WO2014055972A2

WO2014055972A2 - A new chemical entity useful for treating various diseases

Info

Publication number: WO2014055972A2
Application number: PCT/US2013/063616
Authority: WO
Inventors: Theodore Holman; David Maloney; Ganesha Rai Bantukallu; Ajit Jadhav; Anton Simeonov; Steven L. KELLY
Original assignee: The Regents Of The University Of California
Priority date: 2012-10-05
Filing date: 2013-10-07
Publication date: 2014-04-10
Also published as: WO2014055972A3

Abstract

Novel chemical entities that inhibit the activity of human 5-lipoxygenase (5-LOX).

Description

PATENT COOPERATION TREATY OFFICE

INTERNATIONAL PATENT APPLICATION

Title: A new chemical entity useful for treating various diseases

Inventors

Theodore Holman (UCSC) Santa Cruz, CA

David Maloney (NIH) Bethesda, MD

Ganesha Rai Bantukallu (NIH) Bethesda, MD

Ajit Jadhav (NIH) Bethesda, MD

Anton Simeonov (NIH) Bethesda, MD

Steven L Kelly (Swansea University, UK)

Attorneys for the applicant

BELL & ASSOCIATES

58 West Portal Avenue # 121 , San Francisco, California 94127

info@bell-iplaw.com, PTO Customer No. 039843

Statement of support

This invention was made with support of National Institutes of Health, GM56062 (TRH) and S10- RR20939 (UCSC MS Equipment grant). The government has certain rights in the invention.

Assignee

The Regents of the University of California, Office of Technology Transfer, 1 11 1 Franklin Street, 5th floor , Oakland, California 94607 USA

Relation to other applications

This application claims priority to and the benefit of US provisional application No. 61/710,505 filed 05 October 2012 titled A new chemical entity useful for treating various diseases

[001 ] Field of the invention

The invention concerns lipoxygenase (LOX) inhibitors.

[002] Background

[003] Lipoxygenases, such as Human 5-lipoxygenase (5-LOX) has long been considered a possible therapeutic target for inflammatory diseases. Asthma is the principle disease target, however, numerous other diseases have been postulated in the literature as possible targets for 5-LOX inhibition, such as allergic rhinitis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, atherosclerosis, ischemia-reperfusion injury, atopic dermatitis and acne vulgaris. 5-LOX has also been implicated in another skin disease, seborrheic dermatitis (i.e. dandruff). Disclosed are novel compounds that target human 5-lipoxygenase (5-LOX).

[004] Brief description of the drawings

[005] Figure 1 is a graph showing enzyme activity: 1 Λ/max vs. concentration of inhibitor (μΜ) [006] Figure 2 is a graph showing enzyme activity: Km/Vmax vs. concentration of inhibitor (μΜ)

[007] Brief description of the invention

[008] Disclosed are a novel compounds that target human 5-lipoxygenase (5-LOX). These compounds also target fungal CYP51 . The novel compounds are derived from Phenylenediamine, an organic compound with the formula C₆H₄(NH²)2.

[009] Novel phenylenediamine derivatives were created that display highly selective, non- chelative, reductive inhibition of 5-LOX.

[0010] For one derivative, the phenylenediamine core has been translated into the ketoconazole (a widely used anti-fungal agent) structure, generating a novel compound ("Ketaminazole" - Structure 16) which demonstrates dual CYP51 /5-LOX inhibitory properties. Ketaminazole exhibits improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. This new chemical entity, which combines anti-inflammatory and antifungal activities, is presented as a possible novel therapeutic against both the fungal and inflammatory causes of disease.

[001 1 ] A series of potent 5-LOX inhibitors containing a phenylenediamine core, was synthesized the members of which exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1 , and epithelial 15- human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2.

[0012] The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound,

"ketaminazole".

[0013] Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM) and fungal sterol 14odemethylase (Erg1 1 or CYP51 ) (IC50 = 43 nM) in vitro. In addition, ketaminazole selectively inhibits yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. Ketaminazole was tested in human leukocytes and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 μΜ.

[0014] The invention encompasses various compounds and methods.

[0015] Embodiments include a novel compound that inhibits the activity of fungal CYP51 and of 5- lipoxygenase (5-LOX), compound comprising at least the following structure or variants and derivatives of the same:

[0016] Ketaminazole may comprise at least the following novel structure herein called "structure 16".

[0017] In various embodiments the compound exhibits potent inhibition against the activity of both human 5-LOX (IC₅₀ = 700 nM) and fungal sterol 14a-demethylase (Erg1 1 or CYP51 ) (IC₅₀ = 43 nM) in vitro; and that selectively inhibits yeast CYP51 relative to human CYP51 by 17-fold; and that further down- regulates LTB4 synthesis, displaying 45% inhibition at 10 μΜ.

[0018] Another embodiment includes a method of treating a disease by inhibiting 5-LOX, the method comprising administering to a subject the compound "ketaminazole" comprising at least structure 16, or variants and derivatives thereof.

[0019] The disease treated may include any disease. For example it may include a disease where inhibition of the activity of CYP51 and/or 5-lipoxygenase (5-LOX) is desirable and where these enzymes play a role in pathogenesis.

[0020] Various examples of diseases that may be treated with the disclosed compounds include asthma, allergic rhinitis, hayfever, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, atherosclerosis, ischemia-reperfusion injury, atopic dermatitis, acne vulgaris and seborrheic dermatitis (dandruff) or a disease where one symptom of the disease treated is a type II hypersensitivity reaction. Embodiments include a method for treating any of the above diseases, the method comprising administering to a subject the compound "ketaminazole" comprising at least structure 16, or variants and derivatives thereof.

[0021 ] The above list is exemplary and not exclusive and the compounds and methods of the invention may be used to treat and number of suitable diseases.

[0022] In other embodiments the same modification that is performed on ketoconazole to make it a 5-lipoxygenase inhibitor may be applied to other compounds such as azole antifungal compounds, including triazole antifungal compounds.

