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Peptide process for preparation thereof and use thereof |
| RE32992 |
Peptide process for preparation thereof and use thereof
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| Patent Drawings: | |
| Inventor: |
Kuroda, et al. |
| Date Issued: |
July 18, 1989 |
| Application: |
06/611,733 |
| Filed: |
May 18, 1984 |
| Inventors: |
Aoki; Hatsuo (Ikeda, JP)
Aratani; Matsuhiko (Awaji-Shimmachi, JP)
Hashimoto; Masashi (Takarazuka, JP)
Hemmi; Keiji (Yawata, JP)
Iguchi; Eiko (Osaka, JP)
Imanaka; Hiroshi (Osaka, JP)
Kitaura; Yoshihiko (Suita, JP)
Kohsaka; Masanobu (Sakai, JP)
Kuroda; Yoshio (Takatsuki, JP)
Nakaguchi; Osamu (Toyonaka, JP)
Okada; Satoshi (Osaka, JP)
Takeno; Hidekazu (Nara, JP)
Tanaka; Hirokazu (Takarazuka, JP)
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| Assignee: |
Fujisawa Pharaceutical Co., Ltd. (Osaka, JP) |
| Primary Examiner: |
Phillips; Delbert R. |
| Assistant Examiner: |
Moezie; F. T. |
| Attorney Or Agent: |
Oblon, Spivak, McClelland, Maier & Neustadt |
| U.S. Class: |
530/330; 530/331; 530/332; 930/20; 930/21; 930/30; 930/DIG.500 |
| Field Of Search: |
260/112.5; 424/177; 514/18; 514/332; 530/330; 530/331 |
| International Class: |
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| U.S Patent Documents: |
4031069; 4130554; 4261976 |
| Foreign Patent Documents: |
0013856; 2053231 |
| Other References: |
Dezelee; P., et al., Biochemistry, vol. 9, No. 4, pp. 823-831, 1970..
Bulletin de la Societe de Chimie Biologique, vol. 49, pp. 1579-1591, 1967..
Nakamura, T., et al., Agricultural and Biological Chemistry, vol. 41, pp. 763-768, 1977..
Abstracts of the Eleventh International Congress of Chemotherapy, Oct. 1-5, 1979, Abstracts 702..
Werner et al., Immunopotentiating Activities of a Microbial Tetrapeptide after Coupling with Lauric Acid, (a typed draft for presentation at the Eleventh Int. Cong. of Chemotherapy, Oct. 1-5, 1979)..
Elloys, F., et al., Biochem. and Biophys. Res. Comm., vol. 59, pp. 1317-1325, 1974..
Kotani, S., et al., Abstracts of Societe de Chime Biologique International Symposium, Oct. 14-15, 1974, Paris..
Comptes Rendus Hebdomandaires des Seance de l'Academie des Sciences, Paris, 6260, pp. 1302-1304..
Migliore-Samour et al., CR Acad. Sc. Paris, Series D, vol. 289, pp. 473-476..
Migliore-Samour, et al., Life Sciences, vol. 26, No. 11, pp. 883-888..
Kontani, C. A., vol. 83, 1975, item 161981z.. |
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| Abstract: |
The invention deals with novel peptides useful for the therapeutic treatment of infectious diseases caused by pathogenic microorganisms. Included is the peptide FR900156 having the structure: ##STR1## as well as the peptides of the structure ##STR2## wherein R.sup.1 is acyl;R.sub.b.sup.1 is hydrogen, methyl, isopropyl, hydroxymethyl, protected hydroxymethyl or benzyl;R.sup.2 is hydrogen, carboxy, protected carboxy, or a group of the formula: ##STR3## wherein R.sub.a.sup.2 is mono- or di-carboxy lower alkyl or ar(carboxy) lower alkyl whose aryl moiety may be substituted by hydroxy,R.sub.b.sup.2 is hydrogen or lower alkyl;R.sup.p and R.sup.q are each hydrogen, carboxy, protected carboxy, with proviso that when one of R.sup.2 and R.sup.q is hydrogen, then the other is carboxy or protected carboxy;R.sup.r is hydrogen or amino protective group; m is an integer 1 to 3, and n is 1 .Iadd.provided that when R.sup.1 is benzyloxycarbonyl R.sub.5.sup.1 is methyl, m is an integer 2 and n is an integer 1, then R.sup.2 is not protected carboxy, and .Iaddend.provided that when R.sup.1 is .[.hydrogen,.]. t-butoxycarbonyl or N-acetylmuramyl, R.sub.b.sup.1 is methyl, m is an integer 2 and n is an integer 1, then R.sup.2 is hydrogen, protected carboxy or a group of the formula: ##STR4## wherein R.sub.a.sup.2 is mono- or di-carboxy lower alkyl having 1 and 3 to 6, carbon atoms, .[..alpha.-carboxyethyl,.]. .Iadd.--CH.sub.2 --CH.sub.2 --COOH, .Iaddend.ar(carboxy) lower alkyl whose aryl moiety may be substituted by hydroxy and R.sub.b.sup.2 is as defined above, or its pharmaceutically acceptable salt. |
| Claim: |
We claim: .[.1. A compound of the formula:.]. ##STR379## .[.wherein
R.sup.1 is acyl;
R.sub.b.sup.1 is hydrogen, methyl, isopropyl, hydroxymethyl, protected hydroxymethyl or benzyl;
R.sup.2 is hydrogen, carboxy, protected carboxy, or a group of the formula:.]. ##STR380## .[.wherein R.sub.a.sup.2 is mono-or di-carboxy lower alkyl or ar(carboxy)lower alkyl whose aryl moiety may be substituted by hydroxy, R.sub.b.sup.2 ishydrogen or lower alkyl;
R.sup.p and R.sup.q are each hydrogen, carboxy, protected carboxy, with proviso that when one of R.sup.2 and R.sup.q is hydrogen, then the other is carboxy or protected carboxy;
R.sup.r is hydrogen or amino protective group;
m is an integer 1 to 3; and
n is 1, provided that when R.sup.1 is hydrogen, t-butoxycarbonyl or N-acetylmuramyl, R.sub.b.sup.1 is methyl, m is an integer 2 and n is an integer 1, then R.sup.2 is hydrogen, protected carboxy or a group of the formula:.]. ##STR381## .[.whereinR.sub.a.sup.2 is mono- or di-carboxy lower alkyl having 1 and 3 to 6, carbon atoms, .alpha.-carboxyethyl, ar(carboxy)lower alkyl whose aryl moiety may be substituted by hydroxy and R.sub.b.sup.2 is as defined
above, or its pharmaceutically acceptable salt..]. .[.2. A compound according to the claim 1, wherein
R.sup.1 is acyl,
R.sub.b.sup.1 is hydrogen, methyl, isopropyl, hydroxymethyl or benzyl,
R.sup.2 is hydrogen, carboxy or a group of the formula:.]. ##STR382## .[.wherein R.sub.a.sup.2 and R.sub.b.sup.2 is as defined in claim 1,
