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Cardioprotective inhibitors of reperfusion injury

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Author Information

,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 , ,2 ,2 ,1 ,1 ,1 ,2 ,2 ,2 ,1 ,1,* and 3.

1 Sanford-Burnham Center for Chemical Genomics at Sanford-Burnham Medical Research Institute, Orlando, Florida 32827, USA.
2 Sanford-Burnham Center for Chemical Genomics at Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.
3 Albert Einstein College of Medicine of Yeshiva University, USA.
* Corresponding author: Siobhan Malany, Ph.D. gro.mahnrubdrofnas@ynalams

Received: ; Last Update: March 22, 2013.

An understanding of the mechanisms and pathways that mediate cardiac myocyte death is critical for the development of pharmacological therapies to limit heart damage during myocardial infarction (“heart attack”). We have completed the first unbiased cell-based screen of the MLSMR library consisting of ~360,000 compounds to identify small molecule probes that protect cardiac myocytes against oxidative and metabolic stresses, both precipitants of cell death during myocardial infarction. Oxidative stress was modeled with hydrogen peroxide treatment, and metabolic stress by inhibition of glycolysis using 2-deoxyglucose (2-DG). Since cardiac myocytes in vivo are terminally differentiated cells that cannot be expanded to the numbers needed for these studies, two cardiac myocyte model cell lines were used: H9c2 cells, derived fetal rat heart; and HL-1 cells, derived from the hearts of mice with cardiac-specific expression of an SV40 T-antigen transgene. Based on the activities of compounds in each of these cell types in response to each of these death stimuli (4 assays), 10 scaffolds were prioritized for reorder of their powders and available analogs for “analog-by-catalog” (ABC) testing. Two scaffolds advanced through multiple rounds of analog synthesis and structure-activity studies to result in the first probe molecule ML330. ML330 protects both H9c2 and HL-1 cell lines (EC50 0.4–4.0 μM & Emax 42–99 %) from both oxidative and metabolic stress induced by hydrogen peroxide and 2-DG, respectively. A second probe ML331, was identified preferentially protects both cell lines (EC50 0.6–9.0 μM & Emax 39–74 %) from metabolic stress induced by 2-DG. Probe molecules ML330 and ML331 will be useful to map and understand the critical cell death pathways in cardiac myocytes and lead to the development of a pharmacological agent to limit heart damage during myocardial infarction in humans. The probe molecules may also find broad utility in studies to understand relationships that regulate connections between various cell death programs, such as apoptosis and necrosis. Moreover, these findings may also extend to other ischemic syndromes such as stroke.

Assigned Assay Grant #: 1 R03 DA031671-01

Screening Center Name & PI: Sanford Burnham Center for Chemical Genomics (SBCCG) & John C. Reed (PI)

Chemistry Center Name & PI: Sanford Burnham Center for Chemical Genomics (SBCCG) & John C. Reed (PI)

Assay Submitter & Institution: Richard Kitsis, Albert Einstein College of Medicine of Yeshiva University, USA.

PubChem Summary Bioassay Identifier (AID): 588508

Probe Structure & Characteristics

Image ml331fu1

This Center Probe Report describes two cardioprotective compounds; i) ML330, which protects H9c2 & HL-1 cells from cell-death resulting from oxidative damage caused by H2O2 as well as metabolic stress imposed by 2-DG and ii) ML331, which selectively protects H9c2 and HL-1 cells from cell death resulting from metabolic stress imposed by 2-DG. Potency and selectivity characteristics for ML330 and ML331 are described in Table 1.

Table 1. Potency and selectivity characteristics for probes ML330 and ML331.

Table 1

Potency and selectivity characteristics for probes ML330 and ML331.

1. Recommendations for Scientific Use of the Probe

There are two clear uses for the probes at present: (a) to delineate cell death pathways in cardiac myocytes that are relevant to myocardial infarction and (b) to provide a prototype for the development of a pharmacological agent to be used clinically to limit heart damage during human myocardial infarction. We note that heart damage sustained in a myocardial infarction is a primary determinant of long-term cardiac dysfunction, disability, and death. There are approximately ~50 independent labs that study cardiac myocyte death, and another ~200 that study related issues. Prior work employing genetic and pharmacological manipulations to reduce cardiac myocyte death suggest that many of these labs will use the developed probes. One set of uses will focus on understanding the complex molecular circuitry that mediates cell death in the heart – in particular, the largely undefined connections between necrosis and apoptosis. In addition, multiple labs will use the probes as a starting point to generate a drug for myocardial infarction to be used in clinical practice. This study reports one probe, ML330, which protects rat H9c2 and mouse HL-1 cardiac myocytes against both oxidative and metabolic stressors, and another probe, ML331, that protects both cell lines against metabolic stress, but not oxidative stress. These findings are informative in several respects. First, these data suggest that ML330 and ML331 impact different molecular targets. Second, they illustrate the species-independence of these effects, an observation that portends well for the applicability of the findings to humans.

