METHYLENE-CYCLOALKYLACETATE DERIVATIVES AND THEIR USE IN TREATMENT OF NEUROTROPIC CONDITIONS
20200239398 ยท 2020-07-30
Assignee
Inventors
- Dmitry Tsvelikhovsky (Nof Hagalil, IL)
- Philip Lazarovici (Jerusalem, IL)
- Dikla HAHAM (Jerusalem, IL)
- David Lankri (Beit Shean, IL)
Cpc classification
A61P25/28
HUMAN NECESSITIES
C07C69/608
CHEMISTRY; METALLURGY
C07C255/31
CHEMISTRY; METALLURGY
C07D211/70
CHEMISTRY; METALLURGY
International classification
C07C57/26
CHEMISTRY; METALLURGY
C07C255/31
CHEMISTRY; METALLURGY
C07D211/70
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
Abstract
Methylene-cycloalkylacetate compounds and derivatives thereof and their use in methods for treatment of neurotropic conditions.
Claims
1. A compound having the general formula (I), including any stereoisomer or salt thereof: ##STR00009## wherein R.sub.101 is selected from H, straight or branched C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl; m is an integer selected from 1-10; C.sub.(m) is selected from a straight or branched alkylene, straight or branched alkenylene, straight or branched alkynylene; optionally interrupted by at least one heteroatom; R.sub.102 is selected from C(O)R.sub.108, C(S)R.sub.109, C(P)R.sub.110, C(CR.sub.111R.sub.112)R.sub.113, straight or branched C.sub.2-C.sub.10 alkenyl, straight or branched C.sub.2-C.sub.10 alkynyl; each of R.sub.108, R.sub.109, R.sub.110, R.sub.111, R.sub.112 and R.sub.113 are independently is selected from a group consisting of OH, OR.sub.114, NH.sub.2, NHR.sub.115, NR.sub.116R.sub.117; each of R.sub.114, R.sub.115, R.sub.116 and R.sub.117 are independently selected from straight or branched C.sub.1-C.sub.10 alkyl; l is an integer selected from 1-10; C.sub.(l) is selected from a straight or branched alkylene, straight or branched alkenylene, straight or branched alkynylene; optionally interrupted by at least one heteroatom; R.sub.103, R.sub.104, R.sub.105 are each independently selected from H, straight or branched C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl; provided that when R.sub.101 is H (i) at least one of R.sub.103, R.sub.104 and R.sub.105 is different than H; OR (ii) m>1.
2. The compound according to claim 1, wherein R.sub.102 is a straight or branched C.sub.1-C.sub.10 alkyl.
3. The compound according to claim 1, wherein R.sub.102 is H.
4. The compound according to claim 1, wherein m is 1.
5. The compound according to claim 1, wherein m is 2-5.
6. The compound according to claim 1, wherein m is 2, l is 1 and R.sub.102 is C(O)OH.
7. The compound according to claim 1, wherein R.sub.101 is H.
8. The compound according to claim 1, wherein R.sub.101 is a straight or branched C.sub.1-C.sub.10 alkyl.
9. The compound according to claim 1, wherein R.sub.103 is a straight or branched C.sub.1-C.sub.10 alkyl.
10. The compound according to claim 1, wherein R.sub.104 is a straight or branched C.sub.1-C.sub.10 alkyl.
11. The compound according to claim 1, wherein R.sub.105 is a straight or branched C.sub.1-C.sub.10 alkyl.
12. The compound according to claim 1, wherein R.sub.103, R.sub.104, R.sub.105 are each independently a straight or branched C.sub.1-C.sub.10 alkyl.
13. The compound according to claim 1, wherein R.sub.103, R.sub.104, R.sub.105 are H.
14. The compound according to claim 1, wherein l is 1-5.
15. The compound according to claim 1, selected from the following: ##STR00010##
16. A method of treatment of a neurotropic condition in a subject in need thereof; said method comprising administering to said subject a compound of the general formula (II), including any stereoisomer or salt thereof: ##STR00011## wherein Ring A is optionally a saturated or unsaturated ring having optionally at least one heteroatom; and is optionally substituted by at least one group selected from straight or branched C.sub.1-C.sub.10 alkyl, straight or branched C.sub.2-C.sub.10 alkenyl, straight or branched C.sub.2-C.sub.10 alkynyl, CN, OR.sub.4, NR.sub.5R.sub.6, C(O)R.sub.7, halogen; R.sub.4, R.sub.5 and R.sub.6 are each independently selected from H, halogen, straight or branched C.sub.1-C.sub.10 alkyl, straight or branched C.sub.2-C.sub.10 alkenyl, straight or branched C.sub.2-C.sub.10 alkynyl; R.sub.7 is selected from H, straight or branched C.sub.1-C.sub.10 alkyl, straight or branched C.sub.2-C.sub.10 alkenyl, straight or branched C.sub.2-C.sub.10 alkynyl, halogen, OH, O(C.sub.1-C.sub.10)alkyl, NH.sub.2, amine; n is an integer selected from 1-10; C.sub.(n) is selected from a straight or branched alkylene, straight or branched alkenylene, straight or branched alkynylene; optionally interrupted by at least one heteroatom; m is an integer selected from 1-10; C.sub.(m) is selected from a straight or branched alkylene, straight or branched alkenylene, straight or branched alkynylene; optionally interrupted by at least one heteroatom; R.sub.1 is selected from C(O)R.sub.8, C(S)R.sub.9, C(P)R.sub.10, C(CR.sub.11R.sub.12)R.sub.13, straight or branched C.sub.2-C.sub.10 alkenyl, straight or branched C.sub.2-C.sub.10 alkynyl; each of R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are independently is selected from a group consisting of OH, OR.sub.14, NH.sub.2, NHR.sub.15, NR.sub.16R.sub.17; each of R.sub.14, R.sub.15, R.sub.16 and R.sub.17 are independently selected from straight or branched C.sub.1-C.sub.10 alkyl; R.sub.2 is selected from O, S, CR.sub.18R.sub.19; each of R.sub.18 and R.sub.19 is independently selected from H, straight or branched C.sub.1-C.sub.10 alkyl, halogen, CF.sub.3SO.sub.3, OH, C.sub.1-C.sub.10 alkoxy. l is an integer selected from 1-10; C.sub.(l) is selected from a straight or branched alkylene, straight or branched alkenylene, straight or branched alkynylene; optionally interrupted by at least one heteroatom; R.sub.3 is selected from C(O)R.sub.20, OR.sub.21, C(O)OR.sub.22, CF.sub.3SO.sub.3, straight or branched C.sub.2-C.sub.10 alkenyl, straight or branched C.sub.2-C.sub.10 alkynyl; each of alkenyl or alkylenyl groups are optionally substituted by at least one group selected from C(O)R.sub.23, OR.sub.24, halogen, CF.sub.3SO.sub.3; each of R.sub.20, R.sub.21, R.sub.22, R.sub.23 and R.sub.24 is independently selected from H, OH, halogen, straight or branched C.sub.1-C.sub.10 alkyl, straight or branched C.sub.1-C.sub.10 alkoxy, NH.sub.2, amine.
