NZ526120A - 13-membered azalides and their use as antibiotic agents - Google Patents

Abstract

Disclosed is the use of 13-membered azalides of formula I, for which the substituents are defined herein, in methods of treating a bacterial infection or protozoa infection in a non-human mammal, fish, or bird. (62) Divided out of 511199

Description

New Zealand Paient Spedficaiion for Paient Number 526120 526 12 0 iNTELLECTUAL PROPERTY OFFICE OF N.Z 2 6 MAY 2003 RECEIVED NEW ZEALAND PATENTS ACT, 1953 No: Date: Divided out of No. 511199 Dated 9 November 1999 COMPLETE SPECIFICATION 13-MEMBERED AZALIDES AND THEIR USE AS ANTIBIOTIC AGENTS We, PFIZER PRODUCTS INC., a corporation organized under the laws of the State of Connecticut, United States of America of Eastern Point Road, Groton, Connecticut 06340, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page la) I 13-MEMBERED AZALIDES ANO THEIR USE AS ANTIBIOTIC AGENTS Background of the Invention c This invention relates to methods of treating bacterial infections and protozoa infections in non-human mammals, fish and birds by administering novel 13-membered azalides to mammals, fish and birds requiring such treatment.

Macroiide antibiotics are known to be useful in the treatment of a broad spectrum of 10 bacterial infections and protozoa infections in mammals, fish and birds. Such antibiotics include various derivatives of erythromycin A, such as azithromycin. Azithromycin is commercially available, and is referred to in United States patents 4,474,768 and 4,517,359, both of which are incorporated herein by reference in their entirety.

Additional macrolides are referred to in U.S. patent application serial number 60/063676, 15 filed October 29,1997 (Yong-Jin Wu), U.S. application serial number 60/063161, filed October 29, 1997 (Yong-Jin Wu), U.S. application serial number 60/054866, filed August 6,1997 (Hiroko Masamune, Yong-Jin Wu, Takushi Kaneko and Paul R. McGuirk), U.S. application serial number 60/049980, filed June 11,1997 (Brian S. Bronk, Michael A. Letavic, Takushi Kaneko and Bingwei V. Yang), International Application No. PCT/IB98/00839, filed May 29,1998 (Brian S. Bronk, Hengmiao 20 Cheng, E. A. Glazer, Michael A. Letavic, Takushi Kaneko and Bingwei V. Yang), U.S. application serial number 60/049348, filed June 11,1997 (Brian S. Bronk, Hengmiao Cheng, E.A. Glazer, Michael A. Letavic, Takushi Kaneko and Bingwei V. Yang), International Application No. PCT/GB97/01810 filed July 4,1997 (Peter Francis Leadlay, James Stauton, Jesus Cortes and Michael Stephen Pacey), International Application No. PCT/GB97/01819 filed July 4,1997 (Peter 25 Francis Leadlay, James Staunton, and Jesus Cortes), U.S. application serial no., 60/070358, filed January 2,1998 (Yong-Jin Wu), U.S. application serial no. 60/070343, filed January 2.1998 (Dirlam) and U.S. application serial no 60/097075, filed August 19,1998 (Hengmiao Cheng, Michael A. Letavic, Carl B. Ziegler, Jason K. Dutra, Brian S. Bronk), all of which are incorporated herein by reference in their entirety. 3 o Without admitting that the above cited patents and patent applications are prior art to the present application, there remains a need in the art for readily available, 13-membered azafide antibiotic compounds that possess potent activity against a broad range of bacteria and protozoa.

Like azithromycin and other macroiide antibiotics, the novel macroiide compounds disclosed herein possess potent activity against various bacterial and protozoa infections as 35 described below. mil"--.^toperivofhce - 2 DEC 2004 j Summary of the Invention Disclosed is a compound of the formula 1 n(ch3)r2 chj, r k N10 r oh, ch,"" 7112 3 or a pharmaceutical^ acceptable salt thereof, wherein: CH, OH R1 is on , acetyl, 3-N,N-dimethylamino-2-propenoyl, , 1-N-methyl- 5-pyrazolyl, 3-pyrazolyl, 1-methyl-N-3-pyrazolyl, 1-N-benzyI-3-pyrazolyl, 1-N-(3-hydroxybenzyl)-3- rV4 N\\ N—N (R\ pyrazolyl, 3-isoxazolyl, nhr3 or R2 is hydrogen or 0,-C* alkyl; R3 is hydrogen, CrC,0 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, -(CH2)m(C6-C10 aryl), -(CH2)m(C6-C10 heterocyclic) or aryl, each, other than hydrogen, being optionally substituted with 1 to 3 substituents independently selected from halogen, cyano, nitro, trifluoromethyl, azido, -C(O)C,-C10 30 alkenyl, -C(0)C2-Cio alkynyl, -00(0)Ci-0,q alkyl, ~OG(0)02~Ciq alkenyl, - OC(O)C2-C10 alkynyl, -N(hydrogen, C,-C,0 alkyl, C2-C10 alkenyl or C2-C10 alkynyl )C(O)(C,-C,0 alkyl, C2-C10 alkenyl or C2-C10 alkynyl), -C(0)N(hydrogen, 0,-0,0 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl)(hydrogen, C,-C10 alkyl, C2-Cl0 alkenyl or C2-C10 alkynyl), -N(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl)(hydrogen, 0,-0,0 alkyl, C2-C10 alkenyl orC2-C10 alkynyl), C,-C10 alkoxy, C6-C,0 aryl, 5-10 membered heterocyclic, hydroxyl, methoxyl, 0,-0,0 alkyl, C2-C,0 alkenyl, C2-C10 alkynyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-pyridylethyl, 3-pyridylethyl and 4-pyridylethyl; INltLLECTUAL PROPERTY OFFICE OF N2. - 2 DEC 2004 m is an integer ranging from 0 to 4; each R* is hydrogen. -(CH2)m(C6-C,e aryl) or -(CH2)ra(C6-C,0 heterocyclic), each, other than hydrogen, being optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl. azido. -0(0)0,-0.? alkyl. -C(O)C2-C,0 alkenyi. -C(0)C2-C.;. alkynyl. -5 OC(O)Ct-C,0 alkyl, -0C(0)C2-C,c alkenyl. -00(0)C2-C,c alkynyl. -N(hydrogen, C,-C,c alkyl, C2-C,e alkenyl or C2-C,0 a!kynyl)C(O)(C,-Ct0 alkyl. C2-C.c alkenyl or C2-C,„ alkynyi). -C(0)N(hydrogen, 0,-C,0 alkyl, C2-C10 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C,= alkyl. C2-C.; alkenyl or C2-C,0 alkynyl), -N(hydrogen, C,-C,0 alkyl, C--C,0 alkenyl or C2-C13 alkynyl)(hydrogen. C -C-: alkyl, C2-C!0 alkenyl or C2-C10 alkynyl), C,-C.0 alkoxy, C6-C10 aryl and 5-10 membered heterocyclic; n is an integer from 0 to 5; R6 is hydrogen or methyl; each R7 is independently hydrogen, 0,-0^ alkyl, C2-C2o alkenyl. C.-C^ alkynyl, -C(O)C1-C20 alkyl, -C(0)C2-C2o alkenyl. -0(0)0,-0,0 alkynyl. -C(O)N(H)C,-C10 alkyl. -CfOJNfHJCj-C^ alkenyl, -C(O)N(H)C2-C20 alkynyl. -502(0)0,-020 alkyl, -SO2(O)C2-C20 alkenyl, -SO^OXVCjo alkynyl or -P042-.

R8 is hydrogen or methyl; or 4"-oxocladinosyl; and R12 is C,-C10 alkyl, C2-C,0 alkenyl, C2-C,0 alkynyl, cyano, -CH2S(O),C,-C10 alkyl, -CH2S(O)pC2-C,0 alkenyl, -CH^O^Oj-C^ alkynyl, wherein p is an integer ranging from 0 to 2, -CH2O(C,-C10 alkyl), -CH2O(C2-C,0 alkenyl), -CH2O(C2-C10 alkynyl), -CH2N(hydrogen, C,-C10 alkyl, C2-25 C,o alkenyl or 02-0,0 alkynyl)(hydrogen, C,-C,0 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl), -(CH2)m(C6-C10 aryl) or -(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein the alkyl, alkenyi. alkynyl, aryl and heteroaryl moieties of the foregoing are optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido. -C(O)C,-C10 alkyl, -C(O)CrC,0 alkenyl, -C(O)C2-C10 alkynyl, -00(0)0,-0,0 alkyl, -00(0)C2-3 0 C,o alkenyl, -OC(O)C2-C10 alkynyl, -N(hydrogen. C,-C,0 alkyl, C2-C10 alkenyl or Cj-C^ alkynyOCtOKCf-Cto alkyl, C2-C,0 alkenyl or C2-C10 alkynyl), -C(0)N(hydrogen, CrC10 alkyl, C2-C,0 alkenyl or C2-C,0 a!kynyl)(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyl or C2-C,0 alkynyl), -N(hydrogen, C,-C,o alkyl, C2-C,0 alkenyl or C2-C)0 alkynyl)(hydrogen. C,-C-0 alkyl. C2-C,; alkenyl or C2-C,0 alkynyl), C,-C,0 alkoxy, C6-C10 aryl or 5-10 membered heterocyclic, hydroxy, C,-Cs alkyl, C,-C6 alkoxy, C6-C10 35 aryl and 5-10 membered heteroaryl.

R9 is 2.