2. Materials and Methods

The primary assay and secondary assays are relatively simple cytoprotection assays that read-out cell viability indirectly using a commercial cell lysis reagent, ATPLite® (PerkinElmer) to quantify the high steady-state levels of cellular ATP that healthy cells produce to drive ATP-dependent bioluminescence. Cell lines: Rat H9c2 cells and mouse HL-1 are two cell lines that have been used as models of cardiac myocytes. Cytotoxic stressors: Hydrogen peroxide simulates the oxidative stress experienced by cardiac myocytes primarily during the reperfusion phase of ischemia/reperfusion, while 2-deoxyglucose simulates the metabolic stress that occurs primarily during the ischemic phase.

2.1. Assays

The details of the primary HTS and additional assay can be found in the “Assay Description” section in the PubChem BioAssay view under the AIDs as listed in Table 2.

Table 2. Summary of Assays and AIDs.

Table 2

Summary of Assays and AIDs. Summarizes the details for the assays that drove this probe project

2.2. Probe Chemical Characterization

Chemical name of probe compounds. The IUPAC name of the probe ML330 is N-(2-((4-acetyl-phenyl)amino)-2-oxoethyl)-6-methoxy-2-(3,4,5-trimethoxyphenyl)quinoline-4-carboxamide. The IUPAC name of ML331 is 6-(2,3-dichlorobenzyl)-9,10-dimethoxy-6,6a-dihydroisoindolo[2,1-a]quin-azoline-5,11-dione. The specific batches prepared, tested and submitted to the MLSMR are archived as SID SID 144223395, corresponding to CID 60202221 (ML330) and SID 144220864 corresponding to CID 60202247 (ML331).

Figure 1. Chemical structures of ML330 and ML331 and stereochemistry if known.

Figure 1Chemical structures of ML330 and ML331 and stereochemistry if known

Synthesis and Structural Verification Information of probe SID 144223395, corresponding to CID 60202221 (ML330).

Scheme 1. Synthesis of ML330.

Scheme 1Synthesis of ML330

Conditions: i) KOH, ethanol, MW 100 °C, 30 minutes, 77%; ii) HATU, triethylamine, CH2Cl2, 83%; iii) CF3CO2H, CH2Cl2, 15 h, 83%; and iv) HATU, DIEA, CH2Cl2, 2 h, 67%.

Synthesis and Structural Verification Information of probe SID 144220864 corresponding to CID 60202247 (ML331).

Scheme 2. Synthesis of ML331.

Scheme 2Synthesis of ML331

Figure 2. 1H NMR, 13C NMR, and LC-MS spectra of ML330.

Figure 21H NMR, 13C NMR, and LC-MS spectra of ML330

A. 1H NMR (500 MHz, DMSO-d6). B. LC of LC-MS. C. MS of LC-MS.

Figure 3. 1H NMR and LC-MS spectra of ML331.

Figure 31H NMR and LC-MS spectra of ML331

A. 1H NMR Spectrum of ML331 (500 MHz, CDCl3). B. LC of LC-MS for ML331. (Reverse phase C18 column). C. MS of LC-MS.

Solubility and Stability of ML330 and ML331 in PBS at room temperature. The solubility of ML330 and ML331 were investigated in aqueous buffers at room temperature. As noted in the Summary of in vitro ADME/T properties ML330 has low solubility in aqueous buffer at pH 5, 6.2 and 7.4 while the solubility of ML331 approaches 20 μM. To evaluate their potential instability in buffer and in assay media, 1 μM solutions of ML330 and ML331 were prepared either in acetonitrile:PBS (1:1) or assay media which contains 400 μM hydrogen peroxide and were incubated at room temperature. The amount of the parent compounds remaining at various times were analyzed by LC/MS (Figure 4). The results indicate that both probes showed little instability in buffer and in assay media up to 24 hours, whereas prolonged exposure to the oxidative environment of the assay media led to increased decomposition of parent compounds. The stability of ML330 or ML331 in assay media is suitable within the time course of the primary assay (3h).