[0023] For example itraconazole and posaconazole are both antifungals and may be converted into 5 Lox inhibitors using the same methods used here for ketoconazole . Fluconazole, voriconazole and posaconazole may be similarly used. Many other therapeutics could also be made into dual inhibitors by this method and we claim the use of the phenylenediamine moiety as a modification for adding 5- LOX inhibitory potency to any known therapeutic agent.

[0024] The invention also embodies a novel class of compounds comprising a phenylenediamine moiety. Specifically one embodiment is a 5-LOX inhibitory therapeutic compound comprising a phenylenediamine moiety. Another specific embodiment includes an itraconazole derivative comprising a phenylenediamine moiety. Another specific embodiment includes an posaconazole derivative comprising a phenylenediamine moiety.

[0025] Detailed description of the invention

[0026] Human 5-lipoxygenase (5-LOX) has long been considered a possible therapeutic target for inflammatory diseases. Asthma is the principle disease target, however, numerous other diseases have been postulated in the literature as possible targets for 5-LOX inhibition, such as allergic rhinitis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, atherosclerosis, ischemia- reperfusion injury, atopic dermatitis and acne vulgaris^1"6. The role of 5-LOX in the latter disease, acne vulgaris, has been shown to be related to the production of sebum in the derma⁷. 5-LOX has also been implicated in another skin disease, seborrheic dermatitis (i.e. dandruff)⁸. Dandruff is a common chronic skin disorder that affects sebum-rich areas and shares some features with psoriasis and atopic dermatitis. The pathogenesis of dandruff is complex and appears to result from interactions between scalp skin, cutaneous microflora and the cutaneous inflammation⁹. Recently, three inflammation biomarkers (IL-1 alpha, IL-1 RA and IL-8) were associated with development of dandruff⁹. Of these markers, IL-8 has been shown to be induced by the production of leukotriene B4 (LTB4), indicating the involvement of 5-LOX in the cause of dandruff, since LTB4 is a product of 5-LOX. See Figure 1 .

"Structures of LOX inhibitors".

[0027] Ketoconazole is a widely used anti-fungal agent that is currently utilized as an active ingredient in anti-dandruff shampoo. Its mode of action is by inhibiting fungal sterol 14a-demethylase (Erg1 1 or CYP51 ) during ergosterol biosynthesis, thus retarding fungal growth. However, it has been proposed that part of its effectiveness is due to anti-inflammation activity, since it also weakly inhibits 5- LOX. The anti-inflammatory effect of ketoconazole has also been seen for itraconazole, a similar antifungal thereapeutic, which suggests a common theme for effective dandruff agents, dual anti- fungal/anti-inflammatory targeting. Nevertheless, the potency for ketoconazole and itraconazole against 5-LOX is poor, with IC₅₀ values greater than 50 μΜ for both molecules, which indicates a potential for improvement in their anti-dandruff activity.

[0028] Numerous inhibitors for 5-LOX have been found, which can be generally classified into three categories; reductive, iron ligands and competitive/mixed inhibitors (Figure 1 ), however, only one compound has been approved as a drug, Zileuton. Zileuton is a potent and selective 5-LOX inhibitor but its mode of action is unusual for a therapeutic. It contains an N-hydroxyurea moiety, which is proposed to chelate to the active enzyme's ferric ion and reduce it to the inactive ferrous ion. In general, chelation/reduction is not considered a viable mode of inhibition for a therapeutic since metal chelation tends toward promiscuous behavior with other metalloproteins and reductive inhibitors can be chemically inactivated in the cell. Nevertheless, Zileuton has been shown to not only be selective against 5-LOX but also efficacious in the cell, which presents this class of inhibitor as a viable chemotype for 5-LOX inhibition. Other chelative inhibitors, such as nordihydroguaiaretic acid (NDGA) are also reductive due to the facile nature of inner sphere electron reduction. NDGA contains a catechol moiety, which binds to the active site ferric ion, reducing it to the ferrous ion, with the concomitant oxidation of the catechol moiety to the semiquinone. This reactivity has previously been seen with the metalloenzyme, catechol dioxygenase, whose catechol substrate is activated to the semiquinone by the active site ferric ion for oxidation by molecular oxygen. There is also a sub-classification of reductive inhibitors that do not chelate the active site iron. The mechanism for these inhibitors is most likely long- range electron transfer, but no direct proof has been found for this mechanism. Recent efforts by the pharmaceutical industry have focused on non-reductive inhibitors of 5-LOX (see Figure 1 ; MK-0633 and PF-4191834), however, these appear to have been discontinued from during Phase II clinical trials.

[0029] In this disclosure, phenylenediamine derivatives are presented as highly selective, non- chelative, reductive inhibitors towards 5-LOX. For one derivative, the phenylenediamine core has been translated into the ketoconazole structure, generating a novel compound which demonstrates dual CYP51 /5-LOX inhibitory properties.

[0030] This new chemical entity, which combines anti-inflammatory and antifungal activities, is presented as a possible novel therapeutic against both the fungal and inflammatory causes of disease.

[0031 ] Results/Discussion

[0032] A novel 5-LOX inhibitor chemotype, phenylenediamine, was discovered while screening for LOX inhibitors in our lab.

[0033] Due to its chemical nature, the mode of inhibition was postulated to be due to reduction of the active site ferric atom. Reductive inhibition of lipoxygenase is a very effective mode of action, with many reductive inhibitors having sub-micromolar IC₅₀ values and is indicative of both the ease of which the active site ferric ion can be reduced and the importance of the oxidation state of this ion.

[0034] With this in mind, the phenylenediamine parent compound (1 ) was modified to change its reduction potential (Table 1 ). Modifications of the phenylenediamine core, such as atom substitutions of the nitrogens with carbon or oxygen (2 and 3, respectively), or the insertion of two additional nitrogen atoms into the core phenyl of the phenylenediamine (4, 5, and 6), induced complete loss of inhibitory potency. Interestingly, substitution of only one nitrogen into the core phenyl ring (6) did not lower potency dramatically, nor did methylation of the nitrogen (7).

