BENZYLPHOSPHATE AND SUBSTITUTED BENZYLPHOSPHATE PRODRUGS FOR THE TREATMENT OF PULMONARY INFLAMMATION Field of the Invention The current invention relates to the preparation of novel prodrugs of lidocaine and related local anesthetics and corticosteroid for delivery to the lung by aerosolization. In particular, the invention concerns the synthesis, formulation and delivery of benzylphosphate and substituted benzylphosphate prodrugs and combinations of tertiary amine-based compounds such that when delivered to the lung, exogenous enzymes present in the lung tissue and airway degrade the prodrug releasing tertiary amine-based drugs at the site of administration. The benzylphosphate prodrugs and combinations are formulated as either liquids or dry powders and the formulation permits and is suitable for delivery of benzylphosphate prodrugs and combinations to the lung endobronchial space of airways in an aerosol having a mass medium average diameter predominantly between 1 to 5 p.. The formulated and delivered efficacious amount of benzylphosphate prodrugs and combinations is sufficient to deliver therapeutic amounts of tertiary amine-based drugs for treatment of respiratory tract diseases, specifically pulmonary inflammation associated with mild to severe asthma, bronchitis, and chronic obstructive pulmonary disease (COPD).

Background of the Invention Asthma is a chronic inflammatory disease of the airways resulting from the infiltration of pro-inflammatory cells, mostly eosinophils and activated T-lymphocytes (Poston, 1992; Walker, 1991) into the bronchial mucosa and submucosa. The secretion of potent chemical mediators, including cytokines, by these pre-inflammatory cells alters mucosal permeability, mucus production, and causes smooth muscle contraction. All of these factors lead to an increased reactivity of the airways to a wide variety of irritant stimuli (Kaliner, et al., 1988).

Glucocorticoids, which were first introduced as an asthma therapy in 1950 (Carrier, et al., 1950), remain the most potent and consistently effective therapy for this disease, although their mechanism of action is not yet fully understood (Morris, 1985). Available evidence suggests that at least one mechanism by which they exert their potent anti-inflammatory properties is by inhibiting the release and activity of cytokines, which recruit and activate inflammatory cells such as eosinophils (Schleimer, 1990). Ordinarily, eosinophils undergo the phenomenon of apoptosis or programmed cell death, but certain cytokines such as Interleukin 5 (IL-5), Interleukin-3 (IL-3), and granulocyte-macrophage colony stimulating factor (GM-CSF) increase eosinophil survival from 1 or 2 days to 4 days or longer and cause eosinophil activation (Kita, 1992). Wallen, et al. was the first to show that glucocorticoids potently block the cytokine's ability to enhance eosinophil survival in a concentration- dependent manner (Wallen, 1991).

Unfortunately, oral glucocorticoid therapies are associated with profound undesirable side effects such as truncal obesity, hypertension, glaucoma, glucose intolerance, acceleration of cataract formation, bone mineral loss, and psychological effects, all of which limit their use as long-term therapeutic agents (Goodman and Gilman, 10th edition). An obvious solution to systemic side effects would be the delivery of steroid drugs directly to the site of inflammation. Thus, inhaled corticosteroids (ICS) were developed to mitigate the severe adverse effects of oral steroids. While ICSs are very effective in controling inflammation in asthma they too produce unwanted side effects in the mouth (candidiasis) and pharynx (sore throat). The side effects associated with oral glucocorticoid and ICS therapy have led to interest in agents, which exhibit similar anti-inflammatory effects. A variety of such agents have been tested. For example, preparations of cyclosporin (Szczeklik, 1991; Mungan, 1995), methotrexate (Dyer, 1991), troleandomycin (TAO) (Wald, 1986; Shivaram, 1991), and gold (Szczeklik, 1991; Dykewicz, 2001; Bernstein, 1988) have been used in attempts to wean

patients off orally-administered steroids. Similarly, leukotriene receptor antagonists (e. g., montelukast [Singular] and zafirlukast [Accolate]) (Korenblat, 2001; Dykewicz, 2001; Wechsler, 1999), colchicine (Fish, 1997), salmeterol (Lazarus, 2001; Lemanske, 2001), and anti-immunoglobulin E (IgE) (Dykewicz, 2001) have been used with limited success in efforts to wean patients off inhaled steroids. However, to date, no completely satisfactory substitute for glucocorticoid therapy has been identified.

Serendipitously, Ohnishi, et al. (Ohnishi, 1996) discovered that eosinophil survival is inhibited by lidocaine in a potent and concentration-dependent manner similar to that of corticosteroids. Lidocaine was shown to be effective at low concentrations, which can easily be achieved in the airways by nebulization. The activity of lidocaine, combined with its established record of low toxicity when administered to the airways, inspired use of this agent in preliminary clinical trials to determine its effects in patients with severe, glucocorticoid- dependent asthma. Results of these studies demonstrated that treatment with inhaled lidocaine allowed the majority of patients to significantly reduce, or discontinue, their oral glucocorticoid use without any concurrent increase in their asthma symptoms.

In consideration of all problems and disadvantages connected with the local anesthetic properties of lidocaine and related analogs (numbing effect) and the adverse side effect profile of ICS (candidiasis and sore throat), it would be highly advantageous to provide a water soluble prodrug or prodrug P-agonist combination to mask the local anesthetic and oropharyngeal side effects of lidocaine and ICS, respectively. Such a prodrug would be effectively delivered to the endobronchial space and converted to active drug by the action of lung enzymes thereby delivering to the site of inflammation a therapeutic amount of drug.

There would be no activation of the prodrug or combination in the oropharyngeal cavity.

Furthermore, it would be desirable to administer directly to the lung a combination drug that contains in one molecule a bronchodialator such as salbutamol or salmeterol with an anti-

inflammatory agent, (i. e. lidocaine) or a corticosteroid. The (3-agonist-drug molecular combination would provide a therapeutic to dilate the airway, thereby allowing the second component (drug) to effectively penetrate and reach the site of inflammation. It would be highly desired to have a drug combination of a P-agonist and anti-inflammatory compound that produced sustained release of the P-agonist and anti-inflammatory compound at the site of administration. Additionally, it would be highly desirable to have such a drug combination to be poorly absorbed from the lung and water soluble.

It is therefore a primary object of this invention to provide a composition of benzylphosphate and substituted benzylphosphate prodrugs and prodrug combinations of lidocaine, corticosteroids and the like which are stable as a liquid or solid dosage form for nebulization or dry powder delivery. Such composition contains sufficient but not excessive concentration of the drug which can be efficiently aerosolized by metered-dose inhalers, nebulization in jet, ultrasonic, pressurized, or vibrating porous plate nebulizers or by dry powder into aerosol particles predominantly within the 1 to 5 11 size range, and which salinity and pH are adjusted to permit generation of a benzylphosphate prodrug aerosol well tolerated by patients, and which formulation further has an adequate shelf life.

Summary of the Invention The present invention concerns the use of, and formulation for corticosteroid and lidocaine benzylphosphate and substituted benzylphosphate prodrug combinations delivered by inhalation as a method to treat pulmonary inflammation. The prodrug design incorporates polar and charged groups, which block the ability of the lidocaine/corticosteroid prodrug to penetrate cells thereby inhibiting oropharyngeal anesthetic and therapeutic effect. In addition, the prodrug design features a benzylphosphate group which renders the prodrug highly polar/water soluble and imparts affinity to lung DNA and protein thus minimizing rapid absorption. Furthermore, the benzylphosphate prodrug I or II can be modified to

include in one molecule the amino alcohol pharmacophore (i. e. R6 = CHOHCH2CHNHR) that is present in various ß-agonists such as salbutamol (R = t-butyl) and salmeterol (R = (CH2) 60 (CH2) 4Ph. This process is depicted in Scheme III.

1. A compound of the formula I or II

and pharmaceutical acceptable salts thereof, wherein: X is N or

in which case Rl, R2, and R3 are absent, R7 is H or NO2, and Rs is H or F; and when X is N then RI and R2 are independently selected from the group consisting of aryl, loweralkyl and substituted loweralkyl or Ri and R2 can be linked such that a nonaromatic cyclic ring is formed having 2-10 atoms selected from C, O, S and N; R3 is selected from the group consisting of (a)- (CH2)n-CO-NR4-aryl, -(CH2)n-CO-NR4- heterocycle, -(CH2)n-SO2-NR4-aryl or -(CH2)n-SO2-NR4-heterocycle where n is 1-5 and R4 is H or loweralkyl ; (b) -(CH2)n-NR4-CO-aryl, -(CH2)n-O-CO-aryl or -(CH2)n-NR4-CO- heterocycle where n is 1-5 and (c)- (CHZ) nNR4SO2-aryl or- (CH2)"NR4S02-heterocycle where n is 2-5 and R4 is H or loweralkyl ; and R6 is H or where R7 is arylalkyl or

substituted arylalkyl with 1-3 atoms selected from O, S, and N substituted for CH2, loweralkyl or substituted loweralkyl, and the pharmaceutically acceptable salts thereof.

The stereochemistry of the alcohol CHOH is R, S or a combination of R and S stereochemistry such that a ratio of each isomer varies from 0. 01 to 99.99 R and S.

Presently preferred embodiments of this invention include compounds of formula I or II wherein Ri and R2 are methyl or ethyl and R3 is (CH2) nCONH-aryl, wherein n is an integer ranging from 1-4 and aryl is 2,6-dimethylphenyl.

Other specific embodiments of this invention include compounds of formula I or II wherein Ri and R2 are methyl and R3 is (CH2) nOCO-aryl, wherein n is an integer ranging from 1-4 and aryl is 4-alkylaminophenyl.

Other presently preferred embodiments of this invention include compounds of formula I or II wherein Ri and R2 are methyl and R3 is (CH2) nNHCO-aryl, wherein n is an integer ranging from 1-4 and aryl is 2-butoxy-4-quinoline.

Examples of preferred compounds of this invention include: (2, 6-Dimethyl-phenylcarbamoyl)-methyl]-diethyl-(2-phosphonooxyb enzyl)- ammonium trifluoroacetate disodium salt; (2, 6-Dimethyl-phenylcarbamoyl)-metliyl]-diethyl- (4-phosphonooxybenzyl)- ammonium trifluoroacetate disodium salt; Phosphoric acid mono-E2-({L (2, 6-dimethylphenylcarbamoyl)-methyl]-ethyl-amino}- methyl)-4- (l-hydroxy-2-methylamioethyl)-phenyl] ester N-methyl ammonium trifluoracetate salt;

Salmeterol phosphate-corticosteroid quaternary salt; Salmeterol phosphate-fluorocorticosteroid quaternary salt; Salmeterol phosphate-nitrocorticosteroid quaternary salt; Salmeterol phosphate-fluoronitrocorticosteroid quaternary salt; Salmeterol phosphate-corticosteroid N-methyl piperazine quaternary salt; and Salmeterol phosphate-fluorocorticosteroid N-methyl piperadine quaternary salt.

