Bioorganic & Medicinal Chemistry Letters 21 (2011) 4705–4709

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Thrombin inhibitors with lipid peroxidation and lipoxygenaseinhibitory activities

Miloš Ilic a, Christos Kontogiorgis b, Dimitra Hadjipavlou-Litina b, Janez Ilaš a, Danijel Kikelj a,⇑a University of Ljubljana, Faculty of Pharmacy, Aškerceva 7, 1000 Ljubljana, Sloveniab Aristotle University of Thessaloniki, School of Pharmacy, 54124 Thessaloniki, Greece

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 May 2011Revised 16 June 2011Accepted 19 June 2011Available online 25 June 2011

Keywords:Thrombin inhibitorAntithromboticLipid peroxidationLipoxygenase inhibitionRadical scavenging

0960-894X/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.bmcl.2011.06.089

⇑ Corresponding author. Tel.: +386 1 476 9561; faxE-mail address: danijel.kikelj@ffa.uni-lj.si (D. Kikel

Vascular oxidative stress, endothelial injury, and thrombosis are intertwined processes that display asynergistic pathological effect in many cardiovascular diseases. Antithrombotic therapy with anticoagu-lant and/or antiplatelet agents, combined with interventions against vascular oxidative stress and/orinflammation, both boosting endothelial antithrombotic potential, could display a synergistic action inthe treatment of thrombosis. Of the compounds 10a–h and 11a–d, shown to possess thrombin inhibitoryactivity, 11a–d were found to display radical scavenging activity, 10a, 10d, and 10f were demonstrated toinhibit lipid peroxidation of linoleic acid, and 10b and 10h inhibited soybean lipoxygenase. The observedcombination of thrombin inhibition with lipid peroxidation and/or lipoxygenase inhibitory activitymakes compounds 10 and 11 interesting candidates for further investigations towards multiple anti-thrombotic drugs.

� 2011 Elsevier Ltd. All rights reserved.

Arterial and venous thrombosis are the major causes of morbid-ity and mortality worldwide.1 In contrast to hemostasis, by whichthe body stops blood loss whenever a blood vessel is severed orruptured, thrombosis is a pathological process in which hemostaticmechanisms, that is, blood coagulation and platelet aggregation,are activated in the absence of bleeding. The blood clots formedby pathological coagulation and aggregation processes can causemyocardial infarction, ischemic stroke, limb gangrene, deep veinthrombosis, and pulmonary embolism, which can be life threaten-ing. Antithrombotic drugs, including anticoagulants (e.g., warfarin,heparins, thrombin inhibitors, and factor Xa inhibitors) and anti-platelet agents such as acetylsalicylic acid, clopidogrel and glyco-protein IIb/IIIa antagonists, are widely used in clinics, either incombination or alone for the prevention of thrombosis. However,due to the limitations of existing drugs, there is a growing needfor antithrombotic agents with a new mode of action to providealternatives to existing treatment strategies.2 Multitarget anti-thrombotic agents,3a designed to modulate two or more targets3b

involved in the development of thrombosis, pose a challengingemerging strategy for antithrombotic drug discovery.

Under normal circumstances, endothelial cells play a criticalrole in preventing intravascular thrombosis (i) by providing a un-ique surface which prevents activation of coagulation and plateletaggregation and (ii) by releasing mediators that inhibit prothrom-botic processes. However, endothelial cells, which normally have

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: +386 1 425 8031.j).

antithrombotic functions, are vulnerable to oxidative stress initi-ated by reactive oxygen species (ROS) diffusing from leukocytesor generated in the endothelium in the context of inflammation.4

The resulting endothelial injury may lead to exposure of thrombo-genic components of the subendothelial extracellular matrix,which initiates a vicious cycle of edema, leukocyte adhesion andthrombosis.5,6 The pathologically altered vasculature involved ininflammation and oxidative stress is thus an important underlyingcause of thrombosis.