[0035] Table 1. Representative analogues evaluated for esaudojptroxidase activity ^a.

Compound Seductive- I ^ ( Μ}

Activit ·■ S IM:}

The UV-Vis-bassd manual inhibition data (3 rep!icates) were fit as described in the methods section.

[0036] The pseudoperoxidase assay, which requires a reductive inhibitor, was subsequently conducted with these inhibitors to establish their reductive activity against 5-LOX (Table 1 ). From this data, it was demonstrated that the pseudoperoxidase activity paralleled their inhibitor potency, consistent with changes in the reductive potential of the inhibitors. Alterations of the core

phenylenediamine structure were used in a similar manner to determine the relationship between potency and reductive properties. For comparison, Zileuton and Setileuton were screened as positive controls, with Zileuton being reductive and Setileuton being non-reductive, in their mechanism of inhibition. See Table 1 "Representative analogues evaluated for superoxidase activity".

[0037] Interpreting IC₅₀ values for reductive inhibitors is challenging because their relative potency is dependent on a combination of both their reactivity with the active site iron and their binding affinity. The binding affinity was therefore investigated by changing the substituents on either side of the phenylenediamine core. As seen in Table 2, the chemotype core tolerated a large range of modifications, such as changing the steric bulk on either side of the phenylenediamine core.

[0038] Table 2. 5-LOX inhibition of Representative Analogues ^a.

3 1.5 13.23

10' ϊ^'°,ν - >180

11 ^' [l 1.t |β,1β]

« ¾-0"* 2.6 [^β.¾

13 ^ ^- ».5a [0J.7]

14 ^ί^^ί >-C* 6.33 £0,67}

15 9.6 [0.13

³ The UV-Yis-based manual in ibi ion data (3 replicates} were fit as described In the methods section.

[0039] The only modification in this small set that showed a greater than 10-fold decrease in potency, 10, which was surprising given the activity of related oxazoles 8 and 9. The relative lack of potency dependence on inhibitor structure suggests that the active site can accommodate a variety of inhibitor shapes and sizes, consistent with the large size of the 5-LOX active site and the relatively large 5- LOX inhibitors, previously discovered.

[0040] Steady state kinetics were conducted with compound 13, by monitoring the initial formation rate of 5-HPETE as a function of substrate and inhibitor concentration in the presence of 0.01 % Triton X-100. Plotting K_M/V_max and 1 /V_max versus inhibitor concentration yielded linear plots, with K, equaling 0.58 (±0.27) and K,' equaling 1 .72 (±1 .37) μΜ, which are defined as the equilibrium constants of dissociation from the enzyme and enzyme substrate complex, respectively (Supporting information, Figures S1 and S2). These values reflect a mixed-type inhibition, which is common for lipoxygenase inhibitors. Considering that 13 is a reductive inhibitor, as seen by the pseudoperoxidase assay, it is interesting to note that the catalytic site inhibition constant (K,), is similar in magnitude to the IC₅₀ value of 13 (0.57 (±0.07) μΜ). See Table 2. "5-LOX inhibition of Representative Analogues ^".

[0041 ] Selectivity of the inhibitor chemotype was evaluated by screening a variety of LOX isozymes with a small subset of compounds (Table 3). Strong selectivity was displayed against 5- LOX relative to the other isozymes, with selectivity starting from 80-fold for 12-LOX, 75-fold for 15- LOX -1 , and 30-fold for 15- LOX -2 (Table 3).

[0042] The chemotype also displayed strong selectivity when assayed against cyclooxygenase (COX), with a 140-fold selectivity versus COX-1 , and a 240-fold selectivity versus COX-2. These combined results indicate this chemotype has a strong preference/selectivity against 5- LOX versus other arachidonic acid processing enzymes. As controls, Setileuton and Zileuton were utilized as selective inhibitors of 5-LOX, whereas baicalein is a non-selective inhibitor.

[0043] A few inhibitors were then tested for efficacy in whole human blood, which is known to express 5-LOX upon activation by an ionophore. 1 and 13 displayed roughly 50% inhibiton at 10 μΜ drug dosing in the whole blood, while the positive control, Setileuton, was found to inhibit 100% at 10 μΜ (Table 4). Compound 15 was also tested, but the potency was shown to be weak, with less than 10% inhibition at 10 μΜ (Table 4). The cellular inhibition values for 1 , 13 and Setileuton are diminished relative to the isolated-enzyme inhibitor values (Table 1 ). This result, along with other analogues failing to display high potency, could indicate either non-specific interactions or metabolism of the inhibitors by the cell.

[0044]

[0045] The determination that the reductive phenylenediamine core was the key potency component and the fact that addition of large functionalities to either side of the phenylenediamine core were well tolerated led us consider the similarity between the phenylenediamine chemotype and ketoconazole (Table 5). Ketoconazole is a CYP51 inhibitor with an azole that targets the active site heme and is a potent antifungal medication. In addition, ketoconazole was previously determined to inhibit 5-LOX, although weakly. Considering the similarity of ketoconazole to our chemotype, we hypothesized that this low potency was most likely due to the absence of the phenylenediamine core, which can not reduce the active site ferric ion in 5-LOX, as was seen for 3. Thus, we modified the structure of ketoconazole to include a diamine core to generate a novel compound, ketaminazole (16) and found that its potency against 5-LOX increased over 70-fold, compared to ketoconazole by it becoming a reductive inhibitor, as seen by its activity in the pseudoperoxidase assay (Table 5).

