Patent Application
20130029983
The present invention is directed to aminothienopyradazine (ATPZ) compounds and their use for treating tauopathies.
Inclusions comprised of fibrils of the microtubule (MT)-associated protein tau are found in the brains of those with Alzheimer's disease (AD), certain frontotemporal dementias and a host of additional neurodegenerative disorders that are broadly referred to as “tauopathies.” The pathology that is observed in these diseases is believed to result from the formation of toxic tau oligomers or fibrils, and/or from the loss of normal tau function due to its sequestration into insoluble deposits. Hence, small molecules that prevent tau oligomerization and/or fibrillization might have therapeutic value.
Despite the hypothesis that tau aggregation inhibitors may be therapeutically useful for AD and related tauopathies, to date only one compound of this type, methylene blue, has entered clinical trials. Numerous additional classes of compounds have been shown to inhibit tau assembly in vitro, but there are no reports of these molecules being evaluated in vivo for pharmacokinetic (PK) properties or efficacy in models of tauopathy. Because BBB permeability is known to be a major bottleneck that hampers the development of new CNS-active drugs, (Pardridge, W. M. The blood-brain barrier: bottleneck in brain drug development. NeuroRx 2005, 2, 3-14) an early evaluation of the brain penetration of candidate compounds is important, as such studies would permit focus on the most promising compound type.
Compounds and methods for the treatment of an amyloid disease or disorder in a patient are described. These methods comprise administering to the patient a therapeutically effective amount of a compound of formula I:
wherein:
The present invention is directed to methods of treating a tauopathy in a patient. Tauopathies are a class of neurodegenerative diseases resulting from the pathological aggregation of tau protein. Examples of tauopathies include, but are not limited to, frontotemporal dementia, Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal lobar degeneration (Pick's disease). In preferred embodiments, the tauopathy is Alzheimer's disease.
The invention is directed to methods of treating a tauopathy in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I:
wherein:
Also within the scope of the invention are methods of treating a tauopathy in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I, wherein:
Also within the scope of the invention are methods of treating a tauopathy in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I, wherein:
Also within the scope of the invention are methods of treating a tauopathy in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I, wherein:
In preferred embodiments, R1 is H. In other embodiments, R1 is C1-6alkyl, for example, —CH3. In other embodiments, R1 is —C(O)C1-6alkyl, for example, acetyl [—C(O)CH3]. In yet other embodiments, R1 is —C(O)aryl, for example, —C(O)-phenyl. In other embodiments, R1 is —C(O)alkaryl, for example, —C(O)CH2C6H5.
In other embodiments, R2 is H. In other embodiments, R2 is halogen. Preferred halogens for R2 are bromine and chlorine. In yet other embodiments, R2 is C1-6alkyl, for example, —CH3. In still other embodiments, R2 is C3-6cycloalkyl, for example, cyclopropyl.
In some embodiments, R3 is C1-6alkyl, for example, —CH3 or —CH2CH3.
In some embodiments, R3 is aryl, for example, phenyl.
In preferred embodiments, R3 is —C(O)—O—C1-6alkyl, for example, —C(O)—O—CH2CH3 or —C(O)—O—CH3.
In other preferred embodiments, R3 is —COOH.
In other embodiment, R3 is C1-6alkylene-OH.
In some embodiments, R3 is —NHC(O)OC1-6alkyl. Preferably R3 is —NHC(O)OCH2CH3 or —NHC(O)Ot-butyl. In other embodiments, R3 is —NHC(O)OC3-6cycloalkyl, for example, —NHC(O)Ocyclopropyl.
In other embodiments, R3 is —NH2.
In yet other embodiments of the invention, R3 is —C(O)NR3aR3b. Preferably, R3a and R3b are each H or R3a and R3b are each independently C1-6alkyl, for example —CH3, —CH2CH3, or isopropyl. In other embodiments R3a is C1-6alkyl, for example, —CH3, —CH2CH3, or isopropyl, and R3b is C3-6cycloalkyl, for example, cyclopropyl.
In other embodiments, R3a is H. In other embodiments, R3b is H and R3a is C1-6alkyl, for example, —CH3, —CH2CH3, and isopropyl. Also preferred is wherein R3b is H and R3a is C3-6cycloalkyl, for example cyclopropyl. Also preferred is wherein R3b is H and R3a is alkaryl, for example, benzyl. Also preferred is wherein R3b is H and R3a is —NH2. Also preferred is wherein R3b is H and R3a is —C1-6alkylene-OH, for example, —CH2CH2—OH.
In other embodiments, R3a and R3b, together with the nitrogen to which they are attached, form a 5-, 6-, or 7-membered heterocycloalkyl. Preferably, the heterocycloalkyl is piperidyl or morpholinyl.
In preferred embodiments, the compounds of formula I include one R4 substituent. In those embodiments, it is preferred that the R4 substituent is at the 4-position of the ring. In other embodiments, the R4 substituent is at the 3-position of the ring. In other embodiments, R4 substituent is at the 2-position of the ring.
In other embodiments, the compounds of formula I include two R4 substituents. In those embodiments, it is preferred that the R4 substituents are at the 2 and 3 positions of the ring. In other embodiments, the R4 substituents are at the 3 and 4 positions of the ring. In other embodiments, the R4 substituents are at the 2 and 4 positions of the ring.
A preferred substitutent for R4 is hydrogen. Other preferred substituents for R4 include halogen, for example, —F, —Cl, —Br, and —I, preferably, —F, —Cl, and —Br. Yet other preferred substituents for R4 are C1-6alkyl, for example, —CH3, —CH2CH3, and isopropyl. Other preferred substituents for R4 are C1-6alkyl substituted with 1-3 halogen, for example, —CF3. Other preferred substituents for R4 are C1-6alkoxy, for example, —OCH3. Other preferred substituents for R4 are C1-6alkoxy substituted with 1-3 halogen, for example, —OCF3.
Another preferred substituted for R4 is aryl, for example, phenyl.
Other preferred substituents for R4 are —C(O)O—C1-6alkyl, for example, —C(O)O—CH3, —C(O)O—CH2CH3, and —C(O)O-isopropyl.
Another preferred substituent for R4 is —OH. Yet another preferred substitutent for R4 is —NO2. Still another preferred substituent for R4 is —N3.
Still other preferred embodiments are where R4 is —NR4aR4b. In some embodiments, R4a and R4b are each H. In other embodiments, R4a and R4b are each independently C1-6alkyl, for example —CH3, —CH2CH3, and isopropyl. In other embodiments R4a is hydrogen and R4b is C1-6alkyl, for example, —CH2, —CH2CH3, and isopropyl.
Preferred compounds for use in the methods of the present invention are set forth in Table 1:
Preferred compounds for use in the methods of the present invention are set forth in Table 2:
The compounds set forth in Table 2 are also within the scope of the invention.
As used herein, the term “alkyl” refers to a straight-chain, or branched alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, neopentyl, 1-ethylpropyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, hexyl, octyl, etc. The alkyl moiety of alkyl-containing groups has the same meaning as alkyl defined above. A designation such as “C1-C6 alkyl” refers to straight-chain, or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, neopentyl, 1-ethylpropyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, hexyl, etc. Lower alkyl groups, which are preferred, are alkyl groups as defined above which contain 1 to 4 carbons. A designation such as “C1-C4 alkyl” refers to an alkyl radical containing from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl. A designation such as “C1-C3 alkyl” refers to an alkyl radical containing from 1 to 3 carbon atoms, such as methyl, ethyl, propyl, and isopropyl.
As used herein, the term “alkylene” refers to a divalent radical derived from an alkane, for example, —CH2CH2CH2—. A designation such as “C1-C6 alkylene” refers to straight-chain alkylene group having 1 to 6 carbon atoms, such as methylene, ethylene, propylene, butylene, and the like.
As used herein, the term “cycloalkyl” refers to a saturated or partially saturated mono- or bicyclic alkyl ring system containing 3 to 11 carbon atoms. Certain embodiments contain 3 to 10 carbon atoms, other embodiments contain 3 to 7 carbon atoms, other embodiments contain 3 to 6 carbon atoms, and other embodiments contain 5 or 6 carbon atoms. A designation such as “C3-C7 cycloalkyl” refers to a cycloalkyl radical containing from 3 to 7 ring carbon atoms. Examples of cycloalkyl groups include such groups as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, and cycloheptyl.
As used herein, the term “aryl” refers to a substituted or unsubstituted, mono- or bicyclic hydrocarbon aromatic ring system having 6 to 10 ring carbon atoms. Examples include phenyl and naphthyl. As used herein, the term “alkaryl” refers to an alkylene-aryl group, such as, for example, benzyl.
As used herein, the term “heteroaryl” refers to an aromatic group or ring system containing 5 to 10 ring carbon atoms in which one or more ring carbon atoms are replaced by at least one hetero atom such as O, N, or S. Certain embodiments include 5 or 6 membered rings. Examples of heteroaryl groups include pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxathiolyl, oxadiazolyl, triazolyl, oxatriazolyl, furazanyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, picolinyl, imidazopyridinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, isobenzofuranyl, purinyl, quinazolinyl, quinolyl, isoquinolyl, benzoimidazolyl, benzothiazolyl, benzothiophenyl, thianaphthenyl, benzoxazolyl, benzooxadiazolyl, benzisoxazolyl, cinnolinyl, phthalazinyl, naphthyridinyl, and quinoxalinyl. As used herein, the term “alkheteroaryl” refers to an alkylene-heteroaryl group, for example —CH2-heteroaryl.
As used herein, the term “heterocycloalkyl” refers to a cycloalkyl group in which one or more ring carbon atoms are replaced by at least one hetero atom such as O, N, S, SO, and SO2. Certain embodiments include 3 to 6 membered rings, and other embodiments include 5 or 6 membered rings. Examples of heterocycloalkyl groups include azetidinyl, 3H-benzooxazolyl, 1,1-dioxo-thiomorpholinyl, 1,4-diazapinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, pirazolidinyl, pirazolinyl, pyrazalinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, morpholinyl, thiomorpholinyl, dihydrobenzofuranyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrazolopyridinyl, tetrahydro-1,3a,7-triaza-azulenyl, dihydrooxazolyl, dithiolyl, oxathiolyl, dioxazolyl, oxathiazolyl, pyranyl, oxazinyl, oxathiazinyl, and oxadiazinyl. Included within the definition of “heterocycloalkyl” are fused ring systems, including, for example, ring systems in which an aromatic ring is fused to a heterocycloalkyl ring and ring systems in which a heteroaromatic ring is fused to a cycloalkyl ring or a heterocycloalkyl ring. Examples of such fused ring systems include, for example, 2,3-dihydrobenzofuran, 2,3-dihydro-1,3-benzoxazole, phthalamide, phthalic anhydride, indoline, isoindoline, tetrahydroisoquinoline, chroman, isochroman, chromene, and isochromene.
As used herein “halogen” refers to fluorine, chlorine, bromine, and iodine.
A “therapeutically effective amount” (or dose) is an amount that, upon administration to a patient, results in a discernible patient benefit (e.g., provides detectable relief from at least one condition being treated). It will be apparent that the discernible patient benefit may be apparent after administration of a single dose, or may become apparent following repeated administration of the therapeutically effective dose according to a predetermined regimen, depending upon the indication for which the compound is administered.
As used herein, “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
As used herein, “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. 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 carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. These physiologically acceptable salts are prepared by methods known in the art, e.g., by dissolving the free amine bases with an excess of the acid in aqueous alcohol, or neutralizing a free carboxylic acid with an alkali metal base such as a hydroxide, or with an amine.
As used herein, “patient” refers to animals, including mammals, preferably humans.
The terms “treatment” and “treating” as used herein include preventative (e.g., prophylactic), curative and/or palliative treatment.
Full length tau (Tau40; amino acids 1-441) (Goedert, M., Spillantini, M. G., Potier, M. C., Ulrich, J., and Crowther, R. A. (1989) Embo Journal 8, 393-399), tau K18 fragment containing the P301L missense mutation (Goedert, M. (2005) Movement Disorders 20, S45-S52) (K18PL; amino acids 244-369) (Gustke, N., Trinczek, B., Biernat, J., Mandelkow, E. M., and Mandelkow, E. (1994) Biochemistry 33, 9511-9522), and a non-fibrillizing K18 tau construct carrying the K311D missense mutation (K18KD) (Li, W. K. and Lee, V. M. Y. (2006) Biochemistry 45, 15692-15701) were cloned into the pRK172 expression vector, expressed and purified as previously described (Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszolek, Z., Reed, L., Miller, B. I., Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A., Morris, J. C., Wilhelmsen, K. C., Schellenberg, G. D., Trojanowski, J. Q., and Lee, V. M. Y. (1998) Science 282, 1914-1917). To eliminate batch-to-batch variations that might contribute to assay variability, K18PL from several preparations was pooled so that a single homogeneous preparation was used throughout qHTS.
