Synthetic coumarin derivatives with anticoagulation and antiplatelet aggregation inhibitory effects

Thrombosis is the leading cause of illness and mortality worldwide, posing a serious risk to human health and life. Because antithrombotic drugs can prevent the beginning and progression of thrombotic disorders, they are essential in the management of thrombotic diseases. The substantial side effects and unsatisfactory efficacy of present antithrombotic drugs stimulate the search for novel, effective, and safer antithrombotic therapies. Natural and synthetic coumarins have been shown to have antithrombotic action, specifically anticoagulation, and antiplatelet aggregation. In particular, coumarin-based medications like warfarin, phenprocoumon, and cloricromen have long been used to treat thrombosis in clinical settings. The favored structure for creating novel antithrombotic medications with diverse modes of action is coumarin, which has low toxicity. The goal of the current review is to consolidate recent findings on the development of coumarins as antithrombotic agents, with a focus on the connection between these compounds’ chemical compositions and therapeutic potency. It aims to offer promising suggestions for the identification of new coumarin compounds with potent antithrombotic properties.


Introduction
Thromboembolic diseases continue to be the most prevalent causes of death.In hemostatic and thrombotic processes, platelets become key players driven by many physiological mediators.Blood fluidity is regulated by sophisticated and fascinating physiological mechanisms.At locations of vascular injury, blood must remain fluid within the vasculature while yet clotting swiftly when not subjected to endothelial surfaces.A system of fibrinolysis is engaged to restore fluidity when intravascular thrombi do develop.Both thrombosis and hemorrhages are prevented, and physiological fibrinolysis is permitted without excessive pathological fibrinogenolysis, attributable to a fine balance.Platelets assemble at the injured endothelium after an injury.The platelets then transform, activate receptors, and release chemical messengers.Finally, platelets aggregate and connect with one another through receptors.The synthesis, deposition, and binding of fibrin as well as the activation of coagulation factors (secondary hemostasis) are associated with the creation of platelet aggregates (primary hemostasis).In order to halt bleeding, these processes may combine to form clots [1,2].Antiplatelet agents have been proposed as therapeutic tools to prevent thromboembolism through multiple mechanisms of action phosphodiesterase or cyclo-oxygenase inhibitors.

Cyclic adenosine monophosphate (cAMP)
Cyclic adenosine monophosphate (cAMP), a second messenger, is a potent inhibitor of platelet activity.Adenylate cyclase and/or phosphodiesterases regulate the rate of intracellular cAMP generation and/or hydrolysis, respectively (PDEs).Platelets contain members of the PDE2, PDE3, and PDE5 groups of PDEs.A substantial antiplatelet impact is produced when PDE3 isoform, which makes up 80-90% of all platelet PDE, is inhibited [3,4].

Cyclo-oxygenase pathway
Multiple routes regulate platelet aggregation.Through a cyclooxygenase (COX)-dependent process, platelet activation by powerful agonists like thrombin as well as collagen triggers the secretion of secondary agonists like thromboxane A2 (TXA2) and the adenosine diphosphate (ADP) from compact platelet granules.ADP is also secreted as a result of TXA2 interaction to the thromboxane receptor.The aforementioned process causes molecules like TXA2 and ADP to accumulate locally, which is crucial for additional platelet recruitment and intensifying the previously mentioned activation signals.The most significant and extensively researched endogenous platelet aggregator among these is ADP.The presence of calcium ions and fibrinogen is necessary for ADP-induced aggregation process.The platelet membrane's P2Y1 as well as P2Y12 receptors bind ADP.The production of TXA2 is aided by the degradation of phosphoinositide and the activation of calcium ions that occur when the P2Y1 receptor is activated.P2Y12 receptor activation suppresses platelet (cAMP) activation.The activation of glycoprotein IIb/IIIa (GPIIb/GPIIIa) on the membrane is caused by both of these processes, which cause platelet aggregation [5].