R.sup.p and R.sup.q are each hydrogen or carboxy, with proviso that when one of R.sup.2 and R.sup.q is hydrogen, then the other is carboxy, and
R.sup.r is hydrogen..]. .[.3. A compound according to the claim 1, wherein
n is an integer 1 and
m is an integer 1..]. .[.4. A compound according to claim 2 wherein n is 1
and m is 1..]. .[.5. A compound according to the claim 1, wherein
n is an integer 1 and
m is an integer 2..]. .[.6. A compound according to claim 2, wherein n is
1 and m is 2..]. .[.7. A compound according to the claim 1, wherein
n is an integer 1 and
m is an integer 3..]. .[.8. A compound according to claim 2 wherein n is 1
and m is 3..]. .[.9. A compound according to the claim 1, wherein
R.sup.2 is hydrogen or protected carboxy,
R.sup.p and R.sup.q are each hydrogen or protected carboxy, with proviso that when one of R.sup.2 and R.sup.q is hydrogen, then the other is
protected carboxy..]. .[.10. A compound according to the claim 1, wherein the protected carboxy for R.sup.2, R.sup.p and/or R.sup.q is esterified
carboxy..]. 11. A compound of the formula: ##STR383##
12. A compound of the formula: ##STR384##
.Iadd.13. A compound of the formula: ##STR385## wherein R.sup.1 is an aliphatic acyl radical which is unsubstituted alkanoyl or alkenoyl or is alkanoyl of at least two carbon atoms or alkenoyl substituted by amino, halogen, hydroxy, loweralkoxy, or lower alkanoylamino;
R.sub.b.sup.1 is hydrogen, methyl, isopropyl, hydroxymethyl, protected hydroxymethyl or benzyl;
R.sup.2 is hydrogen, carboxy, protected carboxy, or a group of the formula: ##STR386## wherein R.sub.a.sup.2 is mono- or di-carboxy lower alkyl or ar(carboxy)lower alkyl whose aryl moiety is unsubstituted or is substituted by hydroxy,R.sub.b.sup.2 is hydrogen or lower alkyl;
R.sup.p is hydrogen, carboxy, or protected carboxy,
R.sup.q is carboxy or protected carboxy;
R.sup.r is hydrogen or amino protective group;
m is an integer 1 to 3; and
n is 1,
or its pharmaceutically acceptable salt. .Iaddend. .Iadd.14. A compound of the formula: ##STR387## wherein R.sup.1 is acyl,
R.sub.b.sup.1 is hydrogen, methyl, isopropyl, hydroxymethyl or benzyl,
R.sup.2 is hydrogen, carboxy or a group of the formula: ##STR388## R.sub.a.sup.2 is mono- or di-carboxy lower alkyl or ar(carboxy)lower alkyl whose aryl moiety is unsubstituted or is substituted by hydroxy, R.sub.b.sup.2 is hydrogen or loweralkyl;
R.sup.p is hydrogen or carboxy;
R.sup.q is carboxy;
R.sup.r is hydrogen.
m is an integer 1 to 3; and
n is 1, provided that when R.sup.1 is t-butoxycarbonyl or N-acetylmuramyl, R.sub.b.sup.1 is methyl, m is an integer 2 and n is an integer 1, then R.sup.2 is hydrogen, or a group of the formula: ##STR389## wherein R.sub.a.sup.2 is mono- ordi-carboxy lower alkyl having 1 and 3 to 6 carbon atoms, -CH.sub.2 -CH.sub.2 -COOH, ar(-carboxyl)lower alkyl whose aryl moiety is unsubstituted or is substituted by hydroxy and R.sub.b.sup.2 is as defined above, or its pharamaceutically acceptable
salt. .Iaddend. .Iadd.15. A compound according to the claim 13, wherein n is an integer 1 and m is an integer 1. .Iaddend. .Iadd.16. A compound according to claim 14 wherein n is 1 and m is 1. .Iaddend. .Iadd.17. A compound according tothe claim 13, wherein n is an integer 1 and m is an integer 2. .Iaddend. .Iadd.18. A compound according to claim 14, wherein n
is 1 and m is 2. .Iaddend. .Iadd.19. A compound according to the claim 13, wherein n is an integer 1 and m is an integer 3. .Iaddend. .Iadd.20. A compound according to claim 14, wherein n is 1 and m is 3. .Iaddend. .Iadd.21. A compoundaccording to the claim 13, wherein R.sup.2 is hydrogen or protected carboxy, R.sup.p is hydrogen or protected carboxy, and R.sup.q is protected carboxy. .Iaddend. .Iadd.22. A compound according to the claim 13, wherein the protected carboxy for atleast one of R.sup.2, R.sup.p and R.sup.q is esterified carboxy. .Iaddend. |
| Description: |
This invention relates to a new peptide. More particularly, this invention relates to a new peptide and thepharmaceutically acceptable salt thereof, which have pharmacological activities, to processes for the preparation thereof and to a new intermediate for preparing the active peptide, and to the pharmaceutical composition comprising the same and a methodof use thereof.
.Iadd.BRIEF DESCRIPTION OF THE DRAWING FIGURE
The drawing figure is the nuclear magnetic resonance absorption spectrum for the Compound FR-900156. .Iaddend.
Firstly, it is to be noted that this invention is originated from and base on the first and new discovery of the new activepeptide, e.i. FR-900156 substance. That is, the FR-900156 substance was firstly and newly isolated in pure form from a culture broth obtained by fermentation of a new strain belonging to the genus Streptomyces and characterized by the physico-chemicalproperties.
Thereafter, as a result of extensive study, the inventors of this invention succeeded in determining the chemical structure thereof by commanding physical and chemical techniques so that they could give the sequential structure of the formula(If) indicated below to FR-900156 substance and proposed with a firm belief the possible stereoisomeric structure of the formula (Ie) indicated below for the same. ##STR5##
Further, after the structural elucidation of FR-900156 as explained above, the inventors of this invention have continued extensive studies on total syntheses of the compound of the formula (If) including the compound of the formula (Ie) so thatthey have succeeded in completing the industrially advantageous and applicable synthetic processes for preparation of the same, and further have synthesized a lot of related compounds.