Figure 4. Stability of ML330 & ML331 in Buffer.

Figure 4

Stability of ML330 & ML331 in Buffer.

MLS# verifying submission of probe molecule and five related samples submitted to the SMR collection. These probes are not commercially available. Samples of the probe ML330 and ML331 (>25 mg), and five analogs of each (>20 mg), synthesized at SBCCG were submitted to MLSMR (Tables 3 and 4), and 5 mg of the probes were provided to the Assay Provider.

Table 3. ML330 - Probe and Analog Submissions to MLSMR (Evotec) for Cardioprotectants.

Table 3

ML330 - Probe and Analog Submissions to MLSMR (Evotec) for Cardioprotectants.

Table 4. ML331 - Probe and Analog Submissions to MLSMR (Evotec) for Cardioprotectants.

Table 4

ML331 - Probe and Analog Submissions to MLSMR (Evotec) for Cardioprotectants.

2.3. Probe Preparation

ML330 Preparation: [Pfitzinger, W. J. Prakt. Chem. 1886, 33, 100.; Dasgupta, S.; Murumkar, P. R.; Giridhar, R.; Yadav, M. R. Bioorg. Med. Chem. 2009, 17, 3604–3617.]

A mixture of 5-methoxyindoline-2,3-dione (1.0 g, 5.64 mmol) and KOH (1.58 g, 28.2 mmol) were weighed into a 35 mL microwave reaction vessel. Ethanol (5 mL) was added and the mixture was stirred for five minutes at 23 °C. 1-(3,4,5-trimethoxyphenyl)ethanone (1.42 g, 6.77 mmol) was added as a solid and the mixture was heated under microwave conditions at 100 °C for 0.5 h. The solvent was evaporated and residue dissolved in water (100 mL) and extracted with ethyl acetate (3x, 25 mL). The aqueous layer was acidified with conc. HCl (pH ~ 2.0) and the solution cooled at 4 °C for 15 h. The precipitate formed was collected by filtration, washed with water and air-dried to afford the product, 6-methoxy-2-(3,4,5-trimethoxyphenyl)quinoline-4-carboxylic acid (A) as an orange solid (1.6 g, 77%). 1H NMR (500 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.09 (d, J = 9.2 Hz, 1H), 8.06 (d, J = 2.9 Hz, 1H), 7.59 – 7.43 (m, 3H), 3.93 (s, 6H), 3.92 (s, 3H), 3.75 (s, 3H). ESI-MS (+ve): calculated for C20H19NO6, [M+H] = 370.13, observed [M+H] = 370.23.

A mixture of 2-((tert-butoxycarbonyl)amino)acetic acid (400 mg, 2.28 mmol), triethylamine (0.955 mL, 6.85 mmol), and HATU (1.04 g, 2.74 mmol) in dichloromethane (12 mL) was allowed to stir 5 minutes prior to the addition of 1-(4-aminophenyl)ethanone (462 mg, 3.43 mmol). The mixture was stirred overnight and then was diluted with dichloromethane to 50 mL. The mixture was washed with water (20 mL), saturated aqueous sodium bisulfate (10 mL), water (20 mL), and saturated aqueous sodium bicarbonate (20 mL). The organic phase was dried with sodium sulfate and concentrated in vacuo to a yellow residue which was purified on silica gel eluting with 20–50% ethyl acetate in hexanes. The resulting Boc-protected intermediate (555 mg, 83%) was treated with 20 mL of 20% trifluoroacetic acid in dichloromethane for 2 hours. The solvent was removed in vacuo to provide the resulting N-(4-acetylphenyl)-2-aminoacetamide trifluoroacetic acid salt (B). Yield = 580 mg (83%) was used without further purification. 1H NMR (500 MHz, DMSO-d6) δ 10.92 (s, 1H), 8.26 (s, 3H), 7.98 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.7 Hz, 2H), 3.88 (d, J = 12.2 Hz, 2H), 2.55 (s, 3H). ESI-MS (+ve): Calculated for C10H12N2O2, [M+H] = 193.09, observed [M+H] = 193.05.