Production of ROS is increased in many diseases, such as rheu-matoid arthritis, ischemic stroke, atheromatosis, and heart attack.Blood coagulation also stimulates production of reactive oxygenspecies by human neutrophils and ROS have emerged as importantmodulators of integrins in the blood clotting process through bothoutside-in (integrins stimulating ROS production to affect intracel-lular events) and inside-out signaling.7 The major lipidic proinflam-matory mediators involved in thrombosis are leukotrienesgenerated by 5-lipoxygenase, an enzyme expressed mainly by leu-kocytes, and it has been demonstrated that leukocyte mediatedvein injury and thrombosis are reduced by 5-lipoxygenase inhibi-tors.8,9 Besides damaging vascular endothelium, reactive oxygenspecies can also initiate lipid peroxidation, which again is linkedto thrombotic events.10 Vascular oxidative stress, inflammationand thrombosis are thus intertwined processes that display a syn-ergistic pathological effect in many cardiovascular diseases.4 Itcan therefore be anticipated that antithrombotic therapy with anti-coagulant and/or antiplatelet agents, combined with interventionsdirected against vascular oxidative stress and/or inflammation,both boosting endothelial antithrombotic potential, would display

4706 M. Ilic et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4705–4709

a synergistic action in the treatment of thrombosis. New multitar-get antithrombotic drugs, combining in the same molecule antico-agulant and anti-inflammatory activity, would present abreakthrough in the therapy of thromboembolic diseases.

Recently we reported a new type of antithrombotic compoundpossessing both thrombin inhibitory and GPIIb/IIIa receptor antag-onistic activities,11,12 and later systematically varied them in the P3part to improve their thrombin inhibitory activity and fibrinogenreceptor binding affinity. Whereas the resulting compounds 10and 11 (Scheme 1, Table 1) were all found to be submicromolarthrombin inhibitors, their IC50 values for antagonizing binding offibrinogen to GPIIb/IIIa were higher than 100 lM. Based on thewell known involvement of reactive oxygen species, lipid peroxi-dation and 5-lipoxygenase mediated production of proinflamma-tory leukotrienes in the pathology of thrombosis,4,14 as well asthe known antioxidant activities of some benzoxazine15 and mor-pholine16 derivatives, we screened thrombin inhibitors 10 and 11for their radical scavenging potential, inhibition of linoleic acidperoxidation, and inhibition of soybean lipoxygenase, to assesstheir multiple antithrombotic potential. The results demonstrate,surprisingly, that some of our compounds, in addition to thrombininhibition, display antioxidant, and anti-lipid peroxidation activitytogether with soybean lipoxygenase inhibition, which all mightcontribute synergistically to their antithrombotic potential andpave the way for a novel concept of antithrombotic therapy.

The synthesis of the target compounds is given in Scheme 1.Ethyl 2,4-dimethyl-6/7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxa-zine-2-carboxylates 3 were prepared by cyclization of 2-amino-4/

NH

O

O

CH3

COOEtOH

NH2

N

OCH3

CH3

O CNN

OCH3

CH3

O

baO2N O2N O2N

O2N

e

H2N

N

OCH3

CH3

O CNN

R

g

EtOOC

N

OCH3

CH3

O CNN

R

h

EtOOC

O

21

65

8

9 R = 3-F; 4-F; 3,5-d

Scheme 1. Reagents and conditions: (a) Br(CH3)C(COOEt)2, KF, DMF, 60 �C, 6 h; (b) MeIDIAD, THF, reflux, 48 h; (e) H2, Pd/C, THF, rt, 2 h; (f) fluorobenzaldehyde, NaBH(AcO)3, Cchloride, Et3N, THF, rt, overnight; (i) HCl(g), EtOH, 0 �C, 30 min, then NH4OAc, EtOH, rt, 2

5-nitrophenol 1 and subsequent N-methylation.17 Reduction of 3with borane dimethyl sulfide complex afforded alcohols 4 whichwere transformed to ethers 5 with 4-cyanophenol under Mitsun-obu reaction conditions.18 Catalytic reduction of the nitro groupin 5 afforded aromatic amines 6 that were N-alkylated, using var-ious fluorobenzaldehydes and sodium triacetoxyborohydride asreducing agent,12,19 to afford secondary amines 7. Alkylation of 7with ethyl bromoacetate afforded esters 8, and ketoesters 9 wereobtained by acylation of 7 with ethoxalyl chloride. Finally, com-pounds 10a–h and 11a–d were prepared from nitriles 8 and 9,using the Pinner reaction.20 Compounds with N-(benzyl)eth-oxalylamino substituents in position 7 (analogs of 10e–h; struc-tures not shown) were not tested since they could not beobtained pure due to their instability.

