CA2121234A1 - Marcfortine/paraherquamide derivatives useful as antiparasitic agents - Google Patents

Abstract

2121234 9310120 PCTABS00022 There are disclosed 18-thiomarcfortine derivatives of the natural products marcfortine A, B, and C, C-18 thioparaherquamide and derivatives thereof, novel N-1 marcfortines A, B, and C and derivatives thereof, novel N-1 paraherquamide and derivatives thereof useful in the treatment and prevention of helminth and arthropod infections of animals and plants. The synthetic derivatives are of Formula (I).

Description

W0 93/101202 1 2 1 2 3 4 P(~llJS92/U9483 MARCFORTINE/PARAHERQUAMID~ DERIVATIVES
- USEFUL AS ANTIPARASITIC AGENTS
BACKGROUND OF THE INVENTlON
The marcfortines ~re known compounds and are disclosed by Polonsky e~ al. in Joumal 5 of the Chemical Society Chemical Communications 1980 601-602 (Marcfortine A) and Tetrahedron Letters 1981 22 1977-1980 (Marcfortines B and C). The compounds are fungal metabolites of Penicillium roqueforti. The marcfortines are structurally related to the paraherquamides which are also known compounds. The paraherquamides are disclosed by Yamazaki et al. in Tetr~hedron Letters 1981 22 135-136~ and by Blanchflower et al., Joumal of 10 Antibiotics, 1991, 44, 492-497. U.S. Patents 4,866,060 and 4.923,867 disclose the use of the marcfortines A, B, and C, and certain derivatives thereof as useful for the treatment and prevention of parasitic diseases in animals.
Paraherquamide has the following structure:
H3C~ ,CH3 - 20 3~o o C~

Marcfortine A has the following structure:

~,

2 i ~ ~3 22s CH~

WO 93/10120 2 1 2 :1 2 3 ~ PCI/US92/09483 Marcfortine B has the following structure: -~,"
~25 Marcfortine C has the following structure:

3 ,CH3 WO 91/~9961 (published 11 July 1991~ discloses various derivatives of marcfortine and pa~herquamide, and N(12) oxides thereof, well as the production the production of VM 29919 (paraher~uamide) and VM 5S596 (the N(12)oxide of par;lherquamide~ inter alia from Pcnicilli~ln Sp. IMI 332995.
US Patent 4~873.247 discloses derivatives of p.~lcrquamide a~ld a strain of Peni~ v)~
charlessi M~; 5123 (ATCC 20841) for the production of paraherquamide. US Patent 4~978~ 656 (DS well as E~P 390532-A, EP-301742-A) discloses vasious synthedc derivatives ofpa~herquamide as well as the production of paraherguamide from Penicilli~n charlcssi MF
512~ (ATCC 20841).
Paraherquamide is a compound produced under certain conditions by the fungal or~anism Peniclli~n paraherquei. WO 92/00300 (published 9 January 1992) discloses the WO ~3/10120 2 1 2 1 2 3 4 PCI/US92/09483 synthesis p~raherquamide related compounds produced from Penicillium paraherquei, del osiled at the C.A.B. lnternational Mycological lnstitute, Ferry Lane, Kew, London~ under deposit number CMI 68220.
SUMMARY OF T~ INVENTION
This invention is concemed with the synthesis of C-lg thiomarcfortines A. B, and C and derivatives thereof, C-18 thioparaherquamide and derivatives thereof, novel N-l marcfortines A, B, and C and derivatives thereof, novel N-l paraherquamide and derivatives thereof. and the use of these compounds as antiparasitic agents. Thus it is an object of this invention lO descrihe these thiomarcfortine, thioparaherquamide, marcfortine and paraherquamide derivatives. A
further objective of this invention is to describe processes for the preparation of these compounds. A still further objective is to describe the use of the instant compounds as antiparasitic agents in the treatment and prevention of parasitic diseases. A still further object is to describe compositions for the treatment of parasitic diseases which contain the novel compounds of this invention as the active ingredient therec)f. Further o~jectives will t)ecome apparent from a reading of the follo~,ving description.

DESCRIPI`ION OF T~ INVENTION
The compounds of the instant invention are represented by Formula 1:
E3C~ C~3 ()m 0~ ~25 wherein:
n is 0 or 1;
R~4,, and Rl4b~ beinB the same or different, are selected from hydrogen~ hydroxy~ C,-C~
alkyl, Cl-C6 alkoxy~ C~-C6 alkenyl, C~-C6 alkenyl-C,-C6 alkoxy, C~-C6 alkynyl-C,-C6 alkoxy~
C~-C6 alkynoyloxy, poly Cl-C6 alkoxy-C~-C6 alkoxy, phenyl~ phenyl-C,-C6 alkyl, tri-C~-C6 a~cylsilyloxy, diphenylphosphoryloxy and halogen~ or R~4, and Rl~b together fonn the epoxide or 3~ =CH2~ with the pro~risio that when n is 1, then Rl4~ and R,~" are both hydrogen;
m is 0 or I ~preferat ly 0);

WO 93/10120 ~ ~ 2 1 2 ~ 4 PCI/US92/09483 WisOorS;
when W is S, R, is hydrogen. Cl-C7 alkyl, cyclo C3-C8alkyl. henzyl, C2-C, alkanoyl (-C(O)C.-C7alkyl) ~optionally substituted with carboxy (-COOM), C~-C7 alkanoyl. carbo Cl-C7alkoxy (-C(O)OCI-C7alkyl), -NR4R5, aminocarbonyl (-C(O)NR4Rs))~ Cl0-C24all;anoyl 5 (-C(O)C~0-C24alkyl, cyclo C3-C8alk~noyl ~optionally substituted with c~rboxy, C~-C7 alkanoyl.
carboCI-C7alkoxy, -NR4R5, aminocarbonyl~, alkanoyloxymethylene (-CH2OC(O)-C2-C7alkyl), benzoyloxymethlene (-CH2OC(O)phenyl) loptionally substituted with 1 or 2 groups selected from halogen, Cl-C4 alkyl. haloC,-C7alkyl, nitro, cyano and C,-C7alkoxyl. C,0-C24alkenoyl (-C(O)C9-C23alkenyl), benzenesulfonyl (-SO2CH2phenyl) (optionally substituted with I or 2 10 groups selected from halogen. C~-C4 alkyl, haloCI-C7alkyl, nitro. cyano and Cl-C7alkoxy).
Cl-C4alkylaminocarbonyl (-C(O)N(C,-C4alkyl)2), C,-C4alkylaminothiocarbonyl (-C'(S)N
(Cl-C4a~kyl)2), Cl-C7 alkoxycarbonyl, phenoxycarbonyl ~optionally substituted with 1 or 2 groups selected from halogen, C~-C4 alkyl, haloC,-C7alkyl, nitro, cyano and C,-C7alkoxyJ, -C(O)NR'4R'5, -P(=X)(R2)(R3), -SR6 -SO2NR4RS, benzoyl substituted at the 3 or 4 position with 15 -CH7NR4R5, or 2-tetrahydrofuran or bicycloC8-CI2alkanoyl;
when W is O; Rl is selected from the group consisting of:
(a) C2-C7 alkanoyl substituted with carboxy (-COOH), Cl-C7 alkanoyl, carboC~-C7alkoxy (-C(O)OC,-C7alkyl), -NR4R5, aminocarbonyl - (-C(O)NR4Rs);
(b) cyclo C3-C8alkanoyl opdonally substituted with carboxy, Cl-C7 alkanoyl, carboC,-C7alkoxy. -NR4R5, aminocarbonyl;
(c) alkanoyloxymethylene (-CH20C(O)-C2-C7alkyl);
(d) benzoyloxymethlene (-CH20C(O)phenyl) substituted with I or 2 groups selected from halogen, C~-C, aUkyl, haloC,-C7alkyl, nitro, cyano and Cl-C7~L~coxy;
(e) phenoxycarbonyl substituted with 1 or 2 groups selected from halogen, C~-C4 alkyl, haloCI-C7allcyl, nitro, cyano and C,-C7alkoxy;
(f) -C(O)NR'4R'5;
(g) -p(=x)(R2)(R3);
(h) -SR6 (i) C,0-C~alkanoyl (-C(O)C,0-C~alkyl);
(j) C~O-C2~ enoyl (-C(O)Cg-C23~1kcnyl); or O 2-tetr~ydrofuran;
R4 and R5, being the same or different, are selected from hydrogen, Cl-C7 alkyl, phenyl 35 ~optionaUy su~)stituted with I or 2 groups selected from halogen, C,-C~ alkyl, haloCI-C7alkyl, nitro, cyano and Cl-C7alkoxy~ or when taken together with N, form a saturated WO 93/tO120 2 1 2 1 2 3 ~ PCr/US92/09483 or unsaturated heterocyclic amine ring;
R'4 and R'5, being the same or different. are selected from C~ alkyl.
cyclo(C3-C8)alkyl, phenyl loPtionallY substituted with I or 2 groups selected from halo~en, Cl-C4 alkyl, haloCI-C7alkyl, nitr~, cyano and C,-~alkoxyJ or when taken together with 1~, form 5 a saturated heterocyclic amine ring optionally containing I or 2 additional heteroatoms selected from N, O or S;
X is O or S;
R2 and R3, being the same or different, are selected from Cl-C7 alkyl, phenyl loption~lly substituted with I or 2 groups selected from h~logen, Cl-C~ al~yl. haloCI-C,alkyl. nitro, cyano and C,-C7alkoxyl, Cl-C1alkoxy, thio(C,-C7)alkoxy, phenoxy, thiophenoxy, -NR7R8 ~where R7 and R8. being the same or different, are selected from H, C,-C1 all;yl or taken together with N, form a saturated heterocyclic ring), or l~;en together with P
form a 4- to 7-membered heterocyclic ring;
R6 is Cl-C, alkyl, haloC,-C,alkyl, carboC,-C7alkoxy. -NR9RIo where R4 and Rlo, heing the same or different, are Cl-C7 alkyl or phenyl (optionally substituted with I or 2 groups selected from halo, lower alkyl, haloCI-C7alkyl, nitro, cyano, Cl-C7 alkoxy);
Rz4 is hydrogen, halogen or Cl-C7 alkoxy;
R~5 is hydrogen or halogen;
R,8" is hydrogen, Cl-C7 alkyl, C2-C8 alkoxyalkyl, C2-C8 alkenyl, C2-C8 alkynyl or benzyl;
the broken line between ~arbons 24 and 25 represents a single or douhle bond.
The compoùnds of this invention include pharmaceutically accept~le salts thereof ;LS
well as 12a-N-oxides thereof.
Another aspect of this invention are - 10,10"-(1,4-dicarbonylpiperazine) bis(6',7',8',9',10'.10'a-hexahydro-1',1',4,4,12'-penlarnethyl)-~2'S-[2'.alpha.,3'a alpha, 9'a.alpha.,10(2"'R*, 3"'aS*9"'aS*.
lO"'aR*) lO'abeta.~]-Spiro[4H,RH-[ 1 ~4]dioxepino~2,3-g]indole-8~2'(3'H)-[ I H~4H-3a~9a](iminomethano) cyclopenta[b]quinolizinl-9~11'(1()H~-dione - Cpd #120)~
and lO,la'-(1,44ioxo-2-butene)bis(fi',7',R',9'.10'~10'a-he~ahydro-1',1'~4,4,12'-pclltamcthyl)-[2'S-12'~alpha~,3'aalpha.,9'aalpha.~10(2"'R~,3"'aS~9"'aS~,lO"'aR~),lO'~bet~]]-Spirol4H,8H-[1,4]dioxepino[2,3-g]indole-8,2'(3'H)-l lH,4H-3a,9a](imino me~ano)cyclopenta~b] quinolizin]-9,1 I'(IOH) dione, Cpd #12T), which can ~e used in the same mar~er as compounds of Folmula 1.
~5 WO 93/10120 PCr/US92/09483 21?123~ -6-Another aspec~ of ~is invention provides an 18-thiomarcfortine or derivatives of Forrnula 11.
H3C~ ,CH3 ()~n 0~
S ! H3C~ 3 ~ 0~R24 ~N~N--Rl ~N\
S R18a wherein:
m is 0 or I (preferably 0);
Rl is hydrogen~ C,-C7 alkyl, cyclo C3-C8~1kyl, ~enzyl, C~-C7 alkanoyl (-C(O)C2-C7alkyl) ~optionally substitu~ed with c~rboxy (-COOH), C~-C7 alkanoyl. carbo 15 Cl-C7alkoxy (-C(O)OC,-C7alkyl), -NR4R5, aminocarbonyl (-C(O)NR4R5)}, CID-C24alkanoyl (-C(O)C~0-C~4alkyl, cyclo C3-C8alkanoyl ~optionally substituted with carboxy, C~-C7 alkanoyl, carboCI-C7alkoxy, -NR4R5. aminocar~onyl~, alkanoyloxyrnethylene (-CH20C(O)-C2-C7alkyl), benzoyloxymeWene (-CH2OC(O)phenyl) loptionally substituted with 1 or 2 groups selected from halogen, C~-C4 alkyl, haloCI-C7alkyl, nitro, cyano ~nd Cl-C7alkoxy), CtO-C24alkenoyl 20 ~-C(O)C9-C73aL~cenyl), benzenesulfonyl (-SO2CH2phenyl) (optionally substituted with I or 2 groups selected from halogen, C~-C4 alkyl~ haloCI-C7alkyl, nitro, cyano and C,-C7alkoxy)~ Cl-C4 alkylaminocarbonyl (-C(O)N(CI-C4alkyl)2), Cl-C4alkylamiino~!iocarbonyl (-C(S)N(CI-C4alkyl)2), Cl-C7 alkoxycart~onyl, phenoxyc~rbonyl {optionally subsl:inlted with I or ~ groups selected from halogen, Cl-C4 alkyl, haloCI-C~alkyl, nitro, cyano and Cl-C7alkoxy~, -C(O)NR'4R'5, -P(=X)(R2) 2S (R3), -SR6 -SO~R4R5, ~enzoyl substituted at the 3 or 4 position with -CH2NR,R5, 2-tetrahydrofuran, or bicycloC8-CI2aLcanoyl;
R4 and R5, being the same or different, are ~elected fmm hydrogen, C,-C7 alkyl, phenyl (optionally substituted with I or 2 gmul-s selectecl from halc gc.n, C~-C, all;yl, halo C~-C~ ;yl, nitro, cyano and Cl-C7alkoxyl or wh~n l~ lo~ r wittl N, t`~)nn ~ tur.~trd clr un~;ltllr~ltrll 30 heterocyclic ~mine ring;
R'4 and R~s~ being the same or different, are selected from C,-C7 alkyl, cyclo~C~-C8)aLkyl. phenyl loptionally substituted wish I or 2 groups selected from halogen, C~-C~ alkyl, haloCI-C7alkyl, nit~o, cyano and Cl-C7alkoxy~ or when taken together with N, form a sa~ ted heterocyclic amine ring optionally containillg I or 2 addition~l hetero~toms selected 3~ fromN,QorS;
XisOorS;

R2 and R3, being the sarne or different. are selected from C,-C~ alkyl, phenyl ~optionally substituted with 1 or 2 ~roups selected from halogen, C,-C4 alkyl. haloC,-C,alkyl, nitro, cyano and C,-C7alkoxy}, C,-C,alkoxy, thio(C,-C,)alkoxy, phenoxy, thiophenoxy, -NR7R8 ~where R, and R8. being the same or different, are selected from H, C,-C, alkyl S or taken together with N, form a satura ed heterocyclic ring ), or taken together with P
form a 4- to 7-membered heterocyclic ring;
R6 is C,-C, alkyl, haloC,-C7alkyl, carboC,-C7alkoxy, -NR9RIo where R9 and Rlo, being the same or different, are C,-C7 alkyl or phenyl (optionally substituted with I or 2 groups selected from halo, lower alkyl, haloC,-C7alkyl, nitro. cyano, C,-C~ alkoxy);
R24 is hydrogen, halogen or C~-C7 alkoxy;
R2s is hydrogen or halogen;
R,8a is hydrogen, Cl-C7 alkyl, C2-C8 alkoxyalkyl, C2-C8 alkenyl, C2 CR all;ynyl or benzyl; the broken line between carbons 24 and 25 represents a single or double bond.
Another aspect of this invcntion provides an 18-thioparaherquamide or derivatives of Formula nI:

3 ~ ,, 3 20 <~

Rl4a S R18a wherein:
m is 0 or 1 (preferably 0);
R~ is hydrogen. C,-C, alkyl, cyclo C3-C,~alkyl. t~enzyl, C2-C7 alkanoyl (-"(O~C2-C7alkyl) lopdonally subsdtutcd with car~oxy (-COOH), Cl-C7 alkanoyl, carbo Cl-C7alkoxy (-C(O)OC~-C7aLkyl), -NR,IR5, aminoca~bonyl (-C(O)NR~R5)~, C~0-C24alkanoyl (-C(O)CI0-C24alkyl~ cyclo CrC8alkanoyl (optionally subsdtuted with carboxy, Cl-C7 alkanoyl, carboC,-C7alkoxy, -NR~R5, aminocarbonyl), alkanoyloxymethylene (-CH20C(O)-C2-C7alkyl), benzoyloxymethlene (-CH2OC(O)phenyl) lopdonally substituted with 1 or 2 groups selected from halogen, C,-C4 alkyl, haloC,-C7alkyl, nitto, cyano and C,-C7aLkoxy)~ C,0-C~alkenoyl WO 93/10120 PCI/US~2/09483 2~2123~ -8-(-C(O)C9-C23alkenyl~, benzenesulfonyl (-SO2CH2phenyl) ~option~lly substituted with I or 2 groups selected from halogen, C,-C4 alkyl, haloC~-C7aLkyl, nitro, cyano and C,-C,alkoxy~.
C,-C4~lkyl~ninoc~rbonyl (-C(O)N(C,-C4alkyl)2), C,-C4alkylaminothiocarbonyl (-C(S)N
(Cl-C4aL~cyl)2), C,-C, alkoxycart~onyl. phenoxycarbonyl ~option~ly substituted with I or 2 groups selected from halogen, Cl-C4 alkyl, haloCI-C1alkyl, nitro, cyano and C,-C,alkoxyl~
-C(O)NR'4R'5, -P(=X)(R2)(R3), -SR6 -SO2NR4RS. ben~oyl substituted at the 3 or 4 position with -CH2NR4R5, 2-tetrahydrofuran, or hicycloC8-CI2alkanoyl;
R4 and R5, being the same or different, are selected from hydrogen, Cl-C7 alkyl, phenyl (optionally substituted with I or 2 groups selected from halo~en, Cl-C4 alkyl, haloCI-C,alkyl, nitro, cyano and C,-C,alkoxyl or when taken together with N~ form a s~tur~ted or-unsaturated heterocyclic amine ring;
R'4 and R'5~ heing the same or different, are selected from C,-C7 alkyl, cyclo(C3-C8)alkyl, phenyl (optionally substituted with 1 or 2 groups selected from h~logen~
C,-C4 alkyl, haloC,-C7alkyl, nitro, cyano and Cl-C,alkoxy~ or when taken together with N, form a saturated heterocyclic amine ring optionally containing I or 2 addition~l heteroatoms selecled fromN,OorS;
X is O or S;
R2 and R3, being the same or different, are selected from Cl-C7 alkyl, phenyl loptionally substituted with I or 2 groups selected f~om halogen, C,-C~ alkyl, haloCI-C7alkyl, nitro, - 20 cyano and C,-C7alkoxy). C,-C7 alkoxy, thio(C,-C7)alkoxy, phenoxy. thiophenoxy, -NR7R8 lwhere R7 and R8, being the same or different. are selected from H, C,-C, alkyl or taken together with N. form a saturated heterc cyclic ringl, or taken together with P
form a 4- to 7-membered heterocyclic ring;
R6 is Cl-C7 aLkyl, haloCI-C,alkyl, carboCI-C,alkoxy, -NR9R1o where R9 and Rlo, being the same or different. are Cl-C7 alkyl or phenyl (optionally substituted with I or 2 groups selected from halo, lower alkyl, haloCI-C7alkyl, nitro, cyano, Cl-C7 alkoxy);
R24 is hydrogen, halogen or C,-C7 alkoxy;
R25 is hydrogen or halogen;
R~4~ and Rl4b, being the same or diffcrent~ a~ selected f~m hydro~en, hydr~xy~
Cl-C6 alkyl, Cl-C6 alkoxy, C~-C6 alkenyl, Cl-C~ alkenyl-CI-C~ alkoxy, Cl-C~
alkynyl-C~-C6 alkoxy, C~-C6 alkynoyloxy~ poly C~-C6 alkoxy-C~-C6 alkoxy, phenyl~ phenyl-C,-C6 alkyl~ tri-C~-C6 alkylsilyloxy, diphcnylphosphoryloxy and halogen~ or Rl4, and R,4" together form the epoxide or =CH~;
Rlt~ is hydrogen~ Cl-C7 alkyl~ C2-Ct alkoxyalkyl~ C2-C8 alkenyl~ C2-CI alkynyl or benzyl;
the broken line between calbons 24 and 25 represents a single or double bond.

