WO2002078709A2

WO2002078709A2 - Use of pyrido`2,3-d pyrimidin derivatives for the treatment of human cytomegalovirus infections

Info

Publication number: WO2002078709A2
Application number: PCT/US2002/001232
Authority: WO
Inventors: Douglas Wayne Balogh; Joseph Matthew Colacino
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2001-02-16
Filing date: 2002-02-04
Publication date: 2002-10-10
Anticipated expiration: 2003-08-16
Also published as: WO2002078709A3; AU2002306413A1

Abstract

The present invention provides a method for treating HCMV in a human requiring said treatment, which comprises administering to said human an effective amount of a compound of formula I: formula (I); where: Y is CH=CH, O, or S; R3 is hydrogen, chloro, or fluoro; R4 is hydroxy, a carboxy protecting group, or NHC*H(COOH)CH¿2?CH2COOH where the configuration about the carbon atom designated * is S; and R?6¿ is hydrogen or an amino protecting group; and R7 is hydroxy or amino; or a salt or solvate thereof.

Description

ANTIVIRAL METHOD OF USE

Human cytomegalovirus (HCMN) is a herpesvirus commonly found in about 50% of the general population. About 90% of people with HIN carry HCMN. In the general population, the virus usually remains latent in the tissues of the body after the initial infection. It can, however, be shed in the mouth, urine, and genital tract, serving as a source of infection for other people. Infection with HCMN can result in secondary, more severe infections if the immune system becomes compromised for any reason.

Symptoms primarily depend on the age of the person and how strong their immune system is. HCMN may infect a healthy baby in utero. Roughly 5% of infants who get HCMN through vertical transmission have serious birth defects. These can include brain damage, growth failure, blindness, and other defects. This problem usually occurs when the mother becomes infected with HCMN for the first time during pregnancy. When HCMN causes infection in early childhood, it usually results in no symptoms at all. This is thought to be the most common form of HCMN infection. During the teenage and young adult years, infection with HCMN can cause a syndrome similar to that of infectious mononucleosis, or "mono." Here the symptoms include sore throat, fatigue, fevers, and swollen glands and can last for weeks or even months. Full recovery generally occurs without treatment. In the general adult population, HCMN lies dormant, but may be associated with the development of coronary artery disease. Infection with HCMN has been associated with the development of arterial plaques and atherosclerosis.

HCMN can cause serious problems in people with weakened immune systems. This is most commonly a problem in people with AIDS or in those patients on immunosuppressive therapy. HCMN infects between 75 and 100% of HIN positive patients. The most common complications associated with HCMN include chorioretinitis; gastrointestinal tract infections, including hepatitis, esophagitis, colitis, gastritis, and pancreatitis; neurologic involvement, including encephalitis and polyradiculitis; pulmonary involvement; and epididymitis. People with widespread cancer or people who receive organ or bone marrow transplants are commonly affected. Infection may be due to a first time exposure to HCMN or as a result of reactivated HCMN. In transplant and cancer patients, HCMN usually causes pneumonia or a gastrointestinal infection resulting in diarrhea, which may cause death. Furthermore, HCMN contributes to the development of chronic allograft dysfunction in solid organ transplant recipients. The relationship between HCMN disease and the development of bronchiolitis obliterans in lung transplant recipients is well established. Additionally, HCMN is one of a number of risk factors that may lead to allograf injury. Direct viral invasion of the allograph may cause HCMN hepatitis in liver or kidney transplant patients. In addition to direct syndromes produced by HCMN, infection with this virus may increase the risk for fungal and other opportunistic infections, such as Pneumocystis carinii pneumonia and Epstein-Barr virus-related posttransplant lymphoproliferative disease.

Most people have been infected with HCMN by the time they are adults. Anyone receiving a blood transfusion or an organ transplant is at risk for a HCMN infection. Furthermore, people with weakened immune systems and unborn children are at risk for severe disease.

