Substituted TRI-phenyl derivative compounds, compositions comprising same and applications of same

Abstract

This invention provides a glycolic acid substituted tri-phenyl compound and compositions comprising the same for treating various diseases, disorders and conditions with prognostic and therapeutic implications including in personalized medicine applications.

Description

SUBSTITUTED TRI-PHENYU DERIVATIVE COMPOUNDS, COMPOSITIONS COMPRISING SAME AND APPLICATIONS OF SAMEFIELD OF THE INVENTION

[0001] The present invention is directed to a tri-phenyl derivative compound and compositions comprising the same for treating various diseases, disorders and conditions with prognostic and therapeutic implications including in personalized medicine applications.BACKGROUND OF THE INVENTION

[0002] Telomerase is a reverse transcriptase that catalyzes the addition of telomeric repeats to the ends of telomeres thereby elongating telomeres. The catalytic core of telomerase is composed of the RNA subunit TERC (Telomerase RNA Component) and the catalytic protein subunit TERT (Telomerase Reverse Transcriptase). Telomeres are long stretches of repeated sequences that cap the ends of chromosomes. In humans, telomeres are typically 7-10 kb in length and comprise multiple repeats. Telomerase is not expressed in most adult cells, and telomere length decreases with successive rounds of replication.

[0003] Without telomerase the telomeres are shortened at each cell division which leads to senescence, apoptosis and cell death caused by chromosome instability. Telomerase is inactive in somatic cells but active in 90% of cancer cells, where telomerase is reactivated. Although telomerase activation may be dangerous, because it can mimic the cancer development process, telomerase enhancing agents may be theoretically applicable as antiaging agents and clinically useful in certain medical conditions.

[0004] In addition to promoting maintenance of appropriate telomere length, it was recently shown that the TERT protein has various functions in addition to its role in telomere extension. In the nucleus, TERT is involved in gene regulation, chromatin organization and the DNA-damage response.

[0005] During oxidative stress, TERT moves from the nucleus to the mitochondria, where it reduces levels of ROS, DNA damage and apoptosis, and improves mitochondrial membrane potential, respiration and complex I activity, including demonstrations in adult neurons and human brain tissue, in cultivated neurons and in the hippocampal neurons from brain tissue of Alzheimer’s disease patients, where neurons lacking TERT displayed an increased level of oxidative species and increased cellular oxidative damage.

[0006] The interplay between TERT levels and the impact, if any, on the pathogenesis of disease is, as yet, undefined.

[0007] Clearly it remains of interest to provide additional and / or improved compounds that further extend the positive effect on diseases and conditions related to telomerase and any effect on TERT in particular and whether same may as well positively affect disease pathogenesis, which is as yet, elusive.SUMMARY OF THE INVENTION

[0008] This invention, in some embodiments, provides new tri-phenyl substituted compounds, Compounds 2, 3 and 4, of which, in particular, Compound 4 shows remarkably unexpectedly on the one hand greater stability than the structurally related Compound 1, yet paradoxically shows far enhanced rapid hydrolysis in human plasma.

[0009] This invention, for the first time, addresses the effects of Compounds 2, 3 and 4 on TERT expression, with Compound 4 surprisingly significantly outperforming all other compounds in enhancing TERT expression, and in some embodiments, markedly increasing neurofilament expression, and in some embodiments, in particular, in the brain of treated subjects. In some embodiments, Compound 4 surprisingly significantly uniquely and markedly enhanced neuronal plasticity gene expression. In some embodiments, Compound 4 surprisingly significantly outperforms all other compounds in terms of its enhancement of neuronal growth factor and / or neurogenesis related gene expression and in some embodiments, in particular in the brain of treated subjects. In some embodiments, Compound 4 surprisingly significantly uniquely and markedly enhanced neuronal progenitor cell marker expression.

[0010] In still further embodied aspects of the invention, Compound 4 surprisingly significantly uniquely and markedly enhanced TERT and Lag3 expression in the blood, supporting a role for these biomarkers in disease pathogenesis, in some aspects and in treatment outcomes, in other aspects.

[0011] This invention relates, in some embodiments, to tri -phenyl derivative compounds and compositions comprising the same and / or their use in the treatment of, inter alia, diseases or conditions capable of being affected by stimulation or increase of telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

[0012] In one embodiment, this invention provides a Compound 4 characterized by the following formula:Compound 4.

[0013] It will be appreciated that reference to “Compound 4” herein will be understood to refer to a compound characterized by a structure as presented hereinabove.

[0014] In some embodiments, the invention provides a composition comprising a compound as herein described and in some embodiments, the invention provides a pharmaceutically acceptable salt of a compound as herein described, which in some aspects, is a sodium salt.

[0015] In one embodiment, this invention further provides a method of treatment of a disease or condition in a subject that is both treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity, said method comprising contacting a cell or tissue of said subject or administering to said subject a pharmaceutical composition comprising a Compound 4, or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, the invention provides a method of diagnosis, prognosis or a combination thereof of a subject at risk for a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject, said method comprising the steps of: assessing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in a biological sample from said subject prior to and following administering said subject a Compound 4, or a pharmaceutically acceptable salt thereof; andOptionally comparing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in said biological sample in comparison to a standard,whereby alterations in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject following administration of said compound in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said biological sample taken from said subject prior to administration of said compound; or whereby alterations in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said standard, or as a function of administration of Compound 4; serves as an indicator for the diagnosis, prognosis or a combination thereof of a subject at risk for a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

[0017] According to this aspect and in some embodiments, the increase in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject following administration of said compound in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said biological sample taken from said subject prior to administration of said compound indicates said subject is responsive to said administration, thereby being a method of prognosis of a subject at risk for a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

[0018] In other embodiments according to this aspect, assessing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in a biological sample from said subject is performed prior to administering said subject said Compound 4 and TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in said biological sample is compared to a standard, whereby decreased TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject compared to said standard serves as a diagnostic indicator that said subject is suffering from or at risk for a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity.

[0019] In still other embodiments of this invention, there is provided a method of optimizing treatment of Amyotrophic lateral sclerosis (ALS) in a subject, said method comprising: assessing Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject,• administering a Compound 4 to said subject or a pharmaceutically acceptable salt thereof; and• comparing said Lag3 expression and / or activity or a combination thereof in said biological sample before and after administering Compound 4, whereby increased Lag3 expression and / or activity or a combination thereof in said biological sample taken from said subject after said administering indicates said treatment will be effective in said subject, thereby optimizing treatment in said subject.

[0020] According to this aspect, and in some embodiments, the pharmaceutically acceptable salt is a sodium salt of said compound. In other embodiments, according to this aspect, Compound 4 stimulates or enhances neurogenesis, neuroplasticity, increased neurofilament-associated gene expression, increased neuronal growth factor gene expression or protein production, or a combination thereof in said subject.

[0021] In still other embodiments, this invention provides a method of treatment of Amyotrophic lateral sclerosis (ALS) in a subject, said method comprising contacting a cell or tissue of said subject or administering to said subject a Compound 4 characterized by the following structure:Compound 4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same.

[0022] According to this aspect, and in some embodiments, the ALS is positively impacted by neurogenesis, neuroplasticity, increased neurofilament-associated gene expression, increased neuronal growth factor gene expression or protein production, or a combination thereof.

[0023] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0025] In the drawings:

[0026] Figure 1 schematically depicts an overview of the chemical synthesis method to produce embodied compounds of the invention.

[0027] Figures 2A-2C depict the LC-MS and HPLC results, respectively for the embodied described compounds.

[0028] Figure 3 depicts the calibration curve results for Compound 1 in plasma obtained as described herein.

[0029] Figure 4 depicts the calibration curve results for Compound 4, as described herein.

[0030] Figure 5 depicts the results for Compound 1 in murine plasma following treatment with Compound 4.

[0031] Figure 6, similar to Figure 5depicts the results for Compound 1 in plasma following treatment with Compound 4, however, the subjects used for this study were rats.

[0032] Figure 7, similar to Figures 5 and 6, depicts the results for Compound 1 in plasma following treatment with Compound 4, this time in human subjects.

[0033] Figure 8 plots the results of mice SC injected with Compound 1 or DMSO or Compound 4 or UT and sacrificed 12 h post treatment the brain was removed and subjected to RNA extraction and qReal-time PCR analysis with TERT primers. The results are means ±SE,n=27-29 reactions per group and 4 mice per group. P values were calculated by the Graph pad Prism (student T test) software.

[0034] Figures 9A-9C demonstrate increased Neurofilaments genes expression in the mouse brain in mice injected SC with Compound 1 or DMSO or Compound 4 or UT and sacrificed 12 h post treatment, the brain was removed and subjected to RNA extraction and Real-time PCR analysis with NFH,NFL, NFM specific primers. The results are means ±SE, n=17-20 reactions per group and 4 mice per group. P values were calculated by the Graph pad Prism (T test) software.

[0035] Figure 10 depicts Compound 4 and Compound 1 treatments enhanced Gap43 (A) and SYP (B) expression in the mouse brain. The results provide mean ±SE of n=16-17 PCR reactions and 4 mice per group. P value T test or one way ANOVA by the Graph pad Prism software.

[0036] Figure 11 depicts the results of Compound 4 and Compound 1 treatments enhancing BDNF (A) and NGF (B) expression in the mouse brain. The results are mean ±SE of n=12 PCR reactions and 4 mice per group. P value T test or one way ANOVA by the Graph pad Prism software.

[0037] Figure 12 depicts the results of Compound 4 and Compound 1 treatments enhanced DCX (A) and SOX2 (B) expression in the mouse brain. The results are mean ±SE of n=4-5 PCR reactions and 4 mice per group. P value T test or one way ANOVA by the Graph pad Prism software.

[0038] Figure 13 depicts the results of Compound 4 and Compound 1 treatments enhanced beta catenin(A) and cyclin DI (B) expression in the mouse brain. The results are mean ±SE of n=4-5 PCR reactions and 4 mice per group. P value T test or one way ANOVA by the Graph pad Prism software.

[0039] Figure 14 depicts the results of Compound 4 increased TERT expression in the mouse brain in a dose dependent manner. A- The full TERT primers B- TERT primers designated E- primer. The results are mean ±SE of n=15-16 PCR reactions per group and 8 mice per group. P value T test or one way ANOVA by the Graph pad Prism software.

[0040] Figure 15 depicts the results of Compound 4 increased NF expression in the mouse brain in a dose dependent manner. The results are mean ±SE of n=8-16 PCR reactions per group and 8 mice per group. P value T test or one way ANOVA by the Graph pad Prism software.

[0041] Figure 16 depicts the results of Compound 4 increased Gap43 and SYP but not NeuN gene expression in the mouse brain in a dose dependent manner. The results are mean ±SE of n=8-16 PCR reactions per group and 8 mice per group. P value T test or one way ANOVA by the Graph pad Prism software.

[0042] Figure 17 depicts the results of Compound 4 increased NGF gene expression in the mouse brain in a dose dependent manner. The results are mean ±SE of n=15-16 PCR reactionsper group and 8 mice per group. P value T test or one way ANOVA by the Graph pad Prism software.

[0043] Figure 18 depicts the results of Compound 4 increased DCX, SOX2 and CyclinDl genes expression in the mouse brain in a dose dependent manner. The results are mean ±SE of n=8 PCR reactions per group and 8 mice per group. P value T test or one way ANOVA by the Graph pad Prism software

[0044] Figure 19 plots the time- dependent increase of TERT gene expression by Compound 4 in the mouse brain. The results are mean ±SE of n=28 PCR reactions per group and 6 mice per group. P value: ***P<0.0001, **P<0.005, *P<0.05- one way ANOVA by the Graph pad Prism software. UT- control untreated mice.

[0045] Figure 20 plots the time- dependent increase of neurofilament genes expression by Compound 4 in the mouse brain. The results are mean ±SE of n=l 1-16 PCR reactions per group and 6 mice per group. P value by T test or one way ANOVA using the Graph pad Prism software. UT- control untreated mice.

[0046] Figure 21 plots the time- dependent increase of neuronal growth factor (A) and DCX (B) gene expression by Compound 4 in the mouse brain . The results provide mean ±SE of n=8- 12 PCR reactions per group and 6 mice per group. P value by T test or one way ANOVA using the Graph pad Prism software. UT- control untreated mice.

[0047] Figure 22 depicts the results of Compound 4 increased the survival of NSC34 motor neurons cells exposed to oxidative stress (H2O2). The results are mean ±SE of the OD (490nm) from 8 wells (A) and the % of survival was calculated from the control UT cells (B).

[0048] Figure 23 plots Compound 4 effects on TERT expression in the blood of treated ICR mice. The TERT level was examined in murine blood on a per animal basis, before and after treatment, within the same mouse. The % increase in TERT level after treatment was calculated in each mouse (TERT after treatment / TERT before treatment) X 100. Data are mean ±SEM and calculated as % of TERT level in the blood of each treated mouse, relatively to TERT level prior to treatment. n= 6 mice per group, Student T test P values are indicated.

[0049] Figure 24 plots Compound 4 effects on Lag3 expression in the blood of treated ICR mice. Lag3 levels were examined in murine blood on a per animal basis before and after treatment within the same mouse. The % increase in Lag3 level after treatment was calculated in each mouse: (Lag3 after treatment / Lag3 before treatment) X 100. Data are mean ±SEM and calculated as % of Lag3 level in the blood of each treated mouse relatively to Lag3 level prior to treatment level in its blood prior to treatment. n= 6 mice per group, Student T test P values are indicated.

[0050] Figure 25 schematically presents an overview of a synthesis protocol for production of Compound 4.

[0051] Figure 26 depicts the UV Spectrum and LC-UV Chromatogram of Compound 4.

[0052] Figure 27 depicts Mass Spectra and Fragmentation Elucidation of Compound 4.

[0053] Figure 28 depicts LC-UV and LC-HRMS Chromatograms (XICs) of Compound 4 and its Metabolites in Male Plasma.

[0054] Figure 29 depicts Mass Spectra and Fragmentation Elucidation of M5.

[0055] Figure 30 depicts Mass Spectra and Fragmentation Elucidation of M6.

[0056] Figure 31 depicts Mass Spectra and Fragmentation Elucidation of M7.

[0057] Figure 32 depicts Mass Spectra and Fragmentation Elucidation of M8.

[0058] Figure 33 depicts major metabolic pathways in Plasma.

[0059] Figure 34 depicts LC-UV Chromatograms ( : 290-360 nm) of 7-EC and its Target Metabolite(s) in Hepatocytes.

[0060] Figure 35 depicts UV Spectrum and LC-UV Chromatogram.

[0061] Figure 36 depicts Mass Spectra and Fragmentation Elucidation.

[0062] Figure 37 depicts LC-UV and LC-HRMS Chromatograms (XICs) and its Metabolites in Rat Hepatocytes.

[0063] Figure 38 depicts LC-UV and LC-HRMS Chromatograms (XICs) of Compound 4 and its Metabolites in Dog Hepatocytes.

[0064] Figure 39 depicts LC-UV and LC-HRMS Chromatograms (XICs) of Comound 4 and its Metabolites in Human Hepatocytes.

[0065] Figure 40 depicts Mass Spectra and Fragmentation Elucidation of Ml .

[0066] Figure 41 depicts Mass Spectra and Fragmentation Elucidation of M2.

[0067] Figure 42 depicts Mass Spectra and Fragmentation Elucidation of M3.

[0068] Figure 43 depicts Mass Spectra and Fragmentation Elucidation of M4.

[0069] Figure 44 depicts Mass Spectra and Fragmentation Elucidation of M5.

[0070] Figure 45 depicts Mass Spectra and Fragmentation Elucidation of M6.

[0071] Figure 46 depicts Mass Spectra and Fragmentation Elucidation of M7.

[0072] Figure 47 depicts Mass Spectra and Fragmentation Elucidation of M8.

[0073] Figure 48 depicts maj or metabolic pathways in hepatocytes.

[0074] Figure 49 depicts Average Neurological Score of Both Hind Limbs in IM and 2M Treatment Groups.

[0075] Figure 50 depicts Average Neurological Score of Control (IM) and Compound 4 (2M) Treatment Groups after Disease Onset.

[0076] Figure 51 depicts the Average Highest Body Weight in IM (Control) and 2M Mice (Compound 4).

[0077] Figure 52 depicts Duration of Days in the Early Phase of ALS for IM (Control) and 2M Mice.

[0078] Figure 53 depicts Probability of Survival Curve for IM and 2M Mice.

[0079] Figure 54 depicts LAG3 Expression in Blood Samples Collected from ALS Mice Treated with Compound 4.

[0080] Figure 55 depicts Neurological Scores of ALS Mice Treated with Compound 4.

[0081] Figure 56 depicts Duration of the Early Phase of Disease in Compound 4-Treated ALS Mice.

[0082] Figure 57 depicts Linear Correlation Between LAG3 Expression and ALS Disease Progression.

[0083] Figure 58 depicts Linear Correlation Between TERT Expression Levels and ALS Disease Progression.

[0084] Figure 59-60 depicts Linear Correlation Between Disease Onset Versus LAG3 (Figure 59) and TERT Expression Levels (Figure 60). The day of disease onset was determined according to the peak weight measurements throughout the study as the number of days (duration) in the early phase of the disease, which correlated with the increased level of LAG3 and TERT expression 24 hours post first Compound 4 treatment represented as fold from the level of LAG3 and TERT in each mouse prior to treatment.DETAILED DESCRIPTION OF THE INVENTION

[0085] 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.

[0086] The present invention relates, in some embodiments, to the use of a novel class of tri-phenyl derivative compounds and compositions comprising the same and / or their use in the treatment of, inter alia, diseases or conditions capable of being affected by stimulation or increase of telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

[0087] In one embodiment, this invention provides a Compound 4 characterized by the following formula:Compound 4.

[0088] It will be appreciated that reference to “Compound 4” herein will be understood to refer to a compound characterized by a structure as presented hereinabove.

[0089] In some embodiments, the invention provides a composition comprising a compound as herein described and in some embodiments, the invention provides a pharmaceutically acceptable salt of a compound as herein described, which in some aspects, is a sodium salt.

[0090] In one embodiment, this invention further provides a method of treatment of a disease or condition in a subject that is both treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity, said method comprising contacting a cell or tissue of said subject or administering to said subject a pharmaceutical composition comprising a Compound 4, or a pharmaceutically acceptable salt thereof.

