Synthesis and Positive Inotropic Activity of [1,2,4]Triazolo[4,3-a] Quinoxaline Derivatives Bearing Substituted Benzylpiperazine and Benzoylpiperazine Moieties

In an attempt to search for more potent positive inotropic agents, two series of [1,2,4]triazolo[4,3-a] quinoxaline derivatives bearing substituted benzylpiperazine and benzoylpiperazine moieties were synthesized and their positive inotropic activities evaluated by measuring left atrial stroke volume in isolated rabbit heart preparations. Several compounds showed favorable activities compared with the standard drug, milrinone. Compound 6c was the most potent agent, with an increased stroke volume of 12.53% ± 0.30% (milrinone: 2.46% ± 0.07%) at 3 × 10−5 M. The chronotropic effects of compounds having considerable inotropic effects were also evaluated.


Introduction
Glycosides such as digoxin are frequently prescribed cardiotonic agents used for the treatment of congestive heart failure (CHF). Unlike other CHF drugs, they do not increase mortality. However, the narrow safety margins associated with the use of digitalis compounds are serious problems because of the high frequency and severity of digitalis intoxication [1]. The discovery of amrinone led to the synthesis of several agents holding promise for CHF treatment as non-sympathomimetic, non-glycoside agents [2]. The phosphodiesterase-inhibiting agent milrinone has vasodilator and inotropic properties. It was approved for the treatment of CHF more than a decade ago. Nevertheless, the significant ventricular arrhythmias and tachycardia associated with elevated levels of cyclic adenosine monophosphate limit the use of milrinone [3] as well as a newer agent, vesnarinone [4,5]. Therefore, newer positive inotropic agents with fewer side effects are needed [6].

Synthesis
The reaction sequence for the synthesis of 16 new quinoxaline derivatives 6a-k and 7a-e is outlined in Scheme 1. Benzene-1,2-diamine (1) was reacted with diethyl oxalate to afford quinoxaline-2,3(1H,4H)-dione (2), which was subsequently reacted with refluxing hydrazine hydrate to give 3-hydrazono-3,4-dihydroquinoxalin-2(1H)-one (3). Compound 4 was prepared from compound 3 by reaction with ethyl orthoformate. Compound 5 was obtained by reacting 4 with refluxing phosphorus oxychloride (POCl3). The nucleophilic aromatic substitution reactions of 5 with various monosubstituted piperazines in refluxing acetone in the presence of potassium carbonate afforded compounds 6a-k. Finally, compounds 7a-e were obtained in high yield from the reactions of 5 with appropriate different monosubstituted piperazines in refluxing acetone in the presence of potassium carbonate. Newly synthesized derivatives 6a-k and 7a-e were characterized by 1 H-NMR, 13 C-NMR, IR and mass spectral data. In general, IR spectral data in the ranges of 1549-1457 cm −1 and 1638-1620 cm −1 indicated distinctive functional groups such as −C=N and −C=O stand for derivatives 6a-k and 7a-e, respectively. The (M + 1) peaks in the mass spectra for these compounds were in agreement with their molecular formula.

Synthesis
The reaction sequence for the synthesis of 16 new quinoxaline derivatives 6a-k and 7a-e is outlined in Scheme 1. Benzene-1,2-diamine (1) was reacted with diethyl oxalate to afford quinoxaline-2,3(1H,4H)-dione (2), which was subsequently reacted with refluxing hydrazine hydrate to give 3-hydrazono-3,4-dihydroquinoxalin-2(1H)-one (3). Compound 4 was prepared from compound 3 by reaction with ethyl orthoformate. Compound 5 was obtained by reacting 4 with refluxing phosphorus oxychloride (POCl 3 ). The nucleophilic aromatic substitution reactions of 5 with various monosubstituted piperazines in refluxing acetone in the presence of potassium carbonate afforded compounds 6a-k. Finally, compounds 7a-e were obtained in high yield from the reactions of 5 with appropriate different monosubstituted piperazines in refluxing acetone in the presence of potassium carbonate. Newly synthesized derivatives 6a-k and 7a-e were characterized by 1 H-NMR, 13 C-NMR, IR and mass spectral data. In general, IR spectral data in the ranges of 1549-1457 cm −1 and 1638-1620 cm −1 indicated distinctive functional groups such as −C=N and −C=O stand for derivatives 6a-k and 7a-e, respectively. The (M + 1) peaks in the mass spectra for these compounds were in agreement with their molecular formula.

