Detailed Description
Nicotinic acid or nicotinic acid (pyridine-3-carboxylic acid) is a drug generally known for its actions of increasing High Density Lipoprotein (HDL) content and other favorable changes in lipid profile (lowering Very Low Density Lipoprotein (VLDL), Low Density Lipoprotein (LDL), triglycerides, Free Fatty Acids (FFA) and lipoprotein (a) [ lp (a) ]). Niacin increases HDL levels when administered to humans in therapeutically effective doses, e.g., from about 50mg up to about 8g per day. However, niacin is often associated with cutaneous vasodilation, also known as flushing. Flushing usually brings with it redness of the skin, with fever, itching or stinging. It is extremely unpleasant and can be so severe that many patients discontinue niacin treatment. The present invention relates to the treatment, prevention or reversal of atherosclerosis and other diseases and conditions described herein with niacin or a salt or solvate thereof, or another niacin receptor agonist, without significant flushing. In humans, this is achieved by administering niacin or a salt or solvate thereof, or another niacin receptor agonist, with a compound that antagonizes the DP receptor, thereby preventing, reducing, or reducing the frequency and/or severity of flushing events.
There are at least two receptors that interact with prostaglandin D2, referred to as "DP" or "CRTH 2". The present invention relates generally to nicotinic acid or nicotinic acid receptor agonists for use in combination with DP receptor antagonists.
A significant aspect of the invention is a method of treating atherosclerosis in a human patient in need of such treatment, which comprises administering to the patient nicotinic acid or a salt or solvate thereof, or another nicotinic acid receptor agonist, and a DP receptor antagonist in amounts effective to treat atherosclerosis without the occurrence of significant flushing.
Another significant aspect of the invention is a method of increasing serum HDL levels in a human patient in need of such treatment, comprising administering to the patient nicotinic acid or a salt or solvate thereof, or another nicotinic acid receptor agonist, and a DP receptor antagonist, in combination, which is effective in increasing serum HDL levels in the patient without the occurrence of significant flushing.
Another significant aspect of the invention relates to a method of treating dyslipidemia in a human patient in need of such treatment, the method comprising administering to the patient nicotinic acid or a salt or solvate thereof, or another nicotinic acid receptor agonist, and a DP receptor antagonist, in amounts effective to treat dyslipidemia without the occurrence of significant flushing.
Another significant aspect of the invention relates to a method of reducing serum VLDL or LDL levels in a human patient in need of such treatment comprising administering to the patient nicotinic acid or a salt or solvate thereof, or another nicotinic acid receptor agonist, and a DP receptor antagonist in an amount effective to reduce serum VLDL or LDL levels in the patient without the occurrence of significant flushing.
Another significant aspect of the invention relates to a method of reducing serum triglyceride levels in a human patient in need of such treatment comprising administering to the patient nicotinic acid or a salt or solvate thereof, or another nicotinic acid receptor agonist, and a DP receptor antagonist in an amount effective to reduce serum triglyceride levels in the patient without the occurrence of significant flushing.
Another significant aspect of the invention relates to a method of reducing serum lp (a) levels in a patient in need of treatment comprising administering to the patient nicotinic acid or a salt or solvate thereof, or another nicotinic acid receptor agonist, and a DP receptor antagonist in an amount effective to reduce serum lp (a) levels in the patient without the occurrence of significant flushing. Lp (a) as used herein means lipoprotein (a).
A particularly interesting aspect of the present invention relates to the use of nicotinic acid or a salt or solvate thereof in the various methods described above. Of particular interest is the use of nicotinic acid. In another interesting aspect, the DP receptor antagonist selectively modulates the PD receptor in an amount effective to reduce or prevent flushing in a patient.
Another particularly interesting aspect of the present invention relates to the respective methods described above, wherein nicotinic acid is used and the DP receptor antagonist selectively modulates the DP receptor, while essentially not modulating the CRTH2 receptor.
Another particularly interesting aspect of the present invention relates to a method for treating atherosclerosis, dyslipidemia, diabetes or related disorders in a human patient in need of such treatment, comprising administering to the patient nicotinic acid or a salt or solvate thereof, or another nicotinic acid receptor agonist, and a DP receptor antagonist, in amounts effective to treat atherosclerosis, dyslipidemia, diabetes or related disorders without causing significant flushing.
One aspect of the present invention is the use of a DP receptor antagonist compound in combination with niacin or a salt or solvate thereof, or with another niacin receptor agonist, for the treatment of atherosclerosis in humans without the occurrence of significant flushing.
Another particularly interesting aspect of the present invention relates to the above method, wherein the DP receptor antagonist is selected from compounds a to AJ and pharmaceutically acceptable salts and solvates thereof.
Examples of compounds particularly useful for selectively antagonizing DP receptors and inhibiting flushing include the following compounds and their pharmaceutically acceptable salts and solvates:
atherosclerosis, as used herein, is a form of vascular disease characterized by the deposition of atherosclerotic plaques containing cholesterol and lipids on the innermost layers of large and medium-sized arterial walls. Atherosclerosis includes the symptoms of vascular disease that are recognized and understood by physicians working in the relevant medical arts. Atherosclerotic cardiovascular disease (including restenosis following revascularization surgery, coronary heart disease (also known as coronary artery disease or ischemic heart disease)), cerebrovascular disease (including multi-infarct dementia), and peripheral vascular disease (including erectile dysfunction) are all clinical manifestations of atherosclerosis and are therefore included within the terms "atherosclerosis" and "atherosclerotic disease".
"dyslipidemia" is used in the conventional sense to refer to abnormalities in plasma lipid levels, such as HLD (low), LDL (high), VLDL (high), triglyceride (high), lipoprotein (a) (high), FFA (high), and other abnormalities in serum lipid levels, or combinations thereof. It may be an uncomplicated disease or may be part of a specific associated disease or condition such as diabetes (diabetic dyslipidemia), metabolic syndrome, etc. Thus, uncomplicated dyslipidemia and dyslipidemia associated with the underlying disorder are included in the present invention.
The term "patient" includes mammals, especially humans, for the prophylaxis or treatment of medical conditions using the active agents of the invention. Administration to a patient includes self-administration and administration to a patient by another. The patient may be in need of treatment for an existing disease or medical condition, or may be a prophylactic treatment in which it is desirable to prevent or reduce the risk of developing atherosclerosis.
The term "therapeutically effective amount" refers to the amount of a drug that elicits the desired biological or medical response. For example, niacin is often administered in a dosage of about 50mg to about 8g per day.
The terms "prophylactically effective amount" and "amount effective for prevention" refer to an amount of a drug that will prevent or reduce the risk of occurrence of a biological or medical event to be prevented. In many cases, the prophylactically effective amount is the same as the therapeutically effective amount.
The invention described herein includes the administration of the compounds and compositions described herein to prevent or reduce the risk of developing coronary heart disease, cerebrovascular disease, and/or intermittent claudication, or the risk of recurrence that may be present. Coronary events include CHD death, myocardial infarction (i.e., heart attack), and coronary revascularization. Cerebrovascular events include ischemic or hemorrhagic stroke (also known as cerebrovascular accident) and transient ischemic attacks. Intermittent claudication is a clinical manifestation of peripheral vascular disease. The term "atherosclerotic disease event" as used herein includes coronary heart disease events, cerebrovascular events and intermittent claudication. For persons who have previously experienced one or more non-fatal atherosclerotic events, there is a possibility that such events may recur.
Accordingly, the present invention also provides a method of preventing or reducing the first or subsequent occurrence of an atherosclerotic event comprising administering to a patient suffering from such condition a prophylactically effective amount of a compound as described herein while preventing or reducing significant flushing. The patient may already have atherosclerotic disease at the time of administration, or may be at risk of developing the disease.
The method also relates to preventing or slowing the formation of new atherosclerotic lesions or plaques, preventing or slowing the development of existing lesions or plaques, and causing regression of existing lesions or plaques while preventing or reducing visible flushing.
Accordingly, one aspect of the present invention is directed to a method of arresting or slowing the progression of atherosclerosis, including arresting or slowing the progression of atherosclerotic plaques, comprising administering to a patient in need of such treatment a therapeutically effective amount of any of the DP antagonists described herein in combination with niacin or other niacin receptor agonists. This method also includes arresting or slowing the development of existing atherosclerotic plaques (i.e., "existing atherosclerotic plaques") at the time the present treatment is initiated, as well as arresting or slowing the formation of new atherosclerotic plaques in patients with atherosclerosis.
Another aspect of the invention relates to a method of preventing or reducing the risk of atherosclerotic plaque rupture comprising administering to a patient in need of treatment a prophylactically effective amount of any of the compounds described herein and niacin or other niacin receptor agonists. Rupture, as used herein, refers to the rupture loosening of plaque, which may become lodged in a blood vessel. Another aspect of the invention relates to a method of preventing or reducing the occurrence of atherosclerosis comprising administering to a patient in need of treatment a prophylactically effective amount of a compound described herein.
Another aspect of the invention relates to a method of treating or preventing atherosclerosis, dyslipidemia or a related disorder, comprising pre-treating a patient in need of such treatment with a DP receptor antagonist in an amount effective to inhibit or reduce flushing, and then treating the patient with niacin, a salt or solvate thereof, or another niacin receptor agonist, in an amount effective to treat or prevent atherosclerosis, dyslipidemia or a related disorder without causing significant flushing.
Another aspect of the invention relates to the methods described above, further comprising pre-treating or treating the patient with an HMG Co-A reductase inhibitor.
Another aspect of the invention relates to a method of treating or preventing the disorders described above wherein the HMG Co-A reductase inhibitor is simvastatin.
One aspect of the methods described herein involves the use of nicotinic acid or other nicotinic acid receptor agonist compounds in an amount to achieve such effect, as well as DP receptor antagonists that selectively modulate the DP receptor without substantially modulating the CRTH2 receptor. For example, the affinity of the DP receptor antagonist at the DP receptor (i.e., K)j) At least about 10 times higher (K) than the affinity at the CRTH2 receptorjLower values). Any compound that selectively interacts with DP according to these principles is considered "DP selective".
The phrase "does not undergo significant moisturization" refers to the side effects often seen with the administration of therapeutic amounts of niacin. The flushing effect of niacin often becomes less frequent and less severe as patients develop resistance to therapeutic doses of drugs, but flushing still occurs to some extent. Thus, "no significant flushing" means that flushing is reduced in severity, or less frequently, than it otherwise occurs. Preferably, the incidence of flushing is reduced by at least about one-third, more preferably by half, and most preferably by about two-thirds or more. Similarly, the severity is preferably reduced by about one-third, more preferably by at least one-half, and most preferably by at least about two-thirds. It is clearly most preferred that the occurrence and severity of flushing be reduced by 100%, but this is not required.
The specific dosage regimen and level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the patient's symptoms. Consideration of these factors is well within the purview of the ordinarily skilled clinician to determine the therapeutically effective dose or prophylactically effective dose required to prevent, counter, or slow the progression of symptoms. It is contemplated that the compounds will be administered daily for an appropriate period of time in order to treat or prevent the medical conditions associated with the patient, including courses of treatment lasting months, years, or even the life of the patient.
