Journal of Atherosclerosis and Thrombosis Vol.17, No. 7
730
Original Article
Antiplatelet activity and structure-activity relationship study of Pyrazolopyridine Derivatives as potential series for treating thrombotic diseases RB Geraldo 1, ML Bello 2, LRS Dias 2, MAF Vera 2, T Nagashima 1, PA Abreu 1, MB Santos 2, MG Albuquerque 3, LM Cabral 3, ACC Freitas 2, MV Kalil 1, CR Rodrigues 3, and HC Castro 1 1
Laboratório de Antibióticos, Bioquímica e Modelagem Molecular (LABioMol)-Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brazil 2 Laboratório de Química Medicinal (LQMed)-Faculdade de Farmácia, Universidade Federal Fluminense, Niterói, RJ, Brazil 3 Laboratório de Modelagem Molecular e QSAR (ModMolQSAR )-Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Brazil
Aim: Platelets plays a central role in hemostatic processes and consequently are similarly involved in pathological processes, such as arterial thrombosis and atherosclerosis. Herein we described the synthesis, antiplatelet profile and structure-activity relationship (SAR) of a new series of N ’-substitutedphenylmethylene-1H-pyrazolo[3,4-b]pyridine-carbohydrazide derivatives (3a-3k). Methods: These compounds were synthesized in good yield and tested in platelet aggregation assays using collagen, ADP and arachidonic acid as agonists. We also performed a SAR studies using SPARTAN’ 08 program, in silico ADMET screening and the Lipinski “rule of five” using Osiris Property Explorer and molinspiration on-line programs. Results: Interestingly, the new compounds were active against collagen and arachidonic acid (AA) with the two most actives compounds (3a and 3c - IC 50 = 61 μM and 68 μM respectively) almost 5-fold more potent than aspirin (IC 50 = 300 μM). These derivatives showed low theoretical toxicity risks in in silico ADMET screening and fulfilled the Lipinski rule of five, suggesting good oral biodisponibility. Conclusion: This work showed carbohydrazide group as potential for designing new antiplatelets. On that purpose, 3a and 3c may act as prototypes to generate more efficient and safe molecules for treating thrombotic diseases. J Atheroscler Thromb, 2010; 17:730-739. Key words; Antiplatelets, Stroke, Structure-activity relationship (SAR)
Introduction Platelets have a central role in physiological processes (i.e. hemostasis) and in thrombotic diseases related to arterial circulation, leading to heart attack and strokes 1-2). In these diseases, platelet activity is altered Address for correspondence: Dr. Helena Carla Castro, LABioMol, 3o andar, Departamento de Biologia Celular e Molecular, IB-CEG, Universidade Federal Fluminense, CEP 24001-970, Niterói, RJ, Brazil E-mail:
[email protected]. Received: July 12, 2009 Accepted for publication: December 17, 2009
by atherosclerosis formation, which has a multi-factorial pathogenesis involving both the deposition of lipids within the artery wall and the interaction of cellular response characteristics of inflammatory disease. Atherosclerosis promotes increased adhesiveness in the endothelial wall for platelets, and a loss of anticoagulant properties, which contribute to atherosclerotic plaque formation. The rupture of plaques is the most important mechanism for the sudden progression of vascular disease, because this rupture exposes thrombogenic subendothelial matrix protein and collagen, triggering a cascade of platelet activity 3-4). Stroke is responsible for more than 5 million
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Fig. 1. Molecular structure of the known antiplatelet agents (aspirin, clopidogrel, triflusal, tirofiban and eptifibatide) with the CAS Registry Number (CAS#).
deaths and, without intervention, the number of global related deaths will rise to 6.5 million in 2015 and to 7.8 million in 2030, according to the World Health Organization (WHO) 5). The current literature describes many drugs as antiplatelet agents for treating these pathologies (i.e. acetylsalicylic acid, clopidogrel, eptifibatide, triflusal and tirofiban) (Fig. 1); however, some have several collateral effects and resistance in long term therapy, such as the known clinical aspirin resistance 6). In fact, numerous studies have documented inter-individual variability in platelet responsiveness to aspirin and clopidogrel, some of the most used oral antiplatelet drugs 7). The feasibility of using antiplatelet agents to substitute oral anticoagulant treatment has also been discussed in the literature for secondary prevention of further vascular events after limited ischaemic stroke of arterial origin due to their lower risk 8). Thus, international efforts are now being made to develop new antiplatelets agents with low collateral effects 9-12). We have recently reported an efficient method for the synthesis of N ’-substituted-phenylmethylene1H-pyrazolo[3,4-b]pyridine-carbohydrazides 3a, 3c, 3f and 3h compounds by a sequence of 3 reactions from 5-amino-3-methyl-1-phenyl-1H-pyrazole 13). In order to identify the leading compound for developing new antiplatelet agents, and treat thrombotic diseases, we report here seven new pyrazolopiridine derivatives 3b, 3d, 3e, 3g, 3i, 3j and 3k. We also performed a structure-activity relationship (SAR) study
of these compounds by evaluating their antiplatelet profile and comparing with their theoretical steric and electronic parameters, such as HOMO and LUMO (energy, coefficient orbital and density) and clogP, calculated using a molecular modeling approach. Finally, the most active N ’-benzylidene-carbohydrazides derivatives were submitted to in silico ADMET screening to analyze their overall potential as drugs and compared to the current therapeutical agents used against stroke (i.e. acetylsalicylic acid, clopidogrel, eptifibatide, triflusal, cilostazol and tirofiban). Methods Chemistry N ’-substituted-phenylmethylene-1H-pyrazolo [3,4-b]pyridine-4-carbohydrazides 3a, 3c, 3f and 3h were synthesized by the fast sequence of 3 reactions from 5-amino-3-methyl-1-phenyl-1H-pyrazole 14). Melting points were determined in a capillary Thomas Hoover apparatus and are given uncorrected. Infrared (IR) spectra were determined using a Perkin-Elmer 1,420 spectrometer in potassium bromide pellets. 1H and 13C-NMR spectra were recorded using a Varian UNITYplus-300 at 300 MHz spectrophotometer in DMSO-d6. The progress of all reactions was monitored by t.l.c. on Kilsegel 60F 0.2 mm (Merck-Darmstadt) and the chromatograms were monitored at UV range (254−365 nm).
