Moorthy S. S. Palanki

The Effect of a T Cell-Specific NF-κB Inhibitor on In Vitro Cytokine Production and Collagen-Induced Arthritis

NF-κB plays a key role in the production of cytokines in inflammatory diseases. The effects of a novel T cell-specific NF-κB inhibitor, SP100030, were evaluated in cultured Jurkat cells and in murine collagen-induced arthritis (CIA). Chemical libraries were screened for NF-κB-inhibitory activity. SP100030, a compound identified in this process, inhibited NF-κB activation in PMA/PHA-activated Jurkat cells by EMSA at a concentration of 1 μM. Jurkat cells and the monocytic cell line THP-1 were transfected with an NF-κB promotor/luciferase construct and activated. SP100030 inhibited luciferase production in the Jurkat cells (IC50 = 30 nM). ELISA and RT-PCR confirmed that IL-2, IL-8, and TNF-α production by activated Jurkat and other T cell lines were inhibited by SP100030. However, cytokine expression was not blocked by the compound in THP-1 cells, fibroblasts, endothelial cells, or epithelial cells. Subsequently, DBA/1J mice were immunized with type II collagen. Treatment with SP100030 (10 mg/kg/day i.p. beginning on day 21) significantly decreased arthritis severity from onset of clinical signs to the end of the study on day 34 (arthritis score, 5.6 ± 1.7 for SP100030 and 9.8 ± 1.5 for control; p < 0.001). Histologic evaluation demonstrated a trend toward improvement in SP100030-treated animals. EMSA of arthritic mouse ankles in CIA showed that synovial NF-κB binding was suppressed in the SP100030-treated mice. SP100030 inhibits NF-κB activation in T cells, resulting in reduced NF-κB-regulated gene expression and decreased CIA. Its selectivity for T cells could provide potent immunosuppression with less toxicity than other NF-κB inhibitors.

Development of prodrug 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy) phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate (TG100801): A topically administered therapeutic candidate in clinical trials for the treatment of age-related macular degeneration

Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of evidence suggests that inhibitors targeting the VEGFr pathway may be effective for the treatment of AMD. Recent studies using Src/YES knockout mice suggest that along with VEGF, Src and YES play a crucial role in vascular leak and might be useful in treating edema associated with AMD. Therefore, we have developed several potent benzotriazine inhibitors designed to target VEGFr2, Src, and YES. One of the most potent compounds is 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)phenylamino]benzo[1,2,4]triazin-7-yl}phenol ( 5), a dual inhibitor of both VEGFr2 and the Src family (Src and YES) kinases. Several ester analogues of 5 were prepared as prodrugs to improve the concentration of 5 at the back of the eye after topical administration. The thermal stability of these esters was studied, and it was found that benzoyl and substituted benzoyl esters of 5 showed good thermal stability. The hydrolysis rates of these prodrugs were studied to analyze their ability to undergo conversion to 5 in vivo so that appropriate concentrations of 5 are available in the back-of-the-eye tissues. From these studies, we identified 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate ( 12), a topically administered prodrug delivered as an eye drop that is readily converted to the active compound 5 in the eye. This topically delivered compound exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed good efficacy in the laser induced choroidal neovascularization model. On the basis of its superior profile, compound 12 was advanced. It is currently in a clinical trial as a first in class, VEGFr2 targeting, topically applied compound for the treatment of AMD.