K18PL and K18 KD were labeled with Alexa Red (Invitrogen) according to the manufacturer's protocol. Labeled protein was determined to contain 1 mol of dye per 3 mol protein, following the quantification method described in the manufacturer's instructions.
Tau fibril formation reactions were performed essentially as described (Crowe, A., Ballatore, C., Hyde, E., Trojanowski, J. Q., and Lee, V. M. Y. (2007) Biochemical and Biophysical Research Communications 358, 1-6). K18PL (2 μl of a 30 μM stock that also contained 0.24 μM Alexa Red-labeled K18PL) was dispensed into black 1536-well plates (Grenier) using a solenoid-based dispenser. Following transfer of 23 nL of test compound or DMSO vehicle by a pin tool from compound library plates, 2 μl/well of 40 μM heparin in reagent buffer was dispensed and the plate was centrifuged for 15 s at 1000 rpm in a table-top centrifuge. The fibrillization reaction proceeded for 6 hours at 37° C. in a humidified incubator (Kalypsys).
K18PL fibril formation was quantified with a ThT assay as previously described (Crowe, A., Ballatore, C., Hyde, E., Trojanowski, J. Q., and Lee, V. M. Y. (2007) Biochemical and Biophysical Research Communications 358, 1-6). Briefly, 1 μl/well of 62.5 μM ThT (Sigma) in 100 mM glycine, pH 8.5 (12.5 μM final concentration) was dispensed into 1536-well plates after completion of the fibrillization reaction and incubated at room temperature for 1 hour. Plates were read on an Envision fluorescence plate reader with an excitation of 450 nm and an emission setting of 510 nm.
A tau FP method was developed to quantify Alexa Red-labeled K18PL tau incorporation into multimeric tau assemblies based on a similar methodology that was previously employed to measure the oligomerization and fibrillization of α-synuclein (Luk, K. C., Hyde, E. G., Trojanowski, J. Q., and Lee, V. M. Y. (2007) Biochemistry 46, 12522-12529). Although Alexa Red-labeled K18PL tau preparations did not readily assemble into fibrils under the conditions described above, the fluorescently-labeled tau could be incorporated into nascent oligomers and fibrils with normal assembly kinetics when mixed with unlabeled K18PL at a 1:62.5 molar ratio. Under these conditions, a fluorescence polarization increase of >100 polarization units (mP) was obtained upon completion of the fibrillization reaction, as determined on an Envision fluorescence plate reader using a Texas Red FP dual mirror, Texas Red FP excitation filter at 555 nm, and Texas Red FP emission filter (P and S channel) at 632 nm. The detector gains for each emission filter were calibrated to obtain maximum mP values. FP values were calculated from the S and P channel readings according to the equation, mP=1000*(S−G*P)/(S+G*P), with the G value set to one. Total Alexa Red fluorescence emission was calculated using the equation TF=S+2*P.
Compound effects on tau assembly were determined for the ThT and FP assays using the formula 1−(Fcompound−
The determination of compound-induced effects on the amount of tau that remains soluble upon centrifugation, and EM visualization of tau species following incubation with test compounds, were as previously described (Crowe, A., Ballatore, C., Hyde, E., Trojanowski, J. Q., and Lee, V. M. Y. (2007) Biochemical and Biophysical Research Communications 358, 1-6).
The effect of compounds on tau-mediated MT assembly was essentially as described (Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszolek, Z., Reed, L., Miller, B. I., Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A., Morris, J. C., Wilhelmsen, K. C., Schellenberg, G. D., Trojanowski, J. Q., and Lee, V. M. Y. (1998) Science 282, 1914-1917), adapted to a 384-well plate. Wild-type K18 tau was used for MT assembly because K18PL has a decreased ability to promote MT assembly. Lyophilized bovine brain tubulin (Cytoskeleton Inc.) was reconstituted in RAB (100 mM MES, pH 6.9; 1 mM EDTA; 0.5 mM MgSO4) at a concentration of 10 mg/ml. Compounds (50 μM) were added to K18 tau (40 μM) in RAB and pre-incubated at room temperature for 60 minutes. To initiate the MT assembly reaction, 8.25 μl per well of 10 mg/ml tubulin was dispensed on a UV-clear 384-well plate (Corning #3675), followed by 1.0 μl of 100 mM GTP in RAB and 41.25 μl of the compound:tau mixture. This resulted in a final reaction mixture of 30 μM tubulin, 40 μM K18, 50 μM compound and 2 mM GTP. The plate was incubated in a Spectramax M5 plate reader at 37° C. and the absorbance at 340 nm was read every minute for 45 minutes.
Synthetic Aβ(1-42) peptide (rPeptide) was resuspended in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at a concentration of 5 mg/ml for 30 minutes and then air dried in small aliquots, followed by storage at −80° C. For fibrillization assays (46), HFIP-treated Aβ(1-42) aliquots were reconstituted to 2 mg/ml in DMSO and then diluted to 15 μM in 25 mM Tris, pH 7.0 buffer to which test compound was added at 50 μM final concentration, or at several concentrations ranging from 0.16-40 μM. The reaction mixtures were dispensed at 25 μl/well into a 384-well plate (NUNC #262260) and then incubated at 37° C. for 4 hrs. Upon completion of the reaction, 25 μl of 25 μM ThT was added to each well followed by ThT fluorescence readings as described above for K18PL.
Tau40 (50 μM) and heparin (20 μM) were incubated in a PCR tube with or without the compound 5b (100 μM) at 37° C. for 5 days in a final volume of 100 μl of 100 in M sodium acetate buffer, pH 7.0. In the absence of compound, tau40 fibrillization is complete under these conditions, as determined by ThT fluorescence and centrifugation assays. A non-assembly control reaction was also prepared as above with Tau40 and 5b in the absence of heparin. The completed reactions were centrifuged at 40,000 g for 30 minutes to sediment fibrils and the supernatants were applied to a Superdex 200 10/300 column (GE Healthcare) employing an Äkta Basic FPLC unit (GE Healthcare) with a flow rate of 0.4 ml/minute. Tau elution was monitored by absorbance at 280 nm. Under these chromatography conditions, monomeric tau can be separated from oligomeric species (Li, W. K. and Lee, V. M. Y. (2006) Biochemistry 45, 15692-15701).
The solubility of test compounds in the sodium acetate buffer used for subsequent fibrillization reactions was determined by turbidimetric measurements. Compounds were then evaluated in a heparin-induced tau assembly assay, in which the fibrillization of the truncated K18 tau fragment (comprised of four MT-binding repeats) bearing the P301L mutation (K18PL) found in FTDP-17, was monitored by thioflavine-T (ThT) binding and fluorescence (
Notably, compounds also inhibited the aggregation of full-length tau (tau40) as determined by sedimentation assay, which revealed efficacy values in the 50-60% range (cf., Table 5). Compounds E1, 17, 2, and F1-F7 did not appear to interfere with the normal MT-stabilizing function of tau in a MT-polymerization assay (examples shown in
Test compounds underwent preliminary evaluations of brain penetration, in which 5 mg/Kg of each compound was administered intraperitoneally (i.p.) to a group of three normal mice; drug levels in brain and plasma were determined at a single time-point (1 h) by LC-MS/MS using pre-validated calibration curves. The results of these experiments, summarized in Table 5, revealed that with the exception of CNDR-51348 (E1), all other test compounds displayed significant brain uptake as demonstrated by the brain-to-plasma exposure ratios (B/P) above 0.3. The lack of brain penetration of CNDR-51348 was not unexpected given that the carboxylic acid moiety of this compound would be mostly negatively charged at physiological pH, and thus likely result in limited passive diffusion of the compound across the BBB. Conversely, the more lipophilic ester derivatives (CNDR-51346 and CNDR-51371) exhibited comparatively higher B/P ratio. In particular, CNDR-51371 (2) was found to reach significantly higher brain concentrations compared to the corresponding acid CNDR-51348. However, both esters appeared to have relatively short half-lives in plasma, as indicated by the limited amount of parent drug detected after 1 h from administration of the compounds. Moreover, monitoring for the hydrolyzed metabolite (i.e., CNDR-51348) in both brain and plasma revealed a considerable amount of acid CNDR-51348 in plasma, but not in the brain 1 h after administration of either ester. CNDR-51362 was selected for full PK analysis in which brain and plasma drug levels were determined at six time points (i.e., 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h). The results from this experiment, illustrated in
Compounds were serially diluted in DMSO to achieve a 2.5 fold dilution series from 20 mM down 8 points to 3 μM on a 384-well master plate (Costar 3672). Aliquots (2 μL) were transferred to a clear 96-well assay plate (Fisher 12-565-501) containing 198 μL fibrillization buffer (100 mM NaOAc pH 7.0) for a 100-fold dilution (200 μM down to 0.3 μM). The plate was incubated with agitation for 2.5 h, and the absorbance at 550, 600, 650 and 700 nm was measured on a Spectramax M5 spectrophotometer. DMSO (1%) in fibrillization buffer served as a control that was subtracted from each data point. The absorbances were averaged and the concentration at which the average absorbance rose above 0.03 AU was an indication of insoluble compound, with solubility limits reported as the previous concentration in the dilution series. Absorbance scans of compounds in DMSO indicated that the compounds tested here did not have any absorbance peaks in the above range, therefore all absorbance was due to light scattering by insoluble material.
Cells were plated in each well of a 96-well plate in 100 μL of media (20,000 cells; DMEM plus 10% fetal calf serum with antibiotics). After 24 h, half-log serial dilutions of the test compounds, starting at 200 μM, were added in a further 100 μL of media. The cells were cultured for an additional 72 h and then Alamar blue (Biosource, Camarillo, Calif.) was added, with cell viability at each drug concentration measured using a SpectraMax M5 fluorescence plate-reader (Molecular Devices, Sunnyvale, Calif.). CC50 values were generated using Prism software.
Brain tissue was homogenized in 10 mM ammonium acetate, pH 5.7 (1:2; w/v) using a handheld sonic homogenizer. Plasma was obtained from blood that was collected into a 1.5 mL tube containing 0.5M EDTA solution and subjected to centrifugation for 10 min. at 4500 g at 4° C. Aliquots (50 μL) of brain homogenate or plasma were mixed with 0.2 mL of acetonitrile, centrifuged at 15,000 g, and the resulting supernatants were used for subsequent LC-MS/MS analysis. The LC-MS/MS system was comprised of an Aquity HPLC and a TQ MS that was controlled using MassLynx software (Waters Corporation, Milford, Mass., USA). Compounds were detected using multiple reaction monitoring (MRM) of their specific collision-induced ion transitions. Samples (5 μL) were separated on an Aquity BEH C18 column (1.7 μm, 2.1×50 mm) at 35° C. Operation was in positive electrospray ionization mode, with mobile phase A of 0.1% (v/v) formic acid, and B of either acetonitrile or methanol with 0.1% (v/v) formic acid at a flow rate of 0.6 mL/min using a gradient from 5% to 95% B over two min., followed by wash and re-equilibration steps. The MS was operated with a desolvation temperature of 450° C. and a source temperature of 150° C. Desolvation and source nitrogen gas flows were 900 L/hr and 50 L/hr, respectively. Source and MS/MS voltages were optimized for each compound using the MassLynx auto tune utility. Standard curves were generated for each compound from brain homogenate and plasma samples that had compound added at 4, 40, 400 and 4000 ng/mL and extracted as above. Peak areas were plotted against concentration and a 1/x weighted linear regression curve was used to quantify the tissue-derived samples using the average peak area from triplicate injections.