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Biological Activity Assays All reagents were obtained from Sigma Chemical Company (St Louis, MO, USA) and platelet aggregation was monitored by the turbidimetric method using a Chrono-Log aggregometer (Signa), as described by Lima and co-workers 15). The use of rabbit Plateletrich plasma (PRP) enables the investigaton of different pathways as they are more clearly separated in rabbits than humans. In humans, pathways such as those induced by collagen, arachidonic acid and ADP are mixed and cross-react, complicating the analysis. Therefore, we withdrew blood from the rabbit ear central artery and mixed with 3.8% trisodium citrate (9:1 v/v). PRP was prepared by centrifugation at 500 g for 10 min at room temperature. Platelet-poor plasma (PPP) was prepared by centrifugation of the pellet at 2000 g for 10 min at room temperature. PRP (300 mL) was incubated at 37 °C for 1 min with continuous stirring at 900 rpm. Then, platelet aggregation was induced by ADP (5 μM), collagen suspension (5 μg/mL) or arachidonic acid (AA) (100 μM). To test the compounds or vehicle DMSO (0.5% v/v), we pre-incubated each with the PRP samples 1 min before addition of the aggregating agent. DMSO showed neither pro- nor antiplatelet aggregation activity. The results are the mean of at least three experiments in duplicate in at least 4 different animals. They were expressed as a percentage of the inhibition of platelet aggregation induced by the agonists (ADP, AA or collagen). The results were analyzed using one-way ANOVA ( p ＜ 0.05) in the Microcal Origin program. Molecular Modeling Evaluation SAR Studies: All analyses were performed using SPARTAN’ 06 (Wavefunction Inc., Irvine, CA, USA). Molecular modeling studies were initiated with conformational analysis of the unsubstituted compound, as described elsewhere 16-17), which revealed the (E )-diastereomer as more stable (ΔHf = 8.93 kcal/mol) than the (Z)- diastereomer. Thus, the E-isomer of 3c was considered as a single diastereoisomer, and it was used as a template to construct the 3D structures of all derivatives. The compounds were submitted to geometry optimization using the RM1 semi-empirical method. The RM1 method was chosen since it is a reparameterization of AM1 and gives superior results 18). The N ’ -benzylidene-carbohydrazides derivatives were submitted to a single-point ab initio calculation with a 6-31G＊＊ basis set available in the SPARTAN ’06 program (Wavefunction Inc.). We used the lowest-energy conformation of each compound. To evaluate their electronic and structural
properties, such as HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energy values, dipole moment, molecular volume, molecular area, molecular weight and molecular electrostatic potential (MEP), MEP isoenergy surface maps were generated in the range from −100.0 (deepest red) to ＋200.0 (deepest blue) kJ/mol and superimposed onto the molecular surface of constant electron density at 0.002 e/au3. Each point on the three-dimensional molecular surface map expressed the electrostatic interaction energy value evaluated with a probe atom of positive unitary charge providing an indication of the overall molecular size and location of attractive (negative) or repulsive (positive) electrostatic potentials 19). in silico ADMET Screening The theoretical study of drug absorption, distribution, metabolism, excretion and toxicity (ADMET) properties and the Lipinski rule of five were performed using Osiris Property Explorer (http://www.organicchemistry.org/) and the molinspiration on-line program (http://www.molinspiration.com/). The Lipinski rule of five 20) predicts theoretical oral bioavailability by analyzing the molecular properties important for drug pharmacokinetics in the human body, including their absorption, distribution, metabolism, and excretion (ADME). According to Lipinski, the active compound must show at least three of the four rules: ≥ 5 H-bond donors (HBD), ≥ 10 H-bond acceptors (HBA), molecular mass (MM) ≤ 500, and the calculated LogP (cLogP) ≤ 5 20). The drug similarity and drug score were calculated using the Osiris property explorer program. Drug similarity is based on the occurrence frequency of each fragment, which is determined within the collection of traded drugs and within the supposedly non-druglike collection of Fluka compounds, whereas the drug score is related to topological descriptors, fingerprints of molecular drug similarity, structural keys and other properties, such as clog P and molecular weight 21). Results and Discussion Chemistry We synthesized eleven derivatives by reacting 5aminopyrazoles, with aromatic aldehydes and ethyl pyruvate to obtain ethyl 3-methyl-1,6-diphenyl-1Hpyrazolo[3,4-b]pyridine-4-carboxylate derivatives (1) 14). The compound was converted to the N ’-benzylidene-carbohydrazides derivatives (3a-k) using functional group conversion and condensation with the appropriate aromatic aldehydes in good yield (70−
Carbohydrazides: new antiplatelet agents
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Fig. 2. Synthesis of N ’-benzylidene-3-methyl-1-phenyl-6-substituted-1H-pyrazolo[3,4-b] pyridine-4-carbohydrazides. (a) NH2NH2.H2O, reflux, 2h (2 yield = 70%), (b) aromatic aldehyde, HCl, EtOH, reflux (3 yield = 87−97%).
