Use of compounds in the preparation of a medicament for the treatment of antibiotic-resistant bacterial infectionsCross-referencing
This application claims priority to U.S. provisional application serial No. 61/077,293, filed on 1/7/2008, the contents of which are incorporated herein by reference.
Background
Antibiotic resistance of bacteria may be intrinsic or may be obtained by mutation. Bacteria resistant to antibiotics pose a serious threat to public health.
For example, approximately 1% of the world's population is infected with methicillin-resistant staphylococcus aureus (MRSA), a bacterial strain that is resistant to commonly used antibiotics. Most MRSA infections occur in hospitals and health care facilities, such as nursing homes (nursing homes) and dialysis centers (dialsyscenters). It is called health agency related MRSA (HA-MRSA). Elderly or people with weakened immune systems are at high risk of contracting HA-MRSA. Recently, another type of MRSA is found in other healthy people in a wider community, namely community-associated MRSA (CA-MRSA). CA-MRSA causes severe skin and soft tissue infections and severe pneumonia.
The existing antibiotics often fail to cure infections caused by antibiotic-resistant bacteria. Therefore, there is a need to develop new antibiotic drugs.
SUMMARY
The present invention relates to methods of treating infections caused by: methicillin-insensitive bacteria, vancomycin-insensitive bacteria, penicillin-insensitive bacteria, clarithromycin-insensitive bacteria, or metronidazole-insensitive bacteria. The method comprises administering to a subject an effective amount of one of the quinolone compounds represented by the following formula (I):
formula (I)
The above quinolone compound has an asymmetric center. They include all forms of stereoisomers. Two examples of isomeric compounds are:
(3S, 5S) -7- [ 3-amino-5-methyl-piperidinyl ] -1-cyclopropyl-1, 4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid
(3S, 5R) -7- [ 3-amino-5-methyl-piperidinyl ] -1-cyclopropyl-1, 4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid.
These quinolone compounds may be the compounds themselves as well as their salts, prodrugs or solvates. Salts may be formed between anions and positively charged groups (e.g., amino groups) on compounds. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, and maleate. Similarly, salts can also be formed between cations and negatively charged groups (e.g., carboxylates) on the compound. Suitable cations include sodium, potassium, magnesium, calcium and ammonium cations, such as tetramethylammonium. Prodrugs can be esters and other pharmaceutically acceptable derivatives, which, when administered to a subject, provide compounds of formula (I). The solvate refers to a complex formed between the compound shown in the formula (I) and a pharmaceutically acceptable solvent. The pharmaceutically acceptable solvent may be water, ethanol, isopropanol, ethyl acetate, acetic acid and ethanolamine. Thus, the quinolone compounds used in the practice of the present invention may be, for example, the malate salts of the compounds or the hemihydrate of the salts.
The present invention also includes compositions comprising one or more of the above quinolone compounds and a pharmaceutically acceptable carrier for treating infections caused by: methicillin-insensitive bacteria, vancomycin-insensitive bacteria, penicillin-insensitive bacteria, clarithromycin-insensitive bacteria, or metronidazole-insensitive bacteria; and the use of the composition in the manufacture of a medicament for the treatment of said infection. The bacteria can be methicillin-resistant Staphylococcus aureus, efflux-related methicillin-resistant Staphylococcus aureus, vancomycin intermediate-resistant Staphylococcus aureus (vancomycin-intermediate Staphylococcus aureus), hetero-vancomycin (heter-vancomycin) -intermediate-resistant Staphylococcus aureus or vancomycin-resistant Staphylococcus aureus. Examples of infections caused by the above bacteria include, but are not limited to: surgical wound infections, urinary tract infections, bloodstream infections (sepsis), pneumonia (hospital-or community-acquired), diabetic foot infections (diabetic footinfection) and skin infections, such as cellulitis, boils, abscesses, hordeolum, carbuncles and impetigo.
Specifically, in a first aspect of the present invention, there is provided the use of a compound of the formula:
in another preferred embodiment, the infection is caused by methicillin-resistant staphylococcus aureus, efflux-associated methicillin-resistant staphylococcus aureus, intermediate vancomycin-resistant staphylococcus aureus, methicillin-resistant staphylococcus epidermidis, penicillin-resistant streptococcus pneumoniae, clarithromycin-resistant helicobacter pylori, or metronidazole-resistant helicobacter pylori.
In another preferred embodiment, the infection is caused by community-associated methicillin-resistant staphylococcus aureus.
In another preferred embodiment, the infection is a diabetic foot infection, a surgical wound infection, a urinary tract infection, a bloodstream infection, hospital-acquired pneumonia, community-acquired pneumonia, or a skin infection.
In another preferred embodiment, the compound is:
or
In another preferred embodiment, the pharmaceutically acceptable salt of the compound is selected from the group consisting of acetate, malate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate and maleate salts of the compound.
In another preferred embodiment, the pharmaceutically acceptable salt of the compound is in the hemihydrate form.
In a second aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of the formula:
in another preferred embodiment, the compound is:
or
In another preferred embodiment, the pharmaceutically acceptable salt of the compound is selected from the group consisting of acetate, malate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate and maleate salts of the compound.
In a third aspect of the invention, there is provided a method of treating an infection, said method comprising administering to a subject in need thereof an effective amount of a compound of the formula:
wherein the infection is caused by a methicillin-insensitive bacterium, a vancomycin-insensitive bacterium, a penicillin-insensitive bacterium, a clarithromycin-insensitive bacterium, or a metronidazole-insensitive bacterium.
In another preferred embodiment, the infection is caused by methicillin-resistant staphylococcus aureus, efflux-related methicillin-resistant staphylococcus aureus, intermediate-resistant staphylococcus aureus to vancomycin, intermediate-resistant staphylococcus aureus to heteroavancomycin, or vancomycin-resistant staphylococcus aureus.
In another preferred embodiment, the infection is caused by community-associated methicillin-resistant staphylococcus aureus.