WO 93~10120 2 1 2 1 2 3 4 P~/US92/û9483 Another aspect of this invention provides novel N-l marcfortine or denv~tives ofFormula IV:
~13C~ ,CH3 S ~

N\
R18a wherein:
m iS O or 1 (preferably 0);
Rl is selected from the group consisting of:
(a) C2-C7 alkanoyl substituted with carboxy (-COOH), Cl-C7 alkanoyl, casboC,-C,alkoxy (-C(O)OC,-C7alkyl), -NR4Rs, aminocarbonyl (-C(0)~nR4R5);
(b) cyclo C3-C~alkanoyl optionally substituted with carboxy, C~-C7 alkanoyl, caJboCI-C7alkoxy, -NR4Rs, aminocarbonyl;
(c) alkanoyloxymethylene (-CH,OC(O)-C2-C7alkyl);
(d) benzoyloxymethlene (-CH70C(C))phenyl) substituted with I or 2 group~
selected from halogen, C,-C4 al};yl, haloC,-C7alkyl, nitro, cyano and C,-C,~I~oxy;
~e) phenoxycarbonyl substituted with 1 or 2 g~ups selected from halogen, Cl-C4 alkyl, haloCl-C7alkyl, nitr~, cyano and Cl-Cqalkoxy;
(f) -C(0)NR'4R's;
(~) -P(-X)(R~)(R~;
(h) -SR~;
(i) C10-C2~alkanoyl (-C(O)C~0-C.,~alkyl);
(j) C1O-C2~alkenoyl (-C(O)Cg-C23alkenyl); or (k) 2-tetsahydrofuran;
R4 and Rs~ being the s~ne or different, are sele~.ed f~m hydrogen, Cl-C7 alkyl, phenyl 35 ~optionally sul)stituted witn 1 or 2 groups selected from halogen, C,-C~, alkyl, haloCI-C7alkyl~ nitro, cyano and Cl-C7alkoxy) or when taken together with N, fonn a satur~ted WO 93/10120 PCl/US92/09483 ~I~`I2~ ~o or unsaturated heterocyclic amine ring;
R'4 and R'5, being the same or different. are selected from Cl-~ al~;yl, cyclo(C3-C8)~1kyl, phenyl ~optionally substinlted with 1 or 2 groups selected from halo~en, Cl-C4 alkyl, haloC,-C7alkyl, nitro, cyano and Cl-C7alkoxyl or when taken together with N, form a saturated heterocyclic amine ring optionally con~aining I or 2 additional heteroatoms selected from N, O or S;
X is O or S;
R2 ~nd R3, being the same or different, are selected from Cl-C7 alkyl, phenyl (optionally substituted with I or 2 groups selected from halogen. Cl-C~, alkyl. hDloC,-C7;l1kyl, nitro, cy~no and C,-C7alkoxy), C,-C7 alkoxy, thio(C,-C7)alkoxy~ phenoxy, thiophenoxy.
-NR7R" ~where R7 and R8. being the same or different. are selected from H, C~-C7 alkyl or taken together with N, form a saturated heterocyclic ring), or taken together with P
form a 4- to 7-membered heterocyclic ring;
R6 iS Cl-C7 alkyl, haloCI-C7aL~cyl, carboCI-C7alkoxy, -NR9Rlo where R~ and RlU, being the same or different, are Cl-C7 alkyl or phenyl (optionally substituted with I or 2 groups selected from halo, lower alkyl. haloCI-C7alkyl, nitro, cyano, Cl-C7 alkoxy);
R2~ is hydrogen, halogen or C,-C7 alkoxy;
R25 is hydrogen or halogen;
Rl8~ is hydrogen, C,-C7 alkyl, C2-C8 alkoxyalkyl. C2-C8 alkenyl, C2-C8 alkynyl or benzyl;
the ~roken line between carbons 24 and 25 represents a single or double bond.
Another aspect of this invention provides novel N-l paraherquamide or derivatives of Formula V:
~3C~ C~3 O~ R25 ~1 ~3~ ~! 3 ~o~)--R24 wherein:
m is Oor l (preferably O);
R~ is selected from ~e group consisdng of:

WO 93/10120 2 ~ 212 3 4 PCr/US92/09483 (a) C2-C7 alkanoyl substituted with carboxy ~-COOH). Cl-C, ~Ikanoyl, carboCI-C7alkoxy (-C(O)OCI-C7alkyl), -NR4R5, aminocarhonyl (-C(0)NR4R5);
(b) cyclo C3-C~alkanoyl optionally substitu~ed with carboxy. Cl-C7 alkanoyl.
carboCI-C7alkoxy, -NR~R5, aminocarbonyl;
(c) alkanoyloxymethylene (-CH2OC(O~-C2-C7alkyï);
(d) benzoyloxymethlene (-CH20C(O)phenyl) substituted with I or 2 groups selected from halogen, Cl-C4 alkyl, haloC, C7alkyl, nitro. cyano and Cl-C7alkoxy;
(e) phenoxycarbonyl substituted with I or 2 groups selected from halogen, C,-C4 alkyl, haloC~-C7alkyl, nitro, cyano and C,-C7alkoxy;
(f) -C(0)NR'4R'5;
(g) -P(=X)(R2)(R3);
(h) -SR6;
(i) C,O-C24alkanoyl (-C(O)C,O-C24alkyl);
(j) C,O-C24alkenoyl (-C(C))Cg-C23alkenyl); or (k) 2-tetrahydrofuran;
R4 and R5, being the same or different, are selected from hydrogen, Cl-C7 ~Ikyl, phenyl loptionally substituted with 1 or 2 groups selected frc7m halogen, C,-C4 alkyl, haloC,-C7alkyl, nitro, cy~no and Cl-C7alkoxy) or when taken together with N, form a saturated or unsaturated heterocyclic amine ring;
R'4 and R'5, being the same or different, are selected from Cl-C7 alkyl.
cyclo(C3-C8)alkyl, phenyl loptionally substituted with 1 or 2 groups selected from halogen, C,-~ alkyl, haloCI-C7alkyl, nitro, cyano and C,-C7alkoxy) or when ~cen together with N, fonn a saturated heterocyclic amine ring optionally containing I or ~ additional heteroatoms selected from N, O or S;
X is O or S;
R2 and R3, being the same or different, are selectcd fmm Cl-C.7 nll;yl, phenyl ~op~iollnlly substituted with I or 2 g~ouF)s sclcetc(~ fil)m h~lo~cl), Cl-C', nll;yk lullocl-c7llll;yl~ ni~n), cyano and C~ alkoxy), C,-C7 sllkoxy, thio(C,~G7);~1koxy, phcnoxy, tlliophcnc)xy.7R8 (where I~7 ancl R~" being the s~ne or different, are selected from H, Cl-C7 all;yl or taken together with N, form a saturated heterocyclic ringl, or taken together with P
fonn a 4 to 7-membered heterocyclic ring;
R6 iS Cl-C7 alkyl, haloC,-C7alkyl, cart~oC,-C7alkoxy. ~NRqRIo where R9 and Rlo~ being ~5 the same or different, are Cl-C7 alkyl or phenyl (optionally substituted with l or 2 groups selected from halo, lower alkyl. haloC,-C7alkyl, nitro, cyano, Cl-C~ all;oxy);

WO 93/101~ PCr/US92~094~3 2l~ ~3~ -12-R24 is hydrogen, halo~en or C,-C7 alkoxy;
R25 is hydrogen or halo~en;
R~4~ and R,4b, t eing the same or different, are selected from hydrogen, hydroxy, C,-C6 alkyl, C,-C6 alkoxy, C,-C6 alkenyl, C,-C6 alkenyl-CI-C6 alkoxy, C,-C6 alkynyl-C,-C6 alkoxy, 5 Cl-C6 alkynoyloxy, poly C,-C6 alkoxy-CI-C6 alkoxy, phenyl, phenyl-C,-C6 alkyl, tri-C,-C6 alkylsilyloxy, diphenylphosphoryloxy and halogen, or R14a and Rl4b together form the epoxide or =CH2;
R~8a is hydrogen, Cl-c7 alkyk C2-C8 alkoxyalkyl, C2-Cg alkenyl, C2-C8 alkynyl or benzyl;
the broken line between carbons 24 and 25 represents a single or dou~le bond.
l'he carbon content of various hydrocarbon containin~ moieties is indicated ~y a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix Cj-Cj indicates a carhon atoms content of the integer "i" to the integer ";" carbon atoms, inclusive. Thus, Cl-c3 alkyl refers to alkyl of 1-3 carbon atoms, inclusive, or methyl, ethyl, pmpyl, and isopropyl.
With respect to the above, ''Cl-C7 alkyl" is intended to include those alkyl groups of from I to 7 carbon atoms in either a straight or branched represents a double bond between chain. Examples of such lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl and the like.
Cyclo(C3-C8)alkyl is intended to include alkyl rings of 3 to 8 members. Examples of 20 cyclo(C3-C8)alkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, and the like.
Cl-C8 alkoxy is intended to include those alkoxy groups of from I to 8 carton atoms in either a straight or branched chain. Examples of such C,-C8 alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, pentoxy, hexoxy, heptoxy, and the like.C2-C,alkanoyl is intended to include those alkanoyl groups of f~m 2 to 7 carbon atoms in either a straight or branched chain. Examples of such C2-C,alkanoyl groups include acetyl, propionyl, isopropionyl, butyryl, pentanoyl, hexanoyl and the like.
C~O-C24alkanoyl (-C(O)C9-C23alkyl) is intended to include those alk~noyl grou~ of from 10 to 24 ca~on atoms in either a straight or branch~d chain. Exsunples of such C,0-C2,all~anc~yl 30 groups include decanoyl [-C(O)(CH2)9CH3], lauroyl l-C(O)(CH~)IoCH3]~ tridecanoyl [-C(O) (CH2)~1CH3], myristoyl l-C(O)(CH~ CH3], pentadecanoyl [-C(O)(CH~)I9CHJ. palmitoyl l-C(O)(CH~ CH33, magaroyl l-C(O)(CH2)15CH33, s~earoyl [-C(O)(CH2)~6CH3], arachidoyl l-C(O)(CH2)l8CH33. heneicoisanoyl t-C(Q)(CH2)19CH3~, behenoyl l-C(O)(CH2)20CH3], tricosanoyl [-C(O)(CH2)2,CH33, tetracosanoyl 35 [-C(O)(CH2)22CH3], and ~he like.
C~0-C24aLkenoyl (-C(O)C9-C23alkenyl) is intended to include those unsaturated gn)ups of :

W O 93J10120 2 ~ 2 1 2 ~ ~ PC~r/US92/09483 from 10 to 24 carbon atoms in either a straight or branched chain. Ex~mples of such C,~
alkenoyl groups include undecylenoyl [-C(O)(CH2)7CH:CHCH3], oleoyl l-C(O)(CH2), CH:CH(CH2)7CH3], linoloyl [-C(O)(CH2),CH:CH.CH2.CH:CH(CH2)4CH3]. and the like.
The term "C~-C8 alkoxyalkyl" is intended to include those lower alkoxy substituted lower 5 alkyl groups cont~ining from 2 to 8 carbon atoms and from I to 3 oxygen atoms in either a straight or branched chain. Examples of such C2-C8 alkoxyalkyl groups include methoxymethyl, methoxyethoxymethyl, methoxyethoxyethoxymethyl~ ethoxyethyl. and the like. Fxamples of C,-C8 alkoxymethyl are methoxymethyl, ethoxymethyl, propoxymethyl, butoxymethyl,pentoxymethyl, butoxymethyl, pentoxymethyl, hexoxymethyl, and heptoxymethyl, ~nd isomeric 10 forms thereof.
The term alkanoyloxymethylene is intended to include those ~Ikanoyloxy substituted methylenes containing from 2 to 8 carbon atoms in either ~ straight or branched chain.
Examples of such C~-C8 alkanoyloxymethylene groups include acetoxymethyl, tert-butoxymethyl~
n-propoxymethyl, valeroxymethyl and the like.
The term "substituted benzoyloxymethylene" is intended to include those benzoyloxymethyl groups in which the benzene ring is substituted with from 0 to 3 su~stituents selected from lower alkyl, trifluoromethyl, Cl-C7 alkoxy, nitro, or cyano groups, and halogen atoms.
The term "subsdtuted benzenesulfonyl" is intended to include those benzenesulfonyl 20 groups in which the benzene ring is substituted with from 0 to 3 substituents selected from Iawer alkyl, trifluoromethyl, Cl-C7 alkoxy, nitro, or cyano groups, and halogen atoms.
The term `'halogen" is intended to include the halogen atoms fluorine, chlorine, brominc ~nd iodine.
The term "halo Cl-C7alkyl" is intended to include those halogen substituted C~-C7 alkyl 25 groups containing from 1 to 7 carbon atoms in either a straight or blanched chain and f~m 1 to 3 halogen atoms. Examples of haloC,-C7alkyl include fluoromethyl, 2-bromoethyL
3-chlorapropyl, 5-iodopentyl, trifluoromethyl, and ~e like.
The term "C2-C~ alkenyl" is intended to include those lower alkyl groups containin~
from 2 to 8 carbon atoms in either a straipht or branched chain which conta n~ I ~o 2 car~on 30 double bonds~ Examples of such C2-C~ alkenyl groups include allyl, 3-butenyl, 2,4pentadienyl~
hexenyl, and the like~
The term "C2 C, alkynyl" is intended to include those alkynyl groups containing from I
to 8 carbon atoms in either a straight or branched chain which contains 1 to 2 carbon-carbon triple bonds~ Examples of such C2-C~ alkynyl groups include propargyl~ 2-butynyl, 35 2,4pentadiynyl, 5-hexynyl~ and the like.
Bxamples of "alkoxycarbonyl" ( -C(=O)O-(CH2)r CI-C, alkoxy) include ethoxycarbonyh `~::

WO g3/10120 PCI /US92/09483 ~1212.-'~ 14-isopropoxycar~onyl, methoxycarbonyl, butoxycarbonyl, hexoxycarbonyl and the like.
C,-C7 alkanoyl is intended tO include alkyl groups of from 1 to 7 carbon atoms in either a str~ight or branched ch~in. Examples of Cl-C7 alkanoyl include acetyl. propionyl, iso-butyryl, valeryl, 5-methylhexanoyl, and the like.
Examples of aminocarbonyl ( -C(=0)NR4R5) include dimethylaminocarbonyl, propylmethylaminocarbonyl, dibutylaminocarbonyl, isopropyl~minocar~onyl, hexylaminocarbonyl and the like.
Examples of aminothiocarbonyl ( -C(=S)NR4R5) include dimethylaminothiocarbonyl, propylmethylaminothiocarbonyl, dibutylaminothiocarbonyl, isopentylaminothiocarbonyl, hexylaminothiocarbonyl and the like.
Exarnples of the group -P(=X)(R2)(R3) include diethyl thiophosphoryl, phenylmethoxyphosphonyl, 2-thioxo-1,3,2-dioxaphosphorinanyl, N,N-dimethylmethoxyphosphoramidyl, diphenylphosphinyl and the like.
Examples of -SR6 include 2,4-dinitrobenzenesulfenyl, dimethylaminosulfenyl, ethoxycar~onylsulfenyl, trichloromethylsulfenyl, 4morpholinosulfenyl and the like.
Examples of -SO2NR~R5 include dimethylsulfamoyl, phenylmethylsulfamoyl,

4-morpholinosulfamoyl, piperidinylsulfamoyl and the like.
11 te~m "P contaidng heterocydic ring" is intended to include 1,3-dioxa-2-phosphorinane, 1-aza-3-oxa-2-phospholane, 1,3-diaza-2-phospholane, 1-thia-3-oxa-2-20 phospholane, and the like.
Examples of heterocyclic amine rings according to -NR4R5, -hR'4R'5 and -NR7R~ are:
4moTpholine, 4-phenyl-1-piperazine, 4(2-pytidinyl~l-piperazine, 2,~dimethyl~morpholine, l-pynolidine, 4methyl-1-piperazine, l-piperidine, 4phenyl-1-piperidine thiazolidhne, 4-phenyl-1 ,2,3,6-tetrahydropyridine, 4phenylpipuidine, ` ~ ethyl plolinate, tet~ydrofJryl~nine, 3S 3-py~oline, thiazolidine4-calboxylic acid~

WO 93/10120 2 1 2 1 2 3 ~ PCr/US92/09483 _15_ thiomorpholine, nipecotamide, 2-methyl~iperidine, ~-methylpiperidine, 4-methylpiperidine, N -methylpiperazine, methylhomopiperazine, I-acetylpiperazine, N-carboethoxypipemzine, Pharmaceutically acceptable salts means salts useful for administering the compounds of - this invention and include mesylate, hydrochloride, hydro~romide, hydroiodide, sulfate, phosn.hate, acetate~ propionate, lactate, maleate, malate, succinate, tartrate, and the like. These salts may be in hydrated form.
Preferred compounds of this invention are compounds of Formula I (as well a~ Formul~
i~ 15 ll, lII, IV and V) where R~ and R25 ~re hydrogen; R", is hydrogen~ Cl-C4 alkyl, C2-C4 alkoxyalkyl, C2-C~ alkenyl or benzyl; and tne broken line between carbons 24 and 25 represem~
a double bond.
- Examples of the compounds of Formula 11 of this invention are as follows:
18-thiomarcfortine A
` ~ 20 1-acetoxymethyl-18-thiomarcfortine A
diethoxyphosphoryl-18-thiomarcfortine A
I-dimethylsulfamoyl-18-thiomarcfortine A
l-cyclopropylcarbonyl-18-thiomarcfortine A
2-bicyclol2.2.1]heptanoyl- 18-thiomarcfo~ne A
1 -(1 -piperidinyl)thiocarbonyl- 18-thiomarcfortine A
I -succinoyl- 18-~iomarcfortine A
(4-morpholinosulfenyl)-18-thiomarcfortine A
(2,4-dinitrobenzenesulfenyl)-18-thiomarcfortine A
24-propoxy-24,25-dihydro-18-thiomarcforthle A
l-(p toluenesulfonyl)-18-thhmarcfortine A
l-acetyl-18-thiomarcfor~ne A
l-methyl-18-~oma~:fo~ne A
I-benzyl-18-thiomarcfoltine A
14imelhyla~moyl-18-~iomarcfoltine A
1-methoxycarbonyl-18-~iomarcfor~ine A
24~2S-dihyd~18-thiomarcfo~nc B

, .
2 1 2 1 2 3 g PCI/US92/Og483 24-methoxy-24,2S-dihydro- 18-thiom~rcfortine B
I -(p-toluenesulfonyl)- 18-thiomarcforeine B
l-ethyl-18-thiomarcfortine B
I-~enzyl-18-thiomarcfortine B
518a-ethyl- 18-thiomarcfortine B
l8a-benzyl-18-thiomarcfor~ne B
18a-methoxyethoxymethyl- 18-thiomarcfor~ine B
18a-allyl-18-thiomarcfortine B
18a-propargyl- 18-thiomarcfonine B
1018a-ethyl-24methoxy-24,25-dihydro- 18-thiomarcfortine B
:~ 1,18a-bis-ethyl-18-Womarcfortine B
1,18a-bis-benzyl-18-Womarcfonine B
18a-ethyl-24methoxy-18-thiomarcfortine B
I -(2,4-dinitrobenzene sulfonyl~- 18a-ethyl-24-methoxy-24,25-dihydro- 18-thiomarcfortine B
15 18a-ethyl-24,25 dihydlo-18-thiomarcfo~tine B
~,:
24,25 dihyd~l8-thiomarcfortine C
l-(p-bromobenzene sulfonyl~l8-thiomarcfonine C
I-propionyl-18-thiomarcfortine C
I-propyl-18-Womarcfortine C
20 1-benzyl-18-thiomarcfortine C
` ~ 18a-propyl-18-WomarcfortineC
18a-benzyl-18-Womarcfortine C
18a-methoxyethoxymethyl-18-thiomarcfortine C
18-slllyl-18-tbiomarcfor~ne C
25 18a-propargyl-18-thiomar~fortine C
~ 1,18a-bis-propyl-18-thiomarcfortine C
:~ ~ 1,18a-bis-benzyl-18-thiomarcfonine C
18-thiomarcfortine C
I -(4~a~ethoxy- 1,3-thiazo1idinin-3-yl)carbonyl- 18-thiomarcforùne A
30 1-p~nitoyl-18-thiomarcfor~ne A
; l-(~morpholLnoca~onyl)-18-thiomarcfonine A.
Examples of the oompounds of Formula 111 of this invendon are as follows:
18-~iopa~erquamide 24,25-dihyd~18-~iopar~he~uamide 35 140-methyl-18-thiop~3herquamide l~ethyl-18-thiopar~herquamide . ~

