Pteridine-2,4-diamine derivatives as radical scavengers and inhibitors of lipoxygenase that can possess anti-inflammatory properties

Background Reactive oxygen species are associated with inflammation implicated in cancer, atherosclerosis and autoimmune diseases. The complex nature of inflammation and of oxidative stress suggests that dual-target agents may be effective in combating diseases involving reactive oxygen species. Results A novel series of N-substituted 2,4-diaminopteridines has been synthesized and evaluated as antioxidants in several assays. Many exhibited potent lipid antioxidant properties, and some are inhibitors of soybean lipoxygenase, IC50 values extending down to 100 nM for both targets. Several pteridine derivatives showed efficacy at 0.01 mmol/kg with little tissue damage in a rat model of colitis. 2-(4-methylpiperazin-1-yl)-N-(thiophen-2-ylmethyl)pteridin-4-amine (18f) at 0.01 mmol/kg exhibited potent anti-inflammatory activity (reduction by 41%). Conclusion The 2,4-diaminopteridine core represents a new scaffold for lipoxygenase inhibition as well as sustaining anti-inflammatory properties.

Oxidative stress is closely associated with chronic inflammation and plays a crucial role in cancer [1], dyslipidemia [2], atherosclerosis [3] and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis [4]. In many diseases, the rate of production of reactive oxygen species (ROS) is increased compared with normal levels of ROS [5,6]. ROS are produced during the inflammatory process by phagocytic leukocytes that invade the tissue. Under conditions of oxidative stress, ROS including superoxide anion, hydroxyl radical and hydrogen peroxide and their reactive products can attack various biological macromolecules (e.g., proteins, enzymes, DNA and lipids) resulting in DNA mutations, lipid peroxidation and protein oxidation, [7] or they may indirectly interfere with mechanisms of DNA repair [8]. Thus, ROS activity consists of a mixture of deleterious and beneficial roles, depending on the type, concentration and location of the species involved. The breadth of those factors suggests that focused targeting of ROS, probably in a dual-target or multipletarget approach, could be of therapeutic value.
Reactive oxygen species are centrally involved in the cyclooxygenase (COX)-and lipoxygenase (LOX)-mediated conversion of arachidonic acid (AA) into proinflammatory intermediates [9,10]. LOX exerts its biological role via a carbon-centered radical mechanism. LOX metabolism results in many bioregulatory molecules such as leukotrienes, lipoxins and hepoxylins, mediators in the pathophysiology of variety of diseases such as rheumatoid arthritis, bronchial asthma, psoriasis, cancer and other inflammatory diseases [11].
In the search for new antioxidants and anti-inflammatory agents, the pteridine ring ( Figure  1) was selected for study for several reasons: many naturally-occurring derivatives possess important biological activity, including the coenzymes 5,6,7,8-tetrahydrobiopterin and the pterins; the ring system has low toxicity; synthetic pteridine derivatives show a wide range of clinically useful properties, for example, as antioxidants, immunosuppressants, and antiinflammatory and anticancer agents. Consequently, multitarget properties were deemed likely, as were the discovery of new agents for a range of diseases, and also the use of pteridine derivatives to probe the putative targets of diseases.
In addition to the treatment of a variety of cancers, the antifolate methotrexate reduces inflammation in the bowel and is used for treating Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis and other autoimmune diseases. Therefore, novel compounds bearing the pteridine ring could exhibit anti-inflammatory and antioxidant activities and have potential as therapy for a wide range of diseases involving inflammation. The 2,4diaminopteridine core of methotrexate (Figure 1), was used as the scaffold in this study. Alkylamino and dialkylamino substituted 2,4-diaminopteridines were investigated since many have a range of drug-like clogP values (1.0-4.0, Table 1) and because synthesis should afford the required structural diversity. We describe here the synthesis and preliminary evaluation of some novel N-alkylated 2,4-diaminopteridine derivatives as dual-target agents through their radical scavenging properties, inhibition of lipoxygenase and in vivo antiinflammatory activity.
For dissimilar substitution at the 2-and 4-positions of pteridine-2,4-diamine derivatives, a stepwise introduction of those substituents was required. 2,4-dichloropteridine [24] and its derivatives [25] react regioselectively at the 4-position with one equivalent of amine, which makes this approach unsuitable for preparing a range of pteridines with the same 2-amino substituent. Additionally, the use of 2,4,6,7-tetrachloropteridine for related successive displacements with different amines, while succinct, afforded extensive mixtures and with isolation of desired single regioisomers usually in low yields, after chromatography [14]. Since the formation of pteridines from 5-nitrosopyrimidines had proved robust and had delivered single regioisomers, this approach was adopted, in which the 4-amino group would be introduced by displacement of 4-amino-6-chloro-2-(methylthio)pyrimidine 14 ( Figure 5). However, displacement of the chloro group had been reported only for pyrimidine 14, using dimethylamine to install a 4-dimethylamino group [26]. 4,6-Diamino-2methylthiopyrimdines such as 15 would be required, and would be obtained by displacement of 14 with benzylamine derivatives or with heteroarylmethylamines ( Figure 5). Displacement of the 2-methylthio group in 16 by a secondary amine would give the unsymmetrically substituted 2,4,6-triamino-5-nitrosopyrimidines, and hence the corresponding pteridines 18.
4-Amino-6-chloro-2-(methylthio)pyrimidine 14 reacted with a variety of amines (2.1 equivalent) in diglyme at reflux to give the corresponding pyrimidines 15a-f [27] which underwent 5-nitrosation with acidic aqueous sodium nitrite ( Figure 5) [23]. The 2methylthio group of the resulting 5-nitrosopyrimdines 16a-f underwent displacement with 1-methylpiperazine or 4-methyl-1,4-diazepane in ethanol at reflux to give the corresponding 2-amino derivatives 17a-f. Those were subjected to the above reduction with sodium dithionite followed by condensation with aqueous 40% glyoxal to give the pteridine derivatives 18a-g. Compounds 20a and 20b were prepared analogously, the latter by using 4-methyl-1,4-diazepane in ethanol at reflux to give 19b. 1 H NMR spectra of the nitroso compounds 16, 17 and 19 in chloroform are consistent with the presence of two rotamers arising from hydrogen bonding between the C-5 nitroso oxygen atom and the adjacent NH hydrogen atoms at C-4, and also with an NH hydrogen atom at C-6, as established for related pyrimidines [28][29][30].
A representative 5-nitrosopyrimidine, 17f, was transformed into three 6,5-fused systems ( Figure 6) in a brief survey of the relevance of the fused pyrazine ring in the pteridine series. The triazolo[4,5-d]pyrimidine 21 was obtained by hydrogenation of 17f to the corresponding amine, nitrosation and subsequent ring closure at 90°C, following standard methods [31]. The 5-nitrosopyrimdine 17f was also converted into 22 by sodium thiosulfate [32], and into 23 by lead tetraacetate [33]. Synthetic procedures for the new pteridine derivatives are Pontiki

