Carmen M. Baldino

Strategies for the synthesis of novel indole alkaloid-based screening libraries for drug discovery

A series of diverse indole-based chemotypes were synthesized from β-tetrahydrocarboline (β-THC) scaffolds prepared from commercially and readily available tryptamines and α-ketoesters. Diversity can be generated within these chemotypes through the following strategies: (a) appendage of substituents to the β-THC scaffold, prepared in situ or as a template, through further elaboration and (b) skeletal modifications to the β-THC scaffold via ring forming or ring breaking reactions. The strategies described here are amenable to high throughput solution-phase parallel synthesis, providing access to novel indole-based screening libraries for drug discovery.

Access to the noryohimban [6,5,6,5,6] ring system via an intramolecular furan Diels–Alder reaction

Abstract Polycyclic indolic compounds containing the [6,5,6,5,6] ring system were prepared via an intramolecular furan Diels–Alder reaction of α,β-unsaturated amides generated by the N -acylation of 1-(2-furyl)-β-tetrahydrocarbolines. This chemistry can provide access to D(14)-noryohimban derivatives by exploiting the functionality on the C,D,E ring system of the corresponding cycloadducts.

A Practical and Environmentally Friendly Preparation of 3-Carboxycoumarins

Abstract The condensation of salicylaldehydes with Meldrums acid in water at 75°C to produce 3-carboxycoumarin derivatives is described.

Microwave-assisted acylation of 7-amino-5-aryl-6-cyanopyrido[2,3-d]pyrimidines.

A microwave-assisted method is described for monoacylating 7-amino-5-aryl-6-cyanopyrido[2,3-d]pyrimidines using excess acid chlorides in pyridine. A diacylated intermediate is effectively deacylated to the product amide by a macroporous-Tris resin. A small library of 17 amides was prepared to validate the method. The integration of commercial microwave technology into the ArQule chemistry platform is also discussed.

Abstract 5056: PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells

Dysregulation of vitamin D signaling and metabolism enzymes is a frequent change in many cancers and may confound approaches to vitamin -D based therapies. The mechanisms by which aberrant vitamin D signaling and metabolism lead to resistance to vitamin D action are poorly understood. The kidney plays a critical role in vitamin D synthesis and metabolism; in renal cell carcinoma (RCC) cells vitamin D metabolism is dysregulated. Recently, we discovered that an oncogenic protein kinase PIM-1 is involved in vitamin D-induced expression of 24-hydroxylase, a key catalytic enzyme of vitamin D encoded by CYP24A1 gene, in bladder and prostate cancer. To study the impact of PIM1 on vitamin D signaling and vitamin D-mediated anti-tumor activity in RCC, we analyzed 4 human renal cell carcinoma cell lines (Caki-1, ACHN, A498 and 786-O) and 25 samples of human RCC tumor tissue and 13 benign kidney tissues (including 10 matched tumor and benign tissue samples) by qRT-PCR, immunohistochemistry (IHC), Western blot analysis, MTT and clonogenic assays. VDR mRNA and protein were detected in all RCC cell lines. IHC showed that VDR was strongly expressed in the normal kidney tissues, but decreased in RCC tissues. As expected, VDR expression was significantly increased by treatment with 1,25-dihydroxyvitamin D3 (1,25D3) in RCC cell lines. MTT and clonogenic assays showed that 1,25D3 inhibited RCC cell growth (p Citation Format: Wei Luo, Yingyu Ma, Carmen Baldino, Justin Caserta, Swathi Ramakrishnan, Pili Roberto, Candace Johnson, Donald Trump. PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2015-5056