The invention also relates to a pharmaceutically acceptable composition for the treatment of a disorder selected from severe to mild asthma, bronchitis, COPD, and pulmonary inflammation in general which comprises a therapeutically effective amount of at least one compound of formula I or II or a pharmaceutically acceptable salt thereof, and a pharmaceutically accepted carrier and to methods of treating such diseases with therapeutically effective amounts of at least one compound of formula I or II or a pharmaceutically acceptable salt thereof.

The invention also relates to a liquid or dry powder formulation of the corticosteroid, lidocaine, local anesthetic or related prodrug or prodrug combination for the treatment of a disorder selected from severe to mild asthma, bronchitis, and COPD which comprises a therapeutically effective amount of at least one compound of formula I or II or a pharmaceutically acceptable salt thereof.

Brief Description of the Drawings Figure 1 is a graph that shows the aqueous stability of representative benzylphosphates of the present invention.

Figures 2A and 2B are graphs that show the in vitro activation of representative benzylphosphates of the present invention.

Figure 3 is a graph that shows the in vitro activation in rat lung homogenate of a representative benzylphosphate 5 compared to that of lidocaine.

Detailed Description of the Invention As used herein"aryl"is defined as an aromatic ring substituted with 1-3 groups selected from hydrogen amino, hydroxy, halo, 0-alkyl and NH-alkyl. Aryl can be 1 or two rings either fused to form a bicylic aromatic ring system or linear as in biphenyl. The aryl group can be substituted with N, S, or O in the ring to produce a hetrocyclic system.

The term"alkyl"as used herein refers to a branched or straight chain comprising two to twenty carbon atoms which also comprises one or more atoms selected from O, S, or NH.

Representative alkyl groups include methyl, butyl, hexyl, and the like.

As used herein"lower alkyl"includes both substituted or unsubstituted straight or branched chain alkyl groups having from 1 to 10 carbon atoms. Representative loweralkyl groups include for example, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, and the like.

Representative of halo-substituted, amino-substituted and hydroxy-substituted, lower-alkyl include chloromethyl, chloroethyl, hydroxyethyl, aminoethyl, etc.

As used herein, the term"halogen"refers to chloro, bromo, fluoro and iodo groups.

As used herein, the term"benzylphosphate"refers to The term"heterocycle"as used herein refers to an aromatic ring system composed of 5 or 6 atoms selected from the heteroatoms nitrogen, oxygen, and sulfur. The heterocycle may be composed of one or more heteroatoms that are either directly connected such as pyrazole or connected through carbon such as pyrimidine. Heterocycles can be substituted or unsubstituted with one, two or three substituents independently selected from amino, alkylamino, halogen, alkyl acylamino, loweralkyl, aryl, and alkoxy.

The term"substituted heterocycle"or"heterocyclic group"or"heterocycle"as used herein refers to any 3-or 4-membered ring containing a heteroatom selected from nitrogen, oxygen, and sulfur or a 5-or 6-membered ring containing from one to three heteroatoms selected from the group consisting of nitrogen, oxygen, or sulfur; wherein the 5-membered ring has 0-2 double bounds and the 6-membered ring has 0-3 double bounds; wherein the nitrogen and sulfur atom may be optionally oxidized; wherein the nitrogen and sulfur heteroatoms may be optionally quarternized ; and including any bicyclic group in which any of the above heterocyclic rings is fused to a benzene ring or another 5-or 6-membered heterocyclic ring independently defined above. Heterocyclics in which nitrogen is the heteroatom are preferred. Fully saturated heterocyclics are also preferred. Preferred heterocycles include: diazapinyl, pyrryl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazoyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, pyrazinyl, piperazinyl, N-methyl piperazinyl, azetidinyl, N-methylazetidinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl, triazolyl and benzothienyl.

Heterocyclics can be unsubstituted or monosubstituted or disubstituted with substituents independently selected from hydroxy, halo, oxo (C=O), alkylimino (RN=, wherein R is a lower alkyl or alkoxy group), amino, alkylamino, dialkylamino, acylaminoalkyl, alkoxy, thioalkoxy, polyalkoxy, loweralkyl, cycloalkyl or haloalkyl. The most preferred heterocyclics include imidazolyl, pyridyl, piperazinyl, azetidinyl, thiazolyl, triazolyl benzimidazolyl, benzothiazolyl, and benzoxazolyl.

As used herein, the term"pharmaceutically acceptable salts"refers to the nontoxic acid or alkaline earth metal salts of the compounds of formula I or II. These salts can be prepared ira situ during the final isolation and purification of the compounds of formula I or

II, or by separately reacting the base or acid functions with a suitable organic or inorganic acid or base, respectively. Representative acid salts include the hydrochloride, hydrobromide, bisulfate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, citrate, maleate, tartrate and the like. Representative alkali metals of alkaline earth metal salts include sodium, potassium, calcium, and magnesium salts.

As used herein, the term"alkoxy"refers to-O-R wherein R is lower alkyl as defined above. Representative examples of lower alkoxy groups include methoxy, ethoxy, tert- butoxy, and the like.

The term"treating", as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term"treatment", as used herein, refers to the act of treating, as"treating"is defined immediately above.

The term"normal saline"means water solution containing 0.9% (w/v) NaCI.

The term"diluted saline"means normal saline containing 0.9% (w/v) NaCI diluted into its lesser strength.

The term"quarter normal saline"or"1/4 NS"means normal saline diluted to its quarter strength containing 0.225% (w/v) NaCl.

The term"prodrug"as used herein refers to a compound in which specific bond (s) of the compound are broken or cleaved by the action of an enzyme or biological process thereby producing or releasing a drug and compound fragment which is biologically inactive.

The term"prodrug combination"or"combination"as used herein refers to a compound in which specific bond (s) of the compound are broken or cleaved by the action of an enzyme or biological process thereby producing or releasing a drug and compound fragment which is biologically active. The"prodrug combination"or"combination"is biologically inactive.

The compounds of the invention may comprise asymmetrically substituted carbon atoms. Such asymmetrically substituted carbon atoms can result in the compounds of the invention comprising mixtures of stereoisomers at a particular asymmetrically substituted carbon atom or a single stereoisomer. As a result, racemic mixtures, mixtures of diastereomers, as well as single diastereomers of the compounds of the invention are included in the present invention. The terms"S"and"R"configuration, as used herein, are as defined by the IUPAC 1974 RECOMMENDATIONS FOR SECTION E, FUNDAMENTAL STEREOCHEMISTRY, Pure Appl. Chemin. 45: 13-30 (1976). The terms oc and (3 are employed for ring positions of cyclic compounds. The a-side of the reference plane is that side on which the preferred substituent lies at the lower numbered position. Those substituents lying on the opposite side of the reference plane are assigned P descriptor. It should be noted that this usage differs from that for cyclic stereoparents, in which"a"means"below the plane"and denotes absolute configuration. The terms a and ß configuration, as used herein, are as defined by the CHEMICAL ABSTRACTS INDEX GUIDE-APPENDIX IV (1987) paragraph 203.

The present invention also relates to the processes for preparing the compounds of the invention and to the synthetic intermediates useful in such processes, as described in detail below.

I. PREPARATION OF THE COMPOUNDS OF THE INVENTION In general, the compounds of the present invention can be prepared by the processes illustrated in Scheme I. Either 2-hydroxybenzaldehyde (salicyladehyde) or 4- hydroxybenzaldehyde serves as the starting material for the preparation of 2-or 4-substituted benzylphosphates. The excess of unstable di-tert-butyl phosphorobromidate 1 (described by Gajda and Zwierzak, 1976, and Myers et al., 1995) was used for alkylation of the phenolic moiety in presence of diazabicycloundecene (DBU) as a base and dimethylaminopyridine (DMAP) as a catalyst, yielding 69% and 66% of the phosphorylated ortho- (2) and para-

aldehyde (6), respectively. Both aldehydes were reduced by means of excess of sodium borohydride in THF/MeOH (10: 1) mixture cooled to-78°C, yielding respectively 97% and 96% of ortho- (3) and para-alcohol (7). Compounds 3 and 7 were subsequently activated by conversion to mesylates (using methanesulfonyl chloride and pentamethylpiperidine (PMP) as a base in dichloromethane at 0°C), which were used in a crude form for quaternization with lidocaine in the presence of catalytic amount of sodium iodide in acetonitrile at room temperature over 72-96 hours. Crude quaternary ammonium salts isolated by precipitation were deprotected by treatment with 95% aq. TFA. Thus prepared trifluoroacetates of ortho- (4) and para-benzylphosphate (8) (obtained after 3 steps with respective 60% and 68% yields) were formulated as disodium salt by means of 6N NaOH, yielding water-soluble (>40mg/mL) prodrugs 5 (ortho-isomer) and 9 (para-isomer) that were used as the test articles in enzymatic assays in vitro and pharmacokinetic study in vivo.

Scheme I O GBr4, cat. BTEAC O HP-OtBu. P-OtBu OtBu DCM 20% NAOH OtBu 1 OH CHO OH CHO WHO HO DBU/DMAP 1, DBU/DMAP 1 0 0 / P 1 II O (7 I Py/ CHO OS 6 2 ol< 6 NaBH4 NaBH4 A, o 0 OH O OH O XO J"j Ov O i O oh 3 1. CH3SO2CI/PMP 1. CH3SO2CI/PMP 2. lidocaine, cat. Nal 2. lidocaine, cat. Nal 3. 95% aq. TFA 3. 95% aq. TFA g>, OH) r t r HO 0 ,). H t HO"'o H H N p I N I \ ) 8 TFP Q TFA 4 6N NaOH 6N NaOH disodium salt 5 disodium salt 9

Prodrugs combining the 0-agonist and local anesthetic molecules can be prepared by the process illustrated in Scheme II. 5-Bromosalicylaldehyde was phosphorylated using excess of 1 in presence of DBU and catalytic DMAP yielding 73% of aldehyde 10. This intermediate served as an alkylating agent in reaction with N-desethyl-lidocaine (metabolite known as MEGX), prepared from diethylamine and 2-chloro-2', 6'-acetoxylidide. The reductive alkylation was performed by treatment with sodium triacetoxyborohydride in dichloroethane and resulting tertiary amine 11 was obtained in 70% yield. Compound 11 was then lithiated using 3 equivalents of t-butyllithium at-90°C to generate carbon nuclophile to which 2-thiopyridyl ester of Boc-sarcosine was added at-80°C, to yield an aromatic ketone 12. Low temperature reduction with lithium borohydride, followed by prolonged treatment with methyl iodide at room temperature gives quaternary salt, which was deprotected by means of 95% aq. TFA (alternatively TFA/DCM 1: 1) to yield the trifluoroacetate of the desired quaternary ammonium salt 13.