In another preferred embodiment, methicillin-resistant staphylococcus epidermidis causes the infection.
In another preferred embodiment, penicillin-resistant streptococcus pneumoniae causes said infection.
In another preferred example, multi-drug resistant streptococcus pneumoniae causes the infection, wherein the bacteria are resistant to at least one of methicillin, vancomycin and penicillin.
In another preferred embodiment, clarithromycin-resistant helicobacter pylori or metronidazole-resistant helicobacter pylori causes the infection.
In another preferred embodiment, the infection is a diabetic foot infection, a surgical wound infection, a urinary tract infection, a bloodstream infection, hospital-acquired pneumonia, community-acquired pneumonia, or a skin infection.
In another preferred embodiment, the compound is in the form of a salt. Preferably, the compound is in the form of the malate salt. More preferably, the compound is in the form of the malate salt hemihydrate.
In a preferred aspect, the compound is
Preferably, the compound is in the form of a salt; more preferably, the compound is in the form of the malate salt; most preferably, the compound is in the form of the malate salt hemihydrate.
In another preferred embodiment, the infection is caused by methicillin-resistant staphylococcus aureus, efflux-related methicillin-resistant staphylococcus aureus, intermediate-resistant staphylococcus aureus to vancomycin, or vancomycin-resistant staphylococcus aureus. More preferably, the infection is caused by community-associated methicillin-resistant staphylococcus aureus.
In another preferred embodiment, methicillin-resistant staphylococcus epidermidis causes the infection.
In another preferred embodiment, penicillin-resistant streptococcus pneumoniae causes said infection.
In another preferred example, multi-drug resistant streptococcus pneumoniae causes the infection, wherein the bacteria are resistant to at least one of methicillin, vancomycin and penicillin.
In another preferred embodiment, clarithromycin-resistant helicobacter pylori or metronidazole-resistant helicobacter pylori causes the infection.
In another preferred embodiment, the infection is a diabetic foot infection, a surgical wound infection, a urinary tract infection, a bloodstream infection, hospital-acquired pneumonia, community-acquired pneumonia, or a skin infection.
In another preferred aspect, the compound is
In another preferred embodiment, the compound is in the form of a salt. Preferably, the compound is in the form of the malate salt. More preferably, the compound is in the form of the malate salt hemihydrate.
In another preferred embodiment, the infection is caused by methicillin-resistant staphylococcus aureus, efflux-related methicillin-resistant staphylococcus aureus, intermediate-resistant staphylococcus aureus to vancomycin, or vancomycin-resistant staphylococcus aureus.
In another preferred embodiment, the infection is caused by community-associated methicillin-resistant staphylococcus aureus.
In another preferred embodiment, methicillin-resistant staphylococcus epidermidis causes the infection.
In another preferred embodiment, penicillin-resistant streptococcus pneumoniae causes said infection.
In another preferred example, multi-drug resistant streptococcus pneumoniae causes the infection, wherein the bacteria are resistant to at least one of methicillin, vancomycin and penicillin.
In another preferred embodiment, clarithromycin-resistant helicobacter pylori or metronidazole-resistant helicobacter pylori causes the infection.
In another preferred embodiment, the infection is a diabetic foot infection, a surgical wound infection, a urinary tract infection, a bloodstream infection, hospital-acquired pneumonia, community-acquired pneumonia, or a skin infection.
The details of several embodiments of the invention are set forth below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.
Detailed description of the invention
The quinolone compounds useful in the practice of the present invention can be synthesized by conventional methods. The following example 1 describes the synthetic method for preparing the two isomer compounds. Other isomers or other forms of the compounds can be obtained by the skilled person by modifying the synthesis method. Synthetic chemical transformations and protecting group methods (protection and deprotection) for synthesis are known in the art and include, for example, r.larock, Comprehensive organic transformations (Comprehensive organic transformations), VCH Publishers (VCH Publishers) (1989); greene and p.g.m.wuts, Protective Groups in Organic Synthesis, 3 rd edition, John Wiley father (John Wiley and Sons) (1999); fieser and m.fieser, fisher Reagents for Organic Synthesis (Fieser and Fieser's Reagents for Organic Synthesis), john wil father, 1994; and L.Patette et al, Encyclopedia of Organic Synthesis Reagents (Encyclopedia of Reagents for Organic Synthesis), John Willi, Inc. (1995), and subsequent versions thereof.
The compound so synthesized may be further purified by flash column chromatography, high performance liquid chromatography, crystallization, or any other suitable method.
The above quinolone compound inhibits the growth of methicillin-insensitive bacteria, vancomycin-insensitive bacteria, penicillin-insensitive bacteria, clarithromycin-insensitive bacteria, and metronidazole-insensitive bacteria. Accordingly, one aspect of the present invention relates to a method of treating an infection by one of said bacteria by administering to a subject in need thereof an effective amount of one of said quinolone compounds. One embodiment of the method utilizes a quinolone compound to treat an infection caused by multi-drug resistant Streptococcus pneumoniae (Streptococcus pneumoniae) that is resistant to at least one of methicillin, vancomycin, and penicillin.
The term "insensitive" as used herein refers to tolerance to moderate to complete levels of a drug. Methicillin-insensitive bacteria include, but are not limited to: methicillin-resistant Staphylococcus aureus, efflux-associated methicillin-resistant Staphylococcus aureus, community-associated methicillin-resistant Staphylococcus aureus, and methicillin-resistant Staphylococcus epidermidis (Staphylococcus epidermidis). Bacteria that are not susceptible to vancomycin include, but are not limited to: intermediate drug-resistant staphylococcus aureus in heteroavancomycin, intermediate drug-resistant staphylococcus aureus in vancomycin and vancomycin-resistant staphylococcus aureus. Penicillin-insensitive bacteria include, but are not limited to: penicillin-resistant streptococcus pneumoniae. Bacteria that are not susceptible to clarithromycin include, but are not limited to: clarithromycin-resistant Helicobacter pylori (Helicobacter pylori). Metronidazole-insensitive bacteria include, but are not limited to: metronidazole resistant helicobacter pylori.
