HK1139924A - Water-soluble prodrugs of florfenicol and its analogs - Google Patents

Description

Water-soluble prodrugs of florfenicol and its analogs

Cross reference to related applications

This application is a non-provisional application, priority of U.S. provisional application 60/874,864 filed 2006, 12, 13, 35 u.s.c. § 119(e), which is hereby incorporated by reference in its entirety.

Technical Field

[0001] The present invention relates to novel prodrugs of florfenicol (florfenicol) and its analogs. In one particular aspect, the present invention relates to charged nitrogen-containing esters of florfenicol and analogs thereof that exhibit improved water solubility and hydrolytic stability. In a specific embodiment of this aspect, the invention relates to certain charged nitrogen-containing esters of florfenicol.

Background

[0002] Florfenicol (2, 2-dichloro-N- ((1R, 2S) -3-fluoro-1-hydroxy-1- (4- (methylsulfonyl) phenyl) propan-2-yl) acetamide) has the following chemical structure

Florfenicol

[0003] Florfenicol is a broad spectrum antibiotic active against a variety of gram-negative and gram-positive bacteria, including for the prevention and treatment of bacterial infections caused by susceptible pathogens of birds, reptiles, fish, shellfish and mammals. One of the main uses of florfenicol is in the treatment of bovine pulmonary inflammation and concomitant respiratory infections (commonly referred to as bovine respiratory disease or BRD) caused by Mannheimia haemolytica (Mannheimia haemolytica), pasteurella multocida and/or haemophilus somni. It also shows treatment: bovine hoof dermatitis caused by fusobacterium necrophorum and bacteroides melanoides; porcine respiratory disease caused by pasteurella multocida, actinobacillus pleuropneumoniae, streptococcus suis, salmonella choleraesuis and/or mycoplasma; colibacillosis in chickens caused by escherichia coli; intestinal septicemia of catfish caused by edwardsiella catfish; and furunculosis in salmon caused by Aeromonas salmonicida. Other bacterial genera that exhibit susceptibility to florfenicol include Enterobacter, Klebsiella, Staphylococcus, enterococcus, Bordetella, Proteus, and Shigella. In particular, strains of chloramphenicol resistant organisms (e.g., Klebsiella pneumoniae, Enterobacter cloacae, S.typhus, and E.coli) are susceptible to florfenicol.

[0004] Florfenicol is most commonly administered to subjects orally or parenterally, the latter being administered primarily intramuscularly and intravenously. Due to its extremely low water solubility (about 1mg/mL), organic solvents must be added to commercial formulations to achieve the desired product concentration. Due to the need for economical single dose treatment in veterinary medicine, there is a need for new high concentration florfenicol formulations. In addition, there is a need for forms of florfenicol that can maintain effective plasma antibiotic levels for longer periods of time, thereby improving the economics of administration, e.g., to more easily provide single dose treatment, particularly in veterinary medicine. In addition, similar forms of florfenicol analogs are also needed.

[0005] One important application is the administration of drugs to animals via drinking water to treat bacterial infections. This mode of administration provides an effective treatment for bacterial infections because florfenicol is properly absorbed by the gut and achieves the necessary systemic levels of antibiotics. However, as noted above, florfenicol has very low water solubility; florfenicol therefore dissolves very slowly in water. To achieve the desired concentration in drinking water, it is necessary to prepare the pre-dissolved florfenicol in concentrated form in a water-miscible organic solvent. In addition, water-soluble prodrugs of florfenicol or florfenicol analogs that readily dissolve directly in the drinking water of animals are highly desirable.

[0006] The other two common cholalic (phenicol) antibiotics (chloramphenicol and thiamphenicol) contain two hydroxyl groups: one is a primary alcohol type and the other is a secondary alcohol type. A great deal of work has been done to produce water-soluble prodrugs of these two antibiotics by esterification of the more readily available primary alcohol (rather than the hindered secondary alcohol group). The glycine esters of these compounds have been studied extensively. Some examples of such esters are disclosed in U.S. patents 3,740,411 and 3,770,889 (both Akiyama et al), British patent 1,263,116(Sumitomo Chemical Co.), and 3,405,165 and 3,475,470 (both Rebstock et al).

[0007] Much less work has been done to generate water-soluble prodrugs of florfenicol that lack primary alcohol groups. For example, esters of florfenicol are described in U.S. patent 4,311,857 to Nagabhushan. This patent describes primary aliphatic esters of florfenicol and also discloses esters formed from several amino acids, particularly glycine, ornithine and lysine esters. Such amino acid esters are also disclosed in Kohan et al, U.S. patent 6,790,867. U.S. patent application publication No. 2005/014828 to Murthy et al describes a series of florfenicol esters with aliphatic carboxylic acids. U.S. patent application publication No. 2005/0182031 to Hecker et al describes certain phosphate esters of florfenicol. However, there remains a need for alternative forms of florfenicol that have additional beneficial characteristics. (it should be noted that the citation of any reference herein should not be construed as an admission that such reference constitutes "prior art" with respect to the present invention).

Summary of The Invention

[0008] Briefly, the present invention encompasses certain novel prodrugs of florfenicol and/or florfenicol analogs described herein, including prodrugs of pharmaceutically acceptable salts of florfenicol or analogs thereof. These prodrugs include nitrogen-containing esters of the secondary alcohol group of florfenicol and its analogs, which in particular embodiments have sufficient water solubility to provide the desired functionality of the prodrug of florfenicol or of the florfenicol analog. In a more specific embodiment, a particular subclass of compounds (subclasses) also possesses the hydrolytic stability required to maintain the prodrug in solution in the subject system until appropriate conditions arise under which the prodrug can hydrolyze, releasing the florfenicol or florfenicol analog.

[0009] Other aspects of the invention include pharmaceutical formulations of florfenicol or a florfenicol analog, or a prodrug of a pharmaceutically acceptable salt thereof, as described above, further comprising one or more pharmaceutically acceptable excipients or carriers. Other aspects include methods of treating a subject with florfenicol or a florfenicol analog comprising administering to the subject an effective amount of a prodrug of florfenicol or a florfenicol analog or a pharmaceutically acceptable salt thereof according to the present invention, or a pharmaceutical composition comprising the prodrug.

[0010] The present invention further provides pharmaceutical compositions comprising a prophylactically effective amount of a novel florfenicol prodrug, a prodrug of a florfenicol analog, or a pharmaceutically acceptable salt of any of the foregoing. The invention also provides mixtures of these compounds. Furthermore, in certain pharmaceutical compositions, florfenicol itself may be combined with one or more novel florfenicol prodrugs and/or prodrugs of florfenicol analogs of the present invention. The present invention further provides pharmaceutical compositions comprising novel prodrugs for strategic delivery of (metaphalaxis). The pharmaceutical compositions of the present invention may be administered to animals or fish as needed and/or as practical (practicemerites) in prophylactically effective amounts and/or for strategic administration. The invention also provides corresponding methods for administering a prophylactically effective amount of a pharmaceutical composition of the invention and/or for strategic administration, as needed and/or of practical value. The invention also provides methods of treating or preventing a disease or disorder in an animal in need thereof.

Detailed Description

[0011] The present invention provides novel prodrugs of florfenicol and/or florfenicol analogs, including prodrugs of pharmaceutically acceptable salts of florfenicol or its analogs. These prodrugs have one or more advantageous properties, such as: water solubility, hydrolytic stability in aqueous systems, and/or the ability to be released in the stomach of an animal by the action of enzymes. In particular embodiments, the prodrug (when used to improve oral absorption) does not prematurely convert to the parent drug in the dosing solution and/or in the intestine. In a more specific embodiment, administration of a prodrug of the invention to a subject provides a desired rate of conversion to the parent drug when the prodrug reaches its intended site.

[0012] Thus, prodrug molecules containing moieties that increase solubility but do not have sufficient chemical stability upon dissolution may not achieve the desired effect.

[0013] If the parent drug is satisfactorily well absorbed from the gastrointestinal system, the target effect of the prodrug may simply be to increase solubility in order to facilitate administration (e.g., in drinking water). In this case, the parent drug may be released from the prodrug in the gastric system by one or both of two independent mechanisms: enzymatic hydrolysis due to intestinal enzymatic action, or chemical hydrolysis initiated by an increase in the pH present in the intestine relative to the pH of the original dosing solution. Chemical release of the parent drug may be caused by hydrolysis of the ester bond facilitated by elevated pH or by the action of some prodrug moiety capable of intramolecular cyclization. In particular, the release of the parent drug may be caused by intramolecular displacement of the parent drug by the primary or secondary amine of the prodrug induced by a change in pH.

[0014] Accordingly, the present invention provides novel prodrugs of florfenicol or a florfenicol analog having formula (I):

wherein:

r is selected from:

a is oxygen and a is 0 or 1;

l is (a) CH₂And l is an integer from 1 to 6;

(b)CHR₁wherein R is₁Is an amino acid side chain, and l is 1; or

(c)CHR₁NHC(O)CH(NH₂)R₂Wherein R is₁And R₂Is an amino acid side chain, and l is 1;

m is (a) oxygen or sulfur, and M is 0 or 1;

(b)CH₂and m is 0 or an integer from 1 to 4; or

(c) NH, and m is 1;

x is (a) CH₂And x is 0 or an integer from 1 to 4; or

(b) C (O), and x is 1;

y is (a) NH₂；

(b)NHR_xWherein R is_xIs methyl, ethyl, n-propyl or isopropyl;

(c)NR_yR_zwherein R is_yAnd R_zIndependently hydrogen, methyl, ethyl, n-propyl or isopropyl, or R_yAnd R_zTogether form C₂-C₅An alkylene chain, or C further containing a nitrogen or oxygen heteroatom in said chain₂-C₄An alkylene chain;

(d)C(＝NH)NH₂；

(e)N⁺R₄R₅R₆wherein R is₄、R₅And R₆Independently hydrogen, methyl or ethyl, or R₄And R₅Together form C₂-C₅An alkylene chain, or C further containing a nitrogen or oxygen heteroatom in said chain₂-C₄An alkylene chain;

(f) pyridinium salts;

(g) n-methyl or N-ethylpyridinium;

(h) n' -3-methyl-N-1-imidazolium;

(i) is of the formula NR₄R₅Or N⁺R₄R₅R₆A phenyl group substituted with a group of (1), wherein R₄、R₅And R₆As defined above; or

(j)NH-CR₃(═ NH), where R₃Is hydrogen, methyl or amino;

and R₇Selected from the group consisting of dichloromethyl, difluoromethyl, trifluoromethyl, cyanomethyl, azidomethyl and aminomethyl;

with the proviso that the radical A_aL_lM_mX_xY is not an alpha-N-unfunctionalized glycine, ornithine or lysine residue.

[0015] In particular embodiments of these compounds, the nitrogen atom of the prodrug moiety is a charged atom. Furthermore, to modulate the rate of hydrolysis of the ester front moieties (promoieties) containing such charged nitrogen atoms or a nitrogen atom that is sufficiently basic to ensure that the prodrug exists predominantly in charged form at physiological pH, the nitrogen atom may be positioned away from the carbonyl bond of the ester. The same effect can be achieved in carbonate derivatives containing a charged nitrogen atom by linking the charged nitrogen atom at a position further (further) away from the hydrolysable carbonate functionality. A charged nitrogen atom at least two atoms or at least three atoms away from the carbonyl carbon atom of the ester or carbonate group that will hydrolyze upon release of the parent drug is sufficient to achieve the desired hydrolytic stability of the ester or carbonate.

[0016] Some preferred compounds of the invention include formula (II):

wherein:

a is oxygen and a is 0 or 1;

l is (a) CH₂And l is an integer from 1 to 5;

(b)CHR₁wherein R is₁Is an amino acid side chain, and l is 1; or

(c)CHR₁NHC(O)CH(NH₂)R₂Wherein R is₂Is an amino acid side chain, and l is 1; m is (a) oxygen, and M is 0 or 1;

(b)CH₂and m is 0 or an integer from 1 to 4; or

(c) NH, and m is 1;

x is (a) CH₂And x is 0 or an integer from 1 to 4; or

(b) C (O), and x is 1;

y is (a) NH₂；

(b)NHR_xWherein R is_xIs methyl, ethyl, n-propyl or isopropyl;

(c)NR_yR_zwherein R is_yAnd R_zIndependently hydrogen, methyl, ethyl, n-propyl or isopropyl;

(d)C(＝NH)NH₂；

(e)N⁺R₄R₅R₆wherein R is₄、R₅And R₆Independently hydrogen, methyl or ethyl;

(f) n-pyridinium;

(g) n' -3-methyl-N-1-imidazolium; or

(h)NH-CR₃(═ NH), where R₃Is hydrogen, methyl or amino; and

R₇selected from the group consisting of dichloromethyl, difluoromethyl, trifluoromethyl, cyanomethyl, azidomethyl and aminomethyl (wherein R is₇Preferably dichloromethyl);

provided that the sum of a + l + m + x is from 2 to 6, and preferably from 3 to 6;

provided that if a is 1, then M is (CH)₂)_m(ii) a And

with the proviso that the radical A_aL_lM_mX_xY is not an α -N-unfunctionalized glycine, ornithine or lysine residue.