Also disclosed is a compound of the formula 15 . or a pharmaceutical^ acceptable salt thereof, wherein: R11 yo cHj R1 is , acetyl, 3-N,N-dimethylamino-2-propenoyl, ^ , 1-N-methyl-5- pyrazolyl, 3-pyrazolyl, 1-methyl-N-3-pyrazolyl, 1-N-benzyl-3-pyrazolyl, 1 -N-(3-hydroxybenzyl)-3- o r-i* ■Or^ V" pyrazolyl, 3-isoxazolyl, nhk3 or ; 2 5 R2 is hydrogen or C,-C4 alkyl; R3 is hydrogen, C,-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, -(CH2)m(C6-C10 aryl), -(CH2)m(C6-C10 heterocyclic) or aryl, each, other than hydrogen, being optionally substituted with 1 to 3 substituents independently selected from halogen, cyano, nitro, trifluoromethyl, azido, -0(0)0,-0,0 alkyl, -C(O)C2-C10 alkenyl, -C(O)C2-C,0 alkynyl, -00(0)0,-0,0 alkyl, -OC(O)C2-C,0 alkenyl, - 3 0 OC(O)C2-C,0 alkynyl, -N(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl)C(O)(C,-C10 alkyl, C2-C10 alkenyl or C2-C,e alkynyl), -0(0)N(hydrogen, C,-C,0 alkyl, C2-C,0 alkenyl orC2-C,0 alkynyl)(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl), -N(hydrogen, C,-C,0 alkyl, C2-C,0 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl), C,-C,0 alkoxy, C£-C10 aryl, 5-10 membered heterocyclic, hydroxyl, methoxyl, C,-C,0 alkyl, CrC,0 alkenyl, C2-C,Q 35 alkynyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-pyridylethyl, 3-pyridylethyl and 4-pyridylethyl; m is an integer ranging from 0 to 4; INTELLECTUAL PROPERTY OFFICE 0FN2. - 2 DEC 2004 RFrpn/cn each R4 is hydrogen, -{CH2)m(C6-C,0 aryl) or -(CH2)m(C6-C,0 heterocyclic), each, other than hydrogen, being optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro. trifluoromethyl, azido, -C(O)C,-C,0 alkyl, -C(O)C2-C.0 alkenyl. -C(O)C2-C!0 alkynyl, -0C(0)C.-C.c alkyl. -OC(O)C2-C,0 alkenyl. -OC(O)C2-C10 alkynyl. -N(hydrogen. C,-C,e alkyl. C2-C1t 5 alkenyl or C:-C10 alkynyl)C(O)(C1-Ct0 alkyl. C2-C10 alkenyl or C2-C,a alkynyl). -C(0)N(hydrogen, C.-C10 alkyl, C;-C10 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C,0 alkyl. C2-C10 alkenyl or C2-C10 alkynyl), -N(hydrogen, 0,-0,0 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl)(hydrogen, C,-C,0 alkyl, C2-C!0 alkenyl or C2-C10 alkynyl), C,-C,0 alkoxy, C6-C,0 aryl and 5-10 membered heterocyclic; n is an integer from 0 to 5; r6 is hydrogen or methyl; each R7 is independently hydrogen, C,-C20 alkyl, Cj-C^ alkenyl, C2-C2o alkynyl, -C(O)C,-C20 alkyl, -C(0)C2-C2o alkenyl, -C(O)C2-C20 alkynyl, -C(O)N(H)C,-C,0 alkyl, -C(O)N(H)C2-C20 alkenyl, -C(O)N(H)C2-C20 alkynyl, -SO^OK^-C^ alkyl, -SO^OC^-C^ alkenyl, -SO2(O)C2-C20 alkynyl or -P042"; R8 is hydrogen or methyl; R9 is or 4"-oxocladinosyl; R10 is an alpha-branched C2-C„ alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylthioalkyl group, any of which may optionally be substituted by one or more hydroxyl groups; a Cs-C8 cycloalkylalkyl group 25 wherein the alkyl group is an alpha-branched C2-Cs alkyl group; a C3-C8 cycloalkyl or C5-Ce cycloalkenyl group, either of which may optionally be substituted by methyl or one or more hydroxyl, one or more C,-C4 alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more C,-C4 alkyl groups or halo atoms; or R10 is phenyl which may be 30 optionally substituted with at least one substituent selected from C,-C4 alkyl, C,-C4 alkylthio groups, halogen atoms, hydroxyl groups, trifluoromethyl, and cyano; or R10 may be with a formula (a) as shown befow: X>°^CH3 r-,15 '3 wherein Y is O, S or —CH-—, a, b, c, and d is each independently an integer ranging from 0-2 and a 10 + b + c + d<5; R'1 is hydrogen or -OH; and R15 is H, C,-C,0 alkyl, Cj-0,0 alkenyl, C2-C,0 alkynyl, cyano, -CH2S(0)pC,-C10 alkyl, -CH2S(O)pC2-C10 alkenyl, -CH2S(O)pCj-C10 alkynyl, wherein p is an integer ranging from 0 to 2, -0^0(0,-0,0 alkyl), -CH;O(CrC,0 alkenyl), -CH2O(C2-C,0 alkynyl), -CH;N(hydrogen, C,-C,0 alkyl, C2-^5 C,0 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C,0 alkyl, C2-C,0 alkenyl or C;-C10 alkynyl), -(CH2)m(C6-Ci0 aryO or -(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein the alkyl, alkenyl, alkynyl, aryl and heteroaryl moieties of the foregoing are optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido, -0(0)0,-C,o alkyl, -0(0)02-0,q alkenyl, -C(0)C2-C,o alkynyl, -00(0)C,-C,o alkyl, -0C(0)C2~ 2q C,o alkenyl, -OC(O)C2-C,0 alkynyl, -N(hydrogen, 0,-0,0 alkyl, C2-C10 alkenyl or C2-C10 alkynyl)C(O)(C,-C,0 alkyl, C2-Ct0 alkenyl or C2-C,0 alkynyl), -C(0)N(hydrogen, C,-C10 alkyl, C2-C,0 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C10 alkyl, C2-C,0 alkenyl or C2-C;0 alkynyl), -N(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C,0 alkyl, Cj-Ck, alkenyl or C2-C,0 alkynyl), C,-C10 alkoxy, C6-C,0 aryl or 5-10 membered heterocyclic, hydroxy, C,-Cs alkyl, C,-C6 alkoxy, C6-C10 25 aryl and 5-10 membered heteroaryl, except that when R15 is H, R10 is not ethyl.

Also disclosed are compounds of the formula 2 N(CH3)R2 O O * 2 INTELLECTUAL PROPERTY OFFICE OF N.2. -2 DEC 2004 RECEIVED and to pharmaceutically acceptable salts thereof, wherein: X is -0(0)- or -CH(OR7)-; and R2 and R7 are defined above, and R9 is '3 or 4"-oxocladinosyl; and R5 is hydrogen, 0,-0,0 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, cyano, -CH2S(O)rC.-C10 alkyl, -CH2S(0)pC2-C1t, alkenyl, -CH2S(O)pC2-C10 alkynyl, wherein p is an integer ranging from 0 to 2, -CH20(C,-C,o alkyl), -CH2O(C2-C10 alkenyl), -CH2O(C2-C10 alkynyl), -CH2N(hydrogen, C,-C,0 alkyl, C2-C10 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl), -(CH2)m(C6-C10 aryl) or -(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein the alkyl, alkenyl, alkynyl, aryl and heteroaryl moieties of the foregoing are optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, 15 azido, -0(0)0,-0,0 alkyl, -C(O)C2-C10 alkenyl, -C(O)C2-C,0 alkynyl. -00(0)0,-Ct0 alkyl, -0C(0)C2-C)0 alkenyl, -OC(O)C2-C10 alkynyl, -N(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyl or C2-C10 alkynyOCfOXC^C™ alkyl, C2-C,0 alkenyl or C2-C10 alkynyl), -0(0)N(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyl or C2-C,0 alkynyl)(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyi or C2-C,0 alkynyl), -N(hydrogen, C,-C,o alkyl, C2-C10 alkenyl or C2-C,0 alkynyl)(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl), 2 q C,-C,0 alkoxy, C6-C,o aryl or 5-10 membered heterocyclic, hydroxy, C,-C6 alkyl, C,-C6 alkoxy, C6-C,0 aryl and 5-10 membered heteroaryl.

The compounds of formula 1_ and formula 2 are preferably in their isolated or purified form. Also disclosed are pharmaceutical compositions which can be used for the treatment of a bacterial infection or a protozoa infection in a mammal, fish, or bird which comprise a 2 Q therapeutically effective amount of a compound of formula 1^. formula 2, or formula 15 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Thus the invention relates to a method of treating a bacterial infection or a protozoa infection in a non-human mammal, fish, or bird which comprises administering to said mammal, fish or bird a therapeutically effective amount of a compound of formula 1 or formula 15 or a pharmaceutically acceptable salt thereof.

Preferred compounds of formula 2 include those wherein R7 and Re are hydrogen, and R9 is CHf OCH3 INTEllluual HHUPERTY OFFICE OF NZ '2 DEC 2004 In a preferred embodiment, the compound of formula \ is that wherein R1 = CH3 OH ; R6, R7 and R8 = hydrogen; and R9 = 4"-((R13)(R14)NCH2)cladinosyl.

The term "treatment", as used herein, unless otherwise indicated, includes the treatment or prevention of a bacterial infection or protozoa infection as provided in the method of the present invention.

As used herein, unless otherwise indicated, the terms "bacterial infection(s)" and "protozoa 10 infection(s)" include bacterial infections and protozoa infections that occur in mammals, fish and birds as weil as disorders related to bacterial infections and protozoa infections that may be treated or prevented by administering antibiotics such as the compounds disclosed herein. Such bacterial infections and protozoa infections, and disorders related to such infections, include the following: pneumonia, otitis media, sinusitus, bronchitis, tonsillitis, and mastoiditis related to infection 15 by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, or Peptostreptococcus spp.; pharynigitis, rheumatic fever, and glomerulonephritis related to infection by Streptococcus pyogenes, Groups C and G streptococci, Clostridium diptheriae, or Actinobacillus haemolyticum; respiratory tract infections related to infection by Mycoplasma pneumoniae, Legionella pneumophila, Streptococcus pneumoniae, Haemophilus influenzae, or 2 0 Chlamydia pneumoniae; uncomplicated skin and soft tissue infections, abscesses and osteomyelitis. and puerperal fever related to infection by Staphylococcus aureus, coagulase-positive staphylococci (i.e., S. epidermidis, S. hemolyticus, etc.). Streptococcus pyogenes, Streptococcus agalactiae, Streptococcal groups C-F (minute-colony streptococci), viridans streptococci, Corynebacterium minutissimum, Clostridium spp., or Bartonella henselae; uncomplicated acute urinary tract infections 25 related to infection by Staphylococcus saprophytics or Enterococcus spp.; urethritis and cervicitis; and sexually transmitted diseases related to infection by Chlamydia trachomatis, Haemophilus ducreyi, Treponema pallidum, Ureaplasma ureatyticum, or Neiserria gonorrheae; toxin diseases related to infection by S. aureus (food poisoning and Toxic shock syndrome), or Groups A, B, and C streptococci; ulcers related to infection by Helicobacter pylori; systemic febrile syndromes related to 3 0 infection by Borrelia recurrentis; Lyme disease related to infection by Borrelia burgdorferi; conjunctivitis, keratitis, and dacrocystitis related to infection by Chlamydia trachomatis, Neisseria gonorrhoeae, S. aureus, S. pneumoniae, S. pyogenes, H. influenzae, or Listeria spp.; disseminated Mycobacterium avium complex (MAC) disease related to infection by Mycobacterium avium, or Mycobacterium intracellular; gastroenteritis related to infection by Campylobacter jejuni; intestinal 35 protozoa related to infection by Cryptosporidium spp.; odontogenic infection related to infection by viridans streptococci; persistent cough related to infection by Bordetella pertussis; gas gangrene related to infection by Clostridium perfringens or Bacteroides spp.; and atherosclerosis related to INTELLECTUAL PROPERTY OFFICE OF NZ - 2 DEC 20M infection by Helicobacter pylori or Chlamydia pneumoniae. Bacterial infections and protozoa infections and disorders related to such infections that may be treated or prevented in animals include the following: bovine respiratory disease related to infection by P. haem., P. multocida, Mycoplasma bovis, or Bordetella spp.; cow enteric disease related to infection by E. colior protozoa 5 (i.e., coccidia, Cryptosporidia, etc.); dairy cow mastitis related to infection by Staph, aureus, Strep, uberis, Strep, agalactiae. Strep, dysgalactiae, Klebsiella spp., Corynebacterium, or Enterococcus spp.; swine respiratory disease related to infection by A. pleuro., P. multocida, or Mycoplasma spp.; swine enteric disease related to infection by E. coli, Lawsonia intracellularis, Salmonella, or Serpulina hyodyisinteriae; cow footrot related to infection by Fusobacterium spp.; cow metritis 10 related to infection by £. coir, cow hairy warts related to infection by Fusobacterium necrophorum or Bacteroides nodosus; cow pink-eye related to infection by Moraxella bovis; cow premature abortion related to infection by protozoa (i.e. neosporium); urinary tract infection in dogs and cats related to infection by £. coli; skin and soft tissue infections in dogs and cats related to infection by Staph, epidermidis. Staph, intermedius, coagulase neg. Staph, or P. multocida; and dental or mouth 15 infections in dogs and cats related to infection by Alcaligenes spp., Bacteroides spp., Clostridium spp., Enterobacter spp., Eubacterium, Peptostreptococcus, Porphyromonas, or Prevotella. Other bacterial infections and protozoa infections and disorders related to such infections that may be treated or prevented in accord with the method of the present invention are referred to in J. P. Sanford et ai., "The Sanford Guide To Antimicrobial Therapy," 26th Edition, (Antimicrobial Therapy, 20 Inc., 1996). position 11 of formula 1_, or a pharmaceutically acceptable salt thereof, which comprises the step of contacting compound of the formula 5 Also disclosed is a method of preparing a compound of formula 1 particularly wherein R6, R7 and R8 are hydrogen, and R1 is trans with respect to the methyl group at N(CH3), O wherein R9, is as defined for formula 1; R10 is an alpha-branched C2-C8 alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylthioalkyl group, any of which may optionally be substituted by one or more hydroxyl groups: a C5-C8 cycloalkylalkyl group wherein the alkyl group is an alpha-branched C2-C5 alkyl group; a C3-Cs cycloalkyl or Cs-C6 cycloalkenyl group, either of which may optionally be substituted by methyl or one or more hydroxyl, 5 one or more C,-C4 alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more C,-C4 alkyl groups or halo atoms; or R10 is phenyl which may be optionally substituted with at least one substituent selected from C,-C4 alkyl, C,-C4 alkylthio groups, halogen atoms, hydroxyl groups, trifluoromethyl, and cyano; or R10 may be with a formula (a) as 10 shown below: wherein Y is O, S or—CH2—, a, b, c, and d is each independently an integer ranging from 0-2 and a + b + c + d^5; and R11 is hydrogen or —OH, 2 0 with an acid or a base to result in the formation of a compound of formula 1_.