The term "effective amount" refers to the amount of active that will achieve the desired therapeutic effect in a subject. One skilled in the art will recognize that the effective amount may vary depending on the route of administration, the use of excipients, and possibly with other materials. The term "treatment" refers to the administration of one of the aforementioned quinolone compounds to an article having, having a symptom of, or susceptible to, an infection, for the purpose of treating, curing, alleviating, altering, remedying, improving, ameliorating, or affecting the infection, the symptom of the infection, or the propensity to become infected.
To practice the method, the quinolone compound may be administered orally, parenterally, by inhalation spray, or by implantation into a reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
The oral composition may be any orally acceptable dosage form including, but not limited to: tablets, capsules, emulsions and aqueous suspensions, dispersions and solutions. Carriers conventionally used in tablets include lactose and corn starch. Lubricants, such as magnesium stearate, are also commonly added to tablets. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oil phase mixed with emulsifying or suspending agents. If desired, certain sweetening, flavoring or coloring agents may be added.
Sterile injectable compositions (e.g., aqueous or oleaginous suspensions) can be formulated according to the techniques known in the art using suitable dispersing or wetting agents (e.g., tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be utilized are mannitol, water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono-or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents.
Inhalation compositions, which can be prepared as aqueous salt solutions, can be prepared according to techniques well known in the art of pharmaceutical formulation using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
The topical composition may be formulated in the form of an oil, cream, lotion, ointment, or the like. Suitable carriers for use in the compositions include vegetable or mineral oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohols (> C12). Preferred carriers are those in which the active ingredient is soluble. Emulsifiers, stabilizers, wetting agents and antioxidants may also be included, if desired, as may substances imparting color or fragrance. In addition, transdermal penetration enhancers may also be used in these topical formulations. Examples of such accelerators are found in us patents 3,989,816 and 4,444,762. The cream is preferably formulated with a mixture of mineral oil, self-emulsifying beeswax and water in which the active ingredient is mixed dissolved in a small amount of oil, for example almond oil. An example of such a cream is a cream comprising about 40 parts water, about 20 parts beeswax, about 40 parts mineral oil and about 1 part almond oil. Ointments are formulated by mixing a solution of the active ingredient in a vegetable oil (e.g., almond oil) with hot soft paraffin and then cooling the mixture to room temperature. An example of such an ointment is one comprising about 30% by weight almond (oil) and about 70% by weight white soft paraffin.
The carrier in a pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient of the formulation (and preferably capable of stabilizing the formulation) and not deleterious to the subject to be treated. For example, solubilizers, such as cyclodextrins (which form specific, more soluble complexes with one or more of the active compounds of the extract), can be utilized as pharmaceutical excipients for the delivery of the active ingredient. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D & C Yellow # 10.
The efficacy of one of the above compounds in inhibiting bacterial growth can be preliminarily assessed using a suitable in vitro assay. The efficacy of the compounds in treating bacterial infections can also be tested by in vivo assays. For example, the compounds can be administered to an animal with an infection (e.g., a mouse model) and then evaluated for therapeutic efficacy. Based on these results, appropriate dosage ranges and routes of administration can also be determined.
Without further elaboration, it is believed that the invention can be practiced otherwise than as specifically described. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications, including patents, cited herein are hereby incorporated by reference in their entirety.
Example 1
The malate salts of (3S, 5S) -7- [ 3-amino-5-methyl-piperidinyl ] -1-cyclopropyl-1, 4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (compound 1) and (3S, 5R) -7- [ 3-amino-5-methyl-piperidinyl ] -1-cyclopropyl-1, 4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (compound 1') were synthesized as follows:
(A) synthesis of (3S, 5S) - (5-methyl-piperidin-3-yl) -carbamic acid tert-butyl ester (Compound 9) and (3S, SR) - (5-methyl-piperidin-3-yl) -carbamic acid tert-butyl ester (Compound 9):
compound 9 was synthesized as shown in scheme 1 below:
scheme 1
A50-L reactor was charged with Compound 2(5.50kg, 42.60mol), methanol (27L) and cooled to 10-15 deg.C. Thionyl chloride (10.11kg, 2.0 equivalents) was added over a period of 65 minutes via an addition funnel and the temperature was maintained below 30 ℃ with external cooling. The resulting solution was stirred at 25 ℃ for 1.0 hour, and then methanol was removed under reduced pressure. The oily residue was azeotroped with ethyl acetate (3X 2.5L) to remove residual methanol, dissolved in ethyl acetate (27.4L), charged into a 50L reactor, and neutralized by adding triethylamine (3.6kg) slowly at 30 ℃ or lower. The resulting suspension was filtered to remove triethylamine hydrochloride.
The filtrate was charged to a 50L reactor with DMAP (0.53 kg). Di-tert-butyl dicarbonate (8.43kg) was added over a period of 30 minutes via a hot water-heated addition funnel at 20-30 ℃. After 1 hour, the reaction was detected to be complete by TLC analysis. The organic phase was washed with ice cold 1N HCl (2X 7.5L), saturated sodium bicarbonate solution (1X 7.5L), dried over magnesium sulfate and filtered. After removing ethyl acetate under reduced pressure, a crystal slurry was obtained, triturated with MTBE (10.0L), and filtered to obtain compound 3(5.45kg, 52.4%) as a white solid.
C11H17NO5Analytical calculation of (a): c, 54.3; h, 7.04; and N, 5.76. Measured value: c, 54.5; h, 6.96; and N, 5.80. HRMS (ESI)+)C11H18NO5Estimated value of [ M + H ]]244.1185. Found 244.1174;1H NMR(CDCl3，500MHz)：δ＝4.54(dd，J＝3.1，9.5Hz，1H)，3.7(s，3H)，2.58-2.50(m，1H)，2.41(ddd，1H，J＝17.6，9.5，3.7)，2.30-2.23(m，1H)，1.98-1.93(m，1H)，1.40(s，9H)；13C NMR(CDCl3125.70MHz) delta 173.3, 171.9, 149.2, 83.5, 58.8, 52.5, 31.1, 27.9, 21.5. Melting Point 70.2 ℃.