The invention further provides pharmaceutically acceptable salts of these preferred compounds.

[0017]Particular compounds are those of formula (I) or (II): wherein Y contains a positively charged nitrogen atom, i.e. Y is a group N⁺R₄R₅R₆Wherein R is₄、R₅And R₆Independently of one another, hydrogen, methyl or ethyl, e.g. NH₃ ⁺、N⁺H₂(CH₃)、N⁺H(CH₃)₂、N⁺(CH₃)₃、N⁺H₂(C₂H₅)、N⁺H(C₂H₅)₂、N⁺(C₂H₅)₃、1-NH⁺-3-methylimidazolium. Wherein a is 0 is an ester; wherein the compound in which a is 1 is a carbonate. Other particular compounds are those of formula (I) or (II): wherein the radical A_aL_lM_mX_xalpha-N-nonfunctional Y other than alpha-amino acid or aromatic alpha-amino acidA chemotrope (e.g. group A)_aL_lM_mX_xY is not an alpha-N-unfunctionalized phenylalanine residue), or the group A_aL_lM_mX_xY is not an alpha-N-unfunctionalised alpha-amino acid, or the group A_aL_lM_mX_xY is not an alpha-amino acid residue.

[0018]Compounds of the formulae (I) and (II) include those in which the radical A_a-L_l-M_m-X_x-Y_ySuch as the compounds shown below:

1. dipeptide ester R ═ H, amino acid side chain

2. Omega (Omega) amino ester (n-2-6) R-H, Me, Et

3. Omega amidino esters, N-linked (N-2-6) R-H, Me

4. Omega amidino ester, C-linked (n ═ 2-6)

5. Omega guanidine ester (n ═ 2-6)

6. Omega amino acid ester-cyclic amine (n)₁＝1-6；n₂＝2-4)

7. Omega quaternary ammonium esters (n-1-6) R-Me, Et

8. Omega Quaternary ammonium ester-cyclic amine (n)₁＝2-6；n₂＝2-4；R＝Me、Et)

9. Omega quaternary ammonium ester-cyclic diamine (n ═ 2-6; R ═ H, Me, Et)

10. Omega pyridinium salts, C-linkages; (n-2-6; R-Me, Et)

11. Omega imidazolium salts (n-2-6; R-Me, Et)

12. Aminomethyl benzoate (R ═ Me, Et)

13. Quaternary salt of aminomethyl benzoate (R ═ Me, Et)

For types 2-11, also

Type (iv) wherein "charged group" represents a positively charged nitrogen-containing moiety of the type shown.

[0019] Compounds of formula (II) generally include those exemplified below:

A. a carbonate having a terminal amine functional group;

B. esters with an additional α -heteroatom (O, S) in the linker (linker); for example-C (O) CH₂OCH₂CH₂NH₂、C(O)CH₂SCH₂CH₂NH₂；

C. Esters with a further α -heteroatom (N) -, which are amino acid derivatives, but do not carry a protonatable amine in the α position; these being dipeptides, e.g.

-C(O)CH₂NHC(O)CH₂NH₂And

-C(O)CH(Me)NHC(O)CH(Me)NH₂；

D. esters having quaternary nitrogen atoms removed from at least two methylene groups remote from the carbonyl group, e.g. C (O) CH₂CH₂CH₂-N-methylimidazolium and C (O) CH₂CH₂CH₂N⁺Me₃。

[0020] Some preferred compounds are shown below:

[0021] in another aspect, the invention relates to methods of making the novel compounds. In one method, the compounds of the invention can be prepared as esters by reacting florfenicol or a florfenicol analog with a carboxylic acid or derivative thereof having a terminal group W, where W represents a protected primary or secondary amine, which is subsequently deprotected to a free amine, tertiary amine, or a group that is subsequently treated to the desired charged nitrogen functionality. Commercially available activated derivatives of carboxylic acids may be used to form the esters; it can be prepared in a separate reaction step, or it can be prepared in situ in the presence of florfenicol or a florfenicol analog.

[0022] The compounds of the invention may be prepared as carbonates by reacting florfenicol or a florfenicol analog with an alkoxycarbonic acid derivative (e.g., chloroformate) having a terminal group W, where W represents a protected primary or secondary amine, which is subsequently deprotected to a free amine, tertiary amine, or a group that is subsequently treated to the desired charged nitrogen functionality.

The necessary acids or chloroformates, if not commercially available, can be readily prepared by methods known to those skilled in the art. Suitable reaction conditions, solvents, and the like are exemplified below.

[0023] As shown in the above reaction scheme, the secondary alcohol functional group of florfenicol or a florfenicol analog reacts with an activated carboxylic acid or alkoxycarbonic acid reagent through nucleophilic displacement of group Q. The most commonly used reagent of this type employs Q ═ chlorine, but many other leaving groups known in the art may also be used.

As an alternative to chloroformate (Q ═ chloride), other reagents with different leaving groups Q can be used to prepare carbonates of florfenicol alcohol. Representative references are cited for each leaving group, each of which is incorporated herein by reference.

The reaction may be promoted by the addition of a catalyst such as a trialkylamine, pyridine, 4-alkylpyridine, 4-diaminoalkylpyridine, or combinations thereof. The formation of the initial ester or carbonate intermediate may be conveniently carried out in a variety of solvents. Suitable solvents include, for example, chlorinated solvents such as dichloromethane and 1, 2-dichloroethane; ester solvents such as ethyl acetate, isopropyl acetate, isoamyl acetate, ethylene glycol diacetate, propylene glycol diacetate, glycerol triacetate; monoether solvents such as diethyl ether, diisopropyl ether, methyl tert-butyl ether; polyether solvents such as glycol ethers, dimethyl glycol ethers, diethylene glycol dimethyl ether, diethylene glycol diethyl ether; formaldehyde acetal ethers (formaldehydeacetal ethers) such as dimethoxymethane, diethoxymethane, dibutoxymethane; cyclic ethers such as tetrahydrofuran, 1, 3-dioxolane, 1, 4-dioxane; ketone solvents such as acetone, methyl ethyl ketone, methyl isobutyl ketone; mixed ether/ester solvents represented by monoethers of ethylene glycol and diethylene glycol, such as 2-methoxyethyl acetate, 2-ethoxyethyl acetate, 2- (methoxy-ethoxy) ethyl acetate, and 2- (ethoxy-ethoxy) ethyl acetate.

Definition of

As used herein:

[0024]unless otherwise indicated "About"generally refers to values within twenty percent of the values indicated.

[0025]“Amino acids"refers to known natural amino acids, in particular those selected from the group consisting of alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine. "amino acid side chain" and "amino acid residue" refer to a group derived from an alpha-amino acid and denotes NH of an amino acid₂-CH(R_aa)CO₂R in the structure of H_aaRadicals, e.g. of-CH (CH) for valine₃)₂lysine-CH₂CH₂CH₂CH₂NH₂and-CH of serine₂And (5) OH. For proline, it represents-CH₂CH₂CH₂-, the terminal end (digital end) of which is attached to the alpha nitrogen atom. The term "α -N-unfunctionalized" means having an unsubstituted-NH in the α position₂Amino acid residues of radicals, as opposed to functionalized residues in which, for example, the alpha-amino group is a peptideA portion of an amide bond of (a).

[0026]“Alkyl radical"means having the indicated number of carbon atoms (i.e., C)₁-C₁₀Representing 1 to 10 carbons) of a straight or branched chain saturated hydrocarbon moiety. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and the various pentyl, hexyl, heptyl, octyl, and the like groups. Alkyl groups also include those having one or more heteroatoms in the chain, e.g., methoxymethyl (CH)₃OCH₂-), ethoxyethyl, methylthiomethyl (CH)₃SCH₂-) methylaminomethyl (CH)₃NHCH₂-) and the like.

[0027]“Alkylene chain"refers to a divalent hydrocarbon group derived from an alkyl group, i.e., a saturated straight or branched chain hydrocarbon group having both ends attached to the remainder of the molecule. Typical alkylene groups include methylene-CH₂-, ethylene-CH₂CH₂And n-propylene-CH₂CH₂CH₂-. As with the alkyl groups, the alkylene chain may contain one or more heteroatoms, e.g. -CH₂CH₂-NH-CH₂CH₂-. When the alkylene chain is bound to a nitrogen atom (e.g. in a compound of formula NR)_yR_zIn (e) the whole group is a heterocyclic group such as a piperidyl group or the like. When the alkylene chain also contains heteroatoms, the resulting radical NR_yR_zWill be, for example, a cyclic moiety containing two nitrogen atoms, such as piperidinyl.

[0028]“Prodrugs"refers to a compound that is a prodrug that, when administered to a subject, undergoes a chemical transformation, either metabolically or chemically, to yield the active drug, e.g., a carbonate of florfenicol or a florfenicol analog is a prodrug that releases the florfenicol compound in vivo.

[0029]“Pharmaceutical composition"means comprising a compound of the present invention, including pharmaceutically acceptable salts thereof (e.g., florfenicol prodrugs) and pharmaceutically acceptable vehiclesA composition or formulation of agents and/or carriers. In a specific embodiment, the carrier is a solvent (e.g., water).

[0030]“Excipient"refers to inert substances added to pharmaceutical compositions to further facilitate administration of the active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0031]“Administration of"(" Administer "or" administration ") refers to the delivery of a compound or solvate of the present invention, or a pharmaceutical composition containing a compound of the present invention, to an organism for the purpose of treating or preventing a microbial infection.

[0032] As used herein, "therapeutically effective amount" refers to an amount of a prodrug of the present invention that will hydrolyze sufficiently rapidly and with sufficient water to provide a concentration of florfenicol or a florfenicol analog at which the florfenicol or florfenicol analog is capable of alleviating to some extent the symptoms of one or more bacterial infections in a subject. In a particular embodiment, a therapeutically effective amount refers to an amount of a florfenicol prodrug of the present invention that, when administered to a subject, delivers florfenicol or a florfenicol analog to the subject at sufficient plasma concentrations to: (1) reducing and preferably eliminating bacterial cell populations in a subject; (2) inhibiting (i.e., slowing or preferably stopping) the proliferation of bacterial cells; (3) inhibiting (i.e., slowing, preferably stopping) the spread of bacterial infection; and/or (4) reducing, preferably eliminating, one or more symptoms associated with the infection.

[0033]“Florfenicol analogs"means having a substituent other than methanesulfonyl on the benzene ring (represented by" R "in the formula (I) or (II)) and/or wherein R is₇Florfenicol analogs that are groups other than dichloromethyl.

[0034]“A prophylactically effective amount"refers to the amount of a florfenicol prodrug or florfenicol analog prodrug of the present invention that provides sufficient plasma concentration of florfenicol or florfenicol analog by hydrolysis to: (1) health-care productTo reduce the level of bacterial cell populations achieved by pre-administration of a therapeutically effective amount of a prodrug or other suitable drug; (2) maintaining a level of inhibition of bacterial cell proliferation achieved by administration of a therapeutically effective amount of a drug; (3) maintaining the degree of inhibition of the spread of infection achieved by administration of a therapeutically effective amount of the drug; and/or (4) maintaining the level of alleviation of one or more symptoms associated with a bacterial infection, or the absence of symptoms if the symptoms are eliminated, achieved by administration of a therapeutically effective amount of a prodrug of the invention (e.g., florfenicol) or other suitable drug. "prophylactically effective amount" also refers to the amount of a composition comprising a florfenicol prodrug of the present invention or a florfenicol analog prodrug of the present invention that delivers florfenicol or a florfenicol analog at sufficient plasma concentrations to prevent bacteria from accumulating in susceptible organisms to amounts sufficient to cause infection.

[0035]Strategic delivery of drugsRefers to the timely and focused administration of drugs to an entire group of animals to eliminate or minimize the expected outbreak (e.g., in one or more animals at high risk of infection). In a specific embodiment, a high risk cow is a light weight, long haul (long haul) cow with an unknown health history.

[0036]Terms used herein "Minimum inhibitory concentration"can and"MIC"used interchangeably. "MIC₅₀"is the concentration of compound (e.g., a prodrug of the invention) at which 50% of the growth of the isolated population (isolates) is inhibited. Similarly, MIC₉₀Is the concentration of compound at which 90% of the isolate growth is inhibited.

[0037]“Object"refers to animal species or fish, including humans in particular embodiments, that can be infected with a pathogenic bacterium. Suitable animal subjects also include wild animals, livestock (e.g., animals raised for meat, milk, butter, eggs, fur, leather, feathers, and/or wool purposes), work animals, research animals, companion animals, and animals raised for/in zoos, wildlife, and/or circus.

[0038] In a particular embodiment, the "subject" of the invention is an animal that "makes a food". For the purposes of the present invention, the term "food-producing animal" is understood to include all animals (e.g., cows, laying hens, etc.) which are intended for consumption by humans and/or other animals or which are raised for consumption. A non-limiting list of such animals includes birds (chickens, turkeys, geese, ducks, ostriches, etc.), bovines (e.g., cattle, cows, buffalos), ovines (e.g., goats or sheep), porcines (e.g., adult pigs (hog) or pigs (pig)), equines (e.g., horses), etc., as well as aquatic animals, including shellfish and fish (e.g., trout or salmon), as well as other species raised or captured for human consumption. For the purposes of the present invention, the term "fish" is understood to include, but is not limited to, the teleost group of fish, i.e. bony fish. Salmonids (including salmonidae) and perciformes (including echinococcaceae) are included in the bonboneset classification. Examples of possible fish recipients include salmonidae, bass, Pacifidae, Otophagidae, Paralichitiatilis (Parapristinarieleatum), and Pacifidae (Plecostomus spp).