17. A method according to claim 15, wherein said neurotropic condition is associated with neuronal cell death and/or loss of neuronal pathways.
18. A method according to claim 15, wherein said neurotropic condition benefits from mimicking Neuronal Growth Factor (NGF) activity.
19. A method according to claim 15, wherein said neurotropic condition is selected from Parkinson's disease, Alzheimer's disease, traumatic brain injuries, stroke, psychiatric disorders, choreas, ALS, conditions benefiting from neuronal regeneration, conditions benefiting from scaffolds for neural tracks regeneration, conditions benefiting from neural stem cell differentiation, conditions benefiting from tissue engineering, conditions benefiting from regenerative medicine and any combinations thereof.
20. A method according to claim 15, wherein said treatment provides peripheral nerve repair.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:
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[0092] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0093] The compounds of the invention were shown to be able to regenerate neurotropic activity that could be used to treat brain neurological disorders and regenerative medicine. For instance, the neurotropic and NGF-enhanced effects were shown in PC12 model using natural herb products, diterpenenes from the plant Croton yanhuii, sesquiterpenes and iridoids from the plant Valeriana, sargaquinoic acid from the marine brown alga Sargassum macrocarpum, and prenylflavonoids abundant in many plants. Also, neurotropic and NGF enhancing effects were found in PC12 cells using synthetic molecules such as catecholamine precursor dihydroxyphenylalanine (L-DOPA), N-propargyl caffeate amide, pyrimidine heterocyclic compounds, and synthetic peptides.
[0094] A series of compounds 5-21 featuring diverse electronic and steric characteristics were prepared (displaying varying ring architectures and functional group combinations; Scheme 1).
##STR00004## ##STR00005## ##STR00006## ##STR00007##
[0095] To examine whether the compact cycloalkyl scaffolds are endowed with neurotropic activity, the inventors have first screened a library of synthesized compounds 5-13, illustrated in Scheme 1. The functional modifications of general methylene-cycloalkylacetate frame, in this case, were performed on the alkene-bearing chains (5-8), the cyclic domains (9-12) and the length of an acid chain (compound 13).
[0096] Prior to investigation of the activation profile of the compounds, using the PC12 cell neurotropic assay, the inventors evaluated all compounds for cytotoxicity from 0.1 to 100 M. Since the majority of compounds were not cytotoxic at 10 M they were evaluated and compared at this concentration (Table 1).
TABLE-US-00001 TABLE 1 Neurotropic activity of the methylene- cycloalkylacetate novel derivatives Neurotropic effect (mean SE) Two Days Seven Days NGF NGF Compound .sup.a D.sub.f (%) D.sub.f (%) 5 0.42 0.01 91.3 0.20 0.17 43.5 6 0.14 0.15 30.4 0.20 0.17 43.5 7 0.12 0.14 26.1 0.20 0.17 43.5 8.sup.b n.a n.a 9 n.a 0.01 0.01 2.2 10 n.a n.a 11 n.a 0.01 0.01 2.2 12 n.a 0.17 0.16 37.0 13 0.39 0.01 84.8 0.45 0.01 97.8 14 n.a n.a 15.sup.b n.a n.a 16 n.a n.a 17 n.a n.a 18.sup.b n.a n.a 19 n.a n.a 20 n.a n.a 21 n.a 0.01 0.01 2.2 .sup.a All compounds were evaluated at a concentration of 10 M; n.anot active. .sup.bPartial toxicity of about 30% of the culture was observed at 10 M.
[0097] Compounds 5, 6, 7 and 13 were found the most active in inducing fast neurite outgrowth after two days of treatment, while after seven days of treatment a significant neurotropic activity was measured for compounds 5, 6, 7, 12 and 13 (Table 1 and
[0098] By comparing to NGF effect, the most potent compounds were 5>13>6>7 and 13>5, 6, 7>12 after two and seven days of treatment, respectively. Therefore, it was concluded that compounds 5 has a transient, acute effect on induction of neurite outgrowth, losing 50% activity from day two to seven, while for compounds 6, 7, 12 and 13, the neurotropic activity was gradually increased from day two to seven, reminiscent of NGF neurotropic activity time course. Thus, it was found that compact, methylene-cycloalkylacetate-based molecules could induce significant, NGF-like neurotropic activity, in PC12 neuronal cells. Interestingly, no activity was detected for compounds with substituted aromatic anchors (compounds 10 and 11), or 1,4-dimethylenated substrate (compound 9).
[0099] After concluding that structures 5, 6, 7, 12 and 13 are endowed with neurotropic activity, the inventors then examined the functionality of monocyclic variants integrated with heteroatoms. For this purpose, compounds 14-17 were designed according to the reported methodology, 21 and their neurotropic activity was identically measured using the PC12 cell neurotropic assay. As shown in
[0100] Based on these results, a synthetic protocol was applied also to unsaturated variants of cycloalkylacetate substrates. For these series of molecules (compounds 18-21), the cyclic alkene group was substituted by a ketone residue, and their neurotropic activity was again measured using the PC12 cell neurotropic assay. No significant neurotropic activity was detected for the above-mentioned compounds. This observation suggests that an alkene element is indispensable for the neurotropic activity. However, for compounds 14-17, which resemble the topology of active compounds 5, 6, 7 and 12 and retain the double bond, an introduction of heteroatoms within the central ring resulted with complete loss of neurotropic activity. It should be noted that partial cytotoxic activity was detected for compounds 8, 15 and 18. Thus, due to interference with neurotropic activity, these MCA derivatives were excluded from the characterization.
[0101] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.
[0102] Experimental Section
[0103] Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. DMEM medium, fetal calf (FCS) and horse (HS) serums, penicillin and streptomycin were purchased from Biological Industries (Beit Haemek, Afula Israel). Tissue culture grade mouse -NGF, was purchased from Alomone Labs (Jerusalem, Israel). Solvents used in the reactions were distilled from appropriate drying agents prior to use. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 aluminium plates (Merck) and/or gas chromatography-mass spectrometry (GCMS). Visualization of compounds on TLC was accomplished by irradiation with UV light at 254 nm and/or vanillin stain. GCMS Analysis was performed with Agilent 7820A gas chromatograph equipped with Agilent 5975 quadrupole mass selective detector, using Agilent HP-5MS capillary column (30 m, 0.25 mm, 0.25 m film). Column chromatography was performed using silica gel 60 (particle size 0.040-0.063 mm) purchased from Sigma-Aldrich. Proton and carbon NMR spectra were recorded on Varian Mercury 300 MHz or Varian Mercury 500 MHz spectrometer in deuterated solvent. Proton chemical shifts are reported in ppm () relative to tetramethylsilane with the solvent resonance employed as the internal standard (CDCl.sub.3, 7.26 ppm). .sup.13C chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard (CDCl.sub.3, 77.0 ppm). Data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet), integration and coupling constants (Hz). Infrared (IR) spectra were recorded on a Thermo Fischer Scientific NICOLET iS10 spectrometer.