Biological Evaluation
Seven of the 16 compounds tested displayed inotropic effects against isolated rabbit heart preparations (Table 1). Compounds 6c, 6g, 6h, and 7c exhibited more potent effects compared with milrinone (2.46% ± 0.3% at 3 × 10 −5 M), among which compound 6c showed the most potent activity, with an increased stroke volume of 12.53% ± 0.30%. For compounds 6a-k, different substituents on the phenyl ring of the benzyl group at the 4-position of the piperazine ring exerted considerable influence on the inotropic activity. For fluorinated compounds, only para-substituted 6c showed good activity, and clearly exhibited more potent effects compared with lead compound 1 and milrinone, with an increased stroke volume of 12.53% ± 0.30%. Chloro-substituted compounds (6d, 6e, 6j and 6k) did not show any inotropic activity, and the para-chloro-substituted 6f displayed slightly increased activity with an increased stroke volume of 1.01% ± 0.06%. The position of the substituents on the phenyl ring also influenced activity but, in general, a clear pattern for the structure-activity relationship was not found.
We investigated the dynamics of the tested compounds in perfused beating rabbit atria. Compounds 6c, 6g and 6h produced initial increases in stroke volume, whereas longer treatment caused decreases in stroke volume (Figure 2A,B,C). For compound 7c, the stroke volume increased gradually ( Figure 2D).

Biological Evaluation
Seven of the 16 compounds tested displayed inotropic effects against isolated rabbit heart preparations (Table 1). Compounds 6c, 6g, 6h, and 7c exhibited more potent effects compared with milrinone (2.46% ± 0.3% at 3 × 10 −5 M), among which compound 6c showed the most potent activity, with an increased stroke volume of 12.53% ± 0.30%. For compounds 6a-k, different substituents on the phenyl ring of the benzyl group at the 4-position of the piperazine ring exerted considerable influence on the inotropic activity. For fluorinated compounds, only para-substituted 6c showed good activity, and clearly exhibited more potent effects compared with lead compound 1 and milrinone, with an increased stroke volume of 12.53% ± 0.30%. Chloro-substituted compounds (6d, 6e, 6j and 6k) did not show any inotropic activity, and the para-chloro-substituted 6f displayed slightly increased activity with an increased stroke volume of 1.01% ± 0.06%. The position of the substituents on the phenyl ring also influenced activity but, in general, a clear pattern for the structure-activity relationship was not found.
We investigated the dynamics of the tested compounds in perfused beating rabbit atria. Compounds 6c, 6g and 6h produced initial increases in stroke volume, whereas longer treatment caused decreases in stroke volume (Figure 2A-C). For compound 7c, the stroke volume increased gradually ( Figure 2D).

Conclusion
Two series of [1,2,4]triazolo[4,3-a] quinoxaline derivatives bearing substituted benzylpiperazine and benzoylpiperazine moieties were synthesized using A as the lead compound. We tried to ascertain potent compounds for cardiac contractility without increasing the heart rate. Compound 6c exhibited promising cardiovascular properties and potent activities compared with milrinone: 6c was 5.1-fold more active than milrinone. This compound is undergoing further biological tests, including in vivo evaluation, coronary vasodilation tests, and studies into possible mechanisms of action.