[0046]

fi U ~ te >¾$es3 jiiJa ¾ f«tfe «a (3 f p«es were is as- sescf:¾sd ^ t »i

[0047] The selectivity of the ketaminazole (16) was also investigated and found to preferentially inhibit 5-LOX over 100 times better than that of 12-LOX, 15-LOX-1 , 15-LOX-2, COX-1 and COX-2 (Table 5). This is most likely due to the large active site of 5-LOX compared to the other human LOX isozymes. Ketaminazole (16) was also tested in whole human blood and shown to display cellular activity. Its potency is lower relative to in-vitro assay, displaying an approximately 20-fold reduction in potency, which is a similar reduction in potency as seen with Setileuton (Table 4). Ketaminazole (16) displayed a better potency in whole blood relative ketoconazole, however, the difference was not as great as their in vitro data would have indicated. Further studies in seborrheic dermatitis model systems are required to probe this further. See Table 5. IC50 values (μΜ).

[0048] In addition to kinetic data, the importance of the phenylenediamine core for reductive inhibition was further verified using computational methods. Molecular modeling of possible inhibitor binding modes within the active site was initiated by deprotonation of the amine groups at the phenylenediamine core and energy minimization of the compounds with LigPrep. The inhibitors listed in all the Tables above were then docked against the crystal structure of modified protein, Stable-5-LOX (308Y), using Glide's "XP" (extra-precision) mode. Different trials, with varying van der Waals scaling factors and alternating positional or hydrophobic constraints linking the inhibitor to the active site, resulted in the occurrence of high-ranking binding poses depicting the deprotonated amine nitrogen within 10 angstroms of the catalytic iron, for several inhibitors. The docking results of these inhibitors support the hypothesis that the reduction of the ferric iron could be caused by the phenylenediamine core, either through an inner sphere or outer sphere mechanism. Docking of the larger inhibitors, ketoconazole and ketaminazole (16), generated poses with similar Glide docking scores to the other inhibitors studied, suggesting a comparable binding mode despite the differences in IC₅₀ values. In several high-ranking binding poses, the amine/ester core of ketaminazole (16) was observed to be within 5 angstroms of the catalytic iron, supportive of the hypothesis that the phenylenediamine core reduces the active site iron. See figure: "Docking ketoconazole and ketaminazole to the crystal structure of the Stable-5-LOX".

[0049] The docking poses of the phenylenediamine inhibitors not only suggest the amine as a potential conduit of iron reduction, but they also suggest the active site iron-hydroxide moiety could possibly abstract a hydrogen atom from the amine by an inner sphere mechanism, as is seen in the natural mechanism of LOX with its fatty acid substrate. To test this hypothesis, 13 was incubated in buffer constituted with D₂0 to deuterate the phenyldiamine core amine, and its IC₅₀ value compared to the protonated amine, in H₂0. A 2.4-fold increase in the IC₅₀ for 13 was observed in D₂0, which is well below the kinetic isotope effect expected for hydrogen atom abstraction, suggestive of a proton independent outer sphere reductive mechanism. To further verify this proton-independent reductive mechanism, 1 and 7 (containing the protonated and methylated amine, respectively) were also investigated and both were shown to have similar increases in IC₅₀ values in D₂0 relative to H₂0, suggesting the effect does not involve the amine proton.

[0050] In order to evaluate the concept of improved 5-LOX inhibition for an anti-inflammatory effect combined with antifungal potency, we examined the effect of ketoconazole and ketaminazole (16) for selectivity against the human and C. albicans CYP51 proteins, HsCYP51 and CaCYP51 respectively. Binding ketoconazole and ketaminazole (16) with both CaCYP51 and HsCYP51 produced strong type II difference spectra (Figure 2) signifying direct coordination as the sixth ligand of the heme prosthetic group of CYP51 . [0051 ] Ketoconazole and ketaminazole (16) both bound tightly to CaCYP51 with K_d values of 27 (±5) and 43 (±5) nM, respectively. Tight binding is observed when the K_d for the ligand is similar to or less than the concentration of CYP51 present⁴⁶. The similar K_d values obtained for ketoconazole and ketaminazole (16) suggest both azoles would be equally effective as antifungal agents against wild-type CaCYP51 . This is understandable since the CYP51 potency of this class of molecules is predominantly due to their azole moiety, which is quite distant from phenylenediamine core of ketaminazole (16). T [0052] his data also compares with K_d values of 10 to 50 nM previously obtained for clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole and voriconazole with CaCYP51⁴⁷. Ketoconazole bound 17-fold more tightly to HsCYP51 (K_d = 42 (±16) nM) compared to ketaminazole (16) {K_d = 731 (±69) nM), in contrast to the 1 .6-fold difference observed with CaCYP51 , suggesting that ketaminazole would interfere less with the host HsCYP51 and possible other human CYPs than ketoconazole, conferring a therapeutic advantage. This compares with K_d values of <100 nM for clotrimazole, econazole and miconazole, -180 nM for ketoconazole and -70 μΜ for fluconazole with HsCYP51 .

[0053] The IC₅₀ CYP51 reconstitution assay results (Fig. 3) mirrored those of the azole binding results. CaCYP51 was strongly inhibited by both ketoconazole and ketaminazole (16) with IC₅₀ values of -0.5 and -0.9 μΜ, respectively, confirming that both azoles bound tightly to CaCYP51 . Interestingly, at 4 μΜ ketaminazole (16), CaCYP51 retained -15% CYP51 activity suggesting that lanosterol can displace ketaminazole (16) from CaCYP51 leading to ketaminazole (16) being a less effective inhibitor of fungal CYP51 enzymes in vitro than ketoconazole. HsCYP51 was less severely inhibited by both ketoconazole and ketaminazole (16) with IC₅₀ values of -5 and -16 μΜ, respectively, indicating azole binding was less tight and suggested lanosterol can displace ketoconazole and especially ketaminazole (16) from HsCYP51 . At 95 μΜ ketoconazole HsCYP51 was inactivated in contrast to the -30% CYP51 activity remaining in the presence of 155 μΜ ketaminazole (16).