[0104] Methods
[0105] Cell Cultures
[0106] PC12 cells were grown in T-200 flasks in high glucose (4.5 gr/L) DMEM medium supplemented with 7% FCS, 7% horse serum and 1% penicillin and streptomycin. Cells were incubated at 37 C. in a humidified incubator containing 6% CO.sub.2. All experiments were carried out in a clean room, according to ISO7 requirements (10,000 particles/m.sup.3).
[0107] Cell Seeding
[0108] Tissue culture Falcon plates were coated with 200 g/ml collagen type 1 and placed under UV light for 30 minutes for sterilization. Thereafter, one ml of PC12 cell suspension (5000 cells/well) was applied in 12 or 24 well plate, respectively. The cultures were grown in the incubator two days before exposure to investigated compound.
[0109] Neurotropic Activity (Neurite Outgrowth Assay)
[0110] This experimental procedure contained two controls. The first consisted of untreated cells (negative control), representing the random effect: noise signalthe ability of cells to spontaneously grow neurite outgrowth which in seven days are of a length less than two-fold cell diameter. Also, negative controls consisted of cultures treated with 1% DMSO, solvent used to solubilize all compounds. The positive control consisted of cells treated with 50 ng/ml NGF, indicating maximal neurite outgrowth that can be achieved in this model. The experimental group consisted of PC12 cells treated with synthetic compounds. In each experiment, after two and seven days, six cultures were evaluated for neurotropic effect. These consecutive observations allow measurement of the progress of neurite outgrowth elongation and the percentage of cells with neurites..sup.31
[0111] Analysis of the Neurotropic Activity
[0112] In order to assess neurotropic effect, the neurite outgrowth length in each culture was quantified. For this purpose, each culture was placed under an inverted microscope and photographed by the attached camera. Each well was photographed at three to five representative areas. After acquiring the photos, they were analyzed by ImageJ, NIH software. The neurite outgrowth was estimated by fractal dimension (D.sub.f), a statistical parameter that describes the fractional space (area and length) filled by neurite outgrowth. D.sub.f ranged from 0 to 1.20. This method estimates the amount of picsel covered by neurites/cells compared to empty space per square field, and therefore plotting log (number of square boxes) vs. log (size in pixels) relationship generates a linear curve with D.sub.f representing the slope of the curve. Every photograph that was taken, was opened in a Photoshop software and a new layer was generated on it. Using a 5-pixel wide digital pencil tool, all the outgrowths were marked and the layer with the markers (outgrowth network) was saved in a 0-255 gray scale as described in
[0113] Cytotoxicity
[0114] Cell death was evaluated by morphological appearance of the cells and release of LDH into the medium, in the absence and presence of different concentrations of synthetic compounds as previously described..sup.33
[0115] Statistics
[0116] Each experiment was performed in duplicates and repeated three to four times (n=6-8). By using SPSS software, one way ANOVA was performed for the fractional dimension of each compound, in order to evaluate the neurotropic effect. In case of significance, Bonferroni post-hoc analysis was performed. The results were considered significant when p<0.05.
[0117] Acute Tolerance in Mice of HU-MCA-13 (Compound 13 in Scheme 1), Delivered by iv Injection in a Single-Dose
[0118] This pilot study was conducted to obtain acute information of the dose toxicity of HU-MCA-13 (Compound 13 in Scheme 1) in male mice. Male C57BL/6 mice, were injected intravenously with 0.2 ml of HU-MCA-13 in a dose of 250 mg/kg. Mice were monitored for 4 consecutive weeks for autonomic and neurotoxicity symptoms. The mortality and changes on body weight, clinical signs, gross observation, were monitored up to 30 days after treatment. No mortality was found at this high dose of 250 mg/kg. The results obtained in this study might predict that LD.sub.50 after single dose of HU-MCA-13 must be over 2000 mg/kg in male mice. No significant changes on body weight were detected by comparing HU-MCA-13 250 mg/kg to 1% DMSO treated groups: they started with 21 gram body weight and ended with 27 gram body weight. The mice were examined once a week for autonomic symptoms by measuring salivation, urinary delivery, pupillary constriction, heart rate, blood pressure, and hair contraction. However no differences were observed between the HU-MCA-13 250 mg/kg to 1% DMSO treated groups. Occurrences of either flaccid or spastic paralysis of the legs were not observed. At injection of this high dose of 250 mg/kg body weight, the mice did not suffer from visible weakness and/or exhaustion. No paralysis, altered motor activity, or irregular behavior was observed in mice treated, suggesting a lack of neurotoxicity. Cutaneous hematomas around the injection or at distant locations site within 24 hours after injection have not been observed. Furthermore, no mice sudden deaths occurred within 24 hours after the injection or during the 4 weeks of observation. Sensory motor/neurological performance was weekly measured using several routine motor tests, but no neurological losses were observed (Table 2). Sensory motor tests were scored individually and according to the modified neurologic function severity score (mNSS). The data from each individual test and the total score calculated from individual tests that make up the mNSS were measured. The following assays were used: a. beam balance testmice were placed on a 1-inch wide beam for 60 seconds. A normal response is balance with steady posture for 60 seconds (a score of 0). Deficits are scored if the rat: grasps the side of the beam (a score of 1), hugs the beam and one limb falls down from the beam (a score of 2), hugs the beam and two limbs fall off the beam (a score of 3), attempts to balance on the beam, but falls off from 60 seconds (a score of 4), attempts to balance on the beam but falls off from 60 seconds (a score of 5), or falls off with no attempt to balance or hang on the beam in <20 seconds (a score of 6); b. foot fault testforelimb movement dysfunction, while walking on elevated metal grids, was monitored. The animal was placed on horizontal grids (85.526.520) cm with a glass enclosure for observation. With each weight bearing step, the forelimb can fall or slip between the metal support bars, an event recorded as a foot fault. The total number of forelimb steps and the total number of foot faults were recorded. The percentage of forelimb foot faults to the total steps that occurred within 2 minutes marks the test results; c. hind limb placing testThis test monitors reposition of hind limbs placed down and away from the table edge. The ability to retrieve and place the hind limbs back onto the table was scored. Immediate and complete limb retrieval was scored 0, delayed (>2 seconds) limb retrieval and/or interspersed flailing was scored 1, and no limb retrieval was scored 2; d. Increasing platform angle to slide testThis test monitors the strength and stability of resistance to slide down an inclined platform. It deploys a clean wooden board (60 cm by 40 cm) positioned horizontally. Mice were placed on the board with head facing up the increasing incline. The board then swings to increase the degree of inclination the rat experiences until slipping down the board feet first. The degree of the angle at which the rat relinquishes their grip/stance on the board (slips down) constitute the test results; e. the forelimb whole body suspension testThe test measures grip strength by duration of suspension by forepaws. The rat is suspended on a metal bar (diameter 5 mm) which it tightly holds onto by its forelimbs. The time (seconds) during which each rat could sustain its body weight while holding onto the bar is recorded. The mice drop from the bar onto soft material with no harm. The test was repeated three times and a mean result per rat was used as the score. Cumulatively these sensory motor/neurological findings indicate acute tolerability of 250 mg/kg HU-MCA-13 iv injection in Male C57BL/6 mice.