The results of biological assays performed with compounds 10and 11 are collected in Table 1. They were tested for thrombininhibition,13 as well as for their antioxidant activity and their abil-ity to inhibit soybean lipoxygenase in order to assess their anti-thrombotic potential. Taking into account the multifactorialcharacter of oxidative stress, we have evaluated the in vitro antiox-idant activity of the synthesized compounds using two differentantioxidant assays: (i) interaction with the stable 1,l-diphenyl-2-picrylhydrazyl free radical (DPPH)21, and (ii) interaction with thewater-soluble 2,20-azobis(2-amidinopropane) dihydrochloride(AAPH), which has been used extensively as a clean and controlla-ble source of thermally produced alkylperoxy free radicals.22 Theresults obtained were compared to the antioxidant potency of wellknown antioxidant agents, that is, caffeic acid, nordihydroguaiaret-

N

O

O

CH3

COOEt

CH3

c

N

OCH3

CH3

OH

CN N

OCH3

CH3

O CN

O2Nd

HNf

R

N

OCH3

CH3

ON

R

EtOOC

N

OCH3

CH3

ON

R

EtOOC

O

NH

NH

NH2

NH2

i

i

43

7

10

11ifluoro; 4,5-difluoro

, KF, DMF, rt, 12 h; (c) Me2S � BH3, THF, reflux, overnight; (d) 4-cyanophenol, PPh3,lCH2CH2Cl, rt, 6 h; (g) BrCH2COOEt, BTEAC, K2CO3, CH3CN, 60 �C, 24 h; (h) ethoxalyl4 h.

Table 1Thrombin inhibition, interaction with DPPH, inhibition of lipid peroxidation (LP) and in vitro inhibition of soybean lipoxygenase (LO) by compounds 10 and 11.g

No. Compound MW ClogPcalcd

ThrombinKi (lM)

% Reduction of DPPH activitya LP InhibitionIC50 (lM)b

LO Inhibition(percentd/IC50 (lM))e

50 lM 10 lM

20 min 60 min 20 min 60 min

10aN

OO

NH2

NH

N

OEt

F

O

520.6 5.64 0.90 ± 0.21 11 14 10 23 59 9.7d

10bN

O O

NH2

NH

N

OEt

OF

520.6 5.64 0.69 ± 0.21 11 14 11 24 77 73e

10cN

O O

NH2

NH

N

OEt

F

OF

538.6 5.71 0.66± 0.14 11 16 8 21 68 >0d

10dN

OO

NH2

NH

N

OEt

F

O

F

538.6 5.71 0.95 ± 0.20 8 10 8 18 36 41.7d

10eN

OO

NH2

NH

N

OEt

F

OO

534.6 4.97 0.30 ± 0.11 11 14 12 24 90 >0d

10fN

OO

NH2

NH

N

OEt

OOF

534.6 4.97 0.33 ± 0.08 10 12 10 21 51 >0d

10gN

O O

NH2

NH

N

OEt

F

OOF

552.6 5.04 0.37± 0.07 23 28 35 46 61.5 >0d

10hN

OO

NH2

NH

N

OEt

F

OO

F

552.6 5.51 0.29 ± 0.08 23 28 25 36 69 99e

11a

N

ONO

NH2

NH

EtO

F

O

520.6 5.64 0.35 ± 0.08 48 63 71 85 81 >0d

(continued on next page)

M. Ilic et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4705–4709 4707

Table 1 (continued)