[0039] In another embodiment, the subject is a companion animal. For the purposes of the present invention, the term "companion" animal is understood to include domestic cats (felines), dogs (canines), rabbits, horses (equines), rodents (e.g. guinea pigs, squirrels, rats, mice, gerbils and hamsters), primates (e.g. monkeys) and birds, such as pigeons, pigeons (doves), parrots, parakeets, adamantines, canaries and the like.

[0040] Other animals are also contemplated that may benefit from the prodrugs of the invention, including marsupials (e.g., kangaroos), reptiles (e.g., farmed turtles), game birds (game birds), swans, ratites, and other economically important livestock.

[0041] Pharmaceutically acceptable salts of the above compounds include hydrochloride, hydrobromide, methanesulfonate, sulfate, 2-hydroxyethyl sulfate, citrate, and phosphate.

[0042] Table 1 below describes representative compounds of the present invention.

TABLE 1

Compound numbering	Aa	Ll	Mm	Xx	Y
						1	O	CH2	CH2	-	NH3 +[ hydrochloride salt]
2	O	CH2	CH2	-	NH2 +CH3[ hydrochloride salt]
						3	-	CH2	CH2	CH2	NH3 +[ hydrochloride salt]
4	-	(CH2)3	NH	CO	CH(NH3 +)CH3[ hydrochloride salt]
						5	O	CH2	CH2	CH2	NH3 +[ hydrochloride salt]
6	O	CH2	CH2	CH2	NH2 +CH3[ hydrochloride salt]
						7	O	CH2	CH2	-	NH2 +C2H5[ hydrochloride salt]
8	-	CH(NH3 +)	(CH2)2	CH2	NH3 +[ dihydrochloride salt]
						9	-	CH2	NH	CO	CH2NH3 +[ hydrochloride salt]
10	-	(CH2)4	-	-	NH3 +[ hydrochloride salt]
						11	-	CH(CH3)	NH	CO	C(CH3)NH3 +[ hydrochloride salt]
12	-	(CH2)4	-	-	NH2 +CH3[ hydrochloride salt]
						13	O	CH2	CH2	CH2	NH2 +C2H5[ hydrochloride salt]
14	-	CH2	O	(CH2)2	NH3 +[ hydrochloride salt]
						15	-	CH2	CH2	CH2	N(CH3)3 +[ Bromide]
16	-	CH2	CH2	CH2	N-3-methyl-N-1-imidazolium [ bromide ]]
						17	-	CH2	-	-	NH3 +[ hydrochloride salt]

Pharmaceutical composition

[0043]The compounds of the present invention or physiologically acceptable solvates of the compounds can be administered directly to an animal in need thereof or in the form of pharmaceutical compositions wherein the foregoing are in admixture with suitable excipients or carriers. Techniques for formulating or administering drugs are found in the latest edition of Remington's pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. The formulations and techniques discussed in Remington are primarily directed to use by human patients; however, they can be readily adapted for use with non-human patients by techniques well known to those skilled in the veterinary arts. Pharmaceutical compositions containing the novel compounds of the present invention may also contain typical pharmaceutical excipients and additives such as liquid and/or solid carriers, surfactants, dispersants, taste masking agents and the like. Taste masking agents include, for example, those described in U.S. Pat. No. 6,514,492 to Gao et al (incorporated herein by reference in its entirety) for quinolones and derivatives thereof, i.e., ion exchange resins (including both cationic and anionic resins) such as methacrylic acid-divinylbenzene copolymers (e.g., AMBERLITEIRP-64), sodium polystyrene sulfonate resin (e.g., AMBERLITE)IRP-69) and polystyrenesulfonic acid-divinylbenzene resins (e.g., DOWEX)A resin).

[0044] When the compounds of the invention described herein are administered as an ingredient of animal feed, or dissolved or suspended in drinking water, compositions are provided in which the active agent is intimately dispersed in an inert carrier or diluent. An inert carrier is one that does not react with the compounds of the present invention and is safe for administration to an animal. Preferably, the carrier for feed administration is (or may be) an ingredient of an animal ration.

[0045] Suitable compositions include feed premixes or supplements in which the active ingredient is present in relatively large amounts and which are suitable for direct feeding to animals or for addition directly to the feed or to the feed after an intermediate dilution or mixing step. Typical carriers or diluents suitable for use in such compositions include, for example, distillers' dried grains, corn meal, citrus powder, fermentation residues, ground oyster shells, wheat middlings, molasses solubles, corn cob meal, edible soymilk feed, soy grits, ground limestone, and the like. The compounds of the invention are dispersed throughout the support by methods such as milling, stirring, milling or tumbling. Compositions comprising from about 0.05 to about 5.0%, more broadly, from about 0.005 to about 2.0% by weight (w/w) of the compounds of the invention are particularly suitable as feed premixes. A feed supplement for direct feeding animals will contain about 0.0002 to 0.3 wt% of the compound of the invention.

[0046] Such supplements are added to animal feed in amounts to obtain the concentration of active compound required to treat and control susceptible microorganisms in the final feed. Although the desired concentration of the compounds of the present invention will vary depending on the factors described above and the particular derivative used, the compounds will generally be administered in the feed at a concentration of between about 0.0001 to 0.02% or about 0.00001 to about 0.002% (both w/w) to achieve the desired antimicrobial effect.

[0047] The pharmaceutical compositions of the present invention may be prepared by methods well known in the art, for example, using various well-known mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The compositions may be formulated with one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The appropriate formulation will depend on the route of administration chosen.

[0048]For injections (including but not limited to intravenous, intramuscular, and subcutaneous injections), the compounds of the invention may be formulated in polar solvents including but not limited to propylene glycol, alcohols (such as benzyl alcohol or ethanol), polyethylene glycol, and N-methyl-2-pyrrolidone, other pyrrolidones, N-dimethylacetamide, N-dimethylformamide, dimethyl sulfoxide, acetone, triacetin (triacetin), glycerol, formalin, triglycerides (such as Miglyol)Product), optionally water at a concentration of up to 10%, as well as any combination of the foregoing excipients or other materials known to one of ordinary skill. For transmucosal administration, penetrants appropriate to the barrier to be crossed are used in the formulation. Such penetrants are generally known in the art.

[0049] In addition to the above formulations, concentrated compositions of the compounds of the invention may be prepared, which may be diluted at the point of use. The preparation of such concentrated compositions saves the cost of transporting or storing large quantities of diluent, particularly water.

Administration of

[0050] Suitable routes of administration may include, but are not limited to, oral, rectal, topical, transmucosal, intramuscular, subcutaneous, intramedullary, intracapsular, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, intramammary, intra-aural, or intraocular administration.

[0051] As discussed above, one method of administration of the compound is to include it in the subject's drinking water because of its water solubility.

[0052] Alternatively, it may be administered by local rather than systemic means, for example it may be prepared as an ointment or topically applied formulation, for direct application to the affected area or for direct injection of the compound into the affected tissue. In either case, a sustained release formulation may be used.

[0053] Thus, a compound of the invention or a pharmaceutically acceptable solvate thereof, in pure form or in a suitable pharmaceutical composition, may be administered by any acceptable mode of administration of the agent to achieve a similar effect. The route of administration can be any route known to the ordinarily skilled artisan. The compounds of the present invention may be administered to a subject in need thereof in any art-recognized form (i.e., solid, semi-solid, lyophilized powder, or liquid dosage forms, e.g., tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, etc.) and in unit-dose or multi-dose forms suitable for simple administration of precise dosages. The compositions will contain conventional pharmaceutical carriers or excipients and the compounds of the invention as active agents, in addition to other agents, pharmaceutical preparations, carriers, adjuvants and the like.

Dosage form

[0054] A therapeutically effective amount refers to an amount of a compound effective to prevent and/or minimize microbial infection, and/or treat, alleviate and/or ameliorate symptoms caused by microbial infection. Determination of a therapeutically effective amount is well within the ability of those skilled in the art, particularly in light of the disclosure herein.

[0055] For any compound used in the methods of the invention, a therapeutically effective amount may be initially estimated from the known properties of the antibacterial agent released from the prodrug compound of the invention. The dose can then be formulated for use in animal models to achieve a circulating concentration range equal to or greater than the minimum inhibitory concentration ("MIC") known in the art. This information can then be used to more accurately determine the dose that is useful to the patient.

[0056]The therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, the minimum inhibitory concentration ("MIC") can be determined by reference to guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI). Similarly, the toxicity of a compound described herein can be expressed as the LD of that compound₅₀Which is the lethal dose for 50% of the subjects in the group treated with the particular compound.

[0057] The data obtained can be used to formulate a range of dosages suitable for a patient. Of course, the dosage may vary depending on the dosage form and route of administration. The particular dosage form, route of administration and dosage may be selected by The individual physician according to The condition of The patient (see, e.g., Fingl et al, 1975, "The pharmacological basis of Therapeutics", Ch.1p.1). In general, the compounds of the invention are administered to an animal in need of such treatment at a dose that will enable the concentration of antibiotic released in the plasma and body tissues to achieve and/or maintain an effective level against the target (whether to treat and eliminate susceptible infectious microorganisms or prevent new infections) for a length of time that will achieve the intended target. Those skilled in the art will appreciate that the dosage ranges estimated hereinafter may be adjusted according to the clinical response and taking into account the relative amounts of antibiotic released from each respective prodrug compound. For example, for subcutaneous administration, the compounds of the invention are generally administered in a dosage range of about 1mg/kg body weight to about 150mg/kg body weight. The frequency of administration can also vary from a single dose per day to multiple doses per day. For oral administration, the dose is preferably administered once daily.

[0058] The dose and dosage interval can be adjusted individually to provide plasma levels of the compound sufficient to maintain a concentration at or above the MIC, or any other desired level. Such plasma levels are often referred to as the Minimum Effective Concentrations (MECs). The MEC will vary for each compound but can be estimated from in vitro data (e.g. the concentration required to achieve an inhibition of the microbial community of 80% or more). The MEC may be determined by the assay described herein. The dosage required to achieve MEC will depend on the individual characteristics of the compound and/or animal and/or the route of administration. HPLC assays or bioassays can be used to determine the plasma concentration of the compound and/or its corresponding active product.

[0059] The MEC value may also be used to determine the dosing interval. The compound is administered in a regimen that maintains plasma levels above the MEC for 10-90% of the time.

[0060] In the case of topical administration or selective uptake, the effective local concentration of the drug may not be correlated with the plasma concentration, and the correct dosage and interval may be determined using other procedures known in the art.

[0061] The composition may be administered once daily or divided into multiple doses. Usually a single dose is sufficient to treat the infection. In some cases, it is desirable to administer a second dose 48 hours after one dose to treat the animal. As will be appreciated by those of ordinary skill in the art, the exact dosage will depend on the stage and severity of the infection, the susceptibility of the infecting organism to the composition, and the individual characteristics of the animal species being treated.

[0062] The amount of the composition administered will, of course, depend on the patient to be treated, the pathogen or bacteria causing the infection, the severity of the infection, the mode of administration (i.e., oral, intravenous, topical, etc.), and the judgment of the prescribing physician, veterinarian, and the like.

[0063] When the subcutaneous route is employed, the compounds of the present invention will generally be administered to cattle at a dosage ranging from about 1mg/kg body weight to about 150mg/kg body weight. Preferably, the dosage range is from about 20mg/kg body weight to about 70mg/kg body weight. More preferably, the dose is about 60 mg/kg. However, when the compound of the present invention is administered by the Intramuscular (IM) route, the dose is preferably administered twice, and the second dose is administered about 24 to about 48 hours after the first dose.

[0064] For swine, the compounds of the invention are typically administered in a dosage range of about 10mg/kg body weight to about 150mg/kg body weight. Preferably, the dosage range is about 20mg/kg body weight to 70mg/kg body weight. According to the invention, the prodrugs are preferably administered to pigs via their drinking water.

[0065] For avians, the compounds of the invention are typically administered in a dosage range of about 10mg/kg body weight to 150mg/kg body weight. When administered orally, the prodrug will be administered daily in drinking water for a period of time according to clinical instructions, e.g., from about three days to about seven days. In all of the above cases, the prodrug in the drinking water can be administered in a "bucket (bulk tank)" or "dispenser (proposioner)". The final concentration is typically in the range of 50 mg/gallon to 700 mg/gallon. Preferably, the concentration is in the range of about to 100 mg/gallon to 600 mg/gallon. More preferably, the final concentration is about 500 mg/gallon. Administration of such prodrugs in drinking water may last from 1 to 10 days. More preferably, administration may be continued for 5 days, or until symptoms of respiratory disease are controlled.