[0091] In other embodied aspects, the invention provides a method of diagnosis, prognosis or a combination thereof of a subject at risk for, or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject, said method comprising the steps of:assessing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in a biological sample from said subject prior to and following administering said subject a Compound 4 or a pharmaceutically acceptable salt thereof; and-optionally comparing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in said biological sample in comparison to a standard, whereby alterations in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject following administration of said compound in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said biological sample taken from said subject prior to administration of said compound; or whereby alterations in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said standard, or as a function of administration of Compound 4; serves as an indicator for the diagnosis, prognosis or a combination thereof of a subject at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

[0092] According to this aspect and in some embodiments, the increase in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject following administration of said compound in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said biological sample taken from said subject prior to administration of said compound indicates said subject is responsive to said administration, thereby being a method of prognosis of a subject at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

[0093] In other embodiments according to this aspect, assessing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in a biological sample from said subject is performed prior to administering said subject said Compound 4 and TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in said biological sample is compared to a standard, whereby decreased TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject compared to said standard serves as a diagnostic indicator that said subject is suffering from or at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity.

[0094] In some embodiments, the compounds of this invention may be administered at a dosage form, or according to a regimen as described herein. According to this aspect, and in some embodiments, the dosage may be increased or decreased, or otherwise adjusted, or the regimen may be adjusted as a function of the results of dynamic assessment of TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof, as determined from assessing a biological sample taken from the subject, as a function of differences in values in the subject over time, or as compared to a standard, optionally over time, or as compared to a value obtained during the treatment regimen for the patient, or in some embodiments, as compared to a value obtained in a prior treatment regimen, or in some embodiments, reflecting symptoms experienced by the subject, or in some embodiments, any combination of same.

[0095] In some embodiments, such adjustments as described herein are based on results of TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof, as determined from individual patient biological sampling as described herein, and therefore provide for personalized therapy / personalized medicine applications.

[0096] In some embodiments, the compounds of the invention may be provided via different routes of administration, which may be altered, for example, combining two or more routes of administration as a function of the results of dynamic assessment of TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof, as determined from assessing a biological sample taken from the subject, as a function of differences in values in the subject over time, or as compared to a standard, optionally over time, or as compared to a value obtained during the treatment regimen for the patient, or in some embodiments, as compared to a value obtained in a prior treatment regimen, or in some embodiments, reflecting symptoms experienced by the subject, or in some embodiments, any combination of same.

[0097] In other embodiments, the timing of administration of the compounds of this invention may be dynamically altered over time, for example, shortening or lengthening the administration times between administrations, as a function of the results of dynamic assessment of TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof, as determined from assessing a biological sample taken from the subject, as a function of differences in values in the subject over time, or as compared to a standard, optionally over time, or as compared to a value obtained during the treatment regimen for the patient, or in some embodiments, as compared to a value obtained in a prior treatment regimen, or in some embodiments, reflecting symptoms experienced by the subject, or in some embodiments, any combination of same.

[0098] It will be appreciated that any of the concentration, compound composition, excipients used, dosage, administration regimen, route of administration or any combination thereof may be dynamically adjusted as a function of the results of dynamic assessment of TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof, as determined from assessing a biological sample taken from the subject, as a function of differences in values in the subject over time, or as compared to a standard, optionally over time, or as compared to a value obtained during the treatment regimen for the patient, or in some embodiments, as compared to a value obtained in a prior treatment regimen, or in some embodiments, reflecting symptoms experienced by the subject, or in some embodiments, any combination of same.

[0099] The compounds of the invention and their pharmaceutically acceptable salts, in some aspects are structurally related to Compound 1 as described herein, and in addition to the unique new effects of the compounds of the invention on TERT and Lag3 expression, neurofilament expression, and / or effects on neurogenesis-promoting factors and neuroplasticity promoting factors, the enhanced stability and in some embodiments, accompanying enhanced bioavailability as described herein, as well, heralds in some aspects of the invention for the compounds to be effectively used in all the uses in which Compound 1 has been shown to be successful, whereby Compound 4 and salts thereof, and in some aspects, Compound 3 and salts thereof, and in some aspects, Compound 2 and salts thereof, will be useful in stimulating and / or increasing telomerase expression and / or activity in the cells and tissues of a subject, where the activity is decreased, missing, altered or normal. Such disorders include, inter alia, a) Alzheimer’s disease; b) Parkinson’s disease; c) Huntington’s disease; d) nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents; e) stroke; f) diseases or conditions associated with aging, such as for example, aging of the skin such as dermal atrophy and thinning, elastolysis and skin wrinkling, sebaceous gland hyperplasia or hypoplasia, senile lentigo, pigmentation abnormalities, graying of hair and hair loss or thinning (baldness, alopecia), or chronic skin ulcers; g) degenerative joint disease; h) osteoporosis, osteoarthritis and other degenerative conditions of the skeletal system; i) sarcopenia and other degenerative conditions of the musculature; j) age- and stress-related diseases of the vascular system including atherosclerosis, calcification, thrombosis, and aneurysm; k) age-related macular degeneration; 1) AIDS; m) age- and stress-related immune system impairment, including impairment of tissue turnover, which occur with natural aging, cancer, cancer therapy, acute or chronic infections, degenerative inflammatory diseases or with genetic disorders causing accelerated cell turnover, and related anemias and other degenerative conditions; n) healing of wounds, bums, abrasions or other acute or chronic conditions ofepidermis; o) dyskeratosis congenita; p) luteal phase defect; q) premature ovarian failure (primary ovarian insufficiency or hypergonadotropic hypogonadism); and / or r) increasing telomerase expression and / or activity in memory T cells, thereby strengthening immune memory response and response to vaccines; s) increasing telomerase expression and / or activity in healthy tissue, thus elongating the lifespan of a subject while sustaining said subject in good health.Compounds of the Invention:

[0100] In one embodiment, this invention provides the tri -phenyl derivative Compound 4 including providing same in a pharmaceutical composition, as described herein.

[0101] In another embodiment, this invention provides a tri-phenyl derivativeCompound 2, which is characterized by the following structure:Compound 2,

[0102] and in another embodiment, this invention provides a still further tri -phenyl derivative Compound 3, which is described by the following structure:Compound 3,

[0103] and pharmaceutical compositions containing the isolated and / or purified compounds.

[0104] It is noted that Compound 4, Compound 3 and Compound 2 are not compounds found in nature, and their synthesis and formulation is not straightforward.

[0105] In some embodiments, the Compounds of this invention, in particular, Compound 4, Compound 3 and Compound 2 remain soluble even in lower pH, for example, even at a pH of about 6.

[0106] In some embodiments, pharmaceutical compositions comprising the Compounds of this invention, in particular, Compound 4, Compound 3 and Compound 2 will further include a sodium phosphate buffer, or a potassium phosphate buffer, or others, as known to the skilled artisan.

[0107] In some embodiments, the invention provides a pharmaceutical composition comprising a compound as herein described. In some embodiments, the pharmaceutical compositions of the invention comprising, in particular, Compound 4, Compound 3 and Compound 2 will further include pharmaceutically acceptable salt forms of the compounds, which in some embodiments, can be any appropriate salt form, for example, a sodium salt.

[0108] In some embodiments, the compounds / compositions / uses of this invention comprise a stable crystal form of the compounds as herein described.

[0109] In some embodiments of the invention, there is provided an optical isomer, polymorph, crystal or combination thereof of a compound as herein described and / or for use, and same represents envisioned aspects of the invention.

[0110] In one embodiment, the term “isomer” includes, but is not limited to, optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like.

[0111] In one embodiment, the term “isomer” is meant to encompass optical isomers of the tri-phenyl compound. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, which form possesses properties useful in the treatment of telomerase expression and / or activity conditions described herein. In one embodiment, the tri-phenyl compounds are the pure (R)- isomers. In another embodiment, the tri-phenyl compounds are the pure (5 -isomers. In another embodiment, the tri-phenyl compounds are a mixture of the (R) and the (5) isomers. In another embodiment, the tri-phenyl compounds are a racemic mixture comprising an equal amount of the (R) and the (5) isomers. It is well known in the art how to prepare optically- active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

[0112] The invention includes “pharmaceutically acceptable salts” of the compounds of this invention, which may be produced, in one embodiment, to form alkali metal salts and to form addition salts of free acids or free bases. Suitable pharmaceutically-acceptable acid addition salts of compounds of this invention may be prepared from an inorganic acid or from an organic acid. In one embodiment, examples of inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. In one embodiment, organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, oxalic, p- toluenesulphonic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethylsulfonic, benzenesulfonic, sulfanilic, stearic, cyclohexylaminosulfonic, algenic, galacturonic acid. In one embodiment, suitable pharmaceutically-acceptable base addition salts of compounds of this invention include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethyleneldiamine, choline, chloroprocaine,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procain. All of these salts may be prepared by conventional means from the corresponding compounds.

[0113] Pharmaceutically acceptable salts can be prepared, from the phenolic compounds, in other embodiments, by treatment with inorganic bases, for example, sodium hydroxide, or in some embodiments, in a solution of NaHCOs. In another embodiment, esters of the phenolic compounds can be made with aliphatic and aromatic carboxylic acids, for example, acetic acid and benzoic acid esters.

[0114] The invention also includes use of A-oxides of the amino substituents of the compounds described herein.

[0115] This invention provides for the use of derivatives of the compounds as herein described. In one embodiment, “derivatives” includes but is not limited to ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. In another embodiment, this invention further includes use of hydrates of the compounds as described herein. In one embodiment, “hydrate” includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like.

[0116] This invention provides, in other embodiments, use of metabolites of the compounds as herein described. In one embodiment, “metabolite” means any substance produced from another substance by metabolism or a metabolic process.

[0117] This invention provides, in other embodiments, use of pharmaceutical products of the compounds as herein described. The term “pharmaceutical product” refers, in other embodiments, to a composition suitable for pharmaceutical use (pharmaceutical composition), for example, as described herein.Pharmaceutical Compositions

[0118] In some embodiments, this invention provides methods of use which comprise administering a composition comprising the described compounds. As used herein, "pharmaceutical composition" means a “therapeutically effective amount” of the active ingredient, i.e. the compounds of this invention, together with a pharmaceutically acceptable carrier or diluent. A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen.

[0119] In some embodiments, this invention provides compositions which may comprise at least one compound of this invention, in any form or embodiment as describedherein. In some embodiments, the term “a” is to be understood to encompass a single or multiple of the indicated material. In some embodiments, the term “a” or “an” refers to at least one.

[0120] In some embodiments, any of the compositions of this invention will consist of a compound of this invention, in any form or embodiment as described herein. In some embodiments of the compositions of this invention will consist essentially of a compound of this invention, in any form or embodiment as described herein.

[0121] In some embodiments, the term “comprise” refers to the inclusion of the indicated active agent, such as the compounds of this invention, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry. In some embodiments, any of the compositions of this invention will comprise Compound 4, or in some embodiments, Compound 3, or in some embodiments, Compound 2, in any form or embodiment as described herein, or in any combination thereof. In some embodiments, any of the compositions of this invention will consist of the tri-phenyl derivative compounds as herein described, in any form or embodiment as described herein. In some embodiments of the compositions of this invention will consist essentially of the tri-phenyl derivative compounds as herein described, in any form or embodiment as described herein.

[0122] In some embodiments, the term “comprise” refers to the inclusion of the indicated active agent, such as the compound of this invention, or its salt as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry. In some embodiments, the term “consisting essentially of’ refers to a composition, whose only active ingredient is the indicated active ingredient, however, other compounds may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient. In some embodiments, the term “consisting essentially of’ refers to a composition, whose only active ingredient with a comparable mode of action, or comparable molecular target is the indicated active ingredient, however, other active ingredients may be incorporated, with such secondary active ingredients acting on different targets, or in a palliative capacity. In some embodiments, the term “consisting essentially of’ may refer to components which facilitate the release of the active ingredient. In some embodiments, the term “consisting” refers to a composition, which contains a compound as herein described or its salt as the only active ingredient and a pharmaceutically acceptable carrier or excipient.

[0123] In another embodiment, the invention provides a composition comprising the tri-phenyl derivative compounds as herein described, or its isomer, pharmaceutically acceptable salt, pharmaceutical product, polymorph, crystal, or any combination thereof and a suitable carrier or diluent.

[0124] An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts, which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0125] The pharmaceutical compositions containing the compound of this invention can be administered to a subject by any method known to a person skilled in the art, such as orally, parenterally, intravascularly, paracancerally, transmucosally, transdermally, intramuscularly, intranasally, intravenously, intradermally, subcutaneously, sublingually, intraperitoneally, intraventricularly, intracranially, intravaginally, by inhalation, rectally, intratumorally, or by any means in which the composition can be delivered to tissue (e.g., needle or catheter). Alternatively, topical administration may be desired for application to mucosal cells, for skin or ocular application. Another method of administration is via aspiration or aerosol formulation.

[0126] The compositions of the present invention are formulated in one embodiment for oral delivery, wherein the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrup of elixir may contain the active compound, sucrose as a sweetening agent methyl, and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. In addition, the active compounds may beincorporated into sustained-release, pulsed release, controlled release or postponed release preparations and formulations.

[0127] In another embodiment, the compositions of this invention comprise one or more, pharmaceutically acceptable carrier materials.

[0128] In one embodiment, the carriers for use within such compositions are biocompatible, and in another embodiment, biodegradable. In other embodiments, the formulation may provide a relatively constant level of release of one active component. In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. In other embodiments, release of active compounds may be event-triggered. The events triggering the release of the active compounds may be the same in one embodiment, or different in another embodiment. Events triggering the release of the active components may be exposure to moisture in one embodiment, lower pH in another embodiment, or temperature threshold in another embodiment. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly (lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative postponed-release carriers include supramol ecul ar biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as phospholipids. The amount of active compound contained in one embodiment, within a sustained release formulation depends upon the site of administration, the rate and expected duration of release and the nature of the condition to be treated suppressed or inhibited.

[0129] In one embodiment it will be desirable to deliver the compositions disclosed herein subcutaneously, intradermally, orally, parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363, all of which are fully incorporated by reference. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0130] In another embodiment, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. In other embodiments, prolonged absorption of the injectable compositions will be desirable. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin, in the compositions.

[0131] Parenteral vehicles include in certain embodiments sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like

[0132] In some embodiments, the compounds of this invention may be administered at various dosages to a subject, which in one embodiment, is a human subject. In one embodiment, the compounds of this invention are administered at a dosage of 0.1 - 200 mg per day. In one embodiment, the compound of this invention is administered at a dose of 0.1 - 10 mg, or in another embodiment, 0.1 - 25 mg, or in another embodiment, 0.1- 50 mg, or in another embodiment, 0.3 - 15 mg, or in another embodiment, 0.3 - 30 mg, or in another embodiment, 0.5 - 25 mg, or in another embodiment, 0.5 - 50 mg, or in another embodiment, 0.75 - 15 mg, or in another embodiment, 0.75 - 60 mg, or in another embodiment, 1 - 5 mg, or in another embodiment, 1 - 20 mg, or in another embodiment, 3 - 15 mg, or in another embodiment, 1 - 30 mg, or in another embodiment, 30 - 50 mg, or in another embodiment, 30 - 75 mg, or in another embodiment, 100 - 2000 mg. In some embodiments, the compounds of this invention may be administered at different dosages, as a function of time, or disease / symptom / condition severity, or age, or other factors, as will be appreciated by one skilled in the art.

[0133] In one embodiment, the compounds of this invention are administered at a dosage of 0.1 - 200 mg per kg weight. In one embodiment, the compound of this invention is administered at a dose of 0.1 - 10 mg mg / kg, or in another embodiment, 0.1 - 25 mg / kg, or in another embodiment, 0.1- 50 mg mg / kg, or in another embodiment, 0.3 - 15 mg mg / kg, or in another embodiment, 0.3 - 30 mg mg / kg, or in another embodiment, 0.5 - 25 mg mg / kg, or in another embodiment, 0.5 - 50 mg mg / kg, or in another embodiment, 0.75 - 15 mg, or in another embodiment, 0.75 - 60 mg, or in another embodiment, 1 - 5 mg mg / kg, or in another embodiment, 1 - 20 mg mg / kg, or in another embodiment, 3 - 15 mg mg / kg, or in another embodiment, 1 - 30 mg, or in another embodiment, 30 - 50 mg mg / kg, or in another embodiment, 30 - 75 mg mg / kg, or in another embodiment, 10 - 200 mg mg / kg. In some embodiments, the compounds of this invention may be administered at different dosages, as afunction of time, or disease / symptom / condition severity, or age, or other factors, as will be appreciated by one skilled in the art.

[0134] The compounds of this invention may be administered at various dosages. In one embodiment, the compounds of this invention are administered at a dosage of 1 mg. In another embodiment the compounds of this invention are administered at a dosage of 5 mg, or in another embodiment, 3 mg, or in another embodiment 10 mg, or in another embodiment 15 mg, or in another embodiment 20 mg, or in another embodiment 25 mg, or in another embodiment 30 mg, or in another embodiment 35 mg, or in another embodiment 40 mg, or in another embodiment 45 mg, or in another embodiment 50 mg, or in another embodiment 55 mg, or in another embodiment 60 mg, or in another embodiment 65 mg, or in another embodiment 70 mg, or in another embodiment 75 mg, or in another embodiment 80 mg, or in another embodiment 85 mg, or in another embodiment 90 mg, or in another embodiment 95 mg or in another embodiment 100 mg.

[0135] While the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compound, and / or in combination with other agents used in the treatment and / or prevention of the diseases, disorders and / or conditions, as will be understood by one skilled in the art. In another embodiment, the compounds of the present invention can be administered sequentially with one or more such agents to provide sustained therapeutic and prophylactic effects. In another embodiment, the compounds may be administered via different routes, at different times, or a combination thereof.

[0136] In addition, the compounds of the present invention can be used, either singly or in combination, in combination with other modalities for preventing or treating conditions, diseases or disorders. In some embodiments, such other treatment modalities may include without limitation, surgery, radiation, hormone supplementation, diet regulation, wound debridement, etc., as will be appropriate for the condition being treated. These can be performed sequentially (e.g., treatment with a compound of the invention following surgery or radiation) or in combination (e.g., in addition to a diet regimen).