Abstract 5459: Preclinical efficacy of the novel PIM2 kinase inhibitor, JP_11646, in human acute leukemia models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnPIM kinases (PIM1,2, and 3) are constitutively active serine/threonine protein kinases which regulate cell cycle, survival, and drug resistance and have been strongly implicated in cancer cell survival and tumorigenesis. Although expression levels of PIM family members (PIM1, 2, and 3) differ by cancer type, PIM1 and PIM2 kinases are preferentially overexpressed in hematological malignancies. Here we studied the biological effects of treatment with a novel pan-PIM inhibitor, JP_11646 exhibiting potent inhibition of PIM2 kinase activity, in human acute myeloid (AML), acute lymphocytic leukemia (ALL), and lymphoma cell lines.nnHuman AML cell lines (THP-1, KG-1, HL60-VCR, ML2, MV(4;11)) were evaluated for basal mRNA and protein expression of PIM kinases using real time PCR and western blot analysis, respectively. A panel of thirty additional human AML, ALL (Nalm-6), and lymphoma (RAJI) cell lines were exposed to JP_11646 at concentrations ranging from 0.001 to 10 uM for 48-72 hours followed by measurements of cell proliferation by MTT assay and cell viability by CellTiter-Glo assay. The Hill model was used to fit each concentration-response curve for each cell line and endpoint. Nonlinear regression analysis was performed using Phoenix 64 for estimation of IC50 and Imax. AML (HEL) cells after 72 hour of JP_11646 treatment were analyzed for induction of apoptosis measured via annexin V and 7AAD flow cytometry.nnAML cell lines exhibited differential basal mRNA expression of PIM kinases. The cell lines that expressed the highest levels of PIM1, PIM2, and PIM3 mRNA were THP-1 and ML2, respectively. Basal expression of the PIM2 protein revealed the presence of all three PIM2 isoforms in AML (HEL, THP-1) cells. JP_11646 treatment resulted in inhibition of proliferation across the entire panel of AML and ALL cell lines, with the overall potency (IC50) ranging from 0.07 to 0.37 uM and maximal inhibition ranging from 70 to 97.5 percent. As the drug exposure time increased from 48 to 72 hours, the maximal inhibition increased 2 to 3-fold. Inhibition of cell viability across a panel of 30 leukemia and lymphoma cell lines demonstrated a similar IC50 range from 0.05 to 0.25 uM. AML (HEL) cells treated with JP_116464 at doses of 0.1 and 0.5 uM for 72 hours exhibited a concentration-dependent induction of apoptosis of 32% and 89% percent, respectively.nnTaken together, our results demonstrate broad anti-leukemic activity and potency of the novel PIM kinase inhibitor, JP_11646, across a wide panel of human AML and ALL cell lines with induction of apoptosis and cell death at nM concentrations. Further, investigation of the in vivo effects of JP_11646 in representative human AML xenograft models is ongoing.nnCitation Format: Krista E. Pundt, Carmen M. Baldino, Justin Caserta, Stephane Dumas, Yvonne Flanders, Eunice S. Wang, Gerald J. Fetterly. Preclinical efficacy of the novel PIM2 kinase inhibitor, JP_11646, in human acute leukemia models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2014-5459

Abstract 751: Preclinical efficacy of the novel PIM inhibitor, JP_11646, in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is often first diagnosed at an advanced stage with a poor prognosis. Current systemic treatment approaches have limited efficacy, pointing to an urgent need to develop novel therapies. Recent studies have shown that inhibition of Pim-2 activity via JP_11646, a novel pan-PIM inhibitor, resulted in decreased anchorage-independent growth of PDAC. The aim of this work is to examine the ability of JP_11646 alone and in combination with gemcitabine to inhibit the growth of pancreatic cancer cells in vitro and in vivo. Cell viability was assessed using the MTS assay in two pancreatic cancer cell lines, MiaPaCa2 and PANC1. Cells were seeded at an average of 5x10 3 per well on 96-well plates and exposed to JP_11646 in a range of concentrations (0.01 - 5 µM) for 72 hours. Exposure to JP_11646 inhibited PANC1 and MiaPaCa2 cell growth in a dose-dependent manner, with IC 50 values of 0.15 µM and 0.054 µM, respectively. For in vivo experiments, SCID mice were implanted subcutaneously with 3x 10 6 MiaPaCa2 cells. Upon successful establishment of tumors (approximately 100 mm 3 ), animals were randomly divided into groups for drug and control treatments. JP_11646 was given at 5 or 10 mg/kg by intraperitoneal injection (i.p. injection) once a day for 5 consecutive days from Monday to Friday for a total of six weeks. Gemcitabine i.p. was given at 45 mg/kg in combination treatment or 90 mg/kg as a single agent twice a week every Monday and Friday for a total of six weeks. In a dose-dependent manner, JP_11646 at 5 and 10 mg/kg significantly suppressed the growth of the pancreatic xenograft by 36% and 77% respectively, compare with control PBS treatment in SCID mice. Moreover, the combination of JP-11646 5 mg/kg and gemcitabine 45 mg/kg resulted in a greater tumor inhibition (83%) than either single agent alone [JP_11646 5 mg/kg (36%) or gemcitabine 90 mg/kg (69%)]. Taken together, our results suggest that JP_11646 is a potent inhibitor of pancreatic cancer growth with equivalent efficacy to the maximum tolerated dose of gemcitabine, and is synergistic in combination with gemcitabine. Citation Format: Yi Ding, Vun-Sin Lim, Carmen M. Baldino, Justin Caserta, Yvonne Flanders, Stephane Dumas, Gerald Fetterly, Alex A. Adjei. Preclinical efficacy of the novel PIM inhibitor, JP_11646, in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 751. doi:10.1158/1538-7445.AM2014-751