Also disclosed is a method of preparing a compound of formula particularly wherein Rs, R7 and R8 are hydrogen, and R1 is trans with respect to the methyl group at position 11 of formula 1_, or a pharmaceutically acceptable salt thereof, which comprises the step of heating a compound of the formula 5 in the presence of a solvent to result in the formation of a 25 compound of formula^.

Also disclosed is a method of preparing a compound of formula 15 particularly wherein R€, R7 and R8 are hydrogen, and R1 is trans with respect to the methyl group at position 11 of formula 1j>, or a pharmaceutically acceptable salt thereof, which comprises the step of contacting compound of the formula 5 a N(CHfc CH3 O wherein R9 is as defined for formula 15; R10 is an alpha-branched C^-Cg alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylthioalkyl group, any of which may optionally be substituted by one or more hydroxyl groups; a Cs-Ce cycloalkylalkyl group wherein the alkyl group is an alpha-branched C2-C5 alkyl group; a C3-C8 cycloalkyl or CrC8 cycloalkenyl group, either of which may optionally be substituted by methyl or one or more hydroxyl, one or more C,-C4 alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally 2 0 be substituted by one or more C,-C4 alkyl groups or halo atoms; or R10 is phenyl which may be optionally substituted with at least one substituent selected from C,-C4 alkyl, C,-C< alkylthio groups, halogen atoms, hydroxyl groups, trifluoromethyl, and cyano; or R10 may be with a formula (a) as shown below: 3 o wherein Y is O, S or —CH2—, a, b, c. and d is each independently an integer ranging from 0-2 and a + b + c + d<5; and Rt1 is hydrogen or —OH, with an acid or a base to result in the formation of a compound of formula 1j>. a Also disclosed is a method of preparing a compound of formula 15, particularly wherein R6, R7 and R8 are hydrogen, and R1 is trans with respect to the methyl group at position 11 of formula 15, or a pharmaceutically acceptable salt thereof, which comprises the step of heating a compound of the formula 5 in the presence of a solvent to result in the formation of a 5 compound of formula 1j5.

Preferred compounds of formula 5 are those in which R10 is ethyl, isopropyl, cyclopropyi, sec-butyl, cydobutyl, cyciopentyl, methylthioethyl or furyl, and Rn is hydrogen; and those in which R'° is cyclopropyi or cydobutyl, and R11 is -OH.

Compounds of formula 5 are useful in the preparation of the above compounds of formula i or 15 as well as pharmaceutically acceptable salts thereof.

The term "hydroxy protecting group", as used herein, unless otherwise indicated, indudes acetyl, benzyloxycarbonyl, and various hydroxy protecting groups familiar to those skilled in the art include the groups referred to in T. W. Greene, P. G. M. Wuts, "Protective Groups in Organic Synthesis," (J. Wiley & Sons, 1991).

The term "halo", as used herein, unless otherwise indicated, includes fluoro, chloro, bromo or iodo. Preferred halo groups are fluoro, chloro and bromo.

The term "alkyl", as used herein, unless otherwise indicated, indudes saturated monovalent hydrocarbon radicals having straight, cyclic or branched moieties. Such cyclic moieties indude 2 0 cydopropyl, cydobutyl and cyciopentyl. The alkyl group may indude one or two double or triple bonds. It is to be understood that where cyclic moieties are intended, at least three carbons in said alkyl must be present, and for the alkyl group to indude a carbon-carbon double or triple bond, at least two carbon atoms are required in the alkyl group. Where the alkyl moiety is defined as C,-C10 alkyl, this group indudes C6-C10 bicydo groups such as a bicyclo[3.1.1]heptylmethyl group.

The term "alkoxy", as used herein, unless otherwise indicated, includes -O-alkyl groups wherein alkyl is as defined above.

The term "aryl", as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl, as well as benzo-fused carbocydic moieties such as 5,6,7,8-tetrahydronaphthyl. 3 0 The term "4-10 membered heterocyclic", as used herein, unless otherwise indicated, includes aromatic and non-aromatic heterocyclic groups containing one or more heteroatoms each selected from O, S, and N, wherein each heterocyclic group has from 4-10 atoms in its ring system. Non-aromatic heterocyclic groups include groups having only 4 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. The heterocyclic groups include benzo-fused ring systems, and ring systems substituted with one or two oxo moieties. An example of a 5-membered heterocyclic group is thiazolyl, and an example of a 10-membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups are INTELLECTUAL PROPERTY OFFICE OF N.Z. - 2 DEC 2004 pyrrolidinyl, piperidino, morpholino, thiomorpholino and piperazinyl. Non-aromatic heterocyclic groups include saturated and partially unsaturated ring systems. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl and thiazolyl. Heterocyclic groups having a fused benzene ring include chroman, 5 benzodihydrofuran and benzimidazolyl. Heterocyclic groups having one or two oxo moieties include phthalimide and uracil.

The term "5-10 membered heteroaryl", as used herein, unless otherwise indicated, includes aromatic heterocyclic groups containing one or more heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 5 to 10 atoms in its ring system. Examples of suitable 5-10 10 membered heteroaryl groups include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, (1,2,3,)- and (1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl. isoxazolyl, oxazolyl, pyrroly! and thiazolyl.

The term "desosaminyl", as used herein, unless otherwise indicated, refers to the group N(CH3)2 HC The term "cladinosyl", as used herein, unless otherwise indicated, refers to the group ch3o ch3 The term "4"-({R13)(R14)NCH2)ctadinosyr, as used herein, unless otherwise indicated, refers to the group CH3Q CH3 The term "4"-oxocladinosyr, as used herein, unless otherwise indicated refers to the group ch3o ch3 The term "isolated or purified form", as used herein, unless otherwise indicated, means isolated or purified from a reaction mixture, e.g., a reaction mixture containing a 15-membered azalide starting material that is then purified to contain at least about 95% of a compound of formula 2; bacterial culture or broth; or natural, e.g., plant or animal, source, using conventional purification 5 techniques such as chromatography, recrystallization and others known to those skilled in the art, as well as those methods disclosed herein.

The phrase "pharmaceutically acceptable salt(s)", as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds disclosed herein. The compounds disclosed herein that are basic in nature are capable of 10 forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts. Le^, salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, 15 pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (Le., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. The compounds disclosed herein that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned 20 above.

Preferably, the compounds of formula 1_ can be used as antibacterial and an antiprotozoa agents when in admixture with the compounds of formula 5. In such a case, the ratio of a compound of formula 1_ to a compound of formula 5 ranges from about 2:98 to about 40:60.

Those compounds disclosed herein that are acidic in nature are capable of forming 25 base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali metal or alkaline earth metal salts and, particularly, the calcium, magnesium, sodium and potassium salts of the compounds.

Certain compounds that are disclosed herein may have asymmetric centers and therefore exist in different enantiomeric and diastereomic forms. Contemplated is the use of all optical isomers and stereoisomers of the compounds, and mixtures thereof, and to all pharmaceutical compositions and methods of treatment that may employ or contain them.

Also disclosed are the compounds described herein, and the pharmaceutically acceptable salts thereof, wherein one or more hydrogen, carbon or other atoms are replaced by isotopes thereof. Such compounds may be useful as research and diagnostic tools in metabolism pharmacokinetic studies and in binding assays.

INTELLECTUAL PROPERTY OFFICE OF N.Z. - 2 CEC 2004 RECEIVPn The present invention may be understood more fully by reference to the detailed description and illustrative examples which are intended to exemplify non-limiting embodiments of the invention.

Detailed Description of the Invention The compounds disclosed herein may be prepared according to Schemes 1 and 2 5 below, and to the description that follows. In the following Schemes, unless otherwise indicated, substituents R\ R2, R5, R\ R5, R6, R7, R8, R9. R10, R11 ,R12, R13, R14 and R15are as defined above.

Scheme 1 ch3o ch3 INTELLECTUAL PHOPERTY OFFICE OF NI. -2 DEC 2004 received Scheme 1 (Cont) 8 -17-Scheme 2 Cbz N(CH3)2 , wherein R9 = cladinosyl cbz-a DBTA TFAA > Swem ox. (-60°C) CH30 CH3 -18-Scheme 2 (Cont.) Cbz N(CH3)2 h2i Pd/C WTBE N(CH3)2 /' > fOCH3 |3R1</1 N0,„_ < HN(R13)(R14) .5, wherein rs = 4"-{(R13)(R14)NCH2)cladinosyl 12 The compounds disclosed herein are readily prepared. Referring to Scheme 1 above, starting compounds of formula 6 are readily available, either commercially or via ^ ® conventional organic synthesis. A preferred compound of formula 6 is erythromycin A (R10 = ethyl; R11 = -OH). The compounds of formula 6 are converted to compounds of formula 5 wherein R9 = cladinosyi by known means, such as are described in U.S. Patent No. 4,474,768 and 4,517,350. In general, the compounds of formula 6 are treated with hydroxylamine in the presence of base, preferably an inorganic base such as an alkali metal bicarbonate or carbonate, or an alkaline earth carbonate, in the presence of water and a water-soluble organic solvent, to afford the oxime compounds of formula 7. A preferred compound of formula 7 is that in which R10 = ethyl, and R11 = intellectual property office of NZ ' 2 DEC 2004 Dcr»r —OH. Preferably, the inorganic base is sodium carbonate, and the water-soluble organic solvent is methanol. The compounds of formula 7 are then treated with aqueous base and a reagent that converts the oxime hydroxyl group of the compounds of formula 7 into a leaving group, and ultimately provides the iminoether compounds of formula 8. Reagents useful in this regard include, 5 but are not limited to, p-toluenesulfonyl halides or anhydrides, methanesulfonyl halides or anhydrides, trifluoromethanesulfonyl halides or anhydrides, p-bromobenzenesulfonyl halides or anhydrides, and the like. Preferably, the reagent is p-toluenesulfonyl chloride. A preferred compound of formula 8 is that in which R10 = ethyl, and R11 = —OH. The compounds of formula 8 are then reduced with a conventional hydride reducing agent, preferably sodium borohydride, to 10 afford the compounds of formula 5 wherein R9 is cladinosyi. In a preferred embodiment, the compound of formula 5 is desmethylazithromycin.

Compounds of formula 5 are converted to compounds of formula 1_ by methods herein described. It will be understood by those skilled in the art that the compounds of formula 5 are converted to the compounds of formula 2 wherein R1 is trans with respect to the methyl group at 15 position 11 of formula 1, and is ch, oh OH ; R2 = methyl; R6, R7 and R8 = hydrogen; and R9 = cladinosyi. The compounds of ch, oh formuta 1_ wherein R1 = <*• ; R2 = methyl; R6, R7 and R8 = hydrogen; and R9 = cladinosyi can then be converted to other compounds of formula 1_, and to the compounds of formula 2, via conventional organic synthesis and via the methods described herein.

Compounds of formula 5 are converted to compounds of formula 15 by methods herein 25 described. It will be understood by those skilled in the art that the compounds of formula 5 are converted to the compounds of formula 1j5 wherein R1 is trans with respect to the methyl group at CH3 R11 R10. position 11 of formula 15, and is OH ; R2 = methyl; R6, R7 and R8 = hydrogen; and R9 = ch3 r11 cladinosyi. The compounds of formula 1j> wherein R1 = OH ■ p2 = methyl; R6, R7 and R8: hydrogen; and Rs = cladinosyi can then be converted to other compounds of formula 15, and to the 35 compounds of formula 2, via conventional organic synthesis and via the methods described herein.

It will be understood by those skilled in the art that in addition to the compounds of formula 6, other 14-membered macrolides susceptible to a Beckman-type ring expansion, such as for example, erythromycin B, erythromycin C and clarithromycin, can be converted into precursors of 13-membered azalides contemplated herein.

Where it is desired that the compounds of formula 1_ are those wherein R9 = 4"-((R13)(R14)NCH2)cladinosyl, the methods summarized in Scheme 2 can be employed.