97%) (Fig. 2). Biological activity We tested the N-acylhydrazones 3a, 3c, 3f and 3h and new derivatives 3b, 3d, 3e, 3g, 3i, 3j and 3k for the antiplatelet profile against in vitro platelet aggregation induced by collagen (5 μg/mL), as described in Material and Methods. Collagen is one of the most important physiological agonists also involved in pathological conditions, such as stroke 22-23). Interestingly, most of the compounds showed some inhibitory activity, indicating the main structure as a feasible pharmacophoric moiety (Table 1). Apparently, the introduction of any substituent in the N-phenyl ring (A) was significantly deleterious to the antiplatelet activity observed in the collagen-induced assays (i.e. 3i, 3j and 3k) (Table 1). Importantly, compounds 3a (R1 = H and R2 = OH), 3b (R1 = H and R2 = CN), 3c (R1 = R2 = H), 3f (R1 = H and R2 = F) and 3h (R1 = H and R2 = NO2) showed significant antiplatelet profiles (≥ 50%) (Table 1). Thus, we determined the IC50 for the two most promising compounds (3a-IC50 = 61 μM and 3c-IC50 = 68 μM), which were almost five times better than aspirin IC50 (IC50 = 300 μM) (Fig. 3). The literature describes that collagen may active the platelet phospholipase A2 to release arachidonic acid from the platelet membrane 24). Arachidonic acid, a 20-carbon essential fatty acid stored in membrane phospholipids, generates thromboxane A2 (TxA2) as a metabolite, a vasoconstrictor and a potent platelet ag-
onist. TxA2 is synthesized in two subsequent steps, including cycloxigenase (COX), the aspirin target, and the thromboxane A2-synthase (TxA2-synthase), a platelet-exclusive enzyme that is the target of several studies in the literature 24-25). Since collagen induces the release of arachidonic acid during platelet aggregation, we tested the ability of these derivatives to affect the arachidonic acid pathway. Interestingly, compounds 3a, and 3c (100 μM) completely inhibited AA-induced platelet aggregation (100%), which reinforced the arachidonic acid pathway enzymes as feasible targets for these compounds (Fig. 3). Finally, to exclude common steps, such as the receptor GpⅡbⅢa, as a target of these compounds, we used ADP (5 μM), as agonist, since ADP-induced platelet aggregation occurs through an independent pathway different from collagen and AA pathways in rabbits 23). Importantly, no derivative significantly inhibited these assays (≤ 50%), which once more reinforced the AA pathway as a feasible target of these molecules. This result also excluded common steps, such as inhibition through receptor GpⅡbⅢa, which is a receptor involved in the platelet aggregation process induced by all of these agonists. Theoretical analysis Structure Activity Relationship (SAR) analysis of N’-substituted-phenylmethylene-1H-pyrazolo[3,4-b] pyridine-4- carbohydrazide derivatives (3a-k) In structure-activity relationship (SAR) studies, we calculated several descriptors, including HOMO
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Table 1. Comparison of the effects of N ’-substituted-phenylmethylene-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide compounds on in vitro platelet aggregation of citrated rabbit platelet-rich plasma induced by collagen and ADP ( p ＜ 0.05) and their stereoelectronic properties of HOMO energy (eV), LUMO energy (eV), polar surface area (PSA-Å2) and calculated Lipinski parameters of lipophilicity (cLogP), volume (Å3), area (Å2), molecular mass (MM-g/mol), number of hydrogen bond acceptors (HBA) and donor (HBD). All compounds with inhibitory effect higher than 50% are marked (“＊”).
Collagen (5μg/mL)
ADP (5μM)
Derivatives
R1
R2
Aggregation (%)
Inhibition (%)
Aggregation (%)
Inhibition (%)
EHOMO
3a 3b
H H
OH CN
28.6±8.1 49.7±7.9
71.4＊ 50.3＊
86.7±1.9 89.2±2.1
13.3 10.8
−7.82 −7.95
1.83 1.44
3c
H
H
13.6±10.2
86.4＊
89.1±3
10.9
−7.82
3d
H
OCH3
56.7±6.4
43.3
76.6±1.7
24.4
−7.81
3e
H
Cl
78.7±10.1
24.3
86.8±2.3
13.2
3f
H
F
37.1±5
62.9＊
91.9±2
3g 3h
H H
24.1 52.4＊
3i 3j
Cl CN
N(CH3)2 Br
90.4±2.1 69.3±6.8
3k
SCH3
F
53.3±6.2
OCH2CH3 75.9±2.3 NO2 47.6±7.2
nHba
nHbd
cLogP
LogS
μ
MM
455.75 473.9 468.09 485.87
7 7
2 1
4.61 4.70
−6.86 −7.93
5.01 4.43
447.50 456.50
1.84
448.69 465.18
6
1
4.91
−7.15
5.80
431.50
1.84
475.73 494.77
7
1
4.8
−7.17
5.42
461.52
−7.85
1.78
453.37 470.90
6
1
5.52
−7.89
4.93
449.49
8.1
−7.87
1.75
453.75 472.76
6
1
4.97
−7.47
4.64
449.48
88.5±1.3 76.6±2
11.5 23.4
−7.81
1.85 1.07
494.39 515.70 470.49 491.49
7 9
1 1
5.24 4.78
−7.47
5.62 4.51
475.55 476.49
9.6 30.7
81.3±2.6 85.2±3.1
18.7 14.2
−7.70
1.66 1.25
551.77 531.73 586.40 506.39
7 6
1 1
5.52 5.42
−7.92
−8.10
−8.76
6.45 6.04
509.01 535.40
46.7
76.7±2.5
24.3
−7.85
1.75
490.05 510.17
6
1
5.46
−8.32
4.46
495.58
