B264 The identification of novel anti-angiogenic agents with favorable drug-like properties and the ability to induce objective tumor responses (regressions) across multiple tumor types is a major goal of oncological drug discovery efforts. The therapeutic efficacy of anti-angiogenic agents may be improved by inhibition of a combination of multiple angiogenic and tumor-specific targets. CEP-11981, a C3-(2-amino pyrimidine) dihyroindolocarbazole is a potent and highly-cell permeable multiplex inhibitor of human TIE-2, VEGF-R2, and VEGF-R1 receptor tyrosine kinases (IC 50 = 22 ±6, 4 ±1, and 3±1 nM, respectively), in enzyme-based assays, with a kinase selectivity index, S(90) against 217 kinases of 0.25 at 1µM. CEP-11981 has a low nanomolar concentration-related in vitro and ex vivo anti-angiogenesis profile with respect to inhibition of VEGF-A,-C, -D-induced, Angiopoietin-1-induced, and FGF2-induced human and murine endothelial cell proliferation, chemotaxis, migration, and survival in vitro, and microvessel outgrowth and branching in primary rat aortic ring explant cultures ex vivo (EC 50 = 4 ± 1 nM). Sustained oral anti-angiogenic efficacy was observed in VEGF-A-, tumor-and inflammation-induced neo-vascularization models in rodents and dose-related anti-tumor activity demonstrated across a panel of human and murine tumor models at doses of 0.3 to 30 mg/kg orally qd and bid. Dose-related and exposure driven in vivo anti-tumor efficacy was observed in s.c. (melanoma, glioblastoma, prostate carcinoma) and orthotopic human and rodent solid (colon carcinoma, renal carcinoma, and glioblastoma) and hematological (acute leukemia) tumor xenograft models in normal and immunocompromised murine hosts. Depending on the model and dosing regimens, CEP-11981 exhibited dose-related tumor growth inhibitory and anti-angiogenic effects as well as sustained partial and complete tumor regressions when administered as monotherapy. Specific intermittent oral dosing schedules (drug holidays) on qd and bid dosing regimens demonstrated significant anti-tumor efficacy (tumor growth inhibition and partial and complete regressions) in both solid (CT-26 murine colon carcinoma) and hematological (P388 murine leukemia) tumor models, with qd and bid continuous dosing being most effective. CEP-11981 was well-tolerated when administered chronically (>250 days bid p.o.) in mice in combination with cytotoxic agents (e.g. temozolamide, TMZ). The combination of CEP-11981 with TMZ conferred a median survival benefit in orthotopic human glioblastoma-bearing animals relative to that with TMZ alone (253 days versus 160 days; p=0.04). CEP-11981 is currently in Phase I clinical trials in patients with refractory solid tumors.

Understanding the mechanisms of neuronal death in concert with the identification of drugable molecular targets key to this process has held great promise for the development of novel chemical entities (NCEs) to halt neurodegenerative disease progression. Two key targets involved in the apoptotic process identified over the past decade include the mixed lineage kinase (MLK) family and glyceraldehyde phosphate dehydrogenase (GAPDH). Two NCEs, CEP-1347 and TCH346, directed against these respective targets have progressed to the clinic. For each, robust neuroprotective activity was demonstrated in multiple in vitro and in vivo models of neuronal cell death, but neither NCE proved effective Parkinson's disease (PD) patients. These recent clinical failures require a reassessment of both the relevance of apoptosis to neurodegenerative disease etiology and the available animal models used to prioritize NCEs for advancement to the clinic in this area.