WO 93/~0120 2 1 2 1 2 ~ ~ PCI/US92/09483 14-O-butyl- 18-thiopa~herquamide 14-O-~enzyl- 18-thiopar~herquamide 14-O-ally- 18-thiopar~herquamide 14-O-prop~rgyl- 18-thiop~raherquamide

5 14-O-methoxymethyl-18-thioparaherquamide 14-O-methoxy-ethoxy- I 8-thioparaherquamide 14-O-methoxy-ethyoxy-methyl- 18-thioparaherquamide 17-methyl- 18-thioparaherqu~mide 17-methylene- 18-thiop~herquamide 10 1 -N-(p-loluenesulfonyl)- 18-thioparaherquamide 24methoxy-24,25-dihydro- 18-thiop~raherquamide Examples of the compounds of Formula IV of ~his invention are as follows:
~; l~iethoxythiophosphoryl-marcforbine A
I-phenylmethoxyphosphonyl-marcfor~ine A
15 I-cyclohexylcarbonyl-marcfonine A
I-(l-piperidinyl)thiocarbonyl-marcfortine A
I-N-(4-carbethoxy-1,3-thiazolidinin-3-yl)a~rbonyl-marcfortine A
I-succinoyl-m~rcfo tine A
1-(40xopentanoyl)-marcfo~ine A
20 I-dimethylaminoaceql-marcfo~ne A
I-N,N-dimethylsucclnamidoyl-marcfortine A
I-cydopropylcarbonyl-18a-ethyl-marcfortine B
l dimethylaminosulfenyl-18a-N-benzyl-marcfortine B
1 -ethoxycar~onylsulfenyl- I 8a-N-methoxyethoxymcthyl-marcfor~ne B
25 l~iphenylphosphinyl-18a-N-allyl-marcfo~ne B
1-(3-acetoxy)propionyl-18a-N-propa~gyl-marcfortine B
1 -(2,4dichlorophenoxy)carbonyl- 18a-N-ethyl-24-methoxy-24,25-dihydro-marcfor~ne B
I-N-(2,4dinitroba~zenesulfenyl~18a-N-ethyl-marcfortine B
l-N-(p bromobenzenesulfenyl~marcfortine C
30 I-acctoxyme~yl marcfortino C
l~iethoxythiophosphoryl-marcfonino C
I-phenylmethoxyphosphonyl~marcfor~ne C
cyclop~pyl~bonyl-marcfotdne C
~bc~ marcfo~ne C
35 I-(l-pipe~idinyl)tnioarbonyl-marcfo~ine C
I-N~4~ethoxy-l~tlù~olidi~n-3-yl)carbonyl-marcfortine C
, ~ .
,~ ~

WO 93/~0120 PCr/US92/09483 2 1 21 2 3~ -18-I-succinoyl-marcfortine C
1-(4-morpholinosulfenyl~-marcfonine C
l-phenoxycarbonyl-marcfortine C
1-(4-oxopentanoyl)-m~rcfonine C
5 l-dimethylaminoacetyl-marcfonine C
l-N,N-dimethylsuccinamidoyl-marcfortine C
l-palrnitoyl-marcfonine A; preferably I-cyclopropylcarbonyl-marcfonine A
I-phenoxycarbonyl-marcfonine A
10 I-palmitoyl-marcfonine A
-tl(4-nitrophenylPxy]carbonyll-marcfonine A
l-(l-piperidinecarbonyl)-marcfortine A
[4(ethoxycarbonyl)piperazin- 1 -yl~carbonyl]-marcfortine A
I-[[4-(~enzyl)piperazin-I-yl]carbonyl~-marcforline A
15 1-114-(1,3-benzodioxol-5-yl-methyl)piperazin-1-yl]carbonyl]-marcfonine A
4(methYl)piperazm- I -y~]calbonyl]-marcfortine A
1-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)-marcfortine A
4(pyddin-2-yl)piperazin-1-yllcarbonyl]-marcfortine A
; . ~
~: 1-[14-(phenyl)piperazin-1-yl]carbonyl]-marcfortine A
. ~
~ 20 1-[[4(chlorocarbonyl)piperazin-1-yl]carbonyl]-marcfonine A
l-t[4(phenyl)piperidin-1-yl]carbonyl]-marcfonine A
1-[~4-(dimethyl)piperidin-1-yllcarbonyl]-marcfonine A
1-~14-(5-chloropyddazin-3-yl)piperazin-1-yl]carbonyl]-marcfortine A
1-[[4~4chlorophenyl)piperazin-1-yl]carbonyll-marcfortine A
25 1-[t4-(trichloroe~hoxycalbonyl)piperazin-1-yl]carbonyl]-marcfortine A
4-(3-trifluo~methyl-thiadiazol-1-yl)piperazin-1-yllcarbonyl~-marcfortine A
I-acetoxymethyl-marcfortine A
1-(2,4dinitrobenzenesulfenyl)-marcfonine A
1-(4morpholinosulfenyl)-marcfortine A
30 I-(trichloromethylsulfenyl)-marcfortine A
l-(methoxycarbonylsulfenyl)-ma~fortine A
l-(benzenesulfenyl)-matcfortlnc A
1-(2-tetmhydrafur~yl)-marcfortine A~ and ~`r`~ ` ' morc p~erably~ 1-(4morpholinocarbonyl~marcfonine A or 3~ 1-(4-morpholinocarbonyl)-marcfortine A N-oxide.
`` ~ Examples of the compow~ds of Formula V of Ihis in~ention are as follows:
`~"~":
.~
``i :" :

1 -acetoxymethyl-par~herquamide I -diethoxytluophosphoryl-paraherquamide I -phenylmethoxyphosphonyl-paraherquamide I -cyclopropylcarbonyl -par?herqu~mide 5 I-cyclohexylcarbonyl-paraherquamide I -( l-piperidinyl)thiocarbonyl-paraherquamide l-N-(4-carbethoxy- 1,3-thiazolidinin-3-yl)carbonyl-paraherquamide I -succinoyl-paraherquamide I -(~morpholinosulfenyl)-paraherquamide 10 I-phenoxycarbonyl-paraherquamide 1 -(4-oxopentanoyl)-paraherquamide 1 -dimethylaminoacetyl-paraherquamide I -N ,N-dimethylsuccinamidoyl-paraherquamide I -palmitoyl -paraherquamide 15 I-(~morpholinocarbonyl)-paraherquamide.
~he compounds of Formula Il are prepared by the following procedures:
It has been found~ unexpectedly, that treatment of the Marcfortines A, B, and C, or suitably substituted C-24, C-25, N-l and N-18a derivatives thereof, with phosphorous pentasulfide or, preferably, 2,4-bis(methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,~disulfide (Lawesson's ~20 reagent) selecdvely provides the conesponding 18-thio- derivadves. Suitable C-24, C-25, N-l ~nd N-18a substituted marcfortine A, B and C derivatives for this reacdon are readily prepared by the procedures given in U.S. patent 4,923,867, the disclosure of which is incorporated herein hy reference. The reactions are conducted in a suitable inert solvent such as pyridine, collidine, toluene (preferred), xylene, dioxane, tet~ahydrofuran, and the like, at temperatures f~m 10 to 25 180 prefer3bly 80 to 140.
Altematively, the C-24, C-25 and N-1 derivadves can ~e prepared from the 18-thiomarcfo~nes. For example, a large series of 18-thiomarcfortine analogs can be prep~red by alkylation or acylation of N-1 of the N-18a substituted 18-thiomarcfo~tines. These derivadves may be easily prepared by sequential treatment of a soludon of 18-thiomarcfortine A~
30 N-18a-subs~uted 18-thiomarcfordne B, or N-18a-substituted 18-thiomarcfo~inc C in an aprotic or~anic solvent such as tetrahydrofuran, ether, benzene and the like with an excess of a strong base such as potassium hydride (prefenl d)~ sodium hydride~ butyllithium~ potassium tert-butoxide~ and the like followed by a suitable alkylating or acyla~ng agent at temperatures ranging f~m O~C to 50DC for 0.2S to 48 hours. Suitable alkylating agents indude alkyl 5 bromides, alkyl iodides~ alkyl sulfonates, alkenyl iodides, alkynyl bromides~ alkoxyalkyl chlorides, and t~he like. Suitable acylating agents include acyl anhydrides. acyl chlorides, acyl ;

q ~ 3 ~ PCI/US92/09483 bromides, suhstituted benzenesulfonyl chlorides, substituted benzenesulfonic ~nhydrides, sulfenyl chlorides, isocyanates, carbamoyl chlorides. chloroformates, and the like.
An addition~l series of deriv~tives can ~e generated by modification of the C24-C~5 double bond of 18-thiom~rcfortines A, B and C. The 24,25 dihydro ;malogs are readily S prepared by stirring a solution of the appropriate 18-thiomarcfortine in an alcoholic solvent such as meth~nol, ethanol, propanol and the like with a cataiyst such as pall~dium, platinum. tris (triphenylphosphine)-chlororhodium and the like in the presence of hydrogen gas. The product, which is a 24,25-dihydro-18-thiomarcfortine analog, can be isolated and purified by using techniques known to those skilled in the art. Note that the reactions described above for 10 modification of other portions of the 18-thiomarcfortine structure may also t)e applied to 24,25-dihydro 18-thiomarcfortine analogs to prepare the corresponding 24,25-dihydro analogs.
Additional C24-C25 double bond modifled analogs of the 18-thiomarcfortines may t~e prepared via the 24,25 dibromide which is easily prepared by treating a solution of a 18-t~uomarcfortine in a halogenated solvent such as dichloromethane, chloroform, carbon tetrachloride and the li~;c I5 with 1 molar equivalent of bromine at temperatures ranging from -20C to 25~C for 0.25 to 8 hours. This process affords the corresponding 24,25-dibromo 24,25-dlhydro-18-thiomarcfortine derivadve which can be isolated and punfied by using techniques known to those skilled in the art. Note that the 24,25 dichlorD analog may be prepared by substituting chlorine for bromine in the process described above. The 24,25-dibromo 24,25-dihydro-18-thiomarcfortine anaJogs 20 described above are useful intermediates for the preparation of additional derivatives. Thus, treatment of a solution of the dibromide in an alcoholic solvent such as methanol, ethanol, propanol, and the like with a strong base such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) at temperatures ranging from OC to 30C for û.25 to 24 hours affords 24-alkoxy-25 bromo-24,25 dihydro-18-thiomarcfortine analogs which can be isolated and purified by using techniques 25 known to those skilled in the a~t. These 24 alkoxy, 25-bromo derivatives can be debrominated by treatment of a solution of the compound in an aprotic organic solYent such as benzene, toluene, hexane, and the like with a tin hydride reducing agent such as tri-butyl tin hydride, tri-phenyl tin hydride and the like with or without the ~ddition of a radical initiator such as azobis-isobutyronitnle (AIBN) at temperatures ranging fr~m 25C to 120C for 0.S to 48 hours~
30 This process affords the corresponding 24-nlkoxy-18-thiomarcfortine derivadvcs (R2~, = lowcr atkoxy in the general stNcture) which can be isolated and purified by using techniques known lo those skillcd in the arl.
General pmcedures for the preparation of heteroaromatic N-oxides can be found inChapter n of "Chemistry of ~e Heten)cyclic N-Oxides"~ A.R~ Katrit~ky and l.M. Lagowsl;i, ~5 published 1971 Academic Press (Vol. 19 of ORGANlC CHEMISI`RY - A Series of Monographs). Typically the N-oxide is formed by reaction with a percarboxylic acid in an . ~vo 93/10120 2 1 2 1 2 3 4 PC~r/US92/09483 appropriate solvent. Most suitably an aromatic peracid in a non-polar solvent is used. since the reaction may usually be carried out at r~om temperature. Suitable aromatic peracids include perbenzoic acid, chlomperbenzoic acid and perphthalic acid.
The compounds of Formula 111 are prepared by the following procedures:
S Treatment of paraherqu~nide and derivatives thereof, with phosphomus pentasulfide or, preferably, 2,4-bis(methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide (Lawesson's reagent) selectively provides the corresponding 18-thio- derivatives. Suitable paraherquamide derivatives for this reaction are readily prepared by the procedures given in U.S. patent 4,9,8,656 and 4,873,247 as well as WO 91/09961, the disclosures of which are incorporated herein by reference. The reactions are conducted in a suitable ineft solvent such as pyridine, collidine, toluene (preferred), xylene, dioxane, tetrahydrofuran, and the like, at temper~tures from 10 to 180 preferably 80 to 140.
Altematively, the C-24, C-25 and N-l derivatives can be prepared from the 18-thioparaherquamide. For example, a large series of 18-thioparaherquamide analogs can be prepared by alkylation or acylation of N-l of the N-18a substituted 18-thioparaherquamide.
These derivatives may be easily prepared by sequential treatment of a solution of 18-thioparaherquamide in an aprotic organic solvent such as tetrahydrofuran, ether, benzene and the like with an excess of a strong base such as potassium hydride (preferred), sodium hydride, butyllithium, potassium tert-butoxide, and the like followed by a suitable alkylating or acylating ~ 20 agent at temperatures ranging from 0C to 50C for 0.25 to 48 hours. Suitable alkylating agents include alkyl bromides, alkyl iodides, alkyl sulfonates, alkenyl iodides, alkynyl bromides, alkoxyalkyl chlofides, and the like. Suitable acylating agents include acyl anhydrides, acyl chlofides, acyl bromides, substituted benzenesulfonyl chlorides, substituted benzenesulfonic anhydrides, sulfenyl chlorides, isocyanates, carbamoyl chlorides, chloroformates, and the like.
2S An additional series of derivatives can be generated by modification of the C24-C25 double bond of 18-thioparaherquamides. The 24,25 dihydro analogs are readily prepared by stirring a sc~lution of the appropriate 18-thiop~raherquamide in an alcoholic solvent such as methanol~ ethanol, propanol and the like with a catalyst such as palladium, platinum, tris (triphenylphosphine) chlororhodium and the like in the presence of hydrogen gas. The product, which is a 24,254ihydro-18-thioparaherquamide analog, can be isolated and purified by using techniques known to those skilled in the art. Note that the reacdons described above for modification of other portior~s of the 18-thioparaherquamide st!ructure may also be applied to 24,25-dihydro 18-~ioparaherquamide analogs to prep~re the com~onding 24,25-dihydro ~nalogs. Additdonal C24C25 double bond modified anD~logs of the 18-thioparaherquamides ~5 may be prepa~ed via the 24,25 dib~omide which is easily prepared by treating a solution of a 18-thiop~herqu~mide in a halogenated solvent such as dichloromethane, chlotofo~m~ calbon WO 93~10120 PCI`/US92/09483 21212~

tetrachloride ~nd the li~;e with I molar equiv~ens of ~romine ~t tem~er~tures r~nging from -20~C to 25C for 0.25 to 8 hours. This process ~ffords the corresponding 24,25-dibromo 24,25-dihydro-18-thioparaherquarnide derivative which can be isolated and purified ~y using techniques known to those skilled in the ~ Note that ~he 24,25 dichloro analog may be 5 prepared by substituting chlorine for bromine in the process described above. The 24,25-dibromo 24,25-dihydro-18-thioparaherquamide analogs described above are useful intermediates for the preparation of additional derivatives. Thus, treatment of a solution of the dibromide in an alcoholic solvent such as methanol, ethanol, propanol. and the like with a strong base such as 1,8-diazabicyclo~5.4.0]undec-7-ene (DBU) at temperatures ranging from OC
10 to 30C for 0.25 to 24 hours affords 24-alkoxy-25 bromo-24,25 dihydro-18-thioparaherqua nide analogs which c~n be isolated and purified by using techniques known to those skilled in the arl.
These 24 alkoxy, 25-bromo derivatives can be debrominated by`treatment of a solution of the compound in an aprotic organic solven~ such as benzene, toluene, hexane, and the like with a tin hydride reducing agent such as tri-butyl tin hydride, tri-phenyl tin hydride and the like with or 15 without the addidon of a radical inidator such as azobis-isobutyronitrile (AIBN) at temper~tures ~; ranaing from 2SC to 120~C for Q5 to 48 hours. This process affords the corresponding 24-alkoxy-18-thioparaherquamide derivadves (R2~ = lower alkoxy in the general structure) which can be isolated and purified by using techniques known to those skilled in the art.
General procedures for the preparation of heteroaromatic N-oxides can be found in 20 Chapter 11 of "alemistry of the Heterocyclic N-Oxides", A.R. Katritzky and J.M. Lagowski, published 1971 Academic Press (Vol. 19 of ORGANIC CBMlSTR~ - A Series of Monographs). Typically the N-oxide is formed by reaction with a percarboxylic acid in an appropriate solvent. Mpst suitably an aromatic peracid in a non-polar solvent is used, since the reaction may usually be carried out at room temperature. Suitable aromatic peracids include 25 perbenzoic acid, chloroperbenzoic acid and perphthalic acid.
The compounds of Folmula IV are prepared by alkylation or acylation of N-l of marcfortine A and N-18a substituted marcfortines B and C. These derivadves may be easily prepared by sequential t~:atment of a soludon of marcfortine A, or N-18a subsdtuted marcfortines B or C in an aprotic organic solvent such as tctlahydrofuran, ether, bcnzenc and the 30 like wlth an excess of a strong base such as potassium hydride (preferred), sodium hydride butyllithium, potassium tert-butoxide, and the like follnwed by a suhable alkyla~ng or acyl~ting agent at temperatures ranging frwn 0DC to S0C for 0.25 to 48 hours~ Suitable alkyladn~ or acyla~ng agents include alkanoyloxyalkyl bromides, aminosulfenyl chlorides, phosphoryl chlorides, phosphonyl c~orides, acyl anhydrides, acyl chlorides, acyl bromides, substituted 35 benzenesulfenyl chlorides~ ca~moyl chlorides~ substituted phenoxychloroformates, and the like.
Treatment of marcfortine A and N 18a substituted marcfortines B and C and C24C25 modified WO 93/10120 PCr/USg2/09483 212123~ `