For example, the 2' hydroxyl group of the desosaminyl group of the compounds of formula 5 can first be protected with a suitable protecting group, preferably with a benzyloxycarbonyl ("Cbz") group using Cbz-CI, to afford the compounds of formula 9. Such a reaction can be performed at a temperature of about -78°C to about room temperature, preferably at about 0°C. A preferred compound of formula 9 is that in which R10 = ethyl, and R11 = —OH. The 4" hydroxyl group of the cladinosyi group of the compounds of formula 9 can then be oxidized using standard oxidation conditions to afford the compounds of formula 10, which bear a 4"-oxocladinosyl group. A preferred compound of formula 10 is that in which R10 = ethyl, and R11 = —OH. Such oxidation conditions can be found, for example, in the Journal of Antibiotics, 1988, pages 1029-1047. Typical reaction conditions for the oxidation include: (a) Moffatt oxidation which employs N-ethyl-N'-(N,N-dimethylaminopropyl)carbodiimide and DMSO in the presence of pyridinium trifluoroacetate; or (b) Swem oxidation in which oxalyl chloride and DMSO in CH2Cl2 is followed by the addition of triethylamine or alternatively trifiuoracetic anhydride and DMSO in CH2CI2 is followed by the addition of triethylamine. Preferably, the oxidation is a Swem oxidation that is performed in the presence of trifluoroacetic anhydride, at a temperature of about -78°C to about 0°C. More preferably, the Swem 2 0 oxidation is performed at about -60°C.

The carbonyl group of the 4"-oxocladinosyl group of the compounds of formula 10 is then converted into an epoxide, to afford the compounds of formula 1_1. A preferred compound of formula 11 is that in which R10 = ethyl, and R11 = —OH. The compounds of formula 10 may be converted to the compounds of formula 1_1 by at least two methods. In one method (Method A), the compound of 25 formula Ij) is treated with (CH3)3S(0)X2, wherein X2 is halo, -BF4 or -PF6, preferably iodo, in the presence of a base such as potassium tert-butoxide, sodium tert-butoxide, sodium ethoxide, sodium hydride, 1,1,3,3-tetramethylguanidine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicylo[4.3.0]non-5-ene, potassium ethoxide, or sodium methoxide, preferably a sodium-containing base such as sodium hydride, in a solvent such as THF, an ether solvent. 3 0 dimethylformamide (DMF), or methyl sulfoxide (DMSO), or a mixture of two or more of the foregoing solvents, at a temperature within the range of about 0°C to about 60°C; alternatively, with trimethylsulfonium bromide and a strong base, such as potassium /erf-butoxide, in the presence of CH2CI/THF.

In a second method (Method B), the compounds of formula 10 are treated with (CH3)3SX2, 35 wherein X2 is halo, -BF4 or -PF6l preferably -BF„, in the presence of a base such as potassium tert-butoxide, sodium ethoxide, sodium tert-butoxide, sodium hydride, 1,1,3,3-tetramethylguanidine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicylo[4.3.0]non-5-ene, potassium ethoxide, potassium INTELLECTUAL PROPERTY OFFICE OF N2. -2 DEC 2004 hexamethyldisilazide (KHMDS) or sodium methoxide. preferably KHMDS, in a solvent such as THF, an ether solvent, DMF, or DMSO, or a mixture of two or more of the foregoing solvents, at a temperature within the range of about -78°C to about 60°C.

Preferably, Method B is employed, wherein trimethylsulfonium bromide and potassium tert-5 butoxide are used.

The protecting, preferably Cbz, group of the desosaminyl group of the compounds of formula _11_ is hydrogenolyzed in the presence of H2, Pd/C and any suitable organic solvent, preferably methyl fert-butyl ether ("MTBE"), to afford the compounds of formula 12. A preferred compound of formula 12 is that in which R10 = ethyl, and R11 = —OH. Lastly, the epoxide group at 10 the 4" position of the cladinose sugar of the compounds of formula 12 is ring-opened using HN(R,3)(R14), preferably in the presence of potassium iodide, to provide the compounds of formula 5, wherein R9 = 4"-((R13)(R14)NCH2)cladinosyt. Compounds of the formula HN(R13)(R14) include primary and secondary alkyl, alkenyl and alkynyl amines, and are readily obtainable by those skilled in the art. Such a reaction advantageously proceeds at a temperature of about room temperature to 15 about 80°C, preferably at about 30°C to about 60°C. The compounds of formula 5, wherein R9 = 4"-((R13)(R14)NCH2)cladinosyl can be converted to compounds of formulae 1_ and 15 using the methods disclosed herein. it is to be pointed out that the conversion of the compounds of formula 11_ to the compounds of formula 5 wherein R9 = 4"-((R13)(R14)NCH2)cladinosyl can be accomplished in one step by treating 2 0 the compounds of formula 10 with HN(R13)(R14) in the presence of methanol, which removes the protecting group from the desosaminyl group of the compounds of formula 10. Preferably, such a reaction is performed in the presence of potassium iodide.

In order to obtain the compounds of formula 5 wherein R9 = 4"-oxocladinose, the protecting, preferably Cbz, group that resides on the 2'-hydroxyl group of the desosaminyl group of the 25 compounds of formula 10 is simply removed. Procedures for removing such protecting groups can be found, for example, in Greene et al., supra.

Surprisingly and unexpectedly, the present inventors have discovered that the compounds of formula 5, which are 15-membered azalides, are convertible to the compounds of formulae 1 and 15, which are 13-membered azalides. 3 0 The present inventors have discovered that the conversion of the compounds of formula 5 to the compounds of formulae land 15, preferably wherein R6, R7 and R® are hydrogen, and preferably wherein R1 is trans with respect to the methyl group at position 11 of formulae land 15, can be effected by contacting a compound of the formula 5 with an acid or base.

Acids useful in this regard include, but are not limited to, inorganic acids, such as 3 5 hydrochloric, hydrobromic, hydroiodic, hydrofluoric, sulfuric and nitric acids; and organic acids, such as formic, acetic, trifluoroacetic, methanesulfonic, trifluoromethanesulfonic, benzenesulfonic and p-toluenesulfonic acids. The inorganic acids are preferably used in the form of their aqueous ] \ solutions; more preferably, the inorganic acids are used in the form of their dilute, e.g., <2M, aqueous solutions. The organic acids can be used in the form of dilute aqueous or organic solutions, wherein the organic solution comprises a solvent that sufficiently solvates both the organic acid and the compound of formula 5.

Bases useful in this regard include inorganic bases, such as hydroxides of sodium, lithium, potassium, magnesium or calcium; carbonates and bicarbonates of sodium, lithium or potassium; and carbonates of magnesium or calcium bicarbonate or carbonate. Also useful are organic bases, such as triethylamine, ethyldiisopropylamine, pyridine, 4-dimethylaminopyridine, collidine, lutidine, and mixtures thereof. Preferably, the inorganic bases are used in the form of dilute aqueous 10 solutions. Preferably the organic bases are used in the form of dilute organic solutions. Inorganic or organic bases are preferred over inorganic or organic acids.

The compounds of formula 5 can be added to the acid or base, or vice versa. Either way, the reaction of the compounds of formula 5 with the acid or base is facilitated by heating a mixture of a compound of formula 5 and an acid or base at a temperature of about room temperature to about 15 100°C, preferably at a temperature of about room temperature to about 606C, and more preferably at a temperature of about 30°C to about 40°C. Such heating can occur for a period of about 20 minutes to about 48h, preferably for a period of about 1 hour to about 36h.

Also disclosed is a method of preparing a compound of formulae ! and 15, or a pharmaceutically acceptable salt thereof, which comprises the step of heating a compound 2 0 of formula 5 in the presence of solvent.

Such heating is achieved at a temperature of about room temperature to about 100°C, preferably at a temperature of about room temperature to about 60°C, and more preferably at a temperature of about 30°C to about 40°C. The heating can occur for a period of about 20 minutes to about 48h, preferably for a period of about 1h to about 36h. 2 5 Useful solvents are those that sufficiently solvate the compounds of formula 5, and include, but are not limited to, lower alkanols, diethyl ether, acetone, acetonitrile, tetrahydrofuran, ethyl acetate, benzene, toluene, chloroform, metheylene chloride, dimethylformamide, dimethylsulfoxide, N-methylpyrrolidinone, and the like, and mixtures thereof.

However, the present inventors have found that surprisingly and unexpectedly, the 3 0 conversion of compounds of formula 5 to compounds of formulae 1_and 15 proceeds most rapidly in a solvent system that comprises a protic solvent. Useful protic solvents include, but are not limited to, lower alkanols. such as methanol, ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol and sec-butanol; phenolic compounds, such as phenol, halophenols. naphthols and the like; water; and mixtures thereof. It is to be pointed out, however, that the protic solvent is not a carboxylic acid. 3 5 Where the solvent system comprises a protic solvent, the protic solvent is present in an amount of about 10% to about 75% by volume, preferably in an amount of about 25% to about 60% by volume.

INTELLECTUAL PROPERTY OFFICE" OF N.2. -12 DEC 20M received lt will be understood by those skilled in the art that the protic solvent will be miscible in the solvent in which the compound of formula 5 is heated, when heated at the heating temperature.

Preferably, the solvent system comprises acetonitrile. More preferably, the solvent system further comprises a lower alkanol or water. Where the solvent system comprises a lower alkanol, 5 the lower alkanol is preferably methanol.

The compounds of formulae 1_and 15 can be isolated or purified via standard means, e.g., recrystallization; chromatography using a column, preparative plate or CHROMATOTRON® device; or by other means know to those skilled in the art. Where chromatography is employed to isolate or purify the compounds of formulae 1_and 15, the present inventors have discovered that an eluent 10 system that comprises a hydrocarbon solvent and an organic amine provides enhanced separation results, relative to other eluent systems. Hydrocarbon solvents useful in this regard include, but are not limited to, pentane, hexane or hexanes, heptane, petroleum ether, benzene, toluene, xylenes, and the like. Preferably, the hydrocarbon solvent is hexane or hexanes. Useful organic amines include, but are not limited to, diethylamine, triethylamine, ethyldiisopropylamine, pyridine, 4-15 dimethylaminopyridine, collidine, lutidine, and mixtures thereof. Preferably, the organic amine is diethylamine.

Advantageously, the eluent system that comprises a hydrocarbon solvent and an organic amine further comprises a polar organic solvent. The present inventors have discovered that the addition of the polar organic solvent to the eluent system provides a better separation of the 2 0 compounds of formulae 1_and 15 from other compounds, relative to an eluent system that does not comprise a polar organic solvent Useful polar organic solvents include, but are not limited to, lower alkanols, acetonitrile, dimethyfformamide, dimethylsulfoxide, N-methylpyrrolidinone. 1,4-dioxane, tetrahydrofuran, diethyl ether, ethyl acetate, and the like. Preferably, the polar organic solvent is acetonitrile. More preferably, the eluent system comprises hexanes, diethylamine and acetonitrile. 25 The proportions of hydrocarbon solvent, organic amine, and optionally polar organic solvent can vary, but generally, the ratio of hydrocarbon solvent to organic amine will range from about 10:1 to about 1:1, preferably about 7:1 to about 2:1. Where the eluent system further comprises a polar organic solvent, the eluent system will contain the polar organic solvent at between about 1% to about 15% by volume, preferably at between about 1.5% to about 10% by volume. 3 o Preferred compounds of formulae i and 15 are those wherein R1 is acetyl. Especially preferred are compounds of formula 1_ in which R1 acetyl, R6, R7 and Re are hydrogen, and R9 = cladinosyi ("Compound 1B", Table 1); and in which R1 = acetyl, R6 = methyl, R7 and R6 are hydrogen, and R9 is cladinosyi ("Compound 1E", Table 1).

In addition to being useful as antibacterial and antiprotozoa agents, the compounds of 3 5 formulae 1_and 15 wherein R1 is acetyl are useful as intermediates for obtaining other compounds of formulae 1_and 15, as described below.

INTELLECTUAL PROPERTY OFFICE OF NZ - 2 CEC 2004 — RECEIVED In general, compounds of formulae 1_and 15 wherein R1 is acetyl are obtained by oxidizing CH-> OH CH3 compounds of formulae 1 and 15 wherein R1 = , which can be obtained by the methods discussed herein. The oxidation reaction proceeds in the presence of lead tetraacetate, sodium periodate, or any other oxidizing agents that convert 1-methyl-1,2-diols to methyl ketones. Reaction conditions useful for oxidizing a 1-methyl-1,2-diol to a methyl ketone are known to those skilled in the art Preferably, the oxidation reaction proceeds in the presence of about 1.0 to about 1.5 equivalents of lead tetraacetate per equivalent of the compound of formulae 1_and 15, and at the temperature of about -78°C to room temperature, preferably at about -10°C to about 10°C, and for a duration of about 10 minutes to about 6h.