[0137] The additional active agents may generally be employed in therapeutic amounts as indicated in the PHYSICIANS' DESK REFERENCE (PDR) 53rd Edition (1999), or such therapeutically useful amounts as would be known to one of ordinary skill in the art. The compounds of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levelsof the active compounds in the compositions of the invention may be varied to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. The combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

[0138] The pharmaceutical composition can comprise the compounds of this invention alone or can further include a pharmaceutically acceptable carrier and can be in solid or liquid form such as tablets, powders, capsules, pellets, solutions, suspensions, elixirs, emulsions, gels, creams, or suppositories, including rectal and urethral suppositories. Pharmaceutically acceptable carriers include gums, starches, sugars, cellulose materials, and mixtures thereof. The pharmaceutical preparation containing the compounds of this invention can be administered to a subject by, for example, subcutaneous implantation of a pellet; in a further embodiment, the pellet provides for controlled release of the compounds of this invention over a period of time. The preparation can also be administered by intravenous, intraarterial, or intramuscular injection of a liquid preparation, oral administration of a liquid or solid preparation, or by topical application. Administration can also be accomplished by use of a rectal suppository or a urethral suppository. The pharmaceutical composition can also be a parenteral formulation; in one embodiment, the formulation comprises a liposome that includes a complex of a compound of this invention.

[0139] The pharmaceutical composition of the invention can be prepared by known dissolving, mixing, granulating, or tablet-forming processes. For oral administration, the compounds of this invention or their physiologically tolerated derivatives such as salts, esters, A-oxides, and the like are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into a suitable form for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. Examples of suitable inert vehicles are conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, gelatin, or with disintegrating agents such as cornstarch, potato starch, alginic acid, or with a lubricant such as stearic acid or magnesium stearate. Examples of suitable oily vehicles or solvents are vegetable or animal oils such as sunflower oil or fish-liver oil. Preparations can be effected both as dry and as wet granules. For parenteral administration (subcutaneous, intravenous, intraarterial, or intramuscular injection), the compounds of this invention or their physiologically tolerated derivatives such as salts, esters, A-oxides, and the like are converted into a solution, suspension, or emulsion, if desired with the substances customary and suitablefor this purpose, for example, solubilizers or other auxiliaries. Examples are: sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[0140] The preparation of pharmaceutical compositions which contain an active component is well understood in the art. Typically, such compositions are prepared as an aerosol of the polypeptide delivered to the nasopharynx or as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents which enhance the effectiveness of the active ingredient.

[0141] For topical administration to body surfaces using, for example, creams, gels, drops, and the like, the compounds of this invention or their physiologically tolerated derivatives such as salts, esters, 7V-oxides, and the like are prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.

[0142] In another embodiment, the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

[0143] In one embodiment, the present invention provides combined preparations. In one embodiment, the term "a combined preparation" defines especially a "kit of parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners, in some embodiments, can be administered in the combined preparation. In one embodiment, the combined preparationcan be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art.

[0144] It is to be understood that this invention is directed to compositions and combined therapies as described herein, for any disease, disorder or condition, as appropriate, as will be appreciated by one skilled in the art. Certain applications of such compositions and combined therapies have been described hereinabove, for specific diseases, disorders and conditions, representing embodiments of this invention, and methods of treating such diseases, disorders and conditions in a subject by administering a compound as herein described, alone or as part of the combined therapy or using the compositions of this invention represent additional embodiments of this invention.Treatment of Conditions or Diseases Capable of Being Affected bv TERT Expression and / or activity

[0145] Surprisingly, the compounds of this invention are shown herein to significantly increase neurofilament expression in the brain, which in turn will be useful in the treatment, reduction of severity, reduction of incidence, prevention of onset of and otherwise positively impact subjects with or at risk for neurodegenerative diseases.

[0146] According to this aspect and in some embodiments, the compounds / compositions of this invention and uses of same will be useful in the treatment, reduction of severity, reduction of incidence, prevention of onset of and otherwise positively impact subjects with or at risk for amyotrophic lateral sclerosis (ALS).

[0147] Still further according to this aspect and in some embodiments, the compounds / compositions of this invention and uses of same will be useful in the assessment of the potential for positive effects of the treatment, reduction of severity, reduction of incidence, prevention of onset of and positively impact subjects with or at risk for amyotrophic lateral sclerosis (ALS), when subjects provided treatments with the compounds of this invention exhibit increased LAG3 and / or TERT expression following treatment. In some aspects, this invention provides for administering other activators including immune activators such as adjuvants, immune stimulants and cytokines along with the compounds of this invention, in a subject who is not as ideally responsive to treatment with the compounds alone in order to improve their disease prognosis or treatment responsiveness, for example.

[0148] In some embodiments, the compounds / compositions of this invention and uses of same will be useful in the treatment, reduction of severity, reduction of incidence,prevention of onset of and otherwise positively impact subjects with or at risk for multiple sclerosis (MS) Creutzfeldt-Jakob syndrome, Alzheimer’s disease, Stroke, Traumatic Brain injury, Parkinson’s disease, Lewy Body dementia and other related diseases.

[0149] In some embodiments, the invention provides compounds of this invention, salts of the compounds as herein described, compositions comprising the compounds or use of the compound of this invention, for stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in a cell or tissue; and / or treating a condition by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in cells or tissue of a subject; and / or treating an acute or chronic condition which will be improved by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity.

[0150] In some embodiments, this invention shows, the first time, compounds as herein described / compositions comprising same, having an ability to significantly increase neurofilament expression in the brain.

[0151] Without being bound by theory, and representing certain embodied aspects of the invention, the increase in neurofilaments expression is important specifically in neurodegenerative diseases in which a severe degradation of axons, dendrites and the soma structure is observed and the compounds as herein described / compositions comprising same, can positively impact these diseases / conditions.

[0152] According to this aspect and in some embodiments, the invention in particular contemplates treating, lowering the incidence of, reducing the severity of, preventing or delaying onset of, ameliorating the symptoms of, delaying the pathogenesis of, or otherwise positively impacting a subject suffering from or at risk for a disease or condition in a subject that is both treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity, or a disease or condition, which is positively impacted by neurogenesis, neuroplasticity, increased neurofilament-associated gene expression, increased neuronal growth factor gene expression or protein production, or a combination thereof.

[0153] According to this aspect and in some embodiments, the disease or condition is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), myotrophic lateral sclerosis, a disease or condition associated with nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents, or stroke.

[0154] This invention, for the first time, addresses the effects of Compounds 2, 3 and 4 on TERT expression, with Compound 4 surprisingly significantly outperforming all other compounds in enhancing TERT expression, and in some embodiments, markedly increasing neurofilament expression, and in some embodiments, in particular, in the brain of treated subjects.

[0155] In some embodiments, Compound 4 surprisingly significantly uniquely and markedly enhanced neuronal plasticity gene expression.

[0156] In some embodiments, Compound 4 surprisingly significantly outperforms all other compounds in terms of its enhancement of neuronal growth factor and / or neurogenesis related gene expression and in some embodiments, in particular in the brain of treated subjects. In some embodiments, Compound 4 surprisingly significantly uniquely and markedly enhanced neuronal progenitor cell marker expression.

[0157] In still further embodied aspects of the invention, Compound 4 surprisingly significantly uniquely and markedly enhanced TERT and Lag3 expression in the blood, supporting a role for these biomarkers in disease pathogenesis, in some aspects and in treatment outcomes, in other aspects.

[0158] This invention relates, in some embodiments, to tri -phenyl derivative compounds and compositions comprising the same and / or their use in the treatment of, inter alia, diseases or conditions capable of being affected by stimulation or increase of telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

[0159] In still other aspects, in addition to the effect of the compounds of this invention, salts thereof and / or compositions comprising same on diseases or conditions capable of being affected by stimulation or increase of telomerase reverse transcriptase (TERT) expression and / or activity in a subject, same may also positively impact other diseases related thereto.

[0160] According to this aspect, and in some embodiments, the compounds of this invention, salts thereof and / or compositions comprising same may be useful in the treating of age- and stress-related diseases of the vascular system including atherosclerosis, calcification, thrombosis, hypertension or aneurysm; and / or treating age-related macular degeneration and / or age-related diseases of the skin; and / or treating age- and stress-related immune system impairment; and / or strengthening the immune response against infection-resistant organisms.

[0161] In some embodiments, the invention provides compounds as herein described, salts comprising same and compositions comprising the compounds of this invention for treating female infertility -related conditions including luteal phase defect or premature ovarian failure (primary ovarian insufficiency or hypergonadotropic hypogonadism); and / ordiminished granulosa cell telomerase activity; and / or treating male infertility-related conditions including impaired sperm production or impaired sperm delivery; and / or as an adjunct to in vitro fertilization (IVF) techniques; and / or to enhance sperm quality and / or egg quality. For example, and in some embodiments, the compounds of this invention prolong blastocyst viability in ex vivo culture, which in turn enhances implantation efficiency. In some embodiments, the treatment of the population with the compounds as herein described renders them more receptive to other IVF therapeutics, or in some embodiments, allows for the evaluation of combination therapies, or new compounds.

[0162] In one embodiment, this invention provides methods of treatment using a compound of this invention, or composition comprising the same, as herein described. In some embodiments, the invention provides methods of use of a compound of this invention for the treatment of the indicated diseases, disorders or conditions, and includes use of salts of the compounds as herein described and / or compositions comprising the compounds and salts of same.

[0163] In one embodiment, this invention provides methods of treating, suppressing, inhibiting, reducing the severity of, reducing the incidence of, reducing pathogenesis of or delaying onset of, inter alia, (a) Alzheimer’s disease; (b) Parkinson’s disease; ( c) Huntington’s disease; (d) stroke; e) nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents; (f) age- and stress-related immune system impairment, including impairment of tissue turnover, acute or chronic infections, degenerative inflammatory diseases or with genetic disorders causing accelerated cell turnover, and related anemias and other degenerative conditions;, via the administration of any compound as herein described and optionally other therapeutic agents, or compositions comprising the same.

[0164] This invention provides, in some aspects, for a method of treatment of a disease or condition in a subject that is both treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity, said method comprising contacting a cell or tissue of said subject or administering to said subject a pharmaceutical composition comprising a Compound 4, or a pharmaceutically acceptable salt thereof.

[0165] As will be appreciated, Compound 4, or in some embodiments, Compounds 2, or 3 or mixtures of the compounds as herein described or any salt of any compound as herein described is envisioned for use in the described treatment of a disease or condition in a subject that is both treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity, as described, and the compounds, compositions, kits, methods anduses of the invention contemplate use of any of the described compounds, salts, etc. herein, alone or in combinations.

[0166] According to this aspect, and in some embodiments, the disease or condition is positively impacted by neurogenesis, neuroplasticity, increased neurofilament-associated gene expression, increased neuronal growth factor gene expression or protein production, or a combination thereof. According to this aspect and in other embodiments, the disease or condition is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), a disease or condition associated with nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents, or stroke.

[0167] In other embodiments, the disease or condition is a neurodegenerative disease, cognitive disease or disorder, nervous system injury, a disease or disorder associated with aging, a degenerative joint disease, a degenerative disease of the skeletal system, a degenerative disease of the musculature, macular degeneration, age- and stress-related diseases of the vascular system, age- and stress-related immune system impairment, diabetes, cardiac disease, infection, immune system impairment, cancer, degenerative inflammatory disease, a genetic disorder causing accelerated cell turnover, anemia, male or female reproductive disorder, and an acute or chronic disease or disorder of the skin. Still further according to envisioned specific embodiments of the invention, the disease or condition is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), a disease or condition associated with nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents, stroke, an aging disease or a condition associated with aging, sebaceous gland hyperplasia or hypoplasia, senile lentigo, pigmentation abnormalities, graying of hair, hair loss, hair thinning or baldness including alopecia, or chronic skin ulcers, degenerative joint disease, osteoporosis, osteoarthritis, sarcopenia, atherosclerosis, calcification, thrombosis, aneurysm, AIDS, diseases or conditions arising as a consequence of cancer therapy, acute or chronic infections, wounds, burns, abrasions or other acute or chronic conditions of epidermis, dyskeratosis congenita, luteal phase defect, premature ovarian failure, improving fertility including in in vitro fertilization, in subjects exposed to radiation and / or chemotherapy, or any combination of diseases as applicable.

[0168] In some specifically contemplated aspects of the invention, the disease is Amyotrophic lateral sclerosis (ALS).

[0169] In still further aspects of the invention, there is provided a method of diagnosis, prognosis or a combination thereof of a subject at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject, said method comprising the steps of: assessing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in a biological sample from said subject prior to and following administering said subject a Compound 4, or a pharmaceutically acceptable salt thereof; and-optionally comparing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in said biological sample in comparison to a standard, whereby alterations in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject following administration of said compound in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said biological sample taken from said subject prior to administration of said compound; or whereby alterations in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said standard; serves as an indicator for the diagnosis, prognosis or a combination thereof of a subject at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

[0170] In some embodiments, the increase in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject following administration of said compound in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said biological sample taken from said subject prior to administration of said compound indicates said subject is responsive to said administration, thereby being a method of prognosis of a subject at risk for or surffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

[0171] In still other embodied aspects, assessing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in a biological sample from said subject is performed prior to administering said subject said Compound 4 and TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in saidbiological sample is compared to a standard, whereby decreased TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject compared to said standard serves as a diagnostic indicator that said subject is suffering from or at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity.

[0172] As will be appreciated, Compound 4, or in some embodiments, Compounds 2, or 3 or mixtures of the compounds as herein described or any salt of any compound as herein described is envisioned for use in the described diagnosis, prognosis and treatment of a disease or condition in subject as described, immediately hereinabove, and the compounds, compositions, kits, methods and uses of the invention contemplate use of any of the described compounds, salts, etc. herein, alone or in combinations.

[0173] According to this aspect and in some embodiments, the method will further comprise administering Compound 4 to the subject, and any medical use of same, as well as, in some embodiments, Compounds 2, or 3 or mixtures of the compounds as herein described or any salt of any compound as herein described or any combination thereof . In still other embodied aspects, a pharmaceutically acceptable salt of Compound 4 is specifically envisioned for use in which, in some embodiments, a sodium salt of the compound is specifically contemplated.

[0174] According to this aspect and in some embodiments, the disease or condition is positively impacted by neurogenesis, neuroplasticity, increased neurofilament-associated gene expression, increased neuronal growth factor gene expression or protein production, or a combination thereof. According to this aspect and in some embodiments, thedisease or condition is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), a disease or condition associated with nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents, or stroke.

[0175] In one embodiment, the terms “treating” or “treatment” includes preventive as well as disorder remittive treatment. The terms “reducing”, “suppressing” and “inhibiting” have their commonly understood meaning of lessening or decreasing, in another embodiment, or delaying, in another embodiment, or reducing, in another embodiment the incidence, severity or pathogenesis of a disease, disorder or condition. In embodiment, the term treatment refers to delayed progression of, prolonged remission of, reduced incidence of, or amelioration of symptoms associated with the disease, disorder or condition. In one embodiment, the terms“treating” “reducing”, “suppressing” or “inhibiting” refer to a reduction in morbidity, mortality, or a combination thereof, in association with the indicated disease, disorder or condition. In one embodiment, the term “progression” refers to an increasing in scope or severity, advancing, growing or becoming worse. The term “recurrence” means, in another embodiment, the return of a disease after a remission. In one embodiment, the methods of treatment of the invention reduce the severity of the disease, or in another embodiment, symptoms associated with the disease, or in another embodiment, reduces the number of biomarkers expressed during disease.

[0176] In one embodiment, the term “treating” and its included aspects, refers to the administration to a subject with the indicated disease, disorder or condition, or in some embodiments, to a subject predisposed to the indicated disease, disorder or condition. The term “predisposed to” is to be considered to refer, inter alia, to a genetic profile or familial relationship which is associated with a trend or statistical increase in incidence, severity, etc. of the indicated disease. In some embodiments, the term “predisposed to” is to be considered to refer, inter alia, to a lifestyle which is associated with increased risk of the indicated disease. In some embodiments, the term “predisposed to” is to be considered to refer, inter alia, to the presence of biomarkers which are associated with the indicated disease, for example, in cancer, the term “predisposed to” the cancer may comprise the presence of precancerous precursors for the indicated cancer.

[0177] In some embodiments, the term “reducing the pathogenesis” is to be understood to encompass reducing tissue damage, or organ damage associated with a particular disease, disorder or condition. In another embodiment, the term “reducing the pathogenesis” is to be understood to encompass reducing the incidence or severity of an associated disease, disorder or condition, with that in question. In another embodiment, , the term “reducing the pathogenesis” is to be understood to encompass reducing the number of associated diseases, disorders or conditions with the indicated, or symptoms associated thereto.

[0178] In some aspects, reference to the phrase "inhibiting" or "treating" refers to reducing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the stated disease / condition. In some aspects, when referring to the prevention of a disease, such reference is with regard to reduction of incidence of the disease on a population level. In some aspects, such reference may be with regard to a patient suffering from a repeat or relapsing disease, where failure to develop full symptomatology, pathogenesis or severity of the disease as previously occurred in such patient, may serve as an indication of true prevention.

[0179] The term “administering”, in another embodiment, refers to bringing a subject in contact with a compound of the present invention. Administration can be accomplished in vitro, i.e. in a test tube, or in vivo, i.e. in cells or tissues of living organisms, for example humans. In one embodiment, the present invention encompasses administering the compounds of the present invention to a subject.

[0180] In one embodiment, the methods of this invention make use of the described compound of this invention or salts of same, by contacting an affected cell or tissue with a compound as herein described or a salt of same or a composition comprising same in an amount effective to increase TERT activity and / or expression and thereby mediating the described effects. In some embodiments, the methods of this invention may include the preliminary step of identifying a cell or tissue in which an increase TERT activity and / or expression is desired. The cell may be in culture, i.e. in vitro or ex vivo, or within a subject or patient in vivo.

[0181] In some embodiments, this invention provides compounds, salts thereof and pharmaceutical compositions comprising the same for the treatment of conditions and / or diseases capable of being affected by enhanced TERT expression and / or activity. In some embodiment, such TERT expression and / or activity is decreased or absent in the affected subject or subject in need of treatment, resulting in development of, or enhanced pathogenesis or symptoms associated with a disease, disorder or condition in the subject. Such disease, disorder or condition may comprise, inter alia, a) Alzheimer' s disease; b) Parkinson' s disease; c) Huntington' s disease; d) stroke; e) nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents; f) age- and stress-related immune system impairment, including impairment of tissue turnover, which occur with natural aging, cancer, cancer therapy, acute or chronic infections, degenerative inflammatory diseases or with genetic disorders causing accelerated cell turnover, and related anemias and other degenerative conditions; and the treatments, compositions for use, etc. of this invention, provide for sustaining said subject in good health and / or other clinical therapeutic and / or diagnostic areas, including any embodiment of what is encompassed by the term "treating" as described herein.

