in lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% Igepal, supplemented with 1 complete protease inhibitor (Roche) and 4 mM Pefabloc). Lysates were incubated on ice for 10 min, vortexed and sonicated for 4 sec before centrifugation at 21,000g for 10 min at 4 C. Supernatants were saved, protein concentration determined with the Biorad DC assay and stored at 80 C. until use.

[0086] HEK 293 cells were grown in DMEM supplemented with 10% fetal bovine serum, 1%/penicillin/streptomycin and 0.5% glutamine. Transfections were performed using the Fugene reagent (Roche) according to manufacturer's instructions. The transfected DNA encodes amino acids 24-293 of the human caspase 6 enzyme with a C-terminal DDK tag under the control of a CMV promoter (vector pCMVSport6) (SEQ ID NO: 2). The transfected HEK 293 were used to measure the intracellular activity of caspase-6 as evidenced by cleavage of the lamin substrate (described below).

Purification of Htt from COS-7 Cell Lysates

[0087] 50 l HA-agarose beads (EZ-View, Sigma) were mixed with 1 ml lysis buffer, centrifuged at 8200g for 30 sec and the supernatant was discarded. The beads were mixed with 200 l cell lysate diluted to 0.5 g/l in lysis buffer and incubated for 2 h at 4 C. on a rotating wheel. An aliquot of the diluted cell lysate was saved as the input fraction. The sample was centrifuged at 8200g for 30 sec and the supernatant was saved, the beads were washed three times with 100 l lysis buffer and supernatants were saved as wash fractions. Elution was performed by adding 100 l HA peptide (100 g/ml, Sigma) in RIPA buffer to the beads (50 mM Tris pH 8, 150 mM NaCl, 1% Igepal, 0.5% Na-deoxycholate, 0.1% SDS) and incubating for 10 min at 37 C. The elution step was repeated 5 times, all eluates were saved. The beads were then mixed with SDS loading dye and after heat denaturation run together with 10 l aliquots of all fractions on a 3-8% NuPage Tris-Acetate gel (Invitrogen). Gels were either blotted for detection with the BKP1 (Kalchman et al, J Biol Chem (1996), 271 (32):19385)) and HA antibodies with the Odyssey imaging system (Li-cor Biosciences) or stained with Coomassie dye for detection of total protein.

Assessment of Htt Cleavage by Western Blot

[0088] Aliquots of COS-7 lysates expressing 4c Htt corresponding to 25 g protein were diluted with cleavage buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol) and the desired amount of recombinant caspase-6 (BioMol) to 10 l. For experiments including caspase-6 inhibitor compounds, the desired amount of inhibitor was mixed with the caspase before addition to the COS-7 lysate. Samples were incubated for 1 h at 37 C. and analyzed on a 3-8% NuPage Tris-Acetate gel (Invitrogen), followed by Western blotting and detection with the BKP1 and neo-586 antibodies with the Odyssey imaging system (Li-cor Biosciences). The BKP1 and neo-586 antibodies have been described previously (Warby et al. Hum. Mol. Genet 2008. 17(15):2390-404.).

Quantification of Htt Expression by FRET

[0089] Dilution series of cell lysates were prepared in sample buffer (1 PBS without CaCl2 or MgCl2, 0.4% Triton, 1 complete protease inhibitor cocktail (Roche)), Tb-labelled BKP1 antibody and D2-labelled HA antibody (Cisbio) were diluted to 1 ng/l (Tb) and 10 ng/l (D2) in antibody dilution buffer (50 mM NaH2PO4, 0.1% BSA, 0.05% Tween). Antibodies were pre-mixed at a 1:1 ratio, then 10 l cell lysate and 2 l antibody mix were pipetted into each well of a white 384 well plate (Nunc). The plate was centrifuged briefly and FRET was measured on a Victor 3 multilabel plate reader (Perkin Elmer) with the following settings: Excitation: 340 nm, Emission 1: 615 nm, Emission 2: 665 nm, 50 s delay, 200 s window time, 2000 s cycle time. To obtain the final FRET signal, the ratio between Emission 2/Emission 1 (D2/Tb signal) was calculated.

Simultaneous Caspase-6 Cleavage and 586 Fragment Detection by FRET

[0090] Dilution series of cell lysates and caspase-6 (2.2 final concentration) were prepared in FRET cleavage buffer (10 mM HEPES pH 7.4, 100 mM NaCl, 0.05% gelatin, 0.1% CHAPS, 2 mM DTT) since this buffer was previously found to best stabilize caspase-6 in dilute form at room temperature. For negative controls, 22 M zVAD-fmk was added to the caspase-6 dilutions.

[0091] Tb-labelled BKP1 antibody and D2-labelled 586 antibody (Cisbio) were diluted to 1 ng/l (Tb) and 10 ng/l (D2) in FRET cleavage buffer and pre-mixed at a 1:1 ratio.

[0092] In each well of a white 384 well plate (Nunc), 10 l cell lysate were mixed with 10 l caspase and 2 l antibody mix, the plate was centrifuged briefly and incubated in the Victor 3 multilabel plate reader (Perkin Elmer) at 37 C. FRET was measured every 30 min for up to 2 h with the following settings: Excitation: 340 nm, Emission 1: 615 nm, Emission 2: 665 nm, 50 s delay, 200 s window time, 2000 s cycle time. To obtain the final FRET signal, the ratio between Emission 2/Emission 1 (D2/Tb signal) was calculated. After 2 h at 37 C., the assay plate was sealed and after further incubation at 4 C. for 20 h, the FRET signal was read again.

Assessment of Intracellular Htt Cleavage by Western Blot

[0093] COS-7 cells were co-transfected with the 4c htt fragment and the human caspase-6 lacking the pro-domain which leads to fast autoactivation of the caspase-6 enzyme and the generation of the 586 aa Htt cleavage fragment. The co-transfected cells were exposed to either 10 uM of the PG3d compound, 3 uM of the Q-VD-Oph pan-caspase inhibitor or were left untreated. Non-transfected cells were included as a negative control and the purified 586 aa Htt fragment was included as a positive control. Cell lysates were subjected to Western blotting and the 586AA fragment generated intracellularly was detected with Htt antibody 2166 (Millipore). Caspase-6 expression and activation was assessed by Western blotting using antibody HD91 (Ehrnhoefer et al, HMG, 2014. 23(3):717-29).