energy (EHOMO), LUMO energy (ELUMO), polar surface area (PSA), molecular volume, molecular area, molecular weight (MM), number of hydrogen bond acceptors (nHBA), number of hydrogen bond donors (nHBD), dipole moment, lipophilicity (calculated octanol/water partition coefficient-cLogP) and solubility (calculated water solubility-cLogS). In these studies, we calculated several descriptors, including HOMO energy (EHOMO), LUMO energy (ELUMO), polar surface area (PSA), molecular volume, molecular area, molecular weight (MM), number of hydrogen bond acceptors (nHBA), number of hydrogen bond donors (nHBD), dipole moment, lipophilicity (calculated octanol/water partition coefficient-cLogP) and solubility (calculated water solubility-cLogS). These values were compared with the derivatives, inhibitory activity profiles observed against collagen-induced platelet aggregation. The overall analysis of electronic properties, such as HOMO/LUMO energy and PSA values of the 3a−k series, revealed that they varied
−7.97
ELUMO Volume
Area
−7.61
(HOMO −8.10 to −7.70 eV; LUMO 1.07 to 1.85eV and PSA 50.62 to 89.61 Å2) but with low or no correlation with the inhibitory activity of these derivatives in collagen-induced platelet aggregation (Table 2). Differently, all N ’-substituted-phenylmethylene1H-pyrazolo[3,4-b]-pyridine-4-carbohydrazide derivatives, structural descriptors evaluated apparently indicated steric features as being related to the experimental antiplatelet profile (Table 1). In agreement, the cross-correlation matrix, including all calculated descriptors (electronic and structural), and the biological results revealed volume (R = −0.747), area (R = − 0.746), molecular weight (R = −0.647) and lipophilicity (R = −0.718) as the most biologically correlated descriptors (Table 2). These structural features show an inverse correlation coefficient to the inhibitory activity of these compounds, as the higher the values, the worse their biological activity (Table 2). Since stereoelectronic complementarity is essential for interaction with the target, we analyzed the
Carbohydrazides: new antiplatelet agents
A
B
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C
Fig. 3. Inhibitory effect of the most active N ’-substituted-phenylmethylene-carbohydrazide compounds (3a and 3c). A- Comparison of the derivative concentration that inhibits 50% collagen-induced platelet aggregation (IC50) with a market drug (aspirin). B- Representative traces of arachidonic acid-induced rabbit platelet aggregation (AA-control) and the inhibitory effect of the most active compounds (100μM). C- Pathways affected by these derivatives with feasible targets in the AA pathway (cycloxigenase I and thromboxane A2 synthase) boxed with a dashed line (B). Table 2. Linear cross-correlation matrix of collagen-induced platelet aggregation inhibitory activity of N ’-substituted-phenylmethylene-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide compounds and the calculated descriptors HOMO/LUMO Energy (EHOMO, ELUMO-eV), Molecular dipole moment (μ-Debye), volume (Å3), area (Å2), polar surface area (PSA), molecular mass (MM-g/mol), lipophilicity (cLogP) and solubility (LogS). Parameter Inhibition (%) E (HOMO) E (LOMO) PSA μ
Volume cLogP MM LogS Area
Inhibition (%) E (HOMO)
E (LOMO)
PSA
M
Volume
cLogP
1 0.472 0.402 0.413 −0.076 0.453
1 0.546 0.810 −0.489 0.999
1 0.602 −0.688 0.538
MM
LogS
Area
1 −0.091
0.119 0.149 −0.383 −0.747 −0.718 −0.647 0.548 −0.746
1 0.759 −0.536 0.255 0.167 0.091 −0.339 0.536 0.149
1 −0.762
1
0.153 −0.145 0.069 −0.489 0.515 −0.164
−0.291 −0.062 −0.518
0.123 0.053 −0.036
structural properties and electrostatic potential map in attempt to identify more features that could be related to inhibitory activity (Fig. 4). Interestingly, the most active compounds (3a, 3c and 3f ) do not show substituents at R1 position in the A aromatic ring (Table 1 and Fig. 4). In addition, the most active compound (3c) showed no substituent group in the C aromatic ring, (3c, R2 = H) in R2 position. Meanwhile, 3a and 3f showed groups with high electronic density (-OH and -F, respectively) that led to lower activity profiles than 3c (Fig. 4).
1 −0.787
0.815
1 −0.846
1
Importantly, the less active compound was substituted by larger substituents (i.e. 3g, 3i), suggesting that probably due to steric restriction, small substituent groups lead to a better inhibitory profile in collagen-induced platelet aggregation assays (Fig. 4). Analysis of the electrostatic potential map revealed a similar overall profile with negative potential in the B aromatic ring for all derivatives (Fig. 4); however, it is possible to observe a more electronegative charge in the most active compound in the C aromatic ring (3c). This electronegative profile reduces gradually in other derivatives analogous to their inhibitory profile. Al-
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A
B
Fig. 4. Comparison of most stable conformation (A) and molecular electrostatic potential maps (B) of all N ’-substituted-phenylmethylene-1H-pyrazolo[3,4-b]pyridine4-carbohydrazide compounds (3a−k). In A, blue square marks the most active compounds whereas the green circle shows substituents with the largest volume.
though compounds 3d, 3g and 3i showed the electronegative density in the phenyl ring, they presented with a low inhibitory profile, probably due to steric restriction of the bulky substituent in the C ring that is deleterious to good interaction with the platelet target (Fig. 4B).