CaCYFSI ami HisCYF51, ws &imfy.

lA kl iuMi

[0054] The 3-fold higher IC₅₀ value of ketaminazole (16) over ketoconazole with HsCYP51 confirmed that ketaminazole (16) would be less disruptive to the CYP51 function of the host homolog than ketoconazole, conferring a therapeutic advantage for use as an antifungal agent. It should be noted that both itraconazole and posaconazole, both effective anti-fungal agents, could also have a phenylenediamine incorporated into their structures, thus conferring dual anti-fungal/anti-inflammatory properties on these therapeutics as well. We suggest and claim the use of the phenylenediamine moiety as a simple modification for adding 5-LOX inhibitory potency to known therapeutics.

[0055] The fact that ketoconazole is both an anti-fungal and anti-inflammatory molecule is not a new phenomena in the field of anti-fungal therapeutics. Previously, we determined that the common anti-fungal agent, chloroxine, was also a non-specific LOX inhibitor⁴⁹. This fact suggested that the inherent selection process for the search for anti-seborrheic dermatitis agents could be responsible for the dual nature of the anti-fungal/anti-inflammatory therapeutics, such chloroxine and ketoconazole. With this hypothesis in mind, the anti-fungal agent, ciclopirox (trade name Loprox), presented a structure that could be interpreted as a LOX inhibitor, with the N-hydroxyamide being a possible chelator. This was confirmed and ciclopirox was found to be both a potent inhibitor to 5-LOX (IC50 = 1 1 (±1 )) and selective versus other AA processing enzymes (Table 5). This dual nature of many anti- seborrheic dermatitis agents suggest that improving the 5-LOX potency of these therapeutics may be beneficial in their clinical efficacy. Figure 2. Binding properties of ketoconazole and ketaminazole with CaCYP51 and HsCYP51 . See Figure 2. Binding properties of ketoconazole and ketaminazole with CaCYP51 and HsCYP51 , and Fig 3. Figure 3. Determination of IC₅₀ values for ketoconazole and ketaminazole with CaCYP51 and HsCYP51 .

[0056] Conclusion

[0057] In conclusion, the current data indicates that the phenylenediamine chemotype is a robust inhibitor against 5-LOX, which demonstrates high potency, enzyme selectivity and cellular activity. The mechanism of action is via the reduction of the active site ferric ion, similar to that seen for Zileuton, the only FDA approved LOX inhibitor. It is interesting to note, that unlike Zileuton, which chelates the iron through the N-hydroxyurea, the phenylenediamine chemotype lacks an obvious chelating moiety, thus differentiating it from Zileuton. Structural modification around the phenylenediamine core was well- tolerated, however, even relatively minor changes to the phenylenediamine moiety resulted in a loss of activity, presumably due to changes in its reduction potential. This attribute was utilized to modify the structure of ketoconazole to include the phenylenediamine moiety and produce a novel inhibitor, ketaminazole (16).

[0058] This novel compound demonstrated a 40-fold increase in potency against 5-LOX, comparable potency against fungal CYP51 , and improved selectivity against the human CYP51 , relative to ketoconazole. This novel dual nature of ketaminazole (16), both anti-fungal and antiinflammatory activity, could potentially have therapeutic uses for anti-seborrheic dermatitis therapy.

[0059] Methods

[0060] Overexpression and Purification of 5-Human Lipoxygenase, 12-Human Lipoxygenase, and the 15-Human Lipoxygenases. Human reticulocyte 15-lipoxygenase-1 (15-LOX-1 ) and human platelet 12-lipoxygenase (12-LOX) and human prostate epithelial 15-lipoxygenase-2 (15-LOX-2) were expressed as N-terminally, His6-tagged proteins and purified to greater than 90% purity. Human leukocyte 5-lipoxygenase was expressed as a non-tagged protein and used as a crude ammonium sulfate protein fraction, as published previously.

[0061 ] Lipoxygenase UV-Vis-based Manual Assay. The initial one-point inhibition percentages were determined by following the formation of the conjugated diene product at 234 nm (ε = 25,000 M^" ¹cm^"1) with a Perkin-Elmer Lambda 40 UV-vis spectrophotometer at one inhibitor concentration. All reactions were 2 mL in volume and constantly stirred using a magnetic stir bar at room temperature (23°C) with approximately 40 nM for 12-LOX, 20 nM of 15-LOX-1 (by iron content). Reactions with the crude, ammonium sulfate precipitated 5-LOX were carried out in 25 mM HEPES (pH 7.3), 0.3 mM CaCI2, 0.1 mM EDTA, 0.2 mM ATP, 0.01 % Triton X-100, 10 μΜ AA and with 12-hLO in 25 mM Hepes buffer (pH 8.0), 0.01 % Triton X-100, and 10 μΜ AA. Reactions with 15-LOX-1 and 15-LOX-2 were carried out in 25 mM Hepes buffer (pH 7.5), 0.01 % Triton X-100, and 10 μΜ AA. The concentration of AA (for 5-LOX and 12- LOX) and LA (for 15-LOX-1 ) were quantitatively determined by allowing the enzymatic reaction to go to completion. IC₅₀ values were obtained by determining the enzymatic rate at various inhibitor concentrations and plotted against inhibitor concentration, followed by a hyperbolic saturation curve fit. The data used for the saturation curves were performed in duplicate or triplicate, depending on the quality of the data. It should be noted that all of the potent inhibitors displayed greater than 80% maximal inhibition unless stated in the tables. Inhibitors were stored at -20°C in DMSO. As a result of screening with a semi-purified protein there was concern whether the 5-LOX concentration was approaching the inhibitor concentration for our most potent inhibitors, which would affect the Henri- Michaelis-Menten approximation. In order to investigate whether the enzyme concentration was approaching the IC₅₀ value we compared our IC₅₀ values of two high potency 5-LOX inhibitors to that in the literature. Setileuton displayed an IC₅₀ value of 60±6 nM, in good agreement with the literature value of 45±10 nM, and Zilueton displayed an IC₅₀ value of 560±80 nM, in good agreement with the literature value of 500±100 nM (2,13). The solvent isotope effect of the inhibitor IC₅₀ was investigated utilizing the same conditions and methods as stated above. The pH of the buffered D₂0 was dealt with as done previously.

