Synthesis, characterization, molecular docking, analgesic, antiplatelet and anticoagulant effects of dibenzylidene ketone derivatives

In this study dibenzylidene ketone derivatives (2E,5E)-2-(4-methoxybenzylidene)-5-(4-nitrobenzylidene) cyclopentanone (AK-1a) and (1E,4E)-4-(4-nitrobenzylidene)-1-(4-nitrophenyl) oct-1-en-3-one (AK-2a) were newly synthesized, inspired from curcuminoids natural origin. Novel scheme was used for synthesis of AK-1a and AK-2a. The synthesized compounds were characterized by spectroscopic techniques. AK-1a and AK-2a showed high computational affinities (E-value > − 9.0 kcal/mol) against cyclooxygenase-1, cyclooxygenase-2, proteinase-activated receptor 1 and vitamin K epoxide reductase. AK-1a and AK-2a showed moderate docking affinities (E-value > − 8.0 kcal/mol) against mu receptor, kappa receptor, delta receptor, human capsaicin receptor, glycoprotein IIb/IIIa, prostacyclin receptor I2, antithrombin-III, factor-II and factor-X. AK-1a and AK-2a showed lower affinities (E-value > − 7.0 kcal/mol) against purinoceptor-3, glycoprotein-VI and purinergic receptor P2Y12. In analgesic activity, AK-1a and AK-2a decreased numbers of acetic acid-induced writhes (P < 0.001 vs. saline group) in mice. AK-1a and AK-2a significantly prolonged the latency time of mice (P < 0.05, P < 0.01 and P < 0.001 vs. saline group) in hotplate assay. AK-1a and AK-2a inhibited arachidonic acid and adenosine diphosphate induced platelet aggregation with IC50 values of 65.2, 37.7, 750.4 and 422 µM respectively. At 30, 100, 300 and 1000 µM concentrations, AK-1a and AK-2a increased plasma recalcification time (P < 0.001 and P < 0.001 vs. saline group) respectively. At 100, 300 and 1000 µg/kg doses, AK-1a and AK-2a effectively prolonged bleeding time (P < 0.001 and P < 0.01 vs. saline group) respectively. Thus in-silico, in-vitro and in-vivo investigation of AK-1a and AK-2a reports their analgesic, antiplatelet and anticoagulant actions.


Introduction
Pain is an unfavorable sensory and emotional experience that is associated with the potential tissue damage and explained in terms of such damage [1]. Noxious effects such as ulceration, gastrointestinal bleeding by non-steroidal anti-inflammatory drugs and drowsiness, nausea and tolerance by opiates usage limits their use in management of pain [2]. Platelets play vital role in a complex processes which are involved in haemostasis and thrombosis [3]. The most common cause of peripheral artery diseases (PAD) is atherosclerosis and such patients have more chance of myocardial infarction, stroke or death with cardiovascular events and it is 3:1 in comparison to persons without PAD [4]. Antiplatelet agents are used in management of arterial thrombosis. Moreover, anticoagulants inhibit proteases in coagulation cascade [5]. Interference in natural balance among pro-coagulant and anticoagulant due to genetic or any other acquired factors may results in bleeding or thrombotic disorders. Thrombin is a key enzyme of coagulation cascade which has many significant biological functions including platelet

Open Access
Chemistry Central Journal *Correspondence: arif.ullah@riphah.edu.pk 1 Riphah Institute of Pharmaceutical Sciences, Riphah International University, Islamabad, Pakistan Full list of author information is available at the end of the article activation, fibrinogen conversion to fibrin network and feedback amplification of coagulation. Different tissue factors are involved in thrombus formation in order to prevent heamorrhage [6]. Coagulation cascade involves intrinsic and extrinsic pathways [7]. The former has a role in the growth and maintenance of fibrin while the later plays its role in the initiation of fibrin formation. Extrinsic pathway requires tissue factors for its activation which after vascular injury becomes exposed to the blood which ultimately results in thrombin activation [8]. Among antiplatelet agent and anticoagulant drugs which are available commercially, for thrombotic disorders, these agents are associated with certain limitations and side effects [9]. Chemically curcumin is 1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione. It is a yellow-orange colored pigment which is derived from the rhizome of Curcuma longa [10]. The plant has a wide spectrum of pharmacological properties and traditionally it has been used for many ailments since centuries [11]. The reported activities of curcumin are antioxidant, anti-inflammatory, antitumor, antibacterial, antifungal and antiviral [10]. Curcumin also showed inhibition in platelet aggregation and antithrombotic effects [12,13]. Concerning structural aspects, dibenzylidene ketone moieties are considered curcumin analogues, which are compounds of great importance. Structurally, curcuminoids contains two aryl rings connected at the ends of a C 7 carbon-chain where a dienone composes an extended conjugated system. Dibenzylidene ketone derivatives also contain a dienone system connecting two aryl groups at the ends of a C 5 carbon chain. Dienones are good Michel acceptors, allowing its reaction with important biomolecules interfering in biological processes. Previous reported activities of dibenzylidene ketone derivatives include antiparasitic activity, cytotoxicity, antimicrobial activity, analgesic activity [14][15][16]. Based on previous literature studies, two novel dibenzylidene ketone derivatives i.e. (2E,5E)-2-(4-methoxybenzylidene)-5-(4nitrobenzylidene) cyclopentanone (AK-1a) and (1E,4E)-4-(4-nitrobenzylidene)-1-(4-nitrophenyl) oct-1-en-3-one (AK-2a) were synthesized and characterized. AK-1a and AK-2a were investigated for their analgesic, antiplatelet and anticoagulant effects using different pharmacological and computational assays.