A new peptide of this invention is represented by the following formula (I): ##STR6## wherein
R.sup.1 is hydrogen or acyl;
R.sub.b.sup.1 is hydrogen, methyl, isopropyl, hydroxymethyl, protected hydroxymethyl or benzyl;
R.sup.2 is hydrogen, carboxy, protected carboxy, or a group of the formula: ##STR7## wherein
R.sub.a.sup.2 is mono- or di-carboxy lower alkyl or ar(carboxy)lower alkyl whose aryl moiety may be substituted by hydroxy, R.sub.b.sup.2 is hydrogen or lower alkyl;
R.sup.p and R.sup.q are each hydrogen, carboxy, protected carboxy with proviso that when one of R.sup.2 and R.sup.q is hydrogen, then the other is carboxy or protected carboxy; and
R.sup.r is hydrogen or amino protective group,
m is an integer 1 to 3; and
n is an integer 0 to 2, .Iadd.provided that when R.sup.1 is benzyloxycarbonyl, R.sub.b.sup.2 is methyl, m is an integer 2 and n is an integer 1, then R.sup.2 is not protected carboxy, and .Iaddend.provided that when R.sup.1 is hydrogen,t-butoxycarbonyl or N-acetylmuramyl,
R.sub.b.sup.1 is methyl, m is an integer 2 and n is an integer 1, then
R.sup.2 is hydrogen, protected carboxy or a group of the formula: ##STR8## wherein R.sub.a.sup.2 is mono- or di-carboxy lower alkyl having 1 and 3 to 6 carbon atoms,
.alpha.-carboxyethyl,
ar(carboxy)lower alkyl whose aryl moiety may be substituted by hydroxy and R.sub.b.sup.2 is as defined above.
Particulars of the various definitions, which are mentioned hereinabove, and hereinafter and preferred examples thereof are explained in the following.
The term "lower" is intended to mean a group having 1 to 6 carbon atom(s), unless otherwise provided.
(1) Re. Acyl for R.sup.1 and R.sub.c.sup.1
Generally, "acyl" may be an acyl group derived from an acid such as organic carbocylic acid, carbonic acid, or carbamic acid, or the thio acid or imidic acid corresponding to each of the preceeding acids, or an organic sulfonic acid, each ofwhich includes an aliphatic, an aromatic and/or a heterocyclic groups in its molecule; carbamoyl; or carbamimidoyl.
Suitable examples of said acyl are illustrated below.
Aliphatic acyl means an acyl group derived from an aliphatic acid and includes:
alkanoyl (e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, .alpha.-ethylhexanoyl, heptanoyl, lauroyl, stearoyl, docosanoyl, 2-heneicosylpentacosanoyl, etc.);
cycloalkanecarbonyl (e.g. cyclopentanecarbonyl, cyclopropanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl, etc.);
bridgedalicycliccarbonyl such as bicyclic or tricyclic alkane or alkenecarbonyl (e.g. norbornane carbonyl, adamantanecarbonyl, etc.);
alkenoyl (e.g. acryloyl, methacryloyl, crotonoyl, oleoyl, arachidonyl, etc.);
lower alkylthio(lower)alkanoyl (e.g. methylthioacetyl, ethylthioacetyl, etc.);
lower alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, t-pentoxycarbonyl, etc.);
lower alkanesulfonyl(lower)alkoxycarbonyl (e.g. 2-(mesyl)ethoxycarbonyl, etc.);
lower alkythio(lower)alkoxycarbonyl (e.g. 2-(ethylthio)ethoxycarbonyl, etc.);
lower alkenyloxycarbonyl (e.g. allyloxcarbonyl, etc.);
alkatetraenoyl (e.g. 3,7-dimethylnona-2,4,6,8-tetraenoyl, etc);
lower alkynyloxycarbonyl (e.g. 1,1-dimethylpropargyloxycarbonyl, etc.);
lower alkylcarbamoyl (e.g. methylcarbamoyl, etc.);
(N-lower alkyl)thiocarbamoyl [e.g. (N-methyl)thiocarbamoyl, etc.];
lower alkylcarbamimidoyl (e.g. methylcarbamimidoyl, etc.);
oxalo;
alkoxalyl (e.g. methoxalyl, ethoxalyl, propoxalyl, etc.).
In the above exemplified aliphatic acyl, the aliphatic hydrocarbon moiety, particularly the alkyl group and alkane moiety and the alkenyl group and alkene moiety may have optionally one or more suitable substituent(s) such as amino, halogen (e.g.fluorine, chlorine, bromine, etc.), hydroxy, hydroxyimino, carboxy, lower alkoxy (e.g. methoxy, ethoxy, propoxy, etc.), lower alkoxycarbonyl, acylamino such as lower alkanoylamino (e.g. acetylamino, propionylamino, etc.), ar(lower)alkoxycarbonylamino(e.g. benzyloxycarbonylamino, etc.), etc. or acyloxy such as lower alkanoyloxy (e.g. acetoxy, propoxy, etc.), ar(lower)alkanoyloxy (e.g. benzylcarbonyloxy, etc.), aroyloxy (e.g. benzoyloxy, etc.) or 5- or 6-membered cycloalkenyl (e.g.1,3,3-trimethyl-1-cyclohexen-2-yl, etc.).
Preferred examples of aliphatic acyl having such substituents may be exemplified by
hydroxyalkanoyl (e.g. glycoloyl, lactoyl, 2-hydroxybutyl, mycoloyl, etc.),
lower alkoxy(lower)alkanoyl (e.g. methoxyacetyl, 2-methoxypropionyl, etc.),
carboxy(lower)alkanoyl (e.g. carboxyacetyl, carboxypropionyl, etc.),
(lower)alkoxycarbonyl(lower)alkanoyl (e.g. methoxalyl, ethoxalyl, methoxycarbonylacetyl, propoxycarbonylpropionyl, ethoxycarbonylbutyryl, etc.),
halo(lower)alkoxycarbonyl (e.g. chloromethoxycarbonyl, tribromoethoxycarbonyl, trichloroethoxycarbonyl, etc.),
6-membered cycloalkenyl-alkatetraenoyl (e.g. retinoyl, etc.), and the like.
Aromatic acyl means an acyl group derived from an acid having substituted or unsubstituted aryl group, in which the aryl group may include phenyl, tolyl, xylyl, naphthyl and the like, and suitable examples thereof are illustrated as follows:
aroyl (e.g. benzoyl, toluoyl, xyloyl, napththoyl, phthaloyl, etc.);
ar(lower)alkanoyl (e.g. phenylacetyl, diphenylacetyl, etc.);
ar(lower)alkenoyl (e.g. cinnamoyl, etc.);
aryloxy(lower)alkanoyl (e.g. phenoxyacetyl, etc.);
arylthio(lower)alkanoyl (e.g. phenylthioacetyl, etc.);
arylamino(lower)alkanoyl (e.g. N-phenylglycyl, etc.);
arenesulfonyl (e.g. benzenesulfonyl, tosyl, naphthalenesulfonyl, etc.);
aryloxycarbonyl (e.g. phenoxycarbonyl, tolyloxycarbonyl, etc.);
aralkoxycarbonyl (e.g. benzyloxycarbonyl, benzhydryloxycarbonyl, trityloxycarbonyl, .alpha.-naphthylmethoxycarbonyl, etc.);
arylcarbamoyl (e.g. phenylcarbamoyl, tolycarbamoyl, naphthylcarbamoyl, etc.);
arylglyoxyloyl (e.g. phenylglyoxyloyl, etc.);
arylthiocarbamoyl (e.g. phenylthiocarbamoyl, etc.);
arylcarbamimidoyl (e.g. phenylcarbamimidoyl, etc.); and the like.