We have explored the structure-activity relationship (SAR) surrounding the clinically efficacious antiepileptic drug topiramate (1), a unique sugar sulfamate anticonvulsant that was discovered in our laboratories. Systematic structural modification of the parent compound was directed to identifying potent anticonvulsants with a long duration of action and a favorable neurotoxicity index. In this context, we have probed the pharmacological importance of several molecular features: (1) the sulfamate group (6-8, 22-25, 27, 84), (2) the linker between the sulfamate group and the pyran ring (9, 10, 21a,b), (3) the substituents on the 2,3- (58-60, 85, 86) and 4, 5-fused (30-38, 43, 45-47, 52, 53) 1,3-dioxolane rings, (4) the constitution of the 4,5-fused 1,3-dioxolane ring (2, 54, 55, 63-68, 76, 77, 80, 83a-r, 84-87, 90a, 91a, 93a), (5) the ring oxygen atoms (95, 96, 100-102, 104, 105), and (6) the absolute stereochemistry (106 and 107). We established the C1 configuration as R for the predominant alcohol diastereomer from the highly selective addition of methylmagnesium bromide to aldehyde 15 (16:1 ratio) by single-crystal X-ray analysis of the major diastereomer of sulfamate 21a. Details for the stereoselective syntheses of the hydrindane carbocyclic analogues 95, 96, 100, and 104 are presented. We also report the synthesis of cyclic imidosulfites 90a and 93a, and imidosulfate 91a, which are rare examples in the class of such five-membered-ring sulfur species. Imidosulfite 93a required the preparation and use of the novel sulfur dichloride reagent, BocN=SCl2. Our SAR investigation led to the impressive 4,5-cyclic sulfate analogue 2 (RWJ-37947), which exhibits potent anticonvulsant activity in the maximal electroshock seizure (MES) test (ca. 8 times greater than 1 in mice at 4 h, ED50 = 6.3 mg/kg; ca. 15 times greater than 1 in rats at 8 h, ED50 = 1.0 mg/kg) with a long duration of action (>24 h in mice and rats, po) and very low neurotoxicity (TD50 value of >1000 mg/kg at 2 h, po in mice). Cyclic sulfate 2, like topiramate and phenytoin, did not interfere with seizures induced by pentylenetetrazole, bicucculine, picrotoxin, and strychnine; also, 2 was not active in diverse in vitro receptor binding and uptake assays. However, 2 turned out to be a potent inhibitor of carbonic anhydrase from different rat tissue sources (e. g., IC50 of 84 nM for the blood enzyme and 21 nM for the brain enzyme). An examination of several analogues of 2 (83a-r, 85-87, 90a, 91a, 93a) indicated that potent anticonvulsant activity is associated with relatively small alkyl substituents on nitrogen (Me/H, 83a; Me/Me, 83m; Et/H, 83b; allyl/H, 83e; c-Pr/H, 83j; c-Bu/H, 83k) and with limited changes in the cyclic sulfate group, such as 4,5-cyclic sulfite 87a/b. The potent anticonvulsants 83a and 83j had greatly diminished carbonic anhydrase inhibitory activity; thus, inhibition of this enzyme may not be a significant factor in the anticonvulsant activity. The alpha-L-sorbopyranoses 67, 68, and 80, which mainly possess a skew conformation (ref 29), were nearly twice as potent as topiramate (1). The L-fructose enantiomers of 1 (106) and 2 (107), synthesized from L-sorbose, were found to have moderate anticonvulsant activity, with eudysmic ratios (MES ED50 in mice at 4 h, po) of 1:106 = 1.5 and 2:107 = 3.5. The log P values for 1 and 2 were determined experimentally to be 0.53 and 0.42, respectively, which are less than the optimal 2.0 for CNS active agents. However, analogues with more favorable calculated log P (clogP) values, in conjunction with just minor steric perturbation according to the developed SAR profile, such as 47 (clogP = 2.09), 83m (1.93), and 86 (1.50), did not display improved potency: 47 is less potent than 1, 83m is equipotent with 2, and 86 is less potent than 2. Although the measured log P value for diethyl analogue 31 is 1.52, this did not translate into enhanced potency relative to 1. (ABSTRACT TRUNCATED)

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC50values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.

Although the mechanism of neuronal death in Alzheimer's disease (AD) has yet to be elucidated, a putative role for c-jun in this process has emerged. Thus, it was of interest to delineate signal transduction pathway(s) which regulate the transcriptional activity of c-jun, and relate these to alternate gene inductions and biochemical processes associated with beta-amyloid (Abeta) treatment. In this regard, the survival promoting activity of CEP-1347, an inhibitor of the stress-activated/c-jun N-terminal (SAPK/JNK) kinase pathway, was evaluated against Abeta-induced cortical neuron death in vitro. Moreover, CEP-1347 was used as a pharmacologic probe to associate multiple biochemical events with Abeta-induced activation of the SAPK/JNK pathway. CEP-1347 promoted survival and blocked Abeta-induced activation of JNK kinase (MKK4, also known as MEK-4, JNKK and SEK1) as well as other downstream events associated with JNK pathway activation. CEP-1347 also blocked Abeta-induction of cyclin D1 and DP5 genes and blocked Abeta-induced increases in cytoplasmic cytochrome c, caspase 3-like activity and calpain activation. The critical time window for cell death blockade by CEP-1347 resided within the peak of Abeta-induced MKK4 activation, thus defining this point as the most upstream event correlated to its survival-promoting activity. Together, these data link the SAPK/JNK pathway and multiple biochemical events associated with Abeta-induced neuronal death and further delineate the point of CEP-1347 interception within this signal transduction cascade.