Scheme II

The convergent route to corticosteroid-p-agonist prodrugs combinations involves; 1) preparation of the steroid analogs (Schemes III and IV), synthesis of the phosphate- (3-agonist derivative (Scheme V) and alkylation of tertiary amine-functionalized steroids with the activated ß-agonist derivative as described in Schemes VI and VII.

Scheme III OR7 O - fpH R7=H, Rs= H HO, (1 6-alpha-Hydroxyprednisolone) .,,,"OH H R7 = H, R8 = F (triamcinolone) H N 0 O HCI04/1-nitropropane OR7 O - HO' H 14 R7 = H, Rs H H < 15R7=H, R8=F H O HN03/Ac20 16 R7 N02, R8 = H 17R7=NO2, R8=F

Scheme IV OH 16a-Hydroxyprednisolone or triamcinolone Ho cyclohexanecaboxaldehyde ~ \,,. \\\\0 HCI04/1-nitropropane | Rs H 18 R8= H 19 R8= F 1. 2, 4, 6-Triisopropylbenzenesulfonyl chloride/pyridine 2. N-methylpiperazine XN ; N Ha O HO 20 4 i H / < H 21 1R8= F O

Scheme V Scheme VI

26 1. 2, 4, 6-Triisopropylbenzenesulfonyl chloride/pyridine 2. Steroids 14, 15, 16 or 17/cat. Nal Y BocsN-(CH2) 60 (CH2) 4Ph 0 HO 27 R7 = H, R8= H HO/ H R7 28 R7 = H, R8= F HO 29 R7 = N02, R8= H ° C<> uC OPOOBtBu 30 R7 = NO2, R8= F R.., > C" n 1. TMSOTf/lutidine (-1OC)/ 2. TBAF/THF /HN-(CH2) 60 (CH2) 4Ph HO 31 R7 = H, R8= H HO f ! 32R7=H, Rs=F =0 33 R7 = N02, R8= H p R\ H,, v0>."uC 4. H 34 R7 = N02, R$ = F 8 H"u0 \O Scheme VII

28 1. 2, 4, 6-Triisopropylbenzenesulfonyl chloride/pyridine 2. Steroids 20 or 21/cat. Nal O t HotN~ (CH2) 60 (CH2) 4Ph Cl/ O OH 36rif R 36 R8-F P'OtBu //oxo % J.., 1. TMSOTf/lutidine (-lOC) 2. TBAF/THF C H O C H P h HO HN (26 24 0 u JOH 37 R8= H /nul/ o / P,-0 H H/ f0 (OH R, = F 1/00 Stog/o 0

Using the procedure described by Gutterer (2002) 16-a-hydroxyprednisolone, or triamcinolone (9-fluoro-16-a-hydroxyprednisolone) is treated at room temperature with 1- methylpiperidine-4-carboxaldehyde (prepared from isonipecotic acid in 3 steps according to Gray, 1988) in presence of 70% perchloric acid and 1-nitropropane as a solvent. These conditions afford compounds 14 and 15 as the 22R diasteroisomer in good yield and diastereoisomeric purity >90%. The 22S-diastereoisomer could be obtained as the major product by using anhydrous HC1 as the acid catalyst and 1,4-dioxane solvent (Gutterer et al, 1994). Compounds 14 and 15 can be further derivatized to the C-21 nitrate derivatives 16 and 17 by selective nitration according to the procedures of Toth (1972).

Scheme IV describes the preparation of steroid analogs bearing tertiary amine attached at the 21-position. 16-a-Hydroxyprednisolone and triamcinolone are converted to the cyclohexylmethylidene derivatives 18 and 19, respectively using standard methods. The 21-hydroxyl group of 18 or 19 is selectively activated by reaction with sterically hindered 2,4, 6-triisopropylbenzenesulfonyl chloride in pyridine at 0°C, followed by alkylation with N- methylpiperazine, yielding analogs 20 and 21, respectively.

Asymmetric synthesis of the phosphate-functionalized protected salmeterol derivative is shown in Scheme V and starts with the alkylation of bromoacetophenone 22 with the Boc protected amine 23 (Rong 1999). The resulting N-protected amino ketone 24 is obtained in good yield. Alternatively, commercially available racemic salmeterol could be the source of intermediate 24 after protecting the secondary amine with t-butoxycarbonyl, followed by oxidation of both hydroxyls by means of pyridinium chlorochromate. Ketone 24 is s phosphorylated with phosphobromidate 1, analogously to the process described in Scheme I and Example 2 to give compound 25. Reduction of 25 with borane in THF in presence of (R)- tetrahydro-l-methyl-3, 3-diphenyl-lH, 3H-pyrrolo [1, 2-c]- [1, 3,2]-oxazaborole (R-version of

Corey's oxazaborolidine catalyst) proceeds with an enantiomeric ratio exceeding 95%. The aldehyde moiety is simultaneously reduced yielding alcohol 26 which is now ready for activation and coupling with the steroid moiety.

Schemes VI and VII describe the final assembly of the target prodrug combination compounds described in this invention. Steroids 14-17 are alkylated (in presence of sodium iodide as a catalyst) with the 2,4, 6-triisopropylbenzenesulfonate of (3-agonist derivative 26 to give the quaternary ammonium salts 27-30. Deprotection of compounds 27-30 is effected under mild, non-acidic conditions applying trimethylsilyl triflate in presence of lutidine (Sakaitani 1990 and Sekine 1991). Desilylation by means of tetrabutylammonium fluoride in THF, followed by the HPLC purification yields the final compounds 31-34.

The piperazine-bearing steroid analogs 20 and 21 are similarly alkylated with the arylsulfonate ester of alcohol 26 in a manner described above, yielding compounds 35 and 36. Selectivity of the alkylation is achieved due to the steric hindrance imposed by the steroid skeleton and by the deactivating effect of the neighboring C20-keto group. The target compounds 37 and 38 are obtained after two-step deprotection, followed by final purification.

II. IN VITRO ACTIVATION OF LIDOCAlNE BENZYLPHOSPHATE PRODRUGS AND SODIUM CHANNEL BLOCKADE While lidocaine prodrugs are known and have been described in the patent literature (Bodor 1979) all have utilized a formaldehyde spacer having a terminal ester for enzyme- mediated (esterase cleavage) release. A major limitation to the Bodor lidocaine prodrug design is the release of formaldehyde during the cleavage process. Such a process would be highly disadvantageous for a prodrug intended for pulmonary delivery as unwanted and toxic formaldehyde would be released directly to the lung. Related tertiary amine prodrugs are known as reported by Stella et al. (Stella 1999). Stella's approach is closely related to the Bodor design with the exception that phosphate is utilized as the enzyme release trigger.

Again, formaldehyde is produced as a side product of prodrug activation. A solution to the formaldehyde spacer is illustrated in Scheme VIII in which the prodrug design incorporates either a 2-or 4-hydroxybenzyl alcohol as a replacement for formaldehyde (Greenwald 1999).

In addition, the 2-hydroxybenzyl alcohol spacer can be modified at the C-4 position (R6 = CHOHCH2NHt-Bu) thus when a prodrug containing this moiety is cleaved both the drug (lidocaine) and salbutamol are released.

Scheme VIII t t HN HN HO alkaline phosphatase HO HO I \ OP03 OH X-R3 HO salbutamol R2 X-Rg R2 drug OP03 alkaline OH phosphatase OR OP03 OR OH X-R X-R3 Ohm R Oh Oh / OH R2 O H drug OH OH

In vitro studies were performed with prodrugs 5 and 9 to determine their stability under the standard test conditions; 37 °C, pH 7.4, and activation conditions; 37 °C, alkaline phosphatase, pH 7.4. Results from these in vitro studies are shown in Tables 1 and 2, respectively. The data clearly demonstrates that both prodrugs are stable in aqueous solution (Figure 1) and are converted to lidocaine and their respective hydroxybenzyl alcohols under the test conditions (Tables 1 and 2 and Figure 3). The rate of cleavage for compound 9 is about twice the rate of cleavage for 5 (tl/2= 1 hour vs. 2 hours). This finding was unexpected as both compounds are isomeric and have approximately the same steric environment around the phosphate group. Additional activations studies were performed using rat lung homogenate at 37 °C, pH 7.4 to determine the rate of cleavage of compound 5 to lidocaine (Figure 3). As shown in Table 3, compound 5 is converted to lidocaine at the same rate in rat lung homogenate as in aqueous media.

Compounds of the invention do not block voltage-gated sodium channels in Xenopus oocytes. When tested at 100, uM concentration, prodrugs 5 and 9 inhibited the sodium channel by 5% and 1%, respectively. In comparison, lidocaine's IC5o was 290 juM.

III. IN VIVO ACTIVATION OF LIDOCAINE PRODRUGS 5 AND 9 The concentrations of prodrugs 5,9, and lidocaine in lung and plasma were determined after intratracheal administration of 40 mg/kg. While full pharmacokinetic analysis could not be performed due to the irregular intervals when the samples were collected, the Cmax and Tmax were visually estimated from the raw data. Following intratracheal administration of compound 5, the Cmax of both 5 and lidocaine in lungs was 5 minutes, and in plasma it was 60 minutes. Following intratracheal administration of compound 9, the Cmax of 9 in lungs was 5 minutes, and in plasma it was 30 minutes, while the Cmax of lidocaine in lungs was 10 minutes and in plasma 30 minutes. These data support the following results; first, both prodrugs released lidocaine in the lung very rapidly confirming the in vitro results (Table 3), second, charged molecules are retained in the lung as evidenced by the slow release of lidocaine from the lung into plasma.

At the Cmax of 5, the ratio of 5 : lidocaine was 28.70 in the lungs and 10.36 in the plasma. At the Cmax of 9, the ratio of 9: Lidocaine was 0.17 in the lungs and 0.90 in the plasma. With few exceptions, both 5 and 9 showed a steady decrease in the lung: plasma ratios over time, from 364 to 2. 08 for 5 and from 252 to 1.24 for 9. A similar decrease is observed for lidocaine generated from the two prodrugs 5 and 9.

The presence of the hydroxybenzyl alcohol portion of the prodrugs was qualitatively monitored at the 1 hour and 24 hour time points for all animals. Following administration of 5,2-hydroxybenzyl alcohol was present in all lung and plasma samples. Following administration of 9,4-hydroxybenzyl alcohol was present in all lung samples, but was only found in one plasma sample. The detection of 2-and 4-hydroxybenzyl alcohol in lung and plasma support the hypothesis that the enzyme fragmentation reaction involves the enzyme

catylized hydrolysis of the benzylic quaternary ammonium moiety to an alcohol by-product.

