Design and Synthesis of New Pirrol-Indol Derivative with Positive Inotropic Activity

There are studies which indicate that some heterocyclic derivatives have inotropic effect; nevertheless, its inotropic activity is very confusing perhaps to difference in the chemical structure. In order, to clarify these phenomena in this study, some pirrol-indol derivatives (compounds 3, 4, 5 and 6) were synthesized with the objective of to evaluate its biological activity using a biological model. The results indicate that only the compound 4 induce a positive inotropic activity in comparison with the compound 3, 5 and 6. This datasuggest that the different groups involved in the chemical structure of 4 arethe responsible of its positive inotropic activity. In addition, this phenomenon its conditioned by some physicochemical parameters such as LogP, σ, Vm, Rm, Pc and St,which are cumulative effects of the different intra-and intermolecularforces involved in the structural chemistry of the compound 4.

On the other hand, a series of heterociclyc derivatives have been prepared with inotropic activity; for example, the compounds 3-alkyl-4-aryll,5-dihydro-W-pyrrol-2-ones were prepared as potential inhibitors of cardiac cAMP phosphodiesterase 4 .Other data showed the compound DPI 201-106 (piperazinyl-indol derivative) induce positive inotropic effects on left atria of rat using an isolated rat heart model 5 .In addition, other compound (1,3-dihydro-3,3dimethyl-5-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)-2H-indol-2-one) was syn-thesized and its inotropic effect on an in vivo animal model was evaluated 6 .Other results showed the synthesis of 4,5-dihydro-6-(lH-ind01-5-yl)pyridazin-3(2H)-ones which induces a positivity inotropic activity by changes of blood pressure and heart rate in an animal model 7 .Also a series of indol derivatives (5H-Pyridazino [  4,5-b]indole) were prepared and its positive inotropic activity was evaluated on the enzymatic activity of phosphodiesterase-IV which was isolated from dog heart 8 .Other data indicate the synthesis of the compound 3,9-dioxopyrrolo [1,2-a]indole derivatives and the evaluating of its cardiotonic activity using isolated spontaneously contracting right atria from guinea pigs 9 .All these data show that some heterocyclic derivatives induce inotropic effects in the cardiovascular system; nevertheless, the cellular site and molecular mechanism involved in its inotropic activity are very confusing, perhaps this phenomenon is due to differences in the chemical structure of both pirrol andindolderivatives or to the different pharmacological approaches used.Therefore, more data are needed to characterize the activity induced by theseheterocyclic derivatives at cardiovascular level.To provide this information, the present study was designed to investigate the effects of a pirrolindol derivative on perfusion pressure in isolated rat hearts using the Langendorff technique.In addition, to evaluate the chemical characteristic involved in the activity induced by the pirrol-indol derivative on perfusion pressure were used some physicochemical parameters such as LogP, ð, V m , R m , P c and S t .

EXPERIMENTAL
The compounds evaluated in this study were purchased from Sigma-Aldrich Co. Ltd.The melting points for the different compounds were determined on an Electrothermal (900 model).Infrared spectra (IR) were recorded using KBr pellets on a Perkin Elmer Lambda 40 spectrometer.1 H and 13 C NMR spectra were recorded on a Varian VXR-300/5 FT NMR spectrometer at 300 and 75.4 MHz in CDCl 3 using TMS as internal standard.EIMS spectra were obtained with a Finnigan Trace GCPolaris Q. spectrometer.Elementary analysis data were acquired from a Perkin Elmer Ser.II CHNS/0 2400 elemental analyzer.

Biological method
All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal care and use Committee of University Autonomous of Campeche (No. PI-420/12) and were in accordance with the guide for the care and use of laboratory animals 14 .Male Wistar rats; weighing 200-250 g were obtained from University Autonomous of Campeche.

Reagents
All drugs were dissolved in methanol and different dilutions were obtained using Krebs-Henseleit solution (dŠ 0.01%, v/v).

Experimental design
Briefly, the male rat (200 -250 g) was anesthetized by injecting them with pentobarbital at a dose rate of 50 mg/Kg body weight.Then the chest was opened, and a loose ligature passed through the ascending aorta.The heart was then rapidly removed and immersed in ice cold physiologic saline solution.The heart was trimmed of non-cardiac tissue and retrograde perfused via a non-circulating perfusion system at a constant flow rate.The perfusion medium was the Krebs-Henseleit solution (pH = 7.4, 37°C) composed of (mmol); 117.8 NaCl; 6 KCl; 1.75 CaCl 2 ; 1.2 NaH 2 PO 4 ; 1.2 MgSO 4 ; 24.2 NaHCO 3 ; 5 glucose and 5 sodium pyruvate.The solution was actively bubbled with a mixture of O 2 /CO 2 (95:5/5 %).The coronary flow was adjusted with a variable speed peristaltic pump.An initial perfusion rate of 15 ml/min for 5 min was followed by a 15 min equilibration period at a perfusion rate of 10 ml/min.All experimental measurements were done after this equilibration period.