A mixture of A (208 mg, 0.56 mmol), N-ethyl-N-isopropylpropan-2-amine (0.4 mL, 2.3 mmol), and HATU (235 mg, 0.62 mmol) in dichloromethane (15 mL) was allowed to stir 5 minutes prior to the addition of B (248 mg, 0.84 mmol). The mixture was stirred two hours and then was diluted with dichloromethane to 50 mL. The mixture was then washed with water (15mL), saturated aqueous sodium bisulfate (15mL), water (15mL), and saturated aqueous sodium bicarbonate. The organic phase was dried with sodium sulfate and the solvent was removed in vacuo to provide orange foam. The material was purified on silica gel eluting with 0–5% methanol in dichloromethane. Crystallization of the resulting residue from dichloromethane:hexane solution provided N-(2-((4-acetylphenyl)amino)-2-oxoethyl)-6-methoxy-2-(3,4,5-trimethoxyphenyl)quinoline-4-carboxamide as a pale yellow powder (206 mg, 67%). 1H NMR (500 MHz, DMSO-d6) δ 10.58 (s, 1H), 9.24 (t, J = 6.0 Hz, 1H), 8.22 (s, 1H), 8.06 (d, J = 9.2 Hz, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.93 (d, J = 2.8 Hz, 1H), 7.80 (d, J = 8.7 Hz, 2H), 7.60 (s, 2H), 7.49 (dd, J = 9.2, 2.8 Hz, 1H), 4.28 (d, J = 6.0 Hz, 2H), 3.98 (s, 3H), 3.95 (s, 6H), 3.77 (s, 4H), 2.56 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 196.48, 168.17, 167.64, 157.66, 153.25, 152.87, 143.82, 143.23, 141.84, 138.89, 133.90, 131.78, 130.86, 129.60, 124.51, 122.55, 118.30, 116.63, 104.35, 103.90, 60.13, 56.09, 55.59, 43.18, 26.42. HRMS (ESI+ve): Calculated for C30H29N3O7, [M+H] = 544.2078, observed [M+H] = 544.2062.

ML331 Preparation: [Kumar, K. S.; Kumar, P. M.; Kumar, K. A.; Sreenivasulu, M.; Jafar, A. A.; Rambabu, D.; Krishna, G. R.; Reddy, C. M.; Kapavarapu, R.; Shivakumar, K.; Priya, K. K.; Parsa, K. V. L.; Pal, M. Chem. Commun. 2011, 47, 5010–12.]

Isatoic anyhydride (100 mg, 0.61 mmol), 2-carboxy-3,4-dimethoxybenzaldehyde (140 mg, 0.66 mmol), and 2,3-dichlorobenzylamine (116 mg, 0.66 mmol), montmorillonite K-10 (5%, 100 mg) and isopropanol (1.2 mL) were combined in a reaction tube and heated under microwave conditions at 150 °C for 2 h. The semisolid mixture was filtered with assistance from about 10 mL of chloroform. The filtrate was concentrated and the residue was purified via flash chromatography, using 50 mL of silica gel and eluting with 25% ethyl acetate in hexanes. The product ML331 was recovered as an amorphous white solid, 105.7 mg (37%). 1H NMR (500 MHz, CDCl3) δ 8.23 (dd, J = 7.8, 1.5 Hz, 1H), 8.15 (dd, J = 8.2, 1.1 Hz, 1H), 7.78 – 7.64 (m, 1H), 7.46 – 7.33 (m, 2H), 7.11 (t, J = 7.9 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 7.02 – 6.88 (m, 2H), 6.29 – 6.21 (m, 1H), 5.19 (d, J = 17.6 Hz, 1H), 5.09 (d, J = 17.6 Hz, 1H), 4.10 (s, 3H), 3.91 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 164.13, 163.14, 154.45, 148.32, 137.11, 136.42, 133.89, 133.42, 129.74, 129.42, 129.07, 127.55, 125.39, 125.02, 120.55, 120.07, 116.54, 69.63, 62.63, 56.57, 45.09; HRMS (ESI+ve): Calculated for C24H19Cl2N2O4, [M+H] = 469.0716, observed [M+H] = 469.0690. Calculated for C24H18Cl2N2NaO4, [M+Na] = 491.0536, observed [M+Na] = 491.0536.