In the case where R4 is CHOHCH2NHt-Bu the by-product is the 3-agonist, salbutamol.

The appearance of the lidocaine and the 2-and 4-hydroxybenzyl alcohol coincided with the stability of the prodrugs when exposed to lung homogenate. Prodrug 9 showed substantial instability in both plasma and lung homogenate as expected, immediately degrading to lidocaine and 4-hydroxybenzyl alcohol when exposed to the biological matrices at room temperature. The rate of degradation in both matrices was reduced by approximately 26-31% when they were cooled on ice. Compound 5 degraded to lidocaine and 2- hydroxybenzyl alcohol at a much slower rate, and for both matrices the degradation was controlled when they were maintained on ice.

Lung and plasma concentrations of lidocaine prodrugs 5 and 9 and lidocaine are summarized in Tables 1-4.

Table 1 Individual Plasma and Lung Homogenate Concentrations of Compound 5 after Intratracheal Administration to Rats Rat &num Time (min) Plasma Concentration (ng/mL) Lung Concentration (ng/mL 1301 5 7,490 608,000 1302 6,210 724,000 1303 4,470 657,000 1304 10 10,000 5,490 1305 8,430 5,220 1306 5,410 5,110 1307 30 9.13 429 1308 5.54 159 1309 6.35 98.5 1310 60 123 37.6 1311 10,500 30,8000 1312 18,000 5,450 1313 120 0.599 771 1314 0.956 43 1315 833 4,920 1316 240 122 1,680 1317 48.1 1,350 1318 0.692 70.7 1319 480 178 1,800 1320 107 604 1321 BLLOQ 13 1322 1440 190 724 1323 BLLOQ 22.1 1324 409 102

Table 2 Individual Plasma and Lung Homogenate Concentrations of Lidocaine after Intratracheal Administration of Compound 5 to Rats Rat # Time (min) Plasma Concentration (ng/mL) Lung Concentration (ng/mL) 1301 5 986 25,900 1302 495 21,000 1303 400 22,400 1304 10 624 23,900 1305 772 15,300 1306 483 15,300 1307 30 12.3 1,860 1308 4. 83 507 1309 11.6 279 1310 60 13.6 138 1311 1140 19,100 1312 1,610 16,700 1313 120 2.23 1,600 1314 2.7 373 1315 450 1,660 1316 240 351 615 1317 208 505 1318 7.51 180 1319 480 360 586 1320 224 247 1321 6. 27 31. 7 1322 1440 506 363 1323 BLLOQ 29.5 1324 18.8 39.3

Table 3 Individual Plasma and Lung Homogenate Concentrations of Compound 9 after Intratracheal Administration to Rats Rat # Time (min) Plasma Concentration (ng/mLl Lung Concentration (ng/mL) 1325 5 384 13,100 1326 12 1, 980 1327 1,440 3,060 1328 10 2.96 12,200 1329 1,930 3,360 1330 3,520 9,820 1331 30 4,590 139 1332 1,480 122 1333 120 36.3 1334 60 1,100 3,380 1335 3.65 421 1336 47,500 2,070 1337 120 451 35.2 1338 793 16.4 1339 786 15.6 1340 240 1,460 20.2 1341 474 26.2 1342 806 22.3 1343 480 4.76 1.1 1344 N/A 43.9 1345 173 20.4 1346 1440 608 31.7 1347 1350 41.4 1348 2,070 2,370 N/A = no sample available

Table 4 Individual Plasma and Lung Homogenate Concentrations of Lidocaine after Intratracheal Administration of Compound 9 to Rats Rat # Time (min) Plasma Concentration (ng/mL ! Lung Concentration (ng/mL 1325 5 549 68,500 1326 27.3 2,980 1327 792 79,500 1328 10 39.6 790 1329 838 21,500 1330 4,150 78,700 1331 30 3,930 6,370 1332 2,620 7,350 1333 319 607 1334 60 3,330 1,760 1335 48.4 184 1336 52, 800 44,900 1337 120 2,330 1,710 1338 2,540 2,710 1339 2,810 2,540 1340 240 4,210 2,280 1341 3,980 2,880 1342 3,290 2,650 1343 480 31.1 4.38 1344 28.9 2,660 1345 3,180 2,330 1346 1440 3,260 3,350 1347 3,400 2,500 1348 2,040 85,000

IV. AEROSOL DELIVERY DEVICES The use of lidocaine prodrugs with a suitable formulation for liquid nebulization, or as a dry powder provides sufficient prodrug to the lungs for a local therapeutic effect.

Benzylphosphate prodrugs are suitable for aerosolization using jet, electronic, or ultrasonic nebulizers as well as for delivery by dry powder or metered dose inhaler. The pure powder form has long-term stability permitting the drug to be stored at room temperature.

The aerosol formulation comprises a concentrated solution of 10 to 500 mg/mL of pure lidocaine prodrug or its pharmaceutically acceptable salt, dissolved in aqueous solution having a pH between 4.0 and 7.5. Preferred pharmaceutically acceptable salts are inorganic acid salts including hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid as they may cause less pulmonary irritation. The therapeutic amount of the pure lidocaine prodrug is delivered to the lung endobronchial space by nebulization of a liquid aerosol or dry powder having an average mass medium diameter between 1 to 5 g. A liquid formulation may require a separate prodrug salt from the appropriate diluent that can be reconstituted prior to administration because the long-term stability of benzylphosphate prodrugs in aqueous solutions may not provide a commercially acceptable shelf life.

An indivisible part of this invention is a device able to generate aerosol from the formulation of the invention into aerosol particles predominantly in the 1-5 g size range.

Predominantly in this application means that at least 70% but preferably more than 90% of all generated aerosol particles are within the 1-5 li size range. Typical devices include jet nebulizers, ultrasonic nebulizers, vibrating porous plate nebulizers, and energized dry powder inhalers.

A jet nebulizer utilizes air pressure to break a liquid solution into aerosol droplets. An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol

droplets. A pressurized nebulization system forces solution under pressure through small pores to generate aerosol droplets. A vibrating porous plate device utilizes rapid vibration to shear a stream of liquid into appropriate droplet sizes. However, only some formulations of benzylphosphate prodrugs can be efficiently nebulized, as the devices are sensitive to the physical and chemical properties of the formulation. The formulations, which can be nebulized typically, must contain large amounts of benzylphosphate prodrugs, which are delivered in large volumes (up to 5 ml) of aerosol.

IV. UTILITY The compounds of the invention are useful (in humans) for treating pulmonary inflammation and infection.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

This small volume, high concentration formulation of benzylphosphate prodrug can be delivered as an aerosol and be delivered at efficacious concentrations to the respiratory tract in patients suffering from mild to severe asthma. The solid dosage formulation is stable, readily manufactured, and very cost effective. Furthermore, the formulation provides adequate shelf life for commercial distribution. The prodrug masks the anesthetic properties of lidocaine thus numbing in the oral pharyngeal cavity is completely eliminated. The drug is released by enzymes in the lung, specifically alkaline phosphatase, thereby releasing the therapeutic amount of (3-agonist a corticosteroid, lidocaine or related compounds at the site of inflammation.

The foregoing may be better understood from the following examples, which are presented for the purposes of illustration and are not intended to limit the scope of the inventive concepts.

Example 1 Phosphorobromidic acid di-tert-butyl ester Phosphorobromidic acid di-tert-butyl ester was synthesized using a modification of the procedure described by Gajda and Zwierzak (1976). A 500mL 3-neck flask (equipped with dropping funnel and thermometer and immersed in water bath) was charged with carbon tetrabromide (16. 58g, 50mmol) and benzyltriethylammonium chloride (1.14g, 5mmol) dissolved in dichloromethane (50mL). A 20% w/w aqueous solution of NaOH was added (50mL) with vigorous magnetic stirring, followed by dropwise addition (over 20min) of di- tert-butyl phosphite (20. 0g, 99mmol, 96% grade from Lancaster) dissolved in dichloromethane (20mL). The rate of the addition was controlled in order not to exceed 25°C.

Reaction mixture was further stirred for 3 hours, diluted with dichloromethane (20mL) and separated. Organic phase was washed with sat. sodium bisulfite (2 times), water (2 times), dried over anhydrous sodium sulfate, filtered and evaporated at room temperature. After removing most of the solvent residue was pumped under high vacuum for 1 hour to give the crude product as a brown liquid (22.6g, 84%). This material is very unstable and was immediately used (without purification or characterization) for the phosphorylation reactions.

Example 2 Phosphoric acid di-tert-butYl 2-formyl-phenYl ester

Title compound was synthesized based on the procedure described by Myers (1995).

Salicylaldehyde (4.3mL, 40mmol), DBU (6.6mL, 44mmol) and DMAP (0.49g, 4mmol) were

dissolved with stirring in anhydrous THF (40mL) and cooled to 0°C. Phosphorylating agent 1 (22.6g, 83mmol) dissolved in anhydrous THF (20mL) was added dropwise over 1 hour with vigorous stirring and cooling. The reaction mixture was allowed to slowly warm up to room temperature overnight. After 16 hours TLC analysis revealed only traces of starting aldehyde. After concentration the residue was partitioned between ethyl acetate (lOOmL) and 10% aqueous citric acid (75mL), separated and organic phase was washed with 10% citric acid, 0. 5N NaOH (3 times), brine and dried over anhydrous sodium sulfate. Decanted solution was passed through a pad of basic alumina, concentrated and residue was purifed by chromatography using 9% ethyl acetate +1% triethylamine in hexane. Analytically pure aldehyde 2 was obtained as yellowish liquid. Yield: 8.69g (69%); Rf 0.47 (1),'HNMR (CDC13) : 10.45 (d, 1H, J = 0.8 Hz), 7.89 (dt, 1H, J = 8Hz, 1. 2Hz), 7.56-7. 60 (m, 1H), 7.49- 7.51 (m, 1H), 7.23-7. 27 (m, 1H), 1.51 (s, 18H). 3IPNMR (CDC13) : -15.155. LCMS: tR= 6. 901min, MNa+ 337. Anal. Calcd for C15H2305P (Mol. Wt. 314.31) : C, 57.32 ; H, 7.38.

Found: C, 56.64 ; H, 7.13.