One or more additional active agents may be administered with the compounds of the present invention. These additional active agents may be lipid modulating compounds or agents with other pharmaceutical activities, or agents with both lipid modulating and other pharmaceutical activities. Examples of additional such active agents that may be used include, but are not limited to, HGM-CoA reductase inhibitors, including lactonized or dihydroxykohlung statins and pharmaceutically acceptable salts and esters thereof, including, but not limited to, lovastatin (see U.S. Pat. No. 4,342,767), simvastatin (see U.S. Pat. No. 4,444,784), dihydroxykohlung acid or simvastatin, particularly the ammonium or calcium salt thereof, pravastatin, particularly the sodium salt thereof (see U.S. Pat. No. 4,346,227), fluvastatin, particularly the sodium salt thereof (see U.S. Pat. No. 5,354,772), atorvastatin, particularly the calcium salt thereof (see U.S. Pat. No. 5,273,995), pitavastatin, also known as NK-104 (see PCT International patent WO97/23200), and rosuvastatin, also known as ZD-4522(CRESTOR (CREST(ii) a See U.S. Pat. No. 5,260,440); HMG-CoA synthetase inhibitors; a squalene epoxidase inhibitor; a squalene synthetase inhibitor; fatty acyl-coa: cholesterol Acyltransferase (ACAT) inhibitors, including selective inhibitors of ACAT-1 or ACAT-2, as well as dual inhibitors of ACAT-1 and 2; microsomal triglyceride transfer protein (MTP) inhibitors; an endothelial lipase inhibitor; a bile acid sequestrant; an LDL receptor inducer; platelet aggregation inhibitors such as glycoprotein IIb/IIIa fibrinogen receptor antagonists and aspirin; human peroxisome proliferator-activated receptor gamma (PPAR γ) agonists, including compounds commonly referred to as glitazones, such as pioglitazone and rosiglitazone, and including those compounds within the structural class referred to as thiazolidinediones and those PPAR γ agonists outside the thiazolidinedione structural class; PPAR α agonists, e.g., clofibrate, fenofibrate (including micronized fenofibrate), and gemfibrozil; PPAR dual alpha/gamma agonists; vitamin B6(also known as pyridoxine) and pharmaceutically acceptable salts thereof such as the HCl salt; vitamin B12(also known as cyanocobalamin), folic acid or a pharmaceutically acceptable salt or ester thereof, such as the sodium salt and methylglucamine salt; antioxidant vitamins such as vitamin C and E and beta carotene; a beta-blocker; angiotensin II antagonists such as losartan; angiotensin converting enzyme inhibitors such as enalapril and captopril; renin inhibitors, calcium channel blockers such as nifedipine and diltiazem(ii) a An endothelin antagonist; an agent that enhances expression of the ABCA1 gene; cholesteryl Ester Transfer Protein (CETP) inhibiting compounds, 5-lipoxygenase activating protein (FLAP) inhibiting compounds, 5-lipoxygenase (5-LO) inhibiting compounds, Farnesoid X Receptor (FXR) ligands, including antagonists and agonists; liver X Receptor (LXR) -alpha ligands, LXR-beta ligands, bisphosphonate compounds such as alendronate sodium; cyclooxygenase-2 inhibitors such as rofecoxib and celecoxib; and compounds that resolve vascular inflammation.
Cholesterol absorption inhibitors may also be used in the present invention. These compounds block the movement of cholesterol from the intestinal lumen to the intestinal cells of the small intestinal wall, thereby reducing serum cholesterol levels. Examples of cholesterol absorption inhibitors are described in US 5,846,966, 5,631,365, 5,767,115, 6,133,001, 5,886,171, 5,856,473, 5,756,470, 5,739,321, 5,919,672 and PCT applications No. WO 00/63703, WO 00/60107, WO 00/38725, WO 00/34240, WO 00/20623, WO 97/45406, WO 97/16424, WO 97/16455 and WO 95/08532. The most notable cholesterol absorption inhibitor is ezetimibe, also known as 1- (4-fluorophenyl) -3(R) - [3(S) - (4-fluorophenyl) -3-hydroxypropyl) ] -4(S) - (4-hydroxyphenyl) -2-azetidinone, described in U.S. Pat. Nos. 5,767,115 and 5,846,966.
A therapeutically effective amount of the cholesterol absorption inhibitor includes dosages from about 0.01mg/kg to about 30mg/kg body weight per day, preferably from about 0.1mg/kg to about 15mg/kg body weight per day.
For diabetic patients, the compounds used in the present invention may be administered with commonly used diabetes drugs. For example, a diabetic patient treated as described herein may also be administered insulin or an oral antidiabetic agent. One example of an oral antidiabetic agent suitable for the present invention is metformin.
Dosage information
Nicotinic acid as used in the present invention refers to pyridine-3-carboxylic acid. However, for use in the present invention, salts and solvates of nicotinic acid are also included, and many pharmaceutically acceptable salts and solvates of nicotinic acid are useful in the present invention. Alkali metal salts, especially sodium and potassium salts, are suitable salts. Likewise, alkaline earth metal salts, particularly calcium and magnesium salts, are also suitable salts. Various amine salts, such as ammonium and substituted ammonium compounds, also constitute suitable salts. Similarly, the solvated forms of niacin are suitable for use within the scope of the invention. Examples thereof include hemihydrate, mono-, di-, tri-and sesquihydrate. Most interesting for use in the present invention is the free acid, pyridine-3-carboxylic acid.
The DP antagonists described herein are useful for reducing or preventing flushing in mammalian patients, particularly humans, at doses ranging from as low as about 0.01 mg/kg/day up to as high as about 100 mg/kg/day, administered daily, in single or divided doses. Preferably, the dose is from about 0.1 mg/day up to about 1.0 g/day, administered daily in single or divided doses.
Useful niacin doses described herein range from as low as about 50 mg/day up to about 8 g/day, administered in single or divided doses per day. Lower doses may be used initially and increased to further reduce flushing effects.
The dosage of nicotinic acid receptor agonists other than nicotinic acid varies within wide limits. In general, nicotinic acid receptor agonists useful for treating atherosclerosis are administered in single or divided doses ranging from as low as about 0.01 mg/kg/day to as high as about 100 mg/kg/day. A representative dose is from about 0.1 mg/day to about 2 g/day.
The compounds for use in the present invention may be administered by any conventional route of administration. The preferred route of administration is oral.
Nicotinic acid, its salts or solvates, as well as other nicotinic acid receptor agonists and DP antagonists, may be administered together or sequentially in single or multiple daily doses (e.g., two, three or four times daily) without departing from the invention. If a particularly long sustained release is desired, the oral cavity may be administered, for example, in a release form or over 24 hours every other day. However, single daily administration is preferred. Likewise, the medication may be administered in the morning or in the evening.
Pharmaceutical composition
The pharmaceutical compositions described herein generally comprise niacin or another niacin receptor agonist, a DP receptor antagonist, and a pharmaceutically acceptable carrier.
Examples of suitable oral compositions include tablets, capsules, troches, lozenges, suspensions, dispersible powders or granules, emulsions, syrups or elixirs. Examples of carrier ingredients include diluents, binders, disintegrants, lubricants, sweeteners, flavoring agents, coloring agents, preservatives, and the like. Examples of diluents include, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate and sodium phosphate. Examples of granulating and disintegrating agents include corn starch and alginic acid. Examples of binders include starch, gelatin and acacia. Examples of lubricants include magnesium stearate, calcium stearate, stearic acid, and talc. The tablets may be uncoated or they may be coated by known techniques. Such coatings may delay disintegration and thus absorption in the gastrointestinal tract, providing a longer lasting effect over a typical period.
In one embodiment of the invention, niacin or a salt or solvate thereof, or another niacin receptor agonist, is combined with a DP receptor antagonist and a carrier to form an immobilized combination. Such a fixed combination product may be a tablet or capsule for oral administration.
More specifically, in another embodiment of the invention, niacin or a salt or solvate thereof, or another niacin receptor agonist (about 1-1000mg), and a DP antagonist (about 1-500mg), are combined with a pharmaceutically acceptable carrier to form a tablet or capsule for oral administration.
Sustained release over a longer period of time may be particularly important in niacin pharmaceutical formulations. Particularly preferred are sustained release tablets. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. The dosage form may also be coated using techniques described in US 4,256,108, 4,166,452 and 4,265,874 to form osmotic therapeutic tablets for controlled release.
Still other controlled release techniques are available and are included in the present invention. Typical ingredients that can be used to slow the rate of niacin release in sustained release tablets include various cellulosic compounds, such as methyl cellulose, ethyl cellulose, propyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, starch, and the like. Various natural and synthetic materials may also be used in the sustained release formulations. Examples include alginic acid and various alginates, polyvinylpyrrolidone, xanthan gum, locust bean gum, guar gum, gelatin, various long chain alcohols (e.g., cetyl alcohol), and beeswax.
A particularly interesting sustained release tablet uses niacin in combination with one or more of the above cellulose compounds, compressed into a sustained release tablet to form a polymer matrix. The DP antagonist compound may be incorporated into the blend prior to compression or coated onto the outer surface of the matrix.
In a more interesting embodiment, the niacin and matrix-forming material are combined and compressed into a sustained release core, and the DP antagonist compound is mixed with one or more coating agents, coated onto the outer surface of the core.
Optionally and more significant is a tablet as described above, further coated with an HMCCo-A reductase inhibitor, e.g., simvastatin. This embodiment thus contains three active ingredients, an HMG Co-a reductase inhibitor and a DP antagonist which can be released in large amounts upon ingestion, and niacin which can be released over a longer period of time as described above.
Typical release times for sustained release tablets according to the invention are from about 1 hour up to about 48 hours, preferably from about 4 hours to about 24 hours, more preferably from about 8 to 16 hours.
Hard gelatin capsules constitute another type of solid dosage form for oral administration. Such capsules similarly contain the active ingredient in admixture with carrier materials as described above. Soft gelatin capsules comprise the active ingredient in admixture with a water-miscible solvent such as propylene glycol, PEG and ethanol, or with an oil such as peanut oil, liquid paraffin or olive oil.
Aqueous suspensions containing the active substance in admixture with excipients suitable for the manufacture of aqueous suspensions are also contemplated. Such excipients include suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and acacia; dispersing or wetting agents, for example, lecithin; preservatives, such as ethyl or n-propyl p-hydroxybenzoate; coloring agents, flavoring agents, sweetening agents, and the like.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are those already exemplified above.
Can also be formulated as a syrup or elixir
Of particular interest are pharmaceutical compositions which are sustained release tablets comprising nicotinic acid or a salt or solvate thereof and a DP receptor antagonist in combination with a pharmaceutically acceptable carrier.
Another pharmaceutical composition of particular interest is a sustained release tablet comprising nicotinic acid or a salt or solvate thereof, a DP receptor antagonist, and an HMG Co-A reductase inhibitor in combination with a pharmaceutically acceptable carrier.
Another pharmaceutical combination of further interest is a sustained release tablet comprising niacin, a DP receptor antagonist and simvastatin in combination with a pharmaceutically acceptable carrier.
Another pharmaceutical composition of particular interest is a sustained release tablet comprising nicotinic acid and a DP receptor antagonist selected from compounds a to AJ in combination with a pharmaceutically acceptable carrier.
Another pharmaceutical composition of further interest comprises nicotinic acid and one DP antagonist compound selected from the group consisting of compounds A, B, D, E, X, AA, AF, AG, AH, AI and AJ, in combination with a pharmaceutically acceptable carrier.
Of particular interest is another pharmaceutical composition comprising niacin and a DP antagonist compound a in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of further interest comprises niacin and DP antagonist compound B in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of further interest comprises niacin and DP antagonist compound D in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of further interest comprises niacin and DP antagonist compound E in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of interest comprises niacin and a DP antagonist compound X in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of interest comprises niacin and the DP antagonist compound AA in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of further interest comprises niacin and the DP antagonist compound AF in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of further interest comprises niacin and the DP antagonist compound AG in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of further interest comprises niacin and a DP antagonist compound AH in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of further interest comprises niacin and DP antagonist compound AI in combination with a pharmaceutically acceptable carrier.
Yet another pharmaceutical composition of further interest comprises niacin and the DP antagonist compound AJ in combination with a pharmaceutically acceptable carrier.