Assessment of Lamin Cleavage in HEK 293 Cells Overexpressing Caspase-6

[0094] The quantitative assessment of intracellular lamin cleavage in caspase-6 transfected HEK 293 cells was done as described previously (Ehrnhoefer et al. PLoS One, 2011. 6(11):e27680) Briefly, HEK 293 cell lysates were adjusted to 1 g protein/l in lysis buffer, diluted to 0.2 g/l in PBS and 5 l were added to a Multi-Array high-bind 96 well plate (Mesoscale discovery). After incubation at room temperature for 1 h, the wells were blocked by adding 150 l 5% BSA in PBS, followed by further incubation at room temperature for 1 h. Wells were then washed 3 with 150 l PBS+0.05% Tween, and 25 l antibody mix was added (Cell signaling #2036 at 1:100 dilution, Mesoscale discovery goat-anti-rabbit sulfo-tag at 1:500 dilution in PBS with 1% BSA). After lh incubation at room temperature, the wells were washed 3 with 150 l PBS+0.05% Tween, and 150 l/well 2 reading reagent (Mesoscale discovery) was added. The plate was read on a Sector imager 6000 (Mesoscale discovery).

Assessment of Lamin Cleavage in Primary Neuronal Culture

[0095] Cortical neuronal cultures from FVB mice were prepared as described (Metzler et al, J Neurosci (2007) 27(9):2298). At day 10 in vitro, cells were treated with camptothecin, and after 30 h harvested by scraping in PBS supplemented with 1 complete protease inhibitor (Roche) and 4 mM Pefabloc. Cells were lysed by suspension in lysis buffer (50 mM Tris pH8, 150 mM NaCl, 1% Igepal, supplemented with 1 complete protease inhibitor (Roche) and 4 mM Pefabloc). Lysates were incubated on ice for 10 min, vortexed and sonicated for 4 sec before centrifugation at 21 000g for 10 min at 4 C. Supernatants were saved, protein concentration determined with the Biorad DC assay and quantitative assessment of cleaved lamin A in neuronal lysates was performed with the Mesoscale ELISA method as described in Ehrnhoefer et al. PLoS One, 2011. 6(11):e27680.

Assessment of Neuronal Viability During Excitotoxic Stress

[0096] Cortical neuronal cultures from FVB mice were prepared as described (Metzler et al, J Neurosci (2007) 27(9):2298). At day 10 in vitro, cells treated with either 10 uM PG3d or DMSO as a negative control and then were treated with either 25 nM NMDA, 50 uM of NMDA or were left untreated for 20 hr. The measurement of intracellular ATP was used as an assessment of neuronal viability as described previously (Uribe et al, HMG 2012. 21(9):1954-67) using the Cell-titer glo kit from Promega according to manufacturer's instructions.

Assessment of the Binding Interaction Between htt and Caspase 6.

[0097] COS-7 cells were co-transfected with the 1212 amino acid HTT fragment (SEQ ID NO: 3) and the full length human caspase-6 enzyme (SEQ ID NO:4). The co-transfected cells were exposed to either 10 uM of the PG3d compound, 3 uM of the Q-VD-Oph pan-caspase inhibitor or were treated with DMSO as a control. An aliquot of the cell lysates were subjected to Western blotting to detect HTT fragments with the Htt antibody 2166 (Millipore) and the presence of caspase-6 was detected using the HD91 antibody (Ehrnhoefer et al, HMG, 2014. 23(3):717-29). The remaining cell lysates (500 ug protein) were immunoprecipitated with 5 ug of Caspase-6 antibody for 16 hrs at 4 C. The immunoprecipitated proteins were then applied to acrylamide gels and immunoblotted to detect either the HTT fragments or the presence of the active caspase 6 enzyme using the antibodies described above.

Synthetic Methods for the PG3 Compound and its Analogs.

[0098] As described in Table 1, 3-Phenylprop-2-ynamide (1a) was prepared in high yield according to literature procedures by the reaction of 3-phenylprop-2-ynoic acid ester with aqueous ammonia solution (Struebing et al. Tetrahedron (2005) 61:11333). Following this procedure, the corresponding arylpropynamides 1b-e were obtained in good yields by ammonolysis of the crude arylpropynoic ethyl esters, which in turn resulted from esterification of the corresponding arylpropynoic acids with ethanol.

TABLE-US-00005 TABLE 1 Synthesis of Arylpropynamides (1) R Amide 1 Yield (%) Ph 1a 92 2-ClC.sub.6H.sub.4 1b 72 4-ClC.sub.6H.sub.4 1c 78 4-BrC.sub.6H.sub.4 1d 76 4-O.sub.2NC.sub.6H.sub.4 1e 67

[0099] Treatment of 3-phenylprop-2-ynamide (1a) with monosubstituted malonyl chlorides (6a-f) or (chlorocarbonyl)ethylketenes (7a,b) in diethyl ether at 0-5 C. delivered the 4-hydroxy-2-(phenylethynyl)-6H-1,3-oxazin-6-ones (8a-f) in 61-87% yield (as shown in Table 2 and FIG. 1A).

[0100] Theoretically, the heterocycles 8 (R1=CCPh) can adopt the tautomeric forms A-C (FIG. 1B). Mass spectra of similar 4-hydroxy-6H-1,3-oxazin-6-ones (R1=aryl; R2=H, Me) showed the presence of 4-hydroxy-6-oxa, dipolar-ionic and dioxo forms mixture in the gas phase. The presence of a substituent in the para position of the benzene ring has a strong influence on the tautomeric ratio. The quantum-chemical calculations revealed structure A as the most favorable 1,3-oxazine tautomer in the gas phase. Dissolved in tetrahydrofuran and dimethyl sulfoxide-d6 these compounds predominantly exist as tautomerA. (Stanovnik et al. Adv. Heterocycl Chem (2006) 61:1; Zakhs et al. Khim. Geterotsikl. Soedin (1990) 552; Chem. Abstr (1990) 113: 211247; Zakha et al. Khim Geterotsikl (1987) 386: Chem. Abstr. (1988) 108:5938) 1 In the case of 4-hydroxy-6H-1,3-oxazin-6-ones (R1=Bn, Me and R2=Ph) Sheibani et al were able to detect the tautomer B along with the major tautomer A by NMR spectroscopy. (Sheibani et al. ARKIVOC (2005) (xv), 88).