A50-L reactor was charged with Compound 3(7.25kg, 28.8mol), DME (6.31kg) and Bredereck reagent (7.7kg, 44.2 mol). The solution was stirred and heated to 75 ℃. + -. 5 ℃ for 3 hours. The reaction (system) was cooled to 0 ℃ over a period of 1 hour, during which time a precipitate formed. The mixture was maintained at 0 ℃ for one hour, filtered, and dried at 30 ℃ ± 5 ℃ for at least 30 hours in a vacuum oven to give compound 4(6.93kg, 77.9%) as a white crystalline solid.
C14H22N2O5Analytical calculation of (a): c, 56.4; h, 7.43; and N, 9.39. Measured value: c, 56.4; h, 7.32; n, 9.48; HRMS (ESI)+)C14H22N2O5Estimated value of [ M + H ]]299.1607, found 299.1613;1H NMR(CDCl3，499.8MHz)δ7.11(s，1H)，4.54(dd，1H，J＝10.8，3.6)，3.74(s，3H)，3.28-3.19(m，1H)，3.00(s，6H)，2.97-2.85(m，1H)，1.48(s，9H)；13C NMR(CDCl3125.7MHz) delta 172.6, 169.5, 150.5, 146.5, 90.8, 82.2, 56.0, 52.3, 42.0, 28.1, 26.3. Melting point 127.9 ℃.
A10-gallon Pfaudler reactor was charged with ESCAT142(Engelhard Corp) 5% palladium on carbon powder (50% wet, 0.58Kg wet weight), Compound 4(1.89Kg, 6.33mol), and isopropanol (22.4Kg), from Engelhard Corp, N.J.. After stirring at 45 ℃ for 18 hours under a 45-psi hydrogen atmosphere, the reaction mixture was cooled to room temperature and filtered through a pad of Celite (Celite) (0.51 kg). The filtrate was evaporated under reduced pressure to give a thick oil which solidified on standing to give compound 5(1.69kg, 100%) as a 93:7 diastereomeric mixture.
A sample of the product mixture was purified by preparative HPLC to give material for data analysis. C12H19NO5Analytical calculation of (a): c, 56.0; h, 7.44; n, 5.44. Measured value: c, 55.8; h, 7.31; n, 5.44; MS (ESI)+)C12H19NO5Estimated value of [ M + H ]]258.1342. Found 258.1321;1H NMR(CDCl3，499.8MHz)δ＝4.44(m，1H)，3.72(s，3H)，2.60-2.48 (m，2H)，1.59-1.54(m，1H)，1.43(s，9H)，1.20(d，j＝6.8Hz，3H)；13C NMR(CDCl3125.7MHz) delta 175.7, 172.1, 149.5, 83.6, 57.4, 52.5, 37.5, 29.8, 27.9, 16.2. Melting point 89.9 ℃.
A50-L reactor was charged with Compound 5(3.02kg, 11.7mol), absolute ethanol (8.22kg) and MTBE (14.81 kg). Sodium borohydride (1.36kg, 35.9mol) was added in small portions at 0 ℃. + -. 5 ℃. Some blistering was observed. The reaction mixture was warmed to 10 ℃. + -. 5 ℃ and calcium chloride dihydrate (2.65kg) was added in portions over a period of 1 hour at 10 ℃. + -. 5 ℃. The reaction mixture was warmed to 20 ℃. + -. 5 ℃ over a period of 1 hour and stirred at 20 ℃. + -. 5 ℃ for a further 12 hours. After cooling the reaction (system) to-5 ℃. + -. 5 ℃ ice-cold 2N HCl (26.9kg) was slowly added at 0 ℃. + -. 5 ℃. The stirring was stopped. The lower aqueous phase was removed. Saturated aqueous sodium bicarbonate (15.6kg) was added to the reactor over a 5 minute period with stirring. The stirring was stopped again and the lower aqueous phase was removed. Magnesium sulfate (2.5kg) was added to the reactor and stirred for at least 10 minutes. The mixture was filtered through a vacuum suction filter and concentrated under reduced pressure to give compound 6(1.80kg, 66%).
C11H23NO4Analysis calculated value: c, 56.6; h, 9.94; and N, 6.00. Measured value: c, 56.0; h, 9.68; n, 5.96; HRMS (ESI 6)+)C11H24NO4Estimated value of [ M + H ]]234.1705. Found 234.1703;1H NMR(CDCl3，500MHz)δ＝6.34(d，J＝8.9Hz，1H，NH)，4.51(t，J＝5.8，5.3Hz，1H，NHCHCH2OH)，4.34(t，J＝5.3，5.3Hz，1H，CH3CHCH2OH)，3.46-3.45，(m，1H，NHCH)，3.28(dd，J＝10.6，5.3Hz，NHCHCHHOH)，3.21(dd，J＝10.2，5.8Hz，1H，CH3CHCHHOH)，3.16(dd，J＝10.2，6.2Hz，1H，NHCHCHHOH)，3.12(dd，J＝10.6，7.1Hz，1H，CH3CHCHHOH)，1.53-1.50(m，1H，CH3CHCHHOH)，1.35(s，9H，O(CH3)3，1.30(ddd，J＝13.9，10.2，3.7Hz，1H，NHCHCHHCH)，1.14(ddd，J＝13.6，10.2，3.4Hz，1H，NHCHCHHCH)，0.80(d，J＝6.6Hz，3H，CH3)；13C NMR(CDCl3125.7MHz) delta 156.1, 77.9, 50.8, 65.1, 67.6, 65.1, 35.6, 32.8, 29.0, 17.1. Melting Point 92.1 ℃.