Compounds 16-19 were purchased from commercial sources. Compounds 5a-5h and 6a-b were synthesized (Scheme 1) following a modified literature procedure (Ferguson, G. N., Valant, C., Home, J., Figler, H., Flynn, B. L., Linden, J., Chalmers, D. K., Sexton, P. M., Christopoulos, A., and Scammells, P. J. (2008) Journal of Medicinal Chemistry 51, 6165-6172). Commercially available anilines 1a-h were converted to aryl diazonium salts 2, which reacted with β-ketoesters to form hydrazones 3 as a mixture of E/Z isomers. Condensation reactions of the hydrazones with ethyl cyanoacetate gave pyridazines 7. Subsequently reactions of pyridazines 7 with sulfur under Gewald conditions generated 5a-h. Saponification of 5a-b gave the corresponding 6a-b.
Compound 8d was synthesized by a DMC-mediated coupling reaction between acid 6b and diethyl amine. Molecules 8a-c were prepared by a reaction sequence starting from 7a as depicted in Scheme 2. Hydrolysis of 4a under acidic condition yielded 7a. Amide 7b was formed by reacting 4a with ammonium. Coupling reactions of acid 7a with appropriate amines followed by Gewald reaction yielded the desired amides 8a-c.
Compounds 9a-c were prepared by reactions of 5a-b with appropriate acid chlorides, Scheme 3.
The preparation of 14 is illustrated in Scheme 4. EDC-mediated coupling reaction of acid 7a with tert-butylcarbazate followed by Boc-deprotection gave hydrazide 10. The hydrazide reacted with nitrous acid in acetic acid produced carboazide 11. Curtius rearrangement of 11 in ethanol gave ethyl carbamate 13. Under basic condition, the carbamate was converted to the desired amine 14. Compound 15 was prepared by LiAlH4 reduction of ester 5a, Scheme 5.
The synthesis of compounds D1-D24, E1-E6, and F1-15 was achieved via the reaction sequence illustrated in Scheme 6, which entails (a) a diazonium coupling reaction to form hydrazones of general structure B; (b) a Knovenagel-type condensation between B and ethyl cyanoacetate to form pyridazines C;23, 24 and (c) a Gewald aminothiophene synthesis25 to furnish the desired ATPZs of general structure D. Compound 17 was then trans-esterified to the corresponding isopropyl ester, CNDR-51371 (2), upon treatment with Ti(iPrO)4 in isopropanol. Alternatively, compounds of general structure D were saponified, and the resulting acids E employed in series of BOP coupling reactions with various primary and secondary amines to furnish compounds F1-7.
Scheme 6. Reagents and Reaction conditions: a) (i) NaNO2, 37% HCl, ethanol, water, 0° C., 20 min; (ii) ethyl acetoacetate, sodium acetate, ethanol, water, 0° C., 2 h; b) ethyl cyanoacetate, ammonium acetate, acetic acid, 170° C. (microwave irradiation), 4 min; c) ethyl cyanoacetate, 4-aminobutyric acid, 160° C., 2.5 h; d) S8, morpholine, ethanol, 150° C. (microwave irradiation), 15 min; e) LiOH.H2O, tetrahydrofuran, water, rt, 16 h; f) appropriate amine, BOP reagent, N,N-diisopropylethylamine, dimethylsulfoxide, rt, 4 h.
The synthesis of CNDR-51360 was conducted by treating 17 with methyl iodide (Scheme 7).
The synthesis of CNDR-51411 and CNDR-51393 was conducted as depicted in Scheme 8, i.e., by treating 17 with either N-bromosuccinimide to form CNDR-51411, or with N-chlorosuccinimide to form CNDR-51393.
The synthesis of compounds of general structure h was conducted as depicted in Scheme 9, starting from compound 11.
All compounds underwent purity and mass determination using reverse-phase HPLC/MS (ESI+) method with elution monitoring by UV absorbance (220 nm) and evaporative light scatter (ELS). Analyses were performed at a flow rate of 0.5 ml/min on a Waters Acquity HPLC system using a Phenomenex 2.5 μm Luna C18(2)-HST 100×2 mm column at 45° C. A linear 2-100% acetonitrile (0.025% TFA)/H2O (0.05% TFA) gradient was utilized over 2.2 min, with a total run time of 3.0 min. Purity was assessed by integration of chromatograms (UV220 nm and ELSD).
All solvents were reagent grade. All reagents were purchased from Aldrich or Acros and used as received. Thin layer chromatography (TLC) was performed with 0.25 mm E. Merck pre-coated silica gel plates. Flash chromatography was performed with silica gel 60 (particle size 0.040-0.062 mm) supplied by Silicycle and Sorbent Technologies. TLC spots were detected by viewing under a UV light. Infrared (IR) spectra were recorded on a Jasco Model FT/IR-480 Plus spectrometer. All melting points were obtained on a Thomas-Hoover apparatus. Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker AMX-500 spectrometer. Chemical shifts were reported relative to solvents. High-resolution mass spectra were measured at the University of Pennsylvania Mass Spectrometry Center on either a VG Micromass 70/70H or VG ZAB-E spectrometer. Single-crystal X-ray structure determinations were performed at the University of Pennsylvania with an Enraf Nonius CAD-4 automated diffractometer. Analytical reverse-phased (Sunfire™ C18; 4.6×50 mm, 5 mL) high-performance liquid chromatography (HPLC) was performed with a Waters binary gradient module 2525 equipped with Waters 2996 PDA and Waters micromass ZQ. All samples were analyzed employing a linear gradient from 10% to 90% of acetonitrile in water over 8 minutes and flow rate of 1 mL/min, and unless otherwise stated, the purity level was >95%. Preparative reverse phase HPLC purifications were performed on a Gilson instrument (i.e., Gilson 333 pumps, a 215 liquid handler, 845Z injection module, and PDA detector) employing Waters SunFire™ preparative C18 OBD™ columns (5 μm 19×50 or 19×100 mm). Purifications were carried out employing a linear gradient from 10% to 90% of acetonitrile in water for 15 minutes with a flow rate of 20 mL/min. Yields refer to chromatographically and spectroscopically pure compounds.
Prepared as previously reported. (Nagakura, M. et al., J. Med. Chem, 1979, 22, 48-52) Yield: 83%, mp: 84-85° C. (from ethanol, Lit. 81-84° C. Id.). 1H NMR (CDCl3): δ 1.41 (t, J=7.0 Hz, 3H), 2.60 (s, 3H), 4.34 (q, J=7.2 Hz, 2H), 7.34-7.36 ppm (m, 4H). 13C NMR (CDCl3): δ 14.5, 30.9, 61.2, 117.6, 126.5, 129.8, 131.0, 140.4, 164.9, 197.4 ppm. IR: v 3359, 1706, 1617 cm−1. HRMS (ESI+): calculated for C12H13ClN2NaO3+ 291.0518 found 291.0512.
4-Bromoaniline (2.0 g, 11.63 mmol) was dissolved in 37% hydrochloridic acid (2.94 mL), ethanol (1.50 mL) and water (1.50 mL). The reaction mixture was cooled to 0° C. in an ice water bath before a solution of sodium nitrite (0.88 g, 12.75 mmol) in water (2.0 mL) was added dropwise. The resulting mixture was stirred at 0° C. for 20 min. Sodium acetate (3.72 g, 45.3 mmol) in water (5.9 mL) and ethyl acetoacetate (1.51 g, 1.43 mL, 11.63 mmol) were added and the reaction mixture was stirred at 0° C. for 2 h. The precipitated solid was then filtered, washed with water, and dried under high vacuum for 16 h to provide B2 as yellow solid that was crystallized in ethanol and used without further purification in the next step. Yield: 98%.
Prepared as previously reported. (Sotelo, E. et al., Synth Commun. 1997, 27, 2419-23) Yield: 47%, mp: 160-161° C. (from ethanol, Lit. 190° C. Id.). NMR (CDCl3): δ 1.41 (t, J=7.0 Hz, 3H), 2.76 (s, 3H), 4.43 (q, J=7.2 Hz, 2H), 7.48 (d, J=8.5 Hz, 2H). 7.62 ppm (d, J=8.5 Hz, 2H). 13C NMR (CDCl3): δ 14.2, 19.4, 63.0, 112.4, 116.0, 126.3, 129.4, 135.5, 137.5, 138.5, 150.8, 155.8, 162.0 ppm. IR: v 2235, 1726, 1685 cm−1. MS [ESI+]: calculated for C15H13ClN3O3+ 318.06 found 318.07.
A mixture of B2 (2.0 g, 6.39 mmol), 4-aminobutiryc acid (1.33 g, 12.99 mmol), and ethyl cyanoacetate (1.1 g, 1.0 mL, 9.44 mmol) was stirred neat at 160° C. for 2.5 h. After cooling the residue was purified by silica gel flash chromatography (using an ethyl acetate/n-hexane gradient of 5% to 80% of ethyl acetate) to provide C2 as white solid. Yield: 76%, mp: 174-176° C. (from ethanol). NMR (CDCl3): δ 1.41 (t, J=7.0 Hz, 3H), 2.76 (s, 3H), 4.43 (q, J=4.43 Hz, 7.2 Hz, 2H), 7.56 (d, J=9.0 Hz, 2H), 7.64 ppm (d, J=9.0 Hz, 2H). 13C NMR (CDCl3): δ 14.2, 19.4, 62.9, 112.4, 116.0, 123.5, 126.6, 132.3, 137.6, 139.1, 150.8, 155.7, 162.0 ppm. IR: v 2230, 1727, 1681 cm−1.
A mixture of C1 (0.100 g, 0.31 mmol), sulfur (0.015 g, 0.47 mmol), and morpholine (0.55 g, 0.55 mL, 0.63 mmol) was heated to 150° C. using microwave irradiation for 15 min. After cooling, the precipitate which formed was collected and purified by preparative HPLC to give 17 as yellow solid. HPLC-MS retention time 8.20 min. Yield: 64%, mp: 189-191° C. (from ethanol). 1H NMR (CDCl3): δ 1.43 (t, J=7.3 Hz, 3H), 4.44 (q, J=7.2 Hz, 2H), 6.21 (broad s, 2H), 7.25 (s, 1H), 7.42 (d, J=8.5 Hz, 2H), 7.56 ppm (d, J=8.5 Hz, 2H). 13C NMR (CDCl3): δ 14.4, 62.2, 104.9, 106.9, 127.1, 127.2, 128.9, 133.3, 134.0, 139.2, 159.4, 161.8, 163.0 ppm. IR: v 3411, 3303, 1724 cm−1. HRMS (ESI+): calculated for C15H13ClN3O3S+ 350.0366 found 350.0355.
Was synthesized in the same manner as 17 starting with C2. Yield: 55%, mp: 198-200° C. (from ethanol). 1H NMR (CDCl3): δ 1.42 (t, J=7.0 Hz, 3H), 4.44 (q, J=7.2 Hz, 2H), 6.12 (broad s, 2H), 7.25 (s, 1H), 7.51 (d, J=8.5 Hz, 2H), 7.58 ppm (d, J=8.5 Hz, 2H). 13C NMR (CDCl3): δ 14.1, 62.1, 105.9, 106.7, 121.3, 127.1, 127.5, 131.8, 134.0, 139.7, 159.4, 161.9, 163.0 ppm. IR: v 3407, 3310, 1709, 1657 cm−1. HRMS (ESI+): calculated for C15H13BrN3O3S+ 393.9861 found 393.9879.
Lithium hydroxide monohydrate (0.063 g, 1.29 mmol) was added to a solution of 17 (0.150 g, 0.43 mmol) in tetrahydrofuran (3 mL) and water (2 mL). The reaction mixture was stirred at room temperature for 16 h. 1 N HCl was added (pH ˜2), the formed precipitate was filtered and purified by preparative HPLC to afford E1 as yellow solid. HPLC-MS retention time 6.63 min. Yield: 67%, mp: >300° C. (from ethanol). 1H NMR (CD3OD): δ 7.19 (s, 1H), 7.44 (d, J=9.0 Hz, 2H), 7.58 ppm (d, J=9.0 Hz, 2H). 13C NMR (DMSO-d6): δ 104.4, 104.5, 126.8, 128.3, 128.9, 132.0, 134.5, 140.2, 158.9, 163.9, 164.6 ppm. IR: v 3439, 3322, 3065, 1712, 1646, 1590 cm−1. HRMS (ESP): calculated for C13H9ClN3O3S+ 322.0053 found 322.0062.