[0101] The .sup.1H and .sup.13C NMR spectra of the herein described 4-hydroxy-2-(phenylethynyl)-6H-1,3-oxazin-6-ones (8a-f), exhibited only signals consistent with the tautomer A, which in the case of 8d could unambiguously be proven by X-ray crystal structure analysis.

[0102] Prepared 4-hydroxy-2-(phenylethynyl)-6H-1,3-oxazin-6-ones 8a-f have been proved to be unstable in dimethyl sulfoxide solution. The NMR spectra of samples of 8a-f that had been kept in dimethyl sulfoxide-d6 solution for 24 hours at ambient temperature showed in addition to the signals for 1,3-oxazin-6-ones 8a-f, an additional set of signals belonging to hydrolysis products 9a-f (FIG. 2). In the case of 8e the intermediate 9e underwent spontaneous decarboxylation giving imide 10 as the final product (FIG. 2); imide 10 was also obtained on treatment of 4-hydroxy-5-phenyl-2-(phenylethynyl)-6H-1,3-oxazin-6-one (8e) with boiling water. The signals of isolated imide 10 are identical to additional signals in the NMR spectra of 8e that has been kept in dimethyl sulfoxide-d6 solution According to these results, it can be concluded that 1,3-oxazin-6-ones 8a-f are hydrolyzed by traces of water in dimethyl sulfoxide solution (FIG. 2).

TABLE-US-00006 TABLE 2 One-Pot Synthesis of 4-Hydroxy-5-phenyl-2-(phenylethynyl)-6H- 1,3-oxazin-6-ones 8 .sup.a [00002] Product R Yield (%) 8a Me 74 8b Et 70 8c i-Pr 62 8d Bu 65 8e Ph 87 8f Bn 61 .sup.a Reaction conditions: Et.sub.2O, 0-5 C.

3-Phenylprop-2-ynamide (1a)

[0103] Prepared from ethyl phenylpropynoate (8.71 g, 8.26 mL, 50 mmol) according to the literature procedure as colorless crystals; (Struebing et al. Tetrahedron (2005) 61:11333) ] yield: 6.70 g (92%); mp 100-102 C.

[0104] IR (KBr): 3383 and 3179 (NH.sub.2), 2223 (CC), 1654 cm.sup.1 (CO).

[0105] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.18 (s, 1H, NH.sub.2), 7.71 (s, 1H, NH.sub.2), 7.57 (m, 2H, o-CH.sub.Ar), 7.51 (m, 1H, p-CH.sub.Ar), 7.46 (m, 2H, m-CH.sub.Ar).

[0106] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =153.9 (CO), 132.0 (o-CH.sub.Ar), 130.2 (p-CH.sub.Ar), 129.0 (m-CH.sub.Ar), 119.9 (i-C.sub.Ar), 84.2 (CC), 82.9 (CC).

[0107] MS (EI, 70 eV): m/z (%)=145 (64) [M].sup.+, 129 (100) [M-NH.sub.2].sup.+, 75 (13), no other peaks >10%.

[0108] Anal. Calcd for C.sub.9H.sub.7NO: C, 74.47; H, 4.86; N, 9.65. Found: C, 74.59; H, 4.84; N, 9.57.

3-Arylprop-2-ynamides (1b-e); General Procedure

[0109] The corresponding arylpropynoic acid (15 mmol) was heated under reflux with EtOH (60 mmol) in the presence of H.sub.2SO.sub.4 for 5 h. Excess EtOH was removed under reduced pressure and the residue was washed with H.sub.2O and sat. aq NaHCO.sub.3. The organic layer was separated and the aqueous layer was extracted with CHCl.sub.3 (350 mL). The combined organic layers were dried (MgSO.sub.4) and concentrated under reduced pressure to give ethyl 3-arylprop-2-ynoates as oily residues, which were subsequently dissolved in 25% aq NH.sub.3 (60 mmol) and stirred at r.t. for 24 h. The resulting solid products 1b-d were filtered off and recrystallized.

3-(2-Chlorophenyl)prop-2-ynamide (1b) (Mariella et al. Can J. Chem (1965) 43:2426; Unangst et al. J. Heterocycl Chem. (1973) 10: 399)

[0110] Colorless crystals; yield: 1.94 g (72%); mp 121-122 C. (EtOHH.sub.2O, 1:1).

[0111] IR (KBr): 3318 and 3162 (NH2), 2225 (CC), 1655 cm-1 (CO).

[0112] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.25 (s, 1H, NH.sub.2), 7.81 (s, 1H, NH.sub.2), 7.69 (m, 1H, H6), 7.62 (m, 1H, H3), 7.52 (m, 1H, H4), 7.43 (m, 1H, H5).

[0113] 13C NMR (100 MHz, DMSO-d6): =153.4 (CO), 135.1 (C2), 134.0 (C6), 131.6 (C4), 129.5 (C3), 127.5 (C5), 119.8 (C1), 84.5 (CC), 79.2 (CC).

[0114] MS (EI, 70 eV): m/z (%)=181/179 (13/46) [M].sup.+, 165/163 (34/100) [M-NH.sub.2].sup.+, 136 (10), 99 (16), 75 (13), 74 (15), no other peaks >10%.

[0115] Anal. Calcd for C.sub.9H.sub.6ClNO: C, 60.19; H, 3.37; Cl, 19.74; N, 7.80. Found: C, 60.07; H, 3.38; Cl, 19.75; N, 7.87.

3-(4-Chlorophenyl)prop-2-ynamide (1c) (Schmitt J. FR 1305340 (1962); Chem Abstr. (1963) 58:46538)

[0116] Colorless crystals; yield: 2.10 g (78%); mp 186-188 C. (MeCN).

[0117] IR (KBr): 3399 and 3172 (NH.sub.2), 2218 (CC), 1656 cm.sup.1 (CO).

[0118] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.21 (s, 1H, NH.sub.2), 7.75 (s, 1H, NH.sub.2), 7.60 (m, 2H, o-CH.sub.Ar), 7.54 (m, 2H, m-CH.sub.Ar).

[0119] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =153.7 (CO), 135.0 (p-ClC.sub.Ar), 133.8 (o-CH.sub.Ar), 129.1 (m-CH.sub.Ar), 118.8 (i-C.sub.Ar), 85.0 (CC), 81.6 (CC).