marcfortines under these conditions affords 1-N-substituted analogs which c~n he isol~ted ~nd purified ~y using techniques known to those skilled in the ar~
The compounds of Formula V are prepared by alkylation or acylation of N- I of paraherquamide. These derivatives may be easily prepared by sequenliaMreatmenl of a solution S of p~r~herquamide in an aprotic organic solvent such as tetrahydrofuran, ether, benzene and he like with an excess of a strong base such as potassium hydride (preferred), sodium hydride butyllithium, potassium tert-butoxide, and the like followed by a suitable alkylating or acylating agent at temperatures ranging from 0C to 50C for 0.25 to 48 hours. Suitable al~ylating or acylating agents include alkanoyloxyalkyl bromides, aminosulfenyl chlorides, phosphoryl 10 chlorides, phosphonyl chlorides, acyl anhydrides. acyl chlorides, acyl bromides. substituted benzenesulfenyl chlorides, carbamoyl chlorides, substituted phenoxychlorofonnates, and the like.
Treatment of paraherqu~nide under these conditions affords l-N-subslituted ana1Ogs which can be isolated and purified by using techniques known to those skilled in the ar~
General procedures for the preparation of heteroaromatic N-oxides can be found in 15 Chapter 11 of "Chemistry of the Heterocyclic N-Oxides", A.R. Katritzl;y and J.M. Lagowski, published 1971 Academic Press (Vol. 19 of ORGANIC CHEMlSTRY - A Series of Monographs). Typically the N-oxide is formed by reaction with a percarboxylic acid in an appropriate solvent. Most suitably an aromatic peracid in a non-polar solvent is used, since the re~ction may usually be carried out at ~om temperature. Suitabîe aromatic peracids include 20 perbenzoic acid, chloroperbenzoic acid and perphthalic acid.
.
PREPARATlON OF STARTING MATERlAL
Pa~herquamide is isolated from Penicillium Sp. IMI 332g95 and/or Penicillium charlcssi MF 5123 (ATCC 20841) using standard fennentation and isola~ion techniques. The 25 isolation is described in detail in US Patent 4,873,247 and 4,978,656 as well as EP 3905~2-A, EP-301742-A and WO 91/09961 (all of which are inco~orated herein byreference).
PREPARATION OP STARTING MATERlAL
N-18a substituted marcfortines B and C and C24^C25 modified marcfortines arc rcadily 30 prepared by procedures given in U.S. Patent 4,923,867, the disclosure of which is incolpor~ed herein by reference.
Marcfo~#s A, B and C are isola ed, along with the previously known roquefortine, as fungal metabolites of Pcn cillium roqueforn using standard fermentation and isolation ~- ~ techniques. The isol~tion, as well as the analytical and structural characteristics of marcfortine ~5 A~ are descri~ed in detail in Polonsky et al Journal of the Chemical Society Chcrnical .
Conununications 198Q 601-602. The isolation, as well as the analytical and structural WO 93/10120 PCr/US92/09483 %~ 2123~ -24-characteristics of marcfortines B ~nd C, s~re descrihed in detail in Polonsky et al Tetrah~dron Letters 1981, 22, 1977- 1980.
Altematively, and more preferably, Marcfortines A and C msly be isol~ted from Penicillium sp. UC7780 (strain number in Upjohn Culture Collection, UC 7780, The Upjohn S Company, Ksllamsuoo, Ml). This strain ws~ isolated from sl soil sslmple collected in lllinois.
deposited in the U.S. Department of Agriculture patent culture collection in Peoria, IL smd given the accession nu nber NRRL 18887. To further chs~racterize the fungus a taxonomy study ws~s done following the methods and materials described by 1. John Pitt, The Genus Penicillium, Academic Press, London, (1979). Spore smd hyphae surfaces were examined hy scanning electron microscopy according to the methods of Diet~, A. and Matthews, J, Appl. Microbiology 18:694-696 (1969). Intact conidiophores are visualized by light microscopy [A.H.S. Onions et al., Smi~'s Introduction to lndustrial Mycology, John Wiley and Sons, New York, pp 301-302 (1979)] after slide culture(s) are prepared: A glass petri dish containing glass be:ads, microscope slide, and coverslip are sterilized. A small block of pOtslto dextrose agslr is placed on the slide and inoculated on four sides with ~he fungus culture. The coverslip is set on the inoculated ag~r block and sterile water added to maintain moisture. The chamber is incubated for six days at 24 C. A slide is prepared by removing the coverslip and placing it on a drop of lactophenol cotton blue stain.
The characteristics of Penicillium sp. UC 7780 (NRRL 18887) are as follows:
Morphology - a biverticillate penicillus (two branch points between conidium and stipe).
These branches (metulae) support the phialides or conidia bearing structures. Conidiophores (approximately 35 ~m) terminated in verticils of 2-5 (10-14 ~n) metulae. Phialides were ampulliform (like an ancient Greek wine jar) in verticils of 2-5 (7 ~n). Conidisl were smooth and sphe~oidal (2 ~m~ typically appearing in long colulrms. The stipe walls were smooth.
The cul~ure was inoculated onto three petri dishes of Czapek yeast agar (CYA), one being 6 cm in diameter and one each of malt extract agar (MEA~ and 25% glycerol nitrslte agar (G25N). lnoculation was made from a semisolid suspension (0.5 ml of 0.2% agar with 0.05%
Tween 803. An inoculating loop of conidia wsls added to the tube smd mixed. A loop of suspension was inoculated in a pattem of three sites per plate. A needle ws~c used to stslb inocu~ate the 6 cm plate. The incubation regimc wsls: onc CYA pl~te plus thc MEA su~d G. ~N
plates at ~4C, one CYA plate at 37C, and the 6 cm CYA plslle slt S"C. Af`ter seven d~ys the colony diameters~ colors~ and other characters were recorded and are set forth in Table 1. On pota~o dextrose agar (PDA, Mfco) agar, a deep red color on the bonom or reverse of the colony is produced.
~S No sexual stage was noted. This results in the culture (NRRL 18887) being keyed in the Penicillium Key to subgenera. Within the Penicillium Key~ the penicillus type alone WO g3/10120 2 1 2 1 2 3 4 PCI/US92/09483 -25- `?
determines the subgenus to which a species is allocated. This species has several characteristic~
that distinguish it from the Biverticillium subgenus even though its penicillus is bivenicilliate.
The species consistently produces colonies greater than 10 mm in diameter in 7 days on glycerol nitrate agar. The metul~e appear longer than the phialides and are in verticils of 2-5. These S characteristics place this Penicillium sp. (NRRL 18887) into the subgenus Furcatum.
The foregoing d~scription is illustrative of a str~in of Penicillium sp. UC 7780 (NRRl 18887) which can be employed in the production of Marcfortine and derivatives thereof.
However, the present invention also embraces mutants of the above described microorganism.
For example, those mutants which are obtained by natural selection or those produced ~y 10 mutating agents including ionizing radiation such as ultraviolet irradiation, or chemic~l -mutagens such as nitrosoguanidine or the like treatments are also included within the amt i~ of this invention.
This description is also desired and intended to include inter- and intraspecific gcnetic recombinants produced by genetic techniques well known to those skilled in the art, such as. for 15 example, conjugation, transultion and genetic engineering techniques.
Penicilliurn sp. UC 7780 (NML 18887) may be cultivated under aerobic condition in the same manner as commonly employed in the art for cultivation of a known strain of the genus Penicilli~un.
As medium components may be employed any of the well-known nutnent materials for20 PeniciJli~un. For instance, as an assimilable carbon source, glucose, glycerol, maltose, dextrim starch, lactose, sucrose, molasses, soybean oil, cotton seed oil, etc., preferably glucosè and glycerol may be employed and~ as an assimilable nitrogen source, soybean meal, peanut me~l~
cotton seed meal, fish meal, com steep liquor, peptone, rice, br~n, meat extract, yeast, ye~st extrac~, sodium nitrate, ammonium nitrate, ammonium sulfate, etc. may be used. And, such 25 inorganic salts as sodium chloride, phosphates, calcium carbonate, etc. may be added to a culture medium. A minor amount of a metal salt may also be added, if necessary. Further, a minor amount of a heavy metal may be added, if necessary~
Particularly, in cultivating the Penicillium sp. (NRRL 18887) under aerobic condition, ordinary aerobic cultivadon methods such a5, for example, solid culture, culture under ~er~tion 30 and agitation~ shaken culture etc. may be favorably udlized~ Trace metals, for example, zinc.
magnesium, manganese, cobalt~ i~n and the like should be be added to the fermentation medium since reve~osmosis grade water must be supplemented.
ln carrying out cultivation with aeration and agitation~ an anti-foaming agent~ e.g., silicon oil, vegetahle oils, surf~ctants, etc. may be suitably employed.
~5 The pH of the medium may be ususlly within a pH range of 3-9 and preferably within or around neutral range and cultivation tempe~tures may be usually of 20-30C., in particul~r WO 93J10120 PC~r/U~92/09483 ~1~1 234 about 21C being preferred.
Cultivation may be continued until Marcfortine A will be substantially accumul~ted in a culture medium, usually for 20 hours to 240 hours~ preferably for 48 hours to 168 hours and, after cultivation, Marcfortine A may be isolated and recovered from a cultured broth hy ~
5 suitable combination of various method. For example, there may be extraction with an organic solvent, e.g. ether, ethyl acetate or chloroform; dissolution into a more polar solvent, e.g.
acetone or alcohol; removal of impurities with a less polar solvent, e.g. petroleum ether or hexane, adsorptive chromatography with active carbon or silica gel; gel filtration through a col-.lmn of "sephadex" (available from Pharmacia Co., Ltd, U.S.A.); and so on.
Marcfortine B can be prepared from Marcfortine A by biotransformation using a micr~orgainism in an aqueous nutrient medium containing an assimilable source of carbon and an assimilable source of nitrogen under aerobic conditions. The present invention also provides:
Biologically pure cultures of the (Organisms obtained from culture collection asobviously discovered entities) genus Cunninghamella, ch~racterized as species selected from the group consisting of NRRL 1368, 1393 or 3655 and ATCC 8688a A method of using members of genus Cunninghamella, characterized as species selected from the group consisting of NRRL 1368, 1393 or 3655, and ATCC 8688a to produce Marcfortine B which comprises:
adding Marcforline A to metabolizing cultures of the cited microorganisms.
By "biotransformation" is meant the use of microorganisms and/or isolated, partially purified enzymes for the converstion of a given substrate inlo a useful product. H.G. Davies, et al., "Biotransformations in Preparative Organic Chemistry", Academic Press, N.Y., 1989, p. IX.
By "metabolizing" is meant carrying out the p~cesses of metabolism. A definltion of metabolism may be found in A.L. Lehninger, "P~inciples of Biochemistry", Worth Publ.. New 25 York, 1982, p. 333.
By using the new method of the present invention, Marcfortine B is produced by adding Marcfortine A to growing cultures of cited microorgarusms. The genus Cunninghamella is preferled for effecting this transforma~ion, including Cunninghamella echinlllatn subsp. ele~ans (-) NRRL 1368, Cunninghamella bla~eslceana (+) ATCC X688a Cunninghamella echinuluta 30 subsp. elegans NRRL 1393, Cunninghamella echinulata NR~L 3655; the strain NRRL 3655 is particularly prefer~ed.
A subculture of Cunningh~mclla echinulata subsp. elegans (-) was deposited under the provisions of the Budapest Treaty in the permanent collection of the Northem Region Research Center, ARS; U.S. Dept. of Agriculture; Peoria, Illinois~ USA. Its accession number is NRRL
68. A subculture of Cunninghamella eckinula~a subsp. elcgans (-) was also deposited under the provisions of the Budapest Trealy in the permanent collection of the Americ~n Type Culture WO ~3/10120 2 1 2 1 2 3 ~ PCr/US92/09483 Collection, Rockville~ MD, USA. Its accession number is ATCC 8688t).
A subculture of Cunninghamella blakesleeana r+) was deposited under the provisions of the Budapest Tre~ty in the permanent collection of the American Type Culture Collection, Rockville, MD, USA. Its accession number is ATCC 8688a.
A subculture of Cunninghamella echinulata subsp. elegans (-~ was deposiled under the provisions of the Budapest Treaty in the permanent collection of the Northem Region Research Cen~er, ARS; U.S. Dept. of Agriculture; Peoria, Illinois, USA. Its accession number is NRRL
1393. A subculture of Cunninghamella echinulata subsp. elegans (-) was also deposited under the provisions of the Budapest Treaty in the permanent collection of the Arnerican Type Culture Collection, Rockville, MD, USA. Its accession number is ATCC 10028b.
A subculture of Cunninghamella echinulata was deposited under the provisions of the Budapest Treaty in the permanent collection of the Northem Region Research Cenler, ARS; U.S.
Dept. of Agriculture; Peoria, Illinois, USA. lts accession number is NRRL 3655.
The use of these microorganisms in the process of the present invention results in the production of Marcfortine B from Marcfortine A.
Marcfoitine B is produced when Cunninghamella echinula~a subsp. elegans (-) NRRL1368, Cunninghamella blakesleeana (+) ATCC 8688a, Cunnin~hamella echinulata subsp.
elegans (-J NJ~L 1393 or Cunninghamella echinulata NRRL 3655 is fermented in an aqueous ~ :
nutrient medium under submerged aerobic conditions in the presence of Marcforliné A.
20 Typically the microorganism is fermented in a nutrient medium containing a carhon source and a proteinaceous material. Preferred carbon sources include glucose, brown sugar. sucrose, glycerol, starch, cornstarch, lactose, dextrin. molasses, and the like. Preferred nitrogen sources include cnttonseed flour, com steep liquor, yeast, autolyzecl brewer's yeast with milk solids, soybean meD~I, cottonseed meal, cornrneal. milk solids, pancrealic digest of casein. distillers' 25 solids. animal peptone liquors, meat and bone scraps, and the like. Combinations of these car~n and nitrogen sources can be used advantageously. Trace metals, for exarnple, zinc, magnesium, manganese, cobalt, iron and the like need not be added to the fermentation medium since tap water and unpurified ingredients are used as medium components.
Production of Marcfortine E~ can be induced at any temperaturc conducive to satisfactnry 30 growth of the microor6anism between about 23 and 32 C and prefera~ly at about 28C.
Ordinarily, optimum production of Marcfortine B is obta~ned in about I ~o 4 days after addition of Marcfo~ne A to the growing culture, and preferably in about 2 days. The fcrmcntation broth nom~ally remains weakly acid to basic (pH 5.8 - 9.0) during the femlcntation. lhe final pH is dependent~ in p~art, on the buffers present~ if any. and in part, on the initial pH of the culture 35 medium. It is advanta~eously adjusted to about pH 6.S-7.5, and preferably 7.2~ prior to sterili2atiop.

WO g3/10120 PCr/US92/09483 ~12I2?~

When growth is c~rried out in shake flasks or l~rge vessels ~nd tanl;s~ it is prefer;lt)le lo use the vegetative form, rather than the spore form, of the microorg~nism for inocul~tion to avoid a pronounced lag in the production of Marcfortine B and the attend~nt inefficienl udli-zation of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in an S aqueous nutrient medium by inoculating this medium with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to other shake flasks or t~nks. The medium in which the vegetative inoculum is produced can be the same as, or different from, th~t utilized for the production of Marcfortine B, as long as it is such that adequate growth of the microorganism is obtained.
A variety of procedures call ~e employed to isol~te and purify the Marcforline A from the fermentation broth, for example, by chromatographic adsorption procedures followed t-y elution with a suitable solvent, column chromatography, partition chromatography, and crystallization from solvents and combinations thereof.
ln the preferred recovery process, the Marcfortine B is extracted from the whole beer.
15 Column chromatography techniques, preferably over silica gel, are used to perform the initial purificadon. Final purification of marcfortine B is achieved by chromatography and crystallization from organic solvents.
Procedure A further illustrates the biotransformation process of the subject invention.
The instant compounds of this invention are unexpectèdly poten; andparasitic agents 20 against endo and ecto parasites, particularly helminths and artluopods, which cause numerous parasitic diseases in humans, animals, and plants~ ~
. . .
Parasitic diseases may be caused by either endoparasites or ectoparasites~ Endoparasites are those parasites which~ live inside the body of the host, either within an organ (such ~s the stom..ch, lungs, heart, intestines, etc.) or simply under the skin. Ectoparasites are those parasites 25 which live on the outer surface of the host but still draw nutrients f~m the host~
The endoparasitic diseases generally referred to as helminthiasis are due to infection of the host with parasitic worms known as helminths~ Helminthiasis is a prevalent and serious worldwide eronomic pr~blem due to infection of domesticated animals such as swine~ shecp~
horses, cattle, goats, dogs, cats, and poultry. Many of thcse infections arc caused t~y the ~rnup 30 of worms descriW as nematodes which cause diseases in various spccies of animals throughout the world. Thes~ diseases are frequently serious and can result in the death of the infected ~imal. The most common genera of nematodes infecting the animals referred lo above are Haemonchlls, ~richostrongylus, Ostertagia, Nematod~`rus, Cooperia, Ascaris, Bunostom~un, Oesophagostomum, Chabertia, Trichuris, Strongylus. Trichonema, Dictyocaulus, Capillaria~
~5 He~erakis, Toxocara, Ascaridia, O~yuris, Ancylostoma, Uncinaria, Toxascaris, and Parascaris~
Many parasites are species specific (infect only one host) ~nd most also have a prefelTed site o~

, . .

infection within the anim~l. Thus Haemonchus and Ostertagia prim~rily infect the stomach while Nematodirus and Cooperia mostly attack the intestines. Other parasites prefer to reside in the heart, eyes, lungs, blood vessels, and the like while still others are subcut~neous parasites.
Helminthiasis can lead to we~kness, weight loss, anemia, intestinal damage, ~ualnutrition, and S damage to other organs. If left untreated these disea~es can result in the death of the animal.
Infections by ectoparasitic anhropods such as ticks, mites, lice, stable flies, hornflies, -blowflies, fleas, and the like are also a serious problem. lnfection t y these parasites results in loss of blood, skin lesions, and can interfere with normal eating habits thus causing weight loss.
These infections can also result in transmission OI serious diseases such as encephalitis, 10 anaplasmosis, swine pox, and the like which can be fatal.
Animals may be infected by several species of parasite at the same time since infection by one parasite may weaken the animal and make it more susceptible to infection by a second species of parasite. Thus a compound with a broad spectrum of activity is panicularly -~
advantageous in the treatment of these diseases. The compounds of this invention have 15 unexpectedly high activity against these parasites, and in addition, are also active against Diroflaria in dogs, NemaJospiroides and Syphacia in rodents, biting insects and migrating~
dipterous larvae such as Hypoderma sp. in catt~e, and Gastrophilus in horses.
The instant compounds are aiso useful agaLnst endo and ecto parasites which cause parasidc diseases in humans. Examples of such endoparasites which infect man include gastro 20 intestinal parasites of the genera Ancylostorna, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, En~erobius, and the like. Other endoparasites which infect man are found in the blood or in other organs. Examples of such parasites are the filarW worms Wucheria, Brugia, Onchocerca, and the like as well as extra intestinal stages of the intestinal worms Strongylides and Trichinella. Ectoparasites which parasitize man include artl~pods such as 25 ticks, fleas, mites, lice, and the like and, as with domestic animals~ infections by these parasites can result in transmission of serious and even fatal diseases. The instant compounds are a~tive against these endo and ecto parasites and in addition are also active against biting insects ~Id o~er d.aterous pests which annoy humans. The instant compounds when administered orally or parenterally are administered at a dosage rate of from 0.05 to 20 mg/kg of animal body weight.
The instant compounds are also useful against common househo`d pests such as Blatella sp. (cockroacb)~ Tineola sp. (clothes moth), Attagcnus sp. (ca~et beetle), Musca domestica (housefly~ and agalnst Solenopsis Invicta (imported fire ant).
The compounds are fur~ermore useful against agricultural pests such as aphids (Aavrthiosiphon sp.)~ locusts~ and boll weevils as well as against insect pests wbicb ~ttack stored ~5 grains such ~s Tribolium sp. and against immature stages of insects living on plant dssue. The WO 93/10120 PCr/US92/09483 ~1 2 1 23~ ~30-compounds are also useful as a nematocide for the control of soil nematodes which m~y t)e agriculturally important.
For use as an antiparasitic agent in animals the instan~ compounds may be administere~
intemally either orally or by injection, or topically as a liquid drench or as a shampoo.
For oral administration, the compounds may be administered in capsule, tablet, or drench bolus form or altematively they can be mixed in the animals feed. The capsules, tablets, and drenches boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate. These unit dosage forms are psepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, suspending agents, and/or binders such that a uniform mixture solution or suspension is obtained. An inert ingredient is one that will not react with the instant compounds and which is non toxic to the animal being treated. Suitable inelt ingredients include starch, Iactose, talc, magnesium stearate.
vegetable gums and oils, and the like. These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection. The active ingredient may also be administered as an additive to the feed by simply mixing the compound with the feedstuff or by applying the compound to the surface of the feed.
Altematively the active ingredient may be mixed with an inert carrier and the resulting 20 composition may then either be mixed with the feed or fed directly to the animal. Suitable inen carriers include corn meal~ citms meal, fermentation residues, soya gllts, dried grains and the like, The active ingredients are intimately mixed with these inen carriers by grinding, stirring, mllling~ or tumbling such that the fina~ composition contains from 0.001 to 5.0% by weig'ht of the active ingredient.
The compounds may altematively be administered parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either int~unuscular, intraruminal, intratracheal, or subcutaneous. The injectable fonnulation consists of the active ingredient mixed with an appropriate inert liquid carrier. Acceptable liquid carriers include the vegetable oils such as pe~nut oil, cotton sced oil, scs~nc oil ~ld the like as well as organic solvents such as solketal, glycerol fonnal and the like. As an altemative~
aqueous parenteral fonnulations may also be used. The vegetable oils are the prefemd liquid ca~iers, The formulations are prepaled by dissolving or suspending the active ingredient in the liquid carrier such lhat the final formulation con~ains from 0.005 to 20% by weight of the active ingredient.
~S Topical application of the instant compounds is possible tt~ugh the use of a liquid drench or a shampoo containing the instant compounds as an aqueous solution or suspension.