In the above exemplified aromatic acyl, the aromatic hydrocarbon moiety (particularly, aryl moiety) and/or aliphatic hydrocarbon moiety (particularly, alkane moiety) may have optionally one or more suitable substituents(s), such as the same asthose exemplified as the suitable substituent for alkyl group and alkane moiety as mentioned above.
Preferred example of aromatic acyl having such substituents may be exemplified by
hydroxyaroyl (e.g. salicyloyl, etc.)
haloaroyl (e.g. chlorobenzoyl, etc.),
haloar(lower)alkanoyl (e.g. chlorophenylacetyl, etc.),
hydroxyar(lower)alkanoyl (e.g. mandeyl, etc.).
Heterocyclic acyl means an acyl group derived from an acid having heterocyclic group and includes:
heterocyclic carbonyl, in which the heterocycle moiety is 5 to 6 membered heterocycle containing at least one hetero atom selected from nitrogen, oxygen and sulfur (e.g. thenoyl, furoyl, pyrrolecarbonyl, 5-oxo-2-pyrrolidinecarbonyl, nicotinoyl,etc.);
heterocyclo(lower)alkanoyl, in which the heterocycle moiety is 5 to 6 membered heterocycle containing at least one hetero atom selected from nitrogen, oxygen and sulfur (e.g. thienylacetyl, furylacetyl, imidazolylpropionyl, tetrazolylacetyl,2-(2-amino-4-thiazolyl)-2-methoxyiminoacetyl, N-acetylmuramyl, etc.); and the like.
In the above exemplified heterocyclic acyl, heterocycle moiety and/or the aliphatic hydrocarbon moiety may have optionally one or more suitable substituent(s) such as the same as those exemplified as the suitable substituent for aliphatic acyl asmentioned above.
Further, in the above exemplified acyl, in case that these acyls have one or more functional group such as hydroxy, amino, carboxy and the like, such groups may be protected by conventional protective groups(s).
(2) Re. Protected carboxy for R.sup.2, R.sup.p, R.sub.1.sup.p and R.sup.q :
A protective group of the protected carboxy includes a conventional protective group for tentatively protecting a carboxy group which is conventionally used in the field of amino acid and peptide chemistry.
As preferred examples of protected carboxy, there may be exemplified an ester such as an ester with silyl compound (hereinafter referred to as silyl ester), an ester with an aliphatic hydroxy compound (hereinafter referred to as apliphatic ester)and an ester with a hydroxy compound containing an aromatic group (hereinafter referred to as aromatic ester), and a protected carbazoyl of the formula: --COHNNHY (wherein Y is hydrogen or an amino protective group).
Concrete examples of such a protected carboxy are exemplified as follows.
suitable silyl ester such as trialkylsilyl (e.g. trimethylsilyl, triethylsilyl, etc.)ester,
halo-alkylsilyl (e.g. chloro-dimethylsilyl, dichloromethylsilyl, etc.) ester,
trihalosilyl (e.g. trichlorosilyl, etc.) ester,
alkylalkoxysilyl (e.g. methyl-diethoxysilyl, etc.) ester,
trialkoxysilyl (e.g. tris(2-chloroethoxy)silyl, etc.) ester, and the like;
suitable aliphatic hydrocarbon ester such as alkyl (e.g. methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, etc.) ester,
cycloalkyl (e.g. cyclopentyl, cyclohexyl, etc.) ester and the like; and
suitable ester containing an aromatic group such as aryl (e.g. phenyl, tolyl, xylyl, etc.) ester,
aralkyl (e.g. benzyl, diphenylmethyl, phenethyl, etc.) ester,
aryloxyalkyl (e.g. phenoxymethyl, phenoxyethyl, etc.) ester,
aroylaklyl (e.g. phenacyl, toluoylethyl, etc.) ester, and the like.
The ester forming group (e.g. substituted silyl, aliphatic hydrocarbon residue, aryl, aralkyl, aryloxyalkyl, aroylalkyl and the like, as exemplified above) may optionally have one or more appropriate substituent(s) such as alkyl (e.g. methyl,ethyl, etc.), cycloalkyl (e.g. cyclopropyl, cyclohexyl, etc.), alkoxy (e.g. methoxy, ethoxy, etc.), alkanoyloxy (e.g. acetoxy, etc.), alkylthio (e.g. methylthio, etc.), halogen (e.g. chlorine, etc.), cyano, nitro, etc.
Examples of such substituted esters may be mono(di or tri)haloalkyl (e.g. chloromethyl, bromoethyl, dichloromethyl, 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,2,2-trifluoroethyl, etc.) ester, cyanoalkyl (e.g. cyanomethyl, cyanoethyl, etc.)ester, cycloalkyl-substituted-alkyl (e.g. 1-cyclopropylethyl, etc.) ester, mono(di, tri, tetra or penta)halophenyl (e.g. 4-chlorophenyl, 3,5,-1-dibromophenyl, 2,4,5-trichlorophenyl, 2,4,6-trichlorophenyl, pentachlorophenyl, etc.) ester, and the like.
(3) Re. A group of the formula for R.sup.2 : ##STR9##
Suitable example of lower alkyl for R.sub.b.sup.2 and lower alkyl moiety of mono- or di-carboxy(lower)alkyl and of ar(carboxy)lower alkyl for R.sub.a.sup.2 is one having 1-6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl,pentyl, isopentyl and the like.
Suitable example of aryl moiety of ar(carboxy)lower alkyl for R.sub.a.sup.2 is phenyl, tolyl, xylyl, naphthyl and the like. In this respect, it is to be noted that "ar(carboxy)lower alkyl" can alternatively expressed by the wording "lower alkyl"which is substituted by carboxy and aryl group.
Carboxy in "mono- or di-carboxy lower alkyl" for R.sub.a.sup.2 and "ar(carboxy)lower alkyl whose aryl moiety may be substituted by hydroxy" for R.sub.a.sup.2 may be protected by a conventional protective group; namely R.sub.a.sup.2 includeswithin its definition also protected carboxy-lower alkyl and ar(protected carboxy)lower alkyl which may be substituted by hydroxy. Suitable example of such a protected carboxy is the same as that exemplified for R.sup.p and R.sup.q.
Most preferred examples of a group of the formula: ##STR10## are illustrated as follows: ##STR11## and the corresponding group, in which the carboxy group is protected by a conventional carboxy protective group.
(4) Re. Protected hydroxymethyl for R.sub.b.sup.1 :
Hydroxy group of hydroxymethyl for R.sub.b.sup.1 may be protected by a conventional hydroxy protective group; namely, R.sub.b.sup.1 includes protected hydroxymethyl.