[0182] In some embodiments, the invention provides a method of increasing TERT expression and / or activity in a cell or tissue, by contacting the cell or tissue with a composition comprising a compound of this invention. In one embodiment, the methods may include the step of identifying a cell or tissue in which an increase in TERT expression and / or activity is desired.

[0183] The ability of a compound to increase TERT expression and / or activity in a cell can be determined using known methods for detecting gene and protein expression, including methods described further hereinunder.

[0184] In some embodiments, this invention provides methods of treating a condition capable of being affected by TERT expression and / or activation in a subject, the method comprising administering to a subject an effective amount of a compound of this invention, wherein the compound stimulates or enhances TERT expression and / or activity in the cells or tissue of the subject, wherein the compound is, in some embodiments, characterized by the following formula:

[0185] or is an isomer, pharmaceutically acceptable salt, pharmaceutical product, crystal form or polymorph of the compound, including any embodiment of same as described herein. In other embodiments, the methods of this invention include use of a tri-phenyl derivative compound as described herein, characterized by the structure of the formula with regard to Compound 3 or Compound 2, as defined hereinabove.

[0186] In some embodiments, conditions which may be treated by increasing TERT expression and / or activity may comprise use of the compounds as described herein in ex vivo cell therapy, as described further below, employing the appropriate associated cell types. In one embodiment, the condition is Alzheimer's disease, Parkinson's disease, Huntington'sdisease, stroke, nerve damage, motor neuron disease, multiple sclerosis (MS), Amyotrophic lateral sclerosis (ALS) or peripheral and central nervous system injury including spinal injury and cerebral vascular incidents; wherein the cells that may be employed are cells of the central nervous system, including neurons and / or glial cells, e.g., astrocytes, endothelial cells and / or fibroblasts. In another embodiment, the condition is glutamate-induced apoptosis in the cerebellum.

[0187] In another embodiment, the condition is an age- and / or stress-related disease of the vascular system including atherosclerosis, calcification, thrombosis, hypertension and aneurysms; wherein the cells that may be employed are cells of the heart and vascular system, including endothelial cells, smooth muscle cells, and / or adventitial fibroblasts.

[0188] In another embodiment, the condition is acquired immune deficiency or AIDS; wherein the cells that may be employed are T lymphocytes, such as CD4+ or CD8+ T cells. In another embodiment, the condition is congenital immune deficiency.

[0189] In another embodiment, the condition is graft-versus-host disease (GVHD). In another embodiment, the condition is graft-versus leukemia disease (GVL).

[0190] In another embodiment, the condition or disease is related to inflammation or infection, including severe inflammation or infection, and in some aspects, relating particularly to tissue inflammation and tissue destruction. In some embodiments, the condition or disease is related to meningitis, pneumonitis, pyelonephritis, colitis, or any tissue inflammatory condition / infection, wherein tissue scarring following a significant inflammatory response may result in impaired tissue function.

[0191] In another embodiment, the condition is age- and / or stress-related immune system impairment, including impairment of tissue turnover, which occurs with natural aging, cancer, cancer therapy, acute or chronic infections, degenerative inflammatory diseases or with genetic disorders causing accelerated cell turnover, and related anemias and other degenerative conditions, wherein the cells that may be employed are other cells of the immune system, including cells in the lymphoid, myeloid, and erythroid lineages, such as B and T lymphocytes, monocytes, circulating and specialized tissue macrophages, neutrophils, eosinophils, basophils, NK cells, and their respective progenitors.

[0192] In some embodiments, the methods and / or compositions of this invention find application in improving immune function in disease or in health. In some embodiments, the methods and / or compositions of this invention find application in enhancing T cell activity, for example cytotoxic T lymphocyte activity or responsiveness, or rescue from anergy, which mayfind application in treating multiple diseases, for example in infection or neoplasia. In some embodiments, such methods and / or compositions are particularly useful in treating pathogens highly resistant to conventional therapy, particularly virulent organisms, or organisms for which no other therapies are available, for example, multi-drug-resistant organisms.

[0193] In one embodiment, the methods and compositions of the invention may be useful in allogeneic cell therapy, which may include, in one embodiment, stem cell transplantation, donor cell transplantation or cancer immunotherapy. In another embodiment, the methods and compositions of the invention may be useful in the activation and / or mobilization of stem cells. For example, and in some embodiments, the methods and compositions of the invention may be useful in enhancing survival of stem cells in ex vivo culture.

[0194] In some embodiments, the methods and compositions of the invention may prolong culture time, thereby supporting expansion of the cell population, which in turn enhances transplantation efficiency, while in other embodiments, the methods and compositions of the invention may increase cell viability and / or may increase lifespan of the cells at the stem cell stage, prior to cell differentiation. In some embodiments, the treatment of the population with the compounds as herein described renders them more receptive to other stem cell manipulations, for example transformation or transduction.

[0195] In one embodiment, the stem cells may be embryonic stem cells, while in another embodiment, the stem cells may be adult stem cells. In one embodiment, the methods and compositions of the invention may be useful for culturing stem cells in vitro. In another embodiment, the methods and compositions of the invention may be useful in culturing nonstem cells in vitro. In one embodiment, the methods and compositions of the invention may be useful for culturing, inter alia, pancreatic beta cells in vitro. In another embodiment, the methods and compositions of the invention may be useful for tissue engineering. In another embodiment, the stem cells may be cancer stem cells. In one embodiment, the methods and compositions of the invention may be useful in treating aplastic anemia.

[0196] In some embodiments, the invention provides for the use of compounds and / or compositions as herein described as stimulants of (a) cells with TERT responsive capacity in the central nervous system, including astrocytes, endothelial cells, and fibroblasts which play a role in such age-related diseases as Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS) and stroke, or in repairing nerve damage, motor neuron disease, peripheral and central nervous system injury including spinal injury and cerebral vascular incidents (CVI) such as stroke, or in diseases such as multiple sclerosis(MS); (b) cells with finite replicative capacity in the immune system such as B and T lymphocytes, monocytes, neutrophils, eosinophils, basophils, NK cells and their respective progenitors, which may play a role in immune system impairment.

[0197] In some aspects, such conditions / diseases, etc. are effected via the employ of compounds, which stimulate and / or increase TERT expression and / or activity in the tissues and cells of a subject, which in some aspects is via a canonical and in some embodiments, via a non-canonical pathway.

[0198] In some embodiment, such activity is decreased or absent, or in some embodiments, is damaged due to, for example, a disease, disorder or condition in the subject, and / or arising as a result of a treatment of a disease or disorder or condition in the subject.

[0199] In some aspects, the subjects helped by the compounds, compositions and methods of this invention may be afflicted or predisposed to a cancer or precancerous condition. In some aspects, the subjects helped by the compounds, compositions and methods of this invention may be exposed to radiation or other toxic therapy.

[0200] The compounds of this invention can be produced inter-alia, by the methods described hereinunder in , for example, the accompanying examples. It is noted that the synthetic processes for preparation of the compounds / compositions described herein require a high degree of skill and carefully following the guidance provided in the Examples herein as traditional synthetic chemical means were unsuccessful in identifying additional derivative compounds, and the claimed derivative compounds.

[0201] In some aspects as further described and exemplified herein, uniquely, the derivative compounds of this invention were completely soluble at pH 6 and 7, for up to 94 hours, in the buffer systems assessed and at a pH of 8, the Compounds were completely soluble at 24 hours, demonstrating that Compounds 2-4 hydrolyze over time, depending on the conditions evaluated, with Compound 4 providing the greatest solubility.

[0202] Uniquely, the derivative compounds of this invention provide greater solubility in comparison to a structurally related, non-derivatized compound, i.e. Compound 1.

[0203] As further described herein, in fact there is a rapid conversion of Compound 4 to Compound 1 in plasma. Surprisingly, however, and entirely unexpectedly, the conversion in humans was even more rapid than that of rodents, indicating complete hydrolysis of Compound 4 in human plasma, which is an important and unexpected finding.

[0204] Also surprisingly, although it would have predicted that a proline-substituted derivative of Compound 1 would be an ideal candidate, here again, surprisingly, the proline-substituted compound was not stable / difficult to obtain. It was even more unexpected that the glycolic acid substitution would yield the most promising derivative and moreover, that the trisubstituted ester would be the most readily converted in human plasma was completely unpredictable.

[0205] In still other aspects of the invention, uniquely, Compound 4 may be provided at and tolerated at much higher dosages than what was possible for use of e.g. structurally related compounds, such as Compound 1. For example, dosages of up to 150 mg even in a small volume, such as 0.5 or 1 ml is readily delivered when administering Compound 4.

[0206] These in vitro results related to characteristics of the Compounds as described herein, in particular as relating to Compound 4, translate into improved in vivo effects, as well. For Example, and as described further hereinbelow, in vivo administration of Compound 4 to mice resulted in a highlight significant increase in TERT expression in the brains of treated animals and remarkably significantly increased neurofilament expression and neuronal plasticity associated genes in nervous tissue in treated animals, as well.

[0207] Uniquely, the compounds of this invention, in particular Compound 4 may improve cognitive ability, for example, through its effect in enhancing SYP expression and neuronal growth factors expression.

[0208] The effects of the derivative compounds were also shown to be dose- and timedependent.

[0209] 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.

[0210] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of' means “including and limited to”. The term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0211] It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as set forth in the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the claims.

[0212] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of a conflict between the specification and an incorporated reference, the specification shall control. Where number ranges are given in this document, endpoints are included within the range. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges, optionally including or excluding either or both endpoints, in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Where a percentage is recited in reference to a value that intrinsically has units that are whole numbers, any resulting fraction may be rounded to the nearest whole number.

[0213] In the claims articles such as "a,", "an" and "the" mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" or "and / or" between members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides, in various embodiments, all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g. in Markush group format or the like, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in haec verba herein. Certain claims are presented in dependent form for the sake of convenience, but Applicant reserves the right to rewrite any dependent claim in independent format to include the elements or limitations of the independent claim and any other claim(s) on which such claim depends, and such rewritten claim is to be considered equivalent in all respects tothe dependent claim in whatever form it is in (either amended or unamended) prior to being rewritten in independent format.

[0214] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0215] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0216] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.EXAMPLES

[0217] Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.

[0218] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal,"A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.EXAMPLE 1: SYNTHESIS OF VARIOUS DERIVATIVES OF THE TRI-PHENYL COMPOUNDS

[0219] Previously we demonstrated therapeutic benefit in a number of diseases with the use of the Compound (Compound 1) described by the following Formula:Compound 1.

[0220] In order to assess whether derivative compounds could provide a greater therapeutic effect in specific diseases and / or conditions, certain derivative compounds as described herein were prepared and assessed in this context.

[0221] After in silico consideration of the potential derivatives, the lead derivative compounds of interest included proline- (Compound 5) and diglycolic acid- substituted derivatives (Compounds 2, 3 and 4).

[0222] Toward this end, it was perceived that the proline-substituted derivative would be a more stable and effective compound and synthesis of Compound 5 was pursued:Compound 5

[0223] Attempts to prepare the proline-substituted derivative Compound 5, however, provided surprisingly unsatisfactory.

[0224] Synthesis of Compound 5 was attempted according to the following synthetic protocol:

[0225] Compound 1, proline BOC and DMAP were dissolved in DCM and the solution was cooled in an ice bath, followed by DCC addition, and after 5 minutes, the ice bath was removed, whereby the system was stirred at RT for 12 hours and product was subjected to LC- MS, where multiple products were observed, yielding peaks at 6.96, 8.51 and 10.56 minutes.

[0226] Synthesis of Compound 5 was then again attempted, according to the following different synthetic protocol:

[0227] In this synthetic protocol, Compound 1 was dissolved in DCM and the solution was cooled in an ice bath, followed by Proline chloride hydrochloride addition, and after 5 minutes the ice bath was removed.

[0228] The system was then stirred at RT for 12 hours and an LC-MS sample was measured, however, in this instance, the clean product obtained, was not the desired product.

[0229] Given the difficulty in attaining the desired compound at a reasonable stability and purity, different derivative compounds were considered, and synthesis of glycolic acid substituted groups on the phenolic positions of the base Compound 1 (i.e. Compounds 2-4) were prepared.

[0230] The following compounds, characterized by the following formulae, as Compound 2, Compound 3 and Compound 4 were synthesized:Compound 2.

[0231] Compound 3 is described by the following:Compound 3

[0232] Compound 4 is described by the following Formula:

[0233] The Synthesis procedure for the Compounds of Formula 2-4 is as described hereinbelow (everything at standard conditions unless otherwise noted), and a flow diagram is provided in Figure 1. Briefly, the compound of Formula 1, digly colic acid anhydride, triethylamine and DMAP were dissolved in DCM in a 20-mL scintillation vials and the reaction mixture was stirred for at least 12 hours.

[0234] The reaction was checked for completion by HPLC. Completion criteria were less than 1% the starting compound of Formula 1 remaining. 4. 30 mL of hexane were added to precipitate all the product as an oil and the solution was decanted. The oil was washed once with 10 mL hexane and 10 mL of 10% H3PO4 in water was added with the pH normalized to approximately 5 (wherein additional H3PO4 solution was added as required to achieve the desired pH).

[0235] After 10 minutes, the suspension was extracted 5 times with 5 mL of MTBE. After each extraction, the organic phase was collected and the combined organic phases were dried over MgSCU. The dried organic phase was then evaporated to obtain the clean, free acid, product.

[0236] LC-MS results (see Figure 2A) and HPLC results are shown (see Figure 2B- C), indicating the purity of the Compounds. The HPLC of the crystalized material was recorded at 210 nm and shows the peak at a retention time (RT) of 11.05 minutes forCompound 4 and a small peak of the hydrolysis to Compound 2 at 12.26 minutes is seen in Fig- 2B, whereas the HPLC at the end of reaction, was recorded at 210 nm and shows the peak at a retention time (RT) of 11.05 minutes for Compound 4 and no peak at 14.1 minutes of Compound 1 (Fig.2C).

[0237] HPLC was conducted using an Agilent 1100 series instrument, with the Column: xbridge, 3.5 pm , C18, 150*3.0mm. The Mobile phase A was constituted of: HPLC water + 0.1% trifluoro acetic acid, and phase B was constituted of: acetonitrile. Flow was set to 0.5 mL / min, UV Wavelength: 210 nm, Column temperature: 30°C, with Post time: 0 minutes and Run time: 22 minutes for the settings.

[0238] The compounds were then recrystallized as follows:

[0239] The compounds were dissolved in 3 times the product’s weight of MTBE followed by the addition of 6 times the product’s weight of hexane. The solution was stored for 24 hours at -20°C, in order to crystalize the product as an offwhite solid.

[0240] The solid compounds were collected by filtration, washed with 1-time the product’s weight of ice-cold hexane, and dried under vacuum (10 mbar) for 6 hours.EXAMPLE 2ENHANCED SOLUBILITY OF THE DERIVATIVES OF THE TRI-PHENYL COMPOUNDS

[0241] The solubility of Compounds 2, 3 and 4 were visually inspected following adding pre-weighed prodrug to 1 M of either a sodium phosphate and a potassium phosphate buffer, at room temperature, at different pH levels, in order to give a solution of ~40 mg / mL. The suspension was vortexed for 5 minutes at room temperature until dissolved.

[0242] Compounds 2-4 were each soluble at a concentration of at least 40 mg / mL at pH = 6, 7, and 8, in all tested buffers.

[0243] At pH = 5, however, the compounds were considerably less soluble (as noted by the presence of visible solids).

[0244] Further confirmation of the solubility was verified as follows: all samples were filtered through a 0.22 pm syringe filter and their filtrates were analyzed by HPLC against an acetonitrile standard (single point calibration). The HPLC analysis confirmed complete solubility (~40 mg / mL) of the compounds at pH = 6, 7, and 8.

[0245] In contrast, the solubility at pH = 5 was lower for each compound, visible particles were observed, and the solution concentration measured by HPLC was 7 mg / mL. The concentration measured was the total of all ester forms and is summarized in Tables 2 and 3 below.

[0246] The data indicate that the glycolic mono-, di- or tri-substituted Compounds 2-4 exhibit increased solubility in water, as compared to Compound 1.

[0247] In one non-limiting aspect of this invention, the enhanced water solubility provides significantly better results in administering the compounds when administering Compound 4.

[0248] Stability was determined by sampling the abovementioned solutions (pH = 5, 6, 7, and 8, at 40 mg / mL of prodrug, ini M phosphate buffer), stored at room temperature, at several time points over 6 days, until solids were visible.

[0249] HPLC measurements were compared to a solution of the compounds in acetonitrile, which did not exhibit any degradation in the form of either a decrease in total concentration measured, the appearance of hydrolysis products by HPLC analysis, or by appearance of visible solids, throughout the sampling period.

[0250] Table 2 describes the stability determined for the indicated compounds in sodium phosphate buffer solutions (expressed in mg per mL).

[0251] The Compounds were demonstrated to be completely soluble at pH 6 and 7 for up to T=94 hours, however, solids appeared between T=94 hours and T=144 hours. The pH=8 sample was completely soluble at 24 hours, but solids were present at T=94 hours and therefore the sample was not further evaluated at the 144 hours timepoint.

[0252] Table 3 describes the stability data for the compounds in potassium phosphate buffer solutions (expressed as mg per mL).

[0253] As can be understood from the results in Table 3, in the potassium phosphate buffer, as well, at pH 6 and 7, Compounds were completely soluble up to 94 hours, where the appearance of solids occurred between 94 and 144 hours. At a pH of 8, the Compounds were completely soluble at 24 hours, but solids could be discerned by 94 hours, similar to what was seen with sodium phosphate bugger.

[0254] These data indicate that the ester bonds were labile to hydrolysis in aqueous solution, with the bi-ester form growing over time, followed by the appearance of a mono-ester form.

[0255] The half-life for the Compounds in buffered solutions was determined as the time point at which no visible particles were detected, and the concentration of the tri-ester form as measured by HPLC was approximately half of that at the starting soluble concentration. Table 4 describes the half-times of Compound 4 at the indicated pH, in the respective buffers:

[0256] From the results provided hereinabove, Compound 4 (Chemical name: 2, 2', 2"- ((((ethane- 1,1,1 -triyltris(2,6-bis(ethoxymethyl)benzene-4, 1 -diyl))tris(oxy))tris(2-oxoethane- 2,l-diyl))tris(oxy))triacetic acid) emerged as the most stable in its tri -ester form in buffered solutions, at a concentration of approximately 40 mg / mL, for at least 24 hours, without significant hydrolysis occurring.