[0062] Steady-State Inhibition Kinetics. Lipoxygenase rates were determined by monitoring the formation of the conjugated product, 5-HPETE, at 234 nm (e = 25 000 M^"1 cm^"1) with a Perkin-Elmer Lambda 40 UV/vis spectrophotometer. Reactions were initiated by the addition of 5-LOX to a constantly stirring 2mL cuvette containing 40 μΜ AA in 25 mM HEPES (pH 7.3), 0.3 mM CaCI2, 0.1 mM EDTA, 0.2 mM ATP, at varied inhibitor concentrations in the presence of 0.01 % Triton X-100. The substrate concentration was determined by allowing the enzymatic reaction to proceed to completion. Kinetic data were obtained by recording initial enzymatic rates, at varied inhibitor concentrations, and subsequently fitted to the Henri-Michaelis-Menten equation, using KaleidaGraph (Synergy) to determine the microscopic rate constants, Vmax (μιτιοΙ/iTiin/iTig) and Vmax/KM (μιτιοΙ/ιτπη/ιτ^/μΜ). These rate constants were subsequently replotted, 1/V_max and K_M/V_max versus inhibitor concentration, to yield K, and K, , respectively.

[0063] Cyclooxygenase Assay. Ovine COX-1 (Cat. No. 60100) and human COX-2 (Cat. No. 60122) were purchased from Cayman chemical. Approximately 2 g of either COX-1 or COX-2 were added to buffer containing 100 μΜ AA, 0.1 M Tris-HCI buffer (pH 8.0), 5 mM EDTA, 2 mM phenol and 1 μΜ hematin at 37 °C. Data was collected using a Hansatech DW1 oxygen electrode chamber, as described before⁵³. Inhibitor or vehicle were mixed with the respective COX in buffer within the electrode cell, the reaction initiated by the addition of arachidonic acid, followed by monitoring of rate of oxygen consumption. Ibuprofen, aspirin and or indomethacin, and the carrier solvent, DMSO, were used as positive and negative controls, respectively. [0064] Human Blood LTB4 Inhibition Assay. Whole human blood was dispensed in 150 uL samples followed by addition of inhibitor or control (vehicle, DMSO), and incubated for 15 min at 37 °C. The mixture was then stimulated by introduction of the calcium ionophore, A23817, (freshly diluted from a stock 50 mM DMSO stock to 1 .5 mM in Hanks balanced salt solution), and incubated for 30 min at 37 °C. Samples were then centrifuged at 1 ,500 rpm (300g) for 10 min at 4 °C and the supernatant diluted between 20-50-fold (batch dependent) for LTB4 detection, using an ELISA detection kit (Cayman Chemicals Inc.). Inhibitors were added at 10 M or 15 M concentrations (0.5 M for control setilueton)^54"56, IC₅₀ values were generated using a one point IC₅₀ estimation equation.

[0065] Pseudoperoxidase activity assay. The reductive properties of the inhibitors were determined by monitoring the pseudoperoxidase activity of lipoxygenase in the presence of the inhibitor and 13-HPODE. Activity is characterized by direct measurement of the product degradation following the decrease of absorbance at 234 nm using a Perkin-Elmer Lambda 40 UV/Vis spectrometer (50 mM Sodium Phosphate (pH 7.4), 0.3 mM CaCI2, 0.1 mM EDTA, 0.01 % Triton X100, 10 μΜ 13-HPODE). All reactions were performed in 2 ml_ of buffer and constantly stirred with a rotating stir bar (22 °C).

Reaction was initiated by addition of 10 M inhibitor (a 1 to 1 ratio to product), and a positive result for activity reflected a loss of greater than 40% of product absorption at 234 nm. The control inhibitors for this assay were Setilueton and Zilueton, known non-reductive and reductive inhibitors respectively.

[0066] Inhibitor modeling. Grid generation and flexible ligand docking were performed using Glide, while energy minimization and ligand preparation of inhibitors was done with LigPrep. LigPrep and Glide are both products of Schrodinger, Inc., and utilize energy functions to generate and rank models of ligand 3D structures and ligand-protein interactions, respectively. The crystal structure of stable human 5-lipoxygenase (PDB ID: 308Y) was used to generate a Glide grid in which to carry out docking algorithms with our inhibitors. This structure contains several point mutations that remove destabilizing sequences, but since none of these are located at the active site of the enzyme, we find it reasonable to assume the mutant structure holds as an accurate model of the wildtype active site. Positional constraints at the catalytic iron and at hydrophobic pockets within the active site were prepared and utilized intermittently during different docking calculations. Poses generated from ligand docking were ranked according to their GlideScores.

[0067] CYP51 protein studies. C. albicans CYP51 (CaCYP51 ) and Homo sapiens CYP51 (HsCYP51 ) proteins were expressed in E. coli using the pCWori⁺ vector, isolated and purified as previously described to over 90% purity. Native cytochrome P450 concentrations were determined by reduced carbon monoxide difference spectra⁵⁷ based on an extinction coefficient of 91 mM^"1 cm^"1.

Binding of azole antifungal agents to 5 μΜ CaCYP51 and 5 μΜ HsCYP51 were performed as previously described using 0.25 and 0.5 mg ml^"1 stock solutions of ketoconazole and ketaminazole in

DMSO. Azole antifungal agents were progressively titrated against CYP51 protein in 0.1 M Tris-HCI (pH 8.1 ) and 25% (wt/vol) glycerol, with the spectral difference determined after each incremental addition of azole. The dissociation constant (K_d) of the enzyme-azole complex was determined by nonlinear regression (Levenberg-Marquardt algorithm) of

against azole concentration using a rearrangement of the Morrison equation⁶¹ fitted by the computer program ProFit 6.1 .12

(QuantumSoft, Zurich, Switzerland).