TABLE-US-00002 TABLE 2 Summary of sensory motor/neurological-neurobehavioral tests results for each individual animal in the 5-day study HU-MCA-13 DMSO (Compound 13 in Scheme 1) mNSS Day 0 0 0 0 0 0 0 0 Day 1 0 0 0 0 0 0 0 Day 5 0 0 0 0 0 0 0 Foot fault (%) Day 0 0/24 0/33 0/30 0/34 0/41 0/38 1/48 Day 1 0/52 0/59 0/67 0/20 0/15 0/34 0/14 Day 5 0/48 0/54 0/33 2/50 1/47 0/48 0/36 Hind limb Day 0 0 0 0 0 0 0 0 Day 1 0 0 0 0 0 0 0 Day 5 0 0 0 0 0 0 0 Beam balance Day 0 0 0 0 0 0 0 0 Day 1 0 0 0 0 0 0 0 Day 5 0 0 0 0 0 0 0 Self-suspension time (seconds) Day 0 10 7 8 8 10 9 10 Day 1 8 14 10 14 12 15 12 Day 5 9 14 13 13 13 15 14 Angle of increase (degrees) Day 0 35 35 35 35 35 35 35 Day 1 35 35 35 35 35 35 35 Day 5 35 35 35 35 35 35 35
[0119] Tail vein blood samples were taken from DMSO 1% treated control (n=3) and HU-MCA-13-injected mice (n=4) after 24 hours from injection and submitted for hematocrit cell counting and biochemical analysis. Blood analyses are depicted in
[0120] Cumulatively these blood analysis findings indicate acute tolerability of 250 mg/kg HU-MCA-13 upon iv injection in male C57BL/6 mice.
[0121] HU-MCA-13 Safety Profiling Using DiscoverX's SAFETY Scan In Vitro Pharmacological Profiling
[0122] On-target activity refers to the site of action of the test substance (e.g., target receptor or enzyme) that results in the desired pharmacodynamic effect associated with the treatment of disease. Off-target activity refers to all other targets for which the molecule has affinity with the outcome of activation, blockade, or modulation, resulting in a functional effect. In many cases, the off-target activity of the molecule can be sub-clinical and not pose a concern. On the other hand, the off-target activity may result in side effects of the active agent that range from a minor nuisance to a severe adverse event in both preclinical studies and clinical trials. With this background, to further provide the basic information on the safety of HU-MCA-13 and exclude other potential molecular targets for toxicity, Safety74 experiments were performed, to improve off-target liability testing. Safety47 panel includes the receptors and kinases and other cellular targets recommended by major pharmaceutical companies for safety profiling of a novel drug entity. Assessing the specificity of HU-MCA-13 early in development, using highly relevant and predictive assays such as DiscoverX's SAFETYscan analysis, allows more informed decisions about compound safety, ultimately leading to the development of safer and more effective HU-MCA-13 drug.
[0123] In the analysis, 78 assays were performed utilizing the PathHunter enzyme fragment complementation (EFC) technology, FLIPR-based cellular screening assays, KINOMEscan kinase binding assays, and a variety of enzymatic assays, accumulating 780 data points regarding the potential interaction of HU-MCA-13 with a large variety of in vitro pharmacological models of GPCR, tyrosine kinase receptors, second messenger and effector systems, using 10 M of HU-MCA-13. This concentration was chosen since it is not toxic to neurons and since was found very active in stimulating neurite outgrowth in the dopaminergic PC12 cell culture neuronal model (original paper and patent).
[0124] Table 3, last column showing the maximal response measured with 10 M HU-MCA-13, it is evident that HU-MCA 13 is not interacting with the majority of the targets with the exception of the 4 G-protein coupled receptors (GPCRs): 2A-adrenergic receptor ADRA2A (24.5%); Cannabinoid receptor CB1-CNR1 (50.1%) Cannabinoid receptor CB2-CNR2 (49.6%); Histamine H2 receptor HRH2 (26.9%) which are potential targets at high concentrations (doses) of HU-MCA-13. In such a case it is expected that upon using overdoses or toxic doses of HU-MCA-13 this could predict the physiological effects on blood pressure, adrenaline release, sedation, GI motility (ADRA2 related effects), euphoria and dysphoria; anxiety, memory impairment and poor concentration; analgesia, hypothermia weight loss, emesis, depression (CNR1 effects); inflammation and bone mass (CNR2 effects) and gastric acid secretion, emesis, and positive heart inotropic effects (HRH2 effects).
[0125] Cumulatively HU-MCA-13 profiling using pilot DiscoverX's SAFETYscan indicate Off-target safety and predicts some toxic effects over the therapeutic window. These findings strongly support further drug development of HU-MCA-13 pharmacophore.