Abstract 4647: Investigation of the pharmacokinetic profile of the novel PIM2 inhibitor, JP_11646

PIM2 is a serine/threonine protein kinase that has roles in cell growth, proliferation, and apoptosis via the regulation of multiple signal transduction cascades. JP_11646 is a novel PIM2 inhibitor that has shown antitumor activity against breast, colon, liver, lung, and pancreatic solid tumors, as well as multiple myeloma and leukemia. The pharmacokinetics (PK) of JP_11646 has not been described. Therefore, a PK study was conducted to characterize JP_11646 disposition in plasma and various tissues. Normal ICR (CD-1) mice received either single day or five consecutive days of 15 - 25 mg/kg JP_11646 via intraperitoneal (IP) or intravenous (IV) administration. Plasma samples were collected at serial time points (0.5, 1, 2, 4, 6, and 8 hrs) following a single dose of 20 mg/kg IV or 25 mg/kg IP JP_11646 administration. Plasma and various tissues (brain, heart, kidney, lung, liver, muscle, colon, pancreas, and spleen) were collected at serial time points following multiple daily doses of 15 mg/kg IP JP_11646 on day five. Three mice were euthanized per time point on each collection day. JP_11646 concentrations were determined by a validated LC-MS/MS method, with a LLOQ of 1 ng/ml. Noncompartmental PK analysis was performed using Phoenix 64 (Pharsight, WinNonlin 6.3). Following single dose administration of JP_11646 of 20 (IV) and 25 mg/kg (IP), mean plasma Cmax was 6,951 and 6,630 ng/ml, respectively. Mean plasma drug exposures (AUCinf) were 11,065 and 11,024 ng-hr/ml, respectively, resulting in similar drug exposure between the IV and IP route. To compare plasma and tissue exposure following multiple dose administration, JP_11646 was given at 15 mg/kg IP. The mean plasma Cmax was 4,601 ng/ml., while the mean Cmax in colon, pancreas, and spleen were 24,523, 77,811, and 18,835 ng/ml, respectively. The half-life of JP_11646 in plasma and tissues ranged from 0.8 - 3 hrs. Tissue exposure in colon, pancreas, and spleen was 48,792, 157,273, and 40,511 ng-hr/ml, respectively. The pancreas to plasma partition coefficient (Kp) of 20.6 demonstrates that the pancreas receives the highest exposure to JP_11646. The drug also distributes to the colon and spleen, with Kp values of 6.4 and 5.3, respectively. JP_11646 PK appears linear and the half-life ranges from 0.8 - 3.0 hrs. JP_11646 distributes widely to tissues, with the highest drug exposure in the pancreas, spleen, and colon. These results give insight into tumor types with potential for optimal antitumor therapy with JP_11646. Citation Format: Laura B. Pitzonka, Allison Gaudy, Sarah Schihl, Leslie Curtin, Sandra Sexton, Carmen M. Baldino, Justin Caserta, Yvonne Flanders, Stephane Dumas, Gerald Fetterly. Investigation of the pharmacokinetic profile of the novel PIM2 inhibitor, JP_11646. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2014-4647

Abstract 607: Targeting PIM1 kinase enhances 1,25-dihydroxyvitamin D3-mediated anti-tumor activity in bladder cancer cells