WO 93/10120 2 1 2 1 2 3 ~ PCI/US92/û9483 These formulations generally contain a suspending agent such as ~entonite ~nd normally will also contain an antifoarning agent. Formulations containing from 0.005 to 20% by weight of the active ingredient are acceptable. Preferred fonnulations are those containing from 0.5 to 5% ~y weight of the inst~nt compounds.
The instant compounds are primarily useful as antiparasit;c agents for the treatment and/or prevention of helminthiasis in domestic animals such as canle, sheep, horses, dogs~ cats, goats, swine, and poultry. They are also useful in the prevention and treatment of parasitic infections of these animals by ectoparasites such as ticks, mites, lice, fleas and the like. They are also effective in the treatment of parasitic infections of humans. In treating such infections the compounds of this invention may be used individu~lly or in comt-in~tion with each other or with other unrelated antiparasitic agents. The dosage of the instant compounds required for best results depends on several factors such as the species and size of the animal, the ~pe and severity of the infection, the method of administration and the compound used. Oral administration of the instant compounds at a dose level of from 0.005 to 50 mg per kg of animal body weight either in a single dose or in several doses spaced a few days apart, generally gives good results. A single dose of one of the instant compounds normally gives excellent control however repeat doses may be given to combat re-infection or for parasite species which are unusually persistent. The techniques for administering these compounds to animals are known to those skilled in the veterinar~ field.
The compounds of this invendon may also be used to combat agricultural pests which attack crops either in the field or in storage. The compounds are applied for such uses as sprays, dusts, emulsions and the like either to the growing plants or the harvested crops. The techniques for applying these compounds in this maMer are known to those skilled in the agricultural arts.
The following examples are provided in order that this invention might be more fully understood; they are not to be construed as limitative of the invention.
ExamPle 1 Production and lsolation of Marcfortine A
Seed Fermentation Process:
Seed fermentations are inoculated using agar plu~s of isolate Penicillium sp. UC 77xn (NRRL 18887) stored over liquid nitrogcn. Thsee plu~s are thnwed nnd used as inoculum.
GS-7 is composed of glucose and cottonseed flour (sold under the trademark "Pharmamedi~" by Traders P;~tein, Procter ~ Gamble Oilseed Products Co., Memp~is, TN, U.S.A.).
Unsupplemented tap water is used to hydrate the medium components and the medium is adlusted to pH 7.2 with NH,OH. The medium is dispensed into unba~ned closed-system flasks at 300 ml pper 1000 ml flask, and sterilized by autoclaving at 121C for 30 minutes. E~ch closed-system fl~c cont~ining 300 ml of GS-7 medium is inoculated with three agar plugs of -32- ~. .
Penicillium sp. UC 7780 (NRRL 18887) and shaken on a rotary shaker at 250 rpm for 36 hr a 22C.
Secondary Seed Fermentation Process:
The mature seed cultures are used as inoculum for the secondary medium at a 0.~% seed S r~te. The second~ry medium is composed of glucose monohydrate (sold as under the trademar~
Cerelose by C.P.C. International) 25 g, cottonseed flour (sold under the trademark "Pharmamedia") 25 g, MgCI2-6H20 329.8 mg, MnSO4-H20 11.4 mg~ FeSO4-7H10 3.2 mg, Na2MoO4-2H~O 1.8 mg, CaCI2-2H20 ~67.6 mg, NaCI 84.2 mg, KCI 5.8 mg, ZnSO4-7H,O 0.1 mg, CoCI2-6H20 0.1 mg, CuSO4~5H20 3.1 mg, and silicone antifoam (sold under the trademark SAG~71 Antifoam) 0.5 ml per liter of reverse-osmosis grade water. Medium components sufficient for 200 liters of secondary seed medium are hydrated in reverse-osmosis grade water to a q.s. volume of 190 liters in a 25~L fermentor. After formulation, the pH of the medium is ad~usted to pH 7.2 with NH40H. and then the medium is sterilized at 121C for ~0 minutes.
Two closed-system flasks of the mature primary-seed culture are used 3S inoculum at a 0.3%
seed rate. The secondary seed culture is incubated at at 22C, with 125 slm aera~ion, 5 psig backpressure, and 250 rpm for 36 hours.
Production Fermentation Process:
The production medium is composed of beet molasses 50 g, fish meal (sold under the trademark Menhaden Select Pish Meal) 16 g, yeast extract (sold under the trademark Fidco) 10 g, MgCI2-6H20 329.8 m~, MnSO4~H20 11.4 mg,FeSO4-7H20 3.29mg, Na2MoO4-2H20 1.8 mg, CaCI2-2H20 36î.6 mg, NaCI 84.2 mg, KCI 5.8 mg, ZnSO4-7H2d 0.1 mg, CoCI2~6H20 0.1 mg, CuSO4-5H20 3.1 mg, and silicone antifoam (sold under the trademark SAG-471 Antifoam) 0.5 ml per liter of reverse-osmosis grade water.
Medium components sufficient for 5,000 liters of medium are hydrated in reverse-osmosis grade water to a q.s. volume of 4,700 liters in a 5,000 L fermentor. ARer formul~tion, the pH of the medium is adjusted to pH 7.0 with KOH, and then the medium is sterilized at 1~3C for 30 minutes. The mature secondary-seed culture is used as inoculum at a 1.0% seed rate. The culture is incubated at 22C, with 2,SOO slm aeration, 5 psig backpressure, and 250 rpm for 96 hours.
Isolation of Marcfortine A:
The 4900 L fermentation volume is harvested by passing through a high shear mixer to the l~vest vessd. FoUowing transfe~ 4% wt./v. of diatomaceous eanh and 1/2 volume of me~ylale chloride are added. The harvest solution is then filtered using a filter press. The filter cake is washed 2 times with a 10% volwne of methylene chloride.
llhe filtrate obtained is decanted to remove the water (aqueous) phase. The remauung product-rich methylene chloride phase is then concent~ted to a volume of 44 L. T~he WO 93/10120 2 1 21 2 3 4 PCr/US92/09483 concentrate is then polished using a 20% concentr~te volume ( 9 L)of methylene chloride and diatomaceous earth over a filter.
The 53 L polished concentrate is further purified to separate Marcfortine A from other components by silica gel chromatography and crystall32ation.
Before chromatography, the polished concentr~te is divided into four approximately equal aliquots. Each aliquot is chromatographed over a newly packed 9" diameter column prepared from 25 Kg of dry silica gel (bed volume 59 L). The loaded columns are eluted with 120 L of 10% acetone in methylene chloride, 120 L of 20% acetone in methylene chloride~ 120 L of 30% acetone in methylene chloride, 160 L of 40% acetone in methylene chloride. and 130 10 L of acetone collecting the 30 and 40% eluates as 2~ L fractions. Eluates are monitored ~y TLC, using for example a solvent system comprised of 6% isopropanol and 0.3% ammoniumn hydroxide in methylene chloride to develop Whatm;~l LK6DF silica gel plates. Fractions of Marcfortine A (containing a small amount of Marcft3nine D which co-chromatographs with D) are crystallized from acetone. Appropriate fractions ( 40-1001 ) Ire concentrated under reduced 15 pressure to a volume of approximately 5 L. The solution ( or li~ht slurry) is then transferred to a rotatory evaporater and concentration continued under reduced pressure. Several I L portions of acetone are added during the course of the concentration until the methylene chloride is completely displaced. The resulting acetone slurry (approximately I L volurne) is refrigerated ovemight, and the crystals of Marcfortine A are collected and wæhed with several small 20 portions of cold acetone, and dried under vacuum. Such crystals may be contaminated with ..
several percent of Marcfortine D~ Repeated recrystallization from methylene chlonde/acetone (displacing methylene chloride as described) affords pure Marcfortine A.
amnle IA .Production and Isolation of Marcfortines A and C.
Primary Seed Fermentation Process:
Seed fermentations are inoculated using agar plugs of isolate Penicillium sp. UC 7780 (NRRL 18887) stored over liquid nitrogen. Three plugs are thawed and used as inoculum for 100 ml of GS-7 seed medium. GS-7 is composed of glucose and cononseed flour (sold under the trademalk "Pharmamedia" by Traders Protein, P~cter & Gamble Oilseed Praducts Co., Memphis~ TN, U.S.A.) each added at a concentration of 25 g/L of tap watcr. Aftcr formulation.
the pH of GS-7 is ad~usted to 7.2 using NH40H. The medium is autoclaved in 100 ml volumes in 500 ml unbaffled felmentation flasks for 30 min. S~erile GS-7 is inocul~ted as described above and shaken at 2S0 rpm for 3S-58 hr at 23C.
Production Fermentation Process (shaker flask):
The maturc seed cultures are used as inoculum for the production medium at a 1% seed rate. The pro~clion medium is composed of glucose 45 g~ enzymatically digested casein (sold under the trademalk Peptoluzed Milk NutJient by Sheffield Products, Norwich~ N.Y.~ U.S~A.) 2S

W~3 93/10120 PCr/US92/09483 21212~

g, yeast extract (sold under the trademark BACrO Yeast Fxtract Code: 0127 by Difco Laboratories, Detroit, Ml) 2.5 g per liter of tap water. After formulation, the pH of the production medium is adjusted to 7.0 using potassium hydroxide. This medium is then autoclaved for 30 min in 100 ml volumes contained in 500 ml baffled fermentation flasks.
Stcrile production mediwn is inoculated as described above, and sh;lken for 7-14 days at 250 rpm at 21C.
Production Fermentation Process (Labraferm tanks):
The mature seed cultures are used as inoculum for the sterile production medium at a 0.5% seed rate. The production medium is descri~ed above. After pH adjustment to 7.0 using KOH, 10 L of this medium are autoclaved for 90 min in 12 L Labraferm tanks (New Brunswicl;
Scientific Co., Inc.). The tanks are inoculated at a 0.5% seed rate and stirred at 500 rpm at 20C for 5-9 days. The air flow rate is maintained between 10-15 L/min.
Isolation of Marcfortines A and C:
Whole fermentation broth (35 1) is macerated at low speed in a large commercial Waring Blender and then blended with an equal volume of methylene chloride. The mixture is stored overnight under refrigeration and then subjected to centrifugation to break the emulsion. The resulting clear methylene chloride layer is drawn off and evaporated under reduced pressure. A
concentrated solution of the residue (37.4 8) in methylene chloride is applied to a column of silica gel (lKg) slurry packed in methylene chloride~ The column is eluted with increasing concentrations of acetone in methylene chloride (10 %~ 20%, 30%, 409b, and 50% acetone).
Fractions are morlitored by 1 LC and aMropriate fractions evaporated and crystallized from acetone to give Marcfortine A and Marcfortine C.
ExamPle IB Production and Isolation of Marcfortines A and C.
Seed Fermentation Prooess:
Seed fermentations are inoculated using agar plugs of isolate Penicillium sp. UC 7780 (NRRL 18887~ stored over liquid nitrogen. Three plugs are thawed and used as inoculum for 100 ml of GS-7 seed medium. GS-7 is composed of glucose and cottonseed flour (sold under the trademark "Pharmamedia" by Traders Protein, Procter & Gamble Oilseed Products Co.~
Memphis, TN, U.S.A.) each added at a concentration of 25 ~/L of tap w~tcr. Aftcr fonnul~tiol1.
the pH of GS-7 is adjusted to 7.2 using NH40H. The medium is autocl~ved in 100 ml volumes in 500 ml unbaffled fennentation flasks for 30 min. Sterile GS-7 is inoculated as described above and shaken at 250 rpm for 35-58 hr at 23C.
Production Fermentation Process (Shake Flask):
The mature seed cultu es are used as inoculum for the production medium at a 1% seed rate. The production medium is composed of glucose 20 g, glycerol 15 ml~ cononseed flour (sold Imder the tradem~rk "Pharm~media`' by Traders Proteim Procter ~5c Gamble Oilseed WV93/10120 2 1 2 1 2 3 4 P~/US92/09483 Products Co., Memphis, TN, U.S.A.) 20 g, soybean meal 10 g, ~nd K2HPO~, ~ g per liter of t~l water. After formulation, the pH of the production medium is adjusted to 6.8 using potassium hydroxide. This medium is then autoclaved for 30 min in 100 ml volumes contained in 500 ml baffled fermentation flasks. Sterile production medium is inoculated as described above, and 5 shaken for 7-14 days at 250 rpm at 21C.
Production Fermentation Process (Labraferm tanks):
The mature seed cultures are used as inoculum for the sterile production medium at a 0.5% seed rate. The production medium is described above. After pH adjustment to 7.0 usino KOH, 10 L of this medium are autoclaved for 90 min in 12 L Labraferm tan~;s (New Brunswick 10 Scientific Co., Inc.). The tmks are inoculated at a 0.5% seed rate and stirred at 500 rpm at 20C for 5-9 d~ys. The air flow rate is maintained between 10-15 L/min.
Isolation of Marcfortines A and C:
Whole fermentation broth (35 1) is macerated at low speed in a large commercial Waring Blender and then blended with an eoual volume of methylene chloride. The mixrure is store~
15 overnight under refrigeration and tht;n subjected to centrifugation to break the emulsion. The resulting clear methylene chloride layer is drawn off and evaporated under reduced pressure. A
concentrated solution of the residue (37.4 g) in methylene chloride is applied to a column of silica gel (lKg~ slurry packed in methylene chloride. The column is eluted with increasing concentrations of acetone in methylene chliuide (10 %, 20%, 30%, 40%, and 50% acetone).
20 Fractions are monitored ~y TLC and appropriate fractions evaporated and crystallized from acetone to give Marcfortine A and Marcfortine C.
ExamPle 2 18-thiomarcfortine A (Compound ~2) A solution of 30 mg marcfortine A and 22 mg of :2,4-bis(methoxyphenyl)-1,~-dithia-2,4-diphosphetane-2,~disulfide (Lawesson's reagent) in 5 ml of toluene is refluxed under nitrogen 25 for 18 hours. The mixture is cooled and the solvent removed under reduced pressure. The residue is subjected to preparative thin layer chromatography on silica gel plates using 10æ
acetone in methylene chloride as the eluent to give 18-thiomarcfor~ne A, mp 258-260 C.
FAB-MS 494(M'+H) Following the gene~al procedure of Example 2 but employing the ~pmpriate ~0 marcfortine starting material, there are prepared the following othcr 18-~iomarcfortine compounds:
l-methyl-l~-thiomarcfortine A
l-benzyl-18-~iomarcfo tine A
l-ethyl-18-~iomarcfortine B
35 1-benzyl-18-~iomarcfo~ine B
18a-ethyl-18-~iomar~fortine B

WO 93/10120 PCr/US92/09483 21212~ 36 18a-benzyl- 18-thiomarcfortine B
18a-methoxyethoxymethyl- 18-thiomarcfortine B
18a-allyl-18-thiomarcfortine B
18a-propargyl- 18-thiomarcfor~ne B
1,18a-bis-ethyl- 18-thiomarcfortine B
1,18a-bis-benzyl-18-thiomarcfortine B
18a-ethyl-24-methoxy-18-thiomarcfortine B
18-thiomarcfortine B (Compound #2A) FAB-MS 480(M'+H) 18a-ethyl-24,25 dihydro-18-thiomarcfortine B
l-propyl-18-thiomarcfortine C
I-benzyl-18-thiomarcfortint C
18a-propyl- 18-thiomarcfortine C
18a-benzyl-18-thiomarcforline C
18a-methoxyethoxymethyl-18-thiomarcfortine C
18a-allyl-18-thiomarcfortine C
18a-propargyl- 18-thiomarcfortine C
I ~ l 8a-bis-propyl- 18-thiomarcfortine C
1,18a-bis-benzyl- 18-thiomarcfortine C
18-thiomarcfortine C
I-palmitoyl-18-thiomarcfortine A
l-decanoyl-18-thlomarcfortine A
l-decanoyl 18-thiomarcfortine B
Examnle 3 24,25 dihydro-18-thiomarcfortine A
A mixture consisting of 15 mg of 5% palladium on carbon and 18 thiomarcfor~ine A(30 mg, 0~06 mmol) in 1 ml of methanol is stirred vigorously under an atmosphere of hydrogen ~-for 45 minutes. The neaction mixture is filtered through CeUte~ and the filtrate is evaporated under vacuum. Preparative layer chromatography of the residue on a 0.5 mm silic3 gel plate eluted with 3ffo methanol in methylene chloride affords 24,25-dihyd~o 18-thioma~for~ine A.
Exam~le4 24,25-dibromo-24,2S-dihydro 18-thiomarcfortine A
A solution of bromine in chloroform (0.4 ml of 12M solution, 0.048 mmol) is added dtopwise to ~ cold (ice bath) solution of 18-thiomarcfonine A (20 mg, 0.04 mmol) in 2 ml of cl~orofolm. The resuldng yellow solution is stirred at room temper~ture for 15 minutes then evaporated under a steam of nitrogen. Preparative layer chrom~tography of the residue on a 0.5 mm silic~ gel pl~te eluted with 2% methanol in methylene chlo~ide affords 24,25-dib~mo-24,25-dihydro 18-thiomarcfortine A.