Administration to aquatic animals

[0066] One embodiment of the invention includes a method of eliminating, reducing, or preventing bacterial infection in a fish or aquatic invertebrate. The method comprises administering to an aquatic animal in need thereof an effective amount of a compound of the present invention. In aspects of this embodiment, administration can be by administering an effective amount of the subject compound to the animal or by submerging the animal or population of animals in a solution containing an effective amount of the active compound. It is further understood that the compounds of the invention can be administered by applying a drug to the pond or water holding area of the animal, allowing the animal to absorb the compound through their gills, or to take a dose of the compound of the invention. An alternative method of administering the compounds of the invention for individual treatment of a particular animal, such as a particular fish (e.g., in a veterinarian or aquarium), is by direct injection of the compounds of the invention alone or in combination with other agents, or by injection of osmotic delivery devices containing them.

[0067] The dosage of the compounds of the invention effective to reduce, eliminate or prevent bacterial infection in fish or other aquatic species can be determined routinely by veterinarians by the parameters and methods discussed above for other animals, although the dosage will depend on the species of fish being treated, the particular microorganism involved and the extent of the infection. For indications of aquatic organisms, the compounds of the invention may generally be administered in a dose of from about 1mg/kg to about 70mg/kg, preferably from about 10mg/kg to about 30 mg/kg. Suitable routes of administration include: intravenously, subcutaneously, intramuscularly and/or by spraying or instillation as required, and/or by adding the compound directly to a body of water.

[0068] For oral administration, the compounds of the present invention may be administered at the dosages described above for about 10 to about 15 days.

[0069] Although the active ingredient may be administered separately from the food, it is contemplated that in a preferred aspect the active agent may be incorporated into the fish feed. Medicated fish feed can be prepared by mixing appropriate amounts of the compounds of the present invention with commercially available fish feed products to achieve the desired dosage levels. The amount of the compound of the invention mixed with the fish feed will depend on the rate at which the fish are fed. For fish fed at a rate of about 0.2% to 4% biomass/day, the medicated feed is preferably present in an amount of about 50 to about 10000mg/kg feed, more preferably about 100 to about 2000mg/kg feed.

[0070] Although the compound of the present invention may be mixed into the feed mixture prior to pelleting, the medicated feed is preferably formed by coating feed pellets with the compound of the present invention.

[0071] Any fish species, including both freshwater and saltwater species, as well as the invertebrate aquatic species described above, can be treated or prevented from bacterial infection by the compounds of the invention.

In combination with other agents or therapeutic modalities

[0072] It is further contemplated that the compounds of the invention are administered in combination with other useful agents known in the art, either simultaneously or sequentially (e.g., in the same composition or in separate compositions). Such agents include, for example, other antimicrobial agents, such as antibiotics, antifungal agents, antiviral agents, ectoparasiticides, endoparasiticides, and the like, as well as nutritional supplements, feed additives, and the like. For example, it is contemplated that any standard (non-prodrug) cholestyrol known in the art, such as florfenicol, chloramphenicol, or thiamphenicol, may be administered in combination with the compounds of the present invention. Methods for producing these antibiotic compounds, as well as intermediates useful in such methods, are described in U.S. Pat. Nos. 4,311,857, 4,582,918, 4,973,750, 4,876,352, 5,227,494, 4,743,700, 5,567,844, 5,105,009, 5,382,673, 5,352,832, and 5,663,361, which are incorporated herein by reference. Other florfenicol analogs and/or prodrugs have been disclosed and such analogs are also useful in the compositions and methods of the present invention (see, e.g., U.S. patent 7,041,670 and U.S. patent 7,153,842, both of which are incorporated herein by reference in their entirety). When the antibiotic compound is florfenicol, the concentration of the florfenicol is generally from about 10% to about 50%, with preferred levels between about 20% and about 40%, and more preferably at least about 30% (in these cases, w/w for solid compositions and w/v for liquid compositions).

[0073] Another useful antibiotic compound that may be used in combination with the compounds of the present invention is tilmicosin (tilmicosin). Tilmicosin is a macrolide antibiotic of the formula 20-dihydro-20-deoxy-20- (cis-3, 5-dimethylpiperidin-1-yl) -tylosin (desmycosin), which is disclosed in U.S. Pat. No. 4,820,695 (incorporated herein by reference). Also disclosed in U.S. Pat. No. 4,820,695 is an injectable aqueous formulation comprising 50% (by volume) propylene glycol, 4% (by volume) benzyl alcohol and 50-500mg/ml of active ingredient. Tilmicosin may be present as a base or as a phosphate. Tilmicosin has been found to be useful in the treatment of respiratory infections, particularly haemolytic pasteurella infection in cattle, when administered by injection over a treatment period of more than 4 days. Accordingly, tilmicosin may be used to treat, for example, neonatal calf pneumonia and bovine respiratory disease. When tilmicosin is present, it is present in an amount of about 1% to about 50% (w/v), preferably 10% to about 50%, and in particular embodiments 30%.

[0074] Another useful antibiotic compound that may be used in combination with the compounds of the present invention is tolnafcin (tulathromycin). The tolalamectin may be prepared according to the method shown in U.S. patent 6,825,327, which is incorporated herein by reference in its entirety. The tolacin may be present in the injectable dosage form at a concentration level of about 5.0% to about 70% (by weight). Tulathromycin is most suitably administered in single or multiple doses (i.e., from 1 to 4 doses per day) in a dosage range of about 0.2mg/kg body weight per day (mg/kg/day) to about 200 mg/kg/day, and preferably at a dose of 1.25, 2.5 or 5mg/kg once or twice a week, although variations will be required depending on the species, weight and condition of the subject to be treated. The tolacin may be present in the injectable dosage form at a concentration level of about 5.0% to about 70% (by weight, w/v).

Another useful antibiotic that can be used in combination with the compounds of the present invention are fluoroquinolone antibiotics, for example, enrofloxacin, danofloxacin, difloxacin, orbifloxacin and marbofloxacin. Enrofloxacin may be administered at a concentration of about 100 mg/ml. Danofloxacin may be administered at a concentration of about 180 mg/ml.

[0075] Other useful macrolide antibiotics with which the compounds of the present invention may be combined include compounds from ketolides, more particularly from azalides. Such compounds are described, for example, in U.S. Pat. Nos. 6,514,945, 6,472,371, 6,270,768, 6,437,151 and 6,271,255, U.S. Pat. Nos. 6,239,112 and 5,958,888, and U.S. Pat. Nos. 6,339,063 and 6,054,434, the entire contents of which are incorporated herein by reference.

[0076] Other useful antibiotics that may be used in combination with the compounds of the present invention include the tetracyclines, particularly chlorotetracycline and oxytetracycline.

[0077] Other antibiotics include beta-lactams such as one of the penicillins, e.g., penicillin G, penicillin K, ampicillin, amoxicillin, or amoxicillin in combination with clavulanic acid or other beta-lactamase inhibitors. Other specific beta-lactams include cephalosporins, e.g., ceftiofur, cefquinome, and the like.

[0078] In addition, the present invention optionally includes compositions for treating microbial or parasitic infections in animals comprising one or more of the above-listed antibiotics in admixture and/or association with one or more compounds of the present invention, and optionally carriers and/or excipients.

[0079] For all of the methods and compounds of the invention described herein, it is also contemplated that the identified compounds will be used in simple combination with one or more agents known in the art to kill or control various types of parasites, including, for example, all of the ectoparasites and endoparasites described herein. Thus, while the compounds and methods of the present invention are superior to known agents and methods employing known agents, in certain alternative embodiments they are contemplated for use in combination with other known agents or combinations of such known agents (e.g., in the same composition or in separate compositions) for killing or controlling various types of pests, either simultaneously or sequentially.

[0080] Such other agents for use in combination with the compounds of the present invention include anthelmintics known in the art, for example, avermectins (e.g., ivermectin, moxidectin, miticidin), benzimidazoles (e.g., albendazole, trichloroimidazole), anilines of salicylic acid (closantel, hydroxychlorosalicylanilide), substituted phenols (e.g., nitrolofenamide), pyrimidines (e.g., pyrantel), isothiazoles (e.g., levo-thiadiazol), and praziquantel.

[0081] Other agents known in the art that may be used in combination with the compounds of the present invention to kill or control pests include organophosphate insecticides. Such insecticides have a very broad range of activity and, as insecticides, and in particular instances, anthelmintic activity. Organophosphate insecticides include, for example, butylphosphonium, terbufos, dimethoate, diazinon, disulfoton, trichlorfon, baprofos, chlorpyrifos, malathion, methyl oxodemeton, methamidophos, acephate, ethyl parathion, methyl parathion, methamidophos, trithione, vozaphos, a few examples of such compounds. It is also contemplated to include combinations of the present methods and compounds with carbamate-type insecticides, including, for example, carbaryl, carbarfuran, aldicarb, methomyl, and the like, as well as combinations with organochlorine-type insecticides. It is further contemplated to include combinations with biopesticides such as anthelmintics, pyrethrins (and synthetic variants thereof, e.g., allethrin, resmethrin, permethrin, tetrabromthrin), and nicotine commonly used as acaricides. Other contemplated combinations of various insecticides include: bacillus thuringiensis insecticides, acetochlor, formamidine (e.g., altitaz), copper compounds such as copper hydroxide, cupric oxychloride sulfate, cyclotron, cypermethrin, miticide, endosulfan, esenfenvalelate, fenvalerate, lambda cyhalothrin, metolachlor and sulfur.

[0082] Furthermore, for all of the methods and novel compounds described herein, it is further contemplated that the identified compounds may be used in combination with potentiators, such as Piperonyl Butoxide (PBO) and triphenyl phosphate (TPP); and/or in combination with Insect Growth Regulators (IGRs) and Juvenile Hormone Analogs (JHAs) such as florfenicol, cyromazine, metoprolol, and the like, to provide both initial and sustained control of parasites (at all stages of development of the insect, including the egg) in animal subjects and in the environment of the animal subjects.

[0083] Also contemplated in this combination are combinations of cyclic dienes, ryanodine, KT-199 and/or older art-known anthelmintics, e.g., avermectins (e.g., ivermectin, moxidectin, miticides), benzimidazoles (e.g., albendazole, trichloroimidazole), anilines of salicylic acid (closantel, hydroxychlorosalicylanilide), substituted phenols (e.g., nitrolofenamide), pyrimidines (e.g., pyrantel), isothiazoles of imidazoles (e.g., levo-benzisoxazole), praziquantel, and partial organophosphates such as anthelminthin and pyrazofos.

[0084] In particular, other antiparasitic compounds which may be used within the scope of the present invention preferably consist of avermectins. As mentioned above, the avermectins are a series of highly potential antiparasitic agents known to cope with the spectrum of endoparasites and ectoparasites in mammals.

[0085]A preferred compound for use in combination with a compound of the invention within the scope of the invention is ivermectin. Ivermectin is a semi-synthetic derivative of avermectin, and is usually produced as at least 80% of 22, 23-dihydroavermectin B1_aAnd less than 20% of 22, 23-dihydroavermectin B1_bMixtures of ivermectin, and is disclosed in U.S. patent 4,199,569, which is incorporated herein by reference. Since the mid-80's of 20 agents, ivermectin has been used as an antiparasitic agent to treat a variety of animal parasites and parasitic diseases.

[0086]Abamectin is an avermectin disclosed in U.S. Pat. No. 4,310,519 (incorporated herein by reference in its entirety) as avermectin B1_a/B1_b. Abamectin contains at least 80% of avermectin B1_aAnd no more than 20% of avermectin B1_b。

[0087] Another preferred avermectin is doramectin, also known as 25-cyclohexyl-avermectin B1. The structure and preparation of doramectin is disclosed in U.S. patent 5,089,480, which is incorporated herein by reference in its entirety.

[0088] Another preferred avermectin is moxidectin. Moxidectin, also known as LL-F28249alpha, is reported in U.S. Pat. No. 4,916,154 (incorporated herein by reference in its entirety).

[0089] Another preferred avermectin is selamectin. Selamectin is 25-cyclohexyl-25-de- (1-methylpropyl) -5-deoxy-22, 23-dihydro-5- (hydroxyimino) -avermectin B1 monosaccharide.

[0090] Miticidin or B41 is isolated from a fermentation broth of a miticidin-producing strain of streptomyces. The microorganisms, fermentation conditions, and isolation procedures are more fully described in U.S. Pat. Nos. 3,950,360 and 3,984,564.

[0091]Reference is made to U.S. Pat. Nos. 5,288,710 and 5,399,717 for the preparation of emamectin (4 "-deoxy-4" -epi-methylaminoavermectin B1), the two analogs 4 "-deoxy-4" -epi-methylaminoavermectin B1_aAnd 4 "-deoxy-4" -epi-methylaminoavermectin B1_bA mixture of (a). Preferably, salts of emamectin may be used. Non-limiting examples of salts of emamectin that may be used in the present invention include salts described in U.S. patent 5,288,710, for example, salts derived from benzoic acid, substituted benzoic acids, benzenesulfonic acid, citric acid, phosphoric acid, tartaric acid, maleic acid, and the like. More preferably, the emamectin salt used in the present invention is emamectin benzoate.