Bladder cancer is the 5th most common in the United States. 30% of cases are advanced stage requiring multimodality therapy at diagnosis and many which are first diagnosed when superficial will progress to more advanced disease. High recurrence and progression rate and drug resistance highlight the need for new therapeutic strategies to improve clinical outcomes. 1,25-dihydroxyvitamin D3 (1,25D3), the most active vitamin D metabolite, has demonstrated broad spectrum antitumor activities in vitro and in vivo. In addition, 1,25D3 potentiates the antitumor effects of common chemotherapeutic agents in several cancer cell types including bladder cancer. 24-hydroxylase, encoded by the CYP24A1 gene, plays a key role in degrading 1,25D3. Increased expression of CYP24A1 has been found in several human tumors. Several studies reveal that the protein kinase signaling pathways including PIM1 are involved in the regulation of CYP24A1 expression. The PIM kinases are a family of serine/threonine kinases (PIM-1, PIM-2, PIM-3) that have been associated with tumorigenesis and drug resistance. PIM1 has been extensively studied in lymphoma and upregulation of PIM kinase expression has been reported in many malignancies. We analyzed mRNA expression of CYP24A1 and PIM1 in 17 bladder cancer cell lines by qRT-PCR. We found that CYP24A1 expression is increased in 12 out of 17 bladder cancer cell lines and PIM1 expression is increased in 8 out of 17 bladder cancer cell lines compared to expression in a benign bladder cancer cell line. Therefore, we hypothesized that inhibition of PIM kinase activity would suppress CYP24A1 expression and thus enhancing 1,25D3-mediated anti-tumor activity. Using the PIM1 inhibitor, JP11646, we found that inhibition of PIM1 kinase activity reduces CYP24A1 expression at transcriptional level in bladder cancer cells RT112 and RT112D21 which express high level of PIM1 and CYP24A1. We further evaluated the efficacy of the PIM1 inhibitor alone and in combination with 1,25D3. Inhibition of PIM1 kinase activity by JP11646 reduces cell growth in a panel of bladder cancer cell lines in a dose-dependent manner as measured by MTT assays. MTT assays detected a significant inhibition of cell proliferation by 50% compared to the control when cells were treated with 128 nM of JP11646 for 72 h in 6 of bladder cancer cell lines tested. Furthermore, inhibition of PIM1 kinase activity enhances 1,25D3-mediated inhibitory effect on cell growth in bladder cancer cells RT112 and RT112D21. This study provides the first evidence that inhibition of PIM kinase activity may be an attractive therapeutic strategy to facilitate the optimization of antitumor therapy of vitamin D in bladder cancer. This study was supported by NIH/NCI grants 5R01CA067267 and 5R01CA095045 Citation Format: Wei Luo, Carmen M. Baldino, Justin Caserta, Yingyu Ma, Candace Johnson, Donald L. Trump. Targeting PIM1 kinase enhances 1,25-dihydroxyvitamin D3-mediated anti-tumor activity in bladder cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 607. doi:10.1158/1538-7445.AM2014-607

Abstract 1049: PIM2 inhibitor as a targeted therapy for the treatment of multiple myeloma patients with specific genetic signatures.

Jasco Pharmaceuticals has developed a novel and selective pan-PIM inhibitor (JP_11646) that has demonstrated biochemical IC50s of 24, 0.5 and 1 nM for PIM1, PIM2 and PIM3 respectively. Jasco also developed an engineered cell line to measure PIM2 transphosphorylation by over-expressing human PIM2 in human HEK293 cells together with human BAD as the substrate. The cell based assay measures the phosphorylation levels of BAD at Ser112 attributed solely to PIM2. JP_11646 exhibited a 590 nM IC50 in this cell based PIM2 assay confirming the MOA. JP_11646 increases apoptosis and decreases cell viability in multiple myeloma cell lines with the t(14;16) and t(4;14) translocations, which are known markers for poor prognosis. JP_11646 is orally bioavailable and has demonstrated in vivo efficacy, inhibiting tumor growth by >80% in a MM1.S tumor xenograft study. These data provide solid rationale for further development of JP_11646 as a targeted therapy for specific population of multiple myeloma patients that exhibit these genetic markers. Citation Format: Carmen M. Baldino, Justin Caserta, Stephane Dumas, Yvonne Flanders, Jayakumar Nair, Kelvin P. Lee. PIM2 inhibitor as a targeted therapy for the treatment of multiple myeloma patients with specific genetic signatures. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1049. doi:10.1158/1538-7445.AM2013-1049