Inhibitors of ABL and the ABL-T315I Mutation

Chronic myelogenous leukemia (CML) is a hematological stem cell disorder caused by increased and unregulated growth of myeloid cells in the bone marrow, and the accumulation of excessive white blood cells. Abelson tyrosine kinase (ABL) is a non-receptor tyrosine kinase involved in cell growth and proliferation and is usually under tight control. However, 95% of CML patients have the ABL gene from chromosome 9 fused with the breakpoint cluster (BCR) gene from chromosome 22, resulting in a short chromosome known as the Philadelphia chromosome. This Philadelphia chromosome is responsible for the production of BCR-ABL, a constitutively active tyrosine kinase that causes uncontrolled cellular proliferation. An ABL inhibitor, imatinib, was approved by the FDA for the treatment of CML, and is currently used as first line therapy. However, a high percentage of clinical relapse has been observed due to long term treatment with imatinib. A majority of these relapsed patients have several point mutations at and around the ATP binding pocket of the ABL kinase domain in BCR-ABL. In order to address the resistance of mutated BCR-ABL to imatinib, 2(nd) generation inhibitors such as dasatinib, and nilotinib were developed. These compounds were approved for the treatment of CML patients who are resistant to imatinib. All of the BCR-ABL mutants are inhibited by the 2(nd) generation inhibitors with the exception of the T315I mutant. Several 3(rd) generation inhibitors such as AP24534, VX-680 (MK-0457), PHA-739358, PPY-A, XL-228, SGX-70393, FTY720 and TG101113 are being developed to target the T315I mutation. The early results from these compounds are encouraging and it is anticipated that physicians will have additional drugs at their disposal for the treatment of patients with the mutated BCR-ABL-T315I. The success of these inhibitors has greater implication not only in CML, but also in other diseases driven by kinases where the mutated gatekeeper residue plays a major role.

Development of novel benzotriazines for drug discovery

Background: The synthesis of novel benzotriazine heterocycles was developed independently around the same time by Bischler, Bamberger and Arndt. Over the years, different groups have reported the synthesis of benzotriazine based compounds. Objective: This literature review gives an update on recent benzotriazine compounds and their applications. Conclusion: The benzotriazine core has been used in various drug discovery projects including anticancer, anti-inflammatory and antimalarial programs. Recently, the benzotriazine core was used to develop selective kinase inhibitors targeting SRC, VEGFr2, BCR-ABL and BCR-ABL-T315I. Two benzotriazine based compounds, tirapazamine for the treatment of cancer and TG100801 for the treatment of age-related macular degeneration, have entered clinical trials.

Structures of two conformationally defined phenylethanolamines: exo-1,4-epoxy-2-formamido-1,2,3,4-tetrahydro-8-trifluoromethylnaphthalene and exo-1,4-epoxy-2-formamido-1,2,3,4-tetrahydro-6-trifluoromethylnaphthalene

(+/-)-exo-1,4-Epoxy-2-formamido-1,2,3,4-tetrahydro-8- trifluoromethylnaphthalene (1), C12H10F3NO2, Mr = 257.21, Pccn, a = 8.895 (1), b = 19.968 (5), c = 12.656 (3) A, V = 2247.9 (8) A3, Z = 8, Dx = 1.520 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.49 cm-1, F(000) = 1056, T = 297 K, R = 0.0466 for 1481 independent reflections collected. The torsion angle for N(10)-C(2)-C(1)-C(8a) is 167.4 (2) degrees. (+/-)-exo-1,4-Epoxy-2-formamido-1,2,3,4-tetrahydro-6- trifluoromethylnaphthalene, (2), C12H10F3NO2, Mr = 257.21, P2(1)2(1)2(1), a = 8.52 (2), b = 26.15 (2), c = 5.06 (5) A, V = 1127 (11) A3, Z = 4, Dx = 1.516 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.29 cm-1, F(000) = 528, T = 297 K, R = 0.108 for 932 independent reflections collected. The torsion angle N(10)-C(2)-C(1)-C(8a) is 172.5 (6) degrees; the formamido group in both (1) and (2) is exo. X-ray studies on (1) suggest a hydrogen bond between N(10) and O(12) and similarly for (2).

Use of transferred nuclear overhauser effects to determine the conformation of 1-(3,4-dichlorophenyl)-2-aminopropane when bound to the active site of phenylethanolamine N-methyltransferace (PNMT)

Abstract Transferred two dimensional nuclear Overhauser effect spectroscopy (transferred NOESY) was used to show that the side chain of 1-(3,4-dichlorophenyl)-2-aminopropane (1) exists when bound at the active site of PNMT in an extended conformation (τ2=167 – 180°) with the aromatic ring rotated out of the plane of the ethylamine side chain (τ1 = 29 – 45°).