All reactions involving air and moisture-sensitive reagents and solvents were performed under a nitrogen atmosphere using standard chemical techniques. Anhydrous solvents were purchased and freshly used from Sigma-Aldrich or EMD Biosciences. All organic reagents were used as purchased. Analytical thin-layer chromatography was performed on Partisil K6F silica gel 60 Å, 250 μm. Microwave-assisted reactions were performed using a CEM Discover system. 1H and 13C chemical shifts are reported in values in ppm in the corresponding solvent. All solvents used for chromatography on the synthetic materials were Fisher Scientific HPLC grade, and the water was Millipore Milli-Q PP filtered. LCMS analysis of synthetic materials was completed on a Waters Autopurification system, which consists of a 2767 sample manager, a 2545 binary gradient module, a system fluidics organizer, a 2489 UV/vis detector, and a 3100 mass detector, all controlled with MassLynx software. A Sunfire Analytical C18 5 μm column (4.6 × 50 mm) and stepwise gradient {10% [(MeCN + 0.1% TFA) in (water + 0.1% TFA)] to 98% [(MeCN + 0.1% TFA) in (water + 0.1% TFA)] for 9 min.} was used for analytical LCMS of final compounds. The final compounds were purified by silica gel flash chromatography. All NMR spectra for the synthetic materials were recorded on a Bruker Avance II 400 or DRX-500 MHz instrument. MestReNova 7 program was used to process and interpret NMR spectra. High Resolution Mass Spectrometry (HRMS) spectra were carried out on an Agilent 6224A Accurate-Mass Time-of-Flight (TOF) LC/MS system with electron spray ionization (ESI).

3. Results

3.1. Dose Response Curves for Probe

Figures 5A and 5B illuminate the matrix of dose response results for ML330 and ML332.

Figure 5A. EC50 determinations from full dose-response curves tested in quadruplicate runs for ML330 in all assays.

Figure 5A

EC50 determinations from full dose-response curves tested in quadruplicate runs for ML330 in all assays.

Figure 5B. EC50 determinations from full dose-response curves tested in quadruplicate runs for ML331 in all assays.

Figure 5B

EC50 determinations from full dose-response curves tested in quadruplicate runs for ML331 in all assays.

3.2. Cellular Activity

Both probes, ML330 and ML331 are active in cells because the primary, confirmatory and secondary assays were all conducted in cell-based systems. It should also be noted that neither probe shows any significant cytotoxicity (LD50) relative to its potency (EC50) against a human hepatocyte cell line (seeTable 5 ADME/T properties).

Table 5. Summary of in vitro ADME/T Properties of Cardioprotectants ML330 and ML331.

Table 5

Summary of in vitro ADME/T Properties of Cardioprotectants ML330 and ML331.

3.3. Profiling Assays

As a pro forma activity, the SBCCG is committed to profiling all final probe(s) compound(s) and in certain cases key informative analogs in the PanLabs full panel as negotiated by the MLPCN network. Additional commercial profiling services will be considered for funding by SBCCG as deemed appropriate and informative. The nominated probes were evaluated in a detailed in vitro pharmacology screen as shown in Table 5.

While ML330 demonstrates low solubility in aqueous media at pH 5.0, 6.2, and 7.4, ML331 is soluble in excess of its reported IC50 value at all three pH points.

The PAMPA (Parallel Artificial Membrane Permeability Assay) assay is used as an in vitro model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments is measured using UV spectroscopy. Both ML330 and ML331 exhibit high permeability.

Plasma protein binding is a measure of a drug’s efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. ML330 is highly bound to human plasma proteins yet is undetectable in the corresponding mouse study. This observation may be accounted for by the rapid degradation of ML330 in mouse plasma. ML331 is highly bound to both human and mouse plasma proteins.

Plasma stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which strongly can influence the in vivo efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases), and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. Both of the probes are relatively stable in human plasma after 3 hr; however, ML330 is highly metabolized in mouse plasma whereas ML331 is only modestly degraded. This is likely why ML330 was undetectable in the mouse plasma protein binding assay.

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. ML330 shows poor stability (3.9 % & 1.6% remaining at 60 min) in both human and mouse liver homogenates. ML331 shows a similar lack of stability with 4.6 % and 0.4 % remaining, respectively. The lack of microsomal stability may be anticipated based on the presence of multiple methoxy groups in each probe. Unfortunately, in both cases the SAR demanded the presence of multiple methoxy substituents.

Both ML330 and ML331 show no toxicity (>50 μM) toward human hepatocytes.

Profiling against other GPCRs. The two probes, ML330 (CID 60202221) and ML331 (CID 60202247), were submitted to the Psychoactive Drug Screening Program (PDSP) at the University of North Carolina (Bryan Roth, PI) and the data against a GPCR binding assay panel is shown in Figure 6. Preliminary results indicate that both ML330 and ML331 show a low level of promiscuity (at 10 μM). It is not known whether these activities in binding assays are translated into functional modification of the activities of these receptors.