Chemicals
Adenosine diphosphate (ADP) and arachidonic acid (AA) were purchased from Chrono-Log association. Benzaldehyde, cyclopentanone, dimethyl sulfoxide, ethanol and methoxybenzaldehyde were purchased from Merck Millipore., Billerica, MA, USA. Aspirin, calcium chloride (CaCl 2 ), diclofenac sodium, heparin, phosphate buffers solution (PBS) and sodium citrate were obtained from Sigma chemicals., Dt. Louis, MO, USA. The tramadol was acquired from Searle Karachi-Pakistan. All chemicals used were of analytical grade.

Animals
Balb-C mice (25-30 g) of both gender were utilized for this study. All animals were housed according to the standard protocols 25 ± 2 °C, 12 h duration of natural light and dark cycle. Healthy diet was given to mice and water ad libitum. The study was performed in accordance with protocols of Institute of Laboratory Animal Resources, Commission on Life Sciences University, National Research Council (1996) and approved by Riphah Institute of Pharmaceutical Sciences (RIPS) Ethical Committee (Reference No: REC/RIPS/2016/009).

Synthesis of AK-1a and AK-2a
Novel way of synthesis was carried out. The monoarylidene derivative was synthesized by the reaction of cyclopentanone with p-methoxy benzaldehyde. DIM-CARB was utilized as a catalyst in this reaction. DIM-CARB was used in catalytic amount to obtain selective monoarylidene cyclic derivative in a green solvent (EtOH:H 2 O), further second step leads to get an unsymmetrical bis-(arylmethylidene)-cycloalkanones. The synthesis of compound was carried out at room temperature from the reaction of intermediate 1 with p-nitro benzaldehyde. The scheme of the synthesized compound along with its structure is shown in Fig. 1. Chemical characterization was carried out based on the analysis of spectroscopic data. Fourier transform mass spectrometry (FTMS) of AK-1a as shown in Fig. 2. Synthesis of AK-2a was carried in a two-step reaction. In the first step, cycloalkanone was reacted with an aldehyde in a DIMCARB catalysed reaction, while in the second step monoarylidene derivative was reacted with the aldehyde through knoevenagel condensation to get the required product. DIMCARB can be recovered by distillative dissociation-reassociation process in a vacuum or under an atmosphere of CO 2 . The 2-heptanone was reacted with p-nitro benzaldehyde in an acidic medium to get intermediate, and then intermediate yield AK-2a. The scheme of the novel synthesized compound AK-2a along with its structure is shown in Fig. 1. Chemical characterization was carried out based on the analysis of spectroscopic data. Fourier transform mass spectrometry (FTMS) of AK-2a as shown in Fig. 3.

In-silico study
Molecular docking is an informative tool which is used to investigate the affinity between ligand and protein targets. We used Auto Dock Vina program for docking study through PyRx [17,18]. Affinity of best docked pose of ligand and protein target complex was determined by E-value (kcal/mol). It provides prediction of binding free energy and binding constant for docked ligands [19]. 3D-structures of test compounds (AK-1a and AK-2a) were prepared in discovery studio visualiser (DSV) and saved as PDB format. 3D-structures of target proteins were taken from http://www.rcsb.org/ pdb/home/home.

Analgesic models
The analgesic activity was carried out by using two standard protocols i.e. acetic acid-induced writhing test and hot plate test in order to evaluate the peripheral and central effects of analgesia.

Acetic acid-induced writhing test
Mice were divided into five different groups, having five mice in each. After 30 min writhing were induced by an IP injection of 0.1 mL of 0.7% (by volume) acetic acid solution [20]. Drug pretreatment times were chosen so that writhing was counted over a period of maximum analgesic activity. AK-1a and AK-2a in a dose-dependent manner (0.5-100 mg/kg) decreased acetic acidinduced writhes injected through intraperitoneal (IP) route. Perception of pain was recorded in the form of abdominal constrictions and stretches of the hind limb called as a writhe. Some mice showed half writhe. Two half writhes were considered as equal to one full writhe. The writhing episodes were recorded for 20 min. Control group was administered with normal saline (10 mL/ kg). Diclofenac sodium was used as a positive control.