In vitro lipoxygenase inhibition
The substituted pteridines prepared were assayed for inhibition of soybean lipoxygenase (Table 1) [34,35] (Supplementary Information). Many studies have used readily obtainable soybean lipoxygenase, which is a homologue of mammalian lipoxygenase and well examined [36,37]. The availability of soybean LOX and its well-characterized crystal structure [38] led to its use in this study.

Molecular modeling of LOX
Being the most potent inhibitor of soybean LOX of the compounds studied and also possessing efficacy as an antioxidant, pteridine derivative 18d was selected for in silico docking. The molecular modeling study performed (see Supplementary Information for details) provided useful interpretation of the experimental results. The preferred docking orientation for compound 18d is shown in Figure 7. The binding of 18d to soybean LOX (PDB code: 3PZW) has a higher AutoDock Vina score (-8.5 kcal/mol) than any of the other pteridines docked. Pteridine 18d is able to accommodate the extensively hydrophobic cavity close to the active site, incorporating Ile552, Ile553, Ile538 and Leu546 among other residues. Ile553 and especially Leu496 are proximate to the hydrophobic 6,7-flank of the pteridine ring, Ile553 also extending to the hydrophobic C4-C6 region of the pyridine ring in 18d. The increased potency of 18d over its phenyl analog 18a is considered to be due to hydrogen binding, perhaps to Ser747. The simplest explanation is that the extension scaffold of 18dinto the hydrophobic domain blocks approach of substrates to the active site, and hence prevents oxidation by soybean LOX. The docking simulations of NDGA and 18d show a common pattern of interaction with LOX ( Supplementary Figure 1), the terminal rings and central core of each compound showing appreciable overlap. Additionally, Ser747 is engaged in hydrogen bonding with the 3-hydroxyl group of the catechol unit of NDGA, and also with the pyridine nitrogen atom of 18d. The relatively weak antioxidant properties of 18d (Table 2) are also consistent with the main mode of action of LOX inhibition being other than by diminishing general ROS concentrations and hence antioxidant activity [40]. Preliminary screening tests of the pteridines against COX did not present any significant inhibition.