Treating active HCMN in people with weakened immune systems is currently done with antiviral agents, such as ganciclovir, foscarnet, and cidofovir. These drugs may have side effects that include granulocytopenia and anemia for ganciclovir; nephrotoxicity, neurotoxicity, and electrolyte disturbance for foscarnet; and nephrotoxicity, neurotoxicity, and alopecia for cidofovir. Recently, Tomudex® has been found to be active against HCMN. Tomudex® has side effects that consist of rash, diarrhea, decreased production of blood cells by the bone marrow, pyrexia, nausea and vomiting, and loss of appetite. Despite a number of therapeutic approaches, HCMN remains a significant cause of morbidity and mortality in immunosuppressed patients. Antifolates, which are Ν-(6-amino-(pyrido(2,3-- )pyrimidin-3-ylacyl)-glutamic acid derivatives, have been used for a number of years as chemotherapeutic agents in the treatment of cancer. A number of such antifolates are known (see: for example, U.S. Patents 4,684,653 and 4,882,334, incorporated by reference herein). (R)-N-[[5-[2-(2- amino-3,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-- |pyrimidin-6-yl)ethyl]-2- thienyl]carbonyl]-L-glutamic acid, also known as GARFT II; and (6R)-N-[4-[2-(2-amino- 3,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-- ]pyrimidin-6-yl)ethyl]benzoyl]-L-glut-ιmic acid, also known as Lometrexol, are two such compounds. Lometrexol is currently in clinical trials for use as an anticancer treatment in patients exhibiting a wide variety of solid tumors. Extensive research and evaluation has revealed that GARFT II and Lometrexol are potent inhibitors of glycinamide ribonucleotide formyltransferase.

Surprisingly and unexpectedly, we have now found that N-(6-amino-(pyrido(2,3- - )pyrimidin-3-ylacyl)-glutamic acid derivatives are useful as anticytomegalovirus agents.

The present invention provides a method for treating HCMN in a human requiring said treatment, which comprises administering to said human an effective amount of a compound of formula I:

i;

where:

Y is CH*=CH, O, or S; R3 is hydrogen, chloro, or fluoro;

R⁴ is hydroxy, a carboxy protecting group, or NHC*H(COOH)CH2CH₂COOH where the configuration about the carbon atom designated * is S; and

R is hydrogen or an amino protecting group; and

R is hydroxy or amino; or a salt or solvate thereof.

The present invention further provides the use of an effective amount of a compound of formula I for the manufacture of a medicament for the treatment of HCMN.

Additionally, the present invention contemplates a therapeutic package for dispensing to, or for use in dispensing to, a patient being treated for HC-VIN comprising one or more unit doses, each unit dose comprising an amount of a compound of formula I therein such that periodic administration of one or more of said unit doses is effective to treat HCMN; and a finished pharmaceutical container therefore, said container further containing or comprising labeling, said labeling indicating that a compound of formula I is indicated for the treatment of patients with HCMN.

Additionally, the present invention contemplates an article of manufacture comprising packaging material and a compound of formula I contained within said packaging material, wherein a compound of formula I is therapeutically effective for treating HCMN, and wherein the packaging material comprises a label which indicates that a compound of formula I can be used to treat HCMN.

GARFT II, (R)-Ν-[[5-[2-(2-amino-3,4,5,6,7,8-hexahydro-4-oxopyrido[2,3- <f]pyrimidin-6-yl)ethyl]-2-thienyl]carbonyl]-L-glutamic acid, is represented by formula II:

II.

GARFT II was first taught by U.S. Patent No. 4,882,334, the teachings of which are herein incorporated by reference in its entirety.

Lometrexol, (6R)-N-[4-[2-(2-amino-3,4,5,6,7,8-hexahydro-4-oxopyrido[2,3- (f]pyrimidin-6-yl)ethyl]benzoyl]-L-glutamic acid, is represented by formula III:

Lometrexol was first taught by U.S. Patent No. 4,684,653, the teachings of which are herein incorporated by reference in its entirety. The compounds of formula II and III can exist in tautomeric equilibrium with the corresponding 4(3H)-oxo compounds. For illustrative purposes, the equilibrium for the pyridopyrimidine ring system and the numbering thereof, are shown below:

For convenience, the 4(3H)-oxo form is depicted in formula II and III, and the corresponding nomenclature is used throughout this specification. However, it is understood that such depictions include the corresponding tautomeric 4-hydroxy form.