Vitamin K reductase
The carboxylation of the glutamic acid residues on the Nterminal portions of certain clotting factors (II, VII, IX, and X) essentially requires vitamin K as a cofactor.The new amino acid, ɣ-carboxyglutamate, produced by this process causes the proteins to alter in shape by chelating calcium ions.When the clotting cascade is initiated, this alteration in tertiary structure enables the four vitamin K-dependent clotting components to become active and attach to the negatively charged phospholipid membranes.Vitamin K hydroquinone [KH2], the reduced form of Vitamin K, carbon dioxide, as well as molecular oxygen are required as cofactors by the carboxylase enzyme that carboxylates vitamin K-dependent coagulation components.KH2 is converted to vitamin K 2,3-epoxide during this reaction.After a two-step reduction, the epoxide is converted back into the active KH2 form.Initially, vitamin K 2,3-epoxide reductase reduces the epoxide form to vitamin K quinone in the presence of NADH.The vitamin K quinone reductase then further reduces this intermediate quinone back to KH2 [6,7] (Table 1).
One can conclude that a better understanding of the vitamin K cycle and active complexes of VKOR is necessary to improve selectivity and efficacy of coumarin-based drugs.These efforts must be accompanied by synthesis and testing of new members of this family.

Coumarin derivatives in the market
A number of coumarin drugs have been authorized for the treatment of several illnesses.Warfarin 1, Acenocoumarol 2, and Phenprocoumon 3 have been therapeutically used, especially in the treatment of thrombosis.Coumarins' antithrombotic action relies on vitamin K competitive antagonism leading to the suppression of coagulation factors II, VII, IX, and X production [16,17].A number of interesting findings have also demonstrated the ability of coumarin drugs to exhibit their antithrombotic effects by reducing the platelet aggregation induced by adenosine diphosphate (ADP), thrombin (Thr), thromboxane A2 (TXA2), collagen (Coll), as well as arachidonic acid (AA) [18,19].The development, therefore, of safer and more potent antithrombotic coumarin drugs with different multiple mechanisms of action is a promising strategy.

Synthetic coumarins with anti-coagulant activity
Coumarins are a fascinating synthetic target due to their potential for a variety of biological and physical features.In light of this, medicinal chemists have been paying close attention to the development of simple and adaptable synthetic methods for functionalized coumarins.