[0120] MS (EI, 70 eV): m/z (%)=181/179 (17/53) [M].sup.+, 165/163 (32/100) [M-NH.sub.2].sup.+, 99 (15), 75 (13), no other peaks >10%.

[0121] Anal. Calcd for C.sub.9H.sub.6ClNO: C, 60.19; H, 3.37; Cl, 19.74; N, 7.80. Found: C, 60.29; H, 3.37; Cl, 19.64; N, 7.92.

3-(4-Bromophenyl)prop-2-ynamide (1d)

[0122] Colorless crystals; yield: 2.55 g (76%); mp 202-204 C. (dec) (MeCN).

[0123] IR (KBr): 3386 and 3172 (NH.sub.2), 2216 (CC), 1655 cm.sup.1 (CO).

[0124] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.23 (s, 1H, NH.sub.2), 7.75 (s, 1H, NH.sub.2), 7.68 (m, 2H, o-CH.sub.Ar), 7.52 (m, 2H, m-CH.sub.Ar).

[0125] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =153.6 (CO), 133.9 (o-CH.sub.Ar), 132.0 (m-CH.sub.Ar), 123.7 (p-BrC.sub.Ar), 119.1 (i-C.sub.Ar), 85.1 (CC), 81.6 (CC).

[0126] MS (EI, 70 eV): m/z (%)=225/223 (64/65) [M].sup.+, 209/207 (95/100) [M-NH.sub.2].sup.+, 128 (21) [M-NH.sub.2Br].sup.+, 99 (15), 75 (29), 74 (50), no other peaks >10%.

[0127] Anal. Calcd for C.sub.9H.sub.6BrNO: C, 48.25; H, 2.70; Br, 35.66; N, 6.25. Found: C, 48.04; H, 2.83; N, 6.24.

3-(4-Nitrophenyl)prop-2-ynamide (1e)

[0128] Colorless crystals; yield: 1.91 g (67%); mp 192-194 C. (dec) (MeCN).

[0129] IR (KBr): 3406 and 3155 (NH.sub.2), 2220 (CC), 1662 cm.sup.1 (CO).

[0130] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.34 (s, 1H, NH.sub.2), 7.88 (s, 1H, NH.sub.2), 7.30 (m, 2H, o-CH.sub.Ar), 7.85 (m, 2H, m-CH.sub.Ar).

[0131] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =153.2 (CO), 147.8 (p-NO.sub.2C.sub.Ar), 133.3 (o-CH.sub.Ar), 126.6 (i-C.sub.Ar), 124.0 (m-CH.sub.Ar), 87.8 (CC), 80.6 (CC).

[0132] MS (EI, 70 eV): m/z (%)=190 (84) [M].sup.+, 174 (100) [M-NH.sub.2].sup.+, 128 (70) [M-NH.sub.2NO.sub.2].sup.+, 116 (26), 101 (16), 100 (28), 98 (13), 89 (50), 77 (15), 75 (30), 74 (54), 63 (19), 62 (20), 51 (23), 50 (23), no other peaks >10%.

[0133] Anal. Calcd for C.sub.9H.sub.6N.sub.2O.sub.3: C, 56.85; H, 3.18; N, 14.73. Found: C, 56.64; H, 3.24; N, 14.59.

Monosubstituted Malonyl Chlorides (6a-f); General Procedure

[0134] Monosubstituted malonyl chlorides (6a-f) were obtained by refluxing the corresponding malonic acid with SOCl.sub.2 and subsequent distillation. In the case of phenyl-6e and benzylmalonyl chloride 6f the corresponding (chlorocarbonyl)ethylketenes 7a,b were formed as a byproduct, which is in accordance with literature reports. (Nakanishi et al. Org Prep Proced. Int. (1975) 7:155; Friedrichsen et al. Naturforsch. B. Chem Sci (1982) 37:222; Chem. Abstr (1982) 96:199624).

2-(Phenylethynyl)-6H-1,3-oxazin-6-ones (8a-f); General Procedure

[0135] The corresponding malonyl chloride [(chlorocarbonyl)ethylketene] 6a-f (7a,b) (3.1 mmol) was added to a stirred soln of 3-phenylprop-2-ynamide (1a, 0.44 g, 3 mmol) in anhyd Et.sub.2O (30 mL) at r.t. The mixture was cooled in the refrigerator for 12-14 h. During this time a solid deposited that was filtered off, washed with Et.sub.2O, and dried to deliver pure 8a-f.

[0136] For an additional portion of product the filtrate was evaporated under reduced pressure at r.t. and the remaining residue was recrystallized (MeCN).

4-Hydroxy-5-methyl-2-(phenylethynyl)-6H-1,3-oxazin-6-one (8a) (Also Referred to as Compound PG-3a)

[0137] Yellowish crystals; yield: 0.32 g (74%); mp 184-186 C.

[0138] IR (KBr): 2215 (CC), 1747 (CO), 1635 cm.sup.1 (CN).

[0139] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =12.74 (br s, 1H, OH), 7.71 (m, 2H, o-CH.sub.Ar), 7.61 (m, 1H, p-CH.sub.Ar), 7.52 (m, 2H, m-CH.sub.Ar), 1.80 (s, 3H, CH.sub.3).

[0140] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =164.8 (C6), 161.3 (C4), 146.9 (C2), 132.7 (o-CH.sub.Ar), 131.5 (p-CH.sub.Ar), 129.2 (m-CH.sub.Ar), 118.4 (i-C.sub.Ar), 92.8 (C5), 91.2 (C2), 80.7 (C1), 8.1 (CH.sub.3).

[0141] MS (EI, 70 eV): m/z (%)=227 (14) [M].sup.+, 171 (20), 130 (11), 129 (100) [PhCCCO].sup.+, 128 (12), 83 (14), 77 (13) [Ph].sup.+, 75 (29), 74 (14), 70 (12), 63 (11), no other peaks >10%.

[0142] Anal. Calcd for C.sub.13H.sub.9NO.sub.3: C, 68.72; H, 3.99; N, 6.16. Found: C, 68.72; H, 4.01; N, 6.26.

5-Ethyl-4-hydroxy-2-(phenylethynyl)-6H-1,3-oxazin-6-one (8b) (Also Referred to as Compound PG-3b)

[0143] Yellowish crystals; yield: 0.51 g (70%); mp 177-178 C.

[0144] IR (KBr): 2221 (CC), 1744 (CO), 1633 cm.sup.1 (CN).