TABLE-US-00003 TABLE 3 Screening in vitro of HU-MCA-13 effects on selected GPCRs, ion channels, kinases, nuclear hormone receptors, enzymes and neurotransmitter transporters. HU- MCA-13 Target Target Reference Maximal Protein Gene Compound Mode of Functional RC.sub.50 Response name name Name.sup.1 action.sup.2 Assay.sup.3 (M).sup.4 (%).sup.5 GPCRs Adenosine ADORA2A NECA Agonist Calcium 0.01524 0 Receptor A.sub.2A SCH 442416 Antagonist Flux 0.04332 4.09 Adrenergic ADRA1A A61603 Agonist Calcium 0.0011 1.12 Receptor .sub.1A Tamsulosin Antagonist Flux 0.00096 3.06 Adrenergic ADRA2A UK 14304 Agonist cAMP 0.00066 24.55 Receptor .sub.2A Yohimbine Antagonist 0.02617 0 Adrenergic ADRB1 Isoproterenol Agonist cAMP 0.00125 1.92 Receptor .sub.1 Betaxolol Antagonist 0.02278 9.29 Adrenergic ADRB2 Isoproterenol Agonist cAMP 0.0016 1.29 Receptor .sub.2 ICI 118,551 Antagonist 0.00211 8.23 Arginine AVPR1A [Arg.sup.8]- Agonist Calcium 0.00052 0.29 Vasopressin Vasopressin Flux Receptor 1.sub.A SR49059 Antagonist 0.0029 0 Cholecystokinin CCKAR (Tyr[SO.sub.3H]27) Agonist Calcium 0.0002 1.56 Receptor Cholecystokinin Flux A Fragment 26-33 Amide SR 27897 Antagonist 0.0298 0 Muscarinic CHRM1 Acetylcholine Agonist Calcium 0.02359 0 acetylcholine chloride Flux Receptor M.sub.1 Atropine Antagonist 0.00695 7.43 Muscarinic CHRM2 Acetylcholine Agonist cAMP 0.02725 11.68 acetylcholine chloride Receptor M.sub.2 Atropine Antagonist 0.01538 0 Muscarinic CHRM3 Acetylcholine Agonist Calcium 0.04944 0.13 acetylcholine chloride Flux Receptor M.sub.3 Atropine Antagonist 0.00612 8.81 Cannabinoid CNR1 CP 55940 Agonist cAMP 0.00012 50.22 Receptor 1 AM251 Antagonist 0.00466 0.57 Cannabinoid CNR2 CP55940 Agonist cAMP 0.00033 49.59 Receptor 2 SR144528 Antagonist 0.0491 4.11 Dopamine DRD1 Dopamine Agonist cAMP 0.17552 0 Receptor D.sub.1 SCH 39166 Antagonist 0.0026 14.3 Dopamine DRD2S Dopamine Agonist cAMP 0.00321 0 Receptor D.sub.2 Risperidone Antagonist 0.00589 0.51 Endothelin EDNRA Endothelin 1 Agonist Calcium 0.00036 0 Receptor BMS 182874 Antagonist Flux 0.63689 0 Type A Histamine HRH1 Histamine Agonist Calcium 0.0155 0 Receptor H.sub.1 Mepyramine Antagonist Flux 0.01549 1.05 Histamine HRH2 Histamine Agonist cAMP 1.01395 1.78 Receptor H.sub.2 Tiotidine Antagonist 0.06593 26.94 5-Hydroxy HTR1A Serotonin Agonist cAMP 0.01101 7.66 tryptamine Hydrochloride (Serotonin) Spiperone Antagonist 0.08756 0 Receptor 1.sub.A 5-Hydroxy HTR1B Serotonin Agonist cAMP 0.0046 10.47 tryptamine Hydrochloride (Serotonin) SB 224289 Antagonist 0.03853 0 Receptor 1.sub.B 5-Hydroxy HTR2A Serotonin Agonist Calcium 0.0061 1.13 tryptamine Hydrochloride Flux (Serotonin) Altanserin Antagonist 0.01903 0 Receptor 2.sub.A 5-Hydroxy HTR2B Serotonin Agonist Calcium 0.00263 0 tryptamine Hydrochloride Flux (Serotonin) LY 272015 Antagonist 0.00082 0.13 Receptor 2.sub.B Opioid OPRD1 DADLE Agonist cAMP 0.00012 6.34 Receptor Naltriben Antagonist 0.00064 0 Delta .sub.1 Opioid OPRK1 Dynorphin A (1- Agonist cAMP 0.0481 7.89 Receptor 17) Kappa .sub.1 Nor- Antagonist 0.0064 0 Binaltorphimine Opioid OPRM1 DAMGO Agonist cAMP 0.00194 9.31 Receptor Mu .sub.1 Naloxone Antagonist 0.00552 0.38 Ion channels Voltage- CAV1.2 Isradipine Blocker Ion 0.0225 3.82 gated L-type channel calcium channel Gamma- GABAA Picrotoxin Blocker 5.91692 7.74 aminobutyric GABA Opener 8.51412 3.68 acid Receptor A Kv11.1, the hERG Astemizole Blocker 0.07136 0 alpha subunit of a potassium ion channel 5-Hydroxy- HTR3A Bemestetron Blocker 0.00368 0.19 tryptamine Serotonin Opener 0.552217 0 (Serotonin) Hydrochloride Receptor 3.sub.A Kv7.1/ KvLQT1/ XE 991 Blocker 2.63679 0.55 KCNE minK ML-277 Opener 6.71743 0 Potassium voltage- gated channel Nicotinic nAChR Dihydro-AY- Blocker 0.70659 5.95 acetylcholine (a4/b2) erythroidine Receptor- Nicotine Opener 2.18712 0 alpha-4 beta- 2 A NAVI1.5 Lidocaine Blocker 41.01752 0 tetrodotoxin- resistant voltage- gated sodium channel N-methyl-D- NMDAR MK 801 Blocker 0.05277 0 aspartate (1A/2B) L-Glutamic Acid Opener 0.96247 0 (NMDA) Glutamate Receptor 1.sub.A/2.sub.B Kinases Insulin INSR BMS-754807 Inhibitor Binding 0.0009 3.22 Receptor (tyrosine kinase) Lymphocyte LCK Gleevec Inhibitor 0.07191 12.77 Cell-Specific Protein- Tyrosine Kinase (Src family) Rho ROCK1 Staurosporine Inhibitor 0.00034 21.61 Associated Coiled-Coil Containing Protein Kinase 1 (serine- threonine kinase) Vascular VEGFR2 SU-11248 Inhibitor 0.00033 21.68 endothelial growth factor receptor 2 (KDR tyrosine kinase) Nuclear Hormone Receptors Nuclear AR 6a- Agonist Nuclear 0.00646 0 Hormone Fluorotestosterone Hormone Receptor Androgen Geldanamycin Antagonist Translocation 0.08646 5.27 Receptor Nuclear GR Dexamethasone Agonist 0.10198 0 Hormone Mifepristone Antagonist 0.10906 2.28 Glucocorticoid Receptor Enzymes Acetylcholinesterase AChE Physostigmine Inhibitor Enzymatic 0.0282 4.27 Cyclooxygenase COX 1 Indomethacin Inhibitor 0.06281 24.99 1 Cyclooxygenase COX 2 NS-398 Inhibitor 0.07376 7.99 2 Monoamine MAOA Clorgyline Inhibitor 0.00217 1.86 oxidase type A cGMP- PDE3A Cilostamide Inhibitor 0.02113 3.21 inhibited cyclic nucleotide phosphodiesterase 3.sub.A cAMP- PDE4D2 Cilomilast Inhibitor 0.01549 0 specific 3,5- cyclic phosphodiesterase 4D.sub.2 Catecholamine Transporters Dopamine DAT GBR 12909 Blocker Transporter 0.0076 0 transporter Norepinephrine NET Desipramine Blocker 0.01089 3.53 transporter Serotonin SERT Clomipramine Blocker 0.00878 15.45 transporter .sup.1Full chemical names of reference compounds: NECA, 5-(N-Ethylcarboxamido)adenosine; SCH 442416, 2-(2-furyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine; A61603, N-(5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)methanesulfonamide hydrobromide; Tamsulosin, 5-(2-((2-Ethoxyphenoxy)ethyl)amino) propyl-2-methoxybenzenesulfonamide; UK 14304, 5-Bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine; Yohimbine, methyl (1S,15R,18S,19R,20S)-18-hydroxy1,3,11,12,14,15,16,17,18,19,20,21-dodecahydroyohimban-19-carboxylate; Isoproterenol, 4-[1-hydroxy-2-(propan-2-ylamino)ethyl]benzene-1,2-diol; Betaxolol, 1-[4-[2-(cyclopropylmethoxy)ethyl]phenoxyl-3-(propan-2-ylamino)propan-2-ol; Isoproterenol, 4-[1-hydroxy-2-(propan-2-ylamino)ethyl]benzene-1,2-diol; ICI 118551, (2R,3S)-1-[(7-methyl-2,3-dihydro-1H-inden-4-yl)oxy]-3-(propan-2-ylamino)butan-2-ol; hydrochloride. .sup.2The mode of interaction with the biological target; .sup.3The assay describing the major function of the biological target; .sup.4Reference compound effective concentration 50%; .sup.5HU-MCA-13 maximal response at a concentration of 10 M.