A50L reactor was charged with a solution of Compound 6(5.1kg) in isopropyl acetate (19.7 kg). The reaction (system) was cooled to 15 ℃. + -. 5 ℃ and triethylamine (7.8kg) was added at this temperature. The reactor was further cooled to 0 ℃. + -. 5 ℃ and methanesulfonyl chloride (MsCl) (6.6kg) was added. The reaction (system) was stirred for several hours and the completion of the reaction was monitored by HPLC or TLC. The reaction was quenched with saturated aqueous bicarbonate solution. The organic phase was separated and washed successively with cold 10% aqueous triethylamine solution, cold aqueous HCl solution, cold saturated aqueous bicarbonate solution and saturated aqueous brine solution. The organic phase was dried, filtered and concentrated under vacuum at 55 ℃. + -. 5 ℃ to give compound 7 as a solid/liquid slurry which was used in the next reaction without further purification.
After addition of 9.1kg of pure benzylamine, the 50L reactor was warmed to 55 ℃ and 1, 2-dimethoxyethane (14.1kg) of compound 7(8.2kg) was added at this temperature. After addition, the reaction (system) was stirred at 60 ℃. + -. 5 ℃ for several hours, and the completion was monitored by TLC or HPLC. The reaction (system) was cooled to ambient temperature and the solvent was removed in vacuo. The residue was diluted with 11.7kg 15% (v/v) ethyl acetate/hexane solution and treated with 18.7kg 20% (by weight) aqueous potassium carbonate solution while stirring. After standing, a three-phase mixture was obtained. The upper organic layer was collected. The separated intermediate layer was extracted twice more with 11.7kg portions of a 15% (v/v) ethyl acetate/hexane solution. The combined organic layers were concentrated in vacuo to give an oily residue. The residue was purified by chromatography to give compound 8 as an oil.
0.6kg of 50% wet solid palladium on carbon (E101, 10 wt.%) was charged to a 40L pressure vessel under a nitrogen purge. Then 13.7kg of an anhydrous ethanol solution of Compound 8(3.2kg) was added to the reactor under a nitrogen atmosphere. The reactor was purged with nitrogen and then pressurized to 45psi with hydrogen. The reaction (system) was then heated to 45 ℃. Monitored by TLC or LC. After completion, the reaction (system) was cooled to ambient temperature, vented and purged with nitrogen. The mixture was filtered through a pad of celite and the solid was washed with 2.8kg of absolute ethanol. The filtrate was concentrated in vacuo to give compound 9 as a waxy solid.
TLC Rf(silica F)25470:30v/v Ethyl acetate-Hexane, KMnO4Dyed) 0.12;1HNMR(300MHz，CDCl3) δ 5.31(brs, 1H), 3.80-3.68(m, 1H), 2.92(d, J ═ 11.4Hz, 1H), 2.77(AB quart, J)AB＝12.0Hz，v＝50.2Hz，2H)，2.19(t，J＝10.7Hz，1H)，1.82-1.68(m，2H)，1.54(brs，1H)，1.43(s，9H)，1.25-1.15(m，1H)，0.83(d，J＝6.6Hz，3H)；13C NMR(75MHz，CDCl3)δ：155.3，78.9，54.3，50.8，45.3，37.9，28.4，27.1，19.2；MS(ESI+)m/z215(M+H)，429(2M+H)。
Similarly, (3S, 5R) - (5-methyl-piperidin-3-yl) -carbamic acid tert-butyl ester (compound 9') was synthesized as shown in scheme 2.
Scheme 2
(B) Synthesis of 1-cyclopropyl-7-fluoro-8-methoxy-4-oxo-1, 4-dihydro-quinoline-3-carboxylic acid (Compound 10):
compound 10 was prepared according to the procedure described in us patent 6,329,391.
(C) Synthesis of boron ester chelate of 1-cyclopropyl-7-fluoro-8-methoxy-4-oxo-1, 4-dihydro-quinoline-3-carboxylic acid (Compound 11):
scheme 3
c. Toluene, tert-butyl methyl ether
Filtering at 20-50 deg.C
The reactor was charged with boron oxide (2.0kg, 29mol), glacial acetic acid (8.1L, 142mol) and acetic anhydride (16.2L, 171 mol). The resulting mixture was refluxed for at least 2 hours, then cooled to 40 ℃ at which temperature 7-fluoroquinolone acid compound 10(14.2kg, 51mol) was added. The mixture was refluxed for at least 6 hours and then cooled to about 90 ℃. Toluene (45L) was added to the reaction (system). Tert-butyl methyl ether (19L) was added at 50 ℃ to initiate precipitation. The mixture was then cooled to 20 ℃ and filtered to separate the precipitate. The isolated solid was then washed with t-butyl methyl ether (26L) and dried in a vacuum oven at 40 deg.C (50 torr) to afford compound 11 in 86.4% yield.
Raman(cm-1)：3084.7，3022.3，2930.8，1709.2，1620.8，1548.5，1468.0，1397.7，1368.3，1338.5，1201.5，955.3，653.9，580.7，552.8，384.0，305.8。NMR(CDCl3，300MHz)δ(ppm)：9.22(s，1H)，8.38-8.33(m，1H)，7.54(t， J＝9.8Hz，1H)，4.38-4.35(m, 1H), 4.13(s, 3H), 2.04(s, 6H), 1.42-1.38(m, 2H), 1.34-1.29(m, 2H). TLC (Whatman MKC18F silica, 60200 μm), mobile phase: 1:1(v/v) CH3CN: 0.5N NaCl (aqueous), UV (254/366nm) development; rf＝0.4-0.5。
(D) Synthesis of the malate salt of (3S, 5S) -7- [ 3-amino-5-methyl-piperidinyl ] -1-cyclopropyl-1, 4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1) and the malate salt of (3S, 5R) -7- [ 3-amino-5-methyl-piperidinyl ] -1-cyclopropyl-1, 4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1')
Compound 1 was synthesized from compound 9 as shown in scheme 4 below.