Perfusion pressure
Evaluation of measurements of perfusion pressure changes induced by drugs administration in this study were assessed using a pressure transducer connected to the chamber where the hearts were mounted and the results entered into a computerized data capture system (Biopac).

Inotropic activity
Contractile function was assessed by measuring left ventricular developed pressure (LV/ dP), using a saline-filled latex balloon (0.01 mm, diameter) inserted into the left ventricle via the left atrium.The latex balloon was bound to cannula which was linked to pressure transducer that was connected with the MP100 data acquisition system.

Biological evaluation Effects induced by the compound 3, 4, 5 and 6 on perfusion pressure
Changes in perfusion pressure as a consequence of increases in time (3 to 18 min) in absence (control) and presence of the compound 3, 4, 5 and 6 at a concentration of 0.001 nM were determined.The effects were obtained in isolated hearts perfused at a constant-flow rate of 10 ml/ min.

Effect exerted by the compound 4 on left ventricular pressure through synthesis of prostanglandins
The boluses (50 ìl) of the compound 4 [0.001 to 100 nM] were administered and the corresponding effect on the left ventricular pressure was evaluated.The bolus injection administered was done in the point of cannulation.The dose response curve (control) was repeated in the presence of indomethacin at a concentration of 1 nM (duration of the pre-incubation with indomethacin was for a period of 10 min).

Effects of the compound 4 on left ventricular pressure through the calcium channel activation
Intracoronary boluses (50 ìl) of the compound 4[0.001 to 100 nM] were administered and the corresponding effect on the left ventricular pressure was evaluated.The dose-response curve (control) was repeated in the presence of nifedipine at a concentration of 1 nM (duration of the preincubation with nifedipine was for a period of 10 min).

Statistical analysis
The obtained values are expressed as average ± SE, using each heart (n = 9) as its own control.The data obtained were put under Analysis of Variance (ANOVA) with the Bonferroni correction factor 15 using the SPSS 12.0 program.The differences were considered significant when p was equal or smaller than 0.05.

Chemical Synthesis
There are several procedures for the synthesis of both pyrrol and indole derivatives [16][17][18][19][20] ; nevertheless, expensive reagents and special conditions are required; therefore, in this study is reported a straight forward route for the preparation of some pyrrole-indole derivatives (Figure 1 and 2).The first stage was achieved by the synthesis of pyrrolo[3,2-f]indole-1,7-diol (3) by the reaction of 3,5-dinitrobenzoic acid with 1-hexyne in basic medium.The 1 H NMR spectrum of 3 shows signals at 6.10 and 7.70 ppm for both pyrrole rings; at 7.16 and 7.34 for phenyl group; at 10.24 ppm for both hydroxyl groups.The 13 C NMR spectrum of 3 contains peaks at 103.82 and 133.14 ppm for both pyrrol rings; at 104.08-130.30ppm for phenyl group.Finally, the presence of compound 3was further confirmed from mass spectrum which showed a molecular ion at m/z 188.05.
The second stage involved the preparation of 5,5´(pyrrolo[3,2-f]indole-1,7diylbis(oxy))bis(3-nitrobenzoic acid (4) via displacement of nitro group from 3,5-dinitrobenzoic The third stagethe compound5 was synthesized by the reaction of 4 with 4aminoantipyrine to formation of an amide group.It is important to mention that many procedures for the formation of amide groups are known in the literature 22 , the most widely practiced method employs carboxylic acid chlorides as the electrophiles which react with the amino group in the presence of an acid scavenger 23 .Despite its wide scope, the former protocol suffers from several drawbacks; most notable are the limited stability of many acid chlorides and the need for hazardous reagents for their preparation (thionyl chloride) 24 .Other data indicate that boric acid catalyzed amidation of carboxylic acids and amines 25 ; therefore, in this study the compound 5 was synthetized by the reaction of 4 with 4aminoantipyrine using boric acid as catalyst.The 1 H NMR spectrum of 5 shows signals at 2.20-2.90ppm for methyl groups; at 6.22 and 7.94 ppm for protons of both pyrrol groups; at 7.16-7.30and 7.56 ppm forboth phenyl groups bound to both pyrazole ring;   The fourth stage was achieved by the reaction of 5 with ethylenediamine using boric acid   ppm for both phenyl groups bound to both triazole rings.Finally, the presence of compound 6was further confirmed from mass spectrum which showed a molecular ion at m/z 936.35.