Another pharmaceutical composition of further interest comprises niacin, one of the DP antagonists described above, and simvastatin in combination with a pharmaceutically acceptable carrier.
Another pharmaceutical composition of further interest comprises niacin, a DP receptor antagonist selected from compounds A through AJ, and simvastatin in combination with a pharmaceutically acceptable carrier.
Another pharmaceutical composition of further interest comprises niacin, a DP antagonist selected from the group consisting of compounds A, B, D, E, X, AA, AF, AG, AH, AI and AJ, and simvastatin in combination with a pharmaceutically acceptable carrier.
The term "composition" includes, in addition to the above-described pharmaceutical compositions, any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients (active or excipient ingredients), or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Thus, the pharmaceutical compositions of the present invention include any composition made by admixing or combining the compound, any additional active ingredient, and pharmaceutically acceptable excipients.
Another aspect of the invention relates to the use of niacin or a salt or solvate thereof, or another niacin receptor agonist, and a DP antagonist, in the manufacture of a medicament. The medicament has the uses described herein.
More particularly, the invention relates to the use of niacin or a salt or solvate thereof, or another niacin receptor agonist, a DP antagonist and an HMG Co-A reductase inhibitor such as simvastatin, in the manufacture of a medicament. The medicament has the uses described herein.
In addition to nicotinic acid, which is the benchmark for nicotinic acid receptor agonists, a number of nicotinic acid receptor agonists have been mentioned. The following publications disclose compounds that are nicotinic acid receptor agonists:
Lorenzen，A.et al.Molecular Pharmacology 59：349-357(2001)，
Lorenzen，A.et al.Biochemical Pharmacology 64：645-648(2002)，
Soga，T.et al.Biochemical and Biophysical Research Comm.303：364-369(2003)，
Tunaru，S.et al.Nature Medicine 9：352-355(2003)，
wise, A.et al.journal of Biological Chemistry 278: 9869 (2003) 9874(2003), and
Van Herk，T.et al Journal of Medicinal Chemistry 46：3945-3951(2003).
it should be noted that partial agonists of nicotinic acid receptors, such as those disclosed in van Herk et al, are also intended to be included in the compositions and methods of treatment of the present invention.
In addition, nicotinic acid receptors have been identified and characterized in WO 02/084298A2 (published 2002, 10/24) and in the documents of Soga, T, et al, Tunaru, S, et al and Wise, A, et al (cited above).
A number of DP receptor antagonist compounds have been disclosed which are suitable for and included in the methods of the present invention. DP receptor antagonists are available, for example, according to the following patent documents: WO 01/79169 published on day 25O month 1 in 2001, EP 1305286 published on day 2/5/2003, WO 02/094830 published on day 28/11/2002, and WO 03/062200 published on day 31/7/2003. Compound AB can be synthesized according to the instructions in WO01/66520A1 published on 9/13/2001; compound AC can be synthesized according to the description in WO 03/022814a1 published on 3/20/2003, and compounds AD and AE can be synthesized according to the description in WO 03/078409 published on 9/25/2003. Other representative DP antagonist compounds for use in the present invention can be synthesized according to the examples provided below.
Example 1
[5- [ (4-chlorophenyl) thio ] thio]-4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyridineAnd [3, 2-b ]]Indolizin-6-yl]Acetic acid (Compound G)
Step 14-chloronicotinaldehydes
The title compound was prepared as described in f.marsaisi et al, j.heterocyclic chem., 25, 81 (1988).
Step 24- (methylthio) nicotinaldehydes
To a solution of NaSMe (9.5g.135mmol) in MeOH (250ml) was added a solution of step 1's 40 chloronicotinaldehyde (13.5g, 94.4mmol) in MeOH (250 ml). The reaction mixture was kept at 60 ℃ for 15 minutes and then poured onto NH4Cl and EtOAc. Separating the organic phase, washing with water, and adding Na2SO4And (5) drying. The compound was then purified on silica gel with 50% EtOAc/hexanes to provide the title compound.
Step 3(2Z) -2-azido-3- [4- (methylthio) pyridin-3-yl]2-propenoic acidMethyl ester
A solution of 4- (methylthio) nicotinaldehyde (4.8g, 31mmol) and methyl azidoacetate (9.0g, 78mmol) in MeOH (50ml) was added to a 25% NaOMe methanol solution (16.9ml, 78mmol) at-12 ℃. The internal temperature was monitored and maintained at-10 ℃ to-12 ℃ during 30 minutes of addition. The resulting mixture was then stirred in an ice bath for several hours, followed by overnight in an ice bath in a cold room. The suspension was poured onto ice and NH4Cl, the slurry was filtered after stirring for 10 minutes. The product was washed with cold water and then dried in vacuo to give the title compound as a brown-grey solid (7.4g) containing some salt. The compound was then purified on silica gel with EtOAc.
Step 44- (methylthio) -1H-pyrrolo [2, 3-b]Pyridine-2-carboxylic acid methyl ester
A suspension of the compound from step 3 (0.40g, 1.6mmol) in xylene (16ml) was slowly heated to 140 ℃. After 15 minutes at 140 ℃, the yellow solution was cooled to room temperature. Care must be taken because of the possible exotherm due to the formation of nitrogen. The suspension was cooled to 0 ℃, filtered and washed with xylene to give the title compound.
Step 54- (methylthio) -6-oxo-6, 7, 8, 9-tetrahydropyrido [3, 2-b]Indolizine-7-carboxylic acid ethyl ester
To a solution of the compound of step 4 (0.35g, 1.6mmol) in DMF (20ml) at 0 ℃ was added NaH (1.2 eq). After 5 minutes iodine was addedTetra-n-butylammonium chloride (0.10g) and ethyl 4-bromobutyrate (0.40 ml). After 1 hour at room temperature, the reaction mixture was poured into saturated NH4Cl and EtOAc. Separating the organic phase, washing with water, and adding Na2SO4And (5) drying. After evaporation the crude product was purified by flash chromatography. The diester was then dissolved in THF (7.0ml) and 1.06M potassium tert-butoxide in THF (2.2ml) was added at 0 ℃. After 1 hour at room temperature, the reaction mixture was poured into saturated NH4Cl and EtOAc. Separating the organic phase from Na2SO4Drying and evaporation under reduced pressure gave the title compound as a mixture of ethyl and methyl esters.
Step 64- (methylthio) -8, 9-dihydropyrido [3, 2-b ]]Indolizine-6 (7H) -Ketones
To the step 5 compound (0.32g) were added EtOH (8.0ml) and concentrated hydrochloric acid (2.0 ml). The resulting suspension was refluxed for 5 hours. The reaction mixture was partitioned between EtOAc and Na2CO3Among them. The organic phase was separated and evaporated to give the title compound.
Step 7(2E, 2Z) - [4- (methylthio) -8, 9-dihydropyrido [3, 2-b ]]IndoleOxazin-6 (7H) -ylidene]Ethyl acetate
To a solution of triethyl phosphonoacetate (0.45g, 2.17mmol) in DMF (12ml) were added 80% NaH (0.06g, 2.00mmol) and the compound of step 6 (0.22g, 1.00 mmol). After 4 hours at 55 ℃ the reaction mixture was poured into saturated NH4Cl and EtOAc. The organic phase was separated off and evaporated under reduced pressure. The crude product was purified by flash chromatography to afford the title compound.
Step 8[4- (methylthio) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ]]Indolizine-6-Base of]Ethyl acetate
The step 7 compound was dissolved in MeOH-THF with heating. Adding PtO to the previous cooling solution at room temperature2The resulting mixture was maintained under hydrogen at one atmosphere for 18 hours. Reaction mixture with CH2Cl2Carefully filter through celite. Evaporating the filtrate under reduced pressureThe title compound was obtained. Alternatively, the compound of step 7 can be prepared in EtOAc with Pd (OH)2H at 40psi2Hydrogenation for 18 hours.
Step 9[4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ]]Indolizine-6-yl]Ethyl acetate
To a solution of the step 8 compound (0.08g, 0.27mmol) in MeOH (3.0mL) was added Na2WO4(0.10g) and 30% H2O2(600. mu.L). After 1 hour the reaction mixture was partitioned between water and EtOAc. The organic phase is washed with water, separated and evaporated. The title compound was purified by flash chromatography.
Step 10[5- [ (4-chlorophenyl) thio ] thio]-4- (methylsulfonyl) -6, 7, 8, 9-Tetrahydropyrido [3, 2-b ]]Indolizin-6-yl]Ethyl acetate
To a solution of 4, 4' -dichlorodiphenyl disulfide (0.24g) in 1, 2-dichloroethane was added SO2Cl2(50. mu.L). To the compound of step 9 (0.05g) in DMF (2.0mL) was added the previous mixture (. apprxeq.180. mu.L). By using1H NMR followed the reaction and was kept at room temperature until no more starting material remained. The reaction mixture was poured into saturated NaHCO3And EtOAc. The organic phase was separated, evaporated and the title compound purified by flash chromatography.
Step 11[5- [ (4-chlorophenyl) thio ] thio]-4- (methylsulfonyl) -6, 7, 8, 9-Tetrahydropyrido [3, 2-b ]]Indolizin-6-yl]Acetic acid
To the step 10 compound dissolved in a 1: 1 mixture of THF-MeOH, 1N NaOH was added. After 18 hours at room temperature, the reaction mixture is poured onto NH4Cl and EtOAc. Separating the organic phase with Na2SO4Dried and evaporated to give the title compound.
1H NMR (500MHz, acetone-d6)δ11.00(bs，1H)，8.60(d，1H)，7.80(d，1H)，7.20(d，2H)，7.00(d，2H)，4.65(m，1H)，420(m, 1H), 3.75(m, 1H), 3.35(s, 3H), 2.80 to 2.10(m, 6H).
Example 2
[5- [ (4-chlorophenyl) thio ] -4- (methylthio) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ] indolizin-6-yl ] acetic acid (compound H)
The title compound was prepared from the compound of example 1, step 8 in a similar manner to that described in example 1, steps 10 and 11.
Example 3
[5- [ (3, 4-chlorophenyl) thio ] -4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ] indolizin-6-yl ] acetic acid (Compound I)
The title compound was prepared as described in example 1 using bis (3, 4-dichlorodiphenyl) disulfide from step 10.
1H NMR (500MHz, acetone-d6) δ 8.55(d, 1H), 7.85(d, 1H), 7.35(d, 1H), 7.15(s, 1H), 6.95(d, 1H), 4.60(m, 1H), 4.15(m, 1H), 3.80(m, 1H), 3.40(s, 3H), 2.80 to 2.10(m, 6H).
m/z 484.
The enantiomers were separated on a Chiralecel OD column (25 cm. times.20 mm) using 30% isopropanol, 17% ethanol and 0.2% acetic acid in hexane at a flow rate of 8 ml/min. The purity thereof was confirmed by a ChirallecOD column (25 cm. times.4.6 mm) using hexane containing 30% isopropyl alcohol and 0.2% acetic acid at a flow rate of 1.0 ml/min. The fast eluting enantiomer Tr is divided into 9.7 and the slow eluting enantiomer Tr is divided into 11.1.
Example 4
[5- (4-chlorobenzoyl) -4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ] indolizin-6-yl ] acetic acid (Compound J)
Step 1[5- (4-chlorobenzoyl) -4- (methylthio) -6, 7, 8, 9-tetrahydropyridineAnd [3, 2-b ]]Indolizin-6-yl]Ethyl acetate
To a solution of 4-chlorobenzoyl chloride (0.30g, 1.7mmol) in 1, 2-dichloroethane (6.0mL) was added AlCl3(0.24g, 1.8 mmol). After 5 minutes [4- (methylthio) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ] from example 1, step 8 was added to the previous mixture]Indolizin-6-yl]A solution of ethyl acetate (0.15g, 0.47mmol) in 1, 2-dichloroethane (6.0 mL). After 4 hours at 80 ℃, the reaction mixture was partitioned between EtOAc and NaHNO3Among them. Separating the organic phase with Na2SO4Drying and evaporating. The title compound was purified by flash chromatography.