[0126] The additive neurotropic effect between HU-MCA-13 and NGF in the PC12 dopaminergic neuronal model A variety of in vitro central and peripheral neuronal models has been developed to understand the mechanisms underlying the regenerative failure of axons, and to guide pre-clinical development of regeneration-promoting therapeutics. By utilizing a combination of several in vitro models, this could balance the speed, convenience, and mechanistic resolution of simpler models for future in vivo pharmacological investigations on therapeutic effect in polyneuropathies.
[0127] Most neuronal high content screening assays use neurite outgrowth in vitro as a surrogate measure for process extension in vivo. This is due to multiple reasons including: i. the time required for nerves in vivo to grow and reliably express axonal or dendritic protein markers (weeks to months) is much over the optimal assay duration of in vitro neurite outgrowth assays (1-7 days), and ii. axonal markers are typically restricted to the neurite at some distance away from the cell body, making it difficult to automatically connect traced processes to the correct cell body number.
[0128] Accordingly, assayed living neurons are typically visualized by microscopy and fixed neurons by immunostaining for tubulin or tau (MAP) cytoskeleton proteins, and growth of putative axons and dendrites is often assessed simultaneously. Neurite outgrowth assays are among the most commonly utilized phenotypic screens relevant to axon regeneration. They have been successfully used to identify small molecules that can promote regeneration in vivo (Al-Ali et al. Rational Polypharmacology: Systematically Identifying and Engaging Multiple Drug Targets To Promote Axon Growth. ACS Chem. Biol. 2015; 10:1939-51). Neurons are typically grown on an adherent substrate that may be coated with one or more neuronal matrix proteins. Permissive substrates such as collagen I and IV and laminin are used to more easily identify agents that stimulate neurite outgrowth since on these matrices the neurons extend neurites at a physiological rate, making the correct window for detecting stimulation of neurite outgrowth and axon regeneration. For single endpoint assays, neurite outgrowth is measured dynamically in live low-density cultures in a label-free setting, using light microscopy followed by image analysis and data mining.
[0129] The Method:
[0130] PC12 cells were cultured in T-200 flasks in high glucose (4.5 gr/L) DMEM medium supplemented with 7% FCS, 7% horse serum and 1% penicillin and streptomycin. Cells will be maintained at 37 C. in a humidified incubator containing 6% CO.sub.2. All experiments were carried out in a GLP clean room, according to ISO 7 requirements (10,000 particles/m.sup.3). For neurotropic experiments, tissue culture Falcon plates were coated with 200 g/ml collagen type 1 and placed under UV light for 30 minutes for sterilization. Thereafter, one ml of PC12 cell suspension (5000 cells/well) was applied in 12 or 24 well plate, respectively. The cultures were maintained in the incubator two days before exposure to investigated compound. Each neurotropic experiment contained two controls. The first consisted of untreated cells (negative control), representing the random effect: noise signalthe ability of cells to spontaneously grow neurite outgrowth which in seven days are of a length less than two-fold cell diameter. Also, negative controls consisted of cultures treated with 1% DMSO, solvent used to solubilize HU-MCA-13. The positive controls represented neuronal cultures treated with 50 ng/ml NGF, indicating maximal neurite outgrowth that can be achieved in this model. The experimental group consisted of PC12 cells treated with 10 M HU-MCA-13. In each experiment, after two and seven days, six cultures will be evaluated for neurotropic effect. These consecutive observations allow measurement of the progress of neurite outgrowth elongation and the percentage of cells with neurites (Katzir et al. A quantitative bioassay for nerve growth factor, using PC12 clones expressing different levels of trkA receptors. J Mol Neurosci. 2002; 18(3):251-64). In order to assess neurotropic effect, the neurite outgrowth length in each culture was quantified. For this purpose, each culture was placed under an inverted microscope and photographed by the attached camera. Each well was photographed at three to five representative areas. After acquiring the photos, they were analyzed by ImageJ, NIH software. The neurite outgrowth was estimated by fractal dimension (D.sub.f), a statistical parameter that describes the fractional space (area and length) filled by neurite outgrowth. D.sub.f ranged from 0 to 1.20. This method estimated the amount of picsels covered by neurites/cells compared to empty space per square field, and therefore plotting log(number of square boxes) vs. log(size in pixels) relationship generated a linear curve with D.sub.f representing the slope of the curve. Every photograph that was taken, was opened in a Photoshop software and a new layer was generated on it. Using a 5-pixel wide digital pencil tool, all the outgrowths were marked and the layer with the markers (outgrowth network) were saved in a 0-255 gray scale. Thereafter, the saved layer was opened by ImageJ NIH software. The software was skeletonize the layer, measuring the length and complexity of every outgrowth in a fractal box count and calculated the fractional dimension parameter (D.sub.f) (Arien-Zakay et al. Quantitative assessment of neuronal differentiation in three-dimensional collagen gels using enhanced green fluorescence protein expressing PC12 pheochromocytoma cells. J Mol Neurosci. 2009; 37(3):225-37).
[0131] Using an established PC12 neurotropic in vitro assay, the regenerative enhancing effect of HU-MCA-13 when applied concomitantly, together with NGF, a known neurotrophin was investigated. It was found that HU-MCA-13 alone at 10 M, enhanced neurite outgrowth to a D.sub.f value of 0.36 at 2 days and 0.30 at 7 days of treatment. NGF alone, as positive control, at the conventional dose of 50 ng/ml enhanced neurite outgrowth to a D.sub.f value of 0.54 at 2 days and 0.4 at 7 days of treatment (
[0132] HU-MCA-13 Induced Neurite Outgrowth in Dorsal Root Ganglion (DRG) Sensory Neurons
[0133] Enhancing sensory axon regeneration after peripheral nervous system (PNS) and central nervous system (CNS) injury remains a goal of clinicians and scientists. Thus, being able to study the effect of HU-MCA-13 on sensory neuronal cultures obtained from dissociated dorsal root ganglia (DRG) has obvious benefits. A feature that distinguishes DRG neurons from other CNS neurons is that their peripheral axons can regenerate long distances and often re-establish appropriate functional connections after PNS injuries. Furthermore, these neurons become pre-conditioned during such a process, such that their central axons acquire an enhanced regenerative/sprouting response. A clear benefit of using DRG neurons is that all neurites extended from these cells are technically axons, as confirmed by immune staining for cytoskeletal-specific proteins. Furthermore, DRG neurons tend to extend neurites in culture at a much higher rate than other CNS neurons, which can shorten an assay's duration and compress its dynamic range. This is particularly true for neurite outgrowth assays.