Scheme 4
The reactor was charged with Compound 11(4.4kg, 10.9mol), Compound 9(2.1kg, 9.8mol), Triethylamine (TEA) (2.1L, 14.8mol) and acetonitrile (33.5L, 15.7L/kg). The resulting mixture was stirred at about 50 ℃ until the reaction was complete as monitored by HPLC or reverse phase TLC. Cooling to about 35 ℃ reduced the reaction volume by about half by vacuum distillation of acetonitrile at 0-400 torr. After addition of 28.2kg of 3.0N NaOH (aqueous) solution, the reaction mixture was warmed to about 40 ℃ and distilled under vacuum until no more distillate was observed and hydrolyzed at room temperature. After completion of hydrolysis as monitored by HPLC or reverse phase TLC, the reaction mixture was neutralized by adding 4-5kg glacial acetic acid.
The resulting solution was extracted 3 times with 12.7kg (9.6L) of dichloromethane. The organic layers were combined and transferred to another reactor. Evaporation at 40 ℃ reduced the reaction volume by about half. After addition of 20.2kg of 6.0N HCl (aq) solution, the reaction mixture was stirred at 35 ℃ for at least 12 h. After completion of the reaction as monitored by HPLC or reverse phase TLC, stirring was stopped to allow phase separation. The organic phase was removed and the aqueous layer was extracted with 12.7kg (9.6L) of dichloromethane. The aqueous layer was diluted with 18.3kg of distilled water and warmed to about 50 ℃. The methylene chloride was further removed by vacuum distillation (100 ℃ 400 torr).
The pH of the aqueous solution was then adjusted to 7.8-8.1 below 65 ℃ by the addition of about 9.42kg3.0N NaOH (aq). The reaction mixture was stirred at 50 ℃ for at least 1 hour and then cooled to room temperature. The precipitate was separated off by suction filtration, washed twice with 5.2kg of distilled water, dried by suction filtration for at least 12 hours and then dried in a convection oven at 55 ℃ for a further 12 hours. Solid compound 12(3.2kg, 79%) was obtained.
3.2kg of Compound 12 and 25.6kg of 95% ethanol were added to the reactor. 1.1kg of solid D, L-malic acid was added to the reactor. The mixture was refluxed at about 80 ℃. Distilled water (about 5.7L) was added to dissolve the precipitate, and 0.2kg of activated carbon was added. The reaction mixture was passed through a filter. The clear filtrate was cooled to 45 ℃ and allowed to stand for at least 2 hours to crystallize. After the reaction mixture had cooled further to 5 ℃ the precipitate was isolated by suction filtration, washed with 6.6kg of 95% ethanol and dried by suction filtration for at least 4 hours. The solid was dried in a convection oven at 45 ℃ for at least 12 hours to obtain 3.1kg of Compound 1 (yield: 70%).
NMR(D2O，300MHz)δ(ppm)：8.54(s，1H)，7.37(d，J＝9.0Hz，1H)，7.05(d，J＝9.0Hz，1H)，4.23-4.18(m，1H)，4.10-3.89(m，1H)，3.66(brs，1H)，3.58(s，3H)，3.45(d，J＝9.0Hz，1H)，3.34(d，J＝9.3Hz，1H)，3.16(d，J＝12.9Hz，1H)，2.65(dd，J＝16.1，4.1Hz，1H)，2.64-2.53(m，1H)，2.46(dd，J＝16.1，8.0Hz，1H)，2.06(brs，1H)，1.87(d，J＝14.4Hz，1H)，1.58-1.45(m，1H)，1.15-0.95(m，2H)，0.91(d，J＝6.3Hz，3H)，0.85-0.78(m，2H)。
Similarly, compound 1 'was synthesized from compound 9' as shown in scheme 5 below:
scheme 5
Example 2
Compound 1 inhibits methicillin-resistant staphylococcus aureus (MRSA)
MRSA isolates (n 193) were obtained as part of the canadian national intensive care unit (CAN-ICU) study. 19 medical centers with active ICUs in all regions of canada participated in the CAN-ICU study. Each center is asked to collect "clinically significant" samples of only patients suspected of having an infectious disease. Monitoring swabs (surveyability swab), eye, ear, nose and throat swabs were excluded. Anaerobic organisms and fungal organisms are also excluded.
From 9/2005 to 6/2006 (including end points), up to 300 consecutive pathogens (one pathogen/culture site/patient) isolated from blood, urine, tissue/wound and respiratory tract samples of ICU patients were collected at each center. These isolates were transported with Amies charcoal swabs to the reference laboratory of Health Sciences center, wenniband, Canada, and sub-cultured in a suitable medium and stored in skim milk at-80 ℃.
The isolates were confirmed for methicillin resistance using the disc diffusion (disk diffusion) method described by the Clinical and Laboratory Standards Institute. As previously described, all isolates were subjected to mecA PCR and molecular characterization, including PVL analysis and fingerprinting (fingerprinting) to assess whether they were community-related or health agency-related (Christianson et al, J Clin Microbiol.2007, 45 (6): 1904-11; Mulvey et al, J Clin Microbiol.2001, 39 (10): 3481-5; Mulvey et al, emery Infect Dis.2005, 11 (6): 844-50; Oliveira et al, Antimicrob Agents Chemothers.2002, 46 (7): 2155-61). Isolates were also sub-typed using Pulsed Field Gel Electrophoresis (PFGE) according to the Canadian standardization protocol previously described (Mulvey et al, J Clin Microbiol.2001, 39 (10): 3481-5). The obtained PFGE fingerprints were analyzed using bionumerics sv3.5 (position tolerance 1.0 and optimization 1.0) of Applied Maths st. Strain relationships were determined as previously described (Tenover et al, 1995). The fingerprints of these isolates were compared to the national MRSA fingerprint database and assigned to one of the previously described 10 groups of Canadian epidemic MRSAs (CMRSA-1, CMRSA-2, etc.) (Mulvey et al, emery Infect Dis.2005, 11 (6): 844-50). MRSA isolates belong to the following genotypes: CMRSA-1(USA600), CMRSA-2(USA100), CMRSA-4(USA200), CMRSA-7(USA400, MW2) and CMRSA-10(USA 300).