Step 2[5- (4-chlorobenzoyl) -4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyridinePyrido [3, 2-b]Indolizin-6-yl]Ethyl acetate
To [5- (4-chlorobenzoyl) -4- (methylthio) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ]]Indolizin-6-yl]A solution of ethyl acetate (0.12g, 0.27mmol) in MeOH (5.0mL) was added Na2WO4(0.1g) and 30% H2O2(300. mu.L) the reaction mixture was stirred at 55 ℃ for 1 hour and then partitioned between water and EtOAc. Washing the organic phase with water and Na2SO4Drying and evaporating. The title compound was purified by flash chromatography.
Step (ii) of3[5- (4-chlorobenzoyl) -4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyridinePyrido [3, 2-b]Indolizin-6-yl]Acetic acid
Ethyl [5- (4-chlorobenzoyl) -4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ] indolizin-6-yl ] acetate was processed as described in example 1, step 11 to give the title compound.
1H NMR (500MHz, acetone-d6) δ 8.55(d, 1H), 7.90(d, 2H), 7.65(d, 1H), 7.45(d, 2H), 4.55(m, 1H), 4.25(m, 1H), 3.45(m, 1H), 3.20(s, 3H), 2.05 to 3.00(m, 6H).
m/z446.
Example 5
[5- (4-bromophenyl) thio group]-4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyrido[3，2-b]Indolizin-6-yl]Acetic acid (Compound K)
The title compound was prepared as described in example 1 using 4, 4' -dibromodiphenyldisulfide.
1H NMR (500MHz, acetone-d6) δ 8.60(d, 1H), 7.80(d, 1H), 7.35(d, 2H), 7.00(d, 2H), 4.65(m, 1H), 4.20(m, 1H), 3.80(m, 1H), 3.35(s, 3H), 2.80 to 2.10(m, 6H).
EXAMPLE 6 method-1
[9- [ (3, 4-dichlorophenyl) thio group]-1- (methylsulfonyl) -7, 8-dihydro-6H-pyri dinePyrido [3, 4-b]Pyrin-8-yl]Acetic acid (Compound L)
Step 12- (methylthio) nicotinaldehydes
The title compound was prepared from 2-bromonicotinaldehyde (a. numata Synthesis 1999 p.306) as described in example 1, step 2, except that the solution was heated at 55 ℃ for 2 hours.
Step 2(2Z) -2-azido-3- [2- (methylthio) pyridin-3-yl]2-propenoic acidMethyl ester
The title compound was prepared as described in example 1, step 3.
Step 34- (methylthio) -1H-pyrrolo [3, 2-c]Pyridine-2-carboxylic acid methyl ester
Methyl (2Z) -2-azido-3- [2- (methylthio) pyridin-3-yl ] prop-2-enoate (1.00g, 4.00mmol) was heated in 1,3, 5-trimethylbenzene (50mL) at 160 ℃ for 1 hour. The reaction mixture was cooled to room temperature, then to 0 ℃, the precipitate was filtered off and washed with cold 1,3, 5-trimethylbenzene to give the title compound.
Step 41- (methylthio) -8-oxo-7, 8-dihydro-6H-pyrido [3, 4-b]Pyridine (II)(iv) 7-Carboxylic acid methyl ester
To 4- (methylthio) -1H-pyrrolo [3, 2-c]A suspension of pyridine-2-carboxylic acid methyl ester (0.30g, 1.35mmol) in THF (3 mL)/toluene (12.0mL) was added 1.06M potassium tert-butoxide in THF (1.42mL/1.41mmol) and methyl acrylate (300. mu.L). The resulting mixture was heated at 80 ℃ for 18 hours. The mixture was partitioned between EtOAc and NH4Among Cl, filtered through celite. Separating the organic phase with Na2SO4Drying and filtration gave the title compound.
Step 51- (methylthio) -6, 7-dihydro-8H-pyrido [3, 4-b]Pyrin-8-one
1- (methylthio) -8-oxo-7, 8-dihydro-6H-pyrido [3, 4-b ] pyridine-7-carboxylic acid methyl ester was converted to the title compound as described in example 1, step 6.
Step 6[ 8-hydroxy-1- (methylthio) -7, 8-dihydro-6H-pyrido [3, 4-b ]]Pyridine (II)(iv) Proten-8-yl]Acetic acid methyl ester
1- (methylthio) -6, 7-dihydro-8H-pyrido [3, 4-b]A mixture of pyranthin-8-one (0.15g, 0.68mmol), bromoacetic acid methyl ester (0.34mL), Zn-Cu (0.226g) in THF (3.0mL) was sonicated for 2 hours. The mixture was then heated at 60 ℃ for 5 minutes until the reaction was complete. The reaction mixture was partitioned between EtOAc and NH4Among Cl. Separating the organic phase with Na2SO4Dried, filtered and evaporated under reduced pressure to give the title compound. The compound was purified by flash chromatography.
Step 7[1- (methylthio) -7, 8-dihydro-6H-pyrido [3, 4-b ]]Pyrin-8-yl]Acetic acid methyl ester
To 3.2mL CH3NaI (0.300g) in CN was added to TMSCL (0.266 mL). Adding the mixture to [ 8-hydroxy-1- (methylthio) -7, 8-dihydro-6H-pyrido [3, 4-b ] in a water bath]Pyrin-8-yl]Methyl acetate (0.15g, 0.515mmol) in CH3CN (1.5 mL). After 0.5 h the reaction mixture was partitioned between EtOAct and NaHCO3In (1). The organic phase is separated off, washed with sodium thiosulfate and MgSO4Dried and evaporated. The title compound was purified by flash chromatography.
Step 8 [1- (methylsulfonyl) -7, 8-dihydro-6H-pyrido [3, 4-b ]]Pyrin-8-yl]Ethyl acetate
Methyl [1- (methylthio) -7, 8-dihydro-6H-pyrido [3, 4-b ] pyrazin-8-yl ] acetate was converted to the title compound as described in example 1, step 9.
Step 9[9- [ (3, 4-dichlorophenyl) thio]-1- (methylsulfonyl) -7, 8-dihydro-6H-Pyrido [3, 4-b]Pyrin-8-yl]Acetic acid
Methyl [1- (methylsulfonyl) -7, 8-dihydro-6H-pyrido [3, 4-b ] pyridin-8-yl ] acetate was converted to the title compound using bis (3, 4-dichlorophenyl) disulfide in step 10 as described in example 1, steps 10 and 11.
1H NMR (500MHz, acetone-d6)δ8.35(d，1H)7.80(d，1H)，7.35(d，1H)，7.15(s，1H)，6.95(d，1H)，4.55(m，1H)，4.35(m，1H)，3.90(m，1H)，3.30(s，3H)，3.15(m，1H)，3.05(m，1H)，2.80(m，1H)，2.50(m，1H).
Example 6 method-2
[9- [ (3, 4-dichlorophenyl) thio ] -1- (methylsulfonyl) -7, 8-dihydro-6H-pyrido [3, 4-b ] pyrazin-8-yl ] acetic acid
Step 11- (methylthio) -7, 8-dihydro-6H-pyrrolo [3, 4-b)]Pyrrol-8-ol
1- (methylthio) -6, 7-dihydro-8H-pyrido [3, 4-b ] at 0 deg.C to example 6, method-1, step 5]A suspension of pyrin-8-one (0.55g, 0.22mmol) in EtOH (10ml)/THF (1ml) was added NaBH4(0.10g, 2.6 mmol). After 30 minutes at room temperature, the reaction was quenched by addition of acetone. The solvent was evaporated under reduced pressure and EtOAc and water were added to the residue. Separating the organic phase with MgSO4Drying and evaporating. The title compound was washed with EtOAc/hexanes and filtered.
Step 22- [1- (methylthio) -7, 8-dihydro-6H-pyrido [3, 4-b ]]Pyrin-8-yl]Malonic acid dimethyl ester
To 1- (methylthio) -7, 8-dihydro-6H-pyrido [3, 4-b ] at-78 deg.C]A suspension of pyrin-8-ol (0.54g, 2.1mmol) in THF (10mL) was added 1M solution of NaHMDS in THF (2.35mL, 2.4mmol) and diphenyl chlorophosphate (0.53mL, 2.6 mmol). After 30 minutes dimethyl malonate (0.73mL, 6.4mmol) and a 1M solution of NaHMDS/THF (6.8mL, 6.8mmol) were added. The reaction mixture was warmed to 0 ℃ and then to room temperature. Distributing the mixture in an ETOAc and NH4In Cl, the organic phase was MgSO4Drying, filtering and evaporating. The title compound was purified by flash chromatography.
Step 3 [1- (methylthio) -7, 8-dihydro-6H-pyrido [3, 4-b ]]Pyrin-8-yl]Second stepAcid methyl ester
To 2- [1- (methylthio) -7, 8-dihydro-6H-pyrido [3, 4-b ]]Pyrin-8-yl]To a mixture of dimethyl malonate (0.59g, 2.17mmol) and DMSO (4ml) was added NaCl/water (0.45 ml). After 18 hours at 150 ℃, the reaction mixture was partitioned between EtOAc and water. Separating the organic phase with Na2SO4Drying and evaporating. The title compound was subsequently purified by flash chromatography.
Step 4 [9- [ (3, 4-dichlorophenyl) thio ]]-1- (methylsulfonyl) -7, 8-dihydro-6H-Pyrido [3, 4-b]Pyrin-8-yl]Acetic acid
The title compound was obtained from methyl [1- (methylthio) -7, 8-dihydro-6H-pyrido [3, 4-b ] pyrazin-8-yl ] acetate as described in example 6, method-1, steps 8 to 9.
Example 7
[10- [ (3, 4-dihydrophenyl) sulfanyl group]-1- (methylsulfonyl) -6, 7, 8, 9-tetrakisHydropyrido [3, 4-b]Indolizin-9-yl]Acetic acid (Compound M)
Step 1 [1- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyrido [3, 4-b ]]Indolizine-9-Base of]Ethyl acetate
The title compound was prepared from the product of example 6 step 3 in the same manner as described in example 1 steps 5 to 9
Step 2 [10- [ (3, 4-dichlorophenyl) sulfanyl group]-1- (methylsulfonyl) -6, 7, 8, 9-Tetrahydropyrido [3, 4-b ]]Indolizin-9-yl]Acetic acid
The product of step 1 was converted to the title compound in the same manner as in steps 10-11 of example 1 using bis (3, 4-dichlorophenyl) disulfide in step 10.
MS M+1＝485
Example 8
(4- (methylsulfonyl) -5- { [4- (trifluoromethyl) phenyl]Thio } -6, 7, 8, 9-Tetrahydropyrido [3, 2-b ]]Indolizin-6-yl) acetic acid (compound N)
The title compound was prepared as described in example 1 using bis [4- (trifluoromethyl) phenyl ] disulfide.