No. Compound MW ClogPcalcd

ThrombinKi (lM)

% Reduction of DPPH activitya LP InhibitionIC50 (lM)b

LO Inhibition(percentd/IC50 (lM))e

50 lM 10 lM

20 min 60 min 20 min 60 min

11b

N

ONO

NH2

NH

EtO O

F

520.6 5.64 0.31 ± 0.08 62 70 88 93 94.5 >0d

11c

N

ONO

NH2

NH

EtO O

F

F

538.6 5.78 0.43 ± 0.09 70 81 84 90 70 >0d

11d

N

ON O

NH2

NH

EtO O

F

F538.6 5.78 0.48 ± 0.08 54 68 75 86 69 >0d

NDGAf 302.4 3.92 n.d. 68 81 83 83 n.d. n.d.Caffeic acid 180.2 0.82 n.d. n.d. n.d. n.d. 600e

Trolox� 250.3 4.34 n.d. n.d. 63% @ 100 lMc n.d.

a Percent reduction of DPPH optical absorption at 517 nm measured after 20 and 60 min at 50 and 10 lM DPPH final concentration.b IC50 for inhibition of linoleic acid peroxidation.c Percent inhibition of linoleic acid peroxidation at 0.1 mM inhibitor concentration,d Percent inhibition of soybean lipoxygenase.e IC50 for inhibition of soybean lipoxygenase.f NDGA: nordihydroguairetic acid.g Values are means (SD < 10%) of three or four different determinations; n.d. = not determined.

4708 M. Ilic et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4705–4709

ic acid (NDGA) and 6-hydroxy-2,5,7,8-tetramethychroman-2-car-boxylic acid (Trolox�), a water-soluble vitamin E derivative usedin biological and biochemical applications to reduce oxidativestress damage.

Compounds 10a–h and 11a–d displayed moderate to good lev-els of thrombin inhibition in vitro, with Ki values between 290 and900 nM. DPPH has been used for measuring radical scavengingability21 of the tested compounds. Because of its unpaired electron,DPPH has a strong absorption maximum at 517 nm, which is de-creased when the unpaired electron of the radical becomes pairedin the presence of a hydrogen donor. The interaction of the exam-ined compounds with the stable free radical DPPH (measured aspercent reduction in the optical absorption at 517 nm)22 indicatestheir radical scavenging ability in an iron free system. Comparingthe radical scavenging effectiveness of the tested compounds withthat of NDGA, only 7-substituted derivatives (compounds 11a–d),possessing a 1,4-diaminophenyl moiety, displayed high interactionvalues that increased in a time and concentration dependent man-ner. Compounds 10a–h showed low radical scavenging ability,which could be attributed mainly to the absence of easily oxidizedfunctionalities like those present in 11a–d and NDGA (two catecholmoieties). Comparing the interaction values for the couples of m-and p-isomers, it appears that p-F compound 11b is more potentthan 11a that possesses a m-F substituent. The presence of fluorinein the para-position appears to have a favorable effect on the inter-action with DPPH (11b and 11c). Of the 6-substituted oxo deriva-tives 10e–h, the difluoro derivatives were more potent radicalscavengers than their monofluoro analogs (cf. 10e–f vs 10g–h).Lipophilicity, expressed as theoretically calculated ClogP values,23

does not appear to account for the observed activity.In an assay for lipid peroxidation inhibition,24 a water-soluble

azo compound AAPH was used as free radical initiator to follow

oxidation of linoleic acid to conjugated diene hydroperoxide by UVspectrophotometry. The use of AAPH is recommended as moreappropriate for measuring radical-scavenging activity in vitro, be-cause the activity of the peroxyl radicals produced by the action ofAAPH shows a greater similarity to cellular activities such as lipidperoxidation. The results in Table 1 suggest that inhibition of lipidperoxidation is inherent to 6-substituted derivatives 10, amongwhich compounds 10a, 10d, and 10f showed higher inhibition of li-pid peroxidation values than Trolox�, which was used as a referenceinhibitor. Trolox exerts its inhibitory effect on lipid peroxidationmainly through the ability of its 6-hydroxy-5,7,8-trimethylchro-mane moiety to break the radical chain.25 Due to the similarity ofthe structures of compounds 10 to that of Trolox� (Fig. 1), the 6-ami-no-dihydro-1,4-benzoxazine moiety could be made responsible forgeneration of radicals effectively stabilized through resonance tobreak the radical chain reaction.

