Patent Application
20190263765
A new class of compounds has been developed that exhibits cooperative activity with warfarin to quadruple the anticoagulant activity of the coumarin, while not exhibiting significant anticoagulant activity when administered alone.
Various coumarin anticoagulant compositions, and in particular, warfarin, have long been utilized in different human and animal modalities, e.g., for the treatment of atrial fibrillation in humans, and as the active principle of rodenticide baits. However, as a result of the continued use of the compound warfarin as a poison for, in particular, rats and mice, rodents have become less susceptible to the effects of warfarin. In recent years, this resistance to coumarin anticoagulants has also begun to emerge with the second-generation coumarin anticoagulants such as brodifacoum.
The coumarin anticoagulants act by inhibiting the enzyme VKORC1 (Vitamin K Oxidoreductase Complex 1), which is the only enzyme in mammals that is capable of reducing the oxidized form of vitamin K, vitamin K epoxide, to vitamin K itself VKORC1 is therefore a susceptible bottleneck in the vitamin K cycle (
By inhibiting this enzyme, these anticoagulants prevent the recycling of vitamin K, and they thus slow the production of mature clotting factors (e.g., thrombin) from their precursors (e.g, prothrombin) by reducing the total amount of the active form of vitamin K available to the animal. The inhibition of VKORC1 by coumarin anticoagulants is competitive because the anticoagulation can be reversed by high doses of vitamin K. However, the effectiveness of the vitamin K rescue mechanism depends markedly on the coumarin anticoagulant itself: warfarin is cleared from the body in approximately 72-96 hours, while the clearance of (the much more lipophilic) brodifacoum requires months, necessitating long-term treatment with vitamin K to prevent death.
Therefore, it is desirable to develop a composition that can be utilized either as a substitute or as a complement to existing anticoagulant compositions, such as warfarin, to increase the effectiveness of the anticoagulant. It is also desirable to develop a composition that will decompose to inactive compounds following the death of the rodent, or that has a relatively short half-life to permit rescue of accidental intoxication of non-target animals (e.g., fish, family pets, raptors and scavengers, small children). It is also desirable to develop a new composition that will act by a mechanism other than the competitive inhibition of VKORC1 so that this mechanism of resistance to coumarin anticoagulants may be circumvented. One possible alternative target for inhibition is the cytochrome P450, polymorph CYP 2C9, which oxidized warfarin to the inactive 7-hydroxywarfarin. By inhibiting this enzyme, the oxidation of warfarin to inactive 7-hydroxywarfarin may be slowed or completely stopped, resulting in a net increase in the circulating concentration in the blood at the same oral dosage (
Our studies to date have elucidated the probable course of the initial metabolism of these compounds in the stomach and duodenum of the animal. UWEC-K2, the active principle when co-administered with warfarin, is metabolized to a diol under acidic conditions (0.1 M HCl in 50% aqueous acetone, mimicking stomach acid) and to a cyclopropyl ketone on brief exposure to base (potassium carbonate in methanol, mimicking the duodenum). These outcomes are summarized in
Extensive experiments with UWEC-K2 and warfarin under a wide range of conditions has not yielded significant amounts of the conjugate proposed previously. See Stromich, J. J.; Weber, A. K.; Mirzaei, Y. R.; Caldwell, M. D.; Lewis, D. E. Bioorg. Med. Chem. Lett. 2010, 20, 1928-1932, hereby incorporated by reference. Thus, the dominant pathway of action may not be as previously proposed.
According to one aspect of the present invention, a composition has been developed that, when utilized in combination with warfarin or 7-hydroxywarfarin, acts to greatly improve the anticoagulant effects of the coumarin in mammalian subjects.