[0145] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =12.77 (br s, 1H, OH), 7.71 (m, 2H, o-CH.sub.Ar), 7.61 (m, 1H, p-CH.sub.Ar), 7.53 (m, 2H, m-CH.sub.Ar), 2.29 (q, .sup.3J.sub.HH=7.42 Hz, 2H, CH.sub.2), 1.01 (t, .sup.3J.sub.HH=7.42 Hz, 3H, CH.sub.3).

[0146] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =164.6 (C6), 160.8 (C4), 147.2 (C2), 132.7 (o-CH.sub.Ar), 131.7 (p-CH.sub.Ar), 129.1 (m-CH.sub.Ar), 118.4 (i-C.sub.Ar), 98.6 (C5), 91.1 (C2), 80.6 (C1), 16.1 (CH.sub.2), 12.0 (CH.sub.3).

[0147] MS (EI, 70 eV): m/z (%)=241 (28) [M].sup.+, 129 (100) [PhCCCO].sup.+, 128 (19), 75 (15), 51 (10), no other peaks >10%.

[0148] Anal. Calcd for C.sub.14H.sub.11NO.sub.3: C, 69.70; H, 4.60; N, 5.81. Found: C, 69.42; H, 4.53; N, 5.84.

4-Hydroxy-5-isopropyl-2-(phenylethynyl)-6H-1,3-oxazin-6-one (8c) (Also Referred to as Compound PG-3c)

[0149] Colorless crystals; yield: 0.47 g (62%); mp 202-203 C.

[0150] IR (KBr): 2226 (CC), 1744 (CO), 1627 cm.sup.1 (CN).

[0151] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =12.76 (br s, 1H, OH), 7.70 (m, 2H, o-CH.sub.Ar), 7.61 (m, 1H, p-CH.sub.Ar), 7.52 (m, 2H, m-CH.sub.Ar), 3.01 (sept, .sup.3J.sub.HH=7.00 Hz, 1H, CH), 1.17 (d, .sup.3J.sub.HH=7.00 Hz, 6H, CH.sub.3).

[0152] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =164.4 (C6), 160.0 (C4), 147.4 (C2), 132.8 (o-CH.sub.Ar), 131.6 (p-CH.sub.Ar), 129.2 (m-CH.sub.Ar), 118.5 (i-C.sub.Ar), 101.9 (C5), 91.2 (C2), 80.6 (C1), 23.7 (>CH), 19.5 (CH.sub.3).

[0153] MS (EI, 70 eV): m/z (%)=255 (35) [M].sup.+, 240 (54) [M-CH.sub.3].sup.+, 172 (11), 130 (10), 129 (100) [PhCCCO].sup.+, 128 (20), 75 (11), 69 (16), no other peaks >10%.

[0154] Anal. Calcd for C.sub.15H.sub.13NO.sub.3: C, 70.58; H, 5.13; N, 5.49. Found: C, 70.38; H, 5.32; N, 5.43.

5-Butyl-4-hydroxy-2-(phenylethynyl)-6H-1,3-oxazin-6-one (8d) (Also Referred to as Compound PG-3)

[0155] Yellow crystals; yield: 0.53 g (65%); mp 157-159 C.

[0156] IR (KBr): 2222 (CC), 1743 (CO), 1628 cm.sup.1 (CN).

[0157] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =12.71 (br s, 1H, OH), 7.70 (m, 2H, o-CH.sub.Ar), 7.61 (m, 1H, p-CH.sub.Ar), 7.52 (m, 2H, m-CH.sub.Ar), 2.28 (m, 2H, CH.sub.2), 1.42 (m, 2H, CH.sub.2), 1.29 (m, 2H, CH.sub.2), 0.88 (m, 3H, CH.sub.3).

[0158] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =164.9 (C6), 161.0 (C4), 147.2 (C2), 132.7 (o-CH.sub.Ar), 131.5 (p-CH.sub.Ar), 129.2 (m-CH.sub.Ar), 118.4 (i-C.sub.Ar), 97.3 (C5), 91.2 (C2), 80.7 (C1), 29.3 (C2), 22.3 (C1), 21.9 (C3), 13.7 (C4).

[0159] MS (EI, 70 eV): m/z (%)=269 (10) [M].sup.+, 226 (18) [M-C.sub.3H.sub.7].sup.+, 165 (14), 130 (10), 129 (100) [PhCCCO].sup.+, 128 (20), no other peaks >10%.

[0160] Anal. Calcd for C.sub.16H.sub.15NO.sub.3: C, 71.36; H, 5.61; N, 5.20. Found: C, 70.97; H, 5.46; N, 5.14.

4-Hydroxy-5-phenyl-2-(phenylethynyl)-6H-1,3-oxazin-6-one (8e) (Komarov et al. Russ. J. Gen Chem (2005) 75:770) (Also Referred to as Compound PG-3e)

[0161] Yellow crystals; yield: 0.76 g (87%); mp 187-189 C.

[0162] IR (KBr): 2220 (CC), 1747 (CO), 1620 cm.sup.1 (CN).

[0163] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =13.18 (br s, 1H, OH), 7.74 (m, 2H, o-CH.sub.Ar), 7.63 (m, 1H, p-CH.sub.Ar), 7.54 (m, 2H, m-CH.sub.Ar), 7.52 (m, 2H, H2), 7.38 (m, 2H, H3), 7.29 (m, 1H, H4).

[0164] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =164.8 (C6), 160.0 (C4), 147.9 (C2), 132.8 (o-CH.sub.Ar), 131.7 (p-CH.sub.Ar), 130.6 (C1), 130.0 (C2), 129.2 (m-CH.sub.Ar), 127.6 (C3), 127.2 (C4), 118.3 (i-C.sub.Ar), 97.3 (C5), 91.8 (C2), 80.8 (C1).

[0165] MS (EI, 70 eV): m/z (%)=289 (35) [M].sup.+, 218 (14), 190 (12), 172 (12), 130 (10), 129 (100) [PhCCCO].sup.+, 118 (18), 89 (16), 77 (11) [Ph].sup.+, 75 (13), 51 (10) [C.sub.4H.sub.3].sup.+, no other peaks >10%.

[0166] Anal. Calcd for C.sub.18H.sub.11NO.sub.3: C, 74.73; H, 3.83; N, 4.84. Found: C, 74.79; H, 4.01; N, 4.75.