In silico ADME and theoretical toxicity screening We submitted N ’-substituted-phenylmethylene1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide derivatives (3a−k) to in silico ADME theoretical toxicity screening, drug score and drug similarity analysis, also comparing them with drugs currently used in therapy against stroke (i.e. acetylsalicylic acid, clopidogrel, epitifibatide, triflusal, cilostazol and tirofiban) to analyze their overall potential as drugs. Initially, we evaluated the theoretical oral bioavailability of these derivatives using the Lipinski rules, which states that most ‘drug-like’ molecules have clogP ≤ 5, molecular weight (MW) ≤ 500 hydrogen bond acceptors ≤ 10 and donors ≤ 5. Molecules violating more than one of these rules may have problems with bioavailability 18). The results showed that all carbohydrazide derivatives (3a−k) fulfilled the Lipinski rule of five (Table 1). According to theoretical analysis, the lipophilicity (clog P) of the most active derivatives (4.61−4.97) showed a sufficiently hydrophobic profile for penetrating biological membranes in Lipinski rules. We also calculated drug similarity, which evaluates the frequency of each fragment of the analyzed molecules and compares to commercial drugs and non-druglike compounds. The drug score was also predicted as it combines drug similarity, clog P, log S, molecular weight and toxicity risks in one useful value that may be used to judge the drug potential of a compound. Interestingly, the most active N-acylhydrazone derivatives (i.e. 3a and 3c) showed a better profile of drug similarity and drug score than the drugs currently in use on the market (Fig. 5). Our theoretical study showed active N-acylhydrazone derivatives (3a, 3c and 3f ) with a low profile for irritant, tumorigenic, mutagenic and reproductive effects. It is important to notice that the toxicity predicted herein does not guarantee that these compounds are completely free of any toxic effect but reinforces their promising antiplatelet profile 13) (Fig. 4). Conclusion In summary, in this work we showed that this series of N ’-substituted-phenylmethylene-1H-pyrazolo [3,4-b]pyridine-4-carbohydrazide derivatives (3a−k) has a promising antiplatelet profile. Three derivatives (3a, 3c and 3f ) were active against collagen-and arachidonic acid-induced rabbit platelet aggregation, which involve TXA2 formation, with no significant inhibitory activity against ADP. Our theoretical results suggested that the inhibitory activities of these compounds are modulated by
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A
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new anti-platelet drugs against thromboembolic diseases. Chemistry
B
Fig. 5. Comparison of the theoretical toxicity risks (A), drug similarity and drug score profiles (B) of the N ’-substituted-phenylmethylene-1H-pyrazolo[3,4-b]pyridine4-carbohydrazide compounds (3a−k) and some antiplatelet drugs (aspirin, cilostazol, clopidrogrel, tirofiban, triflusal) calculated by Osiris Property Explorer.
steric restriction in both R1 and R2 positions. Analysis of SAR also showed that the structural parameters (volume, area, molecular weight and lipophilicity) were more important for the antiplatelet profile than the electronics parameters. In addition, ADMET evaluation showed 3a, 3c and 3f as having lower theoretical toxicity risk molecules similar to clopidogrel, eptifibatide, triflusal, cilostazol and tirofiban, and better than aspirin, and with better overall potential drug similarity and drug-score values than all of these anti-platelets agents. Our compounds also fulfilled the Lipinski rule of five, reinforcing its promising profile for further in vivo experimental investigations and as a leading compound to design