The compounds of formulae 1_and 15 wherein R1 = acetyl can be converted to the compounds of formulae 1_and 15 wherein R1 = 3-N,N-dimethylamino-2-propenoyl. Such a reaction advantageously proceeds in the presence of an excess of dimethylformamide dimethyl acetal.

Preferably, this reaction is performed in the absence of additional solvent The compounds of formulae 1_and 15 wherein R1 = 3-N,N-dimethylamino-2-propenoyl can be converted to the compounds of formulae 1_and 15 wherein R1 = 1-N-substituted-3-pyrazofyl by treating the compounds of formula 1_ wherein R1 = 3-N,N-dimethylamino-2-propenoyl with about 1 to g about 10 equivalents of a 1 -substituted hydrazine, or acid salt thereof. If an acid salt of a 1 -substituted hydrazine is used, then the reaction mixture containing the acid salt and the compound of formulae 1_and 15 preferably also contains a weak organic, or alkali metal, base to buffer the reaction mixture. Preferable organic bases include diisopropylethylamine, pyridine, 4-dimethytaminopyridine, lutidine, coilidine, and the like, and mixtures thereof. Preferably, the ^ organic base is diisopropylethylamine. The reaction between the compounds of formulae 1_and 15 wherein R1 = 3-N,N-dimethylamino-2-propenoyl and the 1-substituted hydrazine or its acid salt, proceeds in a protic solvent, such as one described above, at a temperature ranging from about 50°C to about 115°C, for a duration of about 1 h to about 5 days. Preferably, the protic solvent is 2-methoxyethano! or 2-propanol.

The compounds of formulae 1_and 15 wherein R1 = 3-N,N-dimethylamino-2-propenoyl can NL /,N be converted to the compounds of formulae 1_and 15 wherein R' = NHR3 according to the procedure used to obtain the compounds of formulae 1_and 1j> wherein R1 = 1-N-substituted-3- 3 5 pyrazolyl, except that R3N(H)C(=NH)NH2 is used in place at the 1-substituted hydrazine. Methods for obtaining R3N(H)C(=NH)NH2 are known to those skilled in the art.

The compounds of formulae 1_and 15 wherein R' = 3-N,N-dimethylamino-2-propenoyl can O II w / N—N '(&)* be converted to the compounds of formulae 1_and 1_5 wherein R5 = by reacting the compound of formulae 1_and 15 wherein R1 = 3-N,N-dimethylamino-2-propenoyl with y—N3 in an aprotic solvent at a temperature ranging from about 50°C to about (R*Vrv /—^ 110°C, for a duration of about 1 hour to about 5 days. Methods for obtaining are known to those skilled in the art. Useful aprotic solvents include, but are not limited to, pentane, hexanes, heptane, toluene, benzene, xylenes, petroleum ether, tetrahydrofuran, 1,4-dioxane and the like. Preferably, the aprotic solvent is toluene.

The compounds of formulae 1_and 15 wherein R1 = 3-N,N-dimethylamino-2-propenoyl can be converted to the compounds of formulae 1_and 15 wherein R1 = 3-isoxazolyl by reacting the compound of formulae 1 and 15 wherein R1 = 3-N,N-dimethyI-2- propenoyl with about 1 to about 10 2° equivalents of hydroxytamine or an acid salt thereof. The reaction used to obtain compounds of formulae 1_and 15 wherein R1 = 3-isoxazolyl is preferably performed in an aprotic solvent, such as one described above, at a temperature at or about room temperature for a duration of about one to about five days. More preferably, the aprotic solvent is 1,4-dioxane.

The compounds of formulae 1_and 15 wherein R1 = acetyl can be converted into the 25 compounds of formula 2 wherein X = —C(O)— by reacting the compound of formulae 1_and 15 wherein R1 = acetyl with an excess of dimethylformamide dimethyl acetal to afford the compounds of formulae 1_and 15 wherein R1 is 3-N,N-dimethylamino-2-propenoyl, described above. The compounds of formulae 1_and 15 wherein R1 is 3-N,N-dimethylamino-2-propenoyl are intramoleculariy cyclized to provide the compounds of formula 2 wherein X = —C(O)—. Such 3 0 intramolecular cyciization advantageously proceeds at high temperature, e.g., at about 110°C or above. Accordingly, intramolecular cyciization is effected by heating a mixture of a high boiling solvent and a compound of formulae 1_and 15 wherein R1 is 3-N,N-dimethylamino-2-propenoyl to a temperature of about 110°C or above for a duration of about 6h to about 48h, preferably for about 12h to about 24h. Suitable high boiling solvents include, but are not limited to, toluene, xylenes, 35 chlorobenzene, dimethylformamide. 2-methoxyethanol, dimethylsulfoxide and the like. Preferably, the high boiling solvent is 2-methoxyethanol.

The compounds of formula 2 wherein X = —C(O)— are converted to the compounds of formula 2 wherein X = —CH(OH)— fay treating the compounds of formula 2 wherein X = —C(O)— with a hydride reducing agent such as NaBH4, LiAlH4, NaAlH4, a SELECTIDE® reducing agent, or another hydride reagent known to those skilled in the art.

The compounds described herein may have asymmetric carbon atoms and therefore exist in different enantiomeric and diastereomeric forms. Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known to those skilled in the art, for example, by chromatography or fractional crystallization. Enantiomers may be separated by converting the enantiomeric mixtures into a diastereomeric 10 mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. The use of all such isomers, including diastereomer mixtures and pure enantiomers, are contemplated.

The compounds disclosed herein that are basic in nature are capable of forming a 15 wide variety of different salts with various inorganic and organic acids. Although such salts must be pharmaceutically acceDtabie for administration to mammals, it is often desirable in practice to initially isolate the compound ~ from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent and subsequently convert the latter free base to a pharmaceutically 2 0 acceptable acid addition salt. The acid addition salts of the base compounds disclosed herein are readily prepared by contacting the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent, such as methanol or ethanoi. Upon careful evaporation of the solvent the desired solid salt is readily obtained. The desired salt can also be precipitated from a solution of the free base in an organic 25 solvent by adding to the solution an appropriate mineral or organic acid.

Those compounds disclosed herein that are acidic in nature are capable of forming base salts with various cations. For compounds that are to be administered to mammals, fish or birds such salts must be pharmaceutically acceptable. Where a pharmaceutically acceptable salt is required, it may be desirable to initially isolate the compound of the present invention from the 3 o reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter to a pharmaceutically acceptable salt in a process analogous to that described above relating to the conversion of pharmaceutically unacceptable acid addition salts to pharmaceutically acceptable salts. Examples of base salts include the alkali metal or alkaline-earth metal salts and particularly the sodium, amine and potassium salts. These salts are all prepared by conventional techniques. 3 5 The chemical bases which are used as reagents to prepare the pharmaceutically acceptable base salts of this invention are those which form non-toxic base salts with the acidic compounds of the present invention. Such non-toxic base salts include those derived from such pharmacologically iNltLLECTUAyWfcHJY OFFICEj m 2 DEC 2004 I D C — acceptable cations as sodium, potassium, calcium, magnesium, various amine cations, etc. These salts can easily be prepared by contacting the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable bases with cations such as sodium, potassium, calcium, magnesium, various amine cations, etc., and then evaporating the resulting 5 solution to dryness, preferably under reduced pressure. Alternatively, they may also be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali metal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before. In either case, stoichiometric quantities of reagents are preferably employed in order to ensure completeness of reaction and maximum yields of the desired final product. 10 The antibacterial and antiprotozoa activity of the compounds disclosed herein against bacterial and protozoa pathogens is demonstrated by the compounds' ability to inhibit growth of defined strains of human or animal pathogens.

Assay I Assay I, described below, employs conventional methodology and interpretation criteria and 15 is designed to provide direction for chemical modifications that may lead to compounds that circumvent defined mechanisms of macroiide resistance. In Assay 1, a panel of bacterial strains is assembled to include a variety of target pathogenic species, including representatives of macroiide resistance mechanisms that have been characterized. Use of this panel enables the chemical structure/activity relationship to be determined with respect to potency, spectrum of activity, and 20 structural elements or modifications that may be necessary to obviate resistance mechanisms. Bacterial pathogens that comprise the screening panel are shown in the table below. In many cases, both the macrolide-susceptibie parent strain and the macrolide-resistant strain derived from it are available to provide a more accurate assessment of the compounds' ability to circumvent the resistance mechanism. Strains that contain the gene with the designation of ermA/ermB/ermC are 25 resistant to macrolides, lincosamides, and streptogramin B antibiotics due to modifications (methylation) of 23S rRNA molecules by an Erm methylase, thereby generally prevent the binding of all three structural classes. Two types of macroiide efflux have been described; msrA encodes a component of an efflux system in staphylococci that prevents the entry of macrolides and streptogramins while mefA/E encodes a transmembrane protein that appears to efflux only 3 o macrolides. Inactivation of macroiide antibiotics can occur and can be mediated by either a phosphorylation of the 2'-hydroxyl (mph) or by cleavage of the macrocyclic lactone (esterase). The strains may be characterized using conventional polymerase chain reaction (PCR) technology and/or by sequencing the resistance determinant. The use of PCR technology in this application is described in J. Sutcliffe et al., "Detection Of Erythromycin-Resistant Determinants By PCR", 3 5 Antimicrobial Agents and Chemotherapy, 40(11), 2562-2566 (1996). The assay is performed in microtiter trays and interpreted according to Performance Standards for Antimicrobial Disk Susceptibility Tests - Sixth Edition; Approved Standard, published by The National Committee for WftLLECTUAL HKUHtHTY OFFICE! of N.Z. I * 2 DEC 2004 J RPrCll/r-. I Ciinical Laboratory Standards (NCCLS) guidelines; the minimum inhibitory concentration (MIC) is used to compare strains. Compounds are initially dissolved in dimethytsutfoxide (DMSO) as 40 mg/ml stock solutions.

Strain Designation Macroiide Resistance Mechanism(s) Staphylococcus aureus 1116 susceptible parent Staphylococcus aureus 1117 ermB Staphylococcus aureus 0052 susceptible parent Staphylococcus aureus 1120 ermC Staphylococcus aureus 1032 msrA, mph, esterase Staphylococcus hemolyticus 1006 msrA, mph Streptococcus pyogenes 0203 susceptible parent Streptococcus pyogenes 1079 ermB Streptococcus pyogenes 1062 susceptible parent Streptococcus pyogenes 1061 ermB Streptococcus pyogenes 1064 ermB Streptococcus agalactiae 1024 susceptible parent Streptococcus agalactiae 1023 ermB Streptococcus pneumoniae 1016 susceptible Streptococcus pneumoniae 1046 ermB Streptococcus pneumoniae 1095 ermB Streptococcus pneumoniae 1175 mefE Streptococcus pneumoniae 0085 susceptible Haemophilus influenzae 0131 susceptible Moraxelia catarrhalis 0040 susceptible Moraxella catarrhalis 1055 erythromycin intermediate resistance Escherichia coli 0266 susceptible Assay II is utilized to test for activity against Pasteurella multocida and Assay III is utilized to test for activity against Pasteurella haemolytica.

Assay II This assay is based on the liquid dilution method in microliter format. A single colony of P. multocida (strain 59A067) is inoculated into 5 ml of brain heart infusion (BHl) broth. The test compounds are prepared by solubilizing 1 mg of the compound in 125 pi of dimethylsulfoxide (DMSO). Dilutions of the test compound are prepared using uninoculated BHl broth. The concentrations of the test compound used range from 200 pg/ml to 0.098 pg/ml by two-fold serial dilutions. The P. multocida inoculated BHl is diluted with uninoculated BHl broth to make a 104 cell suspension per 200 pi. The BHl cell suspensions are mixed with respective serial dilutions of the test compound, and incubated at 37°C for 18 hours. The minimum inhibitory concentration (MIC) is equal to the concentration of the compound exhibiting 100% inhibition of growth of P. multocida as determined by comparison with an uninoculated control.