[0257] Over the 6-day time period tested, and the bis and mono adducts accumulate and eventually, Compound 1 is obtained.EXAMPLE 3COMPARATIVE EVALUATION OF THE TRI-PHENYL COMPOUNDS IN TERMS OF PLASMA STABILITYMATERIALS & METHODS:

[0258] Murine, Rat and pooled Human Plasma samples were purchased from Commercial suppliers (BioIVT (murine and Rat) and TCS Biosciences (human).

[0259] Plasma samples were spiked with the indicated Compounds, prepared as follows:

[0260] Thawed plasma was mixed, then centrifuged for 10 min at 4 C 3000 rpm, then 3 pL of the solution containing the indicated Compounds (2.5 mg / mL) were taken, and dissolved in 1497 pL (15 mL plastic tube) from the prepared plasma and mix, final concentration was 5 pg / mL. Visual inspection for precipitates was inconclusive since the plasma is not completely clear.

[0261] Samples were incubated at 37 C water bath for 0.25, 0.5, 1, 2, 4, 6, and 24 hr (including T-0, with no incubation). Controls: T-0; plasma control - prepared the same as samples but with no analyte:

[0262] 100 pL of plasma was used and dissolve into 300 pL ACN. At each time point, the incubation tube was mixed and 100 pL of sample added to 300 pL ACN in an Eppendorf vial, mixed well, and centrifuged at 5000 rpm speed for 10 min at 4 C. Approximately 300 pL of the processed sample was added into a 350 pL-insert in an HPLC vial and injected, utilizing an injection volume of 100 pL.

[0263] All micro-volumes were taken by means of calibrated pipettors with microtips.

[0264] Additional extraction with acetone, as opposed to ACN was assessed, as well:

[0265] 50 pL of plasma sample was added to 450 pL acetone, mixed well, and centrifuged for 10 minutes at 4 C and 5000 rpm, and a volume of 100 pL were injected for HPLC analysis.

[0266] Additional Extraction with ACN+TFA, as opposed to ACN was assessed, as well:

[0267] The comparison was performed between adding 100 pL of plasma sample to 300 pL ACN and adding 100 pL of plasma sample to 300 pL ACN with TFA (Final TFA concentration was 0.05%).

[0268] A second extraction of the plasma was performed with either ACN (150 pL of PW were added to residual plasma sample, mixed, sonicated for 4 minutes, and mechanically agitated with an Eppendorf tip. 450 pL of ACN were added and mixed, followed by centrifugation for 10 minutes at 4 C and 5000 rpm. A volume of 100 pL was injected for HPLC analysis), or with acetone (150 pL acetone was added to residual plasma sample,mixed, sonicated for 4 minutes, and mechanically agitated with an Eppendorf tip. Samples were centrifuged for 10 minutes at 4 C and 5000 rpm, and a volume of 100 pL was injected for HPLC analysis).

[0269] Prior to injection, samples were evaporated and re-dissolved in a small volume of ACN (45 pL).

[0270] Alkaline hydrolysis of plasma pellet: All liquid was removed from plasma pellet (2 separate samples), and pellet washed once with 60 pL of PW:ACN (1 :3). A volume of 150 pL of 0.5 M NaOH in PW was added to the plasma pellet and mixed well. The mixture was incubated in a water bath at 80°C for 30 min. The vials were allowed to cool, and then formic acid was added to vials: vial (a), 5 pL of formic acid (pH~7), and vial (b), 10 pL of formic acid (pH~6 to 5). Vial (a) underwent a liquid / liquid extraction with chloroform: 50 pL from vial (a) were added to 200 pL chloroform, centrifuged for 10 minutes at 4 C and 3000 rpm; removed chloroform bottom phase and volumes of 40, 4, and of 1 pL were injected for HPLC analysis. Vial (b) underwent c-18 solid-phase extraction: 150 pL of vial (b) were taken for C-18 solid phase extraction: C-18 cartridge washed with 3X volume PW, followed by a wash with 3X volume ACN:PW (95:5), and final wash with PW (cartridge cleaning). Added 500 pL of PW, then 1000 pL of PW:ACN (9: 1), followed twice by 1000 pL of PW:ACN (5:95). Liquid collected after each addition, and a volume of 100 pL from each collection were injected for HPLC analysis. In addition, each of vials with collected liquid (after injections) were evaporated and re-dissolved in 100, 200, or 400 pL of same PW:ACN solutions as used prior to evaporation. Different volumes between 5 pL and 100 pL were injected for HPLC analysis.

[0271] 100 pL of plasma sample was added to a C-18 solid phase extraction syringe.Pressure was applied to remove the liquid; 150 pL of PW was added and pressure applied to expel the liquid; 150 pL of ACN were added in 6 repetitions (pressure applied between each ACN application to expel the liquid); Each ACN addition was collected in an HPLC vial and a volume of 100 pL of each collection was injected for HPLC analysis.

[0272] 50 pL of plasma sample was added to a C-18 solid phase extraction syringe.Pressure was applied to remove the liquid; 300 pL of PW was added and pressure applied to expel the liquid; 1500 pL of ACN solution (95% in PW) were added and pressure applied to expel the liquid; Liquid was collected in an HPLC vial, evaporated, and redissolved with 400 pL of ACN:PW (3: 1). Volumes of 25 pL and of 100 pL were injected for HPLC analysis.

[0273] 50 pL of plasma sample was added to a C-18 solid phase extraction syringe.Pressure was applied to remove the liquid; 500 pL of PW was added and pressure applied to expel the liquid; 200 pL of ACN solution (20% in PW) were added, pressure applied, and liquid collected; 200 pL of ACN solution (95% in PW) were added in 3 repetitions (pressureapplied between each ACN application to expel the liquid); Each ACN addition was collected in an HPLC vial and a volume of 25 pL of each collection was injected for HPLC analysis.

[0274] An HPLC system including auto sampler, DAD UV detector, column temperature controller, and degasser was used, with the following Column: Waters XBridge® C18 3.5pm, 3.0 x 150 mm or an equivalent column.

[0275] The following Gradient was followed:

[0276] The following conditions were met:

[0277] Injection volume 100 pL, Auto sampler at 5 °C, Column temperature 30 °C.RESULTS:

[0278] In Example 2 it was shown that the Compounds 2-4 were hydrolyzed over time, depending on the conditions evaluated, with Compound 4 providing the greatest stability.

[0279] Therefore it was of interest to determine whether the increased stability of Compound 4 would correlate with greater plasma stability, and same was compared to that of Compound 1.

[0280] Stability of the prodrug was tested at a single concentration in mouse, rat, and human plasma. The testing was performed by incubation of Compound 4 in plasma from each species tested at a temperature of 37 C and analyzed at several time points over a period of 24 hours. Stability of Compound 1 was examined in mouse plasma over a period of 6 hours, in which time there was no change in its concentration, indicating that the molecule is stable for the time period tested.

[0281] Analysis of the ester forms was performed qualitatively according to the response / peak area measure of the concentration, whereas the actual concentration of Compound 1 in plasma was calculated using a calibration curve.

[0282] Compound 1 is known to be somewhat insoluble in aqueous solution, and as a result, a calibration curve was prepared by diluting a DMSO stock solution containing Compound 1 in mouse plasma, in order to establish the relationship between the response (as assessed by peak area) and the actual concentration.

[0283] The calibration curve was constructed by preparing dilutions of 1 :2, 1 :5, 1 : 10, and 1 :50 in mouse plasma of a 1.25 pg / mL Compound 1 stock solution (in DMSO) and injecting 100 pL of each dilution for HPLC analysis. A calibration curve was performed inmouse plasma and was used for qualitative calculation of the concentration of Compound 1 in rat plasma as well. Figure 3 plots the calibration curve constructed in the plasma dilution example, as described and Table 3 presents these results, as follows:

[0284] Next, a calibration curve was derived for Formula 4. The calibration curve forCompound 4 was prepared as a 1 :5 dilution of a 5 pg / mL Prodrug stock solution (in buffer), followed by 2 serial dilutions (1 : 10 and 1 :5). The dilutions were prepared in PW [purified water], and a volume of 100 pL of each dilution was injected for HPLC analysis, with the results presented in Figure 4 and Table 4 presenting the following results:

[0285] Tables 5 and 6 further extend these findings.

[0286] Table 5 demonstrates the stability of Compound 4 in mouse plasma at 5pg / mL:

[0287] Table 6 describes the Compound stability using the calibration curves.

[0288] The concentration in mM for Compound 1 produced in plasma over time after spiking the plasma with Compound 4 is further described by Figure 5, where the recovery depicted is equal to the Total moles of Compound 1 and Compounds 2-4 after 24 hours divided by the number of Moles of Compound 4 in PW, times 100.

[0289] Similar studies were then conducted probing the results in Rat Plasma, as depicted in Table 7 and Figure 6.

[0290] Table 7:Cmteenteiitton (pM) using calibrutioit curves

[0291] These studies were still further extended by probing the results in HumanPlasma, as depicted in Table 8-9 and Figure 7.

[0292] Table 8:ND - not detected

[0293] Table 9:* ConceaDatsanx e&kaiated based oss eshbratinn wve of Frodrug with FW dihstksKs w Estimation of emcentadons according t» CMPD I otdbratksi ram in m«e plasma 4,M!Qwentatioas calculated based on calibration curve wth sansas plasma dilutions

[0294] These results demonstrate that Compound 4 is even more rapidly hydrolyzed in human plasma, compared with mouse and rat plasma.

[0295] Although not quantified, the response curve showed a steep increase in the peak area for Compound 1 that plateaus within 2 hours of incubation. This was the identical trend observed for incubations in mouse and rat plasma, where the only difference discerned was the faster kinetics in human plasma.

[0296] The recovery of Compound 1 ultimately from plasma samples incubated with Compound 4 was relatively high (>80%) and comparable to its recovery from samples spiked with Compound 1, itself.

[0297] Together these results demonstrate a rapid conversion of Compound 4 to Compound 1 in plasma. The immediate disappearance of the triester form, concomitant with the appearance of the di- and mono-ester forms, and eventually Compound 1, indicate complete hydrolysis of Compound 4 in plasma, seen under all experimental conditions and for all species tested.EXAMPLE 4 COMPARATIVE EVALUATION OF THE TRI-PHENYL COMPOUNDS IN TERMS OF IN VIVO EFFICACYMATERIALS & METHODS:

[0298] ICR male mice (10 weeks old) were divided to treatment groups (UT, DMSO, Compound 1, Compound 4, 4 mice per group. Mice were treated with a single s.c. injection (into the mouse neck) of Compound 1 (6 mg / kg in 100 pl PBS) or with DMSO 0.5% in PBS, as a vehicle, or with Pro-drug Compound 4 (6mg / kg in 100 pl Phosphate buffer 0.3M- Na2HPO4, NaH2PO4). Twelve hours post treatment, the mice were sacrificed, and Frontal lobe was removed and placed at -80 oC for 2 weeks before analysis.

[0299] Murine frontal lobes were homogenized in a Tri-reagent RNA extraction buffer (Sigma- Aldrich, Rehovot, Israel) and RNA was isolated according to the manufacturer’s protocol. RNA concentration was determined using NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific Inc., Pittsburgh PA, USA). Purification of samples from DNA by DNase reaction, was conducted according to the manufacturer’s protocol. Purified RNA samples (lOOOng) were run in 1.5% agarose gel. The RNA (lOOOng) was converted to cDNA with qScript cDNA Synthesis Kit (Quantabio) according to the manufacturer’s instruct! on s3.

[0300] Real Time -PCR reaction was used to measure genes expression by the appropriate set of premieres using the PerfeCTa SYBR Green FastMix4 (Quantabio) with LightCycler-'® 480 Instrument II (Roche) and were measured by AACt method in which the gene expressions are calculated relative to the housekeeping gene.RESULTS:

[0301] An increase in TERT expression in the Compound 1 treated mice by 120.9±6.19 % p<0.0001 and in the Compound 4 by 140.8±4.8% p<0.0001 compared to DMSO and UT mice respectively, suggesting that the administration of Compound 4 is more efficient in the increase of TERT in the brain compared to Compound 1 (Figure 8). These results support that Compound 4 enhanced TERT expression in the mouse brain more efficiently than Compound 1.

[0302] Neurofilaments (NF) found in the cytoplasm of neurons are protein polymers (10 nm in diameter) and many micrometers in length. Together with microtubules (~25 nm) and microfilaments (7 nm), they form the neuronal cytoskeleton, presumably providing structural support for axons and regulating axon diameter, which influences nerve conduction velocity. Figure 9 demonstrate that treatment with Compound 1 increased the level of expression of neurofilaments subunits: NFH ( byl28.3±4.6% p<0.0001) NFL (by 112.4±3.16% p<0.0001) and NFM (by 116.3±4.1% p<0.0001) compared to DMSO treatment. Treatment with the new Compound 4 revealed a marked increase in neurofilaments expression in the mouse brain: NFH (by 209.7±10.45% p<0.0001) NFL (125.8±4.7% p<0.0001) and NFM (120±4.13% p<0.0001). The results suggest that Compound 4 treatment is more effective in the enhancement of neurofilaments expression in the brain compared to Compound 1.

[0303] Compound 4 enhanced the expression of Gap43 and SYP more efficiently than Compound 1. Figure 10A-B demonstrates that Compound 4 treatment enhanced the expression of neuronal plasticity genes in the mouse brain more efficiently than Compound 1 : Gap43- this gene encodes for a protein that has been termed a 'growth' or 'plasticity' protein because it is expressed at high levels in neuronal growth cones during development and axonal regeneration. This protein is considered a crucial component of an effective regenerative response in the nervous system. Alternatively spliced transcript variants encoding distinct isoforms have been found for this gene that regulated axon growth). Gap-43 was enhanced by Compound 4 treatment by 145.8±10.5%, p=0.0001 and by Compound 1 : 129.7±8.1%, p=0.0003 compared to untreated and vehicle (DMSO) treated respectively (Fig 10A).

[0304] SYP -This gene encodes an integral membrane protein of small synaptic vesicles in brain and endocrine cells. The protein also binds cholesterol and is thought to direct targeting of vesicle-associated membrane protein 2 (synaptobrevin) to intracellular compartments. Mutations in this gene are associated with an X-linked form of cognitive disability. Treatment with Compound 4 enhanced SYP expression by 109.2±2.1, p<0.0001 and by Compound 1 107±4, p=0.04 compared to UT and vehicle (DMSO) treatment respectively (Fig 10B).

[0305] Compound 4 enhances the expression of neuronal growth factors more efficiently than Compound 1. BDNF- belongs to the neurotrophin family of growth factors, and it is related to the canonical nerve growth factor. Neurotrophic factors are found in the brain and the periphery. BDNF acts both on neurons in the CNS and on the peripheral nervous system. BDNF supports the survival of existing neurons and encouraging growth and differentiation of new neurons and synapses.

[0306] Treatment with Compound 4 enhanced BDNF expression by 136.8±7.8 %, pO.OOOl while Compound 1 by 112.2±8.2%, p-0.045 compared to untreated and DMSO treatment respectively (Fig 11 A)

[0307] NGF- is involved the growth, maintenance, proliferation, and survival of neurons. NGF is critical for the survival and maintenance of sympathetic and sensory neurons, as they undergo apoptosis in its absence. NGF may play a critical role in the maintenance and survival of pancreatic beta cells and in the regulation of the immune system. Treatment with Compound 4 increased the expression of NGF in the mouse brain by 120.6±4%, p=0.0003 and Compound 1 by 110.9±4.3 p=0.02 (Fig 11B).

[0308] Compound 4 enhanced the expression of neurogenesis related genes more efficiently than Compound 1. Adult neurogenesis, the process of generating mature neurons from neuronal progenitor cells, makes critical contributions to neural circuitry and brain function in both healthy and disease states. Doublecortin (DCX) has recently been used as amarker for neurogenesis. It is a neuronal progenitor marker. It is considered as a reliable and specific marker that reflects levels of adult neurogenesis and its modulation.

[0309] SOX2 is a transcription factor with established roles in maintaining stem cell / progenitor cell properties in diverse cellular populations. Genetic deletion of Sox2 causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Human Sox2 mutations are associated with anophthalmia, a disorder characterized by cognitive disabilities and defects in hippocampal development

[0310] Compound 4 treatment enhanced the expression of neuronal progenitors cells markers DCX by 156±6.5, p<0.0001 while Compound 1 by 146.6±23 p =0.009 (Figure 12A). SOX2 was enhanced by Compound 4 treatment to 158.3±24, p=0.01 while by Compound 1 to 108.6±13 p=ns (Figure 12B).

[0311] Compound 4 enhanced the expression of genes related to the Wnt / beta catenin signaling pathway more efficiently than Compound 1. Wnt signaling plays a key role in the regulation of neurogenesis and tissue homeostasis in the nervous system. Wnt signaling controls the balance of neuronal stem cells (NSC) proliferation and differentiation via the transcriptional coactivator P-catenin during brain development and adult tissue homeostasis. Disruption of Wnt signaling may result in developmental defects and neurological diseases.

[0312] Compound 4 treatment enhanced the expression of beta-catenin transcription factor by 331.9±77%, p=0.004 and Compound 1 by 183.3±16.7% p<0.0001 (Fig 13A). To examine if the Wnt / beta catenin pathway was indeed activated we investigated one of its major target Cyclin DI. Indeed the expression of cyclin DI was enhanced by Compound 4 treatment by 116.8±4.6% p=0.0022 but not by Compound 1 (Fig 13B).EXAMPLE 5DOSE DEPENDENT EFFECTS OF THE TRI-PHENYL COMPOUNDSMATERIALS & METHODS:

[0313] ICR male mice (10-11 weeks old ) were divided to 4 treatment groups ( UT, Compound 4 treated at 1 mg / kg, 6mg / kg / , 12mg / kg (8 mice per treatment group). Mice were treated with a single s.c. injection (into the mouse neck) of Compound 4 dissolved in 100 pl Phosphate buffer 0.3M- Na2HPO4, NaH2PO4). Twelve hours post treatment, the mice were sacrificed, and Frontal lobe was removed and placed at (-80 °C) for further analysis as described hereinabove.RESULTS:

[0314] The most effective dose for TERT enhancement in the brain is 6 mg / kg. TERT expression in the mouse frontal lobe brain was analyzed by real-time PCR with twodifferent sets of primers. The results revealed that the most effective dose is 6mg / kg doubling the dose (12 mg / kg) did not significantly increased the level of TERT compared to the 6 mg / kg dose. Compound 4 at 1 mg / kg for the F-TERT primers increased TERT expression by 115.9+5.5%, p=0.0059; at 6mg / kg by 134.9+6, p<0.0001 and at 12mg / kg by 133+7, p=0.0002 (Fig 14).