Ethyl 3-methyl-1-phenyl-6-(4”-thiomethylphenyl)1H-pyrazolo[3,4-b]pyridine-4-carboxylate (1d). 43%, mp = 117 ℃. IR (υ-cm−1): 2,980−2,900 (C-H); 1,760 (C = O); 1,240(C-O). 1HNMR: δ1.50 (t, 3H, CH3 CH2O); 2.64 (s, 3H, SCH3); 2.74 (s, 3H, CH3); 4.6 (q, 2H, CH3CH2O); 7.4 (d, 1H, H-4’ J = 6.0 Hz); 7.5 (d, 2H, H-2’, J = 8.4Hz); 7.7 (dd, 2H, H-3’, J = 8.4 Hz, J = 6.0 Hz); 8.2 (s, 1H, H-5); 8.3 (d, 2H, H-2”, J = 9.0 Hz); 8.4 (d, 2H, H-3”, J = 9.0 Hz). 13CNMR: δ13.8 (CH3); 15.4 (CH3CH2O); 61.9 (CH3CH2O); 111.3 (C-3a); 120.6 (C-5); 125.7 (C-4’); 128.7 (C-3’); 128.8 (C-3’’); 134.9 (C-1’’); 136.0 (C-4’’); 138.6 (C-4); 141.9 (C-1’’); 151.1 (C-7a); 154.5 (C-6); 164.6 (C = O). Ethyl 3-methyl-1-phenyl-6-(4”-thiomethylphenyl)1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2d). 70%, mp = 209 ℃. IR (υ-cm−1): 3,300 (N-H); 1,660 (C = O). 1HNMR: δ2.58 (s, 3H, SCH3); 2.54 (s, 3H, CH3); 3.41 (sl, 2H, NH2); 7.33 (d, 1H, H-4’, J = 7.2 Hz); 7.41(d, 2H, H-3”, J = 8.4 Hz); 7.57 (dd, 2H, H-3’, J = 7.8 Hz, J = 7.2 Hz); 7.86 (s, 1H, H-5); 8.19 (d, 2H, H-2”, J = 8.6Hz); 8.30 (d, 2H, H-2’, J = 7.8Hz); 10.06 (sl, 1H, NH). 13CNMR: δ14.3 (CH3); 14.4(CH3); 111.6 (C-3a); 112.5 (C-3’’); 120.4 (C-5, C-2’); 125.8 (C-4’); 127.7 (C-2’’); 129.1 (C-3’); 134.2 (C-3,C-1’’); 139.0 (C-4); 141.3 (C-1’); 151.5 (C-7a); 155.5 (C-4’’,C-6); 164.6 (C = O). N ’-(4’”-cyanophenylmethylene)-1,6-diphenyl3-methyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (3b). 92%, mp = 276-278 ℃. IR (υ-cm−1): 3,200 (N-H); 2,220 (CN); 1,650 (C = O). 1HNMR: δ2.73 (s, 3H, CH3); 7.49 (d, 1H, H-4’, J = 7.5 Hz); 7.58 (d, 2H, H-2”’, J = 8.4 Hz); 7.67−7.76 (m, 3H, H-3’, H-4”); 7.84 (d, 2H, H-3’”, J = 8.4Hz); 8.05−8.12 (m, 2H, H-3”); 8.28 (s, 1H, H-5); 8.42−8.49 (m, 4H, H-2’, H-2”); 8.58 (s, 1H, CH = N); 12.66 (sl, 1H, N-H). 13 CNMR: δ14.4 (CH3); 122.7 (C-4’’’); 113.3 (C-3a); 118.4 (CN); 120.3 (C-5, C-2’); 125.7 (C-4’); 127.4 (C-4’’); 127.8 (C-2’’); 129.0 (C-3’’); 129.2 (C-3’); 130.0 (C-3); 132.7 (C-3’’’); 138.0 (C-4); 138.3 (C-1’’’); 139.1 (C-1’’); 142.1 (C-1’); 147.1 (NHN = CH); 151.0 (C-7a); 155.8 (C-6); 161.9 (C = O). N ’-(4’”-methoxyphenylmethylene)-1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (3d). 97%, mp = 259 ℃. IR (υ-cm−1): 3,220
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(N-H); 1,640 (C = O). 1HNMR: δ2.60 (s, 3H, CH3); 4.0 (s, 3H, OCH3); 6.94 (d, 2H, H-3’”, J = 9.0 Hz); 7.18 (d, 2H, H-3”, J = 6.0 Hz); 7.36 (d, 2H, H-2”’, J = 9.0 Hz); 7.48 (d, 1H, H-4”, J = 6.0 Hz); 7.67−7.77 (m, 3H, H-3’,H-4’); 7.87 (d, 2H, H-2”, J = 6.0 Hz); 8.24 (s, 1H, H-5); 8.44 (d, 2H, H-2’, J = 9.0 Hz); 8.48 (s, 1H, CH = N); 12.20 (sl, 1H, NH). 13CNMR: δ 13.9 (CH3); 54.9 (OCH3); 122.8 (C-3a); 114.0 (C-3’’’); 120.2 (C-5,C-2’); 126.1 (C-4’); 126.8 (C-2’’,C-2’’’); 128.5 (C-4’’); 128.7 (C-3); 129.5 (C-3’); 129.6 (C-3’’); 137.5 (C-4); 138.4 (C-1’’’); 138.5 (C-1’’); 141.8 (C-1’); 148.6 (NHN = CH); 155.8 (C-6); 160.8 (C-4’’’); 161.1 (C = O). N ’-(4’”-chlorophenylmethylene)-1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (3e). 96%, mp ＝ ＞ 310 ℃. IR (υ-cm−1): 3,200 (N-H); 1,650 (C ＝ O). 1HNMR: δ2.79 (s, 3H, CH3); 7.28 (d, 1H, H-4”, J = 8.7 Hz ); 7.35 (d, 2H, H-3”’, J = 7.5 Hz ); 7.55−7.67 (m, 1H, H-4’); 7.75 (d, 2H, H-3’, J = 7.8 Hz ); 7.79 (d, 2H, H-3”, J = 8.4 Hz ); 8.02 (d, 2H, H-2”, J = 8.4 Hz); 8.12 (s, 1H, H-5); 8.32 (d, 2H, H-2’, J = 7.8 Hz); 8.51 (s, 1H, CH ＝ N); 8.85 (d, 2H, H-2’”, J = 8.7 Hz ); 12.25(sl, 1H, NH). 