Hydroxy-coumarins
The 4-hydroxycoumarin bioactive scaffold present in Warfarin 1 and Acenocoumarol An earlier attempt was made by Chen et al. in search for platelet anti-aggregatory agents.4-hydroxycoumarin derivatives were synthesized via alkylation of the hydroxyl group.The platelet anti-aggregatory activity of the synthesized compounds were investigated against collagen(Co1)induced, thrormbin (Thr)-induced, arachidonic acid (AA)induced, and platelet-activating-factor (PAF)-induced platelet aggregation in washed rabbit platelets.Among the synthesized derivatives, compound 5a showed potent platelet anti-aggregatory effects on AA-induced and PAF-induced aggregation with IC 50 values of 8.21 and 103.67 µM, respectively.The platelet anti-aggregatory activity of compound 5a against PAF-induced aggregation is assumed to be further enhanced by adding a proper functional group on the 2-phenyl group on the tetrahydrofuran ring [21].
Later that same year, Chen et al. published new hydroxycoumarin-based platelet aggregation inhibitors, bearing α-methylene-γ-butyrolactones.However, the position of the hydroxyl group was changed from C4 to C7, in addition to adding various substituents to C3, C4 and C8.The synthesized compounds were also evaluated for inhibitory activity against AA-induced, Col-induced, Thr-induced and PAF-induced platelet aggregation in washed rabbit platelets.Among the synthesized derivatives, compound 5b showed the highest inhibition of AA-induced and PAF-induced aggregation, with improved IC 50 values of 3.65 and 16.36 μM, respectively [22].
In a study by Lu et al., the activity of three compounds 6-8, bearing coumarin nucleus, was investigated against (ADP)induced platelet aggregation.The three compounds were shown to inhibit the active form of GPIIb/IIIa complex on the membrane of the platelets, thus platelet aggregation.Additionally, the downstream signal transduction of the ADP receptor, including the release of calcium ions and the regulation of cAMP, were also found to be inhibited by the three derivatives [23].A study by Katerina et al. investigated the antiplatelet activity, induced by AA, collagen and ADP, of previously synthesized simple 4-methylcoumarin derivatives.4-methylcoumarin derivatives bearing hydroxyl groups on position 5 and 7, particularly those containing a lipophilic side chain at C3, 9a and 9b, exhibited platelet antiaggregatory activity similar to that of acetylsalicylic acid on AA-induced aggregation (IC 50 = 16.1 µM) with IC 50 values of 17.5 µM and 23.3 µM, respectively.promising candidates for the extension of the current spectrum of antiplatelet drugs.In addition, compounds 9a, and 9b exhibited COX-1 inhibitory activity.They were found to be more potent inhibitors than Acetylsalicylic Acid [19].Anti-inflammatory Mannich bases and 7-azomethinelinked coumarin derivatives were synthesized by Kontogiorgis et al. and were further repurposed as platelet antiaggregatory agents and antithrombotic activities.Among the synthesized coumarin derivatives, compounds 10a and 10b were shown to exhibit the highest inhibitory activity as well as selectivity of platelet aggregation.Compound 11 also exhibited the highest activity in the clot retraction assay.These results confirmed the importance of target investigation, such as PDE-3.Docking studies were performed by the author against PDE-3.Compound 10b exhibited a very high binding energy score (−9.8 kcal/mol) with strong hydrogen bonding with the active site [24].A group of scientists from University of Genoa and University of Bari in Italy, prepared a series of 4-(1-piperazinyl)coumarins.The synthesized piperazinylcoumarins were evaluated in vitro to assess their inhibitory activities against ADP-as well as collagen-induced platelet aggregation, in addition to Ca 2+ ionophore.The synthesized coumarin derivatives exhibited high inhibitory activity, especially 12a, which proved to be the most effective in vitro platelet antiaggregatory agents, with IC 50 values of 1.9 µM, 1.8 µM and 1.1 µM, respectively [25].The same group continued their search for better 4-(1-piperazinyl) coumarins, where compound 12b, exhibited the highest in vitro inhibitory activity against platelet aggregation with better IC 50 values of 0.98 µM, 0.51 µM and 1.42 µM, respectively [26].Later, novel 4-(1-piperazinyl)coumarin derivatives were reported by the group and were evaluated as human PDE3 inhibitors.Compounds 12b and the newly synthesized 12c exhibited higher potency than milrinone 13 and cilostazol 14 (used as reference drugs), as inhibitors of PDE3, with IC 50 values of 0.037 µM and 0.078 µM, respectively [27].Later in the same year, the same research group was able to synthesize 4-hydroxycoumarin derivatives bearing pyridine moiety at C3. Compounds 15a and 15b demonstrated Prothrombin Time (PT) values of 20.80s and 21.30 s, respectively, which were higher than that of Warfarin 1 (9.16 s).Compound 15a demonstrated increased locomotors activity in mice, in addition to diarrhea, increased eye secretion and eyelid edema.However, only diarrhea was observed with compound 15b.Warfarin 1, exhibited the same toxicity pattern in mice as compound 15a, yet the severity of the symptoms was lower [28,29].

Coumarin carboxamides
A research group from Belgium was the first to synthesize and evaluate 3-carboxamide-coumarins as selective nonpeptidic inhibitors of FXIIa.The IC 50 value for inhibiting FXIIa was used to determine the inhibitory potency of the newly synthesized 3-carboxamide-coumarins.The most effective 3-carboxamide-coumarins among the newly developed compounds were 16a and 16b, having IC 50 values for inhibiting FXIIa of 4.3 and 4.4 µM, respectively [30].
Later in 2011, Sashidhara et al. were able to synthesize novel 3-carboxamide-benzocoumarin derivatives that were evaluated for their antithrombotic activity in vivo.Compound 17 demonstrated the highest antithrombotic activity, which was similar to aspirin or warfarin, in addition to not exhibiting the unwanted increase in bleeding time [31].

17
Other novel coumarin-3-carboxamide derivatives were described in 2016 by Bouckaert et al. and evaluated as new selective FXIIa inhibitors, possessing improved physicochemical properties.Compounds 18a-18c revealed to be the three most active derivatives among all the synthesized derivatives with IC 50 values of 5, 8 and 7 µM, respectively [32].Kathuria et al. described novel N-acetylaminoand acetoxy-coumarins, that were evaluated as substrates for platelet CRTAase (known to catalyze the activation of NOS leading to the inhibition of ADP/Arachidonic acid (AA)dependent platelet aggregation) as well as inhibitors of COX-1.7-N-Acetylamino-4-methylcoumarin 19 exhibited the highest substrate strength to platelet CRTAase and was therefore the most promising antiplatelet agent both in vitro and in vivo.In addition, compound 19 resulted in the suppression of COX-1, which in turn caused the downregulation of TXA2 and the prevention of platelet aggregation.It was also revealed to be beneficial in inhibiting LPS-induced thrombus formation [33].