Example ~ 24-methoxy 24,25-dihydro 18-thiomarcfortine B
A solution of bromine in chloroform (0.6 ml of 0.12M solution, 0,072 mmol) is added dropwise to a cold (ice ~th) solution of 18-thiomarcfortine B (28 mg, 0.06 mmol) in 2 ml of chloroform. The resulting yellow solution is stirred at room temperature for 10 minutes then at S O C for 20 minutes then evaporated under a stream of nitrogen. The yellow solid residue is dissolved in 2 ml of meth~nol then 1,8-diazabicyclo~5~4.0~undec-7-ene (DBU, 0.015 ml, 0.10 mmol) is added. The, solution is stirred at room temperature for 90 minutes then evaporated under vacuum. Preparative layer chromatography of the residue on a 1.0 mm silica gel plate eluted with 2% methanol in methylene chloride affords 24-methoxy-25-bromo-24,25-dihydro-18-10 thiomarcfortine B. The 24-methoxy 25-bromo-24,25-dihydro-18-thiomarcfortine B thus o~tail-ed is dissolved in 2 ml of dry toluene then tributyltin hydride (0.12 ml, 0.45 mmol) is added. The solution is stirred at 100 C. for 16 hours then evaporated under vacuum. Preparative layer chromatography of the resi~e on a 2.0 mm silica gel plate eluted with 2% methanol in methylene chloride affords ''-~-methoxy 24,25-dihydro-18-thiomarcfortine B.
ExamPle 6 24-propoxy 24,25-dihydro-18-thiomarcfortine A
Substitution of propanol for methanol in the procedure described above for 24-methoxy 24,25-dihydro-18-thiomarcfortine B (Example 5) and application of the modified procedure to 20 mg of 18-thiomarcfortine A affords an oily residue. Preparative layer chromatography of the - crude produce on a 0.5 mm silica gel plate eluted with 2% methanol in methylene chloride 20 affords 24-propoxy-24,25-dihydro-18-thiomarcfortine A.
Examnle 7 1-(dimethylcarbamoyl)-18-thiomarcfortine A
Potassium hydride (50 mg of a 25% oil dispersion) is added to a solution of 18-thiomarcfor~ne A (15 mg, 0.030 mmol) in I ml of dry tetrahydrofuran. The solution is stirred at room temperature for 2 hours then dimethylcarbamoyl chloride (0.028 ml, 0.30 mmol) 25 is added. The mixture is stirred at room temperature for 18 hours then partitioned bet~veen 5%
aqueous sodium bicarbonate (1 ml) and methylene chloride (1 ml). The layers ane separated and the. aqueous layer extracted with methylene chloride (2 ml). The combined extracts are dried with magnesium sulfate, filtered, and evaporated under vacuum. Preparative layerchromatography of the residue on a silica gel plate eluted with 259b acetone in hexanc affords 30 1 -(dimethylcarbamoyl)- 18-thiomarcfottine A.
Following the general procedure of Example 7 but employing the appropriate marcfortine starting material, there ase prepared the following other 18-thiomarcfortine compounds:
I -acetoxymethyl- 18-~iomarcfortine A
3~ 1-diethoxyphos?horyl-18-thiomarcfortine A
I -cyclopropylcarbonyl- 18-thiomarcfortine A

WO 93/10120 PC~r/US92/09483 2l21 234 -38-I -dimethylsulfamoyl- 18-thiomarcfor~ine A
1 -( I -piperidinyl)thiocarbonyl- 18-thiomarcfortine A
I -succinoyl- i 8-thiomarcfortine A
I -~2,4-dinitrobenzenesulfenyl)- 18-thiomarcfortine A
5 1-(4-morpholinosulfenyl)- 18-thiomarcfortine A
I -(p-toluenesulfonyl)- 18-thiomarcfortine A
l-acetyl-18-thiomarcfortine A
l-methoxycarbonyl-18-thiomarcfortine A
I -(p-toluenesulfonyl)- 18-thiomarcfortine B
10 1 -(2,4-dinitroben~ene sulfony!)- 18a- ethyl -24-methoxy-24,25-dihydro l-(p~bromobenzene sulfonyl)-18-thiomarcfortine C
I -propionyl- 18-thiomarcfortine C
1 -(4-carbethoxy- 1,3-thiazolidinin-3-yl)carbonyl 18-thiomarcfortine A
ExamDle 8 1-benzyl-18-thio-marcfortine A
15Potassium hydride (75 mg of a 25% oil dispersion) is added to a solution of 18-thio-marcfortine A (15 mg, Q030 mmol) in I ml of dry tetrahydrofuran. l~le solution is stirred at room temperature for 2 hours then benzyl bromide (0.025 ml, 0.21 mmol) is added. The mixture is stirred at room temperature for 3 hours then partidoned hetween water (I ml) and `?
methylene chloride (1 ml). The layers are separated and the aqueous layer extracted with - 20 methylene chloride (2 ml). The combined extracts are dried with magnesium sulfate, filtered, and evaporated under vacuum. Preparative layer chromatography of the residue on a 1.0 mm silica gel plate eluted with 3% methanol in methylene chloride affords I N-benzyl-18-thio-marcfortine A.
Following the general procedure of Example 8 but employing the appropriate 25 marcfordne starting mate~ial, there are prepared the following other 18-thiomarcfortine compounds:
1,18a-bis-ethyl- 18-thiomarcfortine B;
I-allyl-18-thiomarcfor~ne A;
I-methoxymethyl-18-thiomarcfortine A;
30 1-propar~yl-18-thiomarcfortine A;
I-benzyl-18a-ethyl-18~thiomarcfortine B;
Exam~le 9 Hacmonchus contortuslTrichostrongylus colubr~form~s/Jird Assay:
This in vivo assay utilizes 3irds infected with two important tarpet parasites of n~minants~
H. contortus and T. co1ubr~ormis (anthelmintic-sensitive or -resistant worms can be used).
35 lnitially, activity is assessed orly against H. con~ortus as described in G.A. Conder et al.~ J.
Parasitol. 76, 168-170 (1990)~ while follow-up studies examine activity against both species of WO g3/10120 2 1 2 1 2 3 4 Pcr/usg2/o9483 parasites using the techniques outlined in G.A. Conder et ;~ J. Par~sitol. 77, 621-62~ (19~1).
l ne ~ctivity is set forth in Table 11.
ExamPle 10 Following the general procedure of Example 2 but employing the appropriate 5 paraherquamide/~hioparaherquamide starting material, there are prepared the following other 18-thioparaherquamide com~. ~nds:
18-thioparaherquamide l-methyl-18-thioparaherquamide ;1-e hyl-18-thioparaherquamide 10 1-benzyl-18-thioparaherquamide I -palmitoyl- 18-thiop~herquamide I -decanoyl- I 8-thioparaherquamide ExamDle lOa Following the general procedure of Example 7 but employing the appropriate thioparaherquamide starting matedal, there are prepared the following other . . -18-thioparaherquamide compounds:
: I-acetoxymethyl-18-thiomarcfortine l-diethoxgphosphoryl-18-thiopa~herquamide l-cyclopropylcarbonyl-18-thiopa~erquamide ` ~ 20 1-dimethylsulfamogl-18-thiopa~erquam~de piperldinyl)thiocarbonyl-18-thioparaherquamide l-succinoyl-18-thioparaherquamide 1-(2,4-dinitrobenzenesul~enyl)-18-thioparaherquamide 1-(4-morpholinosulfenyl)-18-thioparaherquamide 25 1-(~toluenesulfonyl)-18-thioparaherquamide I -acetyl- 18-thioparaherquamide ~; I-methoxycarbonyl-18-thiopa aherquamide Exam~le 11 Follo~ng the general procedure of Example 8 but cmploying the appropriate 30 pa~aherquamide star~ng materlal, thae are prepared the following other 18-thioparaherquamide compounds:
1,18a-bis~thyl-18-~iopa~er~amide;
I-allyl-18-~iopalaherquamide~;
methoxyme~yl-18-~iopa aherquamide;
35 1-p~opargyl-18-lhiq_~amide;
enzyl- 18a-ethyl- 18-thiop~ahe~uamide~

WO93/lOt20 ~1 2I23~4 PCI/US92/09483 Example 12 I-pyrTolidinylcarbonyl-m~rcfortine A, Cpd #12 Potassium hydnde (50 mg of ~ 25% oil dispersion) is added to a solution of marcfor~ine A (15 mg, 0.030 mmol) in 1 ml of dry tetrahydrofuran. The solution is stirred ~1 room temperature for 2 hours then pyrrolidinylcarbonyl chloride (40 mg, 0.30 mmol) is added.
S The mixture is stirred at room temperature for 18 hours then partitioned between 5% ~queous sodium bicarbonate (I ml) and methylene chloride (I ml). The layers are separated and the aqueous layer extracted with methylene chloride (2 ml). The comhined extr;lcts are dried with magnesium sulfate, filtered, and evaporated under vacuum. Prepar~tive layer chromatography of the residue on a silica gel plate eluted with 25% acetone in hexane affords l-pyrrolidinyl calbonyl-marcfortine A. IH NMR(CDCI3) ~ 0.83(s,3H), 1.21(s,3H), 1.41 (s.3H), 1.45(s,3H), 1.3-2.4(m,17H), 2.38(d,1H), 2.6-2.8(m, 2H), 2.84(d,1H), 3.00(s,3H), 3.4-3.6(m,4H), 3.72(d,1H), 4.85(d,1H), 6.41(d,1H), 6.84(s,2H) Following the general procedure of Example 12 ~ut employing the appropri~te marcfortine starting material and cartonyl chloride, there are prep~red the following other marcfortine compounds:
l-diethoxyphosphoryl-marcfortine A
l-cyclopropylcarbonyl-marcfonine A, Cpd #12A. 'H NMR(CDCI3) ~ 0.82(s,3H), l.O5(s,3H), 1.44(2s,6H), 0.9-1.7(m,8H), 1.7-3.0(m,11H), 3.14(s,3H), 3.80(d,1H), 4.84(d,1H), 6.28(d,1H), 6.88(s,2H) 1-(1-piperidinyl)thiocarbonyl-marcfor~ne A
l-succinoyl-ma~fortine A
l-phenoxycartonyl-marcfortine A, Cpd #12B. 'H NMR(CDCI3) ~ 0.83(s,3H), I.O9(s,3H), 1~3-l.S(m,lH), 1.42(s,3H), 1.46(s,3H), 1.5-3.0(m,13H), 3.14(s,3H), 3.70(d,1H), 4.83(d,1H), 6.27(d,1H~, 6.90(s,2H), 7~2-7~5(m,5H) 1 -(2,4-dichlorophenoxycalbonyl)- 18a-allyl-marcfortine B
1 -(~oxopentanoyl)- 18a-methyl-marcfortine C
l-palmitoyl-marcfortine A, Cpd #12C
FAB-MS 716(M~+H) 1-(4-nitrophenoxycarbonyl)-marcfortine A, Cpd #12D. ~H NMR(CDC13) ~ Q90ts~3H?~
l.O9(s,3H), 1.3-l.S(m,lH), 1.44(s,3H), 1.47(s,3H), 1~5-2.9(m,13H), 3~14(s,3H), 3.70(d,1H~, 4.86(d,1H), 6.23(d,1H), 6~93(ABq,2H~, 7.48~d, 2H), 8.32(d,2H) l-(l-piperidinecarbonyl)-ma~for~ne A, Cpd #12F
FAB-MS 589 (M'+H); HRMS m/z (M~+H, C3~H~,N4O~H,) calc 589.3390, obsd 589.3398 1-[[4-(ethoxyc~onyl)pipera~in-1-yllcarbonyl]-m~rcfordne A, Cpd #12G
FAB-MS 662 (M'+H); 684 (M~+Na); HRMS m/z (M~+H, -WO 93/10120 2 1 2 1 2 ~ ~ PCI'/US92/09483 ~ ~
-41 - , C36H~,7N507~H,), calc 662.3553, obsd 662.3569 1-[[4-(benzyl)piperazin-1-yl]carbonyl]-marcfortine A, Cpd #12H
'H NM~ (300 mHz, CDCI3): 0.80 (s,3H), 1.26 (s,3H), 1.41 (s.3H). 1.45 (s,~H).
1.2-2.9 (m, H), 2.98 (s.3H), 3.4-3.8 (m,8H), 6.41 (d,lH)~ 6.84 (s,2H), 7.2-7.5 S (m,SH). FAE~-MS 680 (M++H); 702 (M~+Na) 1-[[4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl]carbonyl]-marcfortine A, Cpd #121 'H NMR (300 mHz, CDCI3): 0.80 (s,3H), 1.00-2.6n (m,lSH), 1.2 (s,3H), 1.41 (s,3H), 1.45 (s,3H), 2.63 (t,lH),2.84 (d,lH), 2.98 (s,3H), 3.45-3.80 (m,8H), 4.85 (d,lH), 5.95 (s,2H), 6.41 (d,lH), 6.70-6.90 (m.SH) FAB-MS 724 (M~+H) 1-[[4-~methyl)piper~in-1-yl]carbonyl]-marcfortine A, Cpd #12J
IH NMR (300 mHz, CDCI3): 0.82 (s,3H), 1.26 (s,3H), 1.40-2.72 (m,20H), 1.41 (s,3H), 1.44 (s,3H), 2.82 (d,lH), 2.99 (s,3H). 3.40-3.81 (m,SH), 4.85 (d,lH),

6.41 (d,lH), 6.84 (s,2H). FAB-MS 604 (M'+H); HRMS mJz (M'+H, C3,lH~5N50s~H,), calc 604.3499, obsd 604.3506 1-(1,3~ihydro-1,3-dioxo-2H-isoindol-2-yl)-marcfortine A, Cpd #12K
FAB-MS 665 (M~+H) 1 -[ [4-(pyridin-2-yl)pipe~azin- 1 -yl~carbon~l] -marcfortine A, Cpd # 12L
'H NMR (300 mHz, CDCI3): 0;84 (s,3H), 1.10-2.30 (m,12H), 1.41 (s,3H), 1.44 (s,3H), 2.40 (d,2H), 2.67 (t,2H), 2.85 (d,lH), 3.00 (s,3H), 3.45-3.85 (m,9H), 4.85 (d,lH), 6.40 (d,lH), 6.60-6.70 (m,2H), 6.85 (s,2H), 7.52 (t,lH), 8.15;8.22 (m,lH). FAB-MS 667 (M~+H) l-[[~(phcnyl)piperazin-l-yl]carbonyl]-marcfor~ine A, Cpd #12M
IH NMR (300 mHz; CDCI3): Q84 (s,3H), 1.20-2.50 ~m,llH), 1.22 (s,3H), 1.42 (s,3H), 1.45 (s,3H), 2.67 (t,lH), 2.85 (d~lH), 3.00 (s,3H), 3.10-3.91 (m,9H), 4.85 (d,lH), 6.40 (d,lH), 6.85 (s,2H), 6.90-7.00 (m,3H), 7.25-7.38 ~m,2H) 1 -~[~(chloroca~bonyl)piperazin- I -yl]carbonyl] -marcfortine A, Cpd # 12N
'H N~ (300 mHz, CDCl~: 0.74 (s,3H), 1.18 (s.~H)~ .fi~ (m~2l)H), 2~74 (d,lH), 2.81 (s,3H), ~.48-3.90 (m,XH), 4.~0 (d,lH), ~.32 (d,lH)~ 6.80 (s~2H).
FAB-MS 6S2 (M~+H) 10,1~'-(1,4dicarbonylpiperazine)bis(6',7',8',9',10',10'a-hexahydro 1',1',4,4, 12'-pentame~yl)-12'S-12'.alpha,3'aalpha,9'aalpha,10(2 'R*,3"'aS~9 'aS*,lO"'aR~),lO'a~eta]]
-Spiro~4H,8H-11,4]dioxepinol2,3-g]indole-8,2'(3~-llH,4H-3~9a](iminometh~no)cyclopenta~b~
35 quinolizin]-9,11'(lOH)-dione, Cpd #120 FAB-MS 1093(M~+H) WO 93/10120 PC~/VS92/09483 2 1 ~123~ -42-1 -[ [4-(phenyl)pipelidin- 1 -yl]carbonyl]-marcfortine A, Cpd # 12P
'H NMR (300 mHz, CDCI3): 0.76 (s~3H), 1.10-2.30 (m,13H). 1.14 (s,3H), 1.3S
(s,3H), 1.38 (s~3H), 2.33 (d,lH), 2.52-3.00 (m,8H), 3.00-3.20 (m,lH), 3.50-3.75 (m,2H), 4.05 (d,lH), 4.33 (d,lH), 4.79 (dd,lH), ~.35 (dd,lH), 6.78 (s,2H),

7.1~7.30 (m,SH). FAB-MS 665 (M~+H) 1-[[4-(dimethyl)piperidin-1-yl]ca bonyll-m~rcfor~ine A, Cpd #12Q
'H NMR (300 mHz, CDC13): 0.75 (s,3H), 0.90-1.95 (m,13H), 0.92 (s,3H), 0.96 (s,3H), 1.14 (s,3H), 1.34 (s,3H), 1.37 (s,3H), 2.08 (d,lH), 2.20 (brs.,lH), 2.33(d,lH), 2.60 (t,2H), 2.77 (d,lH), 2.92 (s,3H), 3.45-3.75 (m,4H), 4.77 (d,lH), 6.33 (d,lH), 6.77 (s,2H). FAB-MS 617 (M'+H~
1-~[4-(5-chloropyridazin-3-yl)pipe~zin-1-yl~carbonyl]-m~rcfortine A, Cpd #12R
'H NMR (300 mHz, CDCI3): Q83 (s,3H), 1.20-2.30 (m,l IH), 1.22 (~s,3H), 1.42 (s,3H), 1.44 (s,3H). 2.45 (d,lH), 2.65 (t,2H), 2.83 (s,lH), 2.99 (s,3H), 3.60-3.85 (m,8H), 4.86 (d,lH), 6.40 (d,lH), 6.86 (s,2H), 6.92 (d,lH), 7.29 (d,llH).
FAB-MS 702 (M++H) 1-1[4-(4-chlorophenyl)piperazin-1-yl]carbonyll-marcfortine A, Cpd #12S
'H Nl~ (300 mHz, CDCl3): Q84 (s,3H), 1.20-2.30 (m,9H), 1.26 (s,3H), 1.42 (s,3H), 1.45 (s,3H), 2.40 (d,lH), 2.60-2.75 (m,2H), 2.85 (d,lH), 3.00 (s,3H), 3.15-3.30 (m,4H), 3.49 (d,lH), 3.52-3.84 (m,SH), 4.85 (d,lH), 6.39 (d,lH), 6.80-6.90 (m,4H), 7.23 (d,2H).
10,10"-(1,4-dioxo-2-butene)bis(6',7',8',9',10',10'a-hexahydro-1',1',4,4,;2'-pentamethyl)-[2'S-[2'.alpha.,3'aalpha.,9'aalpha,10(2'~,3"'aS~9"'~S*,lO"'aR*),lO'a beta]]-Spirol4H,8H-[1,4]dioxepino[2i3-g]indole-8,2'(3'H)-[ lH,4H-3~9a](iminomethano)cyclopenta[b]
quinolizin]-9,11'(lOH)~ione, Cpd #12T
FAB-MS 1035(M~+H) 1-[[4-(triehloroethoxycarbonyl)pipera~in-1-yl~c~rt~onyl3-m~rcfordne A, Cpd #12U
H NMR (300 mH~, CDCI3): 0.75 (s,3H), 1.1-1.85 (m,8H), 1.15 (s,3H), 1.34 (s,3H), 1.34 (s,3H), 1.92 (d,lH), 2.00 2.40 (m,2H), 2.34 (d,lH), 2.55 (t,2H), 2.76 (d,lH), 2.91 (s,3H), 3.42-3.75 (m,8H), 4.654.77 (m~2H). 4.79 (d~lH), 6.33 (d,lH), 6.79 (s,2H). FAB-MS 764 (M+H); HRMS m/~ (M~+H~
C3~H44CI~N5~7+Hl), calc 764.2384, obsd 764.2366 4-(3-trifluoromethyl-thiadiazol- 1 -yl)pipe~zin- I -yl]carbonyl]-marcfortine A, Cpd #12V
NMR (3ûO mHz, CDCl3): 0.83 (s~3H)~ 1.26 (s~3H), 1.3~2.30 (m~l IH)~ 1.42 (s~3H), 1.45 (s,3H), 2.42 (d,lH), 2.55-2.72 (m~2H)~ 2.84 (d~lH)~ 2.99 (s,3H)~
3.6~3.90 (m~8H), 4.87 (d,lH)~ 6.30 (d~lH)~ 6.88 (s~2H). FAB-MS 742 (M~H);

WO 93/10120 2 ~ 2 1 2 3 ~ PCr/US92/09483 HRMS m/z (M'+H, C36H4~F3N7S,~H,), calc 742.2998, obsd 742.30~8 Example I 3 I-acetoxymethyl-marcfortine A, Cpd #13 Sodium hydride (47 mg of a 60% oil dispersion) is ~dded to a solution of marcfortine A
(850 mg, 1.78 mmol) in 1 ml of dry tetrahydrofuran. The solution is stirred at room temperature for 3 hours then methyl bromoacetate (280 mg, 1.82 mmol) is added. The mixture is stirred at room temperature for 4 hours then additional methyl bromoacetate (280 mg. 1.82 mmol) is added. The mixture is stirred at room temper~ture for 8 hours then panitioned between 5% sodium bicarbonate (I ml) and methylene chloride (I ml). The layers are separated and the aqueous layer is extracted with methylene chloride (2 ml). The combined extracts are dried with magnesium sulfate, filtered, and evaporated under vacuum. Column chromatography of the residue on silica gel plate eluting first with 5% then 10% acetone in methylene chloride afford~
1 acetoxymethyl-marcfortine A as a colorless oil: 'H NMR (CDCI3) ~ 0~75 (~H. s), 1.08 ~, (3H, s), 1.43 (3H, s), 1.45 (3H, s), 2.05 (3H, s), 3.12 (3H, s), 4.86 llH~ d~ J = 8). 5.85 (lH, d~ , 1=8),5.96(1H,d,J=8),6.26(1H,d,~=8),6.77(1H,d,J=7)~6.86(1H,d,J=7).
Following the general procedure of Example 13 but employing the appropriate marcfonine starting material, there are prepared the following other marcfortine compounds:
l-t-butyryloxymethyl-marcfortine A
1-benzoyloxymethyl-marcfottine A
l-acetoxymethyl-18a-benzyl-marcfortine B
1-3,4-dichlorobenzoyloxy-marcfortine C
ExamDle 14 1-(2,4-dinitrobenzenesulfenyl)-marcfortine A, Cpd #14 Marcfortine A (75 mg, 0.15 mmol) is added to a suspension of potassium hydride (180 mg, I 5~) mmol, 35 wt%-in mineral oil) in THF (6 mL) at 5C, followed by 2,4-dinitrobenzene -sulfen~l chloride ~73 mg, 0.30 mmol). The reaction mixture is then stirred for 3h at 5C. After dilu~ng with H20 and extracting with CHCI3, the crude product is cl~matographed on a I mm silica gel preparative thin layer plate eluting with 3096 Acetone in methylene chloride to yield 1-(2,4 dinitrobenzenesulfenyl)-marcfonine A (90 mg, 89.1%) as a yellow solid, mp 155-160C
(dec). 'H N~ (CDCl3): ~ 0.86~.97 (m,3H), 1.13-1.45 (m,lSH), 1.56-1.78 (m,SH), 1.86 (m,lH), 2.00 (m,lH), 2.15 (d,lH), 2.30 (m,lH), 2.45 (d,lH), 2.67 (bd~IH), 2.79 (d,lH), 2.91 (m,lH), 3.08 (s,3H), 3.74 (d,lH), 4.76 (bd,lH), 5.85 td,lH), 6.87 (d,lH), 6.97 (d,lH), 7.~8 ~m,0.5H), 7.62 (m,0,5H), 8.36 (dd,lH)~ 9.16 (d,lH).
Follow~ng the general procedure of Example 14 but employing Ihe app~priate marcfor~ne sta~ng matenal~ ~ere are prepared the follow~ng other marcfoninc compounds:
1-(4-molpholinosulfenyl)-marcfonine A, Cpd #14A
FAB-MS; m/e, 595(m~+H) l-(trichloromethylsulfenyl)-marcfor~ine A, Cpd #14B