13CNMR: δ14.0 (CH3); 113.3 (C-3a); 120.3 (C5, C-2’); 125.7 (C-4’); 127.3 (C-2, C-4’’); 128.2 (C-2’’’); 129.0 (C-3’); 129.1 (C-3’’); 130.0 (C-3); 132.6 (C-1’’’); 138.6 (C-4); 142.2 (C-1’’); 144.2 (C-1’); 147.8 (NHN = CH); 151.1 (C-7a); 155.9 (C-6); 161.7 (C = O). N ’-(4’”-ethoxyphenylmethylene)-3-methyl1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (3g). 87%, mp ＝ 243−245 ℃. IR (υ-cm−1): 3,220 (N-H); 1,640 (C ＝ O). 1HNMR: δ1.55 (t, 3H, CH3CH2O, J = 7.2); 2.50 (s, 3H, CH3); 4.62 (q, 2H, CH3CH2O, J = 7.2); 7.04 (d, 2H, H-3”’, J = 8.4 Hz ); 7.25−7.37 (m, 2H, H-4’, H-4”); 7.47−7.62 (m, 2H, H-3”); 7.60−7.62 (m, 2H, H-3’); 7.73 (d, 2H, H-2”’, J = 8.1 Hz); 8.13 (s, 1H, H-5); 8.28−8.31 (m, 4H, H-2’, H-2”); 8.34 (s, 1H, CH ＝ N). 13CNMR: δ14.3 (CH3); 14.5 (CH3CH2O); 63.3(CH3CH2O); 113.2 (C-3’’’); 114.9 (C-3a); 119.7 (C-2’); 120.3 (C-5); 125.7 (C-4’); 126.3 (C-4’’); 127.4 (C-2’’); 128.0 (C-2’’’); 129.0 (C-1’’’); 129.1 (C-3’’); 129.3 (C-3’); 130.0 (C-3); 138.8 (C-4); 142.2 (C-1’’); 143.4 (C-1’); 149.0 (NHN ＝ CH); 151.1 (C-7a); 156.3 (C-4’’’); 160.5 (C-6); 161.5 (C ＝ O). N ’-(4’”-N,Ndimethylaminophenylmethylene)3-methyl-1-phenyl-6-(4”-chlorophenyl)-1H-pyrazolo [3,4-b]pyridine-4-carbohydrazide (3i). 98%, mp ＝ 212 ℃. IR (υ-cm−1): 3,200 (N-H); 1,650 (C ＝ O). 1HNMR: δ2.76 (s, 3H, CH3); 2.85 (s, 6H, N (CH3)2); 6.97 (d, 2H, H-3”’, J = 8.7 Hz); 7.36 (d, 2H,
H-2”, J = 7.2 Hz); 7.46−7.49 (m, 1H, H-4’); 7.57 (d, 2H, H-2”’, J = 8.7 Hz); 7.62 (d, 2H, H-3’, J = 8.7 Hz); 7.93 (d, 2H, H-3”, J = 7.2 Hz); 8.15 (s, 1H, H-5); 8.38 (d, 2H, H-2’, J = 8.7 Hz); 8.54 (s, 1H, CH ＝ N); 12.30 (sl, 1H, NH). 13CNMR: δ13.1 (CH3); 44.5 (CH3); 107.9 (C-3a); 113,2 (C-3’’’); 120.7 (C-2’); 121.6 (C-4’); 126.0 (C-5, C-1’’’); 128.5 (C-2’’); 128.7 (C-3’); 129.1 (C3’’); 129.9 (C-2’’’); 132.4 (C-1’’); 137.8 (C-4’’); 139,7 (C-1’); 143.3 (C-4); 146.8 (C-3); 142.2 (NHN ＝ CH); 151.8 (C-7a); 154.7 (C-4’’’); 157.7 (C-6); 165.0 (C ＝ O). N ’-(4’”-bromophenylmethylene)-3-methyl-1phenyl-6-(4”-cyanophenyl)-1H-pyrazolo[3,4-b] pyridine-4-carbohydrazide (3j). 93%, mp ＝ 210 ℃. IR (υ-cm−1): 3,200 (N-H); 1,650 (C ＝ O). 1HNMR: δ2.73 (s, 3H, CH3); 7.47(d, 2H, H-3’”, J = 7.2 Hz); 7.69 (d, 2H, H-3’, J = 8.7 Hz); 7.96−8,02 (m, 1H, H-4’); 8.22 (d, 2H, H-2’, J = 8.7 Hz); 8.62 (d, 2H, H-3”, J = 7.2 Hz); 8.75 (s, 1H, H-5); 8.81(d, 2H, H-2’”, J = 7.2 Hz); 8.90 (d, 2H, H-2”, J = 7.2 Hz); 9.12 (s, 1H, CH = N); 12.66 (sl, 1H, NH). 13CNMR: δ13.7 (CH3); 107.9 (C-3a); 112.5 (C-4’); 118.8 (CN); 121.6 (C-2’); 123,0 (C-5),125.1 (C-4’’’); 127.8 (C-4’); 128.7 (C-2’’); 129.1 (C-3’); 130.2 (C-2’’’); 131.2 (C-3’’’); 131.9 (C-3’’); 132.5 (C-1’’’); 139.7 (C-1’); 141.3 (C-1’’); 143.0 (NHN = CH); 143.3 (C-4); 145.1 (C-3); 151.8 (C-7a); 154.7 (C-6); 165.0 (C = O). N ’-(4’”-fluorophenylmethylene)-3-methyl-1phenyl-6-(4”-thiomethylphenyl)-1H-pyrazolo[3,4b]pyridine-4-carbohydrazide (3k). 96%, mp ＝ 264 ℃. IR (υ-cm−1): 3,200 (N-H); 1,640 (C ＝ O). 1HNMR: δ2.68 (s, 3H, SCH3); 2.72 (s, 3H, CH3); 7.46−7.49 (m, 4H, H-3’, H-3”’); 7.46 (d, 2H, H-2”, J = 7.2 Hz); 7.57 (d, 2H, H-2”’, J = 8.7 Hz); 7.73 (d, 1H, H-4’, J = 7.5 Hz); 7.98 (dd, 2H, H-3”, J = 7.2 Hz); 8.22 (s, 1H, H-5); 8.38 (d, 2H, H-2’, J = 8.7 Hz); 8.80 (s, 1H, CH = N); 7.3 (d, H3’’’, J = 8.7), 7.8 (m, H2’’’); 12.39 (sl, 1H, NH). 13CNMR: δ13.1 (CH3); 14.3(CH3); 111.5 (C-3a); 116 (d, C-3’’’, JC, F = 22.0); 120.5 (C-5, C-2’); 125.9 (C-4’); 127.7 (C2’’); 128.7 (C-3’’); 129.5 (d, C-2’’’, JC, F = 8.5); 129.2 (C-3’); 130.5 (C-3); 134.0 (C1’’’); 138.6 (C-4); 139.0 (C-4’’); 139.6 (C-1’’); 142.2 (C-1’); 147.9 (NHN = CH); 151.0 (C-7a); 155.7 (C-6); 161.7 (C = O); 166.4 (d, C-4’’’, JC, F = 204.9). Acknowledgements We are grateful to the analytical centers of NPPN (UFRJ, Brazil) and the Instituto de Química (UFF, Brazil) for the spectroscopic data. We also thank FAPERJ, Capes, CNPq and PROPP-UFF for the in fellowships and financial support.