Coumarin esters
Coumarin esters were described by R. Frederick et al., and were evaluated as inhibitors of on Thr and factor Xa (FXa), as anticoagulant drugs.Compound 20 was identified as a very powerful Thr inhibitor with (k i /KI) ratio of 37,000 M −1 s −1 .Additionally, this derivative possesses 168-fold higher selectivity towards Thr than FXa.The trends seen in the enzymatic assays were subsequently successfully explained by docking experiments, which also successfully supported the hypothesized inhibitory mechanism [34].

20
The same research group continued with the synthesis of novel coumarins, bearing a guanidine or an amine group on the lateral side chain on C3.Compound 21 with, bearing maniline, was identified as a particularly effective Thr inhibitor in the in vitro study.Interestingly, the Thr inhibitory effects were consistently reduced when a guanidine moiety was added.The results of the in vitro assay were explained by docking experiments, which demonstrated the critical involvement of a conserved water molecule in the specificity pocket of Thr during docking simulation, rendering the guanidine moiety inactive [35].

21
Later in 2007, more coumarin esters were described by Frederick et al., where the novel compounds were evaluated as potent selective Thr inhibitors.From this study, coumarin derivative 22, exhibited promising THR activity (ki/KI) 3455 M −1 .s−1 .Therefore, an excellent selectivity profile can be confirmed based on the obtained results against other serine proteases such as factor Xa and trypsin.Docking analysis of this compound into the different protein structures revealed the molecular basis responsible for its potency and selectivity [36].

22
A series of novel coumarin esters were designed by Huang et al., and biologically evaluated as platelet antiaggregatory agents.Compound 23 was shown to possess the strongest inhibitory activity against ADP-induced platelet aggregation in vitro.The antiplatelet activity of 23 was 2.3-fold more potent than that aspirin [37].

Cycloanelated coumarins
Pyrimidino-coumarin derivatives were synthesized by Bruno et al., among other pyrimidino-benzopyran derivatives, and were reported as platelet antiaggregatory as well as antithrombotic agents.Only pyrimidino-coumarin derivatives 24a-24c were able to inhibit ADP, AA-, U46619induced platelet aggregation as well as thrombin-induced clot retraction.The three pyrimidinocoumarin derivatives 24a-24c exhibited higher potency than that of aspirin (reference drug) [38].Novel amide derivatives of benzocoumarin scaffold have been developed by Sashidhara et al. and tested for their antithrombotic properties.Compounds 25a-25c demonstrated a desirable anti-thrombotic profile in vivo, with protection percentage of 40, 40 and 45%, respectively.In addition, the bleeding time of derivatives 25a-25c was significantly increased by 91.5, 96.5 and 112.5%, respectively.These derivatives significantly reduced the amount of platelet aggregation induced by ADP and collagen when added to mouse plasma in vitro [39].Garazd et al. were able to synthesize several 5-hydroxy-, as well as, 7-hydroxy-3,4-cycloannelated coumarin D-glucopyranosides, resembling coumarin glycosides found in nature.Compounds 26-28, were shown to possess strong anticoagulant activity.Results indicate that 26 has anticoagulant activity at the same level as heparin and that 27 and 28 are better anticoagulants than heparin [40].

Coumarin hybrids
Based on the natural product andrographolide, Li et al. were able to design and synthesize 3,15-disuccinate-12-coumarin substituted analogs.These synthetic compounds demonstrated strong antiplatelet aggregation inhibitory effects in vitro in response to thrombin, ADP, AA, and collagen.Thr-, AA-, and Col-induced platelet aggregation were all significantly inhibited by compound 29a simultaneously, and this effect was dose-dependent, according to the in vitro biological evaluation.The ADP-induced platelet antiaggregatory activity was produced by compound 29b [41].
Coumarin-resveratrol hybrids have been developed by Vilar et al.The inhibitory effects of the synthesized hybrids on thrombin-induced platelet aggregation in washed human platelets have also been assessed.Similar to trans-resveratrol (t-RESV), the developed compounds inhibited platelet aggregation.Derivative 30, in particular, exhibited six-fold platelet antiaggregatory activity in comparison with that of (t-RESV) [42,43].