[0315] Compound 4 increased neurofilaments expression in the brain in a dose dependent manner. Treatment of mice with a single injection of Compound 4 at various doses (1,6,12 mg / kg) revealed a significant increase in the expression of the three neurofilaments (NF) subunits (H,M and L) in a dosed dependent manner. The most effective dose was 6mg / Kg. Although doubling the dose (12mg / Kg) revealed a slight increased in the expression level of the NF but this was not statistically significant compared to 6mg / kg (Fig 15). NFH- Img / Kg: 131.3+8% p=0.0002; 6mg / kg: 164.2+10% p<0.0001; 12mg / kg: 183.4+9% p<0.0001. NFM- Img / kg: 107.5±7%: 6mg / kg: 121.5+8% p=0.075: 12mg / kg: 138.4±9% p=0.0008. NFL:- Img / kg: 104.1+4%; 6mg / Kg: 115.5+8% p=0.003; 12mg / kg 121.8+7 p=0.004.

[0316] Compound 4 increased the expression of neuronal plasticity related genes in the brain in a dose dependent manner. Treatment of mice with a single injection of Compound 4 at various doses (1,6,12 mg / kg) revealed a significant increase in the expression of neuronal plasticity related genes (Gap43, SYP jin a dosed dependent manner but did not affect the expression of NeuN. The most effective dose was 6mg / Kg. Although 12mg / kg slightly but not significantly increased Gap43 and SYP expression compared to 6mg / kg (Fig 16). Gap43: Img / kg: 110.2 ± 4.153 % p=0.013; 6mg / kg: 121.1 ± 3.057% p<0.0001; 12mg / kg: 134.3 ± 4.694% p<0.0001. SYP Img / kg: 98.91 ± 3.936%; 6mg / kg: 111.7 ± 7.057% p+0.049; 12mg / kg: 119.7 ± 3.851% p<0.0001.

[0317] Compound 4 increased the expression of neuronal growth factor gene in the brain in a dose dependent manner. Treatment of mice with a single injection of Compound 4 at various doses (1,6,12 mg / kg) revealed a significant increase in the expression of neuronal growth factor (NGF) in a dose dependent manner. Here the most effective dose was 12mg / kg . Img / kg: 121.7 ± 5.299% p<0.0001; 6mg / kg: 114.4 ± 3.617% p=0.0001; 12mg / kg: 151.4 ± 8.041% p<0.0001 (Fig 17).

[0318] Compound 4 increased the expression of genes related to neurogenesis process in the brain at a dose dependent manner. Treatment of mice with a single injection of Compound 4 at various doses (1,6,12 mg / kg) revealed a significant increase in the expression of genes related to neurogenesis process ( DCX. SOX2 and cyclin DI) in a dose dependent manner. Here 12mg / kg of Compound 4 demonstrated the most effective dose (Fig. 18). DCX- Img / kg: 153.7 ± 20.29% p=0.0096; 6mg / kg: 135.7 ± 20.36% p=0.05; 12mg / kg: 291.4 ± 9.970% pO.0001; SOX2- Img / kg: 101.4 ± 9.759 N=4; 6mg / Kg: 126.7 ± 12.69 N=4 p=0.04;12mg / kg: 166.4 ± 8.154 N=4 p<0.0001. Cyclin DI : Img / kg: 95.84 ± 7.296 %; 6mg / kg: 103.4 ± 4.147 %; 12mg / kg: 116.0 ± 5.511 % p=0.005.EXAMPLE 6TIME DEPENDENT EFFECTS OF THE TRI-PHENYL COMPOUNDSMATERIALS & METHODS:

[0319] Twenty four ICR mice were S.C injected with 6mg / kg of Compound 4 (dissolved in 0.3M phosphate buffer) and divided to 4 treatment groups (6 mice per group): Control untreated, sacrificed after 12 h, or 24h, or 48h. The brain was removed and subjected to RNA or protein extractions for further analysis of Compound 4 effects on TERT and other genes and proteins expression.RESULTS:

[0320] Figure 19 demonstrates that there is a time dependent increase of TERT in the mouse brain in treated animals. The maximum increase of TERT was observed 12 h after Compound 4-treatment (127.3±5.8% N=28, p<0.0001) which reduced to its basal level after 48 h.

[0321] Figure 20 demonstrates that treatment of mice with 6mg / kg of Compound 4 significantly increased neurofilament genes expression with time in the mouse brain: NfH - 135.6 ± 6.735 N=17 P <0.0001; 124.9 ± 5.791 N=16 p<0.0001; 125.6 ± 6.707 N=15 p< 0.0005 for 12, 24 and 48h after treatment respectively. NfM- 127.7 ± 5.586 N=16 P<0.0001; 126.5 ± 4.813 N=18 P0.0001; 138.4 ± 5.550 N=15 P<0.0001 for 12, 24 and 48h after treatment respectively. NfL significantly increased 12h after treatment to 112.1 ± 4.768 N=11 p<0.008.

[0322] Figure 21 demonstrates that there is a time dependent increase of neuronal growth factor and genes related to neuronal plasticity and neurogenesis following Compound 4 treatment. Treatment of mice with 6mg / kg of Compound 4 revealed a significant increase with time in the level of NGF gene peaked at 48 h: 123.8 ± 4.547 N=12 p<0.0001 (Fig 21A). DCX gene expression (marker for neurogenesis) significantly increased to 178.2 ± 10.74 N=8 p<0.0001 24 hr after Compound 4 treatment and reduced to its basal level 48 hrs after treatment (Fig 2 IB)

[0323] Figure 22 demonstrates that Compound 4 increased the survival of motor neurons cells (NSC-34) exposed to oxidative stress. NSC-34 motor neurons cells were exposed to 100 M or 300 M of H2O2 for 4 h in a serum free medium. The medium was replaced after 4 h with fresh medium containing 10% FCS and Compound 4 at 50 nM or 200nM for 24 h. Cell survival was measured by XTT assay. The results revealed that exposure of NSC34 cells to oxidative stress reduced survival by 3 folds compared to control cells andtreatment with Compound 4 increased the survival by 1.5-1.7 fold compared to H2O2 treated cells.EXAMPLE 7TRI-PHENYL COMPOUND EFFECTS ON IMMUNE ACTIVATION MARKERSMATERIALS & METHODS:

[0324] Twenty four ICR mice (3 months old) were injected subcutaneously (S.C.) with Compound 4 dissolved in 0.3M phosphate buffer and divided to 4 treatment groups (6 mice per group): Control untreated, sacrificed after 12 h, or 24h, or 48h.

[0325] In addition 20 ICR mice were injected S.C. with 6mg / kg of Compound 4 either as a freshly made solution or where the solution prepared a week prior and kept at -20 °C. Mice were sacrificed 12h or 24 h after treatment.

[0326] Blood samples (100 pl) were collected from each mouse prior and after treatment and RNA extracted using the RNeasy Protect animal blood kit (Qiagen). Real-Time PCR was performed for TERT and Lag3 genes using the appropriate primers and b-actin as reference gene.RESULTS:

[0327] It is of interest to identify reliable biomarkers in the blood that can demonstrate the responsiveness of the treated subjects to Compound 4 administration. As TERT expression was influenced by Compound 4 administration in tissue as described herein (for example, with regard to brain tissue as described hereinabove), toward this end, telomerase reverse transcriptase (TERT) expression in blood was chosen as a first potential biomarker - Since Compound 4 was shown herein to be an activator of TERT expression in tissue, it seemed plausible to suggest that its transcriptional level will increase following treatment with Compound 4.

[0328] Lag 3 is an immune check point protein which is elevated following lymphocytes activation. Since we previously showed that Compound 1 and related compounds are lymphocyte activators, we hypothesized that the Lag3 gene may be activated following administration of the compounds of this invention, as well, and that Lag3 may be a reliable biomarker, therefore.

[0329] TERT expression level increased in the blood of Compound 4 treated mice. A statistically significant increase in the TERT mRNA level was detected by real-time PCR in the blood of Compound 4-treated mice was detected 24 hours after treatment (154.3 ± 21.78 N=6 mice per group). An increase of 133.5 ± 19.18 N=6 was detected 12 hours after treatmentand a slight, but not statistically significant increase of 110.1 ± 29.93 N=6 was observed 48 hours after treatment (Figure 23).

[0330] Lag 3 expression also increased in the blood of Compound 4 treated mice. A statistically significant increase in Lag3 mRNA levels were detected by real-time PCR in the blood of Compound 4-treated mice, as detected 24 hours after treatment (200.4 ± 35.11 N=10 mice per group). An increase of 133.1 ± 19.37 N=9 mice per group was detected 12 hours after treatment and an increase of 165.4 ± 37.66, N=6 mice per group was observed 48 hours after treatment (Figure 24), yielding statistically significant differences in Lag3 expression for the treatment at any of the timepoints evaluated.EXAMPLE 8LARGE SCALE PRODUCTION OF COMPOUND 4 MATERIALS & METHODS:

[0331] Compound 4 can be prepared through a process essentially involving 4 steps, starting from 1,1,1 -tri s(4-hydroxyphenyl) ethane (THPE). Referring to the scheme depicted in Figure 25, Step 1 is designated at 9270 in the figure.

[0332] To a 10L 4-neckedd flask, ethanol was added (2.2 kgl, water (2.3kg), THPE (250g, 0.816 mol, 1.0 equiv., BLD, demthylamine (8.59 g, 40% solution in water 7.62 mol, 9.3 equiv) and paraformaldehyde (215 g, 7.2 mol, 8.8 equiv) and the resulting suspension was heated to reflux (80 °C). After 30 min. the reaction mixture was analyzed by HPLC to confirm >99% conversion. The reaction mixture was concentrated in vacuo with a jacket temperature of 80 °C until 2.8 L distillate had been collected. The resulting suspension was then cooled to room temperature overnight and filtered. The filtercake was washed with water (2.0 kgl and dried in an air ventilated drying cabinet at 40 °C for 24 hours yielding the product as a faint yellow to white solid (498.4 g, 94%).

[0333] Step 2 is designated as 9273.

[0334] Acetic anhydride (3895 g, Merck PN: 8.22278.9025 lot#S5520278 050) was added to a 6L 4-necked flask and heated to 70°C. Compound 501 :

[0335] (55 1 .0 g, 0.85 mol, 1 .0 equiv., lot#8 1 46 1 (495.4g) and lot#8 1 434 (55.6g)) was added in 3 portions over 20 min keeping the temperature between 70-85°C. The resulting solution was heated to 1 00°C and kept at this temperature for 5 hours before HPLC analysis confirmed a conversion >98%.

[0336] The reaction mixture was added into water (5.67 kg) heated to 70°C and the addition was performed over 45 min keeping the temperature between 70-82°C. The mixture was cooled to 20°C and kept stirring overnight. To the mixture was added water (2.69 kg) and toluene (2.90 kg) and the phases were left stirring for 15 min, before they were separated, and the aqueous phase extracted with toluene (2.60 kg). The phases were separated, and the aqueous phase was extracted with toluene (2.60 kg). The combined organic phases were concentrated in vacuo with a water batch temperature of 50°C. The evaporation residue was dissolved in acetic anhydride (3089 g) and heated to 1 00°C and kept at this temperature for 3 hours, where HPLC analysis confirmed < 1 % of mono-deacetylated product. The reaction mixture was cooled to 44°C and addition of water (1 142 g) was performed over 30 min keeping the temperature at 35-45°C. The mixture was kept at 35-45°C for 2 hours before it was cooled to 20°C and kept stirring overnight. To the mixture was added water (2.60 kg) and toluene (2.66 kg) and the phases were left stirring for 15 min, before they were separated, and the aqueous phase extracted with toluene (2.62 kg). The phases were separated, and the aqueous phase was extracted with toluene (2.67 kg). The combined organic phases were washed once with water (3.90 kg), and the phases were separated. The organic phase was concentrated in vacuo with a water batch temperature of 50°C. The evaporation residue was added toluene (867 g) and concentrated in vacuo with a water batch temperature of 50°C. This was repeated twice (total of 3 evaporations performed). The evaporation residue was dissolved in 2.5% MeOH in toluene v / v (500 ml) and purified by flash column chromatography using silica (3.769 kg) packed in 2.5% MeOH in toluene v / v (5.90 kg). The column was eluted with 2.5%-5% MeOH in toluene v / v (47.5 L). Fractions containing Nona acetate were combined and concentrated in vacuo with a waterbath temperature of 50°C. The evaporation residue was dissolved in toluene (715.9 g) to give Nona acetate as a solution in toluene (794 g, 1 08%, - 37% w / w solution in toluene (lot#8 1 576)), with the following structure:

[0337] Step 3 is designated as 9282:

[0338] Nona acetate solution in toluene (1482.8 g, lot#8 1 576, 729 g neat Nona acetate, 0.84 mol, 1.0 equiv.) was concentrated in vacuo and dried overnight at ambient temperature under high vacuum. The residue was dissolved in ethanol (885.7 g) by heating the mixture to 70°C. The solution was transferred to a 20L reactor using ethanol (1 026 g). To the solution was added sodium ethoxide (4209 g of 21% w / w solution in ethanol, 884 g neat, 1 3 mol, 15 equiv. Sigma PN: 230553 lot#STBK9407) over 30 min keeping the temperature at 50-60°C. 16 min after complete addition, HPLC analysis confirmed 1 00% conversion and the reaction was left stirring at 50°C an additional 28 min before water (2260 g) was added and the mixture heated to 60°C. After 25 min the pH was adjusted to 1 using 37% HCI (1422 g) while keeping the temperature at 40-50°C. To the resulting solution was added toluene (242 1 g) and the phases were left stirring for 20 min, before they were separated, and the aqueous phase extracted with toluene (223 1 g). The phases were separated, and the aqueous phase was extracted with toluene (2160 g). The combined organic phase was concentrated in vacuo with a water batch temperature of 50°C. The evaporation residue was added toluene (867 g) and concentrated in vacuo with a water batch temperature of 50°C. This was repeated once with toluene (665 g). The evaporation residue was dissolved in 15% ethyl acetate in toluene v / v (500 ml) and purified by flash column chromatography using silica (4.09 kg) packed in 15% ethyl acetate in toluene v / v (7.2 kg). The column was eluted with 15% ethyl acetate in toluene v / v (35 L). Fractions containing the compound of Formula 1 were combined and concentrated in vacuo with a waterbath temperature of 50°C. The evaporation residue was dissolved in ethanol (1446 g) and transferred to a 1 OL 4-necked flask with ethanol ( 1 895 g). The solution was heated to 50°C and added water (2654 g) keeping the temperature at 46-50°C. The slightly turbid solution was seeded and cooled to 28°C over 4 hours. The resulting thick suspension was cooled to 0°C over 1 .5 hours, and left at 0°C overnight. The suspension was filtered andthe solid material washed with a mixture of ethanol (47 4 g) and water (426 g) at 0°C. The wet solids were dried in an air ventilated drying cabinet at 40°C for 20 hours to afford the compound of Formula 1 (380 g, lot#8 1 609, 69%) as a white to beige to slightly yellow solid.

[0339] Step 4 is designated as 9285:To a solution of a compound of Formula I (163.3 g, 0.25 mol, 1.00 equiv., lot#8 1 609) in ethyl acetate (1753 g) was added diglycolic anhydride (116. 1 g, 1.0 mol, 4.0 eqiuv., TCI PN: D2308, lot#TJKWO-FP). To the resulting solution was added triethylamine (115.2 g, 1. 14 mol, 4.6 equiv.) while keeping the temperature at 1 5-24°C. After 30 minutes HPLC analysis confirms complete reaction and after a total of 60 min the reaction mixture was added water (1248 g) while keeping the temperature at 20-25°C. The reaction mixture was stirred 30 min, before pH was adjusted to 3.5 using 37% aqueous HCI (85.8 g). The phases were left stirring for 1 O min, before they were separated, and the aqueous phase extracted with ethyl acetate (1023 g). The phases were separated, and the combined organic phases was concentrated in vacuo with a water batch temperature of 40°C. The evaporation residue was added ethyl acetate (372 g) and methyl tert butyl ether (753 g) and the resulting solution was polish filtrated through GF / A paper. The solution was cooled to 0°C and continuously seeded until seeding material was not dissolving (8°C). The resulting suspension was left stirring overnight at 0°C, before n-heptane (1300 ml) was added over 6 hours. The suspension was filtered and ' ie solids washed with a mixture of methyl tert butyl ether (246 g) and n-heptane (471 g) and dried in vacuo at 25°C for 1 8 hours, then 24 hours at 30°C. The solids were added ethyl acetate (391 g) and methyl tertbutyl ether (753 g) and heated to 40°C, after which the mixture was cooled to 0°C. n-Heptane (1300 ml) was added over 2.5 hours. After 30 min of stirring at 0°C the suspension was filtered, the solids washed with a cold (0°C) mixture of methyl tert butyl ether (248 g) and n-heptane (472 g) and the solids dried in vacuo at 25° for 8 days affording a compound of Formula 4 (243. 3 g, lot#8 1 67 4). 203 g of the compound of Formula 4 lot#8 1 67 4 was added methyl tert butyl ether (437 g) and stirred at 20°C for 40 min before the suspension was cooled to 0°C and stirred at this temperature for 1 hour. The suspension was filtered and the solids dried in vacuo at 25°C with nitrogen bleed for 24 hours to afford the compound of Formula 4 ( 1 88.0 g, 91%, lot#8 1 738) as a beige / slightly yellow solid.EXAMPLE 9METABOLITE PROFILING AND IDENTIFICATION OF COMPOUND 4 IN PLASMA AFTER SINGLE SUBCUTANEOUS ADMINISTRATION TO SD RATS MATERIALS & METHODS:

[0340] Compound 4 was prepared as described in Examples 1 and 8.Sample Collection and Pooling

[0341] Plasma samples were obtained from a PK study (Study No.: 429821-20230519- RPK) in which rats were single subcutaneous administration dosed with 60mg / kg AGP-499 in group 4, day 1. Plasma samples were pooled according to AUC rule [1, 2] for each animal. Based on the result of the PK study, samples from 0 to 8 hours (0, 0.25, 0.5, 1, 2, 4, 6, 8 h) were selected for pooling. The pooling scheme of plasma samples was shown in Table Al.