The compounds of formula I often can be employed advantageously in the form of a pharmaceutically acceptable salt. Such salts with bases include those formed from the alkali metals, alkaline earth metals, non-toxic metals, ammonium, and mono-, di-, and trisubstituted amines, such as for example the sodium, potassium, lithium, calcium, magnesium, aluimnum, zinc, ammonium, trimethylammonium, triethanolammonium, pyridinium, and substituted pyridinium salts. The mono and disodium salts, particularly the disodium salts, are preferred. In the method according to the invention, the terms "treat" or "treating" bear their usual meaning which includes preventing, prohibiting, alleviating, ameliorating, halting, restraining, slowing or reversing the progression, or reducing the severity of HCMN. hi the method according to the invention, the term "HCMN" refers to human cytomegalovirus. The human cytomegalovirus is a herpesvirus that causes cellular enlargement and formation of eosinophilic inclusion bodies.

In the method according to the invention, the term "effective amount" refers to an amount of a compound or drug, which is capable of performing the intended result. For example, an effective amount of Lometrexol that is administered in an effort to treat HCMN is that amount which is required to prevent, prohibit, alleviate, ameliorate, halt, restrain, slow or reverse the progression, or reduce the severity of HCMN.

The present invention also includes methods employing pharmaceutical formulations, which contain, as the active ingredient, a compound of formula I, in association with pharmaceutical carriers. A skilled artisan would know of such formulations and their manufacture, see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, (16th ed. 1980).

The formulations are preferably formulated in a unit dosage form, each dosage containing from about 0.1 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. GARFT II and Lometrexol are effective over a wide dosage range. For example, dosages per day normally fall within the range of about 0.5 to about 30 mg/kg of body weight. However, it will be understood that the amount of a compound of formula I actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and therefore the above dosage ranges are not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several smaller doses for administration throughout the day.

A compound of formula I can be administered in the form of a pharmaceutical composition in combination with pharmaceutically acceptable carriers or excipients, the proportion and nature of which are determined by the solubility and chemical properties of the compound in the carriers and/or excipients selected, the chosen route of administration, and standard pharmaceutical practice.

Pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, (16th ed. 1980). The carrier or excipient may be a solid, semi-solid, or liquid material, which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art. The pharmaceutical composition may be adapted for oral, inhalation, parenteral, or topical use and may be administered to the patient in the form of tablets, capsules, aerosols, inhalants, suppositories, solution, suspensions, or the like.

The following formulation example is illustrative only and is not intended to limit the scope of the invention in any way. "Active ingredient" means a compound of formula I.

Example 1 active ingredient 4% (total solution)

L-cysteine 0.03% (total solution) a pharmaceutically acceptable excipient water

The pH of the solution is adjusted to 8.5 using sodium hydroxide. The pH adjusted solution is protected from light. The solution is purged with nitrogen for twenty minutes and then sterile filtered. The formulation is dispensed into prewashed, depyrogenated vials and then stoppered with a prewashed, presterilized teflon coated stopper. Caps are attached using a crimper. The sterile filtration and dispensing steps are conducted using a nitrogen isolator (5% v/v Oxygen).

Utility Test Methods The unexpected activity of a compound of formula I as an anticytomegalovirus can be evidenced by tests conducted in human foreskin or fetal lung cells. All materials required for this testing are commercially available. The testing method described is well documented in the literature, see, e.g. Colacino, JM and Lopez C, 1983, "Efficacy and selectivity of some nucleoside analogs as anti-human cytomegalovirus agents." Antimicrob. Agents Chemother. 24:505-508; Colacino, JM and Lopez C, 1985,

"Antiviral activity of 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine against human cytomegalovirus in human skin fibroblasts." Antimicrob. Agents Chemother. 28:252-258; and Mauldin, SC, Paget CJ Jr., Jones CD, Colacino JM, Baxter AJ, Staschke KA, Johansson NG, and Nrang L., 1998, "Synthesis and antiviral activity of prodrugs of the nucleoside l-[2\3'-dideoxy-3'-C-(hydroxymethyl)-beta-D-erythropentofuranosyl] cytosine." Bioorg. Med. Chem. 6:577-585. The compounds of formula I can be prepared by methods well known in the art, see e.g. WO99/41230. A stock suspension of the compound is then prepared in phosphate buffered saline and diluted as appropriate.