Some preferred examples of the hydroxy protective group may be exemplified by an acyl such as substituted or unsubstituted alkanoyl (e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, bromoacetyl, dichloroacetyl, trifluoroacetyl,etc.), substituted or unsubstituted aroyl (e.g. benzoyl, toluoyl, xyloyl, nitrobenzoyl, bromobenzoyl, salicyloyl, etc.), arylalkyl (e.g. benzyl) or the like.
(5) Re. Amino protective group for R.sup.r, R.sub.1.sup.r and Y:
The amino protective group includes a conventional protective group for tentatively protecting an amino group, which is used in the field of amino acid and peptide chemistry. That is, in the peptide synthesis, it is understood that, for bondinga desired "reactive" amino group (--NH.sub.2) with a desired "reactive" carboxy group (--COOH) to form a desired peptide bond (-CONH-) between them, it is preferably to tentatively protect the other undesired "reactive" amino group to convert it into anunreactive or less reactive protected amino group in the reaction in order to avoid the side reaction between the undesired "reactive" amino group and desired "reactive" carboxy groups. Further, it is understood that it is preferable that a protectivegroup in such protected amino group is easily eliminable according to the necessity in the treatment of the object peptide. Accordingly, an amino protective group to meet the above requirements can be used and suitable one should be selected accordingto the kind and property of the component to be used in this invention.
As preferred examples of the amino protective group, the following examples are illustrated:
Acyl, particularly organic acyl, for example, substituted or unsubstituted aliphatic hydrocarbonoxycarbonyl such as
alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, t-pentoxycarbonyl, etc.),
haloalkoxycarbonyl (e.g. chloromethoxycarbonyl, tribromoethoxycarbonyl, trichloroethoxycarbonyl, etc.),
an alkane- or arene- sulfonylalkoxycarbonyl (e.g. 2-(mesyl)ethoxycarbonyl, 2-(p-toluenesulonyl)-ethoxycarbonyl, etc.),
an alkylthio- or arylthioalkoxycarbonyl (e.g. 2-(ethylthio)ethoxycarbonyl, 2-(p-tolylthio)ethoxycarbonyl, etc.),
a monocyclic or fusedcyclic-alicyclic oxycarbonyl (e.g. cyclohexyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl, etc.),
substituted or unsubstituted alkenyloxycarbonyl (e.g. allyoxycarbonyl, etc.),
substituted or unsubstituted alkynloxycarbonyl (e.g. 1,1-dimethylpropargyloxycarbonyl, etc.) or the like,
substituted or unsubstituted aryloxycarbonyl (e.g. phenoxycarbonyl, p-methylphenoxycarbonyl, etc.),
substituted or unsubstituted aralkoxycarbonyl (e.g. benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl, p-(p-methoxyphenylazo)benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,.alpha.-naphthylmethoxycarbonyl, p-biphenylisopropoxycarbonyl, etc.);
substituted or unsubstituted arenesulfonyl (e.g. benzensulfonyl, p-toluenesulfonyl, etc.);
substituted or unsubstituted dialkylphosphoryl (e.g. dimethylphosphoryl, etc.);
substituted or unsubstituted diaralkylphosphoryl (e.g. 0,0-dibenzylphosphoryl, etc.);
substituted or unsubstituted aryloxyalkanoyl (e.g. phenoxyacetyl, p-chlorophenoxyacetyl, 2-nitrophenoxyacetyl, 2-methyl-2-(2-nitrophenoxy)propyonyl, etc.) or the like;
substituted or unsubstituted aryl such as phenyl, tolyl or the like;
substituted or unsubstituted aralkyl such as benzyl, diphenylmethyl, trityl, nitrobenzyl, or the like;
substituted or unsubstituted alkylidene (e.g. ethylene, isopropylidene, etc.) or the like;
substituted or unsubstituted aralkylidene such as benzylidene, 2-hydroxybenzylidene, 2-hydroxy-5-chlorobenzylidene or the like;
substituted or unsubstituted arylthio such as phenylthio, nitrophenylthio, dinitrophenylthio, trichlorophenylthio or the like; and
substituted or unsubstituted aralkylthio such as tritylthio or the like.
A pharmaceutically acceptable salt of the new peptides of the formula (I) may include a salt with an inorganic or organic base such as an alkali metal salt (e.g. sodium salt, potassium salt, etc.), an alkaline earth metal salt (e.g. calsium salt,etc.), ammonium salt, ethanolamine salt, triethylamine salt, dicyclohexylamine salt or the like, and an acid addition salt with organic or inorganic acid such as methane sulfonate, hydrochloride, sulfate, nitrate, phosphate or the like.
With regard to the formula (I) for the compound of this invention the following is to be noted. That is, partial formula: ##STR12## in the formula (I) particularly means R.sup.1, when n is an integer 0, ##STR13## when n is an integer 1, and##STR14## when n is an integer 2. Further, partial formula ##STR15## in the formula (I) particularly means ##STR16## when m is an integer 1, ##STR17## when m is an integer 2, and ##STR18## when m is an integer 3.
The compound (I) of this invention including FR-900156 can be prepared by chemical synthetic methods and fermentation method, details of which will be apparent from the following description. ##STR19##
In the above formulae, R.sub.c.sup.1 is acyl,
R.sub.1.sup.p is hydrogen or protected carboxy,
R.sub.2.sup.p is hydrogen or carboxy,
R.sub.1.sup.q is hydrogen or protected carboxy,
R.sub.2.sup.q is hydrogen or carboxy,
R.sub.1.sup.r is amino protective group and
m, n, R.sup.1, R.sup.2, R.sup.p, R.sup.q and R.sup.r are each as defined above.
Detailed explanation or processes for preparation of a new peptide of the formula (I) will be made in the following.
[1] Synthesis
(1) Process 1: Peptide bond formation
This process relates to a method for preparing Compound (Ia) by reacting Compound (II) or its salt with a Compound (III) or its salt.
The reaction of this process can be conducted as follows. That is, in one case, as the first step, the carboxy group of Compound (II) or its salt is usually activated in a conventional manner, for example, in the form of its acid halide, azide,acid anhydride or a mixed anhydride activated ester, and the like, and is reacted with the Compound (III) to give Compound (Ia), and in the other case, the Compound (II) or its salt is reacted with the Compound (III) or its salt directly in the presenceof a conventional condensing agent such as N,N-dicyclohexylcarbodiimide and the like. Among these activation methods, preferred activation method for the carboxy group of the Compound (II) into its activated form and preferred condensing agent asmentioned above are selected according to kinds of the carboxy protective group(s) of the Compound (II) and (III) and to the reaction conditions (e.g. the kinds of the reaction solvent, reaction temperature and so on).
This reaction is preferably carried out in a solvent such as methylene chloride, chloroform, tetrahydrofuran, dioxane, ethyl acetate, methanol, ethanol, water or the like under ice-cooling to at ambient temperature and the reaction in thepresence of a condensing agent is usually carried out in an anhydrous, but not critical, conditions.