[0342] A similar volume of rat 0 h plasma sample was used as blank control group.Sample Processing

[0343] 1.92 mL of Acetonitrile: Methanol (95:5) was added to the pooled plasma samples to precipitate the protein. After vortex-mixing and centrifugation at 4,000 rpm for 10 min, the supernatants were analyzed by LC-UV-MSn (n=l~2).

[0344] The following Analytical methods were employed:

[0001] LC System: Waters Acquity UPLCColumn: Waters Acquity UPLC HSS T3 1.8 pm, 2. 1 * 100 mmUV Detector: Waters Acquity PDA, I: 190-400 runColumn Temp.: 40°CAutosampler Temp.: 8°CMobile Phase A: 0.1% FA and 2 mM NH4FA in H2O / ACN (v / v, 95:5)Mobile Phase B: 0.1% FA and 2 mM NH4FA in H2O / ACN (v / v, 5:95)Flow Rate: 0.5 mL / minInjection Volume: 5 pLGradient:Step Time (min) A (%) B (%)1 Initial 95 52 3.00 95 53 7.00 70 304 13.00 40 605 16.00 10 906 18.00 10 907 18.10 95 58 20.00 95 5Mass Spectrometer: Orbitrap Exploris 480Data System: XcaliburIon Source Type: H-ESI (+ / -)Spray Voltage: 3500 V (+); 2500 V (-)Ion Transfer Tube Temp.: 325°CVaporizer Temp.: 350°CSheath Gas Flow: 50 ArbAux Gas Flow: 10 ArbSweep Gas Flow: 10 ArbRF-Lens: 50%Normalized Collision Energy (NCE): 30%, 60%, 90%Scan Type: Full MS; ddMS2Scan Range: 100-1500 m / zResolution: Full MS: 60000; MS2: 15000Data Analysis:

[0345] Post-acquisition data analyses were performed using the Xcalibur, and Mass MetaSite, which generated a series of extracted ion chromatograms (XICs). XICs of the blank control and analysis samples were compared.LC-UV-MS" Analysis of Error! Reference source not found.

[0346] Compound 4 was used to optimize LC-UV-MS instrument conditions as well as to generate molecular ion fragmentation patterns, which were used for metabolite structure elucidation. Under the optimized conditions, Compound 4 was eluted with a retention time of 11.18 min in LC-UV chromatogram. The UV spectrum of Compound 4 displayed a characteristic absorbance at 268 nm (Figure 26Error! Reference source not found.). Therefore, the LC-UV chromatograms were extracted at 250-280 nm. The characteristic MS2fragmentation and elucidation of Compound 4 was depicted in Figure 27.Identification of Metabolites

[0347] Under the current experimental conditions, 4 putative metabolites of Compound 4 were detected and tentatively identified in rat plasma.Error! Reference source not found. The relative abundance of Compound 4 and each of the putative metabolites were calculated using peak area (MS peak area) of individual metabolite relative to the total area for all detected drug related components (Table 1).

[0348] Table 1 : Summary of Compound 4 and its Metabolites in Plasma[M+NH1]+Relative Abundance (MS Peak Area %Total)Code [M-H]- Plasma Metabolic Pathways m / z mmMaleM5 (->945.4100 10.98 0.32 Ester hydrolysis and glucuronidation (P^CJECh+CeHsOe)Compound 4Error!Reference (->1001.4001 11.16 4.26 NA source not found.M6 (->829.4005 12.66 0.27 Ester hydrolysis and glucuronidation (P-3 CTECh+CeHsOe)M7 (->885.3892 12.96 1.93 Ester hydrolysis (P-C4H4O4)M8 (+>788.4203 14.74 93.22 Ester hydrolysis (P-2C4H4O4)

[0349] Note:RT: Retention time of LC-MS; NA: Not applicable; P: Parent; (+) Theoretical Mass (m / z) = Exact Mass + 18.0338; (-): Theoretical Mass (m / z) = Exact Mass - 1.0073.„ . > . Peak Area of a Related Component , „„„ ,%Total = - - - x 100%Peak Area of Total Related Component s

[0350] The MS peak area of Compound 4 and each of the putative metabolites were summarized in Table 1. The accurate mass measurements of Compound 4 and its metabolites in rat plasma were summarized in Table 3 where the maximum mass errors between measured and calculated values were within 5 ppm, indicating a high level of confidence.

[0351] Table 1. MS Peak Area of Compound 4 and its Metabolites in Rat plasma.MS Peak AreaCode -MaleM5 196084Compound 4 2568522M6 162998M7 1165897M8 56256222

[0352] Note: Table was the semi-quantitative data of peak area of mass spectrometry.

[0353] Table 2. Accurate Mass Measurements of Compound 4 and its Metabolites in Rat Plasma„ , RT „ ,ri Theoretical Mass Measured Mass Mass ErrorCode , . . Formula Exact Mass , .(min) (m / z) (m / z) (ppm)M5 10.98 C48H660i9 946.4198 (->945.4125 945.4100 -2.64Compound 4 11.16 CsoHseChi 1002.4097 (->1001.4024 1001.4001 -2.30M6 12.66 C44H620I5830.4089 (->829.4016 829.4005 -1.33M7 12.96 C46H620i7 886.3987 (->885.3914 885.3892 -2.48M8 14.74 C42H580i3 770.3877 (+)788.4215 788.4203 -1.52

[0354] Note: Exact Mass: Monoisotopic Mass; (+): Theoretical Mass (m / z) = Exact Mass + 18.0338; (-): Theoretical Mass (m / z) = Exact Mass - 1.0073; RT Retention time of LC-MS. rnrio cci Measured Mass -Thei5r«tic'al MassAL(J 0355JMass Error = - ihscretT-caiMass x 10s

[0356] The LC-UV (X: 250-280 nm) and LC-HRMS chromatograms of Compound 4 and its metabolites in rat plasma were shown in Figure 28. The MS / MS2spectra data of the detected metabolites with their fragmentation were displayed in Figure 29-32.

[0357] Based on the accurate molecular weight and fragment ions, the metabolites were identified as M5: ester hydrolysis and glucuronidation metabolite (MW=947.04, P- 2C4H4O4+C6H8O6); M6: ester hydrolysis and glucuronidation metabolite (MW=830.97, P- 3C4H4O4+C6H8O6); M7: ester hydrolysis metabolite (MW=886.99, P-C4H4O4); M8: ester hydrolysis metabolite (MW=770.91, P-2C4H4O4).

[0358] In male rat plasma, Compound 4 and 4 metabolites (M5-M8) were detected. M8 accounting for 93.22% of the total drug-related components was the top metabolite, while the relative abundance of the other metabolites was less than 2%.

[0359] Compound 4 accounted for 4.26% of the total drug-related components in male rat plasma.Metabolic Pathways

[0360] Based on the identified metabolites, the major formation pathway of metabolites of Compound 4 in rat plasm, a reflects ester hydrolysis. The formation pathways of metabolites of Compound 4 in rat plasma is as depicted in Figure 33.

[0361] Following single subcutaneous administration of Compound 4 to SD rats, in addition to unchanged Compound 4, a total of 4 putative metabolites were tentatively identified in plasma. Based on the identified metabolites, the major formation pathways of metabolites of Compund 4 in SD rat plasma was proposed to reflects ester hydrolysis.EXAMPLE 10METABOLITE PROFILING AND IDENTIFICATION OF COMPOUND 4 IN RAT, DOG AND HUMAN HEPATOCYTESMATERIALS & METHODS:Materials:

[0362] Compound 4 was prepared as described in Examples 1 and 8 and the test article 7-EC was purchased:Other Materials

[0363] HepatocytesSpecies Vendor Lot. No.SD Rat BioIVT KMFBeagle Dog BioIVT KLVHuman BioIVT EUCReagents and ChemicalsName VendorIncubation BioIVTThawing BioIVTTrypan Blue Sigma-AldrichDMSO ENERGYFA AladdinNH4FA CNW TechnologiesMeOH MerckAcetonitrile Merck7-EC AladdinUltra-high purified water Purified with Millipore systemEquipment and Instruments

[0364] The following equipment and instruments were used:EquipmentCO2 Incubator: i250 (Thermo)Shaker: MX100-4A (ALLSHENG)Water bath: SHA-CA (Ronghua) Biosafety Cabinet: BSC-1304IIA2 (AIRTECH)Automated Cell Counter: Countess ™ (Thermo)Centrifuge: 5427R (Eppendorf)Water purified system: Millipore ultra-pure water filter (Milli Q)Instrument LC system: UPLC (Waters)UV detector: PDA (Waters)Mass spectrometer: Orbitrap Exploris 480 (Thermo)

[0365] The methods make use of the following Test System:Test SystemHepatocytes Cell Density: 1.00X 106cells / mLSpecies: Rat, dog and humanTest Article: Compound 4Test Compound Cone. Error! Reference source not found.Incubation Condition: 37°C in 5%CO2 / Saturated humidityIncubation Time: 0, Error! Reference source not found, minIncubation Volume: 400 pL (200 pL x 2)Positive Control: 7 -EC (30 pM)Blank Control: Without Compound 4.Solution preparation:

[0366] The Incubation solution was commercially available from BioIVT. The thawing solution was commercially available from BioIVT. Stock Solution was prepared as follows: 10 mM stock solution of Compound 4 in 0.3M phosphate buffer (pH8.0) and 30 mM 7-EC in DMSO were prepared respectively. The working solution was prepared as follows: the stock solution was diluted with 0.3M phosphate buffer and acetonitrile to 1.00 mM Compound 4 and 3 mM 7-EC as working solution, respectively. The Stop Solution was prepared as follows: Acetonitrile: Methanol (95:5) was prepared as stop solution and stored at 2-8°C before use.Preparation of Hepatocytes in Suspension:

[0367] The cryopreserved hepatocytes vials were removed from the liquid nitrogen container and immediately immersed into 37°C water bath to allow the ice pellets melted. The melted hepatocytes were transferred into the pre-warmed Thawing solution tubes and mixed well by gently inverting the tubes and then centrifuged at 100xg for 5 minutes at room temperature. The supernatants were discarded and cell pellets were re-suspended by adding appropriate volumes of pre-warmed Incubation solution. The cell viability of each species was determined using Trypan Blue exclusion method. Viability should be greater than 70%. Cell suspension was diluted with pre-warmed Incubation Solution to 1.01 * 106cells / mL. Plasma samples were obtained from a PK study.

[0368] The Incubation and Sample Pretreatment of Test Article was as follows:1) An aliquot of 2 pL of Compound 4 working solution and 198 pL of cell suspension (1.01 x 106cells / mL) in incubation solution were added into the incubation plates, and mixed (1.00 x io6cells / mL). The mixture was incubated for Error! Reference source not found, min at 37°C in CO2 incubator with 5% CChand saturated humidity. All samples were prepared in duplicate.2) For blank sample, the Compound 4 working solution was replaced by 2 pL acetonitrile. All samples were prepared in duplicate.3) For 0 min sample, the stop solution (400 pL) was added first to the hepatocytes solution and mixed to quench the reaction before adding Compound 4 d working solution. All samples were prepared in duplicate.4) At the end of the incubation, the reaction was quenched by removing the plates from incubator and adding 400 pL of ice-cold stop solution. The cell viability of each species was determined again from the blank samples before adding the stop solution.5) The plate was shaken on a plate shaker immediately. The duplicate samples were merged into one sample, then centrifuged at 12,700 rpm for 10 min6) The supernatant was transferred into a 96-well plate and analyzed for LC-UV- MS" (n=l~2) analysis.

[0369] The Incubation and Sample Pretreatment of Positive Control was as follows: 7- EC (30 pM) was used as positive control and incubated for 0 andError! Reference source not found, min. The positive control samples were prepared following the same procedure as used for test article.

[0370] Analytical methods included the following:LC-UV-MS Conditions for Test ArticleLC System: Waters Acquity UPLCColumn: Waters Acquity UPLC HSS T3 1.8 pm, 2. 1 * 100 mmUV Detector: Waters Acquity PDA, X: 190-400 runColumn Temp.: 40°CAutosampler Temp.: 8°CMobile Phase A: 0.1% FA and 2 mM NH4FA in H2O / ACN (v / v, 95:5)Mobile Phase B: 0.1% FA and 2 mM NH4FA in H2O / ACN (v / v, 5:95)Flow Rate: 0.5 mL / minInjection Volume: 5 pLGradient:Step Time (min) A (%) B (%)1 Initial 95 52 3.00 95 53 7.00 70 304 13.00 40 605 16.00 10 906 18.00 10 907 18.10 95 58 20.00 95 5Mass Spectrometer: Orbitrap Exploris 480Data System: XcaliburIon Source Type: H-ESI (+ / -)Spray Voltage: 3500 V (+); 2500 V (-)Ion Transfer Tube Temp.: 325°CVaporizer Temp.: 350°CSheath Gas Flow: 50 ArbAux Gas Flow: 10 ArbSweep Gas Flow: 10 ArbRF-Lens: 50%Normalized Collision Energy (NCE): 30%, 60%, 90%Scan Type: Full MS; ddMS2; PRMScan Range: 100-1500 m / zResolution: Full MS: 60000; MS2: 15000LC-UV-MS Conditions for Positive ControlLC System: Waters Acquity UPLCColumn: Waters Acquity UPLC HSS T3 1.8 pm, 2. 1 * 100 mmUV Detector: Waters Acquity PDA, I: 190-400 nmColumn Temp.: 40°CAutosampler Temp.: 8°CMobile Phase A: 0.1% FA and 2 mM NH4FA in H2O / ACN (v / v, 95:5)Mobile Phase B: 0.1% FA and 2 mM NH4FA in H2O / ACN (v / v, 5:95)Flow Rate: 0.5 mL / minInjection Volume: 5 pLGradient:Step Time (min) A (%) B (%)1 Initial 100 02 1.50 100 03 4.00 90 104 5.50 75 255 6.50 75 256 7.50 10 907 8.50 10 908 8.51 100 09 9.50 100 0Mass Spectrometer: Orbitrap Exploris 480Data System: XcaliburIon Source Type: H-ESI (+)Spray Voltage: 3500 V (+)Ion Transfer Tube Temp.: 325°CVaporizer Temp.: 350°CSheath Gas Flow: 50 ArbAux Gas Flow: 10 ArbSweep Gas Flow: 10 ArbRF-Lens: 50%Scan Type: Full MSScan Range: 100-1500 m / zResolution: Full MS: 60000

[0371] Data Analysis included the following:

[0372] Post-acquisition data analyses were performed using the Xcalibur and MassMetaSite, which generated a series of extracted ion chromatograms (XICs). XICs of the blank control and analysis samples were compared.RESULTS:Enzymatic Activities in Hepatocytes Incubation

[0373] The hepatocytes used in the study were enzymatically active with respect to phase I and phase II metabolic reactions. The positive control 7-EC was used to monitor enzymatic activity in the test systems. The metabolic rate of 7-EC in rat, dog and human hepatocytes were shown in the following Table, all within the range required by historical statistical metabolic rates (rat: 60%~100%; dog: 80%~100%; human: 40%~100%) and the LC- UV chromatograms could be found in Figure 34.Metabolic Rate (%) of 7-EC in Error! Reference source not found. Hepatocytes„ . UV Peak Area in TO UV Peak Area in TError! Reference source not MetabolicPeC1Sample found. Sample Rate eS(k: 290-360 nm) (k: 290-360 nm) (%)Rat 11670 0 100.00Dog 10521 0 100.00Huma10359 0 100.00 nNote: Metabolic rate (%) = (1 - UV peak area of TError! Reference source not found. / UV peak area of TO) X 100%

[0374] The targeted metabolites (the deethylation and glucuronidation metabolite and the glutathione conjugation metabolite) were detected in hepatocytes of all three species.LC-UV-MSn Analysis of Compound 4:

[0375] Compound 4was used to optimize LC-UV-MS instrument conditions as well as to generate molecular ion fragmentation patterns, which were used for metabolite structure elucidation. Under the optimized conditions, Compound 4 was eluted with a retention time of 11.19 min in LC-UV chromatogram. The UV spectrum of Compound 4 displayed a characteristic absorbance at 267 nm (Figure 35). Therefore, the LC-UV chromatograms were extracted at 250-280 nm. The characteristic MS / MS fragmentation of Compound 4was depicted in Figure 36.Identification of Metabolites

[0376] 8 putative metabolites of Compound 4were detected and tentatively identified in rat, dog and human hepatocytes. The relative abundance of Compound 4 and each of the putative metabolites were calculated using peak area (UV peak area under the wavelength at 250-280 nm) of individual metabolite relative to the total area for all detected drug related components (Table 1A). The UV peak area of Compound 4 and each putative metabolite was summarized in Table 2A. Accurate mass measurements of Compound 4and its metabolites in rat, dog and human hepatocytes were summarized in Table 3 A, where the maximum masserrors between measured and calculated values were within 5 ppm, indicating a high level of confidence.00377] Table 1A: Summary of Compound 4 and its Metabolites in Error! Reference source not found. Hepatocytes[M+NHI]+Relative Abundance ode [M-H]RT— (UV Peak Area %Total) — Metabolic Pathways m / z,mmRat Dog Human l (->514.1866 6.62 1.78 + + Unknown 2 (->769.2925 6.71 8.40 ND + Unknown. .0<> ->-> o ne , , Ester hydrolysis, O-dealkylation, hydrogenation and glucuronidation 3 (->1005.4340 9.22 8.98 + + (P-CAO^CZH.OZ+TH+CGHSOG) / xom mm , Ester hydrolysis, O-deethylation and glucuronidation 4 (->801.3707 10.64 4.60 + +5(P.SC.H.O.-GH.+C.HA) 5 (-)945.4128 10.97 3.26 + 12.64 Ester hydrolysis and glucuronidation (P-TCdUCE+CeHsOe) ound 4 (->1001.4020 11.18 14.86 56.11 44.48 NA 6 (->829.4014 12.68 58.11 + 35.89 Ester hydrolysis and glucuronidation (P-3 CTECE+CeHsOe) 7 (->885.3920 12.98 + 36.82 + Ester hydrolysis (P-C4H4O4) 8 (+>788.4232 14.77 + 7.07 6.98 Ester hydrolysis (P-2C4H4O4) 00378] Note: RT: Retention time of LC-MS; ND: Not detected; NA: Not applicable; +: Only detected by MS, but too weak to be integrated under the UV avelength at 250-280 nm; P: Parent; (+) Theoretical Mass (m / z) = Exact Mass + 18.0338; (-): Theoretical Mass (m / z) = Exact Mass - 1.0073. . > . Peak Area of a Related Component , Total = - - - x 100%Peak Area of Total Related Component s 00379] Table 3A. UV Peak Area of Error! Reference source not found, and its Metabolites in Error! Reference source not found. epatocytesUV Peak Area (>.: 250-280 nm)CodeRat Dog HumanMl 86 + +M2 407 ND +M3 435 + +M4 223 + +M5 158 + 362Compound 4 720 1739 1274M6 2815 + * 1028M7 + 1141 +M8 + 219 200Note: Table was the semi-quantitative data of peak area of UV under the wavelength at 250-280 nm; *: Background interference had been subtracted by TO; ND: Not detected; +: Only detected by MS, but too weak to be integrated under the UV wavelength at 250-280 nm