Biological evaluation
There are several reports which indicate that somepyrrol and indol derivatives exert effects on cardiovascular system [4][5][6][7][8][9] ; however there are not insufficient data on the activity exerted by these compounds.Therefore, in this study the effect exerted by new pyrrol-indol derivatives (compounds 3and4) on perfusion pressure were evaluated in an isolated rat heart model.The results obtained (Figure 3)show that the compound 4 significantly increases the perfusion pressure over time compared with the compounds 3and the control conditions.These data indicatedthatpossibly thenitrobenzoic acid fragments involved in the chemical structure of 4 could be responsible of the activity exerted by 4 on perfusion pressure.
In order to asses this hypothesis, also the effect induced by compounds 5and6on perfusion pressure was evaluated.The results showed that these compounds have not activity on perfusion pressure; this results indicates that the only the nitrobenzene fragment is the responsible of activity of the compound 4.
In the search of molecular mechanism involved in the activity exerted by the OTBDSestradiol-hexanoic acid derivative on left ventricular pressure, we considered validating the activity induced by the compound 4 on stimulation and secretion of prostaglandins such happening with other type of substances [25].In this sense, in this experimental study, the activity exerted by the compound 4on left ventricular pressure in the absence or presence of indomethacin was evaluated.The results showed that effect induced by the thecompound 4on left ventricular pressure was not blocked by indomethacin.These data indicate that activity exerted by this steroid derivative on left ventricular pressure was not viaprostanoids synthesis and secretion.
Therefore, analyzing the possibility of that compound 4could induce its activity on left ventricular pressure through of activation of other molecular system that involved increase in the intracellular calcium and consequently bring a positive inotropic effect such as happening with other type of substances 26 .In this study, the activity induced by the compound 4on left ventricular pressure was evaluated in the absence or presence of nifedipine.The results showed that effect exerted by the compound 4was inhibited in the presence of nifedipine which indicate that activity exerted by this compound involves activation calcium channel, this phenomenon could be conditioned by interaction between the pirrol-indol derivative and some endogenous substances involved in the heart or by the degree of lipophilicity exerted by the heterocyclic derivative; this effect may depend of some physicochemical parameters involved in its structure chemical such as happening with other type of compounds 27 .

Physicochemical parameters
In order to delineate the structural chemical requirements involved in the degree of lipophilicity of the compounds 3, 4, 5 and 6, some parameters such as the descriptors 28 log P and ð were calculated.Is important to mention that, the descriptor logP estimates the logarithmic octanolwater partition coefficient; therefore, log P represents the lipophilic effects of a molecule which includes the sum of the lipophilic contributions of the parent molecule and its substituent 29 .The difference between the substituted and unsubstituted log P values is conditionated by the ð value for a particular substituent.Several years ago, Hammett showed that ð values measure the free energy change caused by particular substituent to relate to biological activity 30 .Therefore, in this study, the logP and ð parameters were calculated by previously methods reported 31 .The results (Table 2) showed an increase in logP and ð values in the compound 4 with respect to the compounds 3 and 5, this phenomenon is conditioned mainly, by the contribution of all substituent atoms involved in the chemical structure of compounds.However the logP was low in relationship to the compound 6; these data indicate that aliphatic carbons involved in thethe compound 6 contributes to increase the degree of lipophilicity in comparison with the compound 4. All these resultssuggest that different functional groups involved in thechemical structure of 6 induce changes inthe higher degree of lipophilicityin comparison with 4; however, there are other studies which indicate that other type of physicochemical parameters such as the molar volume (V m ) and molar refractivity (R m ) that are steric constantmayinducechanges in some biological activities.
In order to asses this hypothesis,in this work V m and R m werecalculated using ACD/Chem Sketch algorithms 32 .The resultsshowed an increase in bothR m and V m values for 6in comparisonwith 3, 4and 5.These data indicate that steric impediment,conformational preferences and internal rotation of 6 couldinfluence its biological activity in comparison with 4.
On the other hand, it isimportant to mention that there are reports which suggest thatV m is directly related to parachor (P c ) and surface tension (S t ),which are cumulative effects of the different intraand intermolecularforces involved in the structural chemistry of somecompounds 33 .Therefore, in this study P c and S t were alsoevaluated.The results indicate that both values of P c and S t for6were high in comparison with 3, 4and 5(Table 2).These data indicate that these physicochemical parameters can also modifythe biological activityof 6with respect to 4.
In conclusion, the experimental and theoretical data suggest that:(1) The compound 4 induces a positive inotropic effect via activation of calcium channels and this effect depends of its chemical structure; 2) The compound 6 exert higher lipophilicityand steric impediment in comparison with the compound 4 which consequently brings low activity on perfusion pressure of 6 in relationship to 4.

Confiicts of interest
The authors declare that they have no conûict of interest.