30
Novel 6-halo-3-hydroxyphenylcoumarins that are resveratrol-coumarin hybrids, was synthesized by Quezada et al. and were evaluated for their Thr-induced platelet antiaggregatory activities in washed human platelets.Compounds 31a-31c demonstrated anti-platelet aggregation activity that is higher than that of t-RESV.Compound 31b was found to be possess platelet antiaggregatory activity that is thirty-fold higher than that of t-Resv [42,44].Hrubša et al. were able to design and synthesize novel coumarin-indole hybrids.The novel compounds were tested as platelet antiaggregatory agents.Compound 33 demonstrated the highest antiplatelet activity, which was more than that of aspirin.Thromboxane receptor antagonism was found to be the primary mechanism of action.In addition, Thromboxane synthase inhibition was observed at higher concentrations.The investigated compounds did not inhibit cyclooxygenase-1, in contrast to aspirin [46].

Bis-coumarins
Bang et al. successfully prepared both coumarin and biscoumarin derivatives, where the two coumarin moieties were linked at C7 using ether linkages separated by salicylic acid esters [47].Anticoagulant activity of the synthesized compounds was studied in white laboratory mature male mice.Results showed that the Prothrombin Time (PT) of synthesized bis-coumarins 34a and 34b were 1.5-fold greater than that of the Warfarin 1 (reference).They were also found to be the most active among even the monomeric coumarin derivatives.Dimeric coumarins linked by either an alkyne or a triazole ring were described by Shults et al.Compounds were evaluated in vivo as anticoagulant by measuring prothrombin time value.Compounds 35a and 35b, bearing a triazole linking a coumarin to a dihydrofuro-coumarin, exhibited higher anticoagulant activity than that of warfarin at higher doses, in addition to lower toxicity [48].The synthesized bis-coumarins were evaluated as potential anticoagulants in vivo using prothrombin time assay in rabbit model.The measured prothrombin time revealed that bis-coumarins 36a-36d possess a promising anticoagulant activity comparable to that of warfarin 1 (reference) [49].

Conclusions
Thrombosis is one of the major causes of death worldwide, threatening human health and life.Antithrombotic medications are crucial in the management of thrombotic diseases as they can prevent the onset and progression of thrombotic diseases.The discovery of novel, effective, and safer antithrombotic medications is driven by the serious adverse effects and unsatisfactory efficacy of the current antithrombotic medications.It has been demonstrated that synthetic drugs bearing coumarin nucleus exhibit antithrombotic activity, specifically anticoagulation and antiplatelet aggregation.The development of coumarin derivative research in the field of antithrombotic and anticoagulant agents is described in depth in this comprehensive review, demonstrating the critical role coumarins play in enhancing antithrombotic properties.Coumarins' ability to prevent thrombosis through a diverse range of mechanisms suggests that they can be useful in the creation of very potent antithrombotic drugs that have a myriad of targets in a number of different of pathways.

31 31a,
R= Cl, R 1 = 2'-OH 31b, R= Cl, R 1 = 3'-OH 31c, R= Br, R 1 = 4'-OH Maria et al. described new platelet antiaggregatory agents that are pyridazinone-coumarin hybrids.The in vitro Thr-induced antiplatelet activity of the synthesized hybrids was investigated.The new synthesized hybrids exhibited low micromolar IC 50 values.Compound 32 exhibited the most potent platelet antiaggregatory activity among all the synthesized hybrids, with IC 50 value of 3.34 µM.In addition, compound 32 was found to be more active than the reference drug Milrinone (IC 50 = 4.70 µM).Further studies confirmed ability of 32 to inhibit tyrosine kinase activity leading to inhibiting Gp IIb/IIIa activation[42,45].

Twenty 4 -
hydroxycoumarin derivatives were synthesized.Five of them are described for the first time.A comparative pharmacological study of the anticoagulant effect with respect to Warfarin showed that the synthesized compounds have different anticoagulant activities.The most prospective compound is 3,3′-(4-chlorophenylmethylene)bis(4-hydroxy-2H-1-benzopyran-2-one) 40 with low toxicity, very good index of absorption and dose dependent anticoagulant activity[51].40