5-Benzyl-4-hydroxy-2-(phenylethynyl)-6H-1,3-oxazin-6-one (8f) (Also Referred to as PG-3d)

[0167] Yellow crystals; yield: 0.55 g (61%); mp 179-181 C.

[0168] IR (KBr): 2221 (CC), 1746 (CO), 1627 cm.sup.1 (CN).

[0169] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =13.03 (br s, 1H, OH), 7.71 (m, 2H, o-CH.sub.Ar), 7.60 (m, 1H, p-CH.sub.Ar), 7.52 (m, 2H, m-CH.sub.Ar), 7.25 (m, 4H, H2, H3), 7.17 (m, 1H, H4), 3.61 (s, 2H, CH.sub.2).

[0170] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =165.4 (C6), 161.0 (C4), 147.7 (C2), 139.3 (C1), 132.7 (o-CH.sub.Ar), 131.6 (p-CH.sub.Ar), 129.2 (m-CH.sub.Ar), 128.21 and 128.18 (C2 and C3), 126.0 (C4), 118.4 (i-C.sub.Ar), 96.5 (C5), 91.5 (C2), 80.7 (C1), 28.3 (CH.sub.2).

[0171] MS (EI, 70 eV): m/z (%)=302 (55) [M].sup.+, 176 (28), 158 (74), 131 (15), 130 (65), 129 (100) [PhCCCO].sup.+, 128 (20), 103 (12), 102 (15), 91 (27) [PhCH.sub.2].sup.+, 77 (22) [Ph].sup.+, 75 (13), 51 (12) [C.sub.4H.sub.3].sup.+, no other peaks >10%.

[0172] Anal. Calcd for C.sub.19H.sub.13NO.sub.3: C, 75.24; H, 4.32; N, 4.62. Found: C, 75.27; H, 4.32; N, 4.65.

2-[(4-Chlorophenyl)ethynyl]-4-hydroxy-5-phenyl-6H-1,3-oxazin-6-one (Also Referred to as PG-3g)

[0173] Yellow crystals; yield: 0.87 g (90%); mp 185-187 C. (dec).

[0174] IR (KBr): 2220 (CC), 1767 (CO), 1620 cm.sup.1 (CN).

[0175] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =13.16 (br s, 1H, OH), 7.77 (m, 2H, o-CH.sub.Ar), 7.62 (m, 2H, m-CH.sub.Ar), 7.52 (m, 2H, H2), 7.37 (m, 2H, H3), 7.23 (m, 1H, H4).

[0176] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =164.7 (C6), 159.9 (C4), 147.6 (C2), 136.6 (p-ClC.sub.Ar), 134.5 (o-CH.sub.Ar), 130.5 (C1), 130.0 (C2), 129.4 (m-CH.sub.Ar), 127.5 (C3), 127.2 (C4), 117.2 (i-C.sub.Ar), 97.4 (C5), 90.3 (C2), 81.5 (C1).

[0177] MS (EI, 70 eV): m/z (%)=325/324/323 (15/9/47) [M].sup.+, 254/252 (9/22), 225 (10), 224 (22), 206 (16), 165/163 (32/100) [4-ClC.sub.6H.sub.4CCCO].sup.+, 118 (28), 99 (18), 90 (17), 89 (21), 77 (12) [Ph].sup.+, no other peaks >10%.

[0178] Anal. Calcd for C18H10ClNO3: C, 66.78; H, 3.11; N, 4.33. Found: C, 66.89; H, 3.10; N, 4.39.

2-[(2-Chlorophenyl)ethynyl]-4-hydroxy-5-methyl-6H-1,3-oxazin-6-one (Also Referred to as PG-3f)

[0179] Yellowish crystals; yield: 0.46 g (59%); mp 175-178 C.

[0180] IR (KBr): 2225 (CC), 1747 (CO), 1627 cm.sup.1 (CN).

[0181] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =12.83 (br s, 1H, OH), 7.83 (m, 1H, H6), 7.69 (m, 1H, H3), 7.62 (m, 1H, H4), 7.50 (m, 1H, H5), 1.80 (s, 3H, CH.sub.3).

[0182] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =165.0 (C6), 161.3 (C4), 146.6 (C2), 135.8 (C2), 134.9 (C6), 133.1 (C4), 129.9 (C3), 127.8 (C5), 118.6 (C1), 92.8 (C5), 86.9 (C2), 84.9 (C1), 8.3 (CH.sub.3).

[0183] MS (EI, 70 eV): m/z (%)=263/262/261 (9/4/26) [M].sup.+.

[0184] Anal. Calcd for C13H8ClNO3: C, 59.67; H, 3.08; N, 5.35.

3-Oxo-3-[(3-phenylprop-2-ynoyl)amino]propanoic Acids (9a-f); General Procedure

[0185] After keeping a soln of compounds 8a-f in DMSO-d.sub.6 for 24 h at r.t. the .sup.1H and .sup.13C NMR spectra of the 4-hydroxy-2-(phenylethynyl)-6H-1,3-oxazin-6-ones 8a-f showed new signals of the corresponding hydrolysis products 9a-d,f and 10 (as shown in FIG. 2).

2-Methyl-3-oxo-3-[(3-phenylprop-2-ynoyl)amino]propanoic Acid (9a)

[0186] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =12.79 (br s, 1H, OH), 11.62 (s, 1H, NH), 7.66 (m, 2H, o-CH.sub.Ar), 7.58 (m, 1H, p-CH.sub.Ar), 7.50 (m, 2H, m-CH.sub.Ar), 3.79 (q, .sup.3J.sub.HH=7.15 Hz, 1H, H2), 1.27 (t, .sup.3J.sub.HH=7.15 Hz, 3H, CH.sub.3).

[0187] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =171.3 (C1), 170.1 (C3), 151.5 (C5), 132.6 (o-CH.sub.Ar), 131.1 (p-CH.sub.Ar), 129.0 (m-CH.sub.Ar), 119.0 (i-C.sub.Ar), 88.9 (C7), 83.2 (C6), 47.2 (C2), 13.1 (CH.sub.3).

2-[(3-Phenylprop-2-ynoyl)carbamoyl]butanoic Acid (9b)

[0188] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =12.84 (br s, 1H, OH), 11.65 (s, 1H, NH), 7.66 (m, 2H, o-CH.sub.Ar), 7.58 (m, 1H, p-CH.sub.Ar), 7.50 (m, 2H, m-CH.sub.Ar), 3.61 (m, 1H, H2), 1.80 (m, 2H, CH.sub.2), 0.90 (m, 3H, CH.sub.3).