Hot plate test
The latency period of the test compounds were evaluated by hot plate assay according to the protocols as previously used with little modifications [21]. Mice were divided into five different groups, having five mice in each. The animals were placed individually on the hot plate (55 ± 2 °C) and the observations (jumping or licking paws) were recorded at 30, 60, 90 and 120 min. Normal saline (10 mL/kg) was given to control group, tramadol (30 mg/kg) was used as a positive control.

Antiplatelet assay
Antiplatelet activity was performed to check whether the test compounds possess any effect on platelet aggregation. It was determined by whole-blood aggregometry method, which was performed by an impedance aggregometer (Model 591, Chrono-Log) as previously described [22]. Arterial or venous blood samples were collected from healthy volunteers in plastic tubes having 3.2% sodium citrate anticoagulant (9:1). Measurements were performed at 37 °C and 1200 rpm stirring speed. According to the manufacturer recommendations, 0.5 mL of citrated blood was diluted with same volume of normal saline (0.9%) which was prewarmed for 5 min at 37 °C. 30 µL, AK-1a and AK-2a at 1, 3, 10, 30, 100, 300, 1000 µM concentrations were also added to the tube. After placing the electrode, aggregation was induced by Platelet aggregation response was continually monitored for 6 min as an electrical impedance in ohms. Then mean percent platelet inhibition was calculated. Aspirin was used as positive control.

Anticoagulant activity
Anticoagulant activity of the test compounds were performed using following experiments.

Plasma recalcification time (PRT)
Anticoagulant potential of the test compounds were determined by PRT method [23]. The blood samples were obtained from healthy volunteers in tubes containing 3.8% sodium citrate (9:1) in order to prevent the clotting process. Centrifugation (15 min at rate 3000 rpm) was carried out in order to obtain platelet poor plasma. 0.2 mL plasma, 0.1 mL of different concentration of the test compounds (30, 100, 300 and 1000 μM) and 0.3 mL of CaCl 2 (25 mM) were then added together in a clean fusion tube and incubated in a water bath at 37 °C. Heparin (440 μM) was used as positive control. The clotting time was recorded with a stopwatch by tilting the test tubes every 5 s.

Bleeding time (BT)
Anticoagulant potential of AK-1a and AK-2a was also determined by in-vivo tail BT method in mice [24]. AK-1a and AK-2a (100, 300 and 1000 μg/kg) were administered intravenously via tail vein of mice. After 10 min mice were anesthetized using diethyl ether and a sharp cut (3 mm) deep at tip of the tail were made. The tail was then immersed into PBS which was pre warmed to 37 °C. BT was recorded from the time when bleeding started till it stopped completely observation was made up-to 10 min. Heparin (40 μg/kg) was utilized as a positive control.

Statistical analysis
Data were expressed as mean ± standard error of mean (SEM) and analyzed by using one-way analysis of variance (ANOVA), with post hoc Tukey's test. Data were considered significant at P < 0.05. Bar graphs were analyzed using Graph Pad Prism (GraphPad, San Diego, CA, USA).

Molecular docking evaluation
The results of E-values, hydrogen bonds and binding residues of AK-1a and AK-2a with target proteins involved in pain pathways along with standard drugs are shown in Table 1 and Figs. 4 5, 6, 7. The results of E-values, hydrogen bonds and binding residues of AK-1a and AK-2a with target proteins involved in platelet aggregation along with standard drugs are shown in Table 2 and Figs. 7, 8, 9. The results of E-values, hydrogen bonds and binding residues of AK-1a and AK-2a with target proteins involved in coagulation process along with the standard drugs are shown in Table 3 and Figs. 10, 11, 12.