[0092] The chemical name of eprinomectin is known as 4 "-epi-acetylamino-4" -deoxy-avermectin B1. Eprinomectin was developed specifically for all classes and age groups of cattle. It is the first one that has a broad spectrum of activity against endo-and ectoparasites, while forming minimal residual avermectins in meat and milk. It has another advantage of high efficacy in topical delivery.

[0093] The compositions of the invention optionally comprise a combination of one or more of the following antiparasitic compounds (parasiticides):

U.S. patent application publication No. 2005/0182059 (incorporated herein by reference) discloses antiparasitic imidazo [1, 2-b ] pyridazine compounds.

U.S. patent application publication 2005/0182139, incorporated herein by reference, discloses antiparasitic 1- (4-mono-and di-halomethylsulfonylphenyl) -2-amido-3-fluoropropanol compounds.

U.S. patent application publication No. 2006/0063841 (incorporated herein by reference) discloses antiparasitic trifluoromethanesulfonanilide oxime ether derivative compounds.

U.S. patent application publication No. 2006/0128779, incorporated herein by reference, discloses antiparasitic phenyl-3- (1H-pyrrol-2-yl) acrylonitrile compounds.

Antiparasitic N- [ (phenoxy) phenyl ] -1, 1, 1-trifluoromethane sulfonamide and N- [ (phenylsulfanyl) phenyl ] -1, 1, 1-trifluoromethane sulfonamide derivatives disclosed in U.S. patent application publication 2006/0281695, which is incorporated herein by reference.

U.S. patent application publication No. 2007/0238700 (incorporated herein by reference) discloses antiparasitic N-phenyl-1, 1, 1-trifluoromethanesulfonamide hydrazone compounds.

[0094] The compositions of the invention may also be used in combination with flukicides. Suitable flukicides include, for example, trichloroimidazole, fenbendazole, albendazole, clorsulon and oxibendazole. It will be appreciated that the above combinations may further comprise combinations of antibiotics, antiparasitic and flukicide active compounds.

[0095] In addition to the combinations described above, it is also contemplated to provide combinations of the methods and compounds of the invention described herein with other animal health supplements including, for example, trace elements, anti-inflammatory agents, anti-infective agents, hormones, dermatological agents (including microbicides and disinfectants), and immunobiologic agents, such as vaccines and antisera, to provide disease prevention.

[0096] For example, such anti-infective agents include one or more antibiotics, which may optionally be co-administered (e.g., in a combined composition and/or in separate dosage forms) during treatment with the compounds or methods of the present invention. Antibiotics known in the art to be suitable for this purpose include, for example, the antibiotics listed above.

[0097] In addition, it is also contemplated that the methods and compounds of the present invention can be administered in combination with animal health agents known in the art (e.g., trace elements, vitamins, anti-inflammatory agents, anti-infective agents, etc.) in the same or different compositions, either simultaneously or sequentially.

[0098] Suitable anti-inflammatory agents include, for example, both steroidal and non-steroidal anti-inflammatory agents. Nonsteroidal anti-inflammatory drugs, including racemic mixtures or individual enantiomers thereof where applicable, may include ibuprofen, flurbiprofen, ketoprofen, alfrofen (aclofenac), diclofenac, aloprin, aproxen, aspirin, diflunisal, fenoprofen, indomethacin, mefenamic acid, naproxen, phenylbutazone, piroxicam, salicylamide, salicylic acid, sulindac, deoxysulindac, tenoxicam, tramadol, ketoralac, flufenamic acid (flufenisal), salsate, triethanolamine salicylate, aminopyrine, antipyrine, oxyphenbutazone, azapropazone, cinnazolone (cinazone), flufenamic acid, loniceramide, meclofenamic acid, flunixin, colchicine, demeclovir, oxypurinol, oxyphenoxide, benzydamine, difon, indomethamine hydrochloride, indomethacin hydrochloride, Ranitidine hydrochloride, tetrahydroindamide, phenindidine hydrochloride, fluprofen, ibufenac, methylnaproxol (naproxol), fenbufen, cinchofine, diflufenican sodium, phentriazamide (fenamole), fluorodiazine (fludizin), metozamide (metazamide), letimid hydrochloride, nesiridine hydrochloride, otamide, miconazole, neocinchophene, nimazole, pramozolole citrate, oxoquinamide, benzylisoquine (tesimide), tolmetin, and triflumilast.

[0099] In a specific embodiment, the compounds of the present invention are used in combination with flunixin [ see, for example, U.S. patent 6,790,867B2, which is incorporated herein by reference in its entirety ]. In a related embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention and flunixin.

[0100] Steroidal anti-inflammatory agents include, for example, glucocorticosteroids such as dexamethasone, cortisone, hydrocortisone, prednisone, beclomethasone, betamethasone, flunisolide, methylprednisolone, paramethasone, prednisolone, triamcinolone, alclomethasone, amcinonide, clobetasol, fludrocortisone, diflorasone diacetate, fluocinolone, fluorometholone, fludrolone, halcinolone, medroxypsone, mometasone, and pharmaceutically acceptable salts and mixtures thereof.

Package (I)

[0101] If desired, the compositions may be presented in a pack, sachet or dispenser device, for example in an FDA approved kit which may contain one or more unit dosage forms containing the active ingredient. The package may comprise, for example, a metal or plastic foil film, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The package or dispenser may also carry a notice attached to the container in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, the contents of which reflect approval by the agency of the dosage form of the composition or for human or veterinary use. Such notice may be, for example, a label for a prescription drug approved by the U.S. food and drug administration, or an approved product insert. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for treatment of the indication. In an optional embodiment, the package comprises a glass or plastic vial or other container containing multiple doses.

[0102] The following shows preparation examples of the compounds of the present invention. However, they are only for illustrating the present invention and do not limit it.

Synthesis of water-soluble prodrugs

[0103] In the following schemes, "FFC-OH" and "FFC-O" symbols are used for the florfenicol and oxygen-linked florfenicol moieties, respectively:

[0104] florfenicol carbonates and florfenicol esters bearing a nitrogen-based, positively charged nitrogen functionality can be prepared conventionally by reaction of florfenicol with a suitable activated carboxylic or alkoxycarboxylic carbonic acid reagent. These agents can be prepared by well-known methods described in the literature (e.g., "March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", 5 th edition, Michael B. Smith, JerryMarch, 1/15/2001; Wiley-Interscience).

[0105] One strategy for the preparation of florfenicol carbonates and florfenicol esters having a nitrogen-based, positively charged nitrogen functionality employs appropriately protected amino functionality as shown in scheme 1 below (a large selection of protecting Groups PG for protecting amino Groups and methods of using them are described in "Protective Groups in Organic Synthesis", Theodora W.Greene, Peter G.M.Wuts; Wiley-Interscience, 5.15.1999).

Scheme 1

[0106] Florfenicol is reacted with an activated carboxylic acid reagent (e.g., an acid chloride, an acyl imidazole, a carboxylic hydroxysuccinimide ester, a carboxylic pentafluorophenol ester, a carboxylic carbodiimide adduct, etc.) or an activated alkoxycarbonic acid reagent (a chloroformate having a non-chlorine leaving group or an alternative carbonate reagent-see above) wherein the group Q is nucleophilically displaced. The most common reagent of this type uses Q ═ chlorine. The reaction may be promoted by the addition of a catalyst such as a trialkylamine, pyridine, 4-alkylpyridine, 4-diaminoalkylpyridine, or combinations thereof. Alternatively, the carboxylic acid may be activated in situ by adding a suitable activating reagent to the reaction mixture containing florfenicol and the carboxylic acid. The formation of the initial ester or carbonate intermediate may be conveniently carried out in a variety of solvents. Suitable solvents include, for example, chlorinated solvents such as dichloromethane and 1, 2-dichloroethane; ester solvents such as ethyl acetate, isopropyl acetate, isoamyl acetate, ethylene glycol diacetate, propylene glycol diacetate, glycerol triacetate; monoether solvents such as diethyl ether, diisopropyl ether, methyl tert-butyl ether; polyether solvents such as glycol ethers, dimethyl glycol ethers, diethylene glycol dimethyl ether, diethylene glycol diethyl ether; formaldehyde acetal ethers such as dimethoxymethane, diethoxymethane, dibutoxymethane; cyclic ethers such as tetrahydrofuran, 1, 3-dioxolane, 1, 4-dioxane; ketone solvents such as acetone, methyl ethyl ketone, methyl isobutyl ketone; mixed ether/ester solvents represented by monoethers of ethylene glycol and diethylene glycol, such as 2-methoxyethyl acetate, 2-ethoxyethyl acetate, 2- (methoxy-ethoxy) ethyl acetate, 2- (ethoxy-ethoxy) ethyl acetate. The examples provided below illustrate the use of tetrahydrofuran as a solvent.

[0107] Typically, the reaction is carried out by adding a solution of tetrahydrofuran containing 1.5 to 2.0 equivalents of the corresponding chloroformate or other reactive alkoxycarbonic acid derivative to a solution of tetrahydrofuran containing cholestrol, 1.0 equivalents triethylamine and 0.5 equivalents 4-N, N-dimethylaminopyridine at 0 deg.C and allowing the reaction to proceed to completion.

[0108] After formation of the terminal amine-protected florfenicol ester or carbonate, the amine functionality is deprotected by standard deprotection methods, depending on the characteristics of the protecting group. For acid sensitive protecting groups, when deprotection is carried out using a suitable acid, the deprotection can directly form the desired salt form (e.g., hydrochloride). When the deprotection process yields a free amine (e.g. hydrogenolysis to remove the benzyloxycarbonyl group), the desired salt form can be prepared by adding an acid to the reaction mixture or by converting the amine to a suitable salt in a separate step.

[0109] Representative preparation examples of water-soluble florfenicol amine hydrochloride prodrugs of the present invention represent the preparation of the compound of example 1.

[0110]An alternative strategy for making florfenicol carbonate and florfenicol ester prodrugs with a positively charged functional group that is terminally nitrogen-based may involve a nitrogen-containing "masked amine" functional group that is converted to the desired amine in a subsequent stage. Examples of typical nitrogen-containing functional groups (and corresponding methods of conversion to amines) for this purpose are: nitro (reduction), azido (reduction), nitrile (reduction to CH)₂NH₂) And primary amides (Curtius rearrangement).

Scheme 2

[0111] Scheme 2A below shows a method for the preparation of the compound of example 3 using a masked nitrogen functionality that is convenient for this purpose.

Scheme 2A

[0112] Another convenient strategy for making the prodrugs of the present invention involves replacing the leaving group LG attached to the carbonate or ester moiety of florfenicol. This method is particularly convenient for use with prodrug moieties containing quaternary ammonium nitrogens, but it can also be used to introduce other amino functional groups.

Scheme 3

[0113] Scheme 3A below shows the use of such a strategy in the preparation of the prodrugs of examples 15 and 16, where the desired corresponding quaternary ammonium bromide can be obtained directly in the alkylation step.

Scheme 3A

[0114] A convenient method for making amino acid ester prodrugs of florfenicol may employ a combination of the methods of schemes 2A and 3A, shown below, to prepare the compound of example 17.

Scheme 4

[0115] At least two general strategies can be employed for making florfenicol ester prodrugs containing a basic or charged quaternary nitrogen that is part of the alpha-amino acid not directly attached to florfenicol. In one strategy, the terminal amino acid is incorporated before the prodrug moiety is attached to florfenicol, and in another strategy, the attachment of the terminal amino acid can be performed after the first fragment of the prodrug moiety is attached to florfenicol.

[0116] Two strategies that may be conveniently used to prepare such prodrugs are illustrated in scheme 5, which describes the preparation of the compound of example 4.

Scheme 5

Prodrug stability and florfenicol release

Scheme 7

[0117] Non-enzymatic release of florfenicol can occur when there is a possibility of intramolecular displacement of florfenicol through the terminal amine functional group (scheme 7, scheme a). The rate of such processes is highly dependent on the pH of the substrate and solution. To perform this displacement, the terminal amine needs to be a primary or secondary amine and the pH needs to be high enough to allow the unprotonated amine to be present in a meaningful concentration in the unprotonated form. Furthermore, the sum of x + m + l + a needs to be 3 or 4 to allow easy formation of a 5 or 6-membered ring upon cyclization. Substitution of the terminal amine can also significantly affect the rate of release of florfenicol mediated by such cyclization. Acylation of amines completely prevents such cyclization, while alkyl substitution can significantly slow cyclization compared to unsubstituted primary amines. The compounds of examples 1 to 3 and 5 to 14 according to the invention are capable of undergoing cyclisation according to the criteria described above, whereas the compounds of examples 4 and 15 to 17 according to the invention are not capable of undergoing this procedure. The ability to undergo cyclization and release florfenicol greatly determines prodrug stability when the pH of the aqueous solution is adjusted from the original pH of 4.5 (the pH observed for most crude aqueous solutions of the hydrochloride salt of the prodrug) to near physiological pH of 7.4. In many cases, the rate of such cyclization is not fast enough to affect the stability of the prodrug solution in an effective manner (see table 2), but in some cases, cyclization is quite rapid. Half-lives in the range of minutes to 3 hours at pH7.4 were observed for the aqueous solutions of examples 1-2, 7-9, 11 and 14, which could be largely explained by cyclization mediated release of florfenicol.