[0189] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =170.3 (C1), 169.2 (C3), 151.5 (C5), 132.7 (o-CH.sub.Ar), 131.2 (p-CH.sub.Ar), 129.1 (m-CH.sub.Ar), 119.0 (i-C.sub.Ar), 89.0 (C7), 83.1 (C6), 54.2 (C2), 21.4 (CH.sub.2), 11.8 (CH.sub.3).

3-Methyl-2-[(3-phenylprop-2-ynoyl)carbamoyl]butanoic Acid (9c)

[0190] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =12.74 (br s, 1H, OH), 11.60 (s, 1H, NH), 7.66 (m, 2H, o-CH.sub.Ar), 7.58 (m, 1H, p-CH.sub.Ar), 7.51 (m, 2H, m-CH.sub.Ar), 3.46 (m, 1H, H2), 2.28 (m, 1H, CH), 0.97 (m, 3H, CH.sub.3).

[0191] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =169.6 (C1), 168.4 (C3), 151.4 (C5), 132.6 (o-CH.sub.Ar), 131.2 (p-CH.sub.Ar), 129.0 (m-CH.sub.Ar), 119.0 (i-C.sub.Ar), 89.1 (C7), 83.1 (C6), 59.3 (C2), 28.0 (CH), 20.2 (CH.sub.3), 20.0 (CH.sub.3).

2-[(3-Phenylprop-2-ynoyl)carbamoyl]hexanoic Acid (9d)

[0192] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =12.82 (br s, 1H, OH), 11.62 (s, 1H, NH), 7.66 (m, 2H, o-CH.sub.Ar), 7.58 (m, 1H, p-CH.sub.Ar), 7.50 (m, 2H, m-CH.sub.Ar), 3.67 (m, 1H, H2), 1.78 (m, 2H, H1), 1.28 (m, 4H, H2, H3), 0.87 (m, 3H, H4).

[0193] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =170.4 (C1), 169.2 (C3), 151.5 (C5), 132.6 (o-CH.sub.Ar), 131.2 (p-CH.sub.Ar), 129.0 (m-CH.sub.Ar), 119.0 (i-C.sub.Ar), 89.0 (C7), 83.1 (C6), 52.7 (C2), 29.1 (C3), 27.7 (C1), 21.9 (C2), 13.7 (C4).

2-Benzyl-3-oxo-3-[(3-phenylprop-2-ynoyl)amino]propanoic Acid (9f)

[0194] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =13.03 (br s, 1H, OH), 11.62 (s, 1H, NH), 7.65 (m, 2H, o-CH.sub.Ar), 7.59 (m, 1H, p-CH.sub.Ar), 7.52 (m, 2H, m-CH.sub.Ar), 7.25 (m, 5H, H2, H3, H4), 4.07 (t, .sup.3J.sub.HH=7.46 Hz, 1H, H2), 3.12 (d, .sup.3J.sub.HH=7.46 Hz, 2H, CH.sub.2).

[0195] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =169.7 (C1), 168.5 (C3), 151.4 (C5), 138.4 (C1), 132.6 (o-CH.sub.Ar), 131.2 (p-CH.sub.Ar), 129.0 (m-CH.sub.Ar), 128.7 (C2), 128.3 (C3), 126.4 (C4), 118.9 (i-C.sub.Ar), 89.1 (C7), 83.0 (C6), 54.4 (C2), 33.6 (CH.sub.2).

3-Phenyl-N-(phenylacetyl)prop-2-ynamide (10) (Also Referred to as Compound PG-3h)

[0196] A suspension of 4-hydroxy-5-phenyl-2-(phenylethynyl)-6H-1,3-oxazin-6-one (8e, 0.3 mmol) in H.sub.2O (20 mL) was heated under reflux for 30 min until the color of 8e turned from yellow to white. The solid compound 10 was filtered off and recrystallized (toluene) to give colorless crystals; yield: 71 mg (90%); mp 132-134 C.

[0197] IR (KBr): 3219 (NH), 2213 (CC), 1705 (CO), 1683 cm.sup.1 (CO).

[0198] .sup.1H NMR (400 MHz, DMSO-d.sub.6): =11.60 (s, 1H, NH), 7.64 (m, 2H, o-CH.sub.Ar), 7.56 (m, 1H, p-CH.sub.Ar), 7.49 (m, 2H, m-CH.sub.Ar), 7.33 (m, 2H, H3), 7.27 (m, 3H, H2, H4), 3.82 (s, 2H, CH.sub.2).

[0199] .sup.13C NMR (100 MHz, DMSO-d.sub.6): =170.7 (CO), 151.6 (C1), 134.3 (C1), 132.6 (o-CH.sub.Ar), 131.1 (p-CH.sub.Ar), 129.5 (C3), 129.0 (m-CH.sub.Ar), 128.3 (C2), 126.8 (C4), 119.1 (i-C.sub.Ar), 89.0 (C3), 83.3 (C2), 43.1 (CH.sub.2).

[0200] MS (EI, 70 eV): m/z (%)=263 (26) [M].sup.+, 147 (18), 251 (15), 130 (10), 129 (94) [PhCCCO].sup.+, 118 (100), 117 (12), 105 (15), 91 (30) [PhCH.sub.2].sup.+, 90 (22), 75 (13), 65 (11), no other peaks >10%.

[0201] Anal. Calcd for C.sub.17H.sub.13NO.sub.2: C, 77.55; H, 4.98; N, 5.32. Found: C, 77.49; H, 5.07; N, 5.25.

EXAMPLE 1

PG3 Compounds Inhibit Caspase-6 as Measured by the FRET Assay

[0202] FIG. 3 shows the compounds (referred to as PG3a-h) that were tested in dose-response curves with the FRET assay. As shown in FIG. 4A-C, the presence of increasing concentrations of several of the PG3 analogs resulted in increased inhibition of the recombinant caspase-6 enzyme in a cell-free system. Table 3 shows the calculated IC.sub.50 values for the inhibition of the caspase-6 enzyme as measured by the FRET assay.