Example 3 Phosphoric acid di-tert-butyl 2-hydroxymethyl-phenyl ester A solution of aldehyde prepared in Example 2 (6.65g, 21. 1mmol) stirred in anhydrous THF (20mL) was cooled to-78°C (dry ice-acetone bath) and sodium borohydride (2.40g, 63. 5mmol) was added in portions over few minutes, followed by addition of methanol (2mL). The resulting mixture immersed in cooling bath was stirred slowly allowing to warm up to room temeperature. The TLC analysis after 3.5 hours showed that all starting aldehyde was consumed. The reaction mixture was cautiously added to a

vigorously stirred biphasic mixture of dichloromethane (100mL) and 10% aqueous citric acid (75mL). The organic phase was separated, washed with saturated sodium bicarbonate solution (2 times), brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue crystallized upon drying in high vacuum and the title compound was obtained as a white solid. Yield: 6.48g (97%); RfO. 33 (I), IHNMR (CDCl3) : 7.42-7. 45 (m, 1H), 7.27- 7.31 (m, 1H), 7.16-7. 23 (m, 2H), 4.62 (s, 2H), 4.34 (bs, 1H), 1.52 (s, 18H). 3IPNM : R (CDC13) : -13.167. LCMS: tR= 6. 148min, MNa+ 339. Anal. Calcd for ClsHzsOsP (Mol. Wt.

316.33) : C, 56.95 ; H, 7.97. Found: C, 56.85 ; H, 7.50.

Example 4 (2, 6-Dimethyl-phenylcarbamoyl)-methvll-diethyl- (2-phosphonooxy-benzyl)-ammonium trifluoroacetate The alcohol prepared in Example 3 (0.949g, 3mmol) and 1,2, 2,6, 6- pentamethylpiperidine (1. 08mL, 6mmol) were dissolved in dichloromethane (5mL) and cooled in ice bath with stirring. Methanesulfonyl chloride (0.256mL, 3. 3mmol) was then added via syringe and reaction mixture was allowed to warm up to room temperature. TLC analysis after 3 hours showed no starting alcohol and the mixture was concentrated, redissolved in ethyl acetate, washed with 10% citric acid (3 times), sat. sodium bicarbonate (3 times), brine and dried over anhydrous sodium sulfate. After filtration and evaporation, the residue was pumped under high vacuum yielding crude mesylate as a viscous residue (1.103g, 93%). The obtained product was unstable and was used for the next step without further purification and characterization.

Mesylate (0.945g, 2. 4mmol) and lidocaine (0. 562g, 2. 4mmol) were dissolved in anhydrous acetonitrile (6mL) with stirring, followed by addition of NaI (72mg, 0. 48mmol) dissolved in anhydrous acetonitrile (2mL). The reaction mixture turned cloudy and stirring at room temperature was continued with occasional TLC monitoring. After 4 days, the reaction mixture was concentrated and the resulting residue was triturated with diethyl ether. The solids thus formed were filtered off, washed extensively with diethyl ether and dried yielding 1. 137g of creamy solid, which was used directly in step 3.

The LCMS analysis confirmed that crude product is the desired quaternary ammonium salt as a mixture of di-tert-butyl protected derivative (tR = 4. 764min, M+ 533), mono-tert-butyl protected compound (tR = 3. 730min, M+ 477) and a small amount of unprotected product (tR= 3. 045min, M+ 421).

Crude product from the above reaction (1.13g) was treated with 95% aqueous TFA for 1 hour at room temperature. TLC analysis revealed complete deprotection and reaction mixture was concentrated, azeodried by evaporation with toluene and the resulting residue was recrystallized by dissolving in dichloromethane (lOmL) and dropwise addition to vigorously stirred diethyl ether (60mL). Resulting tan solid was filtered off, washed with diethyl ether and dried. Yield: 0.960g (60% after 3 steps); Rf 0.14 (III),'HNMR (DMSO- d6) : 10.08 (s, 1H), 7.55-7. 62 (m, 2H), 7.51-7. 54 (m, 1H), 7.26-7. 31 (m, 1H), 7.09-7. 14 (m, 3H), 4.82 (s, 2H), 4.26 (s, 2H), 3.53 (q, 4H, J = 7.2Hz), 2.17 (s, 6H), 1.40 (t, 6H, J = <BR> <BR> <BR> <BR> 7. 2Hz). 3IPNMR (DMSO-d6) : -5. 611. I9FNMR (DMSO-d6) : -73.857. LCMS: tR= 3. 147min, M+ 421 (Calc for C2lH30N205P+ Mol. Wt. 421.45). Anal. Calcd for TFA salt C23H3oF3N207P (For. Wt. 534.46) : C, 51.69 ; H, 5.66 ; N, 5.24 ; Found: C, 49.54 ; H, 6.22 ; N, 5.33.

Example 5 (2, 6-Dimethvl-phenvlcarbamovl)-methvl1-diethyl- (2-phosphonooxy-benzyl)-ammonium trifluoroacetate disodium salt

The quaternary ammonium salt prepared in Example 4 (0. 500g, 0. 92mmol) was suspended with stirring in water (lOmL) and 6N NaOH was added dropwise until clear solution was obtained. 350microL of NaOH was added (2. 1mmol), which was followed by adjusting the pH to 8 by adding a drop of glacial AcOH. The addition of acetone (50mL) while cooling in ice-bath caused the oily product to separate. After decantation the residue was azeodried by evaporation with toluene and recrystallized from 2-propanol/diethyl ether yielding title compound as an off-white solid. Yield: 0.434g (82%); Rf 0.14 (III), 1HNMR (D2O) : 7.60-7. 62 (m, 1H), 7.51-7. 57 (m, 2H), 7.16-7. 28 (m, 4H), 4.82 (s, 2H), 4.79 (s, 2H), 3.74 (q, 4H, J = 7.2Hz), 2.22 (s, 6H), 1.53 (t, 6H, J = 7. 2Hz). 3IPNMR (D2O) : 0.363.

19FNMR (D2O) : -75.292. LCMS: tR= 3. 147min, M+ 421 (Calc for C2lH30N2Osp+ Mol. Wt.

421.45). Anal. Calcd for TFA disodium salt C23H28F3N2Na207P (For. Wt. 578.43) : C, 47.76 ; H, 4.88 ; N, 4.84, Na, 7.95 ; Found: C, 49.14 ; H, 6.00 ; N, 5.19, Na, 8.29.

Aqueous solubility of the title compound exceeded 46mg/mL.

Example 6 Phosphoric acid di-tert-butyl 4-formyl-phenyl ester

The title compound was synthesized analogously to the compound prepared in Example 2, using 4-hydroxybenzaldehyde (3.73g, 30mmol), DBU (4.94mL, 33mmol) and DMAP (0.367g, 3mmol) dissolved in anhydrous THF (30mL) and phosphorylating agent 1 (17.75g, 65mmol) in anhydrous THF (15mL). Chromatography (9% ethyl acetate + 1% triethylamine in hexane) yielded analytically pure aldehyde 6 as a white solid. Yield: 6. 21g (66%) ; Rf 0.40 (I), 0.40 (II), IHNMR (CDC13) : 9.96 (s, 1H), 7.87 (d, 2H, J = 8.4Hz), 7. 38 (d, <BR> <BR> <BR> <BR> 2H, J = 8.4Hz), 1.52 (s, 18H). 3lPNMR (CDC13) : -15. 581. LCMS: tR= 5. 184min, MNa+ 337.

Anal. CalcdforCw5H23OsP (Mol. Wt. 314.31) : C, 57.32 ; H, 7.38. Found: C, 57.01 ; H, 7.42.

Example 7 Phosphoric acid di-tert-butyl 4-hydroxymethel-phenyl ester

The title alcohol was synthesized according to the procedure described for the compound prepared in Example 3 using aldehyde 6 (5.13g, 16. 3mmol) and sodium borohydride (1.93g, 51 mmol). Compound 7 was obtained as a white solid. Yield: 4.96g (96%); Rf 0.20 (I), 1HNMR (CDCl3) : 7.31 (d, 2H, J = 8.8Hz), 7.20 (d, 2H, J = 8.8Hz), 4.66

(s, 2H), 4.34 (bs, 1H), 1.51 (s, 18H). 31PNMR (CDC13) : -14.818. LCMS: tR = 4. 583min, MNa+ 339. Anal. Calcd for C15H2505P (Mol. Wt. 316. 33) : C, 56.95 ; H, 7.97. Found: C, 56.75 ; H, 7.84.

Example 8 (2, 6-Dimethyl-phenylcarbamo-methyl]-diethyl- 4-phosphonooxy-benzXl)-ammonium trifluoroacetate The title compound was synthesized according to analogous three-step procedure of Example 4.

Alcohol 7 (1.898g, 6mmol), 1,2, 2,6, 6-pentamethylpiperidine (2.17mL, 12mmol) dissolved in dichloromethane (5mL) and methanesulfonyl chloride (0.512mL, 6. 6mmol) were used. Crude mesylate was obtained as a viscous residue (2.206g, 93%). The obtained product is unstable and was used for the next step without further purification and characterization.

Mesylate (2.18g, 5. 54mmol), lidocaine (1. 30g, 5. 55mmol) dissolved in anhydrous acetonitrile (6mL) and NaI (166mg, l. lmmol) in anhydrous acetonitrile (2mL) were used.

After 4 days. The reaction mixture was concentrated and the resulting residue was dissolved in dichloromethane (15mL) and added to vigorously stirred hexane (75mL). The oily residue formed was separated by decantation, redissolved in a mixture of dichloromethane and diethyl ether and evaporated to form a yellowish foam, that was dried in high vacuum yielding 2.755g of crude product, which was used directly.

The LCMS analysis confirmed that the resulting foam is the desired quaternary ammonium salt as a mixture of di-tert-butyl protected derivative (tR= 4. 698min, M+ 533) and mono-tert-butyl protected compound (tR= 3. 335min, M+ 477).

Crude product prepared above (2. 750g) was treated with 95% aqueous TFA according to the procedure described for compound 4. The crude residue was sonicated in 2-propanol, the supernatant decanted and a solid was obtained by trituration of the residue with diethyl ether, filtration, washing with ether and drying to give 1.585g of first batch. A second crop (0.631g) was obtained from combined 2-propanol supernatant and ethereal washes. Yield: 2.216g (68% after 3 steps); Rf 0.06 (III),'HNMR (DMSO-d6) : 10.20 (s, 1H), 7.53 (d, 2H, J = 8.4Hz), 7.30 (d, 2H, J = 8.4Hz), 7.10-7. 16 (m, 3H), 4.76 (s, 2H), 4.16 (s, 2H), 3.50 (q, 4H, J <BR> <BR> <BR> <BR> = 7. 2Hz), 2.17 (s, 6H), 1.41 (t, 6H, J = 7. 2Hz). 3IPNMR (DMSO-d6) : -5. 387."FOR (DMSO-d6):-73. 361. LCMS: tR = 2. 879min, M+ 421 (Calc for C21H3oN205P+ Mol. Wt.

421.45). Anal. Calcd for TFA salt C23H3oF3N207P (For. Wt. 534.46) : C, 51.69 ; H, 5.66 ; N, 5.24 ; Found: C, 52.01 ; H, 6.67 ; N, 5.41.