[0118] When the prodrug solution is orally taken to reach the intestinal tract, the release of florfenicol or a florfenicol analog induced by a pH change is completely acceptable from the point of view of oral bioavailability of florfenicol, since it is well known that florfenicol itself is well absorbed when orally taken. Depending on the release rate of florfenicol after adjustment of the ingested prodrug solution in the intestinal pH, the release and absorption of free florfenicol can result in different bioavailability of florfenicol from different soluble prodrugs. For more stable prodrugs, greater oral bioavailability of florfenicol can be obtained based on rapid systemic release of the intact prodrug after oral administration through an enzymatically mediated process.

[0119] By initial nucleophilic attack of the terminal primary or secondary amine linking the prodrug moiety to the carbonyl function of florfenicol (tertiary and quaternary amine groups cannot participate in such conversion), a competitive process induced by the prodrug solution pH change that is only possible for carbonate prodrugs (a ═ oxygen) can also occur. For carbonates, the formation of rearranged non-cyclic carbamates (scheme 7, scheme B; florfenicol is not released) can compete with the formation of cyclic carbamates (scheme 7, scheme A; florfenicol is released). In the in vitro evaluation of the prodrug, formation of appreciable amounts of rearranged prodrug isomers was observed for examples 1,2 and 7 (see table 2). The carbonate prodrug of example 6 did not show formation of any rearrangement isomers, whereas the rearrangement of the carbonate prodrug of example 5 was slow enough that it produced only a small amount of rearrangement product in the ph7.4 adjusted solution, but no rearrangement was observed in bovine serum.

General procedure (general procedure) I

Preparation of florfenicol carbonate

R ═ Boc protected terminal aminoalkyl groups

[0120] A solution of the starting alcohol A (1.78 molar equivalents) and triethylamine (1.78 molar equivalents) in anhydrous tetrahydrofuran (0.68M) was added dropwise to a solution of triphosgene (0.64 molar equivalents) in anhydrous tetrahydrofuran (0.48M) at 0 ℃ under a nitrogen atmosphere. The resulting mixture was stirred at 0 ℃ for 30 minutes and then rapidly filtered through filter paper to remove ammonium salts. The chloroformate solution B filtrate was used in the next carbonation reaction without further purification.

[0121]A freshly prepared solution of chloroformate B or a commercially available solution of chloroformate B (1.78 molar equivalents, 0.34M) in anhydrous tetrahydrofuran was placed in a dropping funnel and a solution of 2/3 was added dropwise to a solution of florfenicol C (1 molar equivalent, 0.64M), 4-N, N-dimethylaminopyridine (DMAP, 0.5 molar equivalent) and triethylamine (1.5 molar equivalents) in anhydrous tetrahydrofuran at 0 ℃ under a nitrogen atmosphere. The mixture was stirred at 0 ℃ for 30 minutes and the reaction progress was monitored by thin layer chromatography. (if the thin layer chromatography indicates that the reaction is not complete, an additional amount of chloroformate solution is added). The resulting solution was rapidly filtered through filter paper to remove ammonium salts. The filtrate was concentrated and ethyl acetate was added to dissolve the crude product. The resulting solution was successively treated with 1M aqueous HCl and saturated NaHCO₃Washing with saturated aqueous NaCl solution, passing through silica gel and Na₂SO₄The pad filters quickly. The filtrate is concentrated and the resulting crude product is purified by flash column chromatography or recrystallization to afford the desired protected carbonate D.

General procedure II

Preparation of florfenicol ester

R ═ Boc protected terminal aminoalkyl groups

[0122]A solution of N-Boc protected terminal aminoalkylcarboxylic acid E (1.1 molar equivalent), florfenicol C (1.0 molar equivalent), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (2.0 equivalents), and 4-N, N-dimethylaminopyridine (0.5 molar equivalent) was mixed and stirred in dry N, N-dimethylformamide (0.24M) at room temperature under a nitrogen atmosphere overnight. The resulting solution was diluted with ethyl acetate and successively with 1M aqueous HCl, saturated NaHCO₃Washing with saturated aqueous NaCl solution, passing through silica gel and Na₂SO₄The pad filters quickly.The filtrate was concentrated and the crude product was purified by flash column chromatography to afford the desired ester D.

General procedure III

Deprotection of Boc protected intermediates

[0123] A solution of carbonate D (or ester F) in anhydrous tetrahydrofuran (0.14M) was saturated with gaseous HCl at 0 ℃. The resulting solution was stirred at room temperature for 30 minutes to 1 hour and purged with nitrogen to remove excess HCl. The solution was concentrated to dryness and the resulting solid material was dried under vacuum to afford the desired amine hydrochloride product G.

General procedure IV

[0124]A solution of G (1.0 equiv.) and triethylamine (1.0 equiv.) in anhydrous N, N-dimethylformamide (0.38M) was stirred at room temperature under a nitrogen atmosphere for 15 minutes. Boc protected amino acid H (1.03 equiv.), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDAC, 2.0 equiv.) and 1-hydroxybenzotriazole (HOBt, 0.5 equiv.) were added sequentially at room temperature. The final solution was stirred under nitrogen at room temperature overnight and diluted with ethyl acetate. The resulting solution was successively treated with 1M aqueous HCl and saturated NaHCO₃Washing with saturated aqueous NaCl solution, passing through silica gel and Na₂SO₄The pad filters quickly. The filtrate was concentrated and the crude product was purified by flash column chromatography to afford the desired pure product J.

[0125] A solution of J in anhydrous tetrahydrofuran (0.14M) was saturated with gaseous HCl at 0 deg.C. The resulting solution was stirred at room temperature for 30 minutes to 1 hour and purged with nitrogen to remove excess HCl. The solution was concentrated to dryness and the resulting solid material was dried under vacuum to obtain the desired amine hydrochloride product K.

Examples

[0126] The following are representative examples of the preparation of the compounds of the present invention.

Example 1

2-Aminoethylcarbonic acid (1R, 2S) -2- (2, 2-dichloroacetamido) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0127]The title hydrochloride salt was obtained by general procedures I and III from 20g of florfenicol and purified by stirring the solid overnight in a mixture of ethyl acetate and hexane to give 28g (quantitative yield) of a white powder. H¹-NMR(DMSO-d₆)，δ＝3.1ppm(t，2H)，3.2ppm(s，3H)，4.3ppm(m，2H)，4.4-4.7(m，3H)，5.9ppm(d，1H)，6.6ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.2ppm(br，3H)，9.2(d，1H)。

Example 2

2- (methylamino) ethylcarbonic acid (1R, 2S) -2- (2, 2-dichloroacetylamino) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0128]The title hydrochloride salt was obtained by general procedures I and III from 20g of florfenicol without further purification to give 25g (91% yield) of a white powder. H¹-NMR(DMSO-d₆)，δ＝2.5ppm(s，3H)，3.2ppm(s，5H)，4.3ppm(m，2H)，4.3-4.7(m，5H)，5.9ppm(d，1H)，6.6ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，9.1ppm(br，2H)，9.3(d，1H)。

Example 3

4-Aminobutanoic acid (1R, 2S) -2- (2, 2-dichloroacetamido) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0129]The title hydrochloride salt was obtained by general procedures II and III from 9g of florfenicol and dried at 65 ℃ under reduced pressure for 2 days to yield 7g (56% yield) of a white powder. H¹-NMR(DMSO-d₆)，δ＝1.8ppm(p，2H)，2.5ppm(t，2H)，2.8ppm(t，2H)，3.2ppm(s，3H)，4.3-4.7(m，3H)，6.0ppm(d，1H)，6.6ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.1ppm(br，3H)，9.2(d，1H)。

Example 4

4- (2-amino-propionylamino) butanoic acid (1R, 2S) -2- (2, 2-dichloroacetylamino) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0130]A solution of triethylamine (3.1mL, 1.1 equiv.) and methyl 4-aminobutyrate hydrochloride (3.2g, 1.05 equiv.) in 25mL of anhydrous N, N-dimethylformamide was stirred at-10 ℃ under a nitrogen atmosphere for 10 minutes. Boc-L-alanine (3.8g, 1.0 eq), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (4.2g, 1.1 eq), 1-hydroxybenzotriazole (1.8g, 0.6 eq) and 15mL of dry N, N-dimethylformamide were added sequentially at the same temperature. The final solution was stirred under nitrogen at room temperature overnight and with ethyl acetateAnd (6) diluting. The resulting solution was successively treated with 1M aqueous HCl and saturated NaHCO₃Washing with saturated aqueous NaCl solution, passing through silica gel and Na₂SO₄The pad filters quickly. The filtrate was concentrated and dried under reduced pressure to give a crude paste (4.8 g). The paste was stirred at room temperature for 1 hour in a mixture of 50mL tetrahydrofuran, 25mL methanol and 20mL water in the presence of LiOH (0.45g, 1.1 equiv.). The solution was acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The combined organic extracts were washed with saturated aqueous NaCl solution, passed through silica gel and Na₂SO₄The pad filters quickly. The filtrate was concentrated and dried under reduced pressure to give 4- (2-tert-butoxycarbonylamino-propionylamino) butyric acid (4.0g, 72% yield) as a paste; h¹-NMR(DMSO-d₆)，δ＝1.1ppm(d，3H)，1.3ppm(s，9H)，1.6ppm(p，2H)，2.2ppm(t，2H)，3.0ppm(m，2H)，3.8ppm(m，1H)，6.8ppm(d，1H)，7.8ppm(t，1H)。

[0131]This paste was used as starting acid a for general procedure II to give product D as a white solid (6.2g, 71% yield); h¹-NMR(DMSO-d₆)，δ＝1.1ppm(d，3H)，1.3ppm(s，9H)，1.6ppm(p，2H)，2.4ppm(t，2H)，3.0ppm(m，2H)，3.2ppm(s，3H)，3.8ppm(m，1H)，4.2-4.6ppm(m，3H)，6.0ppm(d，1H)，6.4ppm(s，1H)，6.9ppm(d，1H)，7.6ppm(d，2H)，7.8ppm(t，1H)，7.9ppm(d，2H)，8.9ppm(d，1H)。

[0132]Deprotection of the solid by general procedure III gave the prodrug as free amino after purification by gel column chromatography as a white foam (2.4g, 43% yield). H¹-NMR(DMSO-d₆) δ is 1.3ppm (d, 3H), 1.7ppm (p, 2H), 2.4ppm (t, 2H), 3.1-3.2ppm (m, 7H), 3.7ppm (q, 1H), 4.3-4.6(m, 3H), 6.0ppm (d, 1H), 6.7ppm (s, 1H), 7.6ppm (d, 2H), 7.9ppm (d, 2H), 8.5ppm (t, 1H), 9.2(d, 1H). The hydrochloride salt was prepared as described in general procedure III.

Example 5

3-Aminopropylcarbonic acid (1R, 2S) -2- (2, 2-dichloroacetamido) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0133]The title hydrochloride was obtained by general procedures I and III from 28g of florfenicol and purified by stirring the crude solid in ether overnight to yield 33g (83% yield) of a white powder. H¹-NMR(DMSC-d₆)，δ＝1.9ppm(p，2H)，2.8ppm(s，2H)，3.2ppm(s，3H)，4.1ppm(m，2H)，4.3-4.7(m，3H)，5.9ppm(d，1H)，6.6ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.1ppm(br，3H)，9.2(d，1H)。

Example 6

3- (methylamino) propylcarbonic acid (1R, 2S) -2- (2, 2-dichloroacetylamino) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0134]The title hydrochloride was obtained by general procedures I and III from 2.8g of florfenicol and purified by stirring the crude solid in a mixture of tetrahydrofuran and ether for 2 days to yield 2.1g (63% yield) of a white powder. H¹-NMR(DMSO-d₆)，δ＝1.9ppm(p，2H)，2.9ppm(t，2H)，3.2ppm(s，3H)，4.1ppm(t，2H)，4.3-4.7(m，3H)，5.9ppm(d，1H)，6.6ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.9ppm(br，2H)，9.2(d，1H)。

Example 7

2- (ethylamino) ethylcarbonic acid (1R, 2S) -2- (2, 2-dichloroacetamido) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0135]To a stirred solution of di-tert-butyl dicarbonate (24.5g) in 50mL of tetrahydrofuran was added 2- (ethylamino) ethanol (11mL) and triethylamine (16mL) in that order at-10 ℃. The solution was stirred at room temperature for 1.5 hours, diluted with ethyl acetate and washed successively with 1M aqueous HCl and saturated aqueous NaCl. The organic layer was separated and filtered rapidly through a pad of silica gel and sodium sulfate. The filtrate was concentrated and the crude product was dried under reduced pressure to give ethyl- (2-hydroxy-ethyl) -carbamic acid tert-butyl ester as an off-white oil (21g, 99% yield); h¹-NMR(CDCl₃)，δ＝1.1ppm(t，3H)，1.4ppm(s，9H)，2.7ppm(s，1H)，3.2ppm(q，2H)，3.4ppm(t，2H)，3.7ppm(t，2H)。