Compound 1 and other antibiotics were tested for inhibitory activity against MRSA isolates using the broth microdilution (microdilution) guidelines specified by the clinical and laboratory standards institute. The following table shows the Minimum Inhibitory Concentrations (MICs) of compound 1 and various fluoroquinolone antibiotics to inhibit 193 MRSA isolates:
the following table shows the MICs of Compound 1 and various fluoroquinolone antibiotics to inhibit the community-associated MRSA (CA-MRSA) strains (USA300 and USA400) and the health agency-associated MRSA strains (USA200, USA600 and USA 100/800):
compound 1 is effective in inhibiting MRSA. The activity of the compound against community-associated MRSA strains was also found to be higher than against health agency-associated MRSA strains.
Use of Compound 1 for inhibiting multidrug-resistant methicillin-resistant Staphylococcus aureus, enterococcus faecium and enterococcus faecalis (enterococcus faecalis)
Compound 1 was tested for its inhibitory effect against multidrug-resistant methicillin-resistant staphylococcus aureus and enterococcus obtained from 10 medical centers in all regions of taiwan. MIC was determined using the agar dilution method recommended by the clinical and laboratory standards institute (CLSI-M100-S18). The results are shown in the following table:
as shown in the table, compound 1 effectively inhibited ciprofloxacin resistance, intermediate resistance to vancomycin, and daptomycin-insensitive MRSA isolates. It is also effective in inhibiting vancomycin-resistant enterococcus faecium and vancomycin-resistant enterococcus faecalis.
Inhibition of staphylococcal bacteria using compound 1
Compound 1 was tested for its inhibitory effect against the following strains: 26 methicillin-resistant staphylococcus aureus (MRSA) strains, 2 intermediate hetero-vancomycin resistant staphylococcus aureus (hvsa) strains, 24 intermediate vancomycin resistant staphylococcus aureus (VISA) strains, 5 Vancomycin Resistant Staphylococcus Aureus (VRSA) strains and 31 quinolone-resistant vancomycin-sensitive MRSA strains containing a defined mutation in the QRDR. These mutations (gyrA, gyrB, grlA and grlB) were determined by sequencing analysis of QRDR. Efflux tests were performed using the serpentine method (Brenwald et al, Antimicrob. Agents Chemother.1998, 42: 2032-S2035). MIC was determined using the agar dilution method recommended by the clinical and laboratory standards institute (CLSI- -M100-S18) and the results are given in the following Table:
ciprofox: ciprofloxacin; levolfx: levofloxacin; moxiflox: moxifloxacin; vanco: vancomycin; and (5) Teico: teicoplanin; dapto: daptomycin; and (9) Tige: tigecycline; quinu/dalfo: quinupristin/dalfopristin
The compound 1 is effective in inhibiting methicillin-resistant, intermediate-resistant to hetero-vancomycin, intermediate-resistant to vancomycin and vancomycin-resistant staphylococcus aureus. It is also effective in inhibiting quinolone-resistant vancomycin-sensitive MRSA. It has very low MIC (0.06-4. mu.g/ml) against these strains.
Of the 31 MRSA quinolone-resistant strains, 5 strains carry the QRDR mutation [ GyrA (S84L), Gr1A (S80F/Y), Gr1B (L413S, E422K/N, D432N, E471K); GyrA (S84L), Gr1A (580F/Y), GyrB (R404L); GyrA (S84L), Gr1A (S80F/Y); gyra (S84L), Gr1A (S80F/Y, E84V), Gr1B (E422D) and Gyra (S84L), Gr1A (S80F/Y, E84V/K/G or S108N) ]. In this experiment, no efflux (efflux) mutation was found in relation to compound 1.
Inhibition of gram-positive cocci with Compound 1
Between months 1 and 12 in 2007, 12 hospitals in canada filed isolates of patients who went to hospital clinics, emergency rooms, medical and surgical wards, and intensive care units. A total of 7881 isolates including 3473 gram-positive cocci were collected (CANWARD 2007). Sensitivity testing of compound 1 and levofloxacin was performed using broth microdilution, a clinical and laboratory standards association. MIC50And MIC90As follows:
| microorganism (number of isolates) | Compound 1MIC50/MIC90 | Levofloxacin MIC50/MIC90 |
| SPN-All(656) | 0.015/0.015 | 0.5/1 |
| -PenS(519) | 0.015/0.015 | 0.5/1 |
| -PenI(103) | 0.015/0.015 | 0.5/1 |
| -PenR(34) | 0.015/0.03 | 0.5/2 |
| -CipR(29) | 0.03/0.12 | 2/16 |
| MSSA(372) | 0.03/0.12 | 0.25/4 |
| CA-MRSA(23) | 0.25/0.5 | 4/8 |
| HA-MRSA(91) | 4/>4 | >32/>32 |
| MSSE(32) | 0.03/0.5 | 4/>32 |
| MRSE(9) | 2/2 | >32/>32 |
| Enterococcus faecalis (81) | 0.12/1 | 2/>32 |
| *VISA(12)*VRSA(7) | 1/22 | 32/>3232 |
SPN-Streptococcus pneumoniae, MSSA-methicillin-sensitive Staphylococcus aureus, CA-community-related, HA-health organization-related, VISA-vancomycin-intermediate-resistant Staphylococcus aureus, VRSA-vancomycin-resistant Staphylococcus aureus + intermediate-resistant MIC, MSSE-methicillin-sensitive Staphylococcus epidermidis, MRSE-methicillin-resistant Staphylococcus epidermidis
*Isolate obtained by antimicrobial resistant net of staphylococcus aureus (NARSA) project: supported by NIAID, NIH contract number N01-AI-95359.