1H NMR (500MHz, acetone-d6)δ8.55(d，1H)，7.75(d，1H)，7.45(d，2H)，7.15(d，2H)，4.55(m，1H)，4.15(m，1H)，3.80(m，1H)，3.30(s，3H)，2.80 to 2.10(m，6H).
m/z 513(M+1)。
Example 9
[5- [ (2-chloro-4-fluorophenyl) thio group]-4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyridinePyrido [3, 2-b]Indolizin-6-yl]Acetic acid (Compound O)
The title compound was prepared as described in example 1 using bis (2-chloro-4-fluorophenyl) disulfide.
m/z 469(M+1)。
Example 10
[4- (methylsulfonyl) -5- (2-naphthylthio) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ]]Indolizin-6-yl]Acetic acid (Compound P)
The title compound was prepared as described in example 1 using bis (2-naphthyl) disulfide.
m/z 467(M+1)。
Example 11
[5- [ (2, 3-dichlorophenyl) thio group]-4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyridineAnd [3, 2-b ]]Indolizin-6-yl]Acetic acid (Compound Q)
The title compound was prepared as described in example 1 using bis (2, 3-dichlorophenyl) disulfide.
1H NMR (500MHz, acetone-d6)δ8.85(d，1H)，7.80(d，1H)，7.30(d，1H)，7.00(t，1H)，6.60(d，1H)，4.60(m，1H)，4.20(m，1H)，3.80(m，1H)，3.40(s，3H)，2.80 to 2.10(m，6H).
Example 12
[5- [ (4-methylphenyl) thio group]-4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyrido[3，2-b]Indolizin-6-yl]Acetic acid (Compound R)
The title compound was prepared as described in example 1 using p-tolyl disulfide.
1H NMR (500MHz, acetone-d6)δ8.55(d，1H)，7.80(d，1H)，6.95(m，4H)，4.60(m，1H)，4.15(m，1H)，3.80(m，1H)，3.35(s，3H)，2.80 to 2.10(m，6H).
Example 13
[4- (methylsulfonyl) -5- (phenylthio) -6, 7, 8, 9-tetrahydropyrido [3, 2-b ]]Indolizin-6-yl]Acetic acid (Compound S)
The title compound was prepared as described in example 1 using diphenyl disulfide.
1H NMR (500MHz, acetone-d6)δ8.55(d，1H)，7.80(d，1H)，7.15 to 6.90(m，5H)，4.60(m，1H)，4.15(m，1H)，3.75(m，1H)，3.30(s，3H)，2.80 to 2.10(m，6H).
Example 14
[5- [ (2, 4-dichlorophenyl) thio group]-4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyridinePyrido [3, 2-b]Indolizin-6-yl]Acetic acid (Compound T)
The title compound was prepared as described in example 1 using bis (2, 4-dichlorophenyl) disulfide. The disulfide is prepared from 2, 4-dichlorothiophenol with Br2Prepared in diethyl ether.
1H NMR (500MHz, acetone-d6)δ8.55(d，1H)，7.85(d，1H)，7.35(s，1H)，7.00(d，1H) 6.65(d，1H)，4.55(m，1H)，4.15(m，1H)，3.80(m，1H)，3.35(s，3H)，2.80 to 2.10(m，6H).
Example 15
[5- [ (4-chlorophenyl) thio ] thio]-4- (methylsulfonyl) -6, 7, 8, 9-tetrahydropyrido[4，3-b]Indolizin-6-yl]Acetic acid (Compound U)
The title compound was prepared from 3-chloronicotinaldehyde (Heterocycles, p.151, 1993) as described in example 1, but the thermal cyclisation was carried out by adding the azide to decalin under reflux.
1H NMR (500MHz, acetone-d6)δ9.20(s，1H)，8.85(s，1H)，7.20(d，2H)，7.00(d，2H)，4.70 (m，1H)，4.30(m，1H)，3.75(m，1H)，3.35(s，3H)，2.80 to 2.10(m，6H).
Example 16
[9- [ (4-chlorophenyl) thio ] thio]-1- (methylsulfonyl) -7, 8-dihydro-6H-pyrido[3，4-b]Pyrin-8-yl]Acetic acid (Compound V)
The title compound was prepared from the product of example 6, method 1, step 8, following the procedures outlined in example 1, steps 10 and 11, using bis (4-chlorophenyl) disulfide in step 10.
1H NMR (500MHz, acetone-d6)δ8.25-8.3(m，1H)，7.71-7.75(m，1H)，7.12-7.17(m，2H)，6.97-7.04(m，2H)，4.45-4.51(m，1H)，4.32-4.39(m，1H)，3.73-3.80(m，1H)，3.29(s，3H)，3.15-3.21(m，1H)，2.99-3.08(m，1H)，2.66-2.73(m，1H)，2.46-2.54(m，1H).
Example 17
(mono) - [ (4-chlorobenzyl) -7-fluoro-5-methylsulfonyl) -1, 2, 3, 4-tetrahydrocyclopentaneDiene [ b ]]Indol-3-yl]Acetic acid (Compound E)
Step 1: (+/-) - (7-fluoro-1, 2, 3, 4-tetrahydrocyclopenta [ b)]Indol-3-ylEthyl acetate
A solution of 10.00g of 4-fluoro-2-iodoaniline, 6.57g of ethyl 2- (2-oxocyclopentyl) acetate and 121mg of p-toluenesulfonic acid in 100ml of benzene was refluxed for 24 hours under an atmosphere of N2 using a Dean-Stark trap. Followed by distillation to remove benzene. Then 60mL DMF was added to the solution, degassed and then added with 19mL Hu in sequencenig base and 405mg Pd (OAc)2. The solution was heated at 115 ℃ for 3 hours and then cooled to room temperature. To quench the reaction, 300ml of 1N HCl and 200ml of ethyl acetate were added and the mixture was filtered through Celite. The two phases are separated and the acidic phase is extracted 2 times with 200ml of ethyl acetate. The organic layers were combined, washed with brine, and dried over anhydrous Na2SO4Drying, filtering with diatomaceous earth, and concentrating. The crude product was purified by flash chromatography eluting with 100% toluene to afford the title compound.
1H NMR (acetone-d)6)δ9.76(br s，1H)，7.34(dd，1H)，7.03(d，1H)，6.78(td，1H)，4.14(q，2H)，3.57(m，1H)，2.85-2.55(m，5H)，2.15(m，1H)，1.22(t，3H).
Step 2 (+/-) - (7-fluoro-1, 2, 3, 4-tetrahydrocyclopenta [ b)]Indol-3-ylAcetic acid
To a solution of 1.24g of the ester of step 1 in 14mL of Tetrahydrofuran (THF) was added 7mL of MeOH followed by 7mL of 2N NaOH at room temperature. After 2.5 hours, the reaction mixture was poured into a separatory funnel containing ethyl acetate (EtOAc)/1N HCl. The two phases were separated and the acid phase was extracted 2 times with EtOAc. The organic phases were combined, washed with brine and dried over anhydrous Na2SO4After drying, evaporation to dryness gave a crude oil which was used as such in the next step (> 90% purity).
1H NMR (acetone-d)6)δ10.90(br s，1H)，9.77(br s，1H)，7.34(dd，1H)，7.04(dd，1H)，6.79(td， 1H)，3.56(m，1H)，2.90-2.50(m，5H)，2.16(m，1H).MS(-APCI) m/z 232.2(M-H)-.
And step 3: (+/-) - (5-bromo-7-fluoro-1, 2, 3, 4-tetrahydrocyclopenta [ b)]Indole-3-Yl) acetic acid
To a solution of 2.20g of the acid of step 2 (> 90% purity) in 30ml of pyridine was added 6.85g of pyridinium tribromide (90% purity) at-40 ℃. The suspension was stirred at 0 ℃ for 10 minutes and warmed to room temperature for 30 minutes. The solvent was then removed under high vacuum without heating. The crude product was dissolved in 40ml of AcOH and 2.88g of Zn powder was added in portions to the solution cooled to 0 ℃. The suspension was stirred at 15 ℃ for 15 minutes, warmed to room temperature and kept for a further 15 minutes. At this point the reaction was quenched by the addition of 1N HCl and the mixture poured into a separatory funnel with brine/EtOAc. The layers were separated and the organic layer was washed with water, brine and anhydrous Na2SO4Drying and concentrating. This material was used in the next step without purification.
1H NMR (acetone-d)6)δ10.77(br s，1H)，9.84(br s，1H)，7.09(m，2H)，3.60(m，1H)，2.95-2.65(m，4H)，2.56(dd，1H)，2.19(m，1H).
And 4, step 4: (+/-) - [ 5-bromo-4- (4-chlorobenzyl) -7-fluoro-1, 2, 3, 4-tetrahydrocyclopentaneDiene [ b ]]Indol-3-yl]Acetic acid
To a solution of 2.13g of the acid of step 3 in 10mL THF was added an excess of diazomethane ethereal solution until TLC monitored that the acid had been completely consumed. The solvent was then removed under reduced pressure. To a solution of the crude methyl ester thus formed in 20ml of DMF at-78 ℃ was added 539mg of NaH suspension (60% in oil). The suspension was stirred at 0 ℃ for 10 minutes, cooled again to-78 ℃ and treated with 1.70g of 4-chlorobenzyl bromide. After 5 minutes, the temperature was raised to 0 ℃ and the mixture was stirred 20And (4) dividing. At this point 2mL of AcOH was added to quench the reaction and the mixture was poured into a separatory funnel containing 1N HCl/EtOAc. The layers were separated and the organic layer was washed with brine and anhydrous Na2SO4Dried and concentrated. The alkylate was hydrolyzed using the procedure described in step 2. The crude material was further purified by trituration with EtOAc/hexanes to give the title compound.
1H NMR (acetone-d)6)δ10.70(br s，1H)，7.31(d，2H)，7.18(d，1H)，7.06(d，1H)，6.92(d，2H)，5.90(d，1H)，5.74(d，1H)，3.61(m，1H)，3.00-2.70(m，3H)，2.65(dd，1H)，2.39(dd，1H)，2.26(m，1H).MS(-APCI)m/z 436.3，434.5(M-H)-.
And 5: (+) - [ 5-bromo-4- (4-chlorobenzyl) -7-fluoro-1, 2, 3, 4-tetrahydrocyclopentediEne and [ b ]]Indol-3-yl]Acetic acid
780 μ L of (S) - (-) -1- (1-naphthyl) ethylamine was added to a solution of 2.35g of the acid of step 4 in 130ml of EtOH at 80 ℃. The solution was cooled to room temperature and stirred overnight. The recovered salt (1.7g) was recrystallized from 200ml EtOH. After filtration, the resulting white solid salt was neutralized with 1N HCl and the product was extracted with EtOAc. The organic layer was washed with brine and anhydrous Na2SO4Drying and concentrating. The material is added into SiO2Filtration over pad and elution with EtOAc afforded the title enantiomer. The retention times of the two enantiomers were 7.5 min and 9.4 min respectively [ chiralPak AD column, hexane/2-propanol/acetic acid (95: 5: 0.1)]. The more polar enantiomer was 98% ee. ee is 98%; retention time 9.4 min [ ChiralPak AD column: 250X 4.6mm, hexane/2-propanol/acetic acid (75: 25: 0.1)]；[α]D21＝+39.2°(c 1.0，MeOH)。
Step 6: (-) - [4- (4-chlorobenzyl) -7-fluoro-5- (methylsulfonyl) -1, 2, 3, 4-Tetracyclopenta [ b ] s]-indol-3-yl]Acetic acid and sodium salt
The acid of step 5 (15.4g) was first esterified with diazomethane. The ester thus formed was mixed with 16.3g of the sodium salt of methanesulfonic acid and 30.2g of CuI (I) in N-methylpyrrolidone and sulfonated. The suspension is placed in N2Degassed under a stream of air, heated to 150 ℃ and stirred for 3 hours, then cooled to room temperature. For quenching, 500ml of ethyl acetate and 500ml of hexane are added and the mixture is passed over SiO2The pad was filtered and eluted with EtOAc. The organic phase was concentrated. The crude oil was dissolved in EtOAc, washed 3 times with water, 1 time with brine, and anhydrous Na2SO4Drying, filtering and concentrating. The crude material was further purified by flash chromatography, eluting with a gradient from 100% toluene to 50% toluene/EtOAc to give 14g of sulfonated ester. It was hydrolyzed according to the procedure described in step 2. Two successive recrystallizations (isopropyl acetate/heptane, followed by CH)2Cl2Hexane) to yield the title compound.