J Assay III This assay is based on the agar dilution method using a Steers Replicator. Two to five colonies isolated from an agar plate are inoculated into BHl broth and incubated overnight at 37°C with shaking (200 rpm). The next morning, 300 ul of the fully grown P. haemofytica preculture is 5 inoculated into 3 mi of fresh BHl broth and is incubated at 37°C with shaking (200 rpm). The appropriate amounts of the test compounds are dissolved in ethanol and a series of two-fold serial dilutions are prepared. Two ml of the respective serial dilution is mixed with 18 ml of molten BHl agar and solidified. When the inoculated P. haemolytica culture reaches 0.5 McFarland standard density, about 5 pi of the P. haemolytica culture is inoculated onto BHl agar plates containing the 10 various concentrations of the test compound using a Steers Replicator and incubated for 18 hours at 37°C. Initial concentrations of the test compound range from 100-200 pg/ml. The MIC is equal to the concentration of the test compound exhibiting 100% inhibition of growth of P. haemolytica as determined by comparison with an uninoculated control.

Assay IV The in vivo activity of the compounds disclosed herein can be determined by conventional animal protection studies well known to those skilled in the art, usually carried out in mice.

Mice are allotted to cages (10 per cage) upon their arrival, and allowed to acclimate for a minimum of 48 hours before being used. Animals are inoculated with 0.5 ml of a 3 x 103 CFU/ml 20 bacterial suspension (P. multocida) strain 59A006) intraperitonealiy. Each experiment has at least 3 non-medicated control groups including one infected with 0.1X challenge dose and two infected with 1X challenge dose; a 10X challenge data group may also be used. Generally, all mice in a given study can be challenged within 30-90 minutes, especially if a repeating syringe (such as a Cornwall® syringe) is used to administer the challenge. Thirty minutes after challenging has begun, 25 the first compound treatment is given. It may be necessary for a second person to begin compound dosing if all of the animals have not been challenged at the end of 30 minutes. The routes of administration are subcutaneous or oral doses. Subcutaneous doses are administered into the loose skin in the back of the neck whereas oral doses are given by means of a feeding needle. In both cases, a volume of 0.2 ml is used per mouse. Compounds are administered 30 minutes, 4 3 0 hours, and 24 hours after challenge. A control compound of known efficacy administered by the same route is included in each test. Animals are observed daily, and the number of survivors in each group is recorded. The P. multocida model monitoring continues for 96 hours (four days) post challenge.

The PDso is a calculated dose at which the compound tested protects 50% of a group of 3 5 mice from mortality due to the bacterial infection which would be lethal in the absence of drug treatment.

INTELLECTUAL PROPERTY OFFICE of N.Z. - 2 DEC 2004 received / / 1 / J The compounds used show antibacterial activity in one of the above-described assays, particularly in Assay IV.

The compounds disclosed herein^and the pharmaceutically acceptable salts thereof (hereinafter "the active compounds"), may be administered through oral, parenteral, topical, or rectal 5 routes in the treatment of bacterial and protozoal infections. In general, these compounds are most desirably administered in dosages ranging from about 0.2 mg per kg body weight per day (mg/kg/day) to about 200 mg/kg/day in single or divided doses (i.e., from 1 to 4 doses per day), although variations will necessarily occur depending upon the species, weight and condition of the subject being treated and the particular route of administration chosen. However, a dosage level 10 that is in the range of about 4 mg/kg/day to about 50 mg/kg/day is most desirably employed. Variations may nevertheless occur depending upon the species of mammal, fish or bird being treated and its individual response to said medicament, as well as on the type of pharmaceutical formulation chosen and the time period and interval at which such administration is carried out In some instances, dosage levels below the lower limit of the aforesaid range may be more than 15 adequate, while in other cases still larger doses may be employed without causing any harmful side effects, provided that such larger doses are first divided into several small doses for administration throughout the day.

The active compounds may be administered alone or in combination with pharmaceutically acceptable carriers or diluents by the routes previously indicated, and such administration may be 2 0 carried out in single or multiple doses. More particularly, the active compounds may be administered in a wide variety of different dosage forms, i.e., they may be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, and the tike. Such carriers include solid 2 5 diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Moreover, oral pharmaceutical compositions can be suitably sweetened and/or flavored. In general, the active compounds are present in such dosage forms at concentration levels ranging from about 5.0% to about 99% by weight.

For oral administration, tablets containing various excipients such as microcrystalline 3 0 cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (and preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar 3 5 type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the active compound may be / J combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.

For parenteral administration, solutions of an active compound in either sesame or peanut 5 oil or in aqueous propylene glycol may be employed. The aqueous solutions should be suitably buffered (preferably pH greater than 8) if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical 10 techniques will known to those skilled in the art.

Additionally, it is also possible to administer the active compounds disclosed herein topically and this may be done by way of creams, jellies, gels, pastes, patches, ointments and the like, in accordance with standard pharmaceutical practice.

For administration to animals other than humans, such as cattle or domestic animals, the 15 active compounds may be administered in the feed of the animals or orally as a drench composition.

The active compounds may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. 2 0 The active compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylrnethacrylamide phenyl, polyhydroxyethylaspartamide-phenol or polyethyleneoxide-polylysine substituted with palmitoylresidues. Furthermore, the active compounds may be coupled to a class of biodegradable polymers useful in achieving controlled 25 release of a drug, for example, polylactic acid, polyglycotic acid, copolymers of polylactic and polyglycoiic acid, polyepsilon caprolactone, polyhydroxy butyric add, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

The following Examples further illustrate the method and intermediates of the compounds 3 o disclosed herein. It is to be understood that the present invention is not limited to the specific details of the Examples provided below.

The compounds of Examples 1-12 have the general formula 3 below, with the R' and R6 substituents indicated in Table 1, below. The compounds were prepared as described in Examples 1-12.

INTELLECTUAL PROPERTY OFFICE OF N.Z. - 2 DEC 2004 received Table 1 Compound 1A 1B 1C 1D 1E 1F 1G 1H 11 1J 1K 1L 1M OH acetyl 3-N,N-dimethylamino-2-propenoyl 3-pyrazolyl acetyl 3-N ,N-dimethylamino-2-propenoyl 3-pyrazolyl 1 -N-methyl-3-pyrazolyl 1 -N-benzyl-3-pyrazolyl 1 -N-(3-hydroxybenzyl)-3-pyrazolyl (2-{4-fiuorophenyl)-3-pyrimidinyl (2-phenylamino)-3-pyrimidinyl 1 -N-methyl-5-pyrazolyl ! hydrogen hydrogen hydrogen hydrogen methyl methyl methyl methyl methyl methyl methyl methyl methyl Example 1 Compound 1A. Desmethylazithromycin (30 g, 41 mmol) was added to deionized water (2L), and then acetonitrile was added to effect complete dissolution (total volume was approximately 4.5 5 L). The resulting mixture was allowed to stir at ambient temperature for 2 days, at which time HPLC indicated the presence of a new peak (approx. 22% by peak area). The acetonitrile was removed in vacuo. To the resulting residue was added potassium carbonate (30g). followed by methylene chloride (0.3 L). The mixture was shaken, and the lower organic phase removed. The aqueous phase was re-extracted with methylene chloride (2 x 0.3 L). Combined organic phases were dried 10 over sodium sulfate, and then concentrated in vacuo to afford a dry foam (30 g), which was purified on a slurry-packed silica gel column using 5/1/0.5 (v/v/v) hexanes-diethylamine-acetonitrile. During the separation, the solvent system was switched to 4/1/0.1, and. finally, 3/1.5/0.5 hexanes-diethylamine-acetonitrile. Concentration of the appropriate, late-running fractions afforded Compound 1A as a dry foam.

Example 2 Compound 1B. To a solution of Compound 1A (7.63 g, 10.41 mMole) in methylene chloride (100 mL) at 0°C was added in one portion lead(IV) acetate (5.54 g, 12.49 mMole). The resulting mixture was stirred for 30 minutes at 0°C and then quenched with a saturated solution of aqueous sodium bicarbonate (100 mL). The mixture was transferred to a separatory funnel and the 2 0 methylene chloride layer was removed. The aqueous layer was extracted with methylene chloride (2 x 50 mL). The combined methylene chloride fractions were washed with brine (50 mL), dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 0.2% ammonium hydroxide (10% aqueous)/5% methanol/94.8% methylene chloride to afford Compound 1B (5.64 g, 8.43 mMole) as a white solid. 25 Example 3 Compound 1C. Compound 1B (100 mg, 0.15 mMole) was dissolved in dimethylformamide dimethyl acetal (2 mL) and heated to reflux under nitrogen for 8 h. The mixture was allowed to cool to room temperature and then diluted with ethyl acetate (25 mL). The mixture was washed with water (10 mL) and brine (10 mL). The ethyl acetate solution was dried over magnesium sulfate, 30 filtered, and then concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel, eluting with 0.2% ammonium hydroxide (10% aqueous)/10% methanol/methylene chloride to provide Compound 1C (yield: 65 mg, 60%).

Example 4 Compound 1D. Compound 1C (100 mg, 0.14 mMole) and hydrazine monohydrate (5 mL, 0.15 mmole) were dissolved in 2-methoxyethanol (1.5 mL) and heated to 105°C under nitrogen.

After 2 h the mixture was allowed to cool to room temperature, and then concentrated under reduced 5 pressure. The residue was purified by flash chromatography on silica gel, eluting with 0.2% ammonium hydroxide (10% aqueous)/10% methanol/methylene chloride to provide Compound 1D as a white solid (yield: 58 mg, 60%).

Example 5 Compound 1E. To a solution of Compound 1B (3.9 g, 5.8 mMole) in chloroform (58 mL) 10 was added formic acid (330 mL, 869 mMole) and formaldehyde (37% aqueous, 1.3 mL, 17.33 mMole). The mixture was heated to 60°C for 7 h. After cooling to room temperature, the mixture was transferred to a separatory funnel and washed with aqueous sodium bicarbonate (20 mL). The chloroform fraction was dried over magnesium sulfate, filtered and concentrated to provide Compound 1E (yield: 3.9 g, 98%), which was used without further purification. 15 Example 6 Compound 1F. Compound 1E was dissolved in dimethylformamide dimethyl acetal (25 mL) and heated to reflux under nitrogen for 36 h. The mixture was allowed to cool to room temperature, and then concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel, eluting with 0.2% ammonium hydroxide (10 % aqueous)/8% methanol/methylene 2 0 chloride to provide Compound 1F (yield: 1.36 g, 80%).

Example 7 Compound 1G. Compound 1F (250 mg, 0.34 mMole) and hydrazine monohydrate (16 mL, 0.5 mmole) were dissolved in 2-methoxyethanol (3.4 mL) and heated to 105°C under nitrogen. After 4 h, the mixture was allowed to cool to room temperature and then concentrated under reduced 2 5 pressure. The residue was purified by flash chromatography on silica gel. eluting with 0.2% ammonium hydroxide (10% aqueous)/10% methanol/methylene chloride to provide Compound 1G as a white solid.

Example 8 Compound 11. Compound 1F (250 mg, 0.34 mMole), benzyl hydrazine dihydrochloride 30 (73 mL, 0.37 mmole) and diisopropylethylamine (180 pL, 1.02 mMole) were dissolved in 2-methoxyethanol (3.5 mL) and heated to 105°C under nitrogen. After 48 h, the mixture was allowed to cool to room temperature and then concentrated under reduced pressure. The residue was purified by fiash chromatography on silica gel, eluting with 0.2% ammonium hydroxide (10% aqueous)/10% methanol/methylene chloride to provide Compound 11 as a white solid (yield: 35 137 mg, 50%).

Example 9 Compound 1J. Compound 1F (250 mg, 0.34 mMole), 3-hydroxybenzyl hydrazine dihydrochloride (142 mL, 0.68 mmole) and diisopropylethylamine (148 pL, 0.85 mMole) were dissolved in 2-propanol (3.5 mL) and heated to reflux under nitrogen. After 5 h, the mixture was 5 allowed to cool to room temperature and then concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel, eluting with 0.2% ammonium hydroxide (10% aqueous)/10% methanol/methylene chloride to provide Compound 1J as a white solid (yield: 147 mg, 53%).