Prepared in the same manner as E1 starting with D2. HPLC-MS retention time 6.76 min. Yield: 77%, mp: >300° C. (from ethanol). 1H NMR (DMSO-d6): δ 7.12 (s, 1H), 7.52 (d, J=9.0 Hz, 2H), 7.63 (broad s, 2H), 7.66 ppm (d, J=8.5 HZ, 2H). 13C NMR (DMSO-d6): δ 104.4, 104.5, 120.4, 126.8, 128.6, 131.9, 134.5, 140.6, 158.8, 163.9, 164.5 ppm. IR: v 3439, 3322, 3065, 1712, 1646 cm−1. HRMS (ESI4): calculated for C13H7BrN3NaO3S+ 387.9367 found 387.9378.
A mixture of 17 (0.050 g, 0.143 mmol) and titanium (IV) isopropoxide (0.002 g, 2.1 μL, 7 μmol) in isopropanol (0.5 mL) was heated to 170° C. using microwave irradiation for 40 min. After cooling, the solvent was evaporated and the residue was purified by preparative HPLC to provide 2 as yellow solid. HPLC-MS retention time 8.73 min. Yield: 77%, mp: 118-120° C. (from ethanol). 1H NMR (CDCl3): δ 1.41 (d, J=6.5 Hz, 6H), 5.24-5.33 (m, 1H), 6.25 (broad s, 2H), 7.17 (s, 1H), 7.40 (d, J=8.5 Hz, 2H), 7.57 ppm (d, J=8.5 Hz, 2H). 13C NMR (CDCl3): δ 22.0, 70.1, 104.7, 106.8, 127.1, 127.2, 128.8, 133.1, 134.4, 139.2, 159.4, 161.9, 162.5 ppm. IR: v 3408, 3294, 3145, 1716, 1663, 1585 cm−1. HRMS (ESI+): calculated for C16H14ClN3O3NaS+ 386.0342 found 386.0331.
DIPEA (0.012 g, 14 μL, 0.093 mmol) was added to a mixture of E1 (0.020 g, 0.062 mmol), isopropylamine (0.007 g, 11 μL, 0.124 mmol), and BOP reagent (0.041 g, 0.093 mmol) in anhydrous DMSO (1 mL). The reaction mixture was stirred at room temperature for 4 h. Water was added and the resulting mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSO4, filtered and evaporated. The residue was purified by preparative HPLC to give the F3 as yellow solid. HPLC-MS retention time: 8.04 min. Yield: 89%, mp: 197-199° C. (from ethanol). 1H NMR (CD3OD): δ 1.22 (d, J=6.5 Hz, 6H), 4.09-4.19 (m, 1H), 7.25 (s, 1H), 7.46 (d, J=8.5 Hz, 2H), 7.59 ppm (d, J=8.5 Hz, 2H). 13C NMR (CD3OD): δ 19.6, 39.9, 102.9, 103.3, 124.9, 126.1, 126.9, 131.2, 135.1, 138.1, 158.0, 161.4, 162.1 ppm. IR: v 3404, 3294, 3178, 1661 cm−1. HRMS (ESI+): calculated for C16H15ClN4O2NaS+ 385.0502 found 385.0492.
Prepared in the same manner as F3 starting with E1 using methylamine (2.0 M solution in tetrahydrofuran). HPLC-MS retention time: 6.83 min. Yield: 52%, mp: 192-194° C. (from ethanol). 1H NMR (DMSO-d6): δ 2.75 (d, J=5.0 Hz, 3H), 7.24 (s, 1H), 7.51 (d, J=9.0 Hz, 2H), 7.60 (broad s, 2H), 7.69 (d, J=9 Hz, 2H), 8.27 ppm (broad s, 1H). 13C NMR (DMSO-d6): δ 26.4, 104.5, 105.2, 126.6, 128.1, 128.8, 131.6, 136.8, 140.1, 158.9, 163.5, 163.6 ppm. IR: v 3418, 3302, 1656, 1593 cm−1. HRMS (ESI+): calculated for C14H13ClN4O2S+ 333.0213 found 333.0210.
Prepared in the same manner as F3 starting with E1 using ethylamine hydrochloride. HPLC-MS retention time: 7.43 min. Yield: 58%, mp: 203-205° C. (from ethanol). 1H NMR (CDCl3): δ 1.23 (t, J=7.3 Hz, 3H), 341-347 (m, 2H), 6.18 (broad s, 2H), 7.07 (s, 1H), 7.45 (d, J=9 Hz, 2H), 7.51 (d, J=9 Hz, 2H), 7.60 ppm (broad s, 1H). 13C NMR (CDCl3): δ 14.9, 34.3, 106.8, 106.9, 126.7, 127.3, 129.0, 133.3, 135.6, 139.2, 159.5, 161.4, 162.6 ppm. IR: v 3405, 3297, 1652, 1600 cm−1. HRMS (ESI+): calculated for C15H13ClN4NaO2S+ 371.0345 found 371.0341.
Prepared in the same manner as F3 starting with E1 using cyclopropylamine. HPLC-MS retention time: 7.43 min. Yield: 41%, mp: 188-190° C. (from ethanol). 1H NMR (CD3OD): δ 0.58-0.60 (m, 2H), 0.66-0.69 (m, 2H), 2.76-2.81 (m, 1H), 7.19 (s, 1H), 7.50, (d, J=9.0 Hz, 2H), 7.67 (d, J=9.0 Hz, 2H), 8.28 ppm (broad s, 1H). 13C NMR (CD3OD): δ 6.3, 23.2, 104.4, 105.1, 126.6, 128.2, 128.7, 131.6, 137.0, 140.1, 158.9, 163.3, 164.4 ppm. IR: v 3408, 3302, 1654, 1593 cm−1. HRMS (ESI+): calculated for C16H13Cl4O2N4NaS+ 383.0345 found 383.0333.
Prepared in the same manner as F3 starting with E1 using dimethylamine hydrochloride. HPLC-MS retention time: 6.43 min. Yield: 62%, mp: 242-244° C. (from ethanol). 1H NMR (CDCl3): δ 3.14 (s, 3H), 3.16 (s, 3H), 6.18 (broad s, 2H), 6.73 (s, 1H), 7.41 (d, J=9.0 Hz, 2H), 7.52 ppm (d, J=9.0 Hz, 2H). 13C NMR (CD3OD): δ 35.7, 39.0, 104.0, 106.9, 127.0, 128.1, 128.8, 132.9, 139.2, 139.3, 159.1, 161.7, 164.3 ppm. IR: v 3414, 3300, 1646, 1598 cm−1. HRMS (ESI+): calculated for C14H13ClN4NaO2S+ 371.0345 found 371.0366.
Prepared in the same manner as F3 starting with E1 using ethylmethylamine. HPLC-MS retention time 6.85 min. Yield: 47%, mp: 148-150° C. (from ethanol). 1H NMR (CD3OD): δ 1.19-1.26 (m, 3H), 3.48-3.60 (m, 2H), 6.16 (broad s, 2H), 6.70 (s, 1H), 7.40-7.42 (m, 2H), 7.51-7.54 ppm (m, 2H). 13C NMR (CD3OD): δ 12.2, 14.0, 32.9, 36.3, 42.9, 46.1, 103.5, 106.4, 111.3, 117.0, 126.7, 126.8, 127.0, 127.1, 127.8, 127.9, 128.8 (×2), 132.8, 132.9, 159.1 (×2), 162.2, 164.0, 164.5 ppm. IR: v 3409, 3290, 3175, 1635 cm−1. HRMS (ESI: calculated for C16H15ClN4NaO2S+ 385.0502 found 385.0491.
Prepared in the same manner as F3 starting with E2. HPLC-MS retention time 8.20 min. Yield: 68%, mp: 222° C. dec (from ethanol). 1H NMR (CD3OD): δ 1.26 (d, J=5.0 Hz, 6H), 4.13-4.22 (m, 1H), 7.29 (s, 1H), 7.56 (d, J=8.5 Hz, 2H), 7.64 ppm (d, J=8.5 Hz, 2H). 13C NMR (CD3OD): δ 19.6, 58.6, 103.0, 103.3, 119.0, 124.9, 126.3, 129.9, 135.1, 138.6, 157.9, 161.4, 162.1 ppm. IR: v 3412, 3305, 1655, 1598 cm−1. HRMS (ESI+): calculated for C16H15BrN4NaO2S+ 428.9997 found 428.9986.
Prepared as previously reported (Nagakura, M. et al., J. Med. Chem, 1979, 22, 48-52). Yield: 83%, mp: 84-85° C. (from ethanol, Lit. [Nagakura, M. et al., J. Med. Chem, 1979, 22, 48-52] 81-84° C.). NMR (CDCl3): δ 1.41 (t, J=7.0 Hz, 3H), 2.60 (s, 3H), 4.34 (q, J=7.2 Hz, 2H), 7.34-7.36 (m, 4H) ppm. 13C NMR (CDCl3): δ 14.5, 30.9, 61.2, 117.6, 126.5, 129.8, 131.0, 140.4, 164.9, 197.4 ppm. IR: v 3359, 1706, 1617 cm−1. HRMS (ESI+): calculated for C12H13ClN2NaO3+ 291.0518 found 291.0512.
4-Bromoaniline (2.0 g, 11.63 mmol) was dissolved in 37% hydrochloridic acid (2.94 mL), ethanol (1.50 mL) and water (1.50 mL). The reaction mixture was cooled to 0° C. in an ice water bath before a solution of sodium nitrite (0.88 g, 12.75 mmol) in water (2.0 mL) was added dropwise. The resulting mixture was stirred at 0° C. for 20 min. Sodium acetate (3.72 g, 45.3 mmol) in water (5.9 mL) and ethyl acetoacetate (1.51 g, 1.43 mL, 11.63 mmol) were added and the reaction mixture was stirred at 0° C. for 2 h. The precipitated solid was then filtered, washed with water, and dried under high vacuum for 16 h to provide B2 as yellow solid that was crystallized in ethanol and used without further purification in the next step. Yield: 98%.
Prepared as B1 starting from 4-nitroaniline. Yield: 90%; mp: 130-132° C. (from ethanol). LC-MS retention time: 7.52 min. 1H NMR (CDCl3): δ 1.41 (t, J=7.0 Hz, 3H), 2.53 (s. 3H), 4.40 (q, J=7.0 Hz, 2H), 7.41 (d, J=9.0 Hz, 2H), 8.28 (d. J=9.0 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.2, 27.1, 62.3, 115.2, 116.1, 126.0, 130.5, 144.1, 146.8, 163.3, 194.2 ppm.
Was synthesized as compound B1 from 4-i-propylaniline. Yield: 99%; yellow oil. 1H NMR (CDCl3): δ 1.28 (d, J=7.0 Hz, 6H), 1.40 (t, J=7.0 Hz, 3H), 2.76 (s, 3H), 2.98 (sept, J=6.8 Hz, 1H), 4.42 (q, J=7.1 Hz, 2H), 7.35 (d, J=8.5 Hz, 2H), 7.54 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.3, 19.3, 24.0, 34.1, 62.8, 112.6, 115.8, 124.9, 127.2, 137.1, 137.9, 150.5, 150.6, 156.0, 162.2 ppm.
Prepared as B1 starting from 2,4-dimethylaniline. Yield: 94%, mp: 106-108° C. 1H NMR (CDCl3): δ 1.41 (t, J=7.1 Hz, 3H), 2.32 (s, 3H), 2.35 (s, 3H), 2.61 (s, 3H), 4.34 (q, J=7.1 Hz, 2H), 7.00 (s, 1H), 7.08 (d, J=8.3 Hz, 1H), 7.68 (d, J=7.8 Hz, 1H) ppm. 13C NMR (CDCl3): δ 14.6, 17.1, 21.1, 30.8, 61.0, 115.8, 125.6, 126.3, 128.3, 131.6, 135.7, 137.8, 165.4, 196.9 ppm. IR: n 3319, 1698, 1616 cm−1. MS [ESI]+: calculated for C14H19N2O3+ 263.14 found 263.15.