The composition includes a compound or mixture of compounds obtained by hydrolysis of an epoxy-substituted 1,4-naphthalenediol diester derived from a Diels-Alder adduct of 1,4-naphthoquinone and a substituted cyclopentadiene or 1,3-cyclohexadiene, and having general structures I and II:
where R is a hydrogen or saturated or unsaturated alkyl group with up to 6 carbons; and where R1 , R2 , R3, R4 and R5 are hydrogen, alkyl, alkoxy, trialkylsilyl or trialkylsilyloxy; and
R1 is a hydrogen or a saturated or unsaturated alkyl group with up to 6 carbons;
R2 is a hydrogen or a saturated or unsaturated alkyl group with up to 6 carbons;
R3 is a hydrogen or a saturated or unsaturated alkyl group with up to 6 carbons; or
R4 is a hydrogen or a saturated or unsaturated alkyl group with up to 6 carbons; or
R5 is a hydrogen or a trialkylsilyl group (trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl);
or
where R1 and R4 comprise a substituted or unsubstituted, saturated or unsaturated α,ω-alkylene group with up to four carbons; and
R2 is a hydrogen or saturated or unsaturated alkyl group with up to 6 carbons;
R3 is a hydrogen or saturated or unsaturated alkyl group with up to 6 carbons; or
R5 is a hydrogen or a trialkylsilyl group (trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl);
or
where R3 and R4 comprise a substituted or unsubstituted, saturated or unsaturated α,ω-alkylene group with up to four carbons; and
R1 is a hydrogen or a saturated or unsaturated alkyl group with up to 6 carbons;
R2 is a hydrogen or a saturated or unsaturated alkyl group with up to 6 carbons;
R5 is hydrogen or a trialkylsilyl group (trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl).
The hydrolysis mixture contains as a major component, a compound based on the 4,5-benzotricyclo[6.2.1.02,10]undec-4-en-3,6-dione (III) or the 4,5-benzotricyclo[6.2.2.02,11]-dodec-4-en-3,6-dione (IV):
where R1, R2 , R3 and R4 are hydrogen, alkyl, alkoxy, silyl or silyloxy; and
R1 is a hydrogen or saturated or unsaturated alkyl group with up to 6 carbons;
R2 is a hydrogen or saturated or unsaturated alkyl group with up to 6 carbons;
R3 is a hydrogen or saturated or unsaturated alkyl group with up to 20 carbons; or
R4 is a hydrogen or saturated or unsaturated alkyl group with up to 20 carbons; or
R1 and R4 are part of a cyclic or polycyclic ring system; or
R3 and R4 are part of a cyclic or polycyclic ring system.
and where X is a heteratom group chosen from:
an alcohol;
an ether, OR4, where R4 is a hydrogen or saturated or unsaturated alkyl group with up to 6 carbons;
an ester of a carboxylic acid with up to six carbon atoms;
a halide (bromide, chloride or iodide);
a sulfonate ester (methanesulfonate, benzenesulfonate, p-toluenesulfonate, p-bromobenzenesulfonate, p-nitrobenzenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate);
an amine, NR5R6, where R5 and R6 are hydrogen or an alkyl group with up to 6 carbons;
and a coumarin-based anticoagulant or metabolite thereof with the general structure
where R7 is a substituted alkyl group with up to 6 carbons; and
A is a group chosen from an alcohol, an ester, an ether, an ester, or a ketone; and
where R8 is a substituted alkyl group with up to 6 carbons; and
B is a an aryl group (phenyl, biphenyl, furyl, 4′-bromo-4,1′-biphenyl);
or where R7 and R8 constitute a cycloalkyl group with up to 7 carbons; and
A is a group chosen from an alcohol, an ester, an ether, an ester, or a ketone; and
B is a an aryl group (phenyl, biphenyl, furyl, 4′-bromo-4,1′-biphenyl); and
where X is hydrogen or hydroxy; and
where Y is hydrogen or hydroxy.