[0136]This crude material (10g) was used as starting alcohol A for general procedure I to give product D as a white foam (14.7g, 97% yield); h¹-NMR(DMSO-d₆)，δ＝1.0ppm(br，3H)，1.4ppm(s，9H)，3.1ppm(q，2H)，3.2ppm(s，3H)，3.4ppm(m，2H)，4.2ppm(t，2H)，4.3-4.7(m，5H)，5.9ppm(d，1H)，6.4ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，9.0ppm(d，2H)。

[0137]The intermediate obtained above was deprotected by general procedure III and purified by stirring the crude solid in a mixture of 100mL tetrahydrofuran and 20mL ether for 1 hour, isolated by filtration and dried under reduced pressure to give the title hydrochloride salt as a white powder (11g, 84% yield). H¹-NMR(DMSO-d₆)，δ＝1.2ppm(t，3H)，2.9ppm(q，2H)，3.2ppm(s，5H)，4.3-4.7(m，5H)，5.9ppm(d，1H)，6.6ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.9ppm(br，2H)，9.2(d，1H)。

Example 8

1, 4-Diaminopentanoic acid ((1R, 2S) -2- (2, 2-dichloroacetylamino) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester dihydrochloride

[0138]Thionyl chloride (2.8mL, 1.3 equiv.) was added portionwise to a mixture of L-ornithine hydrochloride (5g, 1 equiv.) and 50mL of methanol at-10 ℃ under nitrogen. The resulting solution was stirred at room temperature for 1 hour, and the solvent was removed to give a methyl ester derivative (6.7g) as a white foam. The foam was stirred in a mixture of tetrahydrofuran and methanol (50mL/50mL) in the presence of triethylamine (12mL, 3 equivalents) at room temperature for 20 minutes, then di-tert-butyl dicarbonate (12.5g, 2 equivalents) was added to the mixture. The resulting solution was stirred at room temperature overnight and then filtered with filter paper to remove ammonium salts. The filtrate was concentrated, and the resulting solid was dissolved in ethyl acetate and washed successively with 1M aqueous HCl solution and saturated aqueous NaCl solution. The organic portion was rapidly filtered through a pad of silica gel and sodium sulfate. The filtrate was concentrated to give a crude paste which was stirred in a mixture of tetrahydrofuran (50mL) and 2N aqueous NaOH (13mL) at room temperature for 10 minutes. The solution was acidified with 1M aqueous HCl and extracted with ethyl acetate. The organic layer was separated and filtered rapidly through a pad of silica gel and sodium sulfate. The filtrate was concentrated and dried under reduced pressure to give 2, 5-bis-tert-butoxycarbonylamino-pentanoic acid (8.8g, 83% yield) as a white foam; h¹-NMR(DMSO-d₆)，δ＝1.3-1.8ppm(m，22H)，2.9ppm(q，2H)，3.9ppm(m，1H)，6.8ppm(br，1H)，7.0ppm(d，0.6H)，7.2ppm(d，0.4H)。

[0139]The protected ornithine intermediate obtained above was used as starting acid a in general procedure II to give product D as a white foam (8.7g, 71% yield); h¹-NMR(DMSO-d₆)，δ＝1.3-1.8ppm(m，22H)，2.9ppm(br，2H)，3.2ppm(s，3H)，4.0-4.7ppm(m，4H)，6.0ppm(s，1H)，6.4PPM(S，1H)，6.8ppm(br，1H)，7.3ppm(d，2H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.9ppm(d，2H)。

[0140]Deprotection of the intermediate obtained above followed by general procedure III gave a white solid without further purificationThe title hydrochloride salt as a colored solid (6.8g, 97% yield); h¹-NMR(DMSO-d₆)，δ＝1.8-2.2ppm(m，2H)，2.8ppm(br，2H)，3.2ppm(s，3H)，3.6ppm(m，2H)，4.2-4.7ppm(m，4H)，6.2ppm(s，1H)，6.9ppm(s，1H)，7.7ppm(d，2H)，7.9ppm(d，2H)，8.0ppm(br，3H)，8.8ppm(br，3H)，9.6ppm(d，1H)。

Example 9

(1R, 2S) -2- (2, 2-Dichloroacetamido) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl (1-amino-acetamido) acetate hydrochloride

[0141]Following general procedures II, III and IV, using 24g of florfenicol and 12g of n-Boc-glycine, after purification by filtration after stirring the crude solid in 200mL of warm dichloromethane for 20 minutes, the title hydrochloride salt was obtained as a white powder after drying at 50 ℃ under reduced pressure for 6 days (20g, 56% yield); (ii) a H¹-NMR(DMSO-d₆)，δ＝3.2ppm(m，3H)，3.6ppm(s，2H)，4.1ppm(d，2H)，4.3-4.7ppm(m，3H)，6.0ppm(s，1H)，6.7ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.1ppm(br，3H)，9.0ppm(t，1H)，9.2ppm(d，1H)。

Example 10

5-Aminopentanoic acid (1R, 2S) -2- (2, 2-dichloroacetamido) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0142]The title hydrochloride salt was obtained by general procedures II and III from 8.2g florfenicol and 5-tert-butoxycarbonylamino-pentanoic acid and purified by stirring the crude solid in a tetrahydrofuran mixture for 2 daysConversion gave a white powder (6.2g, 63% yield). H¹-NMR(DMSO-d₆)，δ＝1.5ppm(m，4H)，2.4ppm(m，2H)，2.7ppm(br，2H)，3.2ppm(s，3H)，4.3-4.6(m，3H)，6.0ppm(d，1H)，6.6ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(m，5H)，9.1(d，1H)。

Example 11

(1R, 2S) -2- (2, 2-Dichloroacetylamino) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl 2- (2-amino-propionylamino) propionate hydrochloride

[0143]Following general procedures II, III and IV, using 9g of florfenicol and 5.4g of boc-L-alanine, after rapid precipitation of the crude solid in a mixture of dichloromethane and methanol, isolation by filtration and purification after drying under reduced pressure gave the title hydrochloride salt as a white powder (5.2g, 58% yield); h¹-NMR(DMSO-d₆)，δ＝1.4ppm(d，6H)，3.1ppm(s，3H)，3.9ppm(q，1H)，4.3-4.7ppm(m，4H)，6.0ppm(s，1H)，6.7ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.2ppm(br，3H)，9.2ppm(d，1H)，9.3ppm(d，1H)。

Example 12

5- (methylamino) pentanoic acid (1R, 2S) -2- (2, 2-dichloroacetamido) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0144]A solution of 1-methyl-2-piperidone (5g, 1 eq.) and KOH (5.4g, 2 eq.) in ethanol (30mL) was heated to 90 ℃ overnight. After cooling the solution to room temperature, di-tert-butyl dicarbonate (19.2g, 2 equivalents) and 100mL of ethanol are added. The resulting solution was stirred at room temperatureStir for 30 minutes, then filter through filter paper to remove the solids. The filtrate was concentrated, acidified with 1M aqueous HCl and extracted with ethyl acetate. The organic extracts were combined, concentrated, and purified by gel column chromatography. Two fractions were obtained by chromatography. The more polar fraction contains the desired 5- (tert-butoxycarbonyl-methyl-amino) pentanoic acid, while the less polar fraction is hydrolyzed by aqueous NaOH to give more of the desired acid. The total yield of the acid was 8.3g (81%) as a brown paste; h¹-NMR(CDCl₃)，δ＝1.4ppm(s，9H)，1.6ppm(m，4H)，2.4ppm(t，2H)，2.8ppm(s，3H)，3.2ppm(t，3H)。

[0145]This paste (3g, 1.1 eq.) was used as starting acid A in general procedure II to give product D as a white foam (6.5g, 86% yield); h¹-NMR(DMSO-d₆)，δ＝1.3-1.5ppm(m，13H)，2.5ppm(m，2H)，2.7ppm(s，3H)，3.1-3.2ppm(m，5H)，4.2-4.6(m，3H)，6.0ppm(d，1H)，6.4ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.9ppm(d，2H)。

[0146]Deprotection of the foam by following general procedure III gave the title hydrochloride salt as a yellow foam without further purification (6.1g, quantitative yield); h¹-NMR(DMSO-d₆)，δ＝1.6ppm(br，4H)，2.8ppm(br，2H)，3.2ppm(s，3H)，3.6ppm(s，5H)，4.2-4.6(m，3H)，6.0ppm(d，1H)，6.6ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.8ppm(br，2H)，9.2ppm(d，1H)。

Example 13

(1R, 2S) -2- (2, 2-Dichloroacetamido) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl 3- (ethylamino) propyl carbonate hydrochloride

[0147]A mixture of 3-chloropropanol (4.4mL, 1 eq.) and ethylamine (15mL, 3 eq.) was added in a sealed tubeThe heat was applied for 2 days. After cooling to room temperature, solid K was added₂CO₃. After filtration of the solids, the by-products were removed by distillation at 95 ℃ under reduced pressure to give the crude, pasty desired product. The remaining paste was treated with di-tert-butyl dicarbonate (12g, 1 eq) while stirring in methanol (50mL) in the presence of triethylamine (16mL, 2 eq). The solution was stirred at room temperature for 30 minutes, concentrated, and diluted with ethyl acetate. The organic solution was washed with 1M aqueous HCl and saturated aqueous NaCl. The organic layer was separated and concentrated to give a colorless crude oil which was purified by gel column chromatography to give ethyl- (3-hydroxy-propyl) -carbamic acid tert-butyl ester (10g, 93% yield) as a colorless oil; h¹-NMR(CDCl₃)，δ＝1.1ppm(t，3H)，1.4ppm(s，9H)，1.7ppm(p，2H)，3.2ppm(q，2H)，3.4ppm(t，2H)，3.6ppm(t，2H)。

[0148]The resulting oil was used as starting alcohol a in general procedure I to give product D as a white foam (13.7g, 95% yield); h¹-NMR(DMSO-d₆)，δ＝1.0ppm(t，3H)，1.3ppm(s，9H)，1.8ppm(p，2H)，3.0-3.2ppm(m，7H)，4.0ppm(t，2H)，4.3-4.7(m，3H)，5.9ppm(d，1H)，6.4ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，9.0(d，1H)。

[0149]The title hydrochloride salt was obtained as a white foam after purification of the crude solid by gel column chromatography by deprotecting the foam following general procedure III (11.9g, 97% yield); h¹-NMR(DMSO-d₆)，δ＝1.2ppm(t，3H)，1.9ppm(m，2H)，2.9ppm(m，4H)，3.2ppm(s，3H)，4.1-4.7(m，5H)，5.9ppm(d，1H)，6.5ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.9ppm(br，2H)，9.2(d，1H)。

Example 14

(2-amino-ethoxy) acetic acid (1R, 2S) -2- (2, 2-dichloroacetamido) -3-fluoro-1- (4- (methylsulfonyl) phenyl) propyl ester hydrochloride

[0150]While stirring a solution of 2-aminoethanol (12mL, 1.2 equiv.) and triethylamine (22mL, 1 equiv.) in tetrahydrofuran (150mL) at room temperature, di-tert-butyl dicarbonate (35g, 1 equiv.) is added in portions. The solution was stirred at room temperature for 20 minutes, diluted with ethyl acetate and washed with 1M aqueous HCl and saturated aqueous NaCl. The organic layer was separated and filtered rapidly through a pad of silica gel and sodium sulfate. The filtrate was concentrated to give the crude product tert-butyl-N- (2-hydroxyethyl) carbamate (24g, 92% yield) as a colorless oil after drying under reduced pressure; h¹-NMR(DMSO-d₆)，δ＝1.3ppm(s，9H)，2.9ppm(q，2H)，3.3ppm(q，2H)，4.6(t，1H)，6.6ppm(t，1H)。

[0151]This oil (13.3g, 1 eq) was dissolved in tetrahydrofuran (150mL) and stirred at-78 ℃ while NaH (4.6g, 1.4 eq) was added in portions. The resulting mixture was stirred at room temperature for 1 hour, then ethyl bromoacetate (12.7mL, 1.4 equivalents) was added at-78 ℃. The mixture was stirred at room temperature overnight and diluted with ethyl acetate. The solution was washed with 1M aqueous HCl and concentrated. The resulting crude mixture was purified by gel column chromatography to give the desired product ethyl 2-aminoethoxy-N-tert-butoxycarbonylacetate (10g, 50% yield) as a colorless oil; h¹-NMR(CDCl₃)，δ＝1.3ppm(t，3H)，1.4ppm(s，9H)，3.3ppm(m，2H)，3.6ppm(t，2H)，4.1ppm(s，2H)，4.2ppm(q，2H)，5.1ppm(br，1H)。

[0152]The oil (5g, 1 equiv.) was hydrolyzed with solid NaOH (1g, 1.2 equiv.) in a mixture of tetrahydrofuran (20mL) and water (20mL) at room temperature over 5 minutes. The mixture was extracted with ether to remove impurities, and the resulting mixture was acidified with 6N aqueous HCl and extracted with ethyl acetate. The combined extracts were washed with saturated aqueous NaCl and filtered rapidly through a pad of silica gel and sodium sulfate. The filtrate was concentrated to give the desired product (2-tert-butoxycarbonylamino-ethoxy) acetic acid (4g, 90% yield) as a colorless oil after drying under reduced pressure; h¹-NMR(DMSO-d₆)，δ＝1.3ppm(s，9H)，3.0ppm(q，2H)，3.4ppm(t，2H)，3.9ppm(s，2H)，6.6ppm(t，1H)，12.6ppm(br，1H)。