Prepared as previously reported. (Sotelo, E. et al., Synth Commun. 1997, 27, 2419-23) Yield: 47%, mp: 160-161° C. (from ethanol, Lit. Sotelo, E. et al., Synth Commun. 1997, 27, 2419-23] 190° C.). 1H NMR (CDCl3): δ 1.41 (t, J=7.0 Hz, 3H), 2.76 (s, 3H), 4.43 (q, J=7.2 Hz, 2H), 7.48 (d, J=8.5 Hz, 2H). 7.62 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.2, 19.4, 63.0, 112.4, 116.0, 126.3, 129.4, 135.5, 137.5, 138.5, 150.8, 155.8, 162.0 ppm. IR: v 2235, 1726, 1685 cm−1. MS [ESI+]: calculated for C15H13ClN3O3+ 318.06 found 318.07.
A mixture of B2 (2.0 g, 6.39 mmol), 4-aminobutiryc acid (1.33 g, 12.99 mmol), and ethyl cyanoacetate (1.1 g, 1.0 mL, 9.44 mmol) was stirred neat at 160° C. for 2.5 h. After cooling the residue was purified by silica gel flash chromatography (using an ethyl acetate/n-hexane gradient of 5% to 80% of ethyl acetate) to provide C2 as white solid. Yield: 76%, mp: 174-176° C. (from ethanol). NMR (CDCl3): δ 1.41 (t, J=7.0 Hz, 3H), 2.76 (s, 3H), 4.43 (q, J=4.43 Hz, 7.2 Hz, 2H), 7.56 (d, J=9.0 Hz, 2H), 7.64 (d, J=9.0 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.2, 19.4, 62.9, 112.4, 116.0, 123.5, 126.6, 132.3, 137.6, 139.1, 150.8, 155.7, 162.0 ppm. IR: v 2230, 1727, 1681 cm−1.
Was synthesized as C1 from B5. Yield: 53%; LC-MS retention time: 7.38 min. 1H NMR (CDCl3): δ 1.42 (t, J=7.0 Hz, 3H), 2.79 (s, 3H), 4.45 (d, J=7.1 Hz, 2H), 7.95 (d, J=8.5 Hz, 2H), 8.37 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.2, 19.5, 63.2, 112.1, 116.5, 124.6, 125.8, 138.3, 144.7, 147.6, 151.3, 155.6, 161.7 ppm. MS [ESI]+: calculated for C15H13N4O5+ 329.09 found 329.12.
A solution of the C5 (0.25 g, 0.76 mmol) in acetic acid (5 mL) was heated to 60° C. and iron (0.21 g, 3.8 mmol) was added in small portion. The reaction mixture was stirred at 60° C. for 3 h. After cooling the solvent was evaporated and the residue was dissolved in water and ethyl acetate. The phases were separated and the aqueous layer was extracted with ethyl acetate (×2). The combined organic layers were washed with saturated solution of sodium bicarbonate, brine, dried over MgSO4, filtered and evaporated. The residue was purified by silica gel column chromatography using ethyl acetate-n-hexanes 1:2 as eluent to provide the desired product. Yield: 98%. 1H NMR (CDCl3): δ 1.38 (t, J=7.0 Hz, 3H), 2.72 (s, 3H), 3.98 (broad s, 2H), 4.40 (q, J=7.2 Hz, 2H), 6.68 (d, J=8.5 Hz, 2H), 7.37 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.2, 19.2, 62.7, 112.9, 114.6, 115.2, 126.1, 130.9, 136.8, 147.9, 150.1, 156.2, 162.3 ppm. IR: v 3470, 3373, 2233, 1725, 1671, 1625, 1606 cm−1.
A mixture of C6 (0.04 g, 0.13 mmol), dimethylsulfate (0.03 g, 25 mL, 0.26 mmol) and potassium carbonate (0.07 g, 0.52 mmol) in acetone (5 mL) was stirred at room temperature for overnight. The reaction mixture was diluted with water and then extracted with etyl acetate. The combined organic layers were washed with brine, dried over MgSO4, filtered and evaporated. The residue was purified by silica gel column chromatography using ethyl acetate-hexanes 2:3 as eluent to provide the desired compounds. R=4-NMe2: yield: 33%. 1H NMR (CDCl3): δ 1.40 (t, J=7.0 Hz, 3H), 2.74 (s, 3H), 3.02 (s, 6H), 4.41 (q, J=7.2 Hz, 2H), 6.75 (d, J=8.5 Hz, 2H), and 7.51 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.3, 19.2, 40.6, 62.6, 111.8, 112.9, 115.1, 125.7, 129.4, 136.6, 149.8, 150.8, 156.3, 162.4 ppm. R=4-NHMe: yield: 11%. 1H NMR (CDCl3): δ 1.40 (t, J=7.3 Hz, 3H), 2.74 (s, 3H), 2.88 (s, 3H), 4.41 (q, J=7.2 Hz, 2H), 6.63 (d, J=8.5 Hz, 2H), 7.44 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.2, 19.2, 19.6, 30.6, 62.7, 111.9, 112.9, 115.1, 126.0, 130.0, 136.6, 149.9, 150.1, 156.2, 162.4 ppm.
Prepared as C1 from B10. Yellow oil. 1H NMR (CDCl3): δ 1.40 (t, J=7.1 Hz, 3H), 2.75 (s, 3H), 4.43 (q, J=7.1 Hz, H), 7.37 (t, J=8.1 Hz, 1H), 7.06-7.62 (m, 2H), 7.81 (s, 1H) ppm. 13C NMR (CDCl3): δ 14.1, 19.4, 63.0, 112.4, 116.1, 121.3, 122.5, 128.3, 130.4, 132.6, 137.7, 141.0, 155.7, 161.9, 163.1 ppm. IR: v 2236, 1732, 1685, 1585 cm−1. MS [ESI]+: calculated for C15H13BrN3O3+ 362.01 found 361.96.
Prepared as C1 from B11. Red solid. 1H NMR (CDCl3): δ 1.40 (t, J=7.3 Hz, 3H), 2.43 (s, 3H), 2.76 (s, 3H), 4.43 (q, J=7.2 Hz, 2H), 7.28 (d, J=7.6 Hz, 1H), 7.39-7.42 (m, 2H) ppm. 13C NMR (CDCl3): δ 14.3, 19.4, 21.6, 62.9, 112.6, 115.9, 122.3, 125.7, 129.1, 130.4, 137.3, 139.4, 140.1, 150.6, 156.0, 162.2 ppm. IR: v 2236, 1730, 1684, 1601 cm−1. MS [ESI]+: calculated for C16H16N3O3+ 298.12 found 298.21.
Prepared as C1 from B15. Yellow solid, mp: 93-95° C. (from ethanol). 1H NMR (CDCl3): δ 1.38 (t, J=7.1 Hz, 3H), 2.78 (s, 3H), 4.41 (q, J=7.1 Hz, 2H), 7.40-4.49 (m, 3H), 7.57 (dd, J=7.9 and 1.4 Hz, 1H) ppm. 13C NMR (CDCl3): δ 14.3, 19.6, 63.0, 112.3, 115.9, 128.1, 128.8130.8, 131.5, 131.6, 137.6, 137.8, 151.5, 155.4, 162.0 ppm. IR: v 2235, 1731, 1690 cm−1.
Prepared as C1 from B16. White solid, m.p.: 106-108° C. (from ethanol). 1H NMR (CDCl3): δ 1.38 (t, J=7.1 Hz, 3H), 2.79 (s, 3H), 4.41 (q, J=7.1 Hz, 2H), 7.38=7.42 (m, 2H), 7.48 (dt, J=7.2 and 1.3 Hz, 1H), 7.74 (dd, J=7.6 and 0.9 Hz, 1H) ppm. 13C NMR (CDCl3): δ 14.3, 19.6, 63.0, 112.3, 116.0, 121.1, 128.8, 128.9, 131.7, 134.0, 137.7, 139.2, 151.5, 155.4, 162.0 ppm. IR: v 2235, 1727, 1689, 1586 cm−1. MS [ESI]+: calculated for C15H13BrN3O3+ 362.01 found 361.93.
Prepared as C1 from B17. Yellow oil. NMR (CDCl3): δ 1.27 (t, J=7.1 Hz, 3H), 1.37 (t, J=7.1 Hz, 3H), 2.78 (s, 3H), 4.25 (q, J=7.1 Hz, 2H), 4.39 (q, J=7.1 Hz, 2H), 7.43 (dd, J=7.8 and 0.8 Hz, 1H), 7.59 (dt, J=7.7 Hz and 1.1 Hz, 1H), 7.70 (dt, J=7.7 and 1.4 Hz, 1H), 8.13 (dd, J=7.8 and 1.4 Hz, 1H) ppm. 13C NMR (CDCl3): δ 14.2, 14.3, 61.8, 62.8, 112.5, 115.3, 127.6, 128.2, 130.3, 131.6, 133.6, 137.0, 139.8, 151.4, 156.5, 162.2, 164.4 ppm. IR: v 2234, 1723, 1687, 1603, 1585 cm−1. MS [ESI]+: calculated for C18H18N3O5+ 356.12 found 356.01.
Prepared as C1 from B19. Yellow oil. 1H NMR (CDCl3): δ 1.38 (t, J=7.2 Hz, 3H), 2.18 (s, 3H), 2.78 (s, 3H), 4.40 (q, J=7.2 Hz, 2H), 7.26 (d, J=7.0 Hz, 1H), 7.33-736 (m, 2H), 7.41 (t, J=7.5 Hz, 1H) ppm. 13C NMR (CDCl3): δ 14.2, 17.8, 19.5, 63.2, 112.5, 115.7, 126.9, 127.2, 130.3, 131.5, 134.7, 137.3, 139.2, 151.0, 155.8, 162.2 ppm. IR: v 2234, 1730, 1684 cm−1. MS [ESI]+: calculated for C16H16N3O3+ 298.12 found 297.96.
A mixture of C1 (0.100 g, 0.31 mmol), sulfur (0.015 g, 0.47 mmol), and morpholine (0.55 g, 0.55 mL, 0.63 mmol) was heated to 150° C. using microwave irradiation for 15 min. After cooling, the precipitate which formed was collected and purified by preparative HPLC to give D1 as yellow solid. HPLC-MS retention time 8.20 min. Yield: 64%, mp: 189-191° C. (from ethanol). 1H NMR (CDCl3): δ 1.43 (t, J=7.3 Hz, 3H), 4.44 (q, J=7.2 Hz, 2H), 6.21 (broad s, 2H), 7.25 (s, 1H), 7.42 (d, J=8.5 Hz, 2H), 7.56 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.4, 62.2, 104.9, 106.9, 127.1, 127.2, 128.9, 133.3, 134.0, 139.2, 159.4, 161.8, 163.0 ppm. IR: v 3411, 3303, 1724 cm−1. HRMS (ESI+): calculated for C15H13ClN3O3S+ 350.0366 found 350.0355.
Was synthesized in the same manner as D1 starting with C2. Yield: 55%, mp: 198-200° C. (from ethanol). 1H NMR (CDCl3): δ 1.42 (t, J=7.0 Hz, 3H), 4.44 (q, J=7.2 Hz, 2H), 6.12 (broad s, 2H), 7.25 (s, 1H), 7.51 (d, J=8.5 Hz, 2H), 7.58 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 14.1, 62.1, 105.9, 106.7, 121.3, 127.1, 127.5, 131.8, 134.0, 139.7, 159.4, 161.9, 163.0 ppm. IR: v 3407, 3310, 1709, 1657 cm−1. HRMS (ESI+): calculated for C15H13BrN3O3S+ 393.9861 found 393.9879.
Was synthesized in the same manner as D1 starting with C3. 1H-NMR (500 MHz; CDCl3): δ 1.42 (t, J=7.1 Hz, 3H), 4.44 (q, J=7.1 Hz, 2H), 6.06 (s, 2H), 7.15-7.11 (m, 2H), 7.22 (s, 1H), 7.57-7.54 (m, 2H) ppm.
Was synthesized as D1 from C4. 1H-NMR (500 MHz; CDCl3): δ 1.42 (t, J=7.1 Hz, 3H), 4.44 (q, J=7.1 Hz, 2H), 6.19 (broad s, 2H), 7.25 (s, 1H), 7.40-7.38 (m, 2H), 7.79-7.77 (m, 2H) ppm.