[0134] To perform these experiments, about 10 dorsal root ganglia (DRGs) were excised under sterile conditions and transferred to 0.25% Type IV collagenase (Sigma-Aldrich-Merck) in Dulbecco's Modified Eagle Medium (DMEM) at 37 C. for 45 min, followed by a 20-min incubation with 0.025% trypsin in DMEM, to digest the associated connective tissue surrounding the neurons. After plating the ganglia onto collagen (50 g/ml)/laminin mixture (5 g/ml)-coated tissue culture wells for 24 h, DRG neurons were cultured for additional 1-7 days in fresh Neurobasal A/B27 containing either 50 ng/ml NGF (positive control), blank medium (DMSO 1%) control and HU-MCA-13. The cultures were photographed; thereafter the ganglia were immune-stained for neuronal tubulin and again photographed using a fluorescent microscope. Image analysis was performed by a researcher blinded to the experimental conditions. Neurite outgrowth distance measured in micrometers, was quantified as for PC12 cultures.
[0135] The results in
[0136] HU-MCA-13 Induced Neurite Outgrowth in Adult Rat Spinal Cord Primary Cultures
[0137] To study the effect of HU-MCA-13 on adult rat spinal cord primary cultures in vitro, a purification procedure was developed that yields highly enriched motor neurons cultures. Spinal cord motor neuron cultures are an important tool for the study of mechanisms involved in motor neuron survival, degeneration and regeneration, motor neuron disorders such as amyotrophic lateral sclerosis or spinal muscular atrophy as well as in spinal cord injury. This bioassay was performed using vehicle (1% DMSO) DMEM negative control and 10 M HU-MCA-13, tested compound. Briefly, dissected pieces of adult rat spinal cord tissue were maintained in phosphate-buffered saline (PBS) without Ca.sup.2+ and Mg.sup.2+ and pooled together and transferred to an enzymatic dissociation media containing 20 IU/ml papain in Earle's balanced salt solution (Worthington Biochemical, Freehold, N.J.) and incubated for 30 min at 37 C. After enzymatic dissociation, the papain solution was aspirated and the tissue was mechanically triturated with a fire-polished Pasteur pipette in complete media [Neurobasal Medium with B-27 supplement (Gibco, Grand Island, N.Y.), 100 IU/ml penicillin, 100 mg/ml streptomycin, 3.3 g/ml aphidicolin, 0.5 mM glutamate] containing 2000 IU/ml DNase and 10-mg/ml protease inhibitors. Single-cell suspensions in complete media was plated on pre-coated collagen IV/laminin mixture-coated 96-well plates (Becton-Dickinson, Bedford, Mass.) at a density of 1.010.sup.4 cells/well. The spinal cord motor neurons (red arrows) and accessory Schwann cells (orange) were maintained for one day in culture and then exposed to 1% DMSO-DMEM negative control or 10 M HU-MCA-13 for 7 days. The cultures were photographed, and Image analysis was performed. Neurite outgrowth was measured and quantified as for PC12 cultures, focusing on two parameters: a. SUM-total area covered by neurite outgrowths; b. mean length of a neurite (microns).
[0138] The results in
[0139] In summary, autonomic and sensor-motory tests indicate that HU-MCA-13, delivered by iv injection in a single-dose of 250 mg/kg, is acute-tolerated in mice. Furthermore, blood analysis findings further support acute tolerability of 250 mg/kg HU-MCA-13 upon iv injection in male C57BL/6 mice. A neurotropic effect was found between HU-MCA-13 and NGF in the PC12 dopaminergic neuronal model. HU-MCA-13 induced neurite outgrowth in dorsal root ganglion (DRG) sensory neurons. HU-MCA-13 induced neurite outgrowth in adult rat spinal cord primary cultures. These findings indicate safety of HU-MCA-13 and further extend the neurotropic effect of HU-MCA-13 to spinal cord motor neurons (cholinergic neurons), in addition to DRG neurons (peptidergic neurons) and PC12 cultures (dopaminergic neurons), justifying further drug development.
[0140] Synthesis
[0141] All compounds were prepared according to the general procedures in (a) Mostinski, Y.; Valerio, V.; Lankri, D.; Tsvelikhovsky, D. J. Org. Chem. 2015, 80, 10464-10473. (b) Valerio, V.; Mostinski, Y.; Kotikalapudi, R.; Tsvelikhovsky, D. Chem. Eur. J. 2016, 22, 2640-2647. (c) Albarghouti, G.; Kotikalapudi, R.; Lankri, D.; Valerio, V.; Tsvelikhovsky, D. Chem. Comm. 2016, 52, 3095-3098. As detailed in Scheme 2A-2B below.
##STR00008##
[0142] Methyl 2-(3-(2-methylallyl)-2-methylenecyclohexyl)acetate (5): 4.5 mmol of corresponding starting material (ketone 2; Scheme 2) were employed. Purification of the final crude product by flash column chromatography (5% ethyl acetate in hexane) yielded pure 5 (0.86 g, 87% yield, colorless oil) as mixture of diastereomers. .sup.1H NMR (300 MHz, CDCl.sub.3): 4.82-4.52 (m, 4H), 3.67 (s, 3H), 2.69-2.41 (m, 2H), 2.38-2.23 (m, 2H), 2.17=1.95 (m, 2H), 1.94-1.82 (m, 2H), 1.82-1.73 (m, 1H), 1.70 (s, 3H), 1.64-1.41 (m, 1H), 1.34-0.76 (m, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3): Major diastereomer: 173.6, 155.0, 144.1, 111.69, 101.8, 51.5, 41.2, 41.0, 41.0, 38.0, 35.5, 34.5, 26.0, 22.3. Minor diastereomer, characteristic peaks: 111.5, 105.9, 51.4, 40.9, 37.9, 37.6, 33.8, 32.3, 22.1, 21.1. IR (neat): 2924, 2853, 1739, 1643, 1437, 1164, 885 cm.sup.1.