(2) Process 2: Selective deacylation
This process relates to a method for preparing Compound (Ib) or its salt by removing selectively an acyl group for R.sub.c.sup.1 of Compound (Ia) or its salt.
This process is applied to case that the acyl group for R.sub.c.sup.1 reveals a different chemical property from that of the amino protective group for R.sub.1.sup.r against each kind of the removal methods and can selectively be removable by amethod to be employed.
This reaction is carried out by conventional methods, such as catalytic reduction method, liquidammoniaalkalimetal method, acid method, zinc acid method, base method, hydrazine method and the like. Among these methods, preferred one is selectedaccording to kind of the acyl group for R.sub.c.sup.1 of Compound (Ia).
Each of the above methods is explained as follows.
(i) Catalytic reduction method:
This method is preferably applied to case that the acyl group for R.sub.c.sup.1 of Compound (Ia) are one which is removable by catalytic reduction. As preferred examples of such an acyl group for R.sub.c.sup.1, there may be exemplifiedsubstituted or unsubstituted aralkoxycarbonyl (e.g. benzyloxycarbonyl, p-phenylazobenzyloxycarbonyl, p-(p'-methoxyphenylazo)benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, etc.); substituted or unsubstituted alkenyl- or alkynyloxycarbonyl (e.g.allyloxycarbonyl, 1,1-dimethylproparglyloxycarbonyl, etc.); substituted or unsubstituted aryloxyalkanoyl (e.g. 2-nitrophenoxyacetyl, 2-methyl-2-(2-nitrophenoxy)propionyl, etc.); and the like.
This catalytic reduction is carried out in a conventional manner, and suitable catalysts to be used in catalytic reduction are conventional ones such as platinum catalyst (e.g. platinum plate, spongy platinum, platinum black, colloidal platinum,platinum oxide or platinum wire, etc.), palladium catalyst (e.g. spongy palladium, palladium black, palladium oxide, palladium on carbon, colloidal palladium, palladium or barium sulfate, palladium on barium carbonate, etc.), nickel catalyst (e.g.reduced nickel, nickel oxide, Raney nickel, etc.) cobalt catalyst (e.g. reduced cobalt, Raney cobalt, etc.), iron catalyst (e.g. reduced iron, Raney iron, etc.), copper catalyst (e.g. reduced copper, Raney copper, Ullmann copper, etc.) or the like.
The reduction is usually carried out in a solvent. A suitable solvent to be used may be, e.g. water, methanol, ethanol, propanol, ethyl acetate, tetrahydrofuran, dioxane, N,N-dimethylformamide, acetic acid, a mixture of water and alcohol (e.g.methanol, ethanol, etc.) tetrahydrofuran, dioxane or ethyl acetate, and other conventional organic solvent or a mixture thereof. Further, the reduction is preferably carried out in the presence of an acid such as acetic acid or the like.
The reaction is preferably carried out under somewhat milder conditions such as cooling or warming.
In this method, in case that R.sup.q is a group of the formula: --CONHNHY and an amino protective group for Y is the same as the acyl for R.sub.c.sup.1, then such an amino protective group also is simultaneously removed to give a Compound (Ib)wherein R.sup.q is a group of the formula: --CONHNH.sub.2. Further, in case that a protected carboxy group for R.sup.p is, for example, substituted or unsubstituted aralkyl ester type one (e.g. benzyl ester p-nitrobenzyl ester, p-chlorobenzyl ester,p-phenylazobenzyl ester, etc.), such a protective group also is simultaneously removed in this process to give a Compound (Ib) wherein R.sup.p is carboxy.
(ii) Acid method:
(ii)-1 Method of use of trifluoroacetic acid or formic acid:
This method is preferably applied to case that the acyl group for R.sub.c.sup.1 is one which is removeable by treating with trifluoro-acetic acid or formic acid. Preferred examples of such an acyl group may be exemplified by a group such asbranched- or alicyclicoxycarbonyl, (e.g. t-butoxycarbonyl, t-pentoxycarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl, etc.); substituted or unsubstituted aralkoxycarbonyl (e.g. p-methoxybenzyloxycarbonyl, etc.).
This reaction is conventionally carried out in a solvent such as methylene chloride, chloroform, acetic acid, water and the like in the presence of trifluoroacetic acid or formic acid, and anisole is preferably added thereto.
Trifluoroacetic acid and formic acid are also used as the solvent.
This reaction is usually carried out under ice-cooling to at ambient temperature.
In this method, in case that R.sup.q is a group of the formula: --CONHNHY and an amino protective group for Y is the same as the acyl for R.sub.c.sup.1, then such an amino protective group also is simultaneously removed to give a Compound (Ib)wherein R.sup.q is a group of the formula: --CONHNH.sub.2. Further, in case that a protected carboxy group for R.sup.p is, for example, a branched alkyl ester (e.g. t-butyl ester, etc.), or substituted or unsubstituted aralkyl ester (e.g. diphenylmethylester, p-methoxybenzyl ester, etc.), such a protective group also is simultaneously removed to give a Compound (Ib) wherein R.sup.p is carboxy.
(ii)-2 Method of use of hydrochloric acid or p-toluenesulfonic acid:
This method is preferably applied to case that an acyl group for R.sub.c.sup.1 is one which is removed by treating with hydrochloric acid or p-toluenesulfonic acid.
Preferred examples of such an acyl group may be exemplified by e.g. substituted or unsubstituted branched alkoxycarbonyl (e.g. t-butoxycarbonyl, 1-(p-biphenyl)-1-methylethoxycarbonyl, etc.) and the like in addition to one as illustrated in theabove (ii)-1.
This reaction is carried out in a solvent such as ethyl acetate, methylene chloride, chloroform, tetrahydrofuran and the like in the presence of an inorganic or organic strong acid such as hydrochloric acid, p-toluenesulfonic acid or the like,and anisole is preferably added thereto.
This reaction is preferably carried out under ice-cooling to at ambient temperature.
In this method, in case that R.sup.q is a group of the formula: --CONHNHY and an amino protective group for Y is the same as the acyl for R.sub.c.sup.1, then such an amino protective group also is simultaneously removed to give a Compound (Ib)wherein R.sup.q is a group of the formula: --CONHNH.sub.2. Further, in case that a protected carboxy group for R.sup.p is, for example, a branched alkyl ester (e.g. t-butyl ester etc.) or substituted or unsubstituted aralkyl ester (e.g. diphenylmethylester, p-methoxybenzyl ester, etc.), such a protective group also is simultaneously removed to give a Compound (Ib) wherein R.sup.p is carboxy.
(ii)-3 Method of use of hydrogen bromide:
This method is preferably applied to case that an acyl group for R.sub.c.sup.1 is one which is removable by treating with hydrogen bromide.