A mixture of D4 (25 mg, 0.055 mmol), sodium azide (7.2 mg, 0.11 mmol), sodium ascorbate (1 mg), N,N′-dimethylethylendiamine (1.4 mg, 1.8 μL, 0.016 mmol) and cuprous iodide (5 mg, 0.0275 mmol) in dimethylsulfoxide (2.5 mL) and water (0.5 mL) was heated in sealed tube to 60° C. for 1 h. After cooling, the reaction mixture was filtered through a short path of celite, diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using ethyl acetate-hexanes 1:3 as eluent to furnish the desired compound. Yield: 43%, mp: 169-170° C. 1H-NMR (500 MHz; CDCl3): δ 1.43 (t, J=7.1 Hz, 3H), 4.44 (q, J=7.1 Hz, 2H), 6.19 (s, 2H), 7.11 (d, J=8.7 Hz, 2H), 7.25 (s, 1H), 7.61 (d, J=8.7 Hz, 2H) ppm.
Was synthesized as D1 from C5. Yield: 43%. 1HNMR (DMSO-d6): δ 1.33 (t, J=7.0 Hz, 3H), 4.36 (q, J=7.1 Hz, 2H), 7.12 (s, 1H), 7.78 (broad s, 2H), 7.90 (d, J=8.5 Hz, 2H), 8.35 (d, J=8.5 Hz, 2H) ppm. 13C NMR (DMSO-d6): δ 14.6, 62.2, 103.9, 105.2, 124.5, 126.2, 126.6, 134.6, 145.9, 146.6, 158.7, 162.7, 164.6 ppm.
Was synthesized as D1 from C6. Yield: 77%. 1H NMR (DMSO-d6): δ 1.29 (t, J=7.0 Hz, 3H), 4.30 (q, J=7.0 Hz, 2H), 5.27 (broad s, 2H), 6.60 (d, J=8.5 Hz, 2H), 7.05-7.07 (m, 3H), 7.55 (broad s, 2H) ppm. 13C NMR (DMSO-d6): δ 14.7, 61.8, 103.1, 105.0, 113.7, 126.9, 127.8, 129.9, 132.51, 148.9, 159.2, 163.1, 163.3 ppm. IR: v 3424, 3319, 3259, 3160, 1724, 1704, 1637, 1605 cm−1.
Was synthesized as D1 from C7. Yield: 81%; mp: 211-213° C. (from ethanol). 1H NMR (CDCl3): δ 1.29 (t, J=7.0 Hz, 3H), 2.71 (s, 3H), 4.30 (q, J=7.2 Hz, 2H), 5.84 (broad s, 1H), 6.57 (d, J=8.5 Hz, 2H), 7.06 (s, 1H), 7.15 (d, J=8.5 Hz, 2H), 7.55 (broad s, 2H) ppm.
Was synthesized as D1 from C8. Yield: 81%; mp: 211-213° C. (from ethanol). 1H NMR (CDCl3): δ 1.29 (t, J=7.0 Hz, 3H), 2.93 (s, 6H), 4.31 (q, J=6.7 Hz, 2H), 6.76 (d, J=8.5 Hz, 2H), 7.07 (s, 1H), 7.25 (d, J=8.5 Hz, 2H), 7.56 (broad s, 2H) ppm.
Was synthesized as D1 from C11. Yield: 55%. 1H-NMR (500 MHz; CDCl3): δ 1.43 (t, J=7.1 Hz, 3H), 4.45 (q, J=7.1 Hz, 2H), 7.26 (s, 1H), 7.31 (d, J=8.5 Hz, 2H), 7.67 (d, J=2.0 Hz, 2H) ppm.
Was synthesized as D1 from C10. Yield: 46%. 1H NMR (DMSO-d6): δ 1.29 (t, J=7.2 Hz, 3H), 4.31 (q, J=7.0 Hz, 2H), 6.83 (d, J=8.5 Hz, 2H), 7.07 (s, 1H), 7.25 (d, J=9.0 Hz, 2H), 7.57 (broad s, 2H), 9.68 (broad s, 1H) ppm. 13C NMR (DMSO-d6): δ 14.7, 61.9, 103.5, 104.8, 115.5, 126.8, 128.3, 132.8, 157.3, 159.1, 163.1, 163.5. IR: v 3404, 1639 cm−1. MS [ESI]+: calculated for C15H14N3O4S+ 332.07 found 332.04.
Was synthesized as D1 from C11. Yield: 90%. 1H-NMR (500 MHz; CDCl3): δ 1.43 (t, J=7.1 Hz, 3H), 4.45 (q, J=7.1 Hz, 2H), 7.27 (s, 1H), 7.72 (d, J=8.6 Hz, 2H), 7.80 (d, J=8.4 Hz, 2H) ppm.
Was synthesized as D1 from C12. Yield: 43%. 1H NMR (DMSO-d6): δ 1.33 (t, J=7.0 Hz, 3H), 4.36 (q, J=7.1 Hz, 2H), 7.12 (s, 1H), 7.78 (broad s, 2H), 7.90 (d, J=8.5 Hz, 2H), 8.35 (d, J=8.5 Hz, 2H) ppm. 13C NMR (DMSO-d6): δ 14.6, 62.2, 103.9, 105.2, 124.5, 126.2, 126.6, 134.6, 145.9, 146.6, 158.7, 162.7, 164.6 ppm.
Was synthesized as D1 from C13. Yield: 78%; mp: 163-165° C. (from dichloromethane). 1H NMR (CDCl3): δ 1.43 (t, J=7.1 Hz, 3H), 4.45 (q, J=7.5 Hz, 2H), 6.11 (broad s, 2H), 7.32 (t, J=8.05 Hz, 1H), 7.48 (d, J=7.4 Hz, 1H), 7.58 (d, J=7.9 Hz, 1H), 7.80 (s, 1H) ppm. 13C NMR (CDCl3): δ 14.5, 62.3, 105.1, 106.8, 122.2, 124.7, 127.1, 129.2, 130.0, 130.8, 134.2, 141.8, 159.4, 162.0, 163.1 ppm. IR: v 3419, 3320, 1709, 1646, 1596 cm−1. HRMS [ESI]+: calculated for C15H13BrN3O3S+ 393.9861 found 393.9857.
Was synthesized as D1 from C14. Yield: 81%; mp: 190-191° C. (from dichloromethane). 1H NMR (CDCl3): δ 1.42 (t, J=7.1 Hz, 3H), 2.41 (s, 3H), 4.44 (q, J=7.1 Hz, 2H), 6.21 (broad s, 2H), 7.17 (d, J=6.6 Hz, 1H), 7.22 (s, 1H), 7.32-7.38 (m, 3H) ppm. 13C NMR (CDCl3): δ 14.4, 21.6, 62.1, 104.1, 17.2, 123.4, 126.9, 127.4, 128.7, 128.8, 133.7, 138.9, 140.6, 159.7, 161.7, 163.3 ppm. IR: v 3436, 3329, 1706, 1652, 1598 cm−1. HRMS [ESI]+: calculated for C16H16N3O3S+ 330.0929 found 330.0912.
It was synthesized as D1 from C18. Yield: 87%. 1H NMR (CDCl3): δ 1.27 (m, 3H), 4.29-4.33 (m, 2H), 7.15-7.16 (m, 1H), 7.47 (m, 2H), 7.53-7.60 (m, 3H), 7.64-7.67 (m, 1H) ppm. 13C NMR (CDCl3): δ 14.0, 54.9, 61.4, 103.5, 104.1, 126.0, 128.0, 129.7, 130.4, 130.6, 131.7, 133.0, 138.3, 157.9, 162.2, 163.1 ppm. IR: v 3425, 3303, 1710, 1648, 1587 cm−1. HRMS [ESI]+: calculated for C15H12ClNaN3O3S 372.0186 found 372.0190.
Was synthesized as D1 from C16. Yield: 73%; mp: 159-160° C. (from dichloromethane). 1H NMR (CDCl3): δ 1.38-1.43 (m, 6H), 4.37-4.46 (m, 4H), 6.36 (broad s, 2H), 7.22 (s, 1H), 7.53 (t, J=7.9 Hz, 1H), 7.81 (d, J=7.95 Hz, 1H), 8.03 (d, J=7.8 Hz, 1H), 8.29 (s, 1H) ppm. 13C NMR (CDCl3): δ 14.4, 14.5, 6.1.4, 61.2, 104.8, 106.6, 127.2, 127.3, 128.7, 128.9, 130.4, 131.2, 134.1, 140.9, 159.5, 162.1, 163.1, 166.2 ppm. IR: n 3452, 3345, 1721, 1667, 1600 cm−1. HRMS [ESI]+: calculated for C18H17N3NaO5S+ 410.0787 found 410.0781.
Was synthesized as D1 from C17. NMR (CDCl3): δ 1.41 (t, J=7.1 Hz, 3H), 4.44 (q, J=7.1 Hz, 2H), 6.16 (broad s, 2H), 7.20-7.31 (m, 2H), 7.38-7.43 (m, 1H), 7.48 (dt, J=7.65 and 1.5 Hz, 1H) ppm.
Was synthesized as D1 from C18. NMR (CDCl3): δ 1.27 (m, 3H), 4.29-4.33 (m, 2H), 7.15-7.16 (m, 1H), 7.47 (m, 2H), 7.53-7.60 (m, 3H), 7.64-7.67 (m, 1H) ppm.
Was synthesized as D1 from C19. Yield: 78%; mp: 203-205° C. (from dichloromethane). 1H NMR (CDCl3): δ 1.40 (t, J=7.0 Hz, 3H), 4.43 (q, J=7.0 Hz, 2H), 6.17 (broad s, 2H), 7.26 (s, 1H), 7.31 (t, J=7.3 Hz, 1H), 7.42-7.47 (m, 2H), 7.71 (d, J=8.0 Hz, 1H) ppm. 13C NMR (CDCl3): δ 14.4, 62.2, 105.3, 106.8, 123.0, 127.4, 128.6, 130.3, 130.6, 133.7, 140.2, 139.9, 159.3, 161.7, 163.1 ppm. IR: v 3424, 3305, 1721, 1659, 1585 cm−1. HRMS [ESI]+: calculated for C15H13BrN3O3S+ 393.9861 found 393.9879.
Was synthesized as D1 from C20. Yield: 78%; mp: 176-178° C. (from dichloromethane). 1H NMR (CDCl3): δ 1.10 (t, J=7.1 Hz, 3H), 1.38 (t, J=7.1 Hz, 3H), 4.19 (q, J=7.1H, 2H), 4.40 (q, J=7.1 Hz, 2H), 6.21 (broad s, 2H), 7.21 (s, 1H), 7.46-7.52 (m, 2H), 7.63 (t, J=7.6 Hz, 1H), 8.03 (d, J=7.8 Hz, 1H) ppm. 13C NMR (CDCl3): δ 13.9, 14.4, 61.4, 62.1, 104.7, 106.8, 127.4, 128.7, 128.9, 129.2, 131.2, 133.0, 133.5, 140.1, 160.0, 161.8, 163.2, 165.7 ppm. IR: v 3417, 1712, 1656, 1593 cm−1. HRMS [ESI]+: calculated for C18H17N3NaO5S+ 410.0787 found 410.0799.
Was synthesized as D1 from C21. 1H NMR (CDCl3): δ 1.36 (t, J=7.1 Hz, 3H), 4.36 (broad s, 2H), 6.03 (broad s, 2H), 7.15 (s, 1H), 7.19-7.35 (m, 5H), 7.44-7.52 (m, 4H) ppm.
Was synthesized as D1 from C22. Yield: 75%; mp: 209-210° C. (from dichloromethane). 1H NMR (DMSO-d6): δ 1.28 (t, J=7.2 Hz, 3H), 2.11 (s, 3H), 4.30 (q, J=7.1 Hz, 2H), 7.13 (s, 1H), 7.31-7.37 (m, 4H), 7.57 (broad s, 2H) ppm. 13C NMR (DMSO-d6): δ 14.0, 17.2, 61.3, 103.4, 104.0, 126.2, 126.5, 128.3, 128.5, 130.5, 132.6, 135.4, 139.9, 158.3, 162.4, 162.9 ppm. IR: v 3429, 3317, 3137, 1717, 1641, 1585 cm−1. HRMS [ESI]+: calculated for C16H15NaN3O3S+ 352.0732 found 352.0736.