1H NMR (500MHz acetone-d6)δ10.73(br s，1H)，7.57(d，2H，J＝8.8Hz)，7.31(m，1H)，7.29(m，1H)，6.84(d，2H，J＝8.8Hz)，6.29(d，1H，JAB＝17.8Hz)，5.79(d，1H，JAB＝17.8Hz)，3.43(m，1H)，2.98(s，3H)，2.94(m，1H)，2.85-2.65(m，3H)，2.42(dd，1H，J1＝16.1Hz，J2＝10.3Hz)，2 27(m，1H).13C NMR (125MHz acetone: -d)6)δ173.0，156.5(d，JCF＝237Hz)，153.9，139.2，133.7，133.3，130.0(d，JCF＝8.9Hz)，129.6，128.2，127.5(d，JCF＝7.6Hz)，122.2(d，JCF＝4.2Hz)，112.3(d，JCF＝29.4Hz)，111.0(d，JCF＝22.6Hz)，50.8，44.7，38.6，36.6，36.5，23.3.MS(-APCI)m/z 436.1，434.1(M-H)-.
ee is 97%; retention time 15.3 min [ Chiral Cel OD column: 250X 4.6mm, hexane/2-propanol/ethanol/acetic acid (90: 5: 0.2) ];
[α]D21＝-29.3°(C1.0，MeOH)。Mp 175.0℃。
6.45g (14.80mmol) or more of the acid compound was treated with 14.80ml of 1N NaOH aqueous solution in EtOH (100ml) to prepare a sodium salt. The organic solvent was removed under reduced pressure and the crude solid was dissolved in 1.2L of isopropanol under reflux. The solvent was distilled to a final volume of 500 ml. The sodium salt was crystallized by cooling to room temperature. The crystalline sodium salt was suspended in water for ice-bath freezing and freeze-dried under high vacuum to give the title compound as sodium salt.
1H NMR(500MHz DMSO-d6)δ7.63(dd，1H，J1＝8.5Hz，J2＝2.6Hz)，7.47(dd，1H，J1＝9.7Hz，J2＝2.6Hz)，7.33(d，2H，J＝8.4Hz)，6.70(d，2H，J＝8.4Hz)，6.06(d，1H，JAB＝17.9Hz)，5.76(d，1H，JAB＝17.9Hz)，3.29(m，1H)，3.08(s，3H)，2.80(m，1H)，2.69(m，1H)，2.55(m，1H)，2.18(m，2H)，1.93(dd，1H，J1＝14.4Hz，J2＝9.7Hz).
Example 17A
Another preparation (+/-) - [ 5-bromo-4- (4-chlorobenzyl) -7-fluoro-1, 2, 3, 4-tetrahydro ringPentadieno [ b ]]Indol-3-yl]Process for acetic acid (example 17, step 4)
Step 1: (+/-) -7-fluoro-1, 2, 3, 4-tetrahydrocyclopenta [ b)]Indol-3-yl acetic acidsDicyclohexylamine (DCHA) salts
A0.526M solution of 2-bromo-4-fluoroaniline in xylene was heated to reflux with ethyl (2-oxocyclopentyl) acetate (1.5eq) and sulfuric acid (0.02eq) for 20 hours. Water was removed by azeotropic distillation using a Dean-Stark trap. Following the reaction by NMR, 80-85% conversion to the desired imine intermediate is typically observed after 20 hours. The reaction mixture was washed with 1M sodium bicarbonate (0.2 vol) for 15 min and the organic fraction was evaporated off. The remaining slurry was vacuum distilled (0.5 mmHg). Residual xylene is distilled at 30 ℃ and excess ketone and unreacted aniline are recovered at 50-110 ℃; the imine is recovered as a light brown transparent liquid with a purity of 83% from the 110-180 ℃ component.
This imine intermediate was then added to a degassed mixture of potassium acetate (3eq), tetra-N-butylammonium chloride-hydrate (1eq), palladium acetate (0.03eq) and N, N-dimethylacetamide (final concentration of imine 0.365M). The reaction mixture was heated at 115 ℃ for 5 hours and then cooled to room temperature. 3N KOH (3eq) was added and the mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with water (1.0 vol) and washed with toluene (3X 0.75 vol). The aqueous phase was acidified to pH 1 with 3N HCl and extracted with tert-butyl methyl ether (2X 0.75 vol). The combined organic fractions were washed with water (0.75 vol). Dicyclohexylamine (1eq) was added to the clear light brown solution and the solution was stirred at room temperature for 16 hours. The salt was filtered off, washed with ethyl acetate, tert-butyl methyl ether and dried to give the title compound. And (3) analysis results: 94A percent.
1H MR(500mHz，CDCl3)：δ9.24(s，1H)，7.16-7.08(m，2H)，6.82(t，1H)，6.2(br，2H)，3.6-3.5(m，1H)，3.04-2.97(m，2H)，2.88-2.70(m，3H)，2.66(dd，1H)，2.45-2.37(m，1H)，2.13-2.05(m，2.05)，1.83(d，4H)，1.67(d，2H)，1.55-1.43(m，4H)，1.33-1.11(m，6H).
Step 2: (+/-) - (5-bromo-7-fluoro-1, 2, 3, 4-tetrahydrocyclopenta [ b)]Indole-3-Yl) acetic acid
The slurry of DCHA in dichloromethane (0.241M solution) in step 1 above was cooled to-20 to-15 ℃. Pyridine (2eq) was added in increments, then bromine (2.5eq) was added dropwise to the slurry over 30-45 minutes and the temperature was maintained at-20 ℃ to-15 ℃ (upon addition of about 1/3 bromine the reaction mixture became viscous requiring significant stirring-eventually, the mixture became "loose" again upon completion of the addition of about 1/2 bromine). After the addition was complete, the reaction mixture was aged at-15 ℃ for a further 1 hour. Acetic acid (3.04eq) and zinc dust (3.04eq) were then added in portions over 5 minutes. (some of the zinc was added at-15 ℃ C. and the mixture was aged for about 5 minutes to ensure that the exothermic reaction proceeded (about-15 to-10 ℃ C.). This operation was repeated, and about 5 batches of zinc were charged in about 30 minutes. When the exotherm was no longer exothermic, the remaining zinc was added more quickly. The whole operation takes about 30-45 minutes.
After addition was complete, the batch was warmed to room temperature, aged for 1 hour and concentrated. The reaction mixture was transferred to methyl tert-butyl ether (MTBE, 0.8 vol) and 10% aqueous acetic acid (0.8 vol) was added. The mixture is aged at room temperature for 1 hour (crystallization of salts, e.g. pyridinium) and filtered through solka-floc (a microcrystalline cellulose). The solka-floc pad was rinsed with MTBE (ca. 0.2 vol.) and the filtrate (two phase, MTBE/water) was transferred to the extractor. The organic phase was washed with water (0.8 vol). The MTBE extract was concentrated and transferred to isopropanol (IPA, 0.25 volume) to crystallize the compound. Water (0.25 vol) was added and the batch was aged for 1 hour. Water (0.33 vol) was added over 1 hour. After addition was complete, the batch was aged for an additional hour, filtered and rinsed with 30/70 isopropanol/water (0.15 vol). The crystallized bromo acid was dried in an oven at +45 ℃.
And step 3: (+/-) - [ 5-bromo-4- (4-chlorobenzyl) -7-fluoro-1, 2, 3, 4-tetrahydrocyclopentaneDiene [ b ]]Indol-3-yl]Acetic acid
The bromoacid from step 2 was dissolved in dimethylacetamide (0.416M solution) and 2.5eq of cesium carbonate was added in one portion. 4-chlorobenzyl chloride (2.5eq) was added to the slurry in one portion and heated at 50 ℃ for 20 hours. The batch was cooled to room temperature, 5N NaOH (4.00eq) was added over 5 minutes and the temperature rose to +40 ℃. The reaction mixture was aged at 50 ℃ for about 3 hours, cooled to room temperature, and transferred to an L extractor. The solution was diluted with isopropyl acetate (IPAc, 2 vol) and cooled to +15 ℃. The solution was acidified to pH 2 with 5N HCl. The layers were separated and the organic layer was washed with water (2 × 2 vol). The IPAc solution was concentrated and transferred to IPA (0.8 vol) to crystallize the product. Water (8L) was added over 2 hours and the batch was filtered to give the title compound. The batch can be dried in an oven at +40 ℃ for 24 hours.
Example 18
(+/-) - {4- [1- (4-chlorophenyl) ethyl]-7-fluoro-5-methanesulfonyl-1, 2, 3, 4-tetrakisHydrocyclopenta [ b ]]Indol-3-yl } acetic acid (Compound X)
The title compound was synthesized according to the instructions provided in PCT WO 03/062200 published on 30/7/2003.
Example 19
(+/-) [9- (4-chlorobenzyl) -6-fluoromethanesulfonyl-2, 3, 4.9-tetrahydro-1H-carbazole-1-Base of]Acetic acid (Compound Y)
The title compound was synthesized according to the instructions provided in PCT WO 03/062200 (published on 30/7/2003).
Example 20
[4- (4-chlorobenzyl) -7-fluoro-5-methanesulfonyl-1-oxo-1, 2, 3, 4-tetrahydrocyclopentadieneAnd [ b ]]Indol-3-yl]Acetic acid (Compound Z)
The title compound was synthesized according to the instructions provided in PCT WO 03/062200 published on 30/7/2003.
Example 21
{9- [ (3, 4-dichlorophenyl) thio]-1-isopropyl-7, 8-dihydro-6H-pyrido [3, 4-b]Pyrin-8-yl } acetic acid (Timomer A and Timomer B) (Compound AA)
Step 12-Chlornicotinaldehyde
To a solution of diisopropylamine (110ml, 780mmol) in THF (500ml) was added a 2.5M solution of n-butyllithium (300ml, 750mmol) in hexane at-40 ℃. After 5 minutes the reaction mixture was cooled to-95 ℃ and then DMPU (15ml) and 2-chloropyridine (50ml, 532mmol) were added successively and the resulting mixture was warmed and stirred at-78 ℃ for 4 hours. After this time, the yellow suspension was cooled again to-95 ℃ and DMF (70ml) was then added. The final reaction mixture was warmed to-78 ℃ and stirred at this temperature for 1.5 hours. The reaction mixture was poured into cold aqueous hydrochloric acid (3N, 800ml) and stirred for 5 minutes. The pH was adjusted to 7.5 by adding concentrated ammonia. The aqueous layer was extracted 3 times with EtOAc. Combined organic layer with NH4Washed with aqueous Cl solution and brine, and then with anhydrous Na2SO4Drying, filtering and concentrating. The crude material was further purified on a silica gel pad, eluting with a gradient from 100% hexanes to 100% EtOAc, and the product crystallized in cold hexanes to give the title compound as a pale yellow solid.
Step 2 (2Z) -2-azido-3- (2-chloropyridin-3-yl) prop-2-enoic acid methyl ester
To a 25% sodium methoxide/methanol solution (80ml, 349mmol) was added a solution of 2-chloronicotinaldehyde (20.0g, 139.9mmol) and methyl azidoacetate (32.2ml, 349.7mmol) in methanol (168ml) at-20 ℃. The internal temperature was monitored and maintained at about-20 ℃ over a 30 minute period of addition. The resulting mixture was then stirred in an ice bath for several hours, followed by overnight in an ice bath in a cold room. Pour the suspension on ice and NH4Over a mixture of Cl for 10 minThe slurry is then filtered. The product was washed with cold water and then dried under vacuum. The crude product was dissolved in CH2Cl2Adding MgSO 24. The suspension was filtered through a pad of silica gel and CH was added2Cl2And (6) washing. The filtrate was concentrated under reduced pressure to give a brown-grey precipitate (20g) of the title compound.