Example 9 (2. 6-Dimethyl-phenylcarbamovl)-methyl]-diethyl- (4-phosphonooxy-benzyl)-ammonium trifluoroacetate disodium salt Quaternary ammonium salt 8 (0.770g, 1. 41mmol) was converted to the sodium salt using 6N NaOH (470microL, 2. 82mmol) according to the same procedure as described in Example 5. After adjusting the pH, the reaction mixture was directly frozen and lyophilized

and residue then relyophilized from 50% aqueous acetonitrile. The title compound was obtained as a tan solid (0.820g, quant. ). Yield: 0.820g (100%); Rf 0.06 (IIn, 'HNMR (D20) : 7.49 (d, 2H, J = 8.8Hz), 7.33 (d, 2H, J = 7Hz), 7.22-7. 30 (m, 3H), 4.79 (s, 2H), 4.21 (s, 2H), 3.58-3. 64 (m, 4H), 2.25 (s, 6H), 1.51 (t, 6H, J = 7.2Hz). 3IPNMR (D2O) : 0.713. 19FNMR (D2O) : -75.301. LCMS: tR= 2. 879min, M+ 421 (Calc for C2IH3oN20sP+ Mol. Wt. 421.45).

Anal. Calcd for TFA disodium salt C23H28F3N2Na207P (For. Wt. 578.43) : C, 47.76 ; H, 4.88 ; N, 4.84, Na, 7.95 ; Found: C, 45.70 ; H, 6.10 ; N, 5.00, Na, 6.55.

Aqueous solubility of the title compound exceeded 50 mg/mL.

Example 10 Phosphoric acid 4-bromo-2-formYl-phenel ester di-tert-butyl ester The title compound has been synthesized analogously as compound 2, using 5- bromosalicylaldehyde (8. 04g, 40mmol), DBU (6.58mL, 44mmol) and DMAP (0.489g, 4mmol) dissolved in anhydrous THF (50mL) and phosphorylating agent 1 (23.2g, 85mmol) in anhydrous THF (20mL). Chromatography (9% ethyl acetate + 1% triethylamine in hexane) yielded analytically pure aldehyde 10 as a yellowish solid. Yield: 11. 51g (73%); Rf 0.56 (I), 0.10 (IV), IHNMR (CDC13) : 10. 35 (s, 1H), 7.99 (d, 1H, J = 2.4Hz), 7.67 (dd, 1H, J = 8.8Hz, 2.4Hz), 7.41 (d, 1H, J = 8.8Hz), 1.51 (s, 18H). 3IPNMR (CDC13) : -15.239. LCMS: tR = 8. 036min, MNa+ 415 (exact mass 392.04 calcd for ClsH22BrOsP).

Example 11 Phosphoric acid 4-bromo-2-({[(26-dimethvl-phenylcarbamoyl)-methyl]-ethyl-ami no} methyl !-phenyl ester di-tert-butyl ester N- (2, 6-Dimethyl-phenyl)-2-ethylamino-acetamide 2-Chloro-2', 6'-acetoxylidide (3.953g, 20 mmol) was treated with 2M ethylamine in THF (50mL, 100mmol) and resulting mixture was stirred at room temperature in a tightly sealed flask for 3 days. The precipitate formed (hydrochloride of the amine) was filtered off, the solid was washed with diethyl ether and the combined filtrate was evaporated to give residue, which was recrystallized from diethyl ether/hexane to yield 3.028g of product (73%).

Reductive Alkylation and Preparation of the Title Compound N- (2, 6-Dimethyl-phenyl)- 2-ethylamino-acetamide (1.031g, 5 mmoles) and aldehyde 10 (1.966g, 5mmoles) were stirred in anhydrous 1,2-dichloroethane (lOmL) for 15 minutes at room temperature, which was followed by the addition (in portions) of sodium triacetoxyborohydride (1.673g, 7. 5mmol).

Then, the reaction mixture was diluted with DCE (3mL) and stirred overnight. After 16 hours it was diluted with dichloromethane (20mL) and extracted with 0. 5N NaOH (40mL) with vigorous stirring over 15 minutes. After separation, the organic phase was washed with sat. sodium bicarbonate (2 times), brine, dried over anhydrous magnesium sulfate, decanted and evaporated. The residue was purified by chromatography (20% ethyl acetate + 1 % triethylamine in hexane) to yield title compound as a white solid. Yield: 2.05g (70%); Rf 0.21 (I), 0.29 (II), 1HNMR (CDC13) : 8.65 (s, 1H), 7.58 (d, 1H, J = 2.8Hz), 7.37 (dd, 1H, J =

8. 8Hz, 2. 8Hz), 7.29-7. 32 (m 1H), 7.04-7. 08 (m, 3H), 3.79 (s, 2H), 3.27 (s, 2H), 2.78 (q, 2H, J = 7.2Hz), 2.12 (s, 6H), 1.48 (s, 18H), 1.25 (t, 3H, J = 7. 2Hz). 3IPNMR (CDC13) : - 14.698. LCMS: tR = 5. 851min, MH 583, MNa+ 605 (exact mass 582.19 calcd for C27H4oBrN205P).

Example 12 {2-[4-(Di-tert-butoxy-phosphoryloxy)-3-({[(2,6-dimethyl-phen ylcarbamoyl)-methyl]-ethyl- amino}-methyl)-phenyl]-2-oxo-ethy-methyl-carbamic acid tert-butyl ester Boc-Sarcosine 2-Thiopyridyl Ester A solution of Boc sarcosine (1.9 g, 10 mmol) and 2- pyridyl disulfide (2.2 g, 10 mmmol) in 50 mL dry THF was stirred at 0 C while a solution of 2.6 g (10 mmol) of triphenylphosphine in 20 mL of THF was added. The reaction was stirred at room temperature for 18 hours, concentrated under reduced pressure and the residue redissolved in ethyl acetate. Solids (triphenylphosphine oxide) were filtered and the crude yellow oil was purified by silica gel column chromatography using 30: 70 ethyl acetate: hexane to give 2.0 g of a yellow oil which was used in the next step. LC/MS (M+Na)+ 305.

Metalation of Compound 11 and Preparation of the Title Compound A solution of aryl bromide 11 (1.76 g, 3.0 mmol) in 10 mL of dry THF was cooled to-95 C (liquid nitrogen/diethyl ether) and 3 equivalents of t-butyllithium (1.7 M, 9.09 mmol, 5.3 mL) was slowly added keeping the reaction temperature between-95 and-100 C. After the addition was complete, the reaction was warmed to-80 C and kept at that temperature for 10 min.

The 2-thiopyridyl ester prepared above (855 mg, 3.0 mmol) in 2 mL THF was added in one portion. The reaction temperature warmed to-20 C and water was added. Work up in the usual manner gave a yellow oil which was purified by silica gel chromatography using 500 mL (50: 450: 5) ethyl acetate: hexane: triethylamine and 1L (200: 800: 5) ethyl + acetate: hexane: triethylamine. LC/MS (M+H) 677.

Example 13 Phosphoric acid mono-[2-({[(2,6-dimethyl-phenylcarbamoyl)-methyl]-ethyl-amin o}-methyl)- 4-(1-hydroxy-2-methvlamino-ethyl !-phenyll ester N-methyl ammonium trifluoracetate salt

A solution of 50 mg (0.07 mmol) of the ketone prepared in Example 12 can be reduced to the alcohol with lithium borohydride solution (THF, 0.07 mmol). The crude alcohol can be stirred for 24 hours in neat methyl iodide (1 mL), excess methyl iodide evaporated and the title compound produced upon hydrolysis of the t-butyl protecting groups (TFA/CH2C12, room temperature).

Example 14 16-cc-Hydroxyprednisolone-16 17-o-acetal of N-methyl-4-formvlpiperidine 16-a-Hydroxyprednisolone (1 equiv) can be dissolved in 1-nitropropane (at concentration ca. 0.7M), cooled in an ice-bath and treated with 70% perchloric acid (3 equiv) and l-methylpiperidine-4-carboxaldehyde (1.2 equiv). The reaction mixture can be stirred overnight at room temperature and then quenched with saturated sodium bicarbonate solution. The precipitate formed can be collected, redissolved in DMF and the solution added dropwise with stirring to sodium bicarbonate solution. The precipitate is filtered off, washed with cold water and dried. Title compound 14 should be predominantly (>90%) 22R-epimer.

Example 15 yprednisolone-10a-fluoro-16, 17-ao-acetal of N-methyl-4- fbrmvluiperidine

Compound 15 can be synthesized identically as described in Example 14 using triamcinolone as the starting material.

Example 16 16-a-Hydroxyprednisolone-21-nitrate-16. 17-a (3-acetal of N-methyl-4- formylPiperidine Concentrated (d 1.50) nitric acid (6 equiv) can be cooled to-10°C while acetic anhydride (10 equiv) can be added dropwise with stirring. Then 0.15M solution of steroid 14 (1 equiv) in chloroform is added with stirring and maintaining temperature at-10°C for the following 30 minutes. The reaction mixture can be quenched by addition of ice-cold water, followed by adding ice-cold diluted NaOH solution. After phase separation, aqueous layer can be extracted twice with chloroform and combined organic solutions washed with sat. bicarbonate, brine and dried over anhydrous sodium sulfate. After decanting and evaporation of the chloroform extracts the crude product (TLC homogeneous) can be recrystallized.

Example 17 16-a-Hydroxyprednisolone-l0a-fluoro-2I-nitrate-I6, 17-a (3-acetal ofN-methyl-4-<BR> <BR> formylpiperidine

Compound 17 can be synthesized identically as described in Example 16, using steroid 15 as the starting material.

Example 18 16-o-Hydroxyprednisolone-16 17-a (3-acetal of cYclohexane carboxaldehyde Title compound 18 can be synthesized as described in Example 14, substituting 1- methylpiperidine-4-carboxaldehyde with cyclohexylcarboxaldehyde. After overnight reaction the precipitate formed can be filtered off without earlier quenching with bicarbonate. The Product should have an R/S-epimer ratio 97/3.

Example 19 16-a-Hyyprednisolone-10a-fluoro-16, 17-a3-acetal of cyclohexane carboxaldehvde

Compound 19 can be prepared as described in Example 18, using triamcinolone as the starting material.

Example 20 16-α-Hydroxyprednisolone-21-(4-N-methylpiperazine)-16,17-α ß-acetal of cyclohexane carboxaldehyde

Solution of compound 18 (1 equiv) in dry pyridine can be cooled in an ice-bath, followed by addition of 2, 4,6-triisopropylbenzenesulfonyl chloride (1.2 equiv) in one portion.