The invention consists of a two-component synergistic mixture of a cyclopropyl ketone and a coumarin anticoagulant or its metabolite. The compound designated UWEC-K2 is prepared from the Diels-Alder adduct of 1,4-naphthoquinone and cyclopentadiene by the method of Lewis, et al. See Lewis, D. E.; Caldwell, M. D. “Synergistic anticoagulant composition.” U.S. Pat. No. 8,765,982 B2 (Jul. 1, 2014), hereby incorporated by reference. The treatment of UWEC-K2 with potassium carbonate in methanol for a short period (not more than 30 minutes) results in the formation of the cyclopropyl ketone. The cyclopropyl ketone in
The epoxide is essential for UWEC-K2 activity. In one model, it is postulated that the synergistic anticoagulant activity involves the transesterification of warfarin sodium by UWEC-K2. This process will lead to ring opening of the epoxide to give the cyclopropyl ketone, which makes the otherwise unfavorable equilibrium for the acetyl transfer irreversible (UWEC-K1 does not exhibit this type of biological activity). It is postulated that the products of the transesterification (warfarin acetate and the cyclopropyl ketone) are not anticoagulants, which then provides a reasonable rationalization of the observation that the anticoagulant activity of warfarin (and the anticoagulant activity of UWEC-K1) is reversed (
It follows from this that the hydrolysis of the warfarin acetate at a later time may lead to the generation of a bolus of warfarin that leads to the much higher levels of anticoagulation observed previously. See Stromich, J. J.; Weber, A. K.; Mirzaei, Y. R. ; Caldwell, M. D.; Lewis, D. E. Bioorg. Med. Chem. Lett. 2010, 20, 1928-1932, hereby incorporated by reference. Warfarin acetate is also more lipophilic than warfarin itself, which may increase its half-life in the animal, also rendering it a more potent anticoagulant.
Another model that rationalizes the observed synergistic anticoagulant activity involves an intriguing possibility based on the typical activity of cytochrome P450. In particular, if the cyclopropyl ketone is bound in the active site of the cytochrome, then CYP 2C9 could oxidize this to a free radical that undergoes the normal cyclopropylcarbinyl to homoallyl radical isomerization to generate a naphthoquinone and a simple alkyl radical (
What is particularly enticing is the fact that this free radical could easily react with warfarin or, much more interestingly, 7-hydroxywarfarin, to give a coupled product that may well be a super inhibitor of CYP 2C9 (
The available evidence about this enzyme is that it has a large active site that can exist on an “open” form and a “closed” form. The X-ray crystal structures show that both active sites can accommodate a second molecule in addition to the warfarin. We suggest that the opening to the active site, however, is such that only one molecule at a time may enter, and that once the UWEC-K2 metabolite and the 7-hydroxywarfarin enter the active site of the enzyme, they actually become covalently bonded to each other. This results in a molecule that is too big to exit the active site, and thus leads to inactivation of the enzyme (
The initial reversal of warfarin anticoagulation by UWEC-K2 may be rationalized in terms of the cyclopropyl ketone binding to the active site of CYP 2C9, and making warfarin and 7-hydroxywarfarin bind much more tightly, thus reducing the available warfarin in circulation, making the anticoagulation by warfarin ineffective. Once the CYP 2C9 active site is saturated, however, the effective warfarin concentration will rise, and anticoagulation will increase.
Alternatively, the final conjugate, formed in the active site of CYP 2C9 by coupling of warfarin (or 7-hydroxywarfarin) with the naphthoquinone radical may be a super inhibitor of CYP 2C9. This should gradually lead to a dramatic rise in warfarin anticoagulation because the inactivation of warfarin by CYP 2C9 is prevented as the amount of uninhibited CYP 2C9 decreases. One would assume that there is some threshold level of inhibition required to see the increased effectiveness of warfarin as an anticoagulant.
One especially puzzling result of early in vivo testing of UWEC-K2 was the observation that when the diets were mixed using a small amount of alcohol to add the UWEC-K2 to the soybean oil, the resultant tests were negative for the anticoagulation enhancement. It is difficult to see how so small a change could have such a large effect, but we now believe that the ethanol may have activated esterases to cleave more of the UWEC-K2 in the stomach, with the result that less of the UWEC-K2 entered the duodenum unaltered. Under these conditions, less UWEC-K2 would also enter the liver, where it is metabolized to the active adjuvant.
As can be appreciated, the results described in the above examples support the utility of the composition described and claimed herein that, when utilized in combination with a coumarin anticoagulant, improves the anticoagulant effects of the anticoagulant in mammalian subjects. Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration from the specification and practice of the invention disclosed herein. All references cited herein for any reason, including all journal citations and U.S./foreign patents and patent applications, if present, are specifically and entirely incorporated herein by reference. It is understood that the invention is not confined to the specific compositions, materials, methods, formulations, manufacturing conditions, etc., herein illustrated and described, but embraces such modified forms thereof as come within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application No. 62/634,304, filed Feb. 23, 2018, and hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62634304 | Feb 2018 | US |