Lithium hydroxide monohydrate (0.063 g, 1.29 mmol) was added to a solution of D1 (0.150 g, 0.43 mmol) in tetrahydrofuran (3 mL) and water (2 mL). The reaction mixture was stirred at room temperature for 16 h. 1 N HCl was added (pH ˜2), the formed precipitate was filtered and purified by preparative HPLC to afford E1 as yellow solid. HPLC-MS retention time 6.63 min. Yield: 67%, mp: >300° C. (from ethanol). 1H NMR (CD3OD): δ 7.19 (s, 1H), 7.44 (d, J=9.0 Hz, 2H), 7.58 (d, J=9.0 Hz, 2H) ppm. 13C NMR (DMSO-d6): δ 104.4, 104.5, 126.8, 128.3, 128.9, 132.0, 134.5, 140.2, 158.9, 163.9, 164.6 ppm. IR: v 3439, 3322, 3065, 1712, 1646, 1590 cm−1. HRMS (ESI+): calculated for C13H9ClN3O3S+ 322.0053 found 322.0062.
Prepared in the same manner as E1 starting with D2. HPLC-MS retention time 6.76 min. Yield: 77%, mp: >300° C. (from ethanol). 1H NMR (DMSO-d6): δ 7.12 (s, 1H), 7.52 (d, J=9.0 Hz, 2H), 7.63 (broad s, 2H), 7.66 (d, J=8.5 HZ, 2H) ppm. 13C NMR (DMSO-d6): δ 104.4, 104.5, 120.4, 126.8, 128.6, 131.9, 134.5, 140.6, 158.8, 163.9, 164.5 ppm. IR: v 3439, 3322, 3065, 1712, 1646 cm−1. HRMS (ESI+): calculated for C13H7BrN3NaO3S+ 387.9367 found 387.9378.
Prepared in the same manner as E1 starting with D3. 1H NMR (DMSO-d6): δ 7.12 (s, 1H), 7.29 (t, J=8.7 Hz, 2H), 7.55-7.58 (m, 4H) ppm.
Prepared in the same manner as E1 starting with D11. 1H-NMR (500 MHz; DMSO-d6): δ 6.81 (d, J=8.8 Hz, 2H), 7.08 (s, 1H), 7.26 (d, J=8.8 Hz, 2H), 7.55 (broad s, 2H), 9.65 (s, 1H) ppm.
Prepared in the same manner as E1 starting with D10. 1H-NMR (500 MHz; MeOD): δ 7.17 (s, 1H), 7.35 (d, J=8.5 Hz, 2H), 7.69 (d, J=8.9 Hz, 2H) ppm.
Prepared in the same manner as E1 starting with D16. 1H-NMR (500 MHz; DMSO-d6): δ 7.13 (s, 1H), 7.44-7.42 (m, 1H), 7.50 (t, J=8.0 Hz, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.68-7.65 (m, 3H) ppm.
A mixture of D1 (0.050 g, 0.143 mmol) and titanium (IV) isopropoxide (0.002 g, 2.1 μL, 7 μmol) in isopropanol (0.5 mL) was heated to 170° C. using microwave irradiation for 40 min. After cooling, the solvent was evaporated and the residue was purified by preparative HPLC to provide 2 as yellow solid. HPLC-MS retention time 8.73 min. Yield: 77%, mp: 118-120° C. (from ethanol). 1H NMR (CDCl3): δ 1.41 (d, J=6.5 Hz, 6H), 5.24-5.33 (m, 1H), 6.25 (broad s, 2H), 7.17 (s, 1H), 7.40 (d, J=8.5 Hz, 2H), 7.57 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 22.0, 70.1, 104.7, 106.8, 127.1, 127.2, 128.8, 133.1, 134.4, 139.2, 159.4, 161.9, 162.5 ppm. IR: v 3408, 3294, 3145, 1716, 1663, 1585 cm−1. HRMS (ESI+): calculated for C16H14ClN3O3NaS+ 386.0342 found 386.0331.
DIPEA (0.012 g, 14 μL, 0.093 mmol) was added to a mixture of E1 (0.020 g, 0.062 mmol), isopropylamine (0.007 g, 11 μL, 0.124 mmol), and BOP reagent (0.041 g, 0.093 mmol) in anhydrous DMSO (1 mL). The reaction mixture was stirred at room temperature for 4 h. Water was added and the resulting mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSO4, filtered and evaporated. The residue was purified by preparative HPLC to give the F3 as yellow solid. HPLC-MS retention time: 8.04 min. Yield: 89%, mp: 197-199° C. (from ethanol). 1H NMR (CD3OD): δ 1.22 (d, J=6.5 Hz, 6H), 4.09-4.19 (m, 1H), 7.25 (s, 1H), 7.46 (d, J=8.5 Hz, 2H), 7.59 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CD3OD): δ 19.6, 39.9, 102.9, 103.3, 124.9, 126.1, 126.9, 131.2, 135.1, 138.1, 158.0, 161.4, 162.1 ppm. IR: v 3404, 3294, 3178, 1661 cm−1. HRMS (ESI+): calculated for C16H15ClN4O2NaS+ 385.0502 found 385.0492.
Prepared in the same manner as F3 starting with E1 using methylamine (2.0 M solution in tetrahydrofuran). HPLC-MS retention time: 6.83 min. Yield: 52%, mp: 192-194° C. (from ethanol). 1H NMR (DMSO-d6): δ 2.75 (d, J=5.0 Hz, 3H), 7.24 (s, 1H), 7.51 (d, J=9.0 Hz, 2H), 7.60 (broad s, 2H), 7.69 (d, J=9 Hz, 2H), 8.27 (broad s, 1H) ppm. 13C NMR (DMSO-d6): δ 26.4, 104.5, 105.2, 126.6, 128.1, 128.8, 131.6, 136.8, 140.1, 158.9, 163.5, 163.6 ppm. IR: v 3418, 3302, 1656, 1593 cm−1. HRMS (ESI+): calculated for C14H10ClN4O2S+ 333.0213 found 333.0210.
Prepared in the same manner as F3 starting with E1 using ethylamine hydrochloride. HPLC-MS retention time: 7.43 min. Yield: 58%, mp: 203-205° C. (from ethanol). 1H NMR (CDCl3): δ 1.23 (t, J=7.3 Hz, 3H), 341-347 (m, 2H), 6.18 (broad s, 2H), 7.07 (s, 1H), 7.45 (d, J=9 Hz, 2H), 7.51 (d, J=9 Hz, 2H), 7.60 (broad s, 1H) ppm. 13C NMR (CDCl3): δ 14.9, 34.3, 106.8, 106.9, 126.7, 127.3, 129.0, 133.3, 135.6, 139.2, 159.5, 161.4, 162.6 ppm. IR: v 3405, 3297, 1652, 1600 cm−1. HRMS (ESI+): calculated for C15H13ClN4NaO2S+ 371.0345 found 371.0341.
Prepared in the same manner as F3 starting with E1 using cyclopropylamine. HPLC-MS retention time: 7.43 min. Yield: 41%, mp: 188-190° C. (from ethanol). 1H NMR (CD3OD): δ 0.58-0.60 (m, 2H), 0.66-0.69 (m, 2H), 2.76-2.81 (m, 1H), 7.19 (s, 1H), 7.50, (d, J=9.0 Hz, 2H), 7.67 (d, J=9.0 Hz, 2H), 8.28 (broad s, 1H) ppm. 13C NMR (CD3OD): δ 6.3, 23.2, 104.4, 105.1, 126.6, 128.2, 128.7, 131.6, 137.0, 140.1, 158.9, 163.3, 164.4 ppm. IR: v 3408, 3302, 1654, 1593 cm−1. HRMS (ESI+): calculated for C16H13Cl4O2N4NaS+ 383.0345 found 383.0333.
Prepared in the same manner as F3 starting with E1 using dimethylamine hydrochloride. HPLC-MS retention time: 6.43 min. Yield: 62%, mp: 242-244° C. (from ethanol). 1H NMR (CDCl3): δ 3.14 (s, 3H), 3.16 (s, 3H), 6.18 (broad s, 2H), 6.73 (s, 1H), 7.41 (d, J=9.0 Hz, 2H), 7.52 (d, J=9.0 Hz, 2H) ppm. 13C NMR (CD3OD): δ 35.7, 39.0, 104.0, 106.9, 127.0, 128.1, 128.8, 132.9, 139.2, 139.3, 159.1, 161.7, 164.3 ppm. IR: v 3414, 3300, 1646, 1598 cm−1. HRMS (ESI4): calculated for C14H13ClN4NaO2S+ 371.0345 found 371.0366.
Prepared in the same manner as F3 starting with E1 using ethylmethylamine. HPLC-MS retention time 6.85 min. Yield: 47%, mp: 148-150° C. (from ethanol). 1H NMR (CD3OD): δ 1.19-1.26 (m, 3H), 3.48-3.60 (m, 2H), 6.16 (broad s, 2H), 6.70 (s, 1H), 7.40-7.42 (m, 2H), 7.51-7.54 (m, 2H) ppm. 13C NMR (CD3OD): δ 12.2, 14.0, 32.9, 36.3, 42.9, 46.1, 103.5, 106.4, 111.3, 117.0, 126.7, 126.8, 127.0, 127.1, 127.8, 127.9, 128.8 (×2), 132.8, 132.9, 159.1 (×2), 162.2, 164.0, 164.5 ppm. IR: v 3409, 3290, 3175, 1635 cm−1. HRMS (ESI+): calculated for C16H15ClN4NaO2S+ 385.0502 found 385.0491.
Prepared in the same manner as F3 starting with E2. HPLC-MS retention time 8.20 min. Yield: 68%, mp: 222° C. dec (from ethanol). 1H NMR (CD3OD): δ 1.26 (d, J=5.0 Hz, 6H), 4.13-4.22 (m, 1H), 7.29 (s, 1H), 7.56 (d, J=8.5 Hz, 2H), 7.64 (d, J=8.5 Hz, 2H) ppm. 13C NMR (CD3OD): δ 19.6, 58.6, 103.0, 103.3, 119.0, 124.9, 126.3, 129.9, 135.1, 138.6, 157.9, 161.4, 162.1 ppm. IR: v 3412, 3305, 1655, 1598 cm−1. HRMS (ESI+): calculated for C16H15BrN4NaO2S+ 428.9997 found 428.9986.
Prepared in the same manner as F3 starting with E3. 1H-NMR (500 MHz; CDCl3): δ 1.24 (d, J=6.5 Hz, 6H), 4.21 (dt, J=13.5, 6.7 Hz, 1H), 6.90 (d, J=7.4 Hz, 1H), 7.16 (t, J=8.4 Hz, 2H), 7.51 (dd, J=8.3, 4.8 Hz, 2H), 7.58 (s, 1H) ppm.
Prepared in the same manner as F3 starting with E3 and cyclopropyl amine. 1H-NMR (500 MHz; DMSO-d6): δ 0.58 (m, 2H), 0.69-0.66 (m, 2H), 2.78 (m, 1H), 7.19 (s, 1H), 7.30-7.26 (m, 2H), 7.57 (s, 2H), 7.66-7.63 (m, 2H), 8.25 (d, J=4.2 Hz, 1H) ppm.
Prepared in the same manner as F3 starting with 6b and diethylamine. 1H MNR (CDCl3): δ 1.18 (t, J=7.1 Hz, 3H), 1.26 (t, J=7.1 Hz, 3H), 3.45 (q, J=7.1 Hz, 2H), 3.55 (q, J=7.1 Hz, 2H), 3.83 (s, 3H), 6.20 (broad s, 2H), 6.64 (s, 1H), 6.95 (d, J=8.8 Hz, 2H), 7.42 (d, J=8.8 Hz, 2H) ppm.
Prepared in the same manner as F3 starting with E6. 1H-NMR (500 MHz; MeOD): δ 1.27 (d, J=6.6 Hz, 6H), 4.16-4.12 (m, 1H), 7.28 (s, 1H), 7.38 (dt, J=8.0, 0.9 Hz, 1H), 7.46 (t, J=8.0 Hz, 1H), 7.58 (dt, J=8.0, 0.9 Hz, 1H), 7.71 (d, J=1.9 Hz, 1H) ppm.
Prepared in the same manner as F3 starting with E5. 1H-NMR (500 MHz; CDCl3): δ 1.25 (d, J=6.6 Hz, 6H), 4.25-4.18 (m, 1H), 6.19 (s, 2H), 6.89 (d, J=7.9 Hz, 1H), 7.34 (d, J=8.2 Hz, 2H), 7.62-7.60 (m, 3H) ppm.