In vitro antioxidant activity
Previous work has shown that pterins, their dihydro-and their tetrahydro-derivatives can each be antioxidants or pro-oxidants, depending on the particular conditions [43]. In the present study, several assays were used to assess in vitro antioxidant activity in order to obtain representative information; each method involves the generation of a different radical. The three assays chosen measured in vitro antioxidant activity in terms of: reduction of the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), whose oxidized form possesses an absorption maximum at 517 nm; hydroxyl radical scavenging activity; extent of reduction of the water-soluble 2,2′-azo-bis(2-amidinopropane) dihydrochloride (AAPH) and inhibition of soybean lipoxygenase (Supplementary Information).
Competition of the novel pteridine derivatives with dimethyl sulfoxide for hydroxyl radicals was measured. Hydroxyl radicals were generated using the Fe3+/ascorbic acid system and expressed as a percentage inhibition of formaldehyde production in the presence of each pteridine derivative at 100 μM (Table 2) [34]. Pteridine derivatives 5a, 10b and 18g strongly inhibited the oxidation of dimethyl sulfoxide (33 mM). The majority of the derivatives were excellent scavengers of hydroxyl radicals with activity higher than the reference compound 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox).
Azo compounds that generate free radicals through spontaneous thermal decomposition are useful for in vitro studies of free radical production. The water-soluble AAPH has been extensively used as a clean and controllable source of thermally produced alkylperoxyl free radicals [44]. In this assay, compound 18g (IC 50 = 0.1 μM) was the most potent in protecting against lipid peroxidation; next, and almost equipotent, were the pteridines 13 and 18e, and the triazolo [4,5-d] being moderate, the 1,4-diazepane derivative 20b being poor and the 2-(4-methylpiperazin-1-yl) derivative 18f being very weak. Evidently, the presence of 6,7-dimethyl groups on the pteridine ring greatly enhances protection against lipid peroxidation; the nature of the alkyl group and ring size on the 2-substituent has some, but much less, effect. Of the 4aminopteridines, the 6,7-diphenyl derivative 5c was by far the most potent inhibitor, whereas of the 2,4-bis(4-methylpiperazin-1-yl) derivatives, 13 is more than twice as potent as the 2,4-(3-hydroxydipiperidin-1-yl derivative 9. Reduction of the pteridine ring in compound 9 to the corresponding 5,6,7,8-tetrahy-dropteridines 10a and 10b decreased protection of lipid peroxidation, with potency decreased by 13-fold and 20-fold, respectively. Conversely, reduction of the pteridine ring in the 4-(4-methylpiperazin-1-yl) derivative 18f to give 18i increased potency by a factor of about 2.5. In the 6,5-fused heteroaromatic compounds studied, 22 and 24 showed similar and potent inhibition of lipid peroxidation whereas compound 23 was half as potent. The role of lipophilicity (as assessed from calculated clogP values; Table 1) is not clear, but substituent bulk plays a significant role.