Figure 6. Profile of ML330 and ML331 in Psychoactive Drug Screening Program (PDSP) panel (% inhibition at 10 μM).

Figure 6

Profile of ML330 and ML331 in Psychoactive Drug Screening Program (PDSP) panel (% inhibition at 10 μM).

4. Discussion

Myocardial infarction (“heart attack”) is one of the most common causes of morbidity and mortality in the United States (~1 every 35 seconds in the U.S; of which ~250,000 are fatal)1. This syndrome is precipitated by the acute rupture of an atherosclerotic plaque leading to thrombotic occlusion of a coronary artery. The sudden cessation of blood flow leads within hours to massive death of heart cardiac myocytes by apoptosis and necrosis2. For the most severe type of myocardial infarction, prompt myocardial reperfusion (restoration of blood flow by opening up the vessel via angioplasty/stenting) is currently standard therapy3,4. While reperfusion itself can augment cell death, data in humans has established unequivocally that restoration of blood results in an overall salvage of cardiac myocytes. A major clinical issue is that reperfusion must be accomplished promptly (within the first 4–6 hours) in order to achieve optimal salvage of heart muscle.

Work over the past 25 years in lower organisms and mammals has led to the recognition that a significant portion of cell death – both apoptosis and necrosis – occurs in a deliberate and highly regulated manner58. Extensive genetic, biochemical, and pharmacological studies from the PI2,927 and others have delineated multiple mechanisms that mediate cell death in the heart. Importantly, this work has shown that cardiac myocyte death during myocardial infarction can be inhibited, resulting in the reduction of infarct size and preservation of cardiac function2. Taken together, this research suggests the possibility that inhibition of the cell death process itself may provide a novel approach to limiting heart muscle damage during myocardial infarction.

In addition to their medical uses, the developed probes will aid research efforts to understand apoptotic and necrotic death programs in the heart.2,28 Future work will delineate which death pathway(s) are impacted by these molecules and, ultimately, identify the cellular targets against which these drugs are directed.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Additional recent SciFinder searches by our Center Chemists for prior art found several cardioprotective agents. These blockers of cell death were developed against specific targets in death pathways. These include caspase inhibitors2932, UCF-10133,34 (which inhibits the serine protease activity of Omi/HtrA2), and necrostatin-13537 (which inhibits the serine-threonine kinase activity of RIP1). However, the clinical suitability of these compounds and whether they are directed against the optimal cellular targets remains untested.

In 2007, Modis38 reported on a number of related dopamine D1 receptor agonists found from testing of the commercially available Sigma LOPAC® (Library of Pharacologically Active Compounds) collection based on the parent 7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine compound (APB) provided significant cytoprotection at 3 h, but only the chloro- and bromo-APB and chloro-PB hydrobromide showed a similar effect at 24 h. Full-dose analysis and IC50 determinations of these were not reported, however, in this study, chloro-APB was 40% cytoprotective against H2O2 challenge at 30 μM, suggesting that the potency of these APBs are in the range of 50–100 μM for cytoprotection. PubChem has two entries for this parent compound CID 1225 (racemate) and CID 6603757 (S-enantiomer) and the details therein provide hyperlinks to some commercial sources. Neither compound appears to be available directly or has been deposited in the MLSMR, and neither compound has been reported to be active in cytoprotection.

Image ml331fu2

The probes identified herein are potent and efficacious small molecule probes that represent novel scaffolds, and are the first-in-class examples of probes provided to the research community for cardioprotection that have be deposited to the MLSMR, through an unbiased screen. These new probes will be useful in two respects: (a) as tools to further interrogate death pathways in the heart and (b) as prototypes for potential drug development. Currently there are no drugs directed specifically at inhibiting cell death in heart disease for which efficacy has been demonstrated. Thus, although other targets (e.g. caspase inhibitors) have been considered, there is no existing art with which to compare the probes identified in the current studies.

Accordingly, if validated in subsequent cell culture and whole animal experiments, these probes will function as prototypes for the development of potential drugs to reduce infarct size during human myocardial infarction. The most likely clinical possibility is that these drugs would be used in conjunction with reperfusion therapy (e.g. acute angioplasty/stenting), so as to promote cardiomyocyte rescue through complementary mechanisms of restoring the availability of oxygen and nutrients and impeding intrinsic cell suicide mechanisms. A particularly attractive possibility is that such drugs may sufficiently slow the kinetics of cell death so as to extend the time window in which reperfusuion therapy would be effective.

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