[0153]The resulting material was used as starting acid a in general procedure II to give product D as a white foam (7.8g, 84% yield); h¹-NMR(DMSO-d₆)，δ＝1.3ppm(s，9H)，3.1ppm(q，2H)，3.2ppm(s，3H)，3.4ppm(t，2H)，4.2-4.6(m，5H)，6.0ppm(d，1H)，6.4ppm(s，1H)，6.8ppm(t，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.9ppm(d，1H)。

[0154]The foam (15.3g) was deprotected by following general procedure III, the crude product was extracted sequentially with warm tetrahydrofuran and warm dichloromethane and after drying at 50 ℃ under reduced pressure for 2 days, the title hydrochloride salt was obtained as a white powder (9.9g, 74% yield); h¹-NMR(DMSO-d₆)，δ＝2.9ppm(br，2H)，3.2ppm(s，3H)，3.7ppm(t，2H)，4.2-4.7(m，5H)，6.1ppm(d，1H)，6.6ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.0ppm(br，3H)，9.2(d，1H)。

Example 15

{3- [2- (2, 2-dichloro-acetylamino) -3-fluoro-1- (4-methanesulfonyl-phenyl) -propoxycarbonyl-propyl } -trimethyl-ammonium bromide

[0155]While stirring a solution of florfenicol (8.8g, 1 eq.), diisopropylethylamine (5mL, 1.2 eq.), and 4-N, N-dimethylaminopyridine (1.2g, 0.4 eq.) in tetrahydrofuran (90mL) at-20 deg.C, a solution of 4-bromobutyryl chloride (5g, 1.1 eq.) in tetrahydrofuran (15mL) was added via syringe. The solution was stirred at room temperature overnight under nitrogen and diluted with ethyl acetate. The resulting solution was washed with 1M aqueous HCl, the organic layer was separated, and concentrated to give a brown oil. The crude oil was purified by gel column chromatography to give 4-bromo-butyric acid 2- (2,2-dichloro-acetylamino) -3-fluoro-1- (4-methanesulfonyl-phenyl) -propyl ester (5.6g, 45% yield); h¹-NMR(DMSO-d₆)，δ＝2.1ppm(p，2H)，2.6ppm(t，2H)，3.2ppm(s，3H)，3.5ppm(t，2H)，4.2-4.6(m，3H)，6.0ppm(d，1H)，6.4ppm(s，1H)，7.6ppm(d，2H)，7.9ppm(d，2H)，8.9ppm(d，2H)。

[0156]A solution of the ester (1.1g, 1 eq) in tetrahydrofuran (2mL) was stirred at-78 deg.C while adding pure trimethylamine (0.4mL, 2 eq). The resulting solution was stirred in a sealed tube overnight. The precipitate was collected by filtration and stirred in dichloromethane at room temperature for 24 hours. The purified title ammonium bromide was collected by filtration and dried at 50 ℃ under reduced pressure for 2 days to give an off-white powder (0.8g, 65% yield); h¹-NMR(CDCl₃/CD₃OD)，δ＝2.0ppm(m，2H)，2.6ppm(m，2H)，3.0ppm(s，3H)，3.1ppm(s，9H)，3.4-3.6ppm(m，3H)，4.2-4.4ppm(m，3H)，6.0ppm(d，1H)，6.4ppm(s，1H)，7.6ppm(d，2H)，7.8ppm(d，2H)。

Example 16

1- [3- [2- (2, 2-dichloro-acetylamino) -3-fluoro-1- (4-methanesulfonyl-phenyl) -propoxycarbonyl ] -propyl) -3-methyl-3H-imidazol-1-ium bromide

[0157]4-bromo-butyric acid 2- (2, 2-dichloro-acetylamino) -3-fluoro-1- (4-methanesulfonyl-phenyl) -propyl ester (1.8g, 1 eq) and 1-methylimidazole (0.56mL, 2 eq) were stirred in tetrahydrofuran (6mL) at room temperature overnight. The solution was diluted with ether, the precipitate was collected by filtration and stirred in a mixture of ethyl acetate and hexane (1: 1) at room temperature for 24 hours. The purified title imidazolium methyl bromide was collected by filtration and dried at 50 ℃ under reduced pressure for 7 hours to give an off-white powder (1.2g, 56% yield); h¹-NMR(CDCl₃/CD₃OD)，δ＝2.2ppm(m，2H)，2.6ppm(m，2H)，3.0ppm(s，3H)，3.9ppm(s，3H)，4.24.5ppm(m，5H)，6.0ppm(d，1H)，6.4ppm(s，1H)，7.2ppm(d，1H)，7.4ppm(s，1H)，7.6(d，2H)，7.8ppm(d，2H)，9.4ppm(s，1H)。

Example 17

Amino-acetic acid 2- (2, 2-dichloro-acetylamino) -3-fluoro-1- (4-methanesulfonyl-phenyl) -propyl ester

[0158]After drying the crude solid at 50 ℃ under reduced pressure for 6 days by following general procedures II and III using 12.9g of florfenicol and 6.9g N-Boc glycine, the prodrug was obtained as a white powder (13.2g, 80% yield); h¹-NMR(DMSO-d₆)，δ＝3.2ppm(m，3H)，3.9ppm(s，2H)，4.3-4.7ppm(m，3H)，6.1ppm(s，1H)，6.8ppm(s，1H)，7.7ppm(d，2H)，7.9ppm(d，2H)，8.6ppm(br，3H)，9.4ppm(d，1H)。

[0159] The following examples demonstrate the activity and action of the compounds of the invention.

[0160] The compounds of table 1 were tested for stability in aqueous solution and stability in bovine or porcine serum according to the following manner:

[0161] determination of the stability of the prodrug in aqueous solution:

at pH7.4

The prodrug was dissolved at a concentration of 1mg/mL in phosphate buffer pH 7.4. The clear solution was left at room temperature. Aliquots of 400 μ L of the solution were transferred to plastic tubes containing 400 μ L of dilute HCl solution at 0, 1,2, 8, 24 and 48 hour time points to reach a final pH of about 4.0. Samples were analyzed by HPLC. The peak area of florfenicol was used to determine the rate of parent drug release.

At pH 4.5

The prodrug was dissolved in distilled water at a concentration of 0.5 mg/mL. The pH was adjusted to 4.5 with dilute HCl if necessary. The clear solution was left at room temperature. Samples were taken at 0, 1,2, 4 and 7 day time points for analysis. Samples were analyzed by HPLC. The peak area of florfenicol was used to determine the rate of parent drug release.

[0162] Determination of the stability of the prodrug in bovine or porcine serum:

a10 mg/mL stock of prodrug was prepared in dimethylsulfoxide. 50 μ L of the stock solution was added to 5mL of bovine or porcine serum and mixed. Aliquots of 400 μ L of the above prodrug serum solution were transferred to plastic tubes (one tube at each time point). The solution was incubated at 37 ℃ for a predetermined time. Samples were taken at 0, 1,2, 4,8 and 24 hour time points and 400 μ L acetonitrile was added. The sample was stirred for 30 seconds to precipitate the protein and the reaction was stopped, and centrifuged at 14,000rpm for 5 minutes. The supernatant was collected for HPLC analysis. The peak area of florfenicol was used to determine the rate of parent drug release.

HPLC conditions

Equipment: agilent 1100

Column: c18, 5mm, 2.1mm x 150mm

Flow rate: 0.5mL/min

Temperature: 30 deg.C

A detector: UV 254nm

Injection volume: 10mL

Solvent: gradient with 0.1% formic acid water solution and acetonitrile

[0163] The results are shown in table 2.

TABLE 2

[0164] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0165] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims (21)

1. A compound having the formula (I):

wherein:

r is selected from:

a is oxygen and a is 0 or 1;

l is (a) CH₂And l is an integer from 1 to 6; (b) CHR₁Wherein R is₁Is an amino acid side chain, and l is 1; or (c) CHR₁NHC(O)CH(NH₂)R₂Wherein R is₁And R₂Is an amino acid side chain, and l is 1;

m is (a) oxygen or sulfur, and M is 0 or 1; (b) CH (CH)₂And m is 0 or an integer from 1 to 4; or (c) NH, and m is 1;

x is (a) CH₂And x is 0 or an integer from 1 to 4; or (b) C (O), and x is 1;

y is (a) NH₂；(b)NHR_xWherein R is_xIs methyl, ethyl, n-propyl or isopropyl; (c) NR (nitrogen to noise ratio)_yR_zWherein R is_yAnd R_zIndependently hydrogen, methyl, ethyl, n-propyl or isopropyl, or R_yAnd R_zTogether form C₂-C₅An alkylene chain, or C further containing a nitrogen or oxygen heteroatom in said chain₂-C₄An alkylene chain; (d) c (═ NH) NH₂；(e)N⁺R₄R₅R₆Wherein R is₄、R₅And R₆Independently hydrogen, methyl or ethyl, or R₄And R₅Together form C₂-C₅An alkylene chain, or C further containing a nitrogen or oxygen heteroatom in said chain₂-C₄An alkylene chain; (f) pyridinium salts; (g) n-methyl or N-ethylpyridinium; (h) n' -3-methyl-N-1-imidazolium; (i) is of the formula NR₄R₅Or N⁺R₄R₅R₆A phenyl group substituted with a group of (1), wherein R₄、R₅And R₆As defined above; or (j) NH-CR₃(═ NH), where R₃Is hydrogen, methyl or amino;

and R₇Selected from the group consisting of dichloromethyl, difluoromethyl, trifluoromethyl, cyanomethyl, azidomethyl and aminomethyl;

with the proviso that the radical A_aL_lM_mX_xY not being alpha-an N-unfunctionalized glycine, ornithine or lysine residue.

2. A compound having the formula (II):

wherein:

a is oxygen and a is 0 or 1;

l is (a) CH₂And l is an integer from 1 to 5; (b) CHR₁Wherein R is₁Is an amino acid side chain, and l is 1; or (c) CHR₁NHC(O)CH(NH₂)R₂Wherein R is₂Is an amino acid side chain, and l is 1;

m is (a) oxygen, and M is 0 or 1; (b) CH (CH)₂And m is 0 or an integer from 1 to 4; or (c) NH, and m is 1;

x is (a) CH₂And x is 0 or an integer from 1 to 4; or (b) C (O), and x is 1;

y is (a) NH₂；(b)NHR_xWherein R is_xIs methyl, ethyl, n-propyl or isopropyl; (c) NR (nitrogen to noise ratio)_yR_zWherein R is_yAnd R_zIndependently hydrogen, methyl, ethyl, n-propyl or isopropyl; (d) c (═ NH) NH₂；(e)N⁺R₄R₅R₆Wherein R is₄、R₅And R₆Independently hydrogen, methyl or ethyl; (f) n-pyridinium; (g) n' -3-methyl-N-1-imidazolium; or (h) NH-CR₃(═ NH), where R₃Is hydrogen, methyl or amino; and

R₇selected from the group consisting of dichloromethyl, difluoromethyl, trifluoromethyl, cyanomethyl, azidomethyl and aminomethyl;

provided that the sum of a + l + m + x is from 2 to 6, and preferably from 3 to 6; provided that if a is 1, then M is (CH)₂)_m；

And with the proviso that the radical A_aL₁M_mX_xY is not an alpha-N-non-functionalized glycine, ornithine or lysine residueAnd (4) a base.

3. The compound of claim 2, wherein Y is a positively charged moiety separated from the carbonyl by at least two catenated atoms.

4. The compound of claim 2, wherein a is 0.

5. The compound of claim 1, wherein a is 1.

6. The compound of claim 2, wherein L is CH₂And l is an integer of 1 to 5.

7. The compound of claim 2, wherein M is CH₂And m is an integer of 1 to 4.

8. The compound of claim 2, wherein X is CH₂And x is an integer of 1 to 4.

9. The compound of claim 2, wherein Y is NR₄R₅R₆ ⁺。

10. The compound of claim 2, wherein R₇Is dichloromethyl.

11. A pharmaceutical composition comprising a therapeutically effective amount of a compound or salt of claim 1 in combination with a pharmaceutically acceptable diluent or carrier.

12. The composition of claim 10 comprising a therapeutically effective amount of a compound or salt of claim 1.

13. The composition of claim 10 comprising a therapeutically effective amount of a compound or salt of claim 2.

14. The composition of claim 8, further comprising an effective amount of one or more other agents.

15. The composition of claim 14, wherein the one or more additional agents are selected from the group consisting of microbicides; anthelmintic agents; ectoparasiticides and endoparasiticides; anti-insect attractants; anti-inflammatory agents; an anti-infective agent; a hormone; dermatological preparations, including antibacterial and disinfectant agents; and immunobiologics, including vaccines and antisera.

16. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound of claim 1.

17. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound of claim 2.

18. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the composition of claim 10.

19. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the composition of claim 13.

20. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the composition of claim 14.

21. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the composition of claim 15.