The reaction mixture can be stirred allowing to warm up to room temperature with occasional TLC monitoring of disappearance of steroid starting material. After 2 hours excess (1.5 equiv) of N-methylpiperazine is added and the mixture left stirring overnight at room temperature. The next day after concentration in vacuo, residue can be redissolved in ethyl acetate and washed with sat. sodium bicarbonate, water, brine and dried over anhydrous

magnesium sulfate. The crude material obtained after decantation and evaporation can be purified on silica gel using increasing gradient of methanol in dichloromethane.

Example 21 16-a-Hydroxyprednisolone-10a-fluoro-21-(4-N-methvlpiperazine )-16, 17-a (3-acetal of cyclohexane carboxaldehyde Compound 21 can be prepared identically as described in Example 20, substituting compound 18 with its 9-fluoro analog 19.

Example 22 5-(2-Bromo-acetyl !-2-hydroxv-benzaldehyde The title compound can be prepared from salicylaldehyde and bromoacetyl chloride in presence of aluminium trichloride in refluxing dichloromethane according to Rong and Ruoho (1999).

Example 23 [6-(4-Phenyl-butoxy)-hexyl]-carbamic acid ter-butyl ester

The title compound can be prepared in a three-step process based on publication by Rong and Ruoho (1999). First, the alkoxide generated with NaH from 4-phenylbutanol can be alkylated with 1, 6-dibromohexane in presence of catalytic tetrabutylammonium bromide. The thus obtained bromide can be substituted by heating with sodium azide and catalytic sodium iodide in DMSO at 80°C. The azide can then be reduced by hydrogenation in presence of palladium catalyst and di-t-butyl dicarbonate, obtaining the desired Boc-protected amine 23.

Example 24 <BR> <BR> f2- (3-Formvl-4-hvdroxv-phenvl)-2-oxo-ethvl]-f6- (4-phenyl-butoxv)-hexyl]-carbamic acid<BR> <BR> tert-butyl ester Compound 23 (1 equiv) can be dissolved under nitrogen in anhydrous THF, cooled in an ice-bath and then 95% NaH (l. l. equiv) can be added in small portions with vigorous stirring. After 15 minutes the THF solution of bromo-compound 22 (1 equiv) can be added dropwise with such a rate so that temperature is maintained below 5°C. Then, the mixture is allowed to slowly warm up to room temperature with stirring overnight. The next day the reaction can be quenched by addition of 10% citric acid and diluted with ethyl acetate. After phase separation organic layer can be washed with sat. sodium bicarbonate, brine and dried over anhydrous magnesium sulfate. The crude material obtained after decantation and evaporation can be purified on silica gel.

Example 25 <BR> <BR> f 2-f4- (Di-tert-butoxy-phosphoryloxy-3-formyl-phenyl]-2-oxo-ethyl-l 6- (4-phenyl-butoxy hexyl] -carbamic acid tert-butyl ester

The title compound 25 can be prepared by alkylation of the phenol 24 with the excess of freshly prepared phosphobromidate 1 in presence of DBU as a base and DMAP as a catalyst, analogously to the procedure from Example 2. The crude product can be purified on silica gel eluting with the increasing gradient of ethyl acetate in hexane with 1% triethylamine (to prevent decomposition of the phosphate ester).

Example 26 {2-[4-(Di-tert-butoxy-phosphoryloxy)-3-formyl-phenyl]-2-hydr oxy-ethyl}-[6-(4-phenyl- butoxv)-hexyl]-carbamic acid tert-butyl ester

Compound 26 can be prepared analogously to the procedure published by Coe et al.

(2003). A solution of borane-THE complex in THF (IM, 2 equiv) can be added slowly to a solution of (R)-tetrahydro-1-methyl-3,3-diphenyl-1H,3H-pyrrolo[1,2-c]-[1 , 3, 2]-oxazaborole in toluene (1M, 0.1-0. 2 equiv) at 5°C under nitrogen. After 15 minutes a solution of intermediate 25 (lequiv) in THF can be added slowly. The reaction can be allowed to warm

to room temperature and further stirred for 2 hours. After cooling in an ice-bath reaction mixture can be quenched by dropwise addition of 10% citric acid. The mixture can be partitioned between ethyl acetate and water and the organic layer washed with sat. sodium bicarbonate, brine and dried over anhydrous magnesium sulfate. After filtering off the drying agent and evaporation the residue can be evaporated several times with hexane upon which title compound solidifies.

Example 27 Boc-Salmeterol-di-ter-tbutylphosphate-16-a-Hydroxyprednisolo ne-16, 17-ap-acetal of N-methyl-4-formylpiperidine Quaternary Ammonium Salt Step 1. Intermediate diol 26 (1 equiv) can be dissolved in dry pyridine and cooled in an ice-bath, followed by addition of 2, 4,6-triisopropylbenzenesulfonyl chloride (1.2 equiv) in one portion. The reaction mixture can be stirred allowing to warm up to room temperature with occasional TLC monitoring of disappearance of the starting material. After 2 hours the reaction mixture can be diluted with ethyl acetate, washed twice with 10% citric acid, sat. bicarbonate, brine and dried. The crude product obtained after decantation and evaporation can be azeodried by evaporation with hexane and then used without further purification in the next step.

Step 2. Sulfonate from step 1 and steroid compound 14 (1 equiv as a free base) can be dissolved in anhydrous acetonitrile, which can be followed by addition of acetonitrile solution of NaI (0.2 equiv) with stirring at room temperature. The reaction mixture should

occasionally be monitored by TLC over next 3-4 days. Then the reaction mixture should be concentrated and the resulting residue triturated with diethyl ether. Solids thus formed should be filtered-off, washed with ether and dried.

Example 28 Boc-Salmeterol-di-tert-butvlphosphate-16-a-Hydroxyprednisolo ne-10a-fluoro- 16,17-αß-acetal of N-methyl-4-formylpiperidine Quaternary Ammonium Salt

Compound 28 can be prepared as described in Example 27 using the steroid analog 15 as a substrate in Step 2.

Example 29 Boc-Salmeterol-di-tert-butphosphate-16-a-Hydroxyprednisolone -21-nitrate-16. 17- αß-acetal of N-methyl-4-formylpiperidine Quaternary Ammonium Salt Compound 29 can be prepared as described in Example 27 using the steroid analog 16 as a substrate in Step 2.

Example 30 Boc-Salmeterol-di-tert-butylphosphate-16-a-Hydroxyprednisolo ne-10a-fluoro-21- nitrate-16,17-αß-acetal of N-methyl-4-formylpiperidine Quaternary Ammonium Salt Compound 30 can be prepared as described in Example 27 using the steroid analog 17 as a substrate in Step 2.

Example 31

Salmeterolphosphate-16-oc-Hydroxyprednisolone-16. 17-ocß-acetal of N-methyl-4- formylpiperidine Quaternary Ammonium Salt

Step 1. Compound 27 can be dissolved in dry dichloromethane and cooled under nitrogen to about-10°C. Then 2,6-lutidine (15 equiv) and trimethylsilyl triflate (10 equiv) can be added to the mixture with stirring. After 2 hours reaction (not exceeding 0°C) the mixture can be poured into water. Extraction with dichloromethane, drying, filtration and concentration provides the crude silylated compound to be used in the next step.

Step 2. The crude material from step 1 can be dissolved in anhydrous THF and treated with the 1M solution of tetrabutylammonium fluoride in THF (10-15 equiv) at room temperature. The progress of desilylation should be monitored by LCMS. When the reaction is complete the solvent should be evaporated and the residue triturated with diethyl ether. The precipitate formed should be filtered-off, washed with ether and dried. Target compound can be purified by HPLC and obtained as a trifluoroacetate salt.

Example 32 <BR> <BR> Salmeterolphosphate-16-a-Hydroxyprednisolone-10a-fluoro-16 17-ac3-acetal of N- methyl-4-formylpiperidine Quaternary Ammonium Salt

Compound 32 can be prepared as described in Example 31 using the intermediate 28 as a starting material for the two-step deprotection procedure.

Example 33 Salmeterolphosphate - 16-α-Hydroxyprednisolone-21-nitrate-16,17-αß-acetal of N- methyl-4-formylpiperidine Quaternary Ammonium Salt Compound 33 can be prepared as described in Example 31 using the intermediate 29 as a starting material for the two-step deprotection procedure.

Example 34 <BR> <BR> <BR> Salmeterolphosphate-16-a-H ydroxyprednisolone-10α-fluoro-21-nitrate-16,17-αß acetal of N-methyl-4-formylpiperidine Quaternary Ammonium Salt

Compound 34 can be prepared as described in Example 31 using the intermediate 30 as a starting material for the two-step deprotection procedure.

Example 35 <BR> <BR> <BR> Boc-Salmeterol-di-tertbutylphosphate-16-a-Hydroxyprednisolon e-21- 4-N-<BR> <BR> <BR> methylpiperazine)-16,17-αß-acetal of Cyclohexane carboxaldehyde Quaternary Ammonium Salt Compound 35 can be prepared as described in Example 27 using the steroid analog 20 as a substrate in Step 2.

Example 36 <BR> <BR> Boc-Salmeterol-di-tertbutylphosphate-16-a-Hvdrox. yprednisolone-10a-fluoro-21- (4- N-methylpiperazine)-16,17-αß-acetal of Cyclohexane carboxaldehyde Quaternary Ammonium Salt

Compound 36 can be prepared as described in Example 27 using the steroid analog 20 as a substrate in Step 2.

Example 37 Salmeterolphosphate-16-a-Hvdroxyprednisolone-21- (4-N-methylpiperazine)-16, 17- ap-acetal of Cyclohexane carboxaldehyde Quaternary Ammonium Salt Compound 37 can be prepared as described in Example 31 using the intermediate 35 as a starting material for the two-step deprotection procedure.

Example 38 Salmeterolphosphate-16-oc-Hvdroxyprednisolone-10a-fluoro-21- (4-N- methylpiperazine)-16,17-αß-acetal of Cyclohexane carboxaldehyde Quaternary Ammonium Salt Compound 38 can be prepared as described in Example 31 using the intermediate 36 as a starting material for the two-step deprotection procedure.

Example 39 Conversion of lidocaine benzylphosphates (5 and 9) to lidocaine after exposure to alkaline phosphatase A. Conversion by alkaline phosphatase A 100 RI aliquot of a ~100 ng/lll solution of either benzylphosphate 5 or benzylphosphate 9 in 1: 1 acetonitrile/water was added to 900 RI of 50 mM tris (hydroxymethyl) aminomethane buffer solution, pH 7.4, containing 1 mM ZnCl2 and 1 mM MgCl2. Then, 10 al of 48 ng/gl alkaline phosphatase solution in water was added to the reaction solutions. No enzyme was added to the control solutions. The solutions were incubated at 37 °C for up to 7 hours, and the solutions were analyzed directly by HPLC.