WO g3/lOt20 PCrtUS92/09483 212~23~ 44 FAB-MS; m/e, 626, 628, 630 (Mt~H) I-(methoxycarbonylsulfenyl)-marcfortine A, Cpd #14C) FAB-MS; m/e, 568 (M'+H) I-(benzenesulfenyl)-marcfonine A~ Cpd #14D
FAB-MS; m/e, 586 (M'+H) l-trichloromethylsulfenyl-18a-ethyl-marcfortine B
1 -ethoxycarbonylsulfenyl- 18a-methyl-marcfortine C
Exam~le 15 1-(4-morpholinecarbonyl)-marcfortine A N-oxide, Cpd #15 1-(4-Morpholinecafbonyl)-marcfortine A (Cpd #12F, 15 mg) is treated with 10 3-chloroperoxybenzoic acid (15 mg) in methylene chloride (2 ml) at 0C for 10 minutes. The mixture is partitioned between 5% sodium bicarbonate (2 ml) and methylene chloride (2 ml).
The layers are separated and the aqueous layer is extracted with methylene chloride (2 ml). The combined extracts are dried with magnesium sulfate, filtered and evaporated under vacuum. The residue is chromatographed on a 0.5 mm silica gel prepar~tive thin layer pl~te eluting with 10 15 methanol in methylene chloride to yield the title compound (10 mg) as a solid. FAB-MS;
607(M~+H) Exam~le 16 1-(2-tetrahydrofuranyl)-marcfortine A, Cpd #16 Carbonyldiimidazole (50 mg) is added to a solution of marcfortine A (60 mg) in 6 ml of tetrahydrofuran. The mixture is heated to reflux and potassium hydride (120 mg) is added. The 20 resulting mixture is stirred under refluxing for I hour. The precipitate is filtered off Dd the filtr~te is concentrated. The residue is chromatographed on a silica plate, eluting with 25%
acetone in methylene chloride to yield the title compound (30 mg) as a solid. HRMS (FAB);
m/e(Mt+H, C32H4lN3Os~H), calc. 548.3124, obsd. 548.3086 Examnle 17 1-(4-morpholinecarbonyl)-marcfordne A, Cpd#12E
Marcfortine A (I g, 2.1 mmol) in T~ (2S mL, disdlled from sodium metal and benzophenone) and potassium hydride (35 weight %, 0.5 g, 4A mmol) are sdrred for 0.5 h at r~.~n temperature under nitrogen. Morpholinecarbonyl chloride (1 mL, 8.4 mmol) is then ~dded via syringe. After 3 hr of stirring at room temperature the turbid reaction mixture is cooled in an ice water bath and quenched with dropwise addition of cold saturatcd potassium c;lrbt n~tc soludon (5 mL). The mixture is then dlluted with water (75 mL) and ext~cted into CH2CI2 (175 mL). The organic layer is dried over magnesium sulfate, flltered and concentrated to give the c~ude material which co~ins oil. Silica gel cluomatography t29b to 5% MeOH/CH~CI2) gives l-morpholinecarbonyl-marcfortine A. (Silica gel ch~m~ltog~phy on the clu~malotron (4~O MeOH/CH2CI~) is a ~etter method of purific~tion for l-molpholinecarbonyl-marcfonine A~) ~H N~ (300 MHz~ CDCI3) ~ 0.81 (s, 3H), 1.21 (s, 3H), 1.19-1.94 (m, 8H)~ 1.41 (s~ 3H)~ 1~45 (s~ 3H)~ 1.98 (d~ lH~ 2.08-2.28 (m, 2H)~ 2.40 (d~ IH)~ 2.62 (t~ 2H)~ 2.82 (d~ IH)~ 2~99 (s~ 3H), WO g3/10120 2 1 2 1 2 3 4 PC~/US92/09483 3.S0-3.86 (m, 8H), 4.86 (d, IH), 6.41 (d, lH), 6.85 (s. 2H). FAB-MS 591 (M'+H); HRMS ~VZ
(M~+H, C33H42N406+H,), calc. 591.3182, obsd. 591.3200.
~xamnle lX
Following the general procedure of Example 12 but employing the appropriate paraherquamide starting material, there are prepared the following other paraherquamide compounds:
1 -diethoxyphosphoryl-paraherquamide . .
1 -cyclopropylcarbonyl-p~aherquamide 1-( I-piperidinyl)thiocarbonyl-paraherqu~mide I-succinoyl-paraherquamide -1-(4car~ethoxy-1,3-thiazolidWn-3-yl)carbonyl-par~herquamide I -phenoxycarbony1 -p~raherquamide I -pyrrolidinylcarbonyl -paraherquamide I -piperidinylcarbonyl-paraherquamide 1~4morpholinecarbonyl~paraherquamide Exam~le 19 Following the genoral procedure of Example 13 but employing the appropriate paraherquamide starting material, there are prepared tne following other paraherquamide compounds:
l-t-butyryloxymethyl-paraherquamide bonzoyloxymethyl-paraherquamide ' l-acetoxymothyl-18a-paraherquamide 1 -3.4dichlorobenzoyloxy-paraherquamide Exam~le 20 FoUowing the general procedure of Example 14 but employing the app~priate pa~herquamide starting matorial, there are prepared the following other pa-aherquamide compounds:
I -(2,4dinitrobenzenesulfenyl)-paraherquamide 1 -(~morpholinosulfenyl)-paraherquamide 1-trichloromethylsulfenyl-18a-ethyl-paraherquamide l-ethoxycalbonylsulfenyl-18a-me~yl-paraherquamide l -palmitoyl-pa~e~uamide ocedure A:
`:
3S A. Seed Fermentation :~ Cunningharndla cchinulata subsp~ clegans (-) NRRL 1368, Cunninghamclla WO g3/10120 . PCI`/US92/09483 212~2~

blakesleeana (+) ATCC 8688~ Cunninghamella echinulata subsp. elegans (-) NRRL
1393, Cunninghamella echinulata NRRL 3655 Frozen agar plugs of NRRL 1368 or 1393, ATCC 8688a or preferably NRRL ~655 (stored over liquid nitrogen "LN2") are aseptically transferred into a 100 ml vol of GS-7 (sterile) 5 medium contained in a S00 ml large mouth shake flask. GS-7 is composed of Cerelose and Pharmamedi~, each added at 25 g/l of tap water. The pH of the medium is adjusted to pH 7.2 using ammonium hydroxide. The medium is sterilized by autoclaving for 30 min. Inoculated GS-7 is shaken at 125 to 300 (preferably 250 rpm) at 21-30C, preferably about 28 C, for 24-72 hrs (preferably about 48-72 hours). This fermentation is used to inoculate the 10 biotransformation process at about I to about 5% rate (preferably about 5% r~te).
B. Biotransformation Process 100 ml vols ~f GS-7 contained in S00 ml large mouth fermentation flasks are inocula~ed with the 48-72 hr seed fermentation at about I to about 5% rate (preferably about 59~ rate). ~ -The inoculated GS-7 is shaken at about 250 rpm at 28C for 2448 hrs. At this time 15 Marcfortine A, dissolved in dimethylformamide (DMF), is added to the fermentation in a range between 10-12 mg/flask, preferably at 10 mg per 0.4 ml DMF per 100 ml flask/fermentation.
The fermentations containing Marcfortine A are continued as previously indicated for 24-72 hrs.
The contents of 100 flasks, each containing 100 ml fermentation as described in the `
protocol for biotransformation and which had been shaken at 28C for 24-72 hours following 20 addition of Marcfortine A (Q010 g per fla~c, I g total) in DMF (Q2 ml per flask, 20 ml total) are each treated with CH2CI2 (100 ml). The contents are combined and stirred in a waring ~lender for S minutes. The mixture is centrifuged and the resulting aqueous layer is decanted.
The organic layer is collected, dried (sodium sulfate), filtered and concentr~ted. The components of the crude mixture are separated by a series of column chromatographies using a 25 silica gel - solid extract ratio of 50-125:1. Columns are eluted wi~ either 2-5% methanol in CH2CI2 or 15-60% acetone in CH2CI2. Prom these chomatographic separations there is isolated Marcfortine B. HRMS (FAB); m/e, found, 464.2563; calculated for C27H33N304 ~ Hl, 464.2549.
If desired, marcfor~ne B may be crystallked from methylene chloride/acetone. Forexample, a methylene chloride solution of m~rcfor~ne B is concentrated under p~ial ~acuum 30 on a rotatory evaporator. The concentrate is diluted with portions of acetone during the concentration until a slurry of marcfortine B in acetone remains.

SUBSTlTUTE SHEEl-W 0 93/tO120 2 1 2 ~ 2 3 ~ P ~ /US92~09483 . -47-~aO --= -- - - -I e j 701 ~

~ ~o ~ 'X ~ ~o ~ ~ ~ ~o ~ , o _ ~

~ .~ ,e .~ ~ ~

~ ~ ~ ~ .
_ ~ ~ I ~ o e 3 N N N ~ e c - ~ ~ ~ ~ ~

~ ~ ~

2~123;~ -48- .

_ _ _ _ _ I
ANTHELMINTIC JIRD PHASE 2 SCREEN .
_ _ _ _ % Red HC % Red TC
CPD # Dose # Animals# Surviv.
_ _ 2 1MG/JIRD 3 3 89 100 l _. _ 0.33MGPIRD 3 - 3 23 96 0.11MG/JIRD 3 3 O 0 . _ 2A 0.33MG/JIRD 3 3 76 98 0.11MG/JIRD 3 3 29 72 _ _ __ 1012 1MGflIRD 3 3 100 100 l _ 0.33MG/J~D 3 3 99 100 l _ . . . _ _ O. 11MG/J~D 3 3 73 O
0.037MG/IIRD 3 3 12 30 ¦
. l 0.11MG/I~D 2 2 65 61 0.037MG/JIRD 3 3 8 O
I ~_ . _ I
12A 1MGIJIRD 3 3 ,99 90 l . - I
0.33MG/JIRD 3 3 28 3 . ~-, _ 12B 1MG/JIRD - 3 3 100 lO0 0.33MGtJIRD 3 3 55 85 ~ _ . _ _ 2012C 1MG/JlRD 3 3 100 100 , . _ 0.33MG/JIRD 3 3 99 100 0.11MG/JIRD 3 3 28 43 I _ ~31Jl3S~t'r~J~E SHE~

WO 93/10120 2 12 ~L 2 3 4 PCI/US92/09483 ANTHELMIN~llC JIRD PHASE 2 SCREEN
I
% Red HC % Red TC
CPD # Dose # Animals# Surviv.
_ .
1 2D 1MGnlRD 3 3 94 99 .
0.33 MG/lIRD 3 3 51 70 I .. _ 12E 1 MGnIRD 3 3 99 100 .
0.33 MG/ 3 3 100 100 _ _ _ 0.11 MGn~D 3 3 80 99 _,. _ 0.037.~ 3nIRD 3 2 O 42 12F 1 MGnlRD 3 3 99 100 _ 0 33 i'JlRD 3 3 98 10~
_. _ 0.11MGIJIRD 3 3 74 7$
_ .
12G 1 MG/JlRD 3 3 99 100 0.33MG/~IRD 3 3 95 100 0.11MG/lIRD 3 3 O 61 I _ 12H 0.33MG~JIRD 3 3 99 9g Qll MG~IRD 3 3 2 .61 I _ _ .
12I 1 M&/IIRD 3 3 100 100 _ _ 033 MGnlRD 3 3 100 100 _ .
0.11MG/m~D 3 2 69 87 _ _ 0.11 Mt~/JIRD 3 3 32 99 _ _ 0.037 Mt3/JIRD 3 3 22 44 , _ SUE~STIl UTE SHEET

WO 93/10120 7, 1 21 ~ 3 4 PCI`IUS92/094*3 ;~
-50- :

ANIHELMINllC JIRD PHASE 2 SCREEN
. _ % Red HC % Red TC ¦ :
CPD # Dose # Animals # Surviv.
_ _ _ _ _ 0.33 MG/JIRD 3 3 66 ~4 12L0.33 MG/JI~D 3 3 99 99 ¦ :1 _ . _ ~
0.11 MGI~IRD 3 3 37 56 l I . _ 12M0.33 MG/JIRD 3 3 97 100 l . _ _ 0.11 MG/~IRD 3 3 73 98 ~ _ .
0.11 MG/JIRD 2 2 25 410 0.037 MGII~RD 3 3 11 66 I . _ _ 12N 033 MG/lIRD 3 ; 3 100 100 -0.11 MG/lIRD 3 3 81 g8 _ I
0.11 MG/JI~D 2 2 O 39 ¦
0.037 MG~llRD 3 3 62 50 I I
120 033 MG~JIRD 3 3 100 100 0.11 MGJIIRD 3 3 80 98 ¦ ' 0.11 MG/IIRD 2 2 46 77 _ I
0.037 MG/II~ 3 3 39 46 I, _ 12P 033 MGIIIRD 3 3 100 100 _ 0.11 MG/JIRD 3 3 51 78 SUBSTITUl E SHEET

WO93/10120 2 l 2 1 2 3 ~ P~/US92/09~3 _ ~m~CJ~P~SE2SC~
_ %R~HC %R~TC
CPD# ~se #~im~s #S~iv.
_ _ 12Q 0.33 MG/J~ 3 3 94 _ 0.11 MG/J~ 3 3 53 63 _ S 12R 0.33 MG~ 3 3 100 99 _ .
0.11 MG~ 3 2 96 97 0.11 MG~ 3 3 0 ~-0.037 MG~ 3 3 5 _ _ _ _ _ 0.33 MG/m~ 3 3 99 100 . _ _ 0.11 MG~ 3 3 76 80 . -12S 033 MG~ 3 3 1~ 100 .
. _ 0.11 MG~ 3 3 93 100 0.11 MG~ 3 3 29 . .
0.037 MG~ 3 3 27 0.33 MG~ 3 3 98 1 . _ 0.11 MG~ 3 3 61 97 I ~ ..
12T033 MG~ 3 3 74 93 _ . . . ._ __ 0.11 MG~ 3 3 32 74 I _ _ . . ........... _ 12UQ33 MG~ 3 3 100 99 0.11 MG~ 3 3 72 82 I . _ 12V033 MG~ 3 3 98 98 _ ._ 0.11 MG~ 3 3 87 86 I _ .
131 MG~ 3 3 100 100 033 MG~ 3 3 S5 90 I .

Sl~ ;T;~U~E SilEEl' WO 93/1~120 PCI/US92/09483 2~21~-~4 -52-¦ ANl HELMINTIC JIRD PHASE 2 SCREEN
~ _ .
% Red HC % Red TC
CPD # Dose # Animals# Surviv.

0.33 MG/J~ 3 3 100 100 O.I l MGpIRD 3 3 74 0.037 MGn~`D 3 3 29 0 033 MG/J~ 3 3 99 100 . . _ . _ 14A0.33 MGn 3 3 96 _ ..
0.11 MGnIRD 3 3 81 63 14B1 MGn~ 3 3 100 IOQ
0.33 MG/IIRD 3 3 98 100 .- . .
0.11 MG~ - 3 3 O 76 14C1 MG/lIR~ 3 2 100 _ 0.33 MG~ 3 3 98 100 _ _ 0.11 MG/JIRD - 3 3 29 80 0~037 MG~ 3 3 56 O . -14D1 MG~ 3 3 100 100 _ _ 033 MG~ 3 3 100 100 _ ,_ .
0.11 -MG~ 3 -3 32 67 lS 1 MG~ 3 3 99 100 _ .
033 MG~ 3 3 61 76 I

SU~STITUTE SHEET

W O 93/10120 2 ~ 2 1 2 3 4 PC~r/US92/09483 r AllTHELMnNllC J~RD P}LASE 2SCREE~
. _ ~ % R~ HC % Red TC
CPD # Dose # Anim~s # S~viv.
.

0.33 MG/JDRD 3 3 92 100 ¦
O.11 MG/JnRD 3 3 S0 12 0.33 MG~DRD 3 _ _ 3 98 100 ¦
0.11 MGn~RD 3 3 76 99 l _ _ . .

SU~s~ru~E S~EET

WO 93/10120 PCr/US~2/0~483 2 3 ~1 FO~MULAE
c~3 C~; 1~ ~C33 CompDund ' C~3 ~ C33 Compound 2A

C~3 Compound 12 CompDund 12A

WO93/1012û 212123 I PCI/US92/09483 c~C33 Compound 12H

~o ~1 ..

~C33 Compound 12C

c~3 c~2(CH2) 13C~3 C~3 Compommd 13 C=0 C~3 WO 93/1012~ PCI/US92/09483 2 1 2 1 2 34 -~ -C C~3 ~C~3 Com~ound 14 ~ ~ o~N~S
~3 ~N2 ~ ~

~3C C~3 1-`1~C~3 ~ ~ ~ ~ ~/ Comcound 12D

~3 ca3 ~I ~3C C~3 ~ C~3 ~ Compound 1:;

~N~

SO

WO 93/101202 l 2 1 2 3 4 PCr/US92/094B3 ~ iA ~ ~ ~ ~ C33 Compound 12E

~N~ -~C33 Compound 14B

2~

c~3 c~C33 Compo~md 14D

4~

~C1~3 Com,oound 12F

WO 93/1012~ PCI/US92/09483 2 1~ 1 ~3 ~ -58-C~3 Compound 12G

CH3 f ~
~NJ
-C~C~3 c~3 ~ Compound 121 N
0~ ~ ' c~3 ~C=0 Compoumd 12J

~N~
C~3 ~0 WO 93/10120 212 i 2 3 ~1 PCI`/US92/09483 CH
~ Compouna 121C

0~0 c~3 C33 Compound 14C

C~3 Compound 14A

C~3 ~:

WO 93/10120 PCI`/US92/094B3 21212~ -C~3 Compound 12L

~) ' ~N

c~3 ~c~3 Compound 12M

~N~
..

c~3 _ ~3C C~3 ~fC~C~3 ~ ~ J _6/ Compound 12N

C~O
Cl WO 93/10120 2 1 2 1 2 3 ~ PCI/US9~/09483 c~3 ~0~ Compound 12P

~N7 N\CI =0 ~C33 Compound 12Q

c~3 c~ 3 Compound 12R

N
~NN

Cl WO 93/10120 PCr/llS92/09483 2 1 ~ 1 2 34 -62-c~3 ~0~ Compound 12S

C~3 N
C~3 ~N ~ca3 Compound 12U

C~2-0-C
Cl C~3 -~3C C~3 ~(~C~3 ~ L~ 6/ Compound 12V

~N) N

N~S
=~CF3 ~WO 93/10120 2 1 21 2 3 4 PCI/US92/09483 6~

;~O~C33 Compound 1211 C~3 N

~N~
.-(~ _CE3 Cclmpoumd 120 =C~N~ 3 `N~ ~:

E3C~

.