Prepared in the same manner as F3 starting with E4. 1H-NMR (500 MHz; DMSO-d6): δ 1.14 (d, J=6.6 Hz, 6H), 4.07-4.02 (m, 1H), 6.82 (d, J=8.8 Hz, 2H), 7.16 (s, 1H), 7.34 (d, J=8.8 Hz, 2H), 7.50 (broad s, 2H), 7.85 (d, J=8.1 Hz, 1H), 9.61 (s, 1H) ppm.
Prepared in the same manner as F3 starting with E1 and ethanolamine. 1H MNR (DMSO-d6): δ 3.29-332 (m, 2H), 3.48 (t, J=6.1 Hz, 2H), 4.77 (broad s, 1H), 7.25 (s, 1H), 7.52 (d, J=7.4 Hz, 2H), 7.62 (broad s, 2H), 7.68 (d, J=7.7 Hz, 2H), 8.21 (t, J=5.7 Hz, 1H) ppm.
D1, dissolved in 1 ml EtOH was added with ˜30 eq. of hydrazine monohydrate (˜240 ult). The resulting mixture was heated at 160° C. for 1 h under microwave promoted conditions. After evaporation of volatiles the crude material was purified by preparative HPLC obtaining the title compound. 1H-NMR (500 MHz; DMSO-d6): δ 4.51 (broad s, 2H), 7.12 (s, 1H), 7.51-7.47 (m, 2H), 7.61 (broad s, 2H), 7.75-7.72 (m, 2H), 9.62 (s, 1H) ppm.
A mixture of 17, potassium carbonate and iodometane in acetonitrile was put in a vial and stirred and heated at 70° C. for 10 h. After cooling the reaction mixture was diluted with water and then extracted with ethyl acetate. Organic layer was washed with brine, dried over MgSO4, filtered and evaporated. The residue was purified by silica gel column chromatography using ethyl acetate-n-Hexane 1:3 as eluent to give the title compound. MS (ESI+): calculated for C16H15ClN3O3S+ 364.05 found 364.07.
A mixture 17 (30 mg, 0.086 mmol) and N-chlorosuccinimide (11 mg, 0.086 mmol) in anhydrous N,N-dimethylformamide (0.5 mL) was heated to 80° C. for 30 minutes. After cooling, water was added and the resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by preparative HPLC using a gradient of acetonitrile in water from 10% to 90% in 10 minutes to give the desired compound. 1H-NMR (500 MHz; CDCl3): δ 1.42 (t, J=7.2 Hz, 3H), 4.45 (q, J=7.2 Hz, 2H), 6.27 (broad s, 2H), 7.41 (d, J=8.8 Hz, 2H), 7.53-7.51 (m, 2H) ppm.
Prepared in the same manner as CNDR-51393 starting with 17 and N-bromosuccinimide. 1H-NMR (500 MHz; CDCl3): δ 1.43 (t, J=7.2 Hz, 3H), 4.46 (q, J=7.2 Hz, 2H), 6.34 (broad s, 2H), 7.40 (d, J=8.8 Hz, 2H), 7.52 (d, J=8.8 Hz, 2H) ppm.
A mixture of 11 (30 mg, 0.095 mmol) in absolute ethanol (2 mL) was refluxed for 5 h. The reaction mixture was cooled at room temperature and diluted with water. The resulting mixture was extracted with ethyl acetate and the combined organic layers were washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using ethyl acetate-hexanes 2:3 as eluent to provide the desired compound. 1H NMR (CDCl3): δ 1.33 (t, J=7.0 Hz, 3H), 4.25 (q, J=7.2 Hz, 2H), 6.17 (broad s, 2H), 6.70 (s, 1H), 6.75 (broad s, 1H), 7.39 (d, J=8.5 Hz, 2H), 7.58 (d, J=8.5 Hz, 2H) ppm.
Prepared in the same manner as CNDR-51385 starting with 11 and tert-butanol. 1H NMR (CDCl3): δ 1.52 (s, 9H), 6.18 (broad s, 2H), 6.63 (broad s, 1H), 6.72 (s, 1H), 7.38 (d, J=8.5 Hz, 2H), 7.57 (d, J=8.5 Hz; 2H) ppm.
Compounds 5a and 5b were identified as inhibitors of tau fibril assembly during the screening of a large compound library (Crowe et al., Biochemistry, Epub Jul. 6, 2009). The ATPZ scaffold chemotype has not been identified in previous tau fibrillization screens and as these compounds showed drug-like structural features meriting their further characterization, we assessed whether this class of molecules interfered with the ability of tau to bind and stabilize MTs.
As depicted in
To further investigate the nature of the tau that remained in solution after incubation with an ATPZ inhibitor, aliquots of the post-centrifugation supernatants from full-length tau40 fibril assembly reactions conducted in the presence or absence of 50 μM 5b were analyzed by size-exclusion chromatography (SEC) to allow for the separation of monomeric tau40 from larger oligomeric species (Li, W. K. and Lee, V. M. Y. (2006) Biochemistry 45, 15692-15701). As shown in
The promising properties of the ATPZ class of tau assembly inhibitors led to the synthesis of additional analogs with the aim of evaluating possible SAR. As listed in Tables 3 and 4, a series of analogs were synthesized which exhibited a variety of substitutions of the ATPZ core structure. In addition, the previously tested compounds 5a and 5b were re-synthesized to confirm batch-to-batch reproducibility. All compound identities were confirmed by molecular mass determination, and were found to be least 85% pure. Identities of the synthesized analogs were further confirmed by NMR spectroscopic analysis The various ATPZ analogs were evaluated in the K18PL ThT and sedimentation assays to determine their IC50 values and percent maximal inhibition (Tables 3-5). Interestingly, structural modifications in the fragment linked at C-4 appear to be generally well tolerated. Indeed, compounds bearing R3 modifications have been identified that appear to cause greater maximal inhibition in the ThT and centrifugation assays than the original hits identified during qHTS.
The ATPZ analogs were also examined in the Aβ(1-42) fibrillization assay to determine whether the relative lack of activity observed with the representatives from the screening library (
1Calculated physical-chemical properties, including molecular weight (MW), polar surface area (PSA) and calculated log permeability (cLogP).
2IC50 values for K18PL tau ThT fluorescence inhibition was as described in Experimental Procedures. The percent inhibition represents that obtained with the highest tested concentration (80 μM) of compound.
3Secondary testing of compounds in the K18PL tau centrifugation assay as described in Experimental Procedures.
4Analysis of the compounds in the Aβ(1-42) fibrillization assay as described in Experimental Procedures. The percent inhibition represents that obtained with the highest tested concentration (80 μM) of compound.
2IC50 values for K18PL tau ThT fluorescence inhibition was as described in Experimental Procedures. The percent inhibition represents that obtained with the highest tested concentration (80 μM) of compound.
3Secondary testing of compounds in the K18PL tau centrifugation assay as described in Experimental Procedures.
The solubility of test compounds in the sodium acetate buffer used for subsequent fibrillization reactions was determined by turbidimetric measurements. Compounds were then evaluated in a heparin-induced tau assembly assay, in which the fibrillization of the truncated K18 tau fragment (comprised of four MT-binding repeats) bearing the P301L mutation (K18PL) found in FTDP-17, was monitored by thioflavine-T (ThT) binding and fluorescence (example in
Notably, compounds E1, 17, 2, and F1-F7 also inhibited the aggregation of full-length tau (tau40) as determined by sedimentation assay, which revealed efficacy values in the 50-60% range (cf., Table 5). Compounds E1, 17, 2, and F1-F7 did not appear to interfere with the normal MT-stabilizing function of tau in a MT-polymerization assay (example shown in
Test compounds underwent preliminary evaluations of brain penetration, in which 5 mg/Kg of each compound was administered intraperitoneally (i.p.) to a group of three normal mice; drug levels in brain and plasma were determined at a single time-point (1 h) by LC-MS/MS using pre-validated calibration curves. The results of these experiments, summarized in Table 5, revealed that with the exception of CNDR-51348 (E1), all other test compounds displayed significant brain uptake as demonstrated by the brain-to-plasma exposure ratios (B/P) above 0.3. The lack of brain penetration of CNDR-51348 was not unexpected given that the carboxylic acid moiety of this compound would be mostly negatively charged at physiological pH, and thus likely result in limited passive diffusion of the compound across the BBB. Conversely, the more lipophilic ester derivatives (CNDR-51346 and CNDR-51371) exhibited comparatively higher B/P ratio. In particular, CNDR-51371 (2) was found to reach significantly higher brain concentrations compared to the corresponding acid CNDR-51348. However, both esters appeared to have relatively short half-lives in plasma, as indicated by the limited amount of parent drug detected after 1 h from administration of the compounds. Moreover, monitoring for the hydrolyzed metabolite (i.e., CNDR-51348) in both brain and plasma revealed a considerable amount of acid CNDR-51348 in plasma, but not in the brain 1 h after administration of either ester. CNDR-51362 was selected for full PK analysis in which brain and plasma drug levels were determined at six time points (i.e., 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h). The results from this experiment, illustrated in
Assuming that similar stoichiometric requirements also exist in vivo, any ATPZ candidate for in vivo efficacy study will have to reach free brain concentrations that are comparable to that of the unbound fraction of tau. The total intraneuronal tau concentration (i.e., MT-bound and MT-unbound tau) is estimated to be in the low μM range and, as previously noted, >99% of the protein is bound to MTs under physiological conditions. Although, the concentration of the MT-unbound fraction of tau is likely to increase in diseased neurons, this may be in the sub μM range. Thus, only compounds that readily cross the BBB would be viable candidates for in vivo evaluations of efficacy. Interestingly, preliminary evaluation of brain exposures of ATPZ test compounds revealed that with the exception of the acid derivative CNDR-51348, all other ATPZ congeners exhibited B/P ratios above 0.3. Considering that most CNS-active drugs typically exhibit B/P>0.3-0.5, these results indicate that ATPZs have the potential to achieve appreciable brain concentrations.
Furthermore, selected amide derivatives, such as CNDR-51349, CNDR-51362 and CNDR-51365, were found to reach brain concentrations above 800 ng/g (i.e., >2 μM) 1 h after i.p. administration of 5 mg/Kg. These results were further confirmed by complete PK studies on CNDR-51362, which displayed an average brain-to-plasma AUC exposure ratio of ˜1.6. Equally important, significant brain concentrations were also observed after oral administration of CNDR-51349 or CNDR-51362, with the oral bioavailability of the latter compound being ˜64%. Collectively, the results from the PK studies combined with the promising in vitro activity and safety data suggest that the ATPZ class of tau aggregation inhibitors hold considerable promise as candidate compounds for efficacy testing in transgenic mouse models of tauopathies.
aPhysical-chemical properties (MW: molecular weight; PSA: polar surface area; ClogP: calculated partition coefficient between n-octanol and water);
bTau (K18PL) fibrillization assay monitored by thioflavine (ThT) binding and fluorescence;
cMaximal percent inhibition of the K18PL fibrillization as determined by SDS-PAGE analyses of the soluble and insoluble fractions obtained after centrifugation of the fibrillizing K18PL mixtures;
dMaximal percent inhibition of the tau40 fibrillization as determined by SDS-PAGE analyses of the soluble and insoluble fractions obtained after centrifugation of the fibrillizing tau40 mixtures;
eRatio between brain/plasma drug level, as determined by LC-MS/MS, 1 h after i.p. administration of 5 mg/Kg of test compound;
øIC50 value;
†Detected amount of hydrolysis product (i.e., CNDR-51348)
This application claims the benefit of U.S. Provisional Application Nos. 61/245,045, filed Sep. 23, 2009, and 61/309,554, filed Mar. 2, 2010, the entireties of which are incorporated herein.
This work was supported by grants from the National Institutes of Health (P01 AG09215, P30 AG10124, P01 AG11542, P01 AG14382, P01 AG14449, P01 AG17586, P01 AG19724, P01 NS-044233, U01 AG24904) and the NIH Roadmap for Medical Research and the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health. Pursuant to 35 U.S.C. §202, the government may have rights in any patent issuing from this application.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/49799 | 9/22/2010 | WO | 00 | 9/13/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61245045 | Sep 2009 | US | |
| 61309554 | Mar 2010 | US |