[0068] IC₅₀ determinations were performed using the CYP51 reconstitution assay system previously described containing 1 μΜ CaCYP51 or 0.3 μΜ HsCYP51 , 2 μΜ human cytochrome P450 reductase, 50 μΜ lanosterol, 50 μΜ dilaurylphosphatidylcholine, 4.5% (wt/vol) 2-hydroxypropyl-3- cyclodextrin, 0.4 mg ml^"1 isocitrate dehydrogenase, 25 mM trisodium isocitrate, 50 mM NaCI, 5 mM MgCI₂ and 40 mM MOPS (pH -7.2). Azole antifungal agents were added in 5 μΙ dimethylsulfoxide followed by 5 minutes incubation at 37 °C prior to assay initiation with 4 mM 3-NADPHNa₄ with shaking for a further 10 minutes at 37 °C. Sterol metabolites were recovered by extraction with ethyl acetate followed by derivatization with N, O bis(trimethylsilyl)trifluoroacetamide and tetramethylsilane prior to analysis by gas chromatography mass spectrometry⁶⁴. IC₅₀ in this study is defined as the inhibitor concentration required causing a 50% inhibition of the CYP51 reaction under the stated assay conditions.

[0069] In Vivo studies

[0070] The compounds of the invention have been shown to inhibit 5-lipoxygenase in human macrophages. Since we are hoping to develop topical 5-lipoxygenase inhibitors an animal skin model is also used. Mice have a similar inflammatory response to humans and the arachidonic acid mouse ear inflammatory assay is well established as the easiest method for measuring anti-5-lipoxygenase activity in the skin.

[0071 ] An inflammatory stimulant (AA) is applied to the left ear of a cohort of five mice in one cage, and the inhibitor/AA is applied to the right ear. The same ear is always used for inhibitor/AA application. The ear thickness is measured after one hour. The animals will be held by hand to apply the reagent to the ear, which takes less than a minute. After an hour, the thickness of the ears is measured again. Approximately 10 μΙ_ of arachidonic acid in acetone is applied to the inside and outside of one of the mouse's ear. When testing the 5-lipoxygenase inhibitors, an inhibitor/acetone solution will be applied 30 minutes prior to application of the arachidonic acid solution. Once the AA is applied, the ear will be measured 60 minutes later and measured at the apex of the pinna and approximately 5 mm from the apex. After the procedure, the mouse is replaced in his cage. The measurement of the ear thickness is done with an Oditest caliper. The main stress to the animal is from the handling, and a low level of discomfort to the ear.

[0072] General Representations Concerning the Disclosure [0073] Abbreviations. LOX, lipoxygenase; 5-LOX, human 5-lipoxygenase; 12-LOX, human platelet 12-lipoxygenase; 15-LOX-1 , human reticulocyte 15-lipoxygenase-1 ; 15-LOX-2, human epithelial 15- lipoxygenase-2; soybean LOX-1 , soybean lipoxygenase 1 ; COX-1 , ovine cyclooxygenase-1 ; COX-2, human cyclooxygenase-2; Erg1 1 , fungal sterol 14a-demethylase; CYP51 , human sterol 14a- demethylase; CaCYP51 , C.albicans CYP51 ; HsCYP51 , H. sapiens CYP51 ; 5-HPETE, 5-(S)- hydroperoxyeicosatetraenoic acid; 5-HETE, 5-(S)-hydroxyeicosatetraenoic acid; LA, linoleic acid; 13- HODE, 13-(S)-hydroxyoctadecadienoic acid; LTB4, leukotriene B4; IL-1 alpha, interleukin-1 alpha; IL- 1 RA, interleukin-1 receptor antagonist; IL-8, interleukin-8; V_max, the rate constant of the maximal velocity; V_max/K_M, the rate constant for fatty acid capture; IC₅₀ inhibitor concentration at 50% enzyme inhibition; change in cytochrome P450 spectra; K_d, dissociation constant.

[0074] This specification incorporates by reference all documents referred to herein and all documents filed concurrently with this specification or filed previously in connection with this application, including but not limited to such documents which are open to public inspection with this specification.

[0075] In various structural formulae, the undesignated substituent groups may be, for example, an alkyl or an aryl group, or a hydrogen group, an organic acid, and amine group or a halide or a hydrogen. 'Aryl' refers to any functional group or substituent derived from an aromatic ring, be it phenyl, naphthyl, thienyl, indolyl, etc. In certain embodiments the undesignated group(s) is/are selected from, for example, the following: hydrogen, hydroxyl, carboxylate, alkane, alkene or alkyne groups, substituted or unsubstituted heteroatom, alkyl, alkenyl, alkanoyl, aryl, aroyl, aralkyl, alkylamino cycloalkyl, heterocycloalkyl, heteroaryl, or halogen, azido, fluorophore or polypeptide. In certain embodiments the substituent group may comprise branched or un-branched C1 -C18 alkyl, C1 -C18 substituted alkyl, C1 -C18 alkenyl, C1 -C18 acyl, amino, substituted amino, wherein the alkyl, alkenyl or acyl is linear or branched, and optionally substituted with a hydroxyl, an ester and its derivatives, 5 a carboxyl and its derivatives. In a particular embodiment, Any R group may be a lower hydrocarbon substituted with alkoxy, substituted alkoxy, imidate, arylthio, or (substituted aryl)thio. In other embodiments, Any R group may be a lower alkyl selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, terabutyl and pentyl. In other embodiments, Any R group may be a lower alkenyl selected from vinyl, substituted vinyl, ethynyl, or substituted ethynyl. In other embodiments, Any R group may be a lower alkanoyl selected from formyl, acetyl, propionyl, isopropionyl, butyryl, isobutyryl, tert-butyryl, valeryl, pivaloyl, caproyl, capryl, lauryl, myristyl, palmityl, stearyl, arachidyl, stilligyl, palmitoyl, oleyl, linolenyl, and arachidonyl. In other embodiments, Any R group may be lower aryl selected from phenyl, p-tolyl, pchlorophenyl, p-aminophenyl, p-nitrophenyl, p-anisyl. In yet other embodiments, Any R group may be a lower aroyl selected from benzoyl and naphthoyl. In other embodiments, Any R group may be a lower aralkyl selected from benzyl, benzhydryl, p-chlorobenzyl, m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, or pentaflourobenzyl. In certain other embodiments, Any R group may be a lower alkylamino is selected from monoalkylamino, monoaralkylamino, dialkylamino, diaralkylamino, and benzylamino. [0076] Although the present disclosure refers frequently to seborrhoeic dermatitis therapy, the compounds and methods of the invention may be directed towards any disease in which inhibition of either fungal CYP51 or human 5-lipoxygenase (5-LOX) would be useful or desirable.