[0380] Table 4A. UV Peak Area of Compound 4 and its Metabolites in Error! Reference source not found. Hepatocytes„ , RT _ . „x„ Theoretical Mass Measured Mass Mass Error „Code , . , Formula Exact Mass , , , , , , , , Source(min) (m / z) (m / z) (ppm)Ml 6.62 C30H29NO7 515.1944 (-)514.1871 514.1866 -0.97 *RH, DH, HHM2 6.71 C36H50OI8770.2997 (-)769.2924 769.2925 0.13 *RH, HHM3 9.22 C50H70O21 1006.4410 (-)1005.4337 1005.4340 0.30 *RH, DH, HHM4 10.64 C42H580i5802.3776 (-)801.3703 801.3707 0.50 *RH, DH, HHM5 10.97 C48H660i9 946.4198 (-)945.4125 945.4128 0.32 *RH, DH, HHrror! Reference source not found. 11.18 C50H66O21 1002.4097 (-)1001.4024 1001.4020 -0.40 * Std, RH, DH, HHM6 12.68 C44H620I5830.4089 (-)829.4016 829.4014 -0.24 *RH, DH, HHM7 12.98 C46H620i7 886.3987 (-)885.3914 885.3920 0.68 RH, *DH, HHM8 14.77 C42H580i3 770.3877 (+)788.4215 788.4232 2.16 RH, DH, *HH

[0001] Note: Exact Mass: Monoisotopic Mass; (+) Theoretical Mass (m / z) = Exact Mass + 18.0338; (-): Theoretical Mass (m / z) = Exact Mass - 1.0073; RT: Retention time of LC-MS; Std: Standard compound; *: The species which the data listed in this table were derived from.Measured Mass — Theoretical MassMass Error = - — - - — — - x IS® fheoretjcat Mass

[0002] The LC-UV (X: 250-280 nm) and LC-HRMS chromatograms of Compound 4 and its metabolites in rat, dog and human hepatocytes were shown in Figure 37-Figure 39. The MS spectra of the detected metabolites with their fragmentation were displayed in Figure 40- Figure 47.

[0003] Based on the accurate molecular weight and fragment ions, the metabolites were identified as below: Ml : unknown; M2: unknown; M3: ester hydrolysis, O-dealkylation, hydrogenation and glucuronidation metabolite (MW=1007.09, P-C4H4O4-C2H2O2+2H+C6H8O6); M4: ester hydrolysis, O-deethylation and glucuronidation metabolite (MW=802.91, P-3C4H4O4-C2H4+C6H8O6); M5: ester hydrolysis and glucuronidation metabolite (MW=947.04, P-2C4H4O4+C6H8O6); M6: ester hydrolysis and glucuronidation metabolite (MW=830.97, P-3C4H4O4+C6H8O6); M7: ester hydrolysis metabolite (MW=886.99, P-C4H4O4); M8: ester hydrolysis metabolite (MW=770.91, P-2C4H4O4).

[0004] In rat hepatocytes, Compound 4 and 8 metabolites (M1-M8) were detected. M6 was considered as the top one metabolite with relative abundance of 58.11%. The relative abundance of the other metabolites was less than 9%.

[0005] In dog hepatocytes, Compound 4 and 7 metabolites (Ml, M3-M8) were detected. M6 was considered as the top one metabolite with relative abundance of 36.82%. The relative abundance of the other metabolites was less than 8%.

[0006] In human hepatocytes, Compound 4 and 8 metabolites (M1-M8) were detected. M5 and M6 were considered as the top two metabolites with relative abundance of 12.64% and 35.89%, respectively. The relative abundance of the other metabolites was less than 7%.

[0007] Compound 4 accounted for 14.86%, 56.11% and 44.48% of the total drug related components in rat, dog and human hepatocytes, respectively, after incubation at 37°C for 120 min.Metabolic Pathways

[0008] Based on the identified metabolites, the major metabolic pathways of Compound 4 in hepatocytes was proposed as ester hydrolysis. The minor metabolic pathways were O- dealkylation, hydrogenation and O-deethylation. The metabolic pathways of Compound 4 in hepatocytes were proposed as shown in Figure 48:

[0009] After incubation of Compound 4 with hepatocytes for 120 min, in addition to the unchanged Compound 4, 8 putative metabolites were tentatively identified. The major metabolic pathways of Compound 4 in hepatocytes was proposed as ester hydrolysis. The minor metabolic pathways were O-dealkylation, hydrogenation, and O-deethylation. The metabolites detected in incubation of human hepatocytes were detected in hepatocytes of at least one animal species.EXAMPLE 11COMPOUND 4 DELAYS ONSET, PROGRESSION AND DURATION OF ALS AND ENHANCES SURVIVAL IN ANIMAL MODELS

[0010] Compound 4 was prepared as described in Examples 1 and 8 and the test article 7- EC was purchased:

[0011] Twenty ALS mice (hSODlG93ATg mice) were divided into two groups of 10 each (IM, 2M). The IM group was treated with the control formulation, 0.3 M sodium phosphate buffer pH8, and the 2M group was treated with the test formulation, Compound 4, 6 mg / kg. The Compound 4and control formulations were administered SC daily (Sunday-Friday) into the mouse neck for 64 days. The following parameters were assessed: morbidity and mortality, body weight, clinical observation, neurological scoring (by tail suspension and walking tests and the righting reflex) and motoric test by rotarod. Blood samples for serum preparation were drawn from the mandibular vein (or other vein if necessary) on all mice at each of the following timepoints: Day (-7) (Baseline), Day 20, Day 35 and before termination. On Day 2 blood samples were drawn from the 2M mice only for biomarker collection.

[0012] Three parameters were assessed to determine the neurological score (NS): tail suspension test, walking test, and righting reflex test. The assessments were based on each hind limb (left and right) independently while the overall observations of all tests performed were scored on a scale from 0 to 5.

[0013] Findings are summarized in Figure 49-50 and as below:• Two mice in the control group (IM) were observed as having the first signs of the disease (NS=1) on Day 16 (age 85 days) while the first signs did not appear in the Compound 4 group (2M) until Day 23 (age 92 days).• On Day 37 (age 106 days), 83% of the mice in the control group (IM) were evaluated with a NS of 1.5 compared to 28% of the mice in the Compound 4 group (2M).• From Day 37 on, there was a delay in disease progression in the 2M group compared to the IM group, with significant meaning from Day 37 to Day 57.• At the end of study (Day 65; age 135 days), there were still two mice in the 2M group that exhibited symptoms of the disease (NS=1-).

[0014] In addition to the neurological improvement in mice treated with Compound 4, the treatment was shown to delay disease onset and increase duration in the early phase of the disease.

[0015] Weight loss can be an indicator of the onset of ALS. The average highest body weight was at 104 days in the 2M Compound 4group and at 88 days in the IM control group, which suggests a significantly delayed onset of disease in the Compound 4 treated mice(Figure 51). An assessment of the duration of the days in the early phase of the disease revealed that mice treated with Compound 4spent significantly more days in the early phase of the disease while the control treated mice progressed to the late phase sooner (Figure 52).

[0016] Compound 4 also increased survival of the treated animals. At the conclusion of the study, there were still some control group mice at NS=3.5-4. The probability of survival curve demonstrated that at the end of study, the probability of survival in the control group was ~30 while the Compound 4 group was two-fold higher at ~60 (Figure 53).

[0017] As expected, the results confirmed the efficacy of Compound 4 by delaying the onset and the progression of ALS in the mouse model. In addition, 20% of the Compound 4 treated mice remained in the early stage of disease (NS=1) throughout the duration of the study and did not progress prior to the end of the study.EXAMPLE 12 LAG3 AND TERT EXPRESSION AS BIOMARKERS FOR COMPOUND 4 RESPONSIVENESS IN ALS

[0018] Blood samples were collected from the 2M Compound 4 treated ALS mice. Samples were subjected to RNA extraction using the RNeasy Protect animal blood kit (Qiagen). Real-time PCR was performed for TERT expression and LAG3 gene using the appropriate primers and b-actin as the reference gene. Specifically, RNA extraction was prepared from blood samples collected prior to and 24 hours after AGP-499 treatment. LAG3 gene expression was measured by real-time PCR and the % of LAG3 expression 24 hours post-treatment relative to the expression prior to treatment was calculated for each mouse. A significant increase in the expression of the LAG3 gene at 24 hours post Compound 4 treatment was observed in six mice (numbers (no.) 11, 12, 17, 18, 20 and 21) (Figure 54). No increase in LAG3 was observed in two mice (no. 15 and 22); both mice developed disease symptoms faster compared to the other 2M treatment mice (as observed by the NS measurements during disease progression) and no treatment efficacy was observed in either mouse (Figure 55). Shown are the average neurological scores for Compound 4-treated mice as a function of age, compared to the neurological scores for mouse number 15 and 22, treated with Compound 4.

[0019] In addition, the duration of each treated mouse at the early phase of the disease was calculated. Calculations reflect the delta in body weight from 100% to 90%. The body weight of the first treatment day was considered 100% body weight and the day that a reduction of 10% was measured was determined as 90% body weight. Figure 56 illustrates that the disease progressed faster in mouse no. 15 and no. 22 as the duration in the early phase was shortcompared to other mice in the same treatment group. This suggests that the LAG3 biomarker accurately predicted the response of the mice to treatment with Compound 4.

[0020] When the level of increased LAG3 expression in the AGP-499 treated mice was analyzed against the duration of days in the early phase of the disease, a linear correlation was observed. In this case, disease progression was determined as the duration (no. of days) in the early phase of the disease, which correlated with the increased level of LAG3 expression 24 hours post first Compound 4 treatment represented as fold from the level of LAG3 in each mouse before treatment. The higher the level of LAG3 expression directly correlated with an increased duration of days in the early phase of the disease, which suggests a delay in disease progression (Figure 57).

[0021] The increase in the level of TERT expression in the blood of Compound 4 treated ALS mice was measured for each mouse as compared to TERT expression prior to treatment. Disease progression was determined herein as the number of days (duration) in the early phase of the disease, which correlated with the increased level of TERT expression 24 hours post first Compound 4 treatment represented as fold from the level of TERT expression prior to treatment. A direct correlation between the duration of days in the early phase of the disease and an increase in TERT expression levels was observed (Figure 58).

[0022] LAG3 and TERT Expression Levels Correlate with the Day of Disease Onset. The day of disease onset in each of the Compound 4 treated ALS mice was determined based on the body weight data and compared to the fold increase (relative to the level prior to treatment) of the expression of the LAG3 (Figure 59) and TERT (Figure 60) biomarkers 24 hours post first treatment. A linear correlation was observed between the levels of the biomarkers and the day of disease onset in each Compound 4 treated mouse.

[0023] The increase in the level of the LAG3 and TERT biomarkers following the first administration of Compound 4 to ALS mice correlated directly with the later onset and slower progression of the disease, which demonstrated the efficacy of Compound 4. The mice in the 2M (Compound 4 treatment) group that did not demonstrate an increase in the biomarkers did not experience a delay in the onset and progression of the disease following treatment, providing evidence that there is a correlation between increased biomarker levels and a delay in the onset and progression of ALS.

[0024] Importantly, when assessing the neurological score (average) in ALS Tg mice treated with vehicle phosphate buffer the score increased with time, indicating disease progression, where, uniquely in animals treated with Compound 4, there was a significant delay in the neurological symptoms observed with such treatment.

[0025] Still more remarkable is the finding that in 20% of the Compound 4 treated group ( 2 / 10) the progression of the disease was halted altogether, remaining at a low score, 1-1.5, a finding not found in any previous study, despite similar assessment, for example, of animals treated with Compound 1, a structurally related compound, which, while demonstrating a therapeutic effect, did not yield these remarkable results of halting disease progression (Figure 61).

[0026] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0027] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

[0028] WHAT IS CLAIMED IS:

1. A Compound 4 characterized by the following structure:Compound 4.

2. The compound of claim 1, wherein said compound rapidly undergoes hydrolysis in circulation of a human subject.

3. The compound of claim 1, wherein the compound is in the form of a sodium salt.

4. A pharmaceutical composition comprising the compound of claim 1.

5. The pharmaceutical composition of claim 4, wherein said composition is formulated for intravenous, subcutaneous or parenteral administration.

6. The pharmaceutical composition of claim 4, wherein the compound remains soluble even in a pH of about 6 or less.

7. A method of treatment of a disease or condition in a subject that is both treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity, said method comprising contacting a cell or tissue of said subject or administering to said subject a Compound 4 characterized by the following structure:Compound 4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same.

8. The method of claim 7, wherein said disease or condition is positively impacted by neurogenesis, neuroplasticity, increased neurofilament-associated gene expression, increased neuronal growth factor gene expression or protein production, or a combination thereof.

9. The method of claim 8, wherein said disease or condition is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), a disease or condition associated with nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents, or stroke.

10. The method of claim 8, wherein said disease or condition is a neurodegenerative disease, cognitive disease or disorder, nervous system injury, a disease or disorder associated with aging, a degenerative joint disease, a degenerative disease of the skeletal system, a degenerative disease of the musculature, macular degeneration, age- and stress-related diseases of the vascular system, age- and stress-related immune system impairment, diabetes, cardiac disease, infection, immune system impairment, cancer, degenerative inflammatory disease, a genetic disorder causing accelerated cell turnover, anemia, male or female reproductive disorder, and an acute or chronic disease or disorder of the skin.

11. The method of claim 8, wherein said disease or condition is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), a disease or condition associated with nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents, stroke, an aging disease or a condition associated with aging, sebaceous gland hyperplasia or hypoplasia, senile lentigo, pigmentation abnormalities, graying of hair, hair loss, hair thinning or baldness including alopecia, or chronic skin ulcers, degenerative joint disease, osteoporosis, osteoarthritis, sarcopenia, atherosclerosis, calcification, thrombosis, aneurysm, AIDS, diseases or conditions arising as a consequence of cancer therapy, acute or chronic infections, wounds, bums, abrasions or other acute or chronic conditions of epidermis, dyskeratosis congenita, luteal phase defect, premature ovarian failure, improving fertility including in in vitro fertilization, in subjects exposed to radiation and / or chemotherapy, or any combination of diseases as applicable.

12. The method of claim 8, wherein said disease is Amyotrophic lateral sclerosis (ALS).

13. The method of claim 8, wherein said compound is a sodium salt.

14. A method of diagnosis, prognosis or a combination thereof of a subject at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject, said method comprising the steps of: assessing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in a biological sample from said subject prior to and following administering said subject a Compound 4 characterized by the following structure:Compound 4, or a pharmaceutically acceptable salt thereof; andOptionally comparing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in said biological sample in comparison to a standard, whereby alterations in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject following administration of said compound in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said biological sample taken from said subject prior to administration of said compound; or whereby alterations in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in said standard, or as a function of administration of Compound 4; serves as an indicator for the diagnosis, prognosis or a combination thereof of a subject at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject.

15. The method of claim 14, whereby an increase in said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject following administration of said compound in comparison to said TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in saidbiological sample taken from said subject prior to administration of said compound indicates said subject is responsive to said administration, thereby being a method of prognosis of a subject at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity in said subject16. The method of claim 14, wherein assessing TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in a biological sample from said subject is performed prior to administering said subject said Compound 4 and TERT expression and / or activity, Lag3 expression and / or activity or a combination thereof in said biological sample is compared to a standard, whereby decreased TERT expression and / or activity or Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject compared to said standard serves as a diagnostic indicator that said subject is suffering from or at risk for or suffering from a disease or condition in a subject that is treatable by stimulating or increasing telomerase reverse transcriptase (TERT) expression and / or activity.

17. The method of claim 16, further comprising administering said Compound 4 to said subject.

18. The method of claim 14, wherein said pharmaceutically acceptable salt is a sodium salt of said compound.

19. The method of claim 14, wherein said disease or condition is positively impacted by neurogenesis, neuroplasticity, increased neurofilament-associated gene expression, increased neuronal growth factor gene expression or protein production, or a combination thereof.

20. The method of claim 14, wherein said disease or condition is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), a disease or condition associated with nerve damage, motor neuron disease, multiple sclerosis (MS), peripheral and central nervous system injury including spinal injury and cerebral vascular incidents, or stroke.

21. A method of optimizing treatment of Amyotrophic lateral sclerosis (ALS) in a subject, said method comprising:• assessing Lag3 expression and / or activity or a combination thereof in a biological sample taken from said subject,• administering a Compound 4 to said subject or a pharmaceutically acceptable salt thereof; and• comparing said Lag3 expression and / or activity or a combination thereof in said biological sample before and after administering Compound 4, whereby increased Lag3 expression and / or activity or a combination thereofin said biological sample taken from said subject after said administering indicates said treatment will be effective in said subject, thereby optimizing treatment in said subject.

22. The method of claim 21, wherein said pharmaceutically acceptable salt is a sodium salt of said compound.

23. The method of claim 21, wherein said Compound 4 stimulates or enhances neurogenesis, neuroplasticity, increased neurofilament-associated gene expression, increased neuronal growth factor gene expression or protein production, or a combination thereof in said subject.

24. A method of treatment of Amyotrophic lateral sclerosis (ALS) in a subject, said method comprising contacting a cell or tissue of said subject or administering to said subject a Compound 4 characterized by the following structure:Compound 4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same.

25. The method of claim 24, wherein said ALS is positively impacted by neurogenesis, neuroplasticity, increased neurofilament-associated gene expression, increased neuronal growth factor gene expression or protein production, or a combination thereof.

26. The method of claim 24, wherein said compound is a sodium salt.

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