B. Conversion by rat lung homogenate

1. Methodology Standards and sample preparation To determine the concentration of lidocaine and 5 in rat lung homogenate samples, standards were prepared in 10% acetonitrile : 90% water in neat matrix in order to ensure that no degradation of the compounds occurred. Standards contained 1000,300, 100,30, 10,3, 1 or 0.3 ng/mL of lidocaine and 5. b. Homogenization of rat lung tissue Rat lungs were homogenized using a Polytron homogenizer. Rat lung weights were first recorded and then 4 mL of 20% methanol/80% water was added to the tissue in a 15mL centrifuge tube. Tissues were then homogenized for-1 minute at-7000 rpm. The volume of the resulting homogenate was recorded and then the samples were stored at-80°C.

Sample preparation Lung homogenate samples were prepared by acetonitrile precipitation. A 100 PLL aliquot of plasma or lung homogenate was combined with 300 I1L of acetonitrile spiked with an internal standard (100 pg/mL prilocaine). The solution was mixed for 1 minute at 950 rpm. After mixing, samples were centrifuged at 12000 rpm for ten minutes. 350 JLL of supernatant was then transferred to an eppendorf tube. These samples were then dried under N2 at 40°C. The residue was reconstituted with 85 , L of 10% acetonitrile: 90% water. All samples were vortexed prior to being analyzed by LC/MS/MS.

HPLC conditions Column: Phenomenex HyperClone, BDS C18, 30 x 2.0 mm id Mobile Phase Buffer: 25 mM NH40H to pH 3.5 w/85 % formic acid Reservoir A: 10 % buffer and 90 % water Reservoir B: 10 % buffer and 90 % acetonitrile Gradient Program Flow Rate: 300 N. min Injection Vol. : 10 vil Run Time: 4.5 min

Mass spectrometer conditions Instrument: PE SCIEX API4000 Interface: Electrospray ("Turbo Ion Spray") Mode: Multiple Reaction Monitoring (MRM) Nebulizing Gas: 20 Drying Gas: 30 Curtain Gas: 20 Ion Spray Voltage: 5000 Temperature (TEM): 450 CAD Gas: 7 DP: Declustering Potential EP: Entrance Potential CE: Collision Energy CXP: Collision Cell Exit Potential Lung homogenate samples were prepared and analyzed using the methods described in the methodology (B. 1) section above. In brief, lidocaine and prodrug 5 were extracted from lung homogenate via acetonitrile precipitation and then analyzed by LC/MS/MS. To determine accuracy and precision, the analytical method was subjected to a one-day pre-study validation. The lower limit of quantitation (LLOQ) of the analytical method was 0.3 ng/mL.

No extraordinary peaks other than those corresponding to lidocaine and 5 were observed in chromatograms lung standards and samples.

C. Conversion after intratracheal administration to rats 1. Test Article and Vehicle Preparation To prepare the dosing formulations, the required amount of compound 5 or 9 was weighed into a container and dissolved in vehicle (Sterile Water for Injection, USP). The pH was adjusted to 6.4 for both compounds 5 and 9. The final appearance of each formulation

was a clear, colorless liquid. Prior to use, the formulations were sterilized using a Millipore 0.2 11 filter (lot number R2EN66834, expiration date June, 2005). Formulations were prepared within four hours of dosing the first 12 animals/group, and were used again on the following day to dose the remaining 12 animals/group. Final drug concentration was 40 mg/mL for each prodrug formulation The required amount of vehicle was dispensed for the control group on the day of dosing. The pH was measured as 6.0. Prior to use, the vehicle was sterilized using a Millipore 0.2 u filter (lot number SL6VR25K5, expiration date June, 2005).

2. Animal Acquisition and Acclimation Sixty male Crl : CD@ (SD) IGS BR rats (approximately 8 weeks of age) were received from Charles River Laboratories, Kingston, New York. Rats were individually housed upon arrival. During the seven-day acclimation period, all animals were observed daily for any clinical signs of disease and weighed.

Randomization, Assignment to Study, and Maintenance Prior to selection for study, all animals were weighed and given a detailed clinical examination. Animals considered suitable for study were weighed and randomized into treatment groups using a standard, by weight, simple randomization procedure. All animals placed on study had body weights that were within 20% of the mean body weight. Extra animals obtained for this study, but not placed on study, were euthanized by carbon dioxide inhalation and discarded without further evaluation.

Fifty males (weighing 286 to 313 g at randomization) were assigned to the treatment groups identified in the following table.

Group Assignment Group Number Dose Level Test Article Number of (mg. kg) Male Animals 1 40 Compound 5 24 2 40 Compound 9 24 3 0 Vehicle 2 "Administration of the test articles was staggered over two days, such that 12 animals/group were dosed each day.

Each animal was assigned a unique number (Table 1-4) and was identified by indelible marker on the tail. The individual animal number and study number comprised a unique identification for each animal. Animal cages were identified by the study number, animal number, group number, and sex. Animal numbers were verified throughout the course of the study, as documented in the study data.

The animals were housed individually in suspended, stainless steel wire-mesh type cages. Fluorescent lighting was provided for approximately 12 hours per day via an automatic light timer. On occasion, the dark cycle was interrupted intermittently due to bleeding intervals that occurred during the 12-hour dark cycle. Temperature and humidity were monitored and recorded daily, and maintained between 70 to 78°F and 30 to 40%, respectively.

Diet (block Lab Diet'» certified Rodent DietX #5002, PMI Nutrition International, Inc. ) was available ad libitum to all animals. Tap water was available ad libitum for each animal via an automatic watering system.

Administration The test articles or vehicle were administered to all animals as a single intratracheal dose. Two treated groups of 24 animals per group received one of the test articles,

Lidocaine-Prodrug 5 or 9, at a dose level of 40 mg/kg. A control group of two animals received the vehicle, Sterile Water for Injection, USP. The dose volume for all groups was approximately 1 mL/kg. Individual dose amounts were calculated using the body weights measured prior to dose on Day 1.

The test article formulation was stirred with a magnetic stir bar and stir plate. For dosing, the animals were anesthetized using isoflurane anesthesia, immobilized, and held upright. The calculated amount of dosing formulation was delivered with a sterile ball-tipped needle and a 1 mL sterile disposable syringe. A different needle and syringe were used for each group, and only a single dose was drawn into the syringe at one time. A laryngoscope was used to facilitate correct placement of the needle. After dosing, the animals were left on an incline with their head up for approximately 20 seconds to allow the dosing formulation to settle into the lungs.

3. Sample Collection Blood Collection Whole blood was collected from all animals predose (approximately 1 mL) via the jugular vein, and from three treated animals/group at proposed times of approximately 5,10, and 30 minutes, and 1,2, 4,8, and 24 hours postdose (approximately 2mL) by cardiac puncture following euthanasia by intraperitoneal administration of sodium pentobarbital.

Samples (approximately 2 mL) were also collected from the two control animals at approximately 24 hours postdose by cardiac puncture. The blood was collected into tubes containing EDTA and stored on wet ice until centrifuged for approximately 10 minutes at approximately 4°C and 3000 rpm. The plasma samples were stored frozen at approximately - 20°C and protected from light until shipment for analysis.

Tissue Collection and Animal Disposition Following blood collection at each postdose interval, the lungs were removed, blotted dry, weighed, and stored frozen until shipment for analysis. The remaining carcasses were stored frozen and were disposed of by incineration after the study was completed.

Sample Identification, Storage, and Shipment All samples were identified with the study number, test article, animal number, group number, sample matrix (plasma or lung), sample weight or volume, and collection interval.

All samples were shipped on dry ice to Absorption Systems LP, Exton, Pennsylvania, for analysis of test article concentration. In addition, 10 mg of the following standards were also included in the shipment: lidocaine-Prodrug 5, lidocaine-Prodrug 9, lidocaine, 2-hydroxybenzyl alcohol, and 4-hydroybenzyl alcohol.

Sample Analysis by Absorption Systems LP Pharmacokinetic analysis of plasma concentration versus time data was to be performed using PC WinNonLin or another suitable non-linear modeling program by Absorption Systems, LP. Compartmental or non-compartmental methods were to be applied, as appropriate, for determination of Ty., AUC, Tmax, and Cmax. However, pharmacokinetic analysis could not be performed due to the irregular intervals when the samples were collected. Lung and plasma lidocaine prodrug and lidocaine concentrations are presented in Tables 1-4.

Example 40 Percent inhibition of lidocaine, benzylphosphate prodrugs 5 and 9 verus control in the sodium channel blockade assay Local anesthetics cause numbing by blocking sodium channel activity. Xenopus oocytes were used as an expression system to study the effect of test articles on the alpha subunit of the NAV 1.4 sodium channel derived from human skeletal muscle.

Oocytes were harvested from female Xenopus laevis (Xenopus I, Dexter, MI), previously injected with human chorionic gonadotropin. Frogs were anesthetized by immersion in 0. 2% 3-aminobenzoic acid ethyl ester and the ovarian tubes surgically removed.

Oocytes were dissociated by gentle agitation for 1 hour in 1 mg/ml collagenase D (Boehringer-Mannheim), and then washed extensively in Ca2+ free OR-2 solution (96 mM NaCI, 2 mM KC1, 1 mM MgC12, 5 mM HEPES, pH 7.4). Stage V and VI oocytes were collected with the aid of a dissecting microscope.

Plasmid containing cDNA for the NAV 1.4 alpha subunit of the human skeletal muscle Na channel was linearized, and capped cRNAs synthesized in vitro (Message Machine RNA polymerase kit; Ambio, Austin TX). RNA was purified with an RNAid kit (BiolOl, Vista, CA). Individual oocytes were injected with cRNA (50 nL) and maintained at 18°C in frog saline solution (96 mM NaCl, 1 mM KC1, 1 mM CaC12, mM MgC12, 10 mM Hepes, 1mM theophylline, 2 mM Na pyruvate, pH 7.4, 50U/ml penicillin G, and 50 ug/mL streptomycin.

Electrophysiological recordings were performed at 2 days post-cRNA injection.

Sodium channel currents were recorded from oocytes with a two-electrode voltage clamp using a Geneclamp 500B amplifier (Axon Instruments, Foster City, CA). Voltage-measuring and current passing electrodes were filled with 3 M KC1 and adjusted to a resistance of 0.3 to 1 M. Currents were sampled at 5 kHz and filtered at 1-2 kHz. Oocytes were perfused continuously with an external solution containing 96 mM NaCl, 2 mM KC1, 2 mM CaC12, 1 mM MgC12, 10 mM HEPES, pH 7.4. Oocytes were clamped at-70 mV and step depolarized to-20mV to activated the channels. Compounds were tested with five (5) replicates, and each experiment was repeated in triplicate.

Example IC50 (µM) % Inhibition Concentration SEM(%) 5 - 5 0.1mM 5 9-1 0. 1mM 1 lidocaine 290 98 1.0 mM 9

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