Example 10 Compound 1K. Compound 1F (250 mg, 0.34 mMole), 4-fluorophenyl guanidine carbonate (240 mg, 0.68 mmole) and diisopropylethylamine (148 pL, 0.85 mMole) were dissolved in 2-propanol (3.5 mL) and heated to reflux under nitrogen. After 24 h, the mixture was allowed to cool to room temperature and then concentrated under reduced pressure. The residue was purified by flash chromatography on siiica gel, eluting with 0.2% acetonitrile/20% diethylamine/hexanes to provide 15 Compound 1K as a white solid (yield: 120 mg, 42%).

Example 11 Compound 1L. Compound 1F (125 mg, 0.168 mMole), phenyl guanidine carbonate (84 mg, 0.252 mmole) and potassium carbonate (70 mg, 0.5 mMole) were dissolved in 2-propanol (1.5 mL) and heated to reflux under nitrogen. After 48 h, the mixture was allowed to cool to room temperature 20 and then diluted with methylene chloride (25 mL). The mature was then washed with water (10 mL), dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel, eluting with 0.2% ammonium hydroxide (10% aqueous)/10% methanol/methylene chloride to provide Compound 1L (54 mg, 40%) as a white solid.

Example 12 Compounds 1H and 1M. Compound 1F (260 mg, 0.35 mMole) and methyl hydrazine monohydrate (56 pL, 1.05 mmole) were dissolved in 2-methoxyethanol (3.5 mL) and heated to 115°C under nitrogen. After 6 h, the mixture was allowed to cool to room temperature and then concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel, eluting with 1% acetonitrile/20% diethylamine/hexanes to provide Compound 1H (yield: 42 mg, 30 17%) and Compound 1M (yield: 21 mg, 8%) as white solids.

The compounds of Examples 13-14 have the general formula 4 below, with the X substituents indicated in Table 2, below. The compounds were prepared as described in Examples 13-14. 4 X -C{0 y -CH(OH y Example 13 Compounds 2A and 1C. Compound 1B (1.5 g, 2.23 mMole) was dissolved in 20 dimethylformamide dimethyl acetal (15 ML) and heated to 105°C for 16 h. After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was dissolved In 2-methoxyethanoi (25 mL) and heated to 125°C for 16 h. The mixture was allowed to cool to room temperature and then diluted with ethyl acetate (100 mL). The mixture was washed with water (2 x 20 mL) and brine (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced 2 5 pressure. The residue was purified by flash chromatography on silica gel, eluting with 0.2% ammonium hydroxide (10% aqueous)/10% methanol/methylene chloride to provide Compounds 2A (yield: 221 mg, 15%) and 1C (833 mg, 54%).

Example 14 Compound 2B. To a solution of Compound 2A (150 mg, 0.21 mMole) in ethanol (2 mL) at 3 o 0°C was added in one portion sodium borohydride (33 mg, 0.84 mMole). The mixture was stirred at 0°C for 2 h and then poured slowly into water (25 mL). The mixture was transferred to a separatory funnel and extracted methylene chloride (3 x 20 mL). The combined methylene chloride fractions were dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash chromatography on silica gel eluting with 0.2% ammonium hydroxide (10% aqueous)/5% 35 methanol/methylene chloride to afford Compound 2B (yield: 103 mg, 71%) as a white solid.

Table 2 Compound 2A 2B The compounds of Examples 15-17 have the general formula 14 below, with the R' substituents indicated in Table 3, below. The compounds were prepared as described in Examples 15-17.

CH/ ^(CHak ch3 CH2N(H)CH2CH2CH3 OH CH* OCH3 14 Table 3 Compound ch3 OH CH IN OH O Ph3 Example 15 3 0 Compound 1N (Method A). To a 2 l ertenmeyer flask was added desmethylazithromycin (190.5 g, 259.2 mmol), methylene chloride (572 mL), and magnesium sulfate (38 g). The mixture was stirred for 10 min then filtered into a 5 L round bottom flask. Additional methylene chloride (2285 mL) was added and the solution cooled to 0-5°C. CBZ-CI (58.4 mL) was then added over 10 min. The reaction stirred at -0°C for 6 hrs then at ambient temperature overnight. HPLC analysis 35 indicated the presence of residual starting material such that the reaction was re-cooled to -0®C and additional CBZ-CI (19.5 mL) was added in a single portion. The reaction stirred for 5.5 hrs at 0°C then for 2.5 hrs at ambient temperature. TLC indicated a complete reaction. The reaction was quenched with saturated aqueous sodium bicarbonate (953 mL) and the phases separated. The organic phase was dried over magnesium sulfate, then filtered and concentrated to afford the compound of formula 9 wherein R10 = ethyl, and R11 = —OH.

To a 5 L round bottom flask containing the compound of formula 9 wherein R15 = ethyl, and 5 R11 = —OH (225.3 g) in methylene chloride (901 mL) and DMSO (450 mL) at -65°C was added trifluoroacetic anhydride (82.4 mL). The temperature was maintained at -60°C throughout the addition which was complete in 9 min. The reaction stirred at -65 to -70°C for 20 min. The reaction was quenched with triethylamine (145 mL) then stirred at -60° to -65°C for 20 min. To the reaction mixture was then added water (1127 mL) over 3 min, at which point the temperature rose to -2°C. The reaction mixture was stirred for 10 min and the phases were allowed to separate. The organic phase was washed with water, (675 mL) then with saturated aqueous sodium chloride (675 mL). The organic phase was dried over magnesium sulfate then filtered and organic solvents removed by distillation. MTBE was added and distilled to remove all traces of methylene chloride and DMSO. Additional MTBE was added to a total volume of 3380 mL. Dibenzoyl-D-tartaric acid monohydrate (87.8 g) in MTBE (1126 mL) was added to form a thick slurry. The mixture was heated to reflux and stirred overnight. After cooling to ambient temperature, the solids were collected on a Buchner funnel and rinsed with MTBE. The solids were dried in a drying oven at 40°C to afford 258.3 g of the dibenzoyl tartrate salt of the compound formula 10 wherein R10 = ethyl, and R11 = —OH.

To a 3 L round bottom flask was added methylene chloride (800 mL) and the dibenzoyl 2 0 tartrate salt of the compound of formula 10 wherein R10 = ethyl, and R11 = —OH (188 g). Water (400 mL) and potassium carbonate (45.5 g) were added and the mixture stirred at ambient temperature for 5 min. The organic phase was separated, then washed with water (250 mL) and dried over magnesium sulfate. Drying agent was removed by filtration, and the resultant solution evaporated under a stream of nitrogen to a final volume of 623 mL to afford a free-base ketone. 25 To a 5 L round bottom flask was added THF (623 mL) and trimethylsulfonium bromide (74.7 g). The resultant slurry was cooled to -10°C and potassium tert-butoxide (54.4 g) added. The reaction mixture was stirred for 10 min at -10°C then cooled to -70°C over 5 min. A solution of the free-base ketone was added over 11 min, keeping the temperature between -60 and -65°C. HPLC indicated the reaction was complete after 90 min. The reaction was quenched at -60°C using a 30 solution of ammonium chloride (315 g) in water (1800 mL). The temperature rose to -5°C during the quench. The reaction mixture was warmed to 5-10°C, and the phases separated. The organic phase was dried over sodium sulfate then filtered and concentrated to afford the compound of formula 1_1_ wherein R1C = ethyl, and R11 = —OH, (117.4 g) as a yellow foam. HPLC indicated a purity of 61.4% by peak area.

To a solution of the compound of formula 21 wherein R10 = ethyl and R11 = —OH (275 g, 312 mmol) in dry methanol (2.75 L) was added potassium iodide (518 g, 3.12 mol) and n-propylamine (250 mL, 3.04 mol). The mixture was stirred overnight at 45°C. TLC indicated a complete reaction. The reaction was concentrated on a rotary evaporator and the residue partitioned between water 5 (2.5 L) and methylene chloride (2.5 L). The pH of the aqueous phase was adjusted to 6.7 using 3N aqueous HCI. The extraction was repeated one additional time. Combined aqueous phases were combined with fresh methylene chloride (1.5 L) and the pH of the aqueous phase adjusted to 8.5 using solid potassium carbonate. The phases were separated and the aqueous phase re-extracted twice with additional methylene chloride. Combined organic phases were dried over sodium sulfate, 10 then filtered. The filtrate was concentrated on a rotary evaporator to afford a beige foam (230 g). Purification of the foam was effected on a slurry-packed silica gel column using 19/3 (v/v) hexanes-diethylamine as the mobile phase. In this manner, 125 g of crude product afforded 72 g of the compound of formula 5, wherein R9 = 4"-(propylaminomethyl)cladinosyl, R10 = ethyl, and R11 = — OH, as a white, amorphous foam.

The compound of formula 5 wherein R9 = 4"-(propylaminomethyl)cladinosyl, R10 = ethyl, and R11 = -OH (10 g, 12.4 mmol), was dissolved in acetonitrile (0.5 L) at ambient temperature. Deionized water (1 L) was then added, which caused precipitation. Additional acetonitrile (0.5 L) was then added to afford a homogenous solution which was stirred at ambient temperature for 30 hrs. HPLC analysis indicated the formation of a new component that comprised -20% total peak area. 2 0 Organic solvent was removed on a rotary evaporator. Potassium carbonate (30 g) was added to the aqueous residue followed by methylene chloride (0.3 L). The mixture was shaken and the lower organic phase removed. Two additional extractions (2 x 0.3 L) were also performed. Combined organic phases were dried over sodium sulfate, then filtered and the resultant solution concentrated to a dry foam (-10 g). 2 5 The resultant mixture of the compound of formula 5 wherein, R9 = 4"- (propylaminomethyl)cladinosyl, R10 = ethyl, and R11 = -OH; and Compound 1N, was dissolved in a mixture of methylene chloride and 19/3 (v/v) hexanes-diethylamine, and placed on a slurry-packed silica gel column, then eluted with the 19/3 system. The eluant was switched to 19/6 hexanes-diethylamine in fraction 56. Fraction 9-17 were combined and concentrated to a dry foam which 30 contained only unreacted starting material. Fractions 52-72 were combined and concentrated, and contained Compound 1N (79% purity by HPLC).

Example 16 Compound 1N (Method B). The compound of formula 5 wherein R9 = 4"-(propylaminomethyl)cladinosyl, R10 = ethyl, and R'1 = -OH, was weighed into 6 vials (25 mg/vial). 35 Solvents (0.5 mL per) were added as indicated below: Vial Solvent A 2-propanol B acetonitrile C acetonitrile (0.35 mL)/water (0.35.mL) D acetone E methanol F benzene All vials were then heated to 50°C in an oil-bath for 5 hrs. TLC analysis using 6/1/0.1 (v/v/v) hexanes-diethylamine-acetonitrile) indicated the presence of Compound 1N in all vials. The greatest proportion, however, was in vials C and E which contained protic solvents.

Example 17 Compound 10. A mixture of the compound of formula 5 wherein R9 = 4"- (propylaminomethyl)cladinosyl, R10 = ethyl, and R11 = -OH; and Compound 1N (~15%)(0.8 g, 0.1 mmol) was dissolved in ethyl acetate (30 ml). Potassium carbonate (0.14 g, 1 mmol) and ethylene carbonate (0.5 g, 5.67 mmol) were then added, and the mixture heated to reflux under nitrogen overnight. TLC analysis using 19/3 (v/v) hexanes-diethylamine indicated the absence of both 15 starting materials.

The reaction mixture was then filtered, and the filtrate concentrated to afford a dark oil which was purified under nitrogen on a 4 mm CHROMATOTRON® (Harrison Research, Palo Alto, California) plate using (19/3 (v/v) hexanes-diethyiamine as the eluant. Fractions 8-13 were combined and concentrated; NMR analysis indicated that this product corresponded to the 11,12-2 0 cyclic carbonate of the starting material. Fractions 18-39 contained a less-mobile component that was re-purified on a 2mm plate using 3/1 (v/v) hexanes-diethylamine. Enriched fractions (16-23) were combined and re-run on a 1 mm plate in the above system to afford Compound 10 in fraction 20 (30 mg). TLC and HPLC indicated that the material was highly pure.

The present invention is not to be limited in scope by the specific embodiments disclosed in 25 the examples which are intended as illustrations of a few aspects of the invention and any embodiments which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the appended claims.