[0143] Methyl 2-(3-(3-methylbut-2-en-1-yl)-2-methylenecyclohexyl)acetate (7): 9.7 mmol of corresponding starting material (ketone 2; Scheme 2) were employed. Purification of the final crude product by flash column chromatography (5% ethyl acetate in hexane) yielded pure 7 (1.9 g, 83% yield, colorless oil) as mixture of diastereomers. .sup.1H NMR (300 MHz, CDCl.sub.3): 5.13-5.05 (m, 1H), 4.59 (d, J=23.0 Hz, 2H), 3.65 (s, 3H), 2.65-2.56 (m, 1H), 2.48-2.36 (m, 1H), 2.33-2.16 (m, 2H), 2.01-1.81 (m, 4H), 1.78-1.71 (m, 1H), 1.68 (s, 3H), 1.59 (s, 3H), 1.52-1.21 (m, 1H), 1.12-0.82 (m, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3): 173.6, 155.2, 132.0, 123.3, 101.8, 51.5, 44.4, 40.9, 38.0, 35.5, 34.7, 31.2, 26.1, 25.8, 17.9. IR (neat): 2922, 2853, 1739, 1435, 1164, 887 cm.sup.1.
[0144] Methyl 2-(3-allyl-2-methylene-5-phenylcyclohexyl)acetate (11): 1.4 mmol of corresponding starting material (ketone 2; Scheme 2) were employed. Purification of the crude product by flash column chromatography (5% ethyl acetate in hexane) yielded pure 11 (307 mg, 77% yield, colorless oil) as mixture of diastereomers. .sup.1H NMR (300 MHz, CDCl.sub.3): 7.35-7.12 (m, 5H), 5.93-5.66 (m, 1H), 5.16-4.89 (m, 2H), 4.82-4.60 (m, 2H), 3.68 (s, 3H), 3.15-2.25 (m, 6H), 2.23-1.61 (m, 4H), 1.34-1.15 (m, 1H). .sup.13C NMR (75 MHz, CDCl.sub.3): 173.4, 173.1, 153.8, 152.5, 145.9, 145.1, 137.4, 137.3, 137.1, 128.4, 128.4, 127.1, 126.9, 126.2, 126.2, 125.9, 116.2, 116.0, 115.7, 107.9, 107.7, 106.4, 102.7, 51.6, 51.6, 51.6, 45.2, 44.1, 43.2, 42.5, 41.9, 41.8, 40.8, 40.5, 39.5, 39.1, 39.0, 38.5, 38.4, 38.3, 38.3, 37.8, 37.8, 37.8, 37.5, 37.1, 36.9, 36.9, 35.0, 34.6. IR (neat): 2919, 2851, 1736, 1641, 1436, 1166, 893, 698 cm.sup.1.
[0145] Ethyl-3-allyl-5-(2-methoxy-2-oxoethyl)-4-methylenepiperidine-1-carboxylate (15): 5.1 mmol of corresponding starting material (ketone 2; Scheme 2) were employed. Purification of the final crude product by flash column chromatography (10% ethyl acetate in hexane) yielded pure 15 (447 mg, 31% yield, colorless oil) as mixture of diastereomers. .sup.1H NMR (300 MHz, CDCl.sub.3): 5.87-5.69 (m, 1H), 5.11-5.01 (m, 2H), 4.82-4.70 (m, 2H), 4.17-3.96 (m, 4H), 3.67 (s, 3H), 2.70-2.41 (m, 4H), 2.39-2.26 (m, 2H), 2.21-1.95 (m, 2H), 1.23 (t, J=7.1 Hz, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3): 172.4, 155.4, 150.3, 136.0, 116.8, 106.0, 61.5, 51.8, 50.2, 49.9, 42.1, 39.5, 34.5 (2C), 14.6. IR (neat): 980, 2913, 1738, 1726, 1643, 1434, 1231, 1159, 1126, 994, 897, 767 cm.sup.1.
[0146] Methyl 2-(3-(2-methylallyl)-2-oxocyclohexyl)acetate (18): 25.0 mmol of corresponding starting material (ketone 2; Scheme 2) were employed. Purification of the final crude product by flash column chromatography (10% ethyl acetate in hexane) yielded pure 18 (2.49 g, 44% yield, colorless oil) as mixture of diastereomers. .sup.1H NMR (300 MHz, CDCl.sub.3): 4.75-4.55 (m, 2H), 3.63 (s, 3H), 2.94-2.82 (m, 1H), 2.79-2.68 (m, 1H), 2.57-2.44 (m, 2H), 2.18-2.05 (m, 3H), 1.90-1.72 (m, 3H), 1.64 (s, 3H), 1.44-1.12 (m, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3): Major diastereomer: 211.6, 173.0, 143.3, 111.6, 51.6, 48.2, 47.4, 37.0, 34.9, 34.4, 34.2, 25.2, 22.4. Minor diastereomer, characteristic peaks: 43.6, 39.2, 36.9, 34.3, 30.7, 21.7, 20.1. IR (neat): 2934, 2858, 1736, 1709, 1436, 1165, 888 cm.sup.1.
[0147] Methyl 2-(3-allyl-2-oxocyclohexyl)acetate (20): 31.2 mmol of corresponding starting material (ketone 2; Scheme 2) were employed. Purification of the final crude product by flash column chromatography (5% ethyl acetate in hexane) yielded pure 20 (3.73 g, 57% yield, colorless oil) as mixture of diastereomers. .sup.1H NMR (300 MHz, CDCl.sub.3): 5.84-5.66 (m, 1H), 5.12-4.92 (m, 2H), 3.66 (s, 3H), 2.93-2.72 (m, 2H), 2.57-2.37 (m, 2H), 2.22-2.09 (m, 3H), 1.96-1.73 (m, 3H), 1.45-1.27 (m, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3): 211.4, 173.0, 136.4, 116.2, 51.6, 50.1, 47.3, 34.8, 34.3, 34.1, 33.5, 25.1. IR (neat): 2932, 2859, 1736, 1708, 1436, 1199, 1166, 911 cm.sup.1.
[0148] Methyl 2-(3-(3-methylbut-2-en-1-yl)-2-oxocyclohexyl)acetate (21): 22.0 mmol of corresponding starting material (ketone 2; Scheme 2) were employed. Purification of the final crude product by flash column chromatography (10% ethyl acetate in hexane) yielded pure 21 (2.35 g, 44% yield, colorless oil) as mixture of diastereomers. .sup.1H NMR (300 MHz, CDCl.sub.3): 5.07-4.96 (m, 1H), 3.62 (s, 3H), 2.87-2.68 (m, 2H), 2.41-2.22 (m, 2H), 2.16-2.04 (m, 3H), 1.95-1.67 (m, 3H), 1.62 (s, 3H), 1.53 (s, 3H), 1.43-1.15 (m, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3): 211.8, 173.1, 132.8, 122.0, 51.5, 51.0, 47.2, 34.8, 34.3, 34.2, 27.5, 25.7, 25.2, 17.7. IR (neat): 2928, 2857, 1737, 1709, 1436, 1173, 1109, 996, 857 cm.sup.1.
[0149] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.
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