A ~ CE3 Compummd 12T

N
CH3 ~
O=C~ o H3C~ o R3C~o)[~N~
~3 WO 93/1012~ PCr/USg2J09483 2~2123 1 -64-~\ ~l ~Oo~C~3 Compoun~l 16 ~N N
3 ~0 CN3 I.larcfonine D

Claims (25)

-65-

1. A compound of Formula I
wherein:
n is 0 or 1;
R14a and R14b, being the same or different, are selected from hydrgen hydroxy, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkenyl, C1-C6 alkenyl-C1-C6 alkoxy, C1-C6 alkynyl-C1-C6 alkoxy, C1-C6 alkynoyloxy, poly C1-C6 alkoxy,C1-C6 alkoxy, phenyl, phenyl-C1-C6 alkyl, tri-C1-C6 alkylsilyloxy, diphenylphosphoryloxy and holgen, or R14a and R14b together form the epoxide or =CH2, with the proviso that when n is 1, then R14a and R14b are both hydrogen;
m is 0 or 1;
W is O or S;
when W is S, R1 is hydrogen, C1-C7 alkyl, cyclo C3-C8alkyl, benzyl, C2-C7 alkanoyl (-C(O)C2-C7alkyl) (optionally substituted with carboxy (-COOH), C1-C7 alkanoyl, carbo C1-C7alkoxy (-C(O)OC1-C7alkyl), -NR4R5, aminocarbonyl (-C(O)NR4R5)], C10-C24alkanoyl (-C(O)C10-C24alkyl, cyclo C3-C8alkanoyl [optionally substituted with carboxy, C1-C7 alkanoyl, carboC1-C7alkoxy, -NR4R5, aminocarbonyl], alkanoyloxymethylene (-CH2OC(O)-C2-C7alkyl), benzoyloxymethlene (-CH2OC(O)phenyl) (optionally substituted with 1 or 2 groups selected from halogen, C1-C4 alkyl, haloC1-C7alkyl, nitro, cyano and C1-C7alkoxy], C10-C24alkenoyl (-C(O)C9-C23alkenyl), benzenesulfonyl (-SO2CH2phenyl) {optionally substituted with 1 or 2 groups selected from halogen, C1-C4 alkyl, haloC1-C7alkyl, nitro, cyano and C1-C7alkoxy], C1-C4alkylaminocarbonyl (-C(O)N(C1-C4alkyl)2), C1-C4alkylaminothiocarbonyl (-C(S)N
(C1-C4alkyl)2), C1-C7 alkoxycarbonyl, phenoxycarbonyl [optionally substituted with 1 or 2 groups selected from halogen, C1-C4 alkyl, haloC1-C7alkyl, nitro, cyano and C1-C7alkoxy], -C(O)NR'4R'5, -P(=X)(R2)(R3), -SR6, -SO2NR4R5, benzoyl substituted at the 3 or 4 position with fro -CH2NR4R5, 2-tetrahydrofuran, or bicycloC8-C12alkanoyl;
when W is O; R1 is selected from the group consisting of:
(a) C2-C7 alkanoyl substituted with cart)oxy (-COOH), C1-C7 alkanoyl.
carboC,-C7alkoxy (-C(O)OC1-C7alkyl), -NR4R5, arninocarbonyl (-C(O)NR4R5);
(b) cyclo C3-C8alkanoyl optionally substituted with carboxy. C1-C7 alkanoyl, carboC1-C7alkoxy, -NR4R5, aminocarbonyl;
(c) alkanoyloxymethylene (-CH2OC(O)-C2-C7alkyl);
(d) benzoyloxymethlene (-CH2OC(O)phenyl) substituted with 1 or 2 groups selected from halogen, C1-C4 alkyl, haloC1-C7alkyl. nitro. cyano and C1-C7alkoxy;
(e) phenoxycarbonyl substituted with 1, or 2 groups selected frolm halogen, C1-C4 alkyl, haloC1-C7alkyl, nitro, cyano and C1-C7alkoxy;
(f) -C(O)NR'4R'5;
(g) -P(=X)(R2)(R3);
(h) -SR6;
(i) C10-C24alkanoyl (-C(O)C1O-C24alkyl);
(j) C10-C24alkenoyl (-C(O)C9-C23alkenyl); or (k) 2-tetrahydrofuran;
R4 and R5, being the same or different, are selected from hydrogen, C1-C7 alkyl, phenyl [optionally substituted with 1 or 2 groups selected from halogen. C1-4 alkyl, haloC1-C7alkyl, nitro, cyano and C1-C7alkoxy] or when taken together with N, form a saturated or unsaturated heterocyclic amine ring;
R'4 and R'5, being the same or different. are selected from C1-C7 alkyl, cyclo(C3-C8)alkyl. phenyl {optionally substituted with 1 or 2 groups selected from halogen, C1-C4 alkyl, haloC1-C7alkyl, nitro, cyano and C1-C7alkoxy} or when taken together with N, form a saturated heterocyclic amine ring optionally containing 1 or 2 additional heteroatoms selected from N, O or S;
X is O or S;
R2 and R3, being the sa ne or different, are selected from C1-C7 alkyl, phenyl {optionally substituted with 1 or 2 groups selected from halogen, C1-C4 alkyl, haloC1-C7alkyl, nitro, cyano and C1-C7alkoxy], C1-C7alkoxy, thio(C1-C7)alkoxy, phenoxy, thiophenoxy, -NR7R8 {where R7 and R8, being the same or different, are selected from H, C1-C7 alkyl or taken together with N, form a saturated heterocyclic ring}, or taken together with P
form a 4- to 7-membered heterocyclic ring;
R6 is C1-C7 alkyl. haloC1-C7alkyl, carboC1-C7alkoxy, -NR9R10 where R9 and R10, being the same or different. are C1-C7 alkyl or phenyl (optionally substituted with 1 or 2 groups selected from halo, lower alkyl, haloC1-C7alkyl, nitro, cyano, C1-C7 alkoxy);
R24 is hydrogen, halogen or C1-C7 alkoxy;
R25 is hydrogen or halogen;
R18a is hydrogen, C1-C7 alkyl, C2-C8 alkoxyalkyl, C2-C4 alkenyl, C2-C8 alkynyl or benzyl;
the broken line between carbons 24 and 25 represents a single or double bond; and pharmaceutically acceptable salts and hydrates thereof; with the overall proviso that, when W is O, R'4 and R'5 are not both C1-C7 alkyl.

2. A compound according to Claim 1 where m is 0.

3. A compound according to claim 2 where R24 and R25 are hydrogen; R18a is hydrogen, C1-C7 alkyl, C1-C8 alkoxymethyl. C2-C8 alkenyl or benzyl; and the broken line represents a double bond between carbons 24 and 25.

4. A compound according to claim 1 where W is S and n is 0.

5. A compound according to claim 4 where m is 0, R24, and R25 are hydrogen; R18a is hydrogen, C1-C7 alkyl, C1-C8 alkoxymethyl. C2-C8 alkenyl or benzyl; and the broken line represents a double bond between carbons 24 and 25.

6. A compound according to claim 5 which is selected from 18-thiomarcfortine A;
1-acetoxymethyl-18-thiomarcfortine A;
1-diethoxyphosphoryl-18-thiomarcfortine A;
1-dimethylsulfamoyl-18-thiomarcfortine A;
1-cyclopropylcarbonyl-18thiomarcfortine A;
1-(1-piperidinyl)thiocarbonyl-18-thiomarcfortine A;
1-succinoyl-18-thiomarcfortine A;
1-(2,4-dinitrobenzenesulfenyl)-18-thiomarcfortine A;
1-(4-morpholinosulfenyl)-18-thiomarcfortine A;
24-propoxy-24,25-dihydo-18-thiomarcfortine A;
1-(p-toluenesulfonyl)-18-thiomarcfortine A;
1-acetyl-18-thiomarcfortine A;
1-methyl-18-thiomarcfortine A;
1-benzyl-18-thiomarcfortine A;

1-dimethylcalbamoyl-18-thiomarcfortine A;
1-methoxycarbonyl-18-thiomarcfortine A;
18-thiomarcfortine B;
24,25-dihydro-18-thiomarcfortine B;
24-methoxy-24,25-dihydro-18-thiomarcfortine B;
1-(p-toluenesulfonyl)-18-thiomarcfortine B;
1-acetyl-18-thiomarcfortine B;
1-ethyl-18-thiomarcfortine B;
1-benzyl-18-thiomarcfortine B;
18a-ethyl-18-tniomarcfortine B;
18a-benzyl-18-thiomarcfortine B;
18a-methoxyethoxymethyl-18-thiomarcfortine B;
18a-allyl-18-thiomarcfortine B;
18a-propargyl-18-thiomarcfortine B;
18a-ethyl-24-methoxy-24,25-dihydro-18-thiomarcfortine B;
18 ethyl-18-benzyl-18-thiomarcfortine B;
18a-ethyl-24-methoxy-18-thiomarcfortine B;
1-(2,4-dinitrobenzene sulfonyl)-18a-ethyl-24-methoxy-24,25-dihydro-18-thiomarcfortine B;
18a-ethyl-24,25 dihydro-18-thiomarcfortine B;
18-thiomarcfortine C;
24,25 dihydo-18-thiomarcfortine C;
1-(p-bromobenzene sulfonyl)-18-thiomarcfortine C;
1-propionyl-18-thiomarcfortine C;
1-propyl-18-thiomarcfortine C, 1-benzyl-18-thiomarcfortine C;
18a-propyl-18-thiomarcfortine C;
18a-benzyl-18-thiomarcfortine C, 18a-methoxyethoxymethyl-18-thiomarcfortine C;
18a-allyl-18-thiomarcfortine C;
18a-propargyl-18-propyl-18-benzyl-18-thiomarcfortine C;
1-(4-carbethoxy-1,3-thiazolidinin-3-yl)carbonyl-18-thiomarcfortine A;
1-palmitoyl-18-thiomarcfortine A; or 1-(4-morpholinocarbonyl)-18-thiomarcfortine A.

7. A compound according to claim 1 where W is S and n is 1.

8. A compound according to claim 7 where m is 0; R24 and R25 are hydrogen; R18a is hydrogen, C1-C7 alkyl, C1-C8 alkoxymethyl, C2-C8 alkenyl or benzyl; and the broken line represents a double bond between carbons 24 and 25.

9. A compound according to Claim 8 where R14a and R14b are both hydrogen.

10. A compound according to Claim 8 where R14a is methyl, R14b is hydroxy.

11. A compound according to claim 8 which is selected from 18-thioparaherquamide 24,25-dihydro-18-thioparaherquamide 14-O-methyl-18-thioparaherquamide 14-O-ethyl-18-thioparaherquamide 14-O-butyl-18-thioparaherquamide 14-O-benzyl-18-thioparaherquamide 14-O-ally-18-thioparaherquamide 14-O-propargyl-18-thioparaherquamide 14-O-methoxymethyl-18-thioparaherquamide 14-O-methoxy-ethoxy-18-thioparaherquamide 14-O-methoxy-ethyoxy-methyl-18-thioparaherquamide 17-methyl-18-thioparaherquamide 17-methylene-18-thioparaherquamide 1-N-(p-toluenesulfonyl)-18-thioparaherquamide 24-methoxy-24,25-dihydro-18-thioparaherquamide

12. A compound according to (: laim 1 wherein W is O and n is 0.

13. A compound according to claim 12 where n: is 0; R24 and R25 are hydrogen; R18a is hydrogen, C1-C7 alkyl, C1-C8 alkoxymethyl, C2-C8 alkenyl or benzyl: and the broken line represents a double bond between carbons 24 and 25.

14. A compound according to claim 12 which is selected from 1-(1-piperidinyl)thiocarbonyl-marcfortine C;
1-(1-piperidinyl)thiocarbonyl-marcfortine A;
1 -(2,4-dichlorophenoxy)carbonyl-18a-N-ethyl-24-methoxy-24,25-dihydro-marcfortine B;
1-(2,4-dichlorophenoxycarbonyl)-18a-allyl-marcfortine B;

1-(2,4-dinitrobenzenesulfenyl)-marcfortine A;
1-(3-acetoxy)propionyl-18a-N-propargyl-marcfortine B;
1-(4-carbethoxy-1,3-thiazolidine-3-yl)carbonyl-marcfortine A:
1-(4-morpholinosulfenyl)-marcfortine C;
1-(4-oxobutyryl)-18a-methyl-marcfortine C;
1-(4-oxopentanoyl)-marcfortine A;
1-(4-oxopentanoyl)-marcfortine C;
1-3,4-dichlorobenzoyloxy-marcfortine C;
1-acetoxymethyl marcfortine C;
1-acetoxymethyl-18a-benzyl-marcfortine B;
1-benzoyloxymethyl-marcfortine A;
1-cyclohexylcarbonyl-marcfortine A;
1-cyclohexylcarbonyl-marcfortine C;
1-cyclopropylcarbonyl-marcfortine A;
1-cyclopropylcarbonyl-18a-ethyl-marcfortine B;
1-cyclopropylcarbonyl-marcfortine C;
1-diethoxyphosphoryl-marcfortine A;
1-diethoxythiophosphoryl-marcfortine A;
1-diethoxythiophosphoryl-marcfortine C;
1-dimethylaminoacetyl-marcfortine C;
1-dimethylaminoacetyl marcfortine A;
1-dimethylaminosulfenyl-18a-N-benzyl-marcfortine B;
1-diphenylphosphinyl-18a-N-allyl-marcfortine B;
1-ethoxycalbonylsulfenyl-18a-methyl-marcfortine C;
1-ethoxycarbonylsulfenyl-18a-N-methoxyethoxymethyl-marcfortine B;
1-N-(2,4-dinitrobenzenesulfenyl)-18a-N-ethyl-marcfortine B;
1-N-(4-carbethoxy-1,3-thiazolidinin-3-yl)catbonyl-marcfortine C;
1-N-(4-carbethoxy-1,3-thiazolidinin-3-yl)carbonyl-marcfortine A;
1-N-(p-bromobenzenesulfenyl)-marcfortine C;
1-N,N-dimethylsuccinamidoyl-marcfortine A;
1-N,N-dimethylsuccinamidoyl-marcfortine C;
1-phenoxycarbonyl-marcfortine C;
1-phenylmethoxyphosphonyl-marcfortine A;
1-phenylmethoxyphosphonyl-marcfortine C;
1-succinoyl-marcfortine A;
1-succinoyl-marcfortine C;

1-t-butyryloxymethyl-marcfortine A;or 1-trichloromethylsulfenyl-18a-ethyl-marcfortine B.

15. A compound according to Claim 1 selected from prederably 1-cyclopropylcarbonyl-marcfortine A
1-phenoxycarbonyl-marcfortine A
1-palmitoyl-marcfortine A
1-[[4-nitrophenyl)oxy]carbonyl]-marcfortine A
1-(1-poperidinecarbonyl)-marcfortine A
1-[[4-(ethoxycarbonyl)piperazine-1-yl]carbonyl]-marcfortine A
1-[[4-(benzyl)piperazine-1-yl]carbonyl]-marcfortine a 1-[[4-(1,3-benzodioxol-5-yl-methyl)piperazin-1-yl]carbonyl]-marcfortine A
1-[[4-(methyl)piperazin-1-yl]carbonyl]-marcfortine A
1-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl-marcfortine A
1-[[4-(pyridin-2-yl)piperazin-1-yl]carbonyl]-marcfortine A
1-[[4-(phenyl)piperazin-1-yl]carbonyl]-marcfortine A
1-[[4-(chlorocarbonyl)piperazin-1-yl]carbonyl]-marcfortine A
1-[[4-(phenyl)piperidin-1-yl]carbonyl]-marcfortine A
1-[[4-(phenyl)(dimethyl)piperidin-1-yl]carbonyl]-marcfortine A
1-[[4-(5-chloropyridazin-3-yl)piperazin-1-yl]carbonyl]-marcfortine A
1-[[4-(4-chlorophenyl)piperazin-1-yl]carbonyl]-marcfortine A
1-[[4-(trichloroethoxycarbonyl)piperazin-1-yl]carbonyl]-marcfortine A
1-[[4-(3-trifluoromethyl-thiadiazol-1-yl)piperazin-1-yl]carbonyl]-marcfortine-A
1-acetoxymethyl-marcfortine A
1-(2,4-dinitrobenzenesulfenyl)-marcfortine A
1-(4-morpholinosulfenyl)-marcfortine A
1-(trichloromethylsulfenyl)-marcfortine A
1-(methoxycarbonylsulfenyl)-marcfortine A
1-(benzenesulfenyl)-marcfortine A
1-(2-tetrahydrofuranyl)-marcfortine A, 1-(4-morpholinocarbonyl)-marcfortine A or 1-(4-morpholinocarbonyl)-marcfortine A N-oxide.

WO 93/10120 PCT/US92/09483 1-(1-piperidinyl)thiocarbonyl-paraherquamide 1-succinoyl-paraherquamide 1-(4-carbethoxy-1,3-thiazolidinin-3-yl)carbonyl-paraherquamide 1-phenoxycarbonyl-paraherquamide 1-pyrrolidinylcarbonyl-paraherquamide 1-t-butyryloxymethyl-paraherquamide 1-benzoyloxymethyl-paraherquamide 1-acetoxymethyl-18a-paraherquamide 1-3,4-dichlorobenzoyloxy-paraherquamide 1-(2,4-dinitrobenzenesulfenyl)-paraherquamide 1-(4-morpholinosulfenyl)-paraherquamide 1-trichloromethylsulfenyl-18a-ethyl-paraherquamide 1-ethoxycarbonylsulfenyl-18a-methyl-paraherquamide 1-palmitoyl-paraherquamide 1-piperidinylcarbonyl-paraherquamide 1-(4-morpholinecarbonyl)-paraherquamide

17. A compound according to any preceding claim, wherein R1 is -C(O)NR'4NR'5 and NR'4R'5 is a saturated heterocyclic ring optionally containing 1 or 2 additional heteroatoms selected from N, O and S.

18. A compound according to claim 17, wherein the saturated heterocyclic ring is selected from 4-morpholine 4-phenyl-1-piperazine 4-(2-pyridinyl)-1-piperazine 2,6-dimethyl-4-morpholine 1-pyrrolidine 4-methyl-1-piperazine 1-piperidine 4-phenyl-1-piperidine thiazolidine 4-phenyl-1,2,3,6-tetrahydropyridine 4-phenylpiperidine ethyl prolinate tetrahydrofurylamine 3-pyrroline thiazolidine-4-carboxylic acid thiomorpholine nipecotamide 2-methylpiperidine 3-methylpiperidine 4-methylpiperidine N-methylpiperidine 1-methylhomopiperazine 1-acetylpiperazine and N-carboethoxypiperazine.

19. Use of a compound according to any preceding claim, for the manufacture of a medicament for use in the treatment or prevention of helminth or arthropod infections in domesticated mammals.

20. A method for the treatment of insect or nematode plant pests, which comprises applying a compound according to any of claims 1 to 18 to the plants or the soil in which they grow.

21. A biologically pure culture of Penicillium sp. NRRL
18887.

WO 93/10120 PCr/US92/09483

22. A process for producing a compound of the formula:
which comprises:
the biotransformation of a compound of the formula:
using a microorganism in an aqueous nutrient mediun containing an assimilable source of carbon and an assimilable source of nitrogen under aerobic conditions.

23. The process of Claim 22 wherein the microorganism is a species of Cunninghamella or a mutant thereof.

24. The process of Claim 22 wherein the strain is selected from the group consisting of NRRL 1368, NRRL 1393, NRRL 3655 or ATCC 8688a.

WO 93/10120 PCr/US92/09483

25. A compound selected from the group consisting of 10,10"-(1,4-dicarbonylpiperazine)bis(6',7',8',9',10',10'a-hexahydro-1',1',4.4,12'-pentamethyl) -[2'S-[2'.alpha.,3'a,alpha.,0'a.alpha.,10(2'''R*,3'''aS*9'''aS*,10'''aR*),10'a.beta.]]
-Spiro[4H,8H-[1,4]dioxepino[2,3-g]indole-8.2'(3'H)-[1H,4H-3a,9a](iminomethano) cyclopenta[b]quinolizin]-9,11'(10H-dione; or 10,10"-(1,4-dioxo-2-butene)bis(6',7',8',9',10',10'a-hexahydro-1',1',4,4,12'-pentamethyl)-[2' S-[2'.alpha.,3'a.alpha.,9'a.alpha.,10(2'''R*,3'''aS*9'''aS*,10'''aR*),10'a.beta.]]
-Spiro[4H,8H-[1,4]dioxepino[2,3-g]indole-8,2'(3'H)-[1H,4H-3a,9a]
(iminomethano)cyclopenta[b] quinolizin]-9,11'(10H)-dione.