Claims (4)

WHAT WE CLAIM IS:

1. A method of treating a bacterial infection or a protozoa infection in a non-human mammal, fish, or bird which comprises administering to said mammal, fish or bird a therapeutically effective amount of a compound of the formula 1 N(CH3)R2 10 ch3 or a pharmaceutically acceptable salt thereof, wherein: CHj OH 15 CHj R1 is °n , acetyl, 3-N,N-dimethylamino-2-propenoyl, x , 1-N-methyl- 20 5-pyrazolyl, 3-pyrazolyl, 1-methyl-N-3-pyrazolyl, 1-N-benzyl-3-pyrazo!yl, 1 -n-(3-hydroxybenzyl)-3- o ■ N N\\ N—N 25 pyrazolyl, 3-isoxazolyl, NHR3 or ; R2 is hydrogen or C,-C4 alkyl; R3 is hydrogen, C,-C,0 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, -(CH2)m(C6-C10 aryl), -(CH2)m(C6-C,0 heterocyclic) or aryl, each, other than hydrogen, being optionally substituted with 1 to 3 substituents independently selected from halogen, cyano, nitro, trifluoromethyl, azido, -C(O)C,-C,0 30 alkyl, -C(O)C2-C10 alkenyl, -C(O)C2-C10 alkynyl, -00(0)0,-0,0 alkyl, -OC(O)C2-C10 alkenyl, - OC(O)C2-C10 alkynyl, -N(hydrogen, C,-C,0 alkyl, C2-C1c alkenyl or C2-C10 alkynyl)C(O)(C,-C,0 alkyl, C2-C10 alkenyl or C2-C10 alkynyl), -C(0)N(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl)(hydrogen, C.-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl), -N(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl), C,-C10 alkoxy. C6 C10 aryl, 5-10 membered heterocyclic, hydroxyl, methoxyl, C,-C,0 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-pyridylethyl, 3-pyridylethyl and 4-pyridylethyl; INTELLECTUAL PROPERTY OFFICE OF HZ. - 2 DEC 2004 -42- m is an integer ranging from 0 to 4; each R4 is hydrogen, -(CH2)m(C6-C10 aryl) or -(CH2)m(C6-C,0 heterocyclic), each, other than hydrogen, being optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido, -0(0)0,-0,0 alkyl, -C(O)C2-C,0 alkenyl, -C(0)C2-CK alkynyl, -5 OC(O)C,-C,0 alkyl, -OC(O)C2-C10 alkenyl, -OC(O)C2-C10 alkynyl, -N(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl)C(O)(C,-C)0 alkyl, C2-C,0 alkenyl or C2-C10 alkynyl), -C(0)N(hydrogen, C,-C10 alkyl, C2-C,0 alkenyl or C2-C,0 alkynyl)(hydrogen, 0,-0,0 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl), -N(hydrogen, C,-C,0 alkyl, C2-C,0 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C,0 alkyl, C2-C10 alkenyl or C2-C10 alkynyl), C,-C,0 alkoxy, C6-C10 aryl and 5-10 membered heterocyclic; 10 n is an integer from 0 to 5; R6 is hydrogen or methyl; each R7 is independently hydrogen, 0,-020 alkyl, alkenyl, C2-C2o alkynyl, -0(0)0,-020 alkyl, -0(0)02-020 alkenyl, -C(0)C2-C2o alkynyl, -C(O)N(H)C,-C,0 alkyl, -C(O)N(H)C2-c20 alkenyl, -C(0)N(H)C2-C2o alkynyl, -S02(0)C^-C2Q alkyl, -302(0)02*020 alkenyl, -302(0)02-020 alkynyl or -P042';;15 R8 is hydrogen or methyl;;or 4"-oxociadinosyl; and;R12 is C,-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, cyano, -CH2S(0)pC,-C10 alkyl, -CH2S(0)pC2-C,o alkenyl, -CH2S(O)pC2-C10 alkynyl, wherein p is an integer ranging from 0 to 2, -CH2O(C,-C,0 alkyl), -CH,0(C2-C10 alkenyl). -CH2O(C2-C10 alkynyl), -CH2N(hydrogen, C,-C10 alkyl, C2-25 C,o alkenyl or C2-C,0 alkynyl)(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or 02-0,0 alkynyl), -(CH2)m(C6-C,o aryl) or -(CH2)m(5-10 membered heteroaryl),.wherein m is an integer ranging from 0 to 4, and wherein the alkyl, alkenyl, alkynyl, aryl and heteroaryl moieties of the foregoing are optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido, -C(0)C,-C10 alkyl, -C(O)C2-Ci0 alkenyl, -C(O)C2-C10 alkynyl, -OC(O)C,-C10 alkyl, -OCfOJCj-3 0 C10 alkenyl, -OC(O)C2-C,0 alkynyl, -N(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyi or C2-C,0;alkynyl)C(O)(C,-C,0 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl), -C(0)N(hydrogen, C,-C,0 alkyl, C2-C10 alkenyl or 'C2-C10 alkynyl)(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyl or C2-C,0 alkynyl), -N(hydrogen, 0,-C,o alkyl, C2-C10 alkenyl or C2-C,0 alkynyl)(hydrogen, C,-C,0 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl). C,-C10 alkoxy. C6-C,0 aryl or 5-10 membered heterocyclic, hydroxy, C,-C6 alkyl, C,-C6 alkoxy, C6-C,0 35 aryl and 5-10 membered heteroaryl.;R9 is;20;-43-;

2. A method of treating a bacterial infection or a protozoa infection in a non-human mammal, fish, or bird which comprises administering to said mammal, fish or bird a therapeutically effective amount of a compound of the formula 15;N(CH3)R2;10;oh,1'";15;35;15;or a pharmaceutically acceptable salt thereof, wherein:;CH3 R11;R10-;20;R1 is ON , acetyl, 3-N,N-dimethylamino-2-propenoyl, / , 1-N-methyl-5-;pyrazolyl, 3-pyrazoiyl, 1 -methyl-N-3-pyrazolyl, 1-N-benzyl-3-pyrazolyl, 1 -N-(3-hydroxybenzyl)-3-;o II;ii I N—N;25;pyrazolyl, 3-isoxazolyl, ^ or R2 is hydrogen or C,-C4 alkyl;;R3 is hydrogen, C,-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, -(CH2)m(C6-C,0 aryl), -(CH2)m(C6-C10 heterocyclic) or aryl, each, other than hydrogen, being optionally substituted with 1 to 3 3 0 substituents independently selected from halogen, cyano, nitro, trifluoromethyl, azido, -C(O)C,-C10 alkyl, -C(O)C2-C10 alkenyl, -C(O)C2-C10 alkynyl, -00(0)C,-C,o alkyl, -00(0)02-0,o alkenyl, -OC(O)C2-C10 alkynyl, -N(hydrogen, C,-C10 alkyl, C2-C,0 alkenyl or C2-C10 alkynyl)C(O)(C,-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl), -C(0)N(hydrogen, C,-C,0 alkyl, C2-C10 alkenyl or C2-C10 alkynyl)(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyl or C2-C10 alkynyl), -N(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl)(hydrogen, C,-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl), 0,-0,0 alkoxy, C6-C10 aryl, 5-10 membered heterocyclic, hydroxyl, methoxyl, 0,-0,0 alkyl, C2-C,0 alkenyl, C2-C,0 alkynyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-;INTELLECTUAL PROPERTY OFFICE OF N.Z.;-2CEC 20G^;Rppcu/cr* -44- pyridylmethyi, 2-pyridylethyl. 3-pyridylethyl and 4-pyridylethyl; m is an integer ranging from 0 to 4; each R4 is hydrogen. -(CH2)m(C6-C,Q aryl) or -(CH2)m(C6-C10 heterocyclic), each, other than hydrogen, being optionally substituted with 1 to 3 substituents independently selected from halo, 5 cyano, nitro, trifluoromethyl, azido, -C(0)C,-C„ alkyl. -C(O)C2-C10 alkenyl. -C(O)C2-C10 alkynyl, -OC(O)C,-C,0 alkyl, -OC(O)C2-C,0 alkenyl, -00(0)C2-C10 alkynyl, -N(hydrogen, C,-C,0 alkyl, C2-C10 alkenyl or C2-C,0 alkynyl)C(O)(Cl-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl), -C(0)N(hydrogen, C,-C10 alkyl, C2-C,0 alkenyl or C2-C10 alkynyl)(hydrogen, C,-C10 alkyl, C,-C10 alkenyl or C2-C10 alkynyl), -N(hydrogen, 0,-0,0 alkyl. C2-C10 alkenyl or C2-C,0 alkynyl)(hydrogen, C,-C.0 alkyl, C2-C10 alkenyl or 10 0,-0,0 alkynyl), C,-C,o alkoxy, C6-C10 aryl and 5-10 membered heterocyclic; n is an integer from 0 to 5; R6 is hydrogen or methyl; each R7 is independently hydrogen, C,-C;>o alkyl, C^-C^ alkenyl, C2-C20 alkynyl, -0(0)0,-020 alkyl, -C(O)C2-C20 alkenyl, -C(O)C2-C20 alkynyl, -C(O)N(H)C,-C10 alkyl, -C(O)N(H)C2-C20 alkenyl, -15 0(O)N(H)C2-C2o alkynyl. -S02(0)C,-C2o alkyl, -302(0)02-020 alkenyl, -S02(0)C2-C2o alkynyl or -P042" R8 is hydrogen or methyl; R9 is or 4"-oxocladinosyl; 25 R'° is an alpha-branched C2-C8 alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylthioalkyl group, any of which may optionally be substituted by one or more hydroxyl groups; a C5-C8 cycloalkylalkyl group wherein the alkyl group is an alpha-branched C2-Cs alkyl group; a C3-CB cycloalkyl or Cs-C8 cydoalkenyl group, either of which may optionally be substituted by methyl or one or more hydroxyl, one or more 0,-04 alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing 3 o heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more C,-C4 alkyl groups or halo atoms; or R10 is phenyl which may be optionally substituted with at least one substituent selected from C,-C4 alkyl, C,-C4 alkylthio groups, halogen atoms, hydroxyl groups, trifluoromethyl, and cyano; or R10 may be with a formula (a) as shown below: 35 -45- wherein Y is O, S or —CH2—, a, b, c, and d is each independently an integer ranging from 0-2 and a + b + c + d^5; R11 is hydrogen or -OH; and R15 is H, 0,-0,0 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, cyano, -CH2S(O)pC1-Ci0 alkyl, -CH2S(O)pC2-C10 alkenyl, -CH2S(O)pC2-C10 alkynyl, wherein p is an integer ranging from 0 to 2, -CH2O{C,-C10 alkyl), -CH2O(C2-C10 alkenyl), -CH2O(C2-C10 alkynyl), -CH2N(hydrogen, C,-C,0 alkyl, C2-15 C10 alkenyl or C2-C,0 alkynyl)(hydrogen, 0,-0,0 alkyl, 02-C10 alkenyl or C2-C,0 alkynyl), -(CH2)m(C6-C,o aryl) or -(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein the alkyl, alkenyl, alkynyl, aryl and heteroaryl moieties of the foregoing are optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido, -C(O)C,-C10 atkyi, -C(O)C2-C,0 alkenyl, -C(O)C2-C,0 alkynyl, -00(0)0,-0,0 alkyl, -OCfOXV 2 q C10 alkenyl, -OC(O)C2-C10 alkynyl, -N(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyl or 0^-010 alkynyl)C(O)(C1-C10 alkyl, C2-C,Q alkenyl or C2-Ct0 alkynyl), -C(0)N(hydrogen, C,-C10 alkyl, C2-C,0 alkenyl or C2-C,0 alkynyl)(hydrogen, C,-C,0 alkyl, C2-C10 alkenyl or C2-C10 alkynyl), -N(hydrogen, C,-C,0 alkyl. C2-C10 alkenyl or C2-C,0 alkynyl)(hydrogen, 0,-0,0 alkyl, C2-C,0 alkenyl or C2-C,0 alkynyl), C,-C,o alkoxy, C6-C,0 aryl or 5-10 membered heterocyclic, hydroxy, C,-C6 alkyl, C,-Cs alkoxy, C6-C10 25 aryl and 5-10 membered heteroaryl, except that when R15 is H, R10 is not ethyl.

3. A method according to any one of claims 1 or 2 wherein the compound is in isolated or purified form.

4. A method according to claim 1 or claim 2 substantially as herein described with reference to any example thereof. INTELLECTUAL PROPERTY OFFICE OF N.Z. - 2 DEC 2004 RECEIVED