In vivo anti-inflammatory activity
LOX has also been associated with inflammation and ulcerative colitis [45]. In the present study, a model for colitis involving intracolonic administration of aqueous 4% acetic acid in the rat was used, leading to acute inflammatory reaction [46]. Treated rats presented partial to diffuse petechial bleeding, single erosion and limited ulceration indicating an overall healing effect of the compounds. Substituted pteridines presenting a satisfactory combination of activities 5a, 18a, 18d and 18f were tested using this in vivo model characterized by diffuse exfoliated mucosa as well as multiple and extended erosion and  (Table 3). No mortality was encountered. Pteridine 5a (score 1-2) was the most potent in this series, followed by its 4-(N-benzyl) analog, 18a. Rats treated with 5a or 18a showed less loss in body weight compared with the control group.
6-chloro-2-(4-methylpiperazin-1-yl)-N-(thiophen-2-ylmethyl)quinazolin-4-amine possesses in vivo anti-inflammatory properties in the rat [47], so on the basis of its close structural analogy with pteridine 18f, that latter was tested for anti-inflammatory effects using the carrageenin paw edema model ( Table 4). The incipient pattern of this edema is characterized by the effects of histamine and 5-hydroxytryptamine. After 1 h, reduction of edema in the rat paw achieved by the pteridine 18f was appreciably greater than the reduction induced by the reference compound indomethacin, a nonselective COX-inhibitor and commonly used nonsteroidal anti-inflammatory drug. Thus, 18f and some related pteridines offer significant protection against reactive oxygen species produced in a model of colitis, probably on account of their properties as antioxidants and radical scavengers.

Conclusion
A general synthetic approach to N, N,N′-trialkylated and N,N,N′N′-tetraalkylated 2,4diaminopteridines has been described. 2,4-diaminopteridine derivatives have been identified as a new and promising class of radical scavengers, anti-inflammatory agents and inhibitors of LOX. Potent inhibitors of soybean lipoxygenase include 9, 10a and especially 18d (IC 50 = 0.1 μM). To our knowledge, 2,4-aminopteridine is a novel scaffold for LOX inhibitors, although the extent of any LOX isoform selectivity remains to be established. Many of the pteridine derivatives studied displayed potent radical-scavenging activity, especially 5c, 9, 13, 18e, 18g, 22 and 24, of which 18g is the most potent (IC 50 = 0.1 μM) in the linoleic acid peroxidation assay. Several pteridine derivatives showed efficacy at 0.01 mmol/kg with little tissue damage in a rat model of colitis. 2-(4-methylpiperazin-1-yl)-N-(thiophen-2ylmethyl)pteridin-4-amine (18f) at 0.01 mmol/kg showed 60% greater reduction of edema in rat paw than that achieved by the anti-inflammatory agent indomethacin, a nonselective COX-inhibitor. Accordingly, this study demonstrates that some pteridine derivatives have at least a dual-target action. These results prompt a more detailed structural, mechanistic and medicinal investigation of substituted 2,4-diaminopteridines, whose therapeutic potential might lead to new agents for the treatment of inflammatory bowel disease, among other inflammatory diseases.

Future perspective
Inflammation is a multifactorial phenomenon that is implicated in a wide range of diseases. Enhanced formation of ROS by phagocytic leukocytes during the process of inflammation leads to tissue dysfunction and damage in a number of pathological conditions. ROS oxidize lipids generating peroxides and aldehydes that have pronounced biological effects including damage to DNA and protein, selective alterations in cell signaling and cytotoxicity [42].
Oxidative stress evidently plays a crucial role in those processes.
Given the importance of radical species in inflammation there is an unmet and timely need for new radical-scavenging agents. In addition, multiple-target anti-inflammatory agents

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have potential for the control of a range of diseases including arthritis, cancer and atherosclerosis. The wide-ranging biological activities of pteridine derivatives, including the reduction in reperfusion injury by the 4-amino analog of tetrahydrobiopterin [48], suggest that pteridines may find therapeutic applications in unexplored or little-charted areas. New pteridine derivatives could also be of value as probes of specific biological oxidation.

Supplementary data
Refer to Web version on PubMed Central for supplementary material.

Executive summary
• Reactive oxygen species are associated with inflammation implicated in cancer, atherosclerosis and autoimmune diseases. The pteridine ring, suitably substituted, offers a diverse array of biological activity with potential for the treatment of those diseases.  Table 3 In vivo colitis studies † .
Future Med Chem. Author manuscript; available in PMC 2017 June 07.  Table 4 Inhibition of carrageenin-induced rat paw edema.