Preferred examples of such an acyl group may be exemplified by substituted or unsubstituted aralkoxycarbonyl (e.g. benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-tolyloxycarbonyl, p-phenylazobenzyloxycarbonyl,.alpha.-naphthylmethoxycarbonyl, etc.) and an alkoxycarbonyl (e.g. isopropoxycarbonyl, etc.) in addition to one as illustrated in the above (ii)-1 and (ii)-2.
This reaction is usually carried out in a solvent such as ethyl acetate, acetic acid, trifluoroacetic acid or the like in the presence of hydrogen bromide.
This reaction is preferably carried out under ice-cooling to at ambient temperature.
In this method, in case that R.sup.q is a group of the formula: --CONHNHY and an amino protective group for Y is the same as the acyl for R.sub.c.sup.1, then such an amino protective group also is simultaneously removed to give Compound (Ib)wherein R.sup.q is a group of the formula: --CONHNH.sub.2. Further, in case that a protected carboxy group for R.sup.p is, for example, a branched alkyl ester or (e.g. t-butyl ester, etc.), substituted or unsubstituted aralkyl ester (e.g. diphenylmethylester, p-methoxybenzyl ester, etc.), such a protective group is simultaneously removed to give a Compound (Ib) wherein R.sup.p is carboxy.
(iii) Liquid-ammonia-alkali metal method:
This method is preferably applied to case that the acyl group for R.sub.c.sup.1 is one which is removable by treating with liquid ammonia-alkali metal. As preferred examples of such an acyl group, there may be exemplified substituted orunsubstituted aralkoxycarbonyl (e.g. benzyloxycarbonyl, etc.), substituted or unsubstituted aryloxycarbonyl (e.g. phenoxycarbonyl, p-methylphenoxycarbonyl, etc.), an arenesulfonyl (e.g. benzenesulfonyl, p-toluenesulfonyl, etc.) and the like.
This reaction is usually carried out by dissolving Compound (Ia) into liquid ammonia and then alkali metal is added thereto.
This reaction is preferably carried out at a lower temperature, e.g. at -78.degree. C. to at boiling point of liquid ammonia.
In this method, in case that R.sup.q is a group of the formula: --CONHNHY and amino protective group for Y is the same as the acyl for R.sub.c.sup.1, then such an amino protective group also is simultaneously removed to give a Compound (Ib)wherein R.sup.q is a group of the formula: --CONHNH.sub.2.
(iv) Hydrazine method:
This method is preferably applied to case that the acyl group for R.sub.c.sup.1 is one which is removable by treating with a hydrazine compound or an amine compound. As preferred examples of such an acyl group, there may be exemplifiedphthaloyl, formyl, acetoacetyl, etc.
Preferred examples of hydrazine compound are exemplified by hydrazine, methylhydrazine, phenylhydrazine and the like and those of amine compound are exemplified by hydroxylamine, dialkylaminoalkylamine (e.g. N,N-dimethylaminopropylamine, etc.)and the like.
This reaction is usually carried out by treating Compound (Ia) with the hydrazine compound or amine compound in a solvent such as water, alcohol (e.g. methanol, ethanol, etc.) tetrahydrofuran, dioxane or the like at ambient temperature to underreflux.
In this method, in case that R.sup.q is a group of the formula: --CONHNHY and an amino protective group for Y is the same as the acyl for R.sub.c.sup.1, then such an amino protective group also is simultaneously removed to give a Compound (Ib)wherein R.sup.q is a group of the formula --CONHNH.sub.2.
(v) Zinc-acid method:
This method is preferably applied to case that the acyl group for R.sub.c.sup.1 is one which is removable by treating with zinc acid.
As preferred examples of such an acyl group, there may be exemplified trichloroethoxycarbonyl, 4-piperidyloxycarbonyl, 1-methyl-1-(4-pyridyl)ethoxycarbonyl and the like.
This method is carried out by treating Compound (Ia) with zinc in the presence of a weak acid such as formic acid, acetic acid and the like. The reaction may be carried out in a solvent such as methylene chloride, chloroform, tetrahydrofuran,ethyl acetate, alcohol (e.g. methanol, ethanol, etc.), dimethylformamide and the like, and in this case a weak acid as mentioned above is added to such a solvent. The reaction is usually carried out at -10.degree. C. to ambient temperature.
In this reaction, in case that R.sup.q is a group for the formula: --CONHNHY and an amino protective group for Y is the same as the acyl for R.sub.c.sup.1, then such an amino protective group also is simultaneously removed to give a Compound (Ib)wherein R.sup.q is a group of the formula: --CONHNH.sub.2. Further, in case that a protected carboxy group for R.sup.p of Compound (Ia) is, for example, a halo-alkyl ester type of group (e.g. trichloroethyl, etc.), such a carboxy protective group alsois simultaneously removed to give a Compound (Ib) wherein R.sup.p is carboxy.
(vi) Base method:
This method is preferably applied to case that the acyl group for R.sub.c.sup.1 is one which is removable by treating with a base. As preferred examples of such an acyl group, there may be exemplified haloalkanoyl (e.g. trifluoroacetyl, etc.),substituted or unsubstituted alkoxycarbonyl (e.g. 2-(p-toluenesulfonyl)ethoxycarbonyl, 2-(p-tolylthio)ethoxycarbonyl, etc.), substituted or unsubstituted aryloxycarbonyl (e.g. 2-nitrophenoxycarbonyl, etc.) and the like.
This method is carried out in the presence of a base under ice-cooling to at ambient temperature.
Suitable base is an inorganic base such as alkali metal hydroxide or alkaline earth metal hydroxide, or the corresponding carbonate or bicarbonate (e.g. sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, lithiumcarbonate, sodium bicarbonate, calcium hydroxide, magnesium hydroxide, etc.), ammonium hydroxide or the like; an organic base such as an alkoxide or phenoxide of the above metal (e.g. sodium ethoxide, sodium methoxide, lithium phenoxide, etc.), an aminesuch as mono-, di- or trialkylamine (e.g. methylamine, ethylamine, propylamine, isopropylamine, butylamine, N,N-dimethyl-1, 3-propanediamine, trimethylamine, triethylamine, atc.), unsubstituted, mono- or disubstituted arylamine (e.g. aniline,N-methylaniline N,N-dimethylaniline, etc.), a heterocyclic base (e.g. pyrrolidine, morpholine, N-methylmorpholine, N-methylpiperidine, N,N-dimethylpiperazine, pyridine, etc.) or the like; a basic ion exchange resin and the like.
This method is preferably conducted under somewhat milder conditions such as cooling or warming and usually in any solvent which does not have an adverse influence on the reaction, e.g. water, a hydrophilic solvent such as alcohol (e.g. methanol,ethanol, propanol, etc.), N,N-dimethylformamide, tetrahydrofuran, dioxane, dimethylsulfoxide, etc. or a mixture thereof. In case that the above-mentioned bases are in liquid, they can also be used as a solvent.