Recent evidence suggests that the activation of the Jun N-terminal kinase (JNK) signal transduction pathway may be important in neuronal responses to stresses such as trophic factor deprivation. Preventing the activation of JNK and expression of c-Jun may, therefore, be neuroprotective. Here, we report that the small molecule CEP-1347, which has been shown to inhibit the JNK signalling pathway, promotes cholinergic activity in cultured embryonic septal neurones. In vivo, we have shown that CEP-1347, administered either by sub-cutaneous (s.c.) injection or by continuous infusion, is partially neuroprotective, for cholinergic neurones in the medial septum, following fimbria-fornix transection. These data suggest that small molecules such as CEP-1347 may have beneficial effects in treating neurodegenerative diseases.

New antipsychotic drugs are needed because current therapy is ineffective for many schizophrenics and because treatment is often accompanied by extrapyramidal symptoms and dyskinesias. This paper describes the design, synthesis, and evaluation of a series of related (aminomethyl)benzamides in assays predictive of antipsychotic activity in humans. These compounds had notable affinity for dopamine D2, serotonin 5-HT1A, and alpha1-adrenergic receptors. The arylpiperazine 1-[3-[[4-[2-(1-methylethoxy)phenyl]-1-piperazinyl]methyl]benzoyl]p ipe ridine (mazapertine, 6) was chosen because of its overall profile for evaluation in human clinical trials. The corresponding 4-arylpiperidine derivative 67 was also highly active indicating that the aniline nitrogen of 6 is not required for activity. Other particularly active structures include homopiperidine amide 14 and N-methylcyclohexylamide 31.

Three structurally unrelated compounds, all of which induce nerve growth factor (NGF) in cell culture systems, were assessed for their ability to induce NGF mRNA in adult rat brain using a highly sensitive RNAse protection assay. Interleukin-1 beta (0.5-1 pmol) and 1,25-dihydroxyvitamin D3 (25-25,000 pmol) were extremely potent inducers of NGF mRNA, being respectively at least 50,000 and 4000 times more potent than 4-methylcatechol. These compounds elicited an approximate twofold increase in NGF mRNA in both the hippocampus and cortex, without altering beta-actin mRNA levels after a single intracerebroventricular injection. The duration of NGF induction was dependent on the compound administered. For example, the elevation of NGF mRNA elicited by interleukin-1 beta peaked at 8 h and lasted for at least 24 h. In contrast, the induction of NGF after 1,25-dihydroxyvitamin D3 and 4-methylcatechol administration peaked between 4 and 8 h and was not apparent 24 h after injection. These results demonstrate induction of NGF mRNA in vivo by administration of physiological or pharmacological agents and differentiate these agents by potency and duration of action. Further, these findings indicate that pharmacological induction of NGF may be a viable strategy for the treatment of neurodegenerative disorders such as Alzheimer's disease.

Motor neuronal disorders, such as the loss of spinal cord motor neurons in amyotrophic lateral sclerosis or the degeneration of spinal cord motor neuron axons in certain peripheral neuropathies, present a unique opportunity for therapeutic intervention with neurotrophic proteins. Normally, such proteins do not cross the blood-brain barrier, but spinal cord motor neuron axons and nerve terminals lie outside the barrier and thus may be targeted by systemic administration of protein growth factors. Insulin-like growth factor-I (IGFI) receptors are present in the spinal cord, and, like members of the neurotrophin receptor family, IGF-I receptors mediate signal transduction via a tyrosine kinase domain. IGF-I was found to prevent the loss of choline acetyltransferase activity in embryonic spinal cord cultures, as well as to reduce the programmed cell death of motor neurons in vivo during normal development or following axotomy or spinal transection. Consistent with earlier reports that IGF-I enhances motor neuronal sprouting in vivo, subcutaneous administration of IGF-I increases muscle endplate size in rats. Subcutaneous injections of IGF-I also accelerate functional recovery following sciatic nerve crush in mice, as well as attenuate the peripheral motor neuropathy induced by chronic administration of the cancer chemotherapeutic agent vincristine in mice. Doses of IGF-I that accelerate recovery from sciatic nerve crush in mice result in elevated serum levels of IGF-I which are similar to those obtained following subcutaneous injections of formulated recombinant human IGF-I (Myotrophin) in normal human subjects. Based on these findings, together with evidence of safety in animals and man, clinical trials of recombinant human IGF-I have been initiated in patients with amyotrophic lateral sclerosis and are planned to begin soon in patients with chemotherapy-induced peripheral neuropathies.