TABLE-US-00007 TABLE 3 The PG3 compound and its analogs (PG-3a-h) have inhibitory effects on recombinant caspase-6 Compound FRET IC.sub.50 [M] PG-3 13.8 PG-3a 8.2 PG-3b 8.0 PG-3c 12.2 PG-3d 5.6 PG-3e 3.9 PG-3f 57.3 PG-3g 7.5 PG-3h 8.1

EXAMPLE 2

PG3 Compounds Inhibit Intracellular Caspase-6 as Measured by Western Blot

[0203] COS-7 cells were co-transfected with the 4c htt fragment and the human caspase-6 lacking the pro-domain which leads to fast autoactivation of the caspase-6 enzyme and the generation of the 586 aa Htt cleavage fragment. As shown in FIG. 5B, the co-transfected cells were exposed to either 10 um of the PG-3d compound, 3 uM of the Q-VD-Oph pan-caspase inhibitor or were left untreated. Non-transfected cells were included as a negative control and the purified 586 aa Htt fragment was included as a positive control. Cells that were exposed to the PG-3d compound show a significant reduction in the generation of the 586AA fragment. The cell lysates were also assayed for the levels of active caspase 6 enzyme as shown in the lower panel of FIG. 5B. The presence of the PG-3d compound resulted in a decreased amount of active enzyme which was also achieved in the presence of pan-caspase inhibitor. FIG. 5A shows a graphical representation from the Western blot, and indicated that the presence of PG-3d resulted in a reduced production of the 586 aa fragment.

EXAMPLE 3

PG3 Compounds Inhibit Intracellular Caspase-6 as Measured by Lamin Cleavage

[0204] The PG3 analog compounds were also tested for their ability to inhibit caspase-6 within cultured cells. HEK 293 cells were transfected with human caspase 6 lacking the pro-domain and were incubated with increasing concentrations of the PG3 analogs. After a 24 hr incubation, the excess compound was washed away, the cells were lysed and the amount of cleaved lamin A protein was quantified by the Mesoscale ELISA method. As shown in FIGS. 6A and 6B, several of the PG3 compounds demonstrated a dose-dependent ability to inhibit the caspase-6-mediated cleavage of lamin. Table 4 shows the calculated IC.sub.50 values for the intracellular inhibition of caspase-6 for the PG3 analogs.

TABLE-US-00008 TABLE 4 The PG3 analogs have an inhibitory effect on caspase-6 within cultured cells Compound HEK intracellular IC.sub.50 [M] PG-3 n.a. PG-3a >15 PG-3b 12.1 PG-3c >15 PG-3d 2.6 PG-3e 2.5 PG-3f n.a. PG-3g 3.9 PG-3h 4.8

EXAMPLE 4

PG3 Compounds Inhibit Neuronal Caspase-6 as Measured by Lamin Cleavage

[0205] Next, it was determined whether the PG-3d compound could inhibit caspase-6 within neuronal cells in an in vitro neuronal system which may be more relevant for the testing of therapeutics for neurodegeneration. Briefly, primary cortical neuronal cultures from FVB/N mice were treated with 10 uM camptothecin for 30 h starting at DIV10 in the presence or absence of 10 uM of the PG-3d compound. Camptothecin treatment leads to the activation of caspase-6 which was then quantified by the cleavage of lamin A. As shown in FIG. 7, the presence of the PG3d compound in the neuronal medium significantly reduces the level of caspase-6 activity as evidenced by the reduction in cleaved lamin.

EXAMPLE 5

PG3 Compounds Improve the Viability of Neuronal Cells During Excitotoxic Stress

[0206] It has been previously been shown in HD animal models that neuronal cells expressing mHTT have an increased vulnerability to excitotoxic stress (Graham et al. 2005. Neurobiol. Dis. 21:444-455) and that this may be mediated by caspase 6 (Graham et al. 2006. Cell 6(13):1179-1191 and Uribe et al HMG 2012; 21(9):1954-67). The link between NMDA-induced toxicity and other neurodegenerative diseases has been reviewed by Lipton et al., Nat Rev Drug Discovery 2006. 5:160-170. Therefore, the effect of the presence of the PG-3d compound on the ability of neuronal cells to survive during excitotoxic stress via its ability to inhibit the caspase-6 enzyme was investigated. As shown in FIG. 8, the presence of the PG-3d compound in the cell culture medium resulted in a significant improvement in cell viability in neuronal cells that were exposed to NMDA (25 uM or 50 uM).

EXAMPLE 6

PG3d Inhibits the Interaction Between Caspase-6 and Htt in Mammalian Cells

[0207] Cos-7 cells were co-transfected with Htt 1-1212AA (SEQ ID NO:3) and full-length human caspase-6 (SEQ ID NO:6), and exposed to either the pan-caspase inhibitor Q-VD-OPh, the PG3d compound or DMSO as a negative control. As shown in the upper panel of FIG. 9A, control cells that received DMSO alone (lanes labelled n.t.) show the presence of the Htt 1212AA protein and the Htt proteolytic cleavage fragments including the 586, 552 and 513 amino acid fragments. As expected, cells treated with the pan-caspase inhibitor Q-VD-OPh show a reduction in the amount of Htt cleavage fragments most markedly in the 513AA fragment derived from Caspase 3 activity. Cells treated with the PG3d compound show a reduction in the level of the 586AA fragment derived from caspase 6 activity (FIG. 9A, upper panel).

[0208] FIG. 9B shows the results following immunoprecipitaton with a caspase-6 antibody. As expected, the DMSO control lysates reveal a variety of Htt fragments within the immunocomplexes, including 513AA fragment generated by caspase-3 cleavage, the full-length 1212AA Htt and the 586AA fragment generated by caspase-6 cleavage. When the cells are treated with the pan-caspase inhibitor Q-VD-OPh, the amount of Htt cleavage fragments interacting with caspase-6 is reduced and it is evident that caspase-6 is primarily interacting with the 1212 AA Htt. Treatment with 10 uM PG3d significantly reduces the interaction between the 1212AA Htt and caspase-6 which also results in a reduced amount of co-immunoprecipitated Htt proteolytic fragments. These findings were analyzed by densitometric analysis of the immunoblot results in FIG. 9B and confirm that that presence of PG3d leads to a significant reduction in the amount of Htt 1212 AA that is co-immunoprecipitated using a Caspase-6 antibody as compared to the negative control DMSO-treated cells or cells treated with the Q-VD-OPh peptide inhibitor.

[0209] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.