Effect on latency time
The latency time of saline group (

Discussion
In this study, we synthesized and chemically characterized two new dibenzylidene ketone derivatives. The in-silico study carried out to get an initial information about the affinity of any compound before the start of in-vivo experiment. Docking is a preliminary tool used to check the affinity of ligands to their respective protein targets. Molecular docking has an ambient role in drug discovery and development including structure based evaluation and finding target specificity and binding affinity [25]. These interactions may exist in the form of hydrogen bonds, hydrophobic interactions and Van der Waal forces. Auto Dock Vina program was used through PyRx. It uses gradient optimization  method and it improves accuracy of binding mode predictions [26]. Hydrogen bonding is reported to be very significant in the formation of ligand protein complex [27]. Further we assessed affinity of ligands through E-value and number of hydrogen bonds against protein targets that influence analgesic, antiplatelet and anticoagulant effect. AK-1a and AK-2a showed highest binding affinity against PAR-1. AK-1a order of binding affinity against target proteins was found as: VKOR > COX-1 > COX-2 > F-IX > PG-I 2 > HCR > mu receptor > GPIIb/IIa > F-II > P 2 Y 12 > kappa receptor > F-X > delta receptor > AT-III > P2X3 > GP-VI. AK-2a order of binding affinity against target proteins was found as: COX-1 > COX-2 > HCR > mu receptor > kappa receptor > GPIIb/ I I Ia > P G -I 2 > AT-I I I > F -I X > F -X > F -I I > d el t a receptor > VKOR > P 2 Y 12 > P 2 X 3 > GPVI. We can infer that our compounds have analgesic, antiplatelet and anticoagulant actions. The analgesic activity was studied using two standard protocols i.e. acetic acid induced writhing method and hot plate assay to evaluate the peripheral and central effects of analgesia [28]. Basically writhing is an abdominal constriction caused by the release of different types of mediators after the i.p injection of acetic acid. This noxious response can be prevented by drugs which have the ability to stop the synthesis of these chemicals. The reduction in the number of writhes in treated group explains the same phenomenon of blocking the production of mediators by inhibiting COX-2 by the test compounds. Analgesic actions of AK-1a and AK-2a are proposed as inhibition of prostanoid release from cyclooxygenase involved in visceral nociception induced by acetic acid [29]. The central nociceptive effects were validated through hotplate assay [30]. AK-1a and AK-2a showed dosedependent analgesic response, while AK-2a is found to be potent, as dose ≥ 10 mg/kg cannot be used for the analgesic activity. Significant response against acetic acid-induced writhing and hotplate assay by AK-1a and AK-2a explains central as well as peripheral activity of dibenzylidene ketone derivatives [31]. In acetic acid-induced writhing at higher dose AK-2a showed significant response, it can be further checked for antiinflammatory response. The nociceptive behavior in the acetic acid-induced writhing test occurs due to synthesis of pain mediators including prostaglandins due to induction of COX-2 that results increased in pain sensitivity after acetic acid injection [32,33]. Acetic acid produces nociception by releasing chemical mediators such as serotonin, histamine, prostaglandins, bradykinins and substance P due to induction of COX-2 that results in increased pain sensitivity after acetic acid injection. The acetic-induced writhing test is also

Table 4 Inhibitory effect of (2E,5E)-2-(4-methoxybenzylidene)-5-(4-nitrobenzylidene) cyclopentanone (AK-1a) and (1E,4E)-4-(4-nitrobenzylidene)-1-(4-nitrophenyl)oct-1-en-3-one (AK-2a) and aspirin against arachidonic acid (AA) and adenosine diphosphate (ADP)-induced platelet aggregation
Values shown as mean ± SEM, n = 4  heparin. Data expressed as mean ± SEM, n = 5, ***P < 0.001 vs. saline group, one way ANOVA with post hoc Tukey's test sensitive to adrenoceptor agonists and opioid agonists which through appropriate receptor stimulation in the peritoneal cavity cause reduction in pain perception [34,35]. This test involves both central and peripheral mechanisms in the early phase of the test [36]. However, hot plate test is regarded as a suitable model for the involvement of central mechanisms [37,38]. PAR-1 activation leads to stimulation of arachidonic acid release and thrombin signaling. Arachidonic acid enhances the activation of platelet aggregation cascade [39,40]. This can be a proposed mechanism of action for AK-1a and AK-2a as antiplatelet and anticoagulant agents. As per computational study results, both can be a potential antagonist of PAR-1 which was further validated. Curcumin analogues inhibit platelet aggregation and repress thrombosis. Dibenzylidene ketone derivatives used in this study, having ketone moiety showed significant antiplatelet and anticoagulant response [41] presence of methoxy group in AK-1a enhanced its biological activity [42]. Previous studies revealed role of curcumin derivatives as a vitamin k antagonist [43], so as these dibenzylidene ketone derivatives. Anticoagulant actions of AK-1a and AK-2a were also validated by the presence of hydrophobic groups [44].

Conclusions
The present study reports t newly synthesized dibenzylidene ketone derivatives AK-1a and AK-2a showed high binding affinities against different protein targets involved in mediation of pain, platelet aggregation and blood coagulation process. The pharmacological investigations based on in-silico, in-vitro and in-vivo studies revealed their analgesic, antiplatelet and anticoagulant actions. These are promising findings, since the production of dibenzylidene compounds is a simple, cheap and feasible process. Data expressed as mean ± SEM, n = 4, **P < 0.01 and ***P < 0.001 vs. saline group, one way ANOVA with post hoc Tukey's test