Cell Culture

An accepted procedure for detecting activity of different classes of drugs against HCMN for which there is a good correlation with the treatment of HCMV in humans is the inhibition of virus replication in cell cultures and comparing virus replication in the presence of a compound of formula I to virus replication in the absence of a compound of formula I.

Human fibroblast cells, foreskin or fetal lung cells, are infected with human cytomeglo virus. The cells are incubated with standard cell culture media containing no or increasing concentrations of a compound of formula I. After a period of time, the number of plaques observed in the non-treated infected cell control cultures is compared to the number of plaques observed in the treated infected cell cultures. Additionally the yield of virus produced in the non-treated infected cell control cultures is compared to the yield of virus produced in the treated infected cell cultures.

The percent inhibition of virus replication can be determined and the concentration of molecule required to inhibit viral replication by 50% (IC50) and 90% (IC90) can be calculated using linear regression analysis. Percent inhibition of 50 or higher at concentrations that are not cytotoxic denotes selective antiviral activity and indicates that the drug is useful for the treatment of infections caused by HCMN. As an example, GARFT II, a compound of formula I, had an inhibition of greater than 50% at a concentration of 0.0078 μM (IC50) and inhibited cell replication by 50% at a concentration of 15.64 μM (TC50) yielding a selective index (TC50/IC50) of 2005. Results:

IC50 = 50% --nhibitory Concentration of HCMN strain AD169 induced plaque formation in MRC-5 Cells.

TC50 = 50% Cytotoxic Concentration as measured in MRC-5 Cells with Promega's Cell Titer 96 Aqueous One Solution

TI (Therapeutic Index) = TC50/IC50

Claims

We Claim:

1. A method for treating HCMN in a human requiring said treatment, which comprises administering to said human an effective amount of a compound of formula I:

where:

Y is CH=CH, O, or S;

R3 is hydrogen, chloro, or fluoro; R⁴ is hydroxy, a carboxy protecting group, or ΝHC*H(COOH)CH2CH₂COOH where the configuration about the carbon atom designated * is S; and

R6 is hydrogen or an amino protecting group; and

R^ is hydroxy or amino; or a salt or solvate thereof.

2. A use of a compound of formula I:

I; where: Y is CH-CH, O, or S;

R3 is hydrogen, chloro, or fluoro;

R⁴ is hydroxy, a carboxy protecting group, or NHC*H(COOH)CH₂CH₂COOH where the configuration about the carbon atom designated * is S; and R6 is hydrogen or an amino protecting group; and

R*⁷ is hydroxy or amino; or a salt or solvate thereof; for the manufacture of a medicament for the treatment of HCMN.

3. A therapeutic package for dispensing to, or for use in dispensing to, a patient being treated for HCMN comprising one or more unit doses, each unit dose comprising an amount of a compound of formula I:

I; where:

Y is CH=CH, O, or S;

R3 is hydrogen, chloro, or fluoro;

R⁴ is hydroxy, a carboxy protecting group, or ΝHC*H(COOH)CH2CH COOH where the configuration about the carbon atom designated * is S; and

R6 is hydrogen or an amino protecting group; and

R is hydroxy or amino; or a salt or solvate thereof; therein such that periodic administration of one or more of said unit doses is effective to treat HCMN; and a finished pharmaceutical container therefor, said container further containing or comprising labeling, said labeling indicating that a compound of formula I is indicated for the treatment of patients with HCMN.

4. An article of manufacture comprising packaging material and a compound of formula I:

i; where:

Y is CH-CH, O, or S; R3 is hydrogen, chloro, or fluoro;

R⁴ is hydroxy, a carboxy protecting group, or NHC*H(COOH)CH2CH₂COOH where the configuration about the carbon atom designated * is S; and R6 is hydrogen or an amino protecting group; and

R is hydroxy or amino; or a salt or solvate thereof; contained within said packaging material, wherein a compound of formula I is therapeutically effective for treating HCMN, and wherein the packaging material comprises a label which indicates that a compound of formula I can be used to treat HCMN.