Step 34-chloro-1H-pyrrolo [3, 2-c]Pyridine-2-carboxylic acid methyl ester
A solution of methyl (2Z) -2-azido-3- [ 2-chloropyridin-3-yl ] prop-2-enoate (21g, 88mmol) in 1,3, 5-trimethylbenzene (880ml) was heated under reflux for 1 hour. The reaction mixture was cooled to room temperature, then to 0 ℃, and the precipitate was filtered off and washed with cold hexane. The material was stirred overnight in 1: 20 ethyl acetate/hexane to give the title compound as a pale yellow solid after filtration (13.2 g).
Step 41-chloro-8-oxo-7, 8-dihydro-6H-pyrido [3, 4-b]Pyridine-7-carboxylic acid methyl esterEsters
To 4-chloro-1H-pyrrolo [3, 2-c)]To a suspension of pyridine-2-carboxylic acid methyl ester (12.5g, 59mmol) in THF (116 ml)/toluene (460ml) were added a 1.0M potassium tert-butoxide/THF solution (64ml, 64mmol) and methyl acrylate (55ml, 611 mmol). The resulting mixture was heated at 100 ℃ for 18 hours. After this the suspension was cooled to room temperature and NH was poured in4A mixture of saturated aqueous Cl (400ml) and hexane (400 ml). The solid was decanted, filtered and washed with water and hexanes to give the title compound.
Step 51-chloro-6, 7-dihydro-8H-pyrido [3, 4-b ]]Pyrin-8-one
To the compound of the previous step, isopropanol (8.0ml) and concentrated hydrochloric acid (2.0ml) were added and heated at 100 ℃ for 1 hour. The reaction mixture was partitioned between EtOAc and Na2CO3Among them. The organic phase was separated and evaporated to give the title compound.
Step 61-isopropenyl-6, 7-dihydro-8H-pyrido [3, 4 ]-b]Pyrin-8-one
To 1-chloro-6, 7-dihydro-8H-pyrido [3, 4-b ]]Tributylisopropenylstannane (9.60g, 29.00mmol) was added to a mixture of pyrantel-8-one (5.0g, 24.3mmol), tris (dibenzylideneacetone) dipalladium (O) (1.0g, 1.09mmol) and triphenylarsine (2.70g, 8.82mmol) in DMF (100 ml). The resulting mixture was degassed and heated at 78 ℃ for 18 hours. The solvent was distilled off under reduced pressure. Adding CH to the resulting mixture2Cl2And celite, then filtered through celite. The title compound was purified by flash chromatography (50% to 100% EtOAc/hexanes).
Step 7 (2E) - (1-isopropenyl-6, 7-dihydro-8H-pyrido [3, 4-b)]Pyridine-8-Subunit) acetic acid ethyl ester
To 1-isopropenyl-6, 7-dihydro-8H pyrrolo [3, 4-b ] at-78 DEG C]A solution of pyranin-8-one (0.60g, 2.8mmol) and triethyl phosphonoacetate (1.00g, 4.46mmol) in THF (24ml) was added 80% NaH (0.12g, 4.00mmol) and the reaction mixture was warmed to 0 deg.C and then to room temperature. The reaction mixture was poured into saturated NH4Cl and EtOAc. Separating the organic phase from Na2SO4Dried and evaporated. The title compound was purified by flash chromatography (40% EtOAc/hexanes).
Step 8 (1-isopropyl-7, 8-dihydro-6H-pyrido [3, 4-b)]Pyrin-8-yl) acetic acidEthyl ester
To (2E) - (1-isopropenyl-6, 7-dihydro-8H-pyrido [3, 4-b)]Ethyl-8-pyrazin-ylidene) acetate (0.40g, 1.4mmol) in MeOH (20ml) was added Pd (OH)2(0.20 g). The mixture is brought to 1 atmosphere H2Stirred for 3 hours. The mixture was filtered through celite and evaporated to give the title compound.
Step 9 {9- [ (3, 4-dichlorophenyl) thio]-1-isopropyl-7, 8-dihydro-6H-pyridineAnd [3, 4-b ]]Pyrin-8-yl ethyl acetate
To two(3, 4-dichlorophenyl) disulfide (0.24g, 0.67mmol) in CH2Cl2(5.6ml) solution in SO2Cl2(0.036 ml). The resulting yellow mixture was stirred at room temperature for 1 hour. Adding the solution to (1-isopropyl-7, 8-dihydro-6H-pyrido [3, 4-b ] at 0 deg.C]A solution of ethylpyran-8-yl) acetate (0.15g, 0.52mmol) in DMF (5.6 ml). After 1.5 hours at 0 ℃ the reaction mixture was poured into saturated NaHCO3Solution and EtOAc. Separating the organic phase with Na2SO4Dried, filtered and evaporated. The title compound was purified by flash chromatography (30% to 40% EtOAc/hexanes).
Step 10 {9- [ (3, 4-dichlorophenyl) thio]-1-isopropyl-7, 8-dihydro-6H-pyri dinePyrido [3, 4-b]Pyrin-8-yl acetic acid
To {9- [ (3, 4-dichlorophenyl) thio]-1-isopropyl-7, 8-dihydro-6H-pyrido [3, 4-b]A solution of ethyl-pyrazol-8-yl } acetate (0.23g, 0.50mmol) in THF (5ml) and MeOH (2.5ml) was added 1.0M NaOH (1.5ml, 1.5 mmol). After stirring at room temperature for 18 hours, HOAc (0.25ml) was added and the solvent was distilled off. The residue was taken up in EtOAc/water and the organic layer was washed with water and brine. With Na2SO4After drying, the solution was filtered and evaporated. The residue was stirred with 1: 1 ethyl acetate/hexane to give the title compound as a white solid after filtration.
1H NMR(MeOH-d4)δ1.14-1.26(m，6H)，2.47-2.56(m，1H)，2.56-2.64(m，1H)，2.94-3.05(m，2H)，3.81-3.89(m，1H)，4.22-4.30(m，1H)，4.33-4.44(m，2H)，6.93-6.99(m，1H)，7.14-7.19(m，1H)，7.33-7.39(m，1H)，7.54-7.59(m，1H)，8.16-8.21(m，1H).
Using CH for the product of the step 102N2Converted to its methyl ester and the ester was separated by HPLC on a chiral stationary phase (Chiralcel OD column 2X 25cm) eluting with 12% 2-propanol/hexane at a flow rate of 6 ml/min. The retention time for enantiomer a (less polar) was 31.9 minutes and for enantiomer B (more polar) was 35.5 minutes. Both A and B are as in example 17Hydrolysis as in 10 gave the enantiomers a and B of the title compound.
Example 22
((1R) -6-fluoro-8- (methylsulfonyl) -9- { (1S) -1- [4- (trifluoromethyl) phenyl]Ethyl } -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl) acetic acid (compound AJ)
Step 1 Chlorination of 2- (2-bromo-4-fluorophenyl) hydrazineOnium salts
To a suspension of 2-bromo-4-fluoroaniline in concentrated hydrochloric acid (1.5M) was slowly added 10.0M NaNO at-10 deg.C2Aqueous solution (1.1 eq). The mixture was stirred at 0 ℃ for 2.5 hours. Then SnCl is slowly added2A cold (-30 ℃) solution (3.8M) in concentrated hydrochloric acid while maintaining the internal temperature below 10 ℃. The resulting mixture was mechanically stirred at 10 ℃ for 20 minutes and then at room temperature for 1 hour. The thick slurry was filtered and the solid air dried overnight. Resuspended in cold hydrochloric acid and filtered again. The dry material was suspended in ether, stirred for 10 minutes, filtered and air dried overnight to give the title compound as a brown-grey solid.
Step 2 (+/-) (8-bromo-6-fluoro-2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl) acetic acid ethyl esterEsters
To a suspension of the compound of step 1(1 eq) in AcOH (0.5M) was added ethyl (2-oxocyclohexyl) acetate (1 eq). The mixture was stirred at reflux for 16 h, cooled and the AcOH was removed by evaporation under reduced pressure. The residue was diluted with EtOAc, water and saturated NaHCO3And (4) washing with an aqueous solution. Na for organic layer2SO4Dried and concentrated. The residue was subsequently purified on a pad of silica gel, eluting with toluene. The filtrate was concentrated, stirred in hexane and filtered to give the title compound as a white solid. MS (+ APCI) m/z 354.2(M+H)+。
And step 3: (+/-) - [ 6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazole-1-Base of]Ethyl acetate
To a solution of compound (1eq) in anhydrous DMSO (0.28M) from step 2 was added sodium methylsulfinate (3eq) and copper iodide (3 eq). Bubbling N into the mixture2Gas for 5 minutes, then stirred at 100 ℃ under nitrogen. After 12 hours, sodium methylsulfinate (2eq) and ketone iodide (2eq) were added. The mixture was stirred at 100 ℃ for an additional 12 h, cooled, diluted with EtOAc and acidified by addition of 1N HC 1. The suspension was stirred for 30 minutes and filtered through celite. The filtrate was washed with water and Na2SO4Dried and concentrated. The residue was filtered through a pad of silica gel eluting with toluene to remove non-polar impurities and then the desired product was eluted with a 2: 1 mixture of hexane/EtOAc. The filtrate, eluted with a hexane/EtOAc mixture, was concentrated to the title compound as a pale yellow solid. MS (-APCI) M/z 352.1(M-H)-。
And 4, step 4: [ (1R) -6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazole-1-yl]Ethyl acetate
The racemic mixture of step 3 was resolved by preparative HPLC on a Chiralpak AD preparative column eluting with a 15% isopropanol/hexane mixture. The title compound was identified as the highly polar enantiomer (long retention time) based on the activity of the final product.
Step 5[ (1R) -9- [ (1S) -1- (4-chlorophenyl) ethyl]-6-fluoro-8- (methylsulfonyl)Acyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Ethyl acetate
To a solution of the compound of step 4 (1eq), triphenylphosphine (1.5eq) and (1R) -1- (4-chlorophenyl) ethanol (1.5eq, prepared according to the general procedure described in reference example 1) in THF (0.175M) was added di-tert-butyl azodicarboxylate (2.1M in THF, 1.5eq) over 10 minutes. The mixture was stirred at room temperature for 2 hours and concentrated. The residue was purified by flash chromatography on silica gel eluting with 7% EtOAc/toluene to give the desired product (purity-90%) which was used as such in the next reaction.
Step 6 [ (1R) - [ (1S) -1- (4-chlorophenyl) ethyl]-6-fluoro-8- (methylsulfonyl)-2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Acetic acid and [ (1S) -9- [ (1S) -1) -1- (4-Chlorophenyl) ethyl group]-6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Second stepAcid(s)
To a solution of the compound of step 5 in a 2: 1 THF/methanol mixture (0.1M) was added 1N aqueous LiOH (3 eq). The mixture was stirred at room temperature for 2 hours, AcOH was added and the solvent was removed by evaporation. The residue was taken up in EtOAc/water and the organic layer was washed with brine and Na2SO4Drying, filtering and concentrating. The residue was shaken in 30% EtOAc/hexanes, the product suspended in diethyl ether sonicated for 45 minutes, filtered, and dried under vacuum at 50 ℃ for 24 hours to give the title compound as a white solid. MS (-APCI) M/z 462.1 (M-H).