In the meta-fluoro analogs, the presence of an oxo group nextto the ethyl ester moiety decreased the lipid peroxidation inhib-itory potency [cf. compound 10a (IC50 = 59 lM) compared to10e (IC50 = 90 lM) and 10d (IC50 = 36 lM) compared to 10h(IC50 = 69 lM)]. In contrast, in the the p-F analog 10b exhibitingan IC50 of 77 lM, introduction of an oxo goup in the position ato the ester carbonyl to give compound 10f increased the lipidperoxidation inhibitory potency to IC50 of 51 lM. Lipophilicityagain does not seem to influence the results.

The synthesized compounds were evaluated further for theirability to inhibit soybean lipoxygenase (LO), using a UV absorbancebased enzyme assay.25 Although the results (Table 1) cannot be di-rectly extrapolated to the inhibition of mammalian 5-lipoxygenase,it has been shown that inhibition of plant lipoxygenase activity bynon-steroidal anti-inflammatory agents is qualitatively similar totheir inhibition of the rat mast cell lipoxygenase.25 The soybean

N

OCH3

CH3

ON

R

EtOOCX

NH

NH2

O

HO

COOH

CH3CH3

H3C

CH3

Trolox

10a-d: X = H, H10e-h: X = O

R = 3-F;4-F; 3,5-difluoro; 4,5-difluoro

Figure 1. Structures of Trolox� and compounds 10a–h.

M. Ilic et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4705–4709 4709

lipoxygenase inhibition assay may therefore be used as a simplequalitative screen for lipoxygenase inhibitory activity. Althoughmost of the LO inhibitors are antioxidants or free radical scaveng-ers,26 the relationship between LO inhibition and the ability of theinhibitors to chelate and reduce the active site Fe3+, or competewith arachidonic acid for binding to the enzyme active site, is wellknown.14 Perusal of the soybean LO inhibition values (Table 1)shows that, with the exception of 10b, 10d, and 10h, which displaybetter inhibition than caffeic acid, the standard inhibitor, othercompounds were devoid of activity. The active compounds werecharacterized by moderate values of interaction with AAPH, butlow interaction values with DPPH, showing that lipid peroxidationactivity is not always accompanied by DPPH radical scavengingability and vice versa.27 This can be attributed to the differentchemical reactions involved in the two assays. Thus, although com-pounds 11a–d interacted strongly with DPPH, they did not inhibitsoybean lipoxygenase. Although it appears that LO inhibition isconnected to the 6-substitution pattern, further experiments areneeded in order to explain the absence of activity in other deriva-tives of the 6-substituted series.

In conclusion, of the compounds 10a–h and 11a–d possessingthrombin inhibition activity, 11a–d were found to possess goodradical scavenging activity, 10a, 10d, and 10f inhibited lipid perox-idation of linoleic acid, and 10b, 10d, and 10h inhibited soybean

lipoxygenase activity. Compounds that combine in the same mole-cule thrombin inhibitory activity with lipid peroxidation andlipoxygenase inhibition would be expected to display a synergisticaction in the treatment of thrombosis. This makes compounds 10and 11 interesting candidates for further investigations towardsmultiple antithrombotic drugs.

Acknowledgments

This work was supported by Slovenian Research Agency GrantNo. P1-208. We wish to thank Dr. C. Hansch and Biobyte Corp.,201 West 4th Str., Suite 204, Claremont, CA 91711, USA, for free ac-cess to the C-QSAR program and Dr. Roger Pain from J. Stefan Insti-tute, Ljubljana for critical reading of the manuscript.

References and notes

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