In this method, in case that R.sup.q is a group of the formula: --CONHNHY and an amino protective group for Y is the same as the acyl for R.sub.c.sup.1, then such an amino protective group also is simultaneously removed to give a Compound (Ib)wherein R.sup.q is a group of the formula: --CONHNH.sub.2. Further, in case that a protected carboxy group for R.sup.p of Compound (Ia) is, for example, an alkyl ester type of group (e.g. methyl ester, ethyl ester, etc.), an aralkyl ester type group(e.g. benzyl ester, etc.), such a protective group also is simultaneously removed to give a Compound (Ib) wherein R.sup.p is carboxy.
(3) Process 3: Compound (Ib) R.sub.c.sup.1 --OH Compound (Ia)
This process relates to a method for preparing Compound (Ia) by reacting Compound (Ib) with an acylating agent.
The acylating agent to be used in this reaction includes an organic acid (R.sub.c.sup.1 --OH) wherein R.sub.c.sup.1 is acyl group) such as monobasic or dibasic organic carboxylic acid, an organic carbonic acid or an organic carbamic acid and thecorresponding thio acid or imidic acid; and an organic sulfonic acid, and more particularly , aliphatic, aromatic or heterocyclic carboxylic acid, and the corresponding carbonic, carbamic, thiocarboxylic, thiocarbonic, thiocarbamic, carboximidic,carbamimidic acid, and sulfonic acid; their reactive derivatives; and also includes an isocyanate (e.g. potassium-, alkyl- or aryl- isocyanate), isothiocyanate (e.g. alkyl isothiocyanate) and an isothiourea (e.g. ethyl isothiourea). Suitable examples ofthese organic acid (R.sub.c.sup.1 --OH wherein R.sub.c.sup.1 is acyl group) are the corresponding organic acid to those comprising the acyl group as exemplified hereinabove in details in the descriptions of suitable examples of acyl groups for R.sup.1and R.sub.c.sup.1 of the compound (I).
Said organic acid as an acylating agent can be used in the form of an activated organic acid, ie.. as a reactive derivative of the acid. As such reactive derivatives of said organic acids, there may be exemplified an acid halide, an acid azide,an acid anhydride, an activated amide, an activated ester, etc., and additionally isocyanate and isothiocyanate can be preferably be used used as reactive derivatives of carbamic and thiocarbamic acids, respectively.
Preferred examples of such reactive derivatives are illustrated by:
an acid halide (e.g. acid chloride, acid bromide etc.);
an acid azide;
an acid anhydride including a mixed acid anhydride with an acid such as dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, dibenzylphosphoric acid, halogenated phosphoric acid, dialkylphosphorous acid, sulfurous acid,thiosulfuric acid, sulfuric acid, monoalkylcarbonic acid, aliphatic carboxylic acid (e.g. acetic acid, pivalic acid, pentanoic acid, isopentanoic acid, 2-ethylbutyric acid, trichloroacetic acid, etc.), aromatic carboxylic acid (e.g. benzoic acid, etc.)or the like, and symmetrical acid anhydride;
an activated amide with pyrazole, imidazole, 4-substituted imidazole, dimethylpyrazole, triazole or tetrazole; and
an activated ester such as substituted or unsubstituted alkylthio ester (e.g. methythio ester, carboxymethyl thioester, etc.), substituted or unsubstituted aryl thioester, (e.g. phenyl thioester, p-nitrophenyl thioester, p-cresyl thioester,etc.), heterocyclic ester (e.g. pyranyl ester, pyridyl ester, piperidyl ester, 8-quinolyl thioester, etc.) or ester with N,N-dimethylhydroxylamine, 1-hydroxy-2-(1H)-pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide or 1hydroxy-6-chlorobenzotriazole,or the like.
The above reactive derivative is selected according to the kind of the acid to be used.
In the reaction, when free acid is used as an acylating agent, the acylation reaction may preferably be conducted in the presence of a condensing agent such as carbodiimidic compound (e.g. N,N'-dicyclohexylcarbodiimide,N-cyclohexyl--N'-morpholinoethylcarbodiimide, N-cyclohexyl-N'-(4-diethylaminocyclohexyl)carbodiimide, N,N'-diethylcarbodiimide, N,N'-diisopropylcarbodiimide, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, etc.), N,N'-carbonyldi(2-methylimidazole),pentamethyleneketene-N-cyclohexylimine, diphenylketene-N-cyclohexylimine, alkoxyacetylene, 1-alkoxy-1-chloroethylene, trialkyl phosphite, ethyl polyphosphate, isopropyl polyphosphate, phosphorus compound (e.g. phosphorus oxychloride, phosphoroustrichloride, etc.), thionyl chloride, oxalyl chloride, 2-ethyl-7-hydroxybenzisoxazolium salt, 2-ethyl-5-(m-sulfophenyl)isoxazolium hydroxide, (chloromethylene)dimethylammonium chloride,2,2,4,4,6,6,-hexachloro-1,3,5,2,4,6-triazatriphosphorine,1-benzensulphonyloxy-6-chloro-1H-benzotriazole, p-toluenesulfonyl chloride, isopropoxybenzenesulfoxyl chloride or the like; or a mixed condensing agent such as a mixture of triphenylphosphine and a carbon tetrahalide (e.g. carbon tetrachloride, carbontetrabromide, etc.), a complex of N,N-dimethylformamide with phosphoryl chloride, phosgene or thionyl chloride, etc., or the like.
The reaction is usually conducted in a solvent such as water, alcohol (e.g. methanol, ethanol, propanol, etc.), acetone, ethyl ether, dioxane, acetonitrile, ethylacetate, N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran, dichloromethane,chloroform, etc. or pyridine, N-methylmorpholine, N-methylpyrrolidine or other conventional solvents, or a mixture thereof.
The reaction can also be conducted preferably in the presence of an organic or inorganic base such as alkali metal (e.g. sodium, potassium, etc.), alkaline earth metal (e.g. calcium, etc.), alakali or alkaline earth metal hydride (e.g. sodiumhydride, calcium hydride, etc.), alkali or alkaline earth metal hydroxide (e.g. sodium hydroxide, potassium hydroxide, calcium hydroxide, etc.), alkali or alkaline earth metal carbonate or bicarbonate (e.g. sodium carbonate, potassium carbonate, sodiumbicarbonate, lithium carbonate, etc.), alkali or alkaline earth metal alkoxide (e.g. sodium ethoxide, lithium methoxide, magnesium methoxide, etc.), trialkylamine (e.g. triethylamine, etc.), pyridine, bicyclodiaza compound (e.g.1,5-diazabicyclo[3,4,0]nonene-5, 1,5-diazabicyclo[5,4,0]undecene-5, etc.) or the like. Among said base, a liquid one can also be used as a solvent.
There is no limination to this reaction temperature, and this reaction may preferably be conducted within the range of cooling to ambient temperature.
(4) Process: Elimination of protective groups
This process relates to a method for preparing Compound (Id) by subjecting Compound (Ic) to