Alternatively, alkylation using ethyl (+/-) [ 6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl ] acetate in step 5 gave a mixture of two diastereomers: ethyl [ (1R) -9- [ (1S) -1- (4-chlorophenyl) ethyl ] -6-chloro-8- (methylsulfonyl) -2, 3, 4.9-tetrahydro-1H-carbazol-1-yl ] acetate and ethyl [ (1S) -9- [ (1S) -1- (4-chlorophenyl) ethyl ] -6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl ] acetate. The diastereomeric mixture is selectively hydrolyzed using the following procedure to give the desired [ (1R) -9- [ (1S) -1- (4-chlorophenyl) ethyl ] -6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl ] acetic acid.
Dismantling
Reacting [ (1R) -9- [ (1S) -1- (4-chlorophenyl) ethyl]-6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Ethyl acetate and [ (1S) -9- [ (1S) -1- (4-chlorophenyl) ethyl ] ethyl]-6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Diastereomer mixture of ethyl acetate (1eq) dissolved in 3.51 in a THF/MeOH mixture (0.25M) and cooled at 0 ℃. LiOH 1N aqueous solution (1eq) was slowly added and the mixture was stirred at 0 ℃ for 12 hours or to [ (1R) -9- [ (1S) -1- (4-chlorophenyl) ethyl]-6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]The ethyl acetate was almost completely hydrolyzed and the other diastereomer was only slightly hydrolyzed under these conditions. AcOH was added, the solvent was evaporated, the residue was taken up in EtOAc/water, the organic layer was washed with brine and Na2SO4Dried, filtered and concentrated. Subjecting [ (1S) -9- [ (1S) -1- (4-chlorophenyl) ethyl group to flash chromatography]-6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Ethyl acetate and [ (1R) -9- [ (1S) -1- (4-chlorophenyl) ethyl ] ethyl]-6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Acetic acid was separated and eluted with EtOAc in 1% AcOH in hexane to give the desired [ (1R) -9- [ (1S) -1- (4-chlorophenyl) ethyl ] ethyl]-6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Acetic acid, de > 90%, was stirred in 30% EtOAc/hexanes to give the desired compound as a white solid, de > 95%.
Step 7[ (1R) -6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Acetic acid methyl ester
To [ (1R) -9- [ (1S) -1- (4-chlorophenyl) ethyl group]-6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Acetic acid ([ alpha ])]D= 226 °, MeOH) in MeOH (0.1M) was added 10% palladium on carbon (10% wt/wt). Bubbling N into the mixture2And the air flow is for 5 minutes. At room temperature and H2The reaction mixture was stirred under atmosphere (gas cylinder) for 24 hours, filtered through a pad of celite, and filtered through CH2Cl2And (4) eluting. The solvent was removed by evaporation under reduced pressure and the residue was transferred to MeOH to give the compound [ (1R) -6-fluoro-8- (methylsulfonyl) -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl]Methyl acetate.
Step 8((1R) -6-fluoro-8- (methylsulfonyl) -9- { (1S) -1- [4- (trifluoromethyl)Phenyl radical]Ethyl } -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl) acetic acid (compound AJ)
To a solution (0.2M) of the compound of step 7 (1eq), triphenylphosphine (1.5eq) and (1R) -1- [4- (trifluoromethyl) phenyl ] ethanol (1.5eq) in THF was added a solution of di-tert-butyl azodicarboxylate (1M in THF, 1.5eq) over 20 minutes. The mixture was stirred at room temperature for 2 hours and concentrated. The residue was purified by flash chromatography on silica gel eluting with 10% EtOAc/toluene to give methyl ((1R) -6-fluoro-8- (methanesulfonyl) -9- { (1S) -1- [4- (trifluoromethyl) phenyl ] ethyl } -2, 3, 4, 9-tetrahydro-1H-carbazol-1-yl) acetate (purity-90%) which was used as such in the next reaction.
To a 0 ℃ solution (0.25M) of the above ester (1eq) in a 3.5/1 mixture of THF/MeOH was slowly added 1N aqueous LiOH (1eq) and the mixture was stirred at 0 ℃ for 16 h or until the ester was almost completely hydrolyzed. Under these conditions, the other, less diastereomer hydrolyzed much more slowly. AcOH was added and the solvent was removed under reduced pressure. The residue was taken up in EtOAc/water and the organic layer was washed with brine and Na2SO4Drying, filtering and concentrating. To remove unreacted methyl ester, the residue was filtered through a pad of silica gel eluting first with 10% EtOAc/toluene and then with 60% EtOAc/toluene containing 1% AcOH. The residue was stirred in EtOAc/hexanes and dried at 50 ℃ under high vacuum for 16 h to give the title compound as a white solid, de and ee > 95% (checked by chiral HPLC). MS (-APCI) M/z 496.0(M-H)-。[α]D-181 ° (in MeOH).
Biology of the species
Compounds useful in the invention as selective DP antagonists generally have affinity for DP (K)i) Comparison of affinity to the CRTH2 receptor (K)i) At least about 10 times higher. Typical DP antagonists used in the present invention are at least about 10-fold more selective for the DP receptor than the CRTH2 receptor. More particularly, the selective DP receptor antagonistsThe selectivity of the anti-agent for the DP receptor is at least 100-fold greater than the selectivity for the CRTH2 receptor. In particular, the selective DP antagonist compounds are at least about 800-fold more selective for the DP receptor than for the CRTH2 receptor, i.e., have an affinity (K) for the DP receptor that is greater than about 1000-foldi) Comparison of affinity to the CRTH2 receptor (K)i) 800 times higher and 1000 times higher.
As used herein, when a compound "selectively modulates DP receptors," the compound binds to and antagonizes DP receptors at a therapeutically achievable concentration, while at such a therapeutically achievable concentration, there is substantially no modulation of CRTH 2.
In general, the DP antagonist used in the present invention has affinity (K) for the CRTH2 receptori) About 0.5 micromolar or more. Those compounds having a binding affinity for the CRTH2 receptor of about 0.5 micromolar or greater and a selectivity for the DP receptor that is at least about 10-fold greater than for the CRTH2 receptor are useful for inhibiting flushing observed with niacin without such selective DP antagonists.
Affinity and selectivity assays for compounds at recombinant human DP and CRTH2 receptors
Receptor affinity and selectivity of compounds on DP and CRTH2 using Abramovitz M et al, biochem. biophysis. acta (2000) 1483: 285, 293, and Sawyer N et al, Br. J. pharmacol. (2002) 137: 1163-1172, by the radioligand binding assay. Briefly, stable cell lines expressing human DP and CRTH2 receptors, respectively, were established using Human Embryonic Kidney (HEK)293EBNA (epstein barr virus nuclear antigen) cells, referred to as HEK 293E cell line. Membrane fractions prepared from these recombinant cell lines were used in equilibrium competition radioligand binding assays to determine the affinity and selectivity of compounds at the DP and CRTH2 receptors.
DP and CRTH2 cDNAs corresponding to the full-length coding sequence were subcloned into the appropriate sites of the mammalian expression vector pCEP4(Invitrogen) and expressed in HEK 293E cells. Using differential centrifugation (1000Xg for 10 min, then 160,000Xg30 minutes, all at 4 ℃), and then the cells were lysed by nitrogen cavitation at 800psi for 30 minutes on ice in the presence of protease inhibitors (2mM AEBSF, 10. mu.M leupeptin and 0.05mg/ml pepstatin) to prepare cell membranes. 160,000Xg of the pellet was resuspended in 10mM HEPES/KOH (pH7.4) containing 1mM EDTA according to approximately 5-10mg/ml protein using Dounce homogenization (Dounce A; 10 strokes), frozen in liquid nitrogen and stored at-80 ℃. The receptor binding assay was performed in the presence of 1mM EDTA, 10mM MnCl2And 0.7nM [2 ]3H]PGD2(200Ci/mmol) in 10mM HEPES/KOH (pH7.4) in a final culture volume of 0.2 ml. The reaction was initiated by the addition of membrane proteins obtained from 160,000Xg fractions (about 30. mu.g for DP, about 10. mu.g for CRTH 2). The ligand was added in dimethyl sulfoxide (DMSO) and was constant at 1% (v/v) in all cultures. At 10. mu.M nonradioactive PGD2Nonspecific binding was determined in the presence of the buffer. Incubate on a micro orbital shaker at room temperature for 60 minutes. Affinity assays were terminated by rapid filtration through 96-well Unifilter GF/C (Canberra packard) previously wetted in EDTA-free assay culture buffer (at 4 ℃) using a Tomtec MachIII 96-well semi-automated cell harvester. The filter was washed with 3 to 4ml of the same buffer and dried at 55 ℃ for 90 minutes. The residual radioactivity bound to each filter was determined by scintillation counting using a 1450 MicroBeta (Wallac) counter with the addition of 50. mu.L of Ultima Gold F (Canberra Packard).
Maximum specific binding is defined as the total amount of binding minus the amount of non-specific binding in the absence of competitor. Specific binding was determined at each concentration of compound and expressed as a percentage of maximal specific binding. The percentage of maximum specific binding was expressed as a function of test compound concentration to construct a sigmoidal equilibrium competition curve, which was analyzed to determine the inflection point (InPt) using a custom designed software package that employed a one-way driven non-linear least squares curve fitting routine based on a four-parameter equation. From equation KiInPt/1+ ([ radioligand)]/Kd) Calculating the equilibrium inhibition constant (K)i) Determining the binding affinity of the test compound, wherein KdIs a radioligand-acceptor phaseEquilibrium dissociation constant of the interaction. When InPt is not determined, IC is used50(i.e., the concentration of test compound required to inhibit 50% of the maximum specific binding).
Compounds for use in the invention are generally K for the DP receptoriFrom as low as about 0.4nM to as high as about 16.3 nM. Furthermore, the compounds used in the present invention are generally K for the CRCH2 receptoriFrom as low as about 180nM to as high as about 22,000nM and higher.
Effect of Compounds on Niacin-induced vasodilation in mice
The efficacy of the selective DP antagonists described herein can be demonstrated by measuring the flushing-inhibiting effect using a human murine model of niacin-induced flushing. Blood flow (a measure of vasodilation, an important component of human flushing) in the ears of mice was measured following administration of niacin to mice pretreated with vehicle (as control) or DP antagonist. Specifically, male C57BL/6 mice were used in this study. 5 mice were evaluated in each test group. Pentobarbital was diluted with water to a final concentration of 5mg/ml and injected intraperitoneally 0.3ml per mouse. The DP antagonist was dissolved in 5% hydroxypropyl β -cyclodextrin to a final concentration of 5 mg/ml. The compound was administered intraperitoneally (40 mpk) in a volume of 0.2 ml/rat. Nicotinic acid was dissolved in 5% hydroxypropyl beta cyclodextrin to a final concentration of 12.5 mg/ml. The stock niacin solution was adjusted to pH7.4 with alpha N NaOH and injected subcutaneously (-100 mpk) at 0.2 ml/mouse.
Perfusion of the skin of the mouse ear was monitored every 30 seconds for 30 minutes using a laser Doppler perfusion imager (PeriScan PIM II, Perimed, Sweden) starting 5 minutes before application of niacin. The percent change in mean perfusion over a 10 minute period following vehicle or niacin administration was calculated and the percent change in mean perfusion was plotted over time for each animal. The area under the curve (AVC,% Δ x points) for mean perfusion was calculated for each graph, and the results are expressed as mean AVC ± SEM for each group.
Compound D inhibited PGD-2-induced vasodilation in mice (figure 1). The DP antagonist tested inhibited niacin-induced vasodilation in mice. Data for selected compounds are provided in fig. 2 and 3.
All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. Although some preferred embodiments have been described in detail, many alternative embodiments are within the scope of the invention.