A selective non-responsiveness to the analgesic effects of opioid mu receptor-, but not opioid delta receptor-, mediated antinociception in the tail-flick test has been identified in C57BL/6J-bg(J) (beige-J) mice. The beige-J mutation is also known to give rise to multiple immunological disorders and immune cell dysfunctions. A link between these apparently disparate manifestations has been examined in a series of studies using, for example, adoptive transfer of spleen cells. The findings appear to have broad implications for the link between the immune and opioid systems.

The present studies were an attempt to examine the mechanism of action of the novel antinociceptive compound RWJ-22757, (±)-trans-3-(2-bromophenyl)-octa-hydroindolizine (McN-5195). Intracerebroventricular (i.c.v.) administration of RWJ-22757 produced dose-related antinociception in the mouse tail-flick (48°C) and rat hot-plate (51°C) tests (ED50 = 243.3 and 261.3 μg, respectively). In contrast, intrathecal (i.t.) administration was without effect. The antinociception produced by peripherally (i.p.) or centrally (i.c.v.) administered RWJ-22757 was attenuated by i.t. administration of 2 μg phentolamine, 5 μg yohimbine, or 10 μg methysergide. I.t. administration of naloxone, at a dose (0.5 μg) that significantly attenuated the antinociceptive effects of peripherally or centrally administered morphine, had no effect on RWJ-22757-induced antinociception. We conclude from these results, coupled with the overall pharmacological and neurochemical profile of RWJ-22757, that the data are consistent with the hypothesis that RWJ-22757 produces antinociception predominantly at a site or sites located supraspinally with little or no activity at the spinal level and that RWJ-22757 activates adrenergic and serotonergic descending inhibitory pathways, increasing the tonic activity of endogenous antinociceptive systems.

The interaction of loreclezole (a structurally novel non-benzodiazepine anticonvulsant), diazepam, phenobarbital sodium, and ethosuximide with the benzodiazepine antagonists beta-CCE, Ro15-1788, and CGS 8216 and the purine antagonist aminophylline were examined in the rat i.v. metrazol seizure test. The anticonvulsant activity of loreclezole was inhibited by beta-CCE and CGS 8216 but not by Ro15-1788 or aminophylline. The inhibition produced by CGS 8216 was dose dependent (ID50 = 0.04 mg/kg i.p. CGS 8216) and surmountable. When the interactions of the various antagonists with loreclezole were compared to those of diazepam, phenobarbital sodium, and ethosuximide, a clear differentiation of activity was evident. Beta-CCE blocked the anticonvulsant activity of all four compounds whereas Ro15-1788 blocked only the anticonvulsant effects of diazepam. CGS 8216blocked the anticonvulsant effects of diazepam and loreclezole in a similar manner while being much less effective against the anticonvulsant effects of ethosuximide and ineffective against phenobarbital sodium. Aminophylline blocked the anticonvulsant effects of only ethosuximide. Based on these data and the anticonvulsant profile of loreclezole, we suggest that loreclezole represents a new class of agents which modulate GABAergic transmission via a unique interaction with the GABA receptor complex.

Vaught, Jeffry L.; Connelly, Charlene D.; Martinez, Rebecca P.; Codd, Ellen E.; Raffa, Robert B. Author Information

Rats were injected intraperitoneally with loceclezole (R 72063), diazepam, or scopolamine 60 min prior to acquisition of a passive avoidance task and tested 18 h later for retention of the learned (passive) behavior. The known impairment of performance produced by diazepam in this test is believed to be a model for the clinically observed diazepam-induced anterograde amnesia in humans. We report in this study that (1) consistent with the literature, pretreatment with diazepam (2.0 –16.0 mg/kg i.p.) or scopolamine (3.0 mg/kg i.p.) produced impairment in passive avoidance performance of rats (anterograde amnesia), but (2) pretreatment with loreclezole (R 72063) (2.0–80.0 mg/kg i.p.) did not impair the acquisition, retention, or the retrieval (tested 18 h later) of passive avoidance behavior by rats at any dose. The results suggest that the anticonvulsant activity of loreclezole is mediated by a mechanism distinct from the one coupled to diazepam-like disruption of cognitive functions involved in the acquisition or posttraining information processing of passive avoidance behavior.