General Information
Melting points were determined in open capillary tubes and were uncorrected. Chemical reactions were monitored by thin-layer chromatography on silica gel precoated F254 plates (Merck, Whitehouse Station, NJ, USA). Developed plates were visualized by ultraviolet light (254 nm). Column chromatography was undertaken with 200-mesh silica gel (Merck). IR spectra were recorded (in KBr) on a FT-IR 1730 system. 1 H-NMR spectra were measured on an AV-300 Spectrometer (Bruker, Billerica, MA, USA) using trimethylsilane as an internal standard. Mass spectra were measured on an HP1100LC system (Agilent Technologies, Santa Clara, CA, USA). Chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and Fluka (Milwaukee, WI, USA).

General Experimental Procedure for the Synthesis of [1,2,4]Triazolo[4,3-a] Quinoxaline-Bearing Substituted Benzylpiperazine Moieties (6a-k)
Compounds 1-5 were synthesized by the previously described method [8][9][10][11][12]. A mixture of 5 (0.20 g, 1.0 mmol), monosubstituted piperazine (2.0 mmol) and anhydrous potassium carbonate in acetone was heated at reflux with stirring for 5 h. The solvent was evaporated under reduced pressure, and the resulting residue dissolved in dichloromethane (DCM). The DCM solution was washed sequentially with water and brine, dried over MgSO 4 , and distilled to dryness under reduced pressure. The resulting residue was purified by silica gel column chromatography with DCM and methanol (30:1). The yield, melting point, and spectral data of each compound were recorded.  164.60, 161.35, 145.67, 140.59, 139.92, 136.90, 135.52, 129.67, 127.90, 126.94,  124.60, 123.70, 121.62, 115.91, 115.63, 62.43, 53.24, 46.20; MS m/z: 363 (M + 1).   145.69, 139.94, 136.91, 135.56, 135.28, 134.47, 130.93, 129.55, 128.40, 128. A mixture of 5 (0.20 g, 1.0 mmol), monosubstituted piperazine (2.0 mmol) and anhydrous potassium carbonate in acetone was heated at reflux with stirring for 8 h. The solvent was evaporated under reduced pressure, and the resulting residue dissolved in DCM. The DCM solution was washed sequentially with water and brine, dried over MgSO 4 , and distilled to dryness under reduced pressure. The resulting residue was purified by silica gel column chromatography with DCM and methanol (20:1). The yield, melting point, and spectral data of each compound were recorded. was set up, transmural electrical field stimulation with a luminal electrode was started at 1.5 Hz (duration, 0.3-0.5 ms; voltage, 30 V). Changes in LA stroke volume were monitored by reading the lowest level of the water column in the calibrated atrial cannula during end diastole. Atria were perfused for 60 min to stabilize the stroke volume. The atrial beat rate was fixed at 1.5 Hz, LA stroke volume recorded at 2 min intervals, and the stimulus effect of the sample recorded after one circulation in the control group. Each circulation was 12 min. Compounds were investigated using the single-dose method at 3 × 10 −5 M. Samples were dissolved in DMSO and diluted with HEPES buffer to 0.1% DMSO. Biological data for these compounds were expressed in mean percentage values of increased stroke volume (Table 1). Heart-rate measurements for selected compounds were carried out in isolated rabbit hearts by recording the electrocardiogram in the volume conduction model. To assess differences, repeated measurements were compared by an ANOVA test followed by Bonferroni's multiple-comparison test. p < 0.05 was considered significant and data are the mean ± SE.

Conclusions
Two series of [1,2,4]triazolo[4,3-a] quinoxaline derivatives bearing substituted benzylpiperazine and benzoylpiperazine moieties were synthesized using A as the lead compound. We tried to ascertain potent compounds for cardiac contractility without increasing the heart rate. Compound 6c exhibited promising cardiovascular properties and potent activities compared with milrinone: 6c was 5.1-fold more active than milrinone. This compound is undergoing further biological tests, including in vivo evaluation, coronary vasodilation tests, and studies into possible mechanisms of action.