Carbohydrazides: new antiplatelet agents
References 1) Wohner N: Role of cellular elements in thrombus formation and dissolution. Cardiovasc Hematol Agents Med Chem 2008; 6: 224-228 2) Andrews RK and Berndt MC: Platelet physiology and thrombosis. Thromb Res 2004; 114: 447-453 3) Fuster V, Baldimon L, Baldimon JJ, Chesebro JH: The pathogenesis of coronary artery disease and the acute coronary syndromes. Part Ⅰ. N Engl J Med 1992; 326: 242250 4) Fuster V, Baldimon L, Baldimon JJ, Chesebro JH: The pathogenesis of coronary artery disease and the acute coronary syndromes. Part Ⅱ. N Engl J Med 1992; 326: 310318 5) World Health Organization (WHO) http://www.who.int/ cardiovascular_diseases/en/. Accessed in 2008 6) Zimmermann N and Hohlfeld T: Clinical implications of aspirin resistance. Thromb Haemost 2008; 100: 379-390 7) Chen WH: Antiplatelet resistance with aspirin and clopidogrel: is it real and does it matter? Curr Cardiol Rep 2006; 8: 301-306 8) Algra A, De Schryver EL, van Gijn J, Kappelle LJ, Koudstaal PJ: Oral anticoagulants versus antiplatelet therapy for preventing further vascular events after transient ischaemic attack or minor stroke of presumed arterial origin. Cochrane Database Syst Rev 2006; 3: CD001342 9) Koo MH, Nawarskas JJ, Frishman WH: Prasugrel: a new antiplatelet drug for the prevention and treatment of cardiovascular disease. Cardiol Rev 2008; 16: 314-318 10) Krötz F, Sohn HY and Klauss V: Antiplatelet drugs in cardiological practice: established strategies and new developments. Vasc Health Risk Manag 2008; 4: 637-645 11) Alexandrov AV, Alagona P: Stroke and atherothrombosis: an update on the role of antiplatelet therapy. Int J Stroke 2008; 3: 175-181 12) Angiolillo DJ, Guzman LA: Clinical overview of promising nonthienopyridine antiplatelet agents. Am Heart J 2008; 156: S23-S28 13) Dias LRS, Santos MB, Souza AMT, Freitas ACC, DiVaio MAV, Castro HC, Rodrigues CR: Synthesis, in vitro evaluation, and SAR studies of a potential antichagasic 1Hpyrazolo [3,4-b] pyridine series. Bioorg Med Chem 2007; 15: 211-219 14) Lima LM, Frattani FS, Dos Santos JL, Castro HC, Fraga CA, Zingali RB, Barreiro EJ: Synthesis and anti-platelet activity of novel arylsulfonate--acylhydrazone derivatives, designed as antithrombotic candidates. Eur J Med Chem
739
2008; 43: 348-356 15) Vera-Divaio MA, Freitas AC, Castro HC, de Albuquerque S, Cabral LM, Rodrigues CR, Albuquerque MG, Martins RC, Henriques MG, Dias LR: Synthesis, antichagasic in vitro evaluation, cytotoxicity assays, molecular modeling and SAR/QSAR studies of a 2-phenyl-3-(1-phenyl-1Hpyrazol-4-yl)-acrylic acid benzylidene-carbohydrazide series. Bioorg Med Chem 2009; 17: 295-302 16) Pinheiro LC, Abreu PA, Afonso IF, Leal B, Corrêa LC, Borges JC, Marques IP, Lourenço AL, Sathler P, Dos Santos AL, Medeiros CA, Cabral LM, Júnior ML, Romeiro GA, Ferreira VF, Rodrigues CR, Castro HC, Bernardino AM: Identification of a potential lead structure for designing new antimicrobials to treat infections caused by Staphylococcus epidermidis-resistant strains. Curr Microbiol 2008; 57: 463-468 17) Rocha GB, Freire RO, Simas AM, Stewart JJ: RM1: a reparameterization of AM1 for H, C, N, O, P, S, F, Cl, Br, and I. J Comput Chem 2006; 27: 1101-1111 18) Bernardino AR, Castro HC, Frugulhetti ICPP, Loureiro NIV, Azevedo AR, Pinheiro LC, Passamani F, Magalhães UO, Albuquerque MG, Cabral LM, Rodrigues CR: SAR of a series of anti-HSV-1 acridone derivatives, and a rational acridone-based design of a new anti-HSV-1 3Hbenzo[b]pyrazolo[3,4-h]-1,6-naphthyridine series. Bioorg Med Chem 2008; 16: 313-321 19) Lipinski CA, Lombardo F, Dominy BW, Feeney PJ: Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 2001; 46: 3-26 20) Tekto Ⅳ: Computing chemistry on the web. Drug Discov Today 2005; 22: 1497-1500 21) Alberio L, Dale GL: Review article: platelet-collagen interactions: membrane receptors and intracellular signalling pathways. Eur J Clin Invest 1999; 29: 1066-1076 22) Varga-Szabo D, Pleines I, Nieswandt B: Cell adhesion mechanisms in platelets. Arterioscler Thromb Vasc Biol 2008; 28: 403-412 23) Cross PE, Dickinson RP: Thromboxane synthetase inhibitors and antagonists. Ann Rep Med Chem 1987; 22: 95105 24) Nakahata N: Thromboxane A2: physiology/pathophysiology, cellular signal transduction and pharmacology. Pharmacol Ther 2008; 118: 18-35 25) Lewis GP, Watts IS: Prostaglandin endoperoxides, thromboxane A2 and adenosine diphosphate in collagen-induced aggregation of rabbit platelets. Br J Pharm 1982; 75: 623-631