Compound 1 inhibits gram-positive cocci including MRSA, VISA, VRSA, MRSE, PenI-SPN, PenR-SPN and CipR-SPN more active than levofloxacin.
Inhibition of helicobacter pylori using compound 1
The inhibitory effects of compounds 1, ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin against 200 helicobacter pylori isolates (2000-2007) obtained from 10 medical centers in all regions of Taiwan were examined. MIC was determined using the agar dilution method recommended by the clinical and laboratory standards institute (CLSI-M100-S18).
Of 200 isolates of H.pylori, 2%, 6%, 29%, 2% and 2% were resistant to amoxicillin (MIC ≥ 0.5. mu.g/mL), clarithromycin (MIC ≥ 1. mu.g/mL, CLSI), metronidazole (MIC ≥ 8. mu.g/mL), ciprofloxacin (MIC ≥ 1. mu.g/mL) and levofloxacin (MIC ≥ 1. mu.g/mL), respectively. MIC range, MIC of 5 quinolone drugs tested50And MIC90As follows:
compound 1 is effective in inhibiting H.pylori isolates. The table above shows that compound 1 inhibits helicobacter pylori isolates more effectively than ciprofloxacin, levofloxacin and moxifloxacin, comparable to gemifloxacin.
Inhibition of antibiotic-resistant bacteria using compound 1
Compounds 1', ciprofloxacin and levofloxacin were tested against the inhibitory effect of methicillin-resistant staphylococcus aureus and methicillin-resistant streptococcus pneumoniae at various concentrations between 0.008-8 μ g/ml over 10 different days. Staphylococcus aureus and streptococcus pneumoniae isolates were obtained from 10 medical centers in all regions of taiwan. MIC was determined by broth microdilution method. As shown in the table below, compound 1 was also very effective in inhibiting staphylococcus aureus and streptococcus pneumoniae.
MRSA-CIP (R): clinical isolates of MRSA-ciprofloxacin resistant strains.
MRSA-CIP (S): clinical isolates of MRSA-ciprofloxacin sensitive strains.
MRSP-Levo (R): clinical isolates of methicillin-resistant levofloxacin-resistant strains of streptococcus pneumoniae.
MRSP-Levo (S): clinical isolates of methicillin-resistant Streptococcus pneumoniae levofloxacin-sensitive strains.
As shown in the above table, compound 1 is effective in inhibiting methicillin-resistant staphylococcus aureus and streptococcus pneumoniae.
Pharmacokinetic testing
Blood samples were collected from subjects dosed with compound 1 at 0 hours (pre-dose) and at 0.5, 1, 1.5, 2, 4, 6, 8, 12, 16 and 24 hours (post-dose) on day 10. 5ml of each sample was transferred to a heparin sodium tube and immediately placed on ice. The plasma was centrifuged at about 4 ℃ and transferred to appropriately labeled polypropylene sample containers (two tubes containing 1-1.5ml of plasma each) and frozen at about-70 ℃ until use.
The pharmacokinetic experiments were validated and blood samples were analyzed. The details of this experimental validation are shown in the table below.
| Analyte | Type of test | LLOQ | Accuracy (% deviation) | Accuracy (% CV) |
| Compound (I) | In blood plasma | 5.0 | -1.8～2.2％ | 4.3～7.5％ |
LLOQ: lower limit of quantitative determination (LLOQ)
CV: coefficient of Variation (CV)
Pharmacokinetic tests were performed by Charles River Laboratories, Worcester, MA, worsted. C was determined from plasma concentration-time data using a non-compartmental method (WinNonlin version 4.1, Pharsight Corporation, Calif.)max(Peak concentration of Compound 1 in plasma) and AUC0 to 24 hours(the area under the plasma concentration-time curve 0-24 hours after administration, calculated by the linear/log trapezoidal method).
Protein binding was also detected as follows: the above heparinized human plasma containing Compound 1 was centrifuged at about 3000rpm (30 minutes, about 37 ℃) in a molecular weight cut-off ultrafiltration unit (30,000Da) to obtain an Ultrafiltrate (UF) sample. UF samples (0.025ml) were mixed with O as an internal standard solution13CD3Compound-1 (. about.800 ng/mL, 0.050mol) (OCH in Compound 1)3Radical is O13CD3Is substituted by radicals, thereby obtaining O13CD3Compound-1) was mixed, diluted 20-fold and analyzed by reverse phase HPLC on a 3.5 micron C-18 column. Quantitative determination was performed by cationic Turbo-ion spray ionization using MRM (multiple reaction monitoring) method. The amount of unbound drug in the plasma quality control sample and the unknown sample was determined quantitatively using ultrafiltrate standards. Non-specific protein binding (NSB) (NSB ═ 0.0415) was detected and used as a correction factor to determine the final percent protein binding. The nominal range (nominal range) for the quantitative determination of analytes is 50-10,000 ng/ml. A0.400 ml human plasma sample was used in the assay. Weighted linearity (1/x) of calibration curves generated using spiked UF standards2) The sample concentration was determined by regression (back-calculation). In a linear mannerTo this extent, the intra-batch CV% for compound 1 was 4.9% -11.8%.
The following table shows the AUC for subjects taking 500mg, 750mg and 1000mg of Compound 1 per day0-24、CmaxAnd protein binding values. Free C shown in the TablemaxAnd free AUC0-24Values are those corrected for plasma protein binding. Also shown in the table is free CmaxThe ratios of/MIC and free AUC/MIC, which can be used to predict clinical and microbiological outcomes and bacterial resistance development. For antibiotic drugs, free C is preferredmaxa/MIC greater than about 8 and a free AUC/MIC greater than about 100.
Other embodiments
All of the features disclosed in this specification may be combined in any combination. Other features serving the same, equivalent or similar purpose may be substituted for the features disclosed in this specification. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
In view of the foregoing description it will be evident to a person skilled in the art that various modifications and improvements can be made to the invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.