[0077] The term "substituent" refers to an atom or group of atoms substituted in place of a hydrogen atom on the parent molecule.

[0078] The term "derivative" or "derivative compound" or "derivatized compound" refers to a compound having a chemical structure that contains a common core chemical structure as a parent or reference compound, but differs by having at least one structural difference, e.g., by having one or more substituents added and/or removed and/or substituted, and/or by having one or more atoms substituted with different atoms.

[0079] A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May- 07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides.

[0080] The terms "formulation", "drug formulation or "pharmaceutical formulation," refers to a drug combined with a non-drug such as a carrier material designed not to have a pharmaceutical activity, such as pharmaceutical excipient, filler, or carrier material that may be used to modify or improve the drug release, improve its physical and/or chemical stability, dosage form performance, processing, manufacturing, etc.

[0081 ] The terms "drug" or "therapeutic agent" mean any substance meant to affect the physiology of a subject. Examples of drugs are described in well-known literature references such as the Merck Index and the Physicians' Desk Reference.

[0082] The term "therapeutically effective amount" means an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent, effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the formulation to be administered, and a variety of other factors that are appreciated by those of ordinary skill in the art.

[0083] As used in this specification, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a part" includes a plurality of such parts, and so forth. The term "comprises" and grammatical equivalents thereof are used in this specification to mean that, in addition to the features specifically identified, other features are optionally present.

Claims

1. A Composition that specifically inhibits the activity of 5-lipoxygenase (5-LOX), the compound comprising a phenylenediamine moiety.

2. The Composition of claim 1 comprising a reductive phenylenediamine core.

3. The Composition of claim 2 exhibiting a half-maximal inhibitory concentration (IC50) against 5-LOX of no more than 700 nM.

4. The Composition of claim 3 that further specifically inhibits the activity of fungal CYP51 .

5. The Composition of claim 2 com rising the following structure

wherein R1 , R2, R3 and R4 may, each individually, be a Hydrogen or an alkyl or aryl moiety.

6. The Composition of claim 5 that exhibits potent inhibition against the activity of both human 5-LOX (IC50 = 700 nM) and fungal sterol 14a-demethylase (Erg1 1 or CYP51 ) (IC50 = 43 nM) in vitro; and that selectively inhibits yeast CYP51 relative to human CYP51 by at least 17-fold; and that further down-regulates LTB4 synthesis, displaying 45% inhibition at 10μΜ.

7. The Composition of claim 5 further comprising an antifungal compound wherein the

phenylenediamine core has been translated into the antifungal compound.

8. The Composition of claim 7 comprising derived from phenylenediamine and ketoconazole, wherein a phenylenediamine core has been translated into ketoconazole.

9. The Composition of claim 8 comprising the following structure (compound 16, 'ketaminazole' ):

10. The Composition of claim 7 comprising an azole antifungal compound.

11 . The Composition of claim 7 comprising a triazole antifungal compound.

12. The Composition of claim 7 comprising a phenylenediamine core translated into a compound selected from the group consisting of ketoconazole, itraconazole, posaconazole, fluconazole, and voriconazole.

13. A method for enhancing or providing 5-LOX inhibitory activity in a compound, the method comprising translating a phenylenediamine moiety into the compound so as to provide a core having the following structure,

14. The method of claim 13 for enhancing or providing 5-LOX inhibitory activity in a compound, wherein the compound is an antifungal compound, and wherein the phenylenediamine core is translated into the antifungal compound.

15. The method of claim 14 for enhancing or providing 5-LOX inhibitory activity in a compound wherein a phenylenediamine core is translated into ketoconazole to provide the following structure (compound 16, 'ketaminazole' ):

16. The method of claim 14 for enhancing or providing 5-LOX inhibitory activity in a compound, wherein the compound is an azole or triazole antifungal compound.

17. The method of claim 14 for enhancing or providing 5-LOX inhibitory activity in a compound, wherein the compound is selected from the group consisting of ketoconazole, itraconazole, posaconazole, fluconazole, and voriconazole.

18. A method for treating a disease by inhibiting 5-LOX, the method comprising (1 ) identifying a subject having a disease treatable by inhibition of 5-LOX, (2) administering to said subject a Composition that specifically inhibits the activity of 5-lipoxygenase (5-LOX), the composition comprising a phenylenediamine moiety translated into an antifungal compound to produce the following structure,

19. The method of claim 18 for treating a disease wherein the antifungal compound is an azole or triazole antifungal compound.

20. The method of claim 18 for treating a disease wherein the antifungal compound is selected from the group consisting of ketoconazole, itraconazole, posaconazole, fluconazole, and voriconazole.

21 . The method of claim 18 wherein the antifungal compound is ketoconazole and the Composition that specifically inhibits the activity of 5-lipoxygenase (5-LOX) has following structure (compound 16, 'ketaminazole' ):

22. The method of claim 18 wherein R1 , R2, R3 and R4 may, each individually, be H or a linear or cyclic hydrocarbon moiety wherein the disease is selected from the group consisting of: a type II hypersensitivity reaction, asthma, allergic rhinitis, hayfever, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, atherosclerosis, ischemia-reperfusion injury, atopic dermatitis, acne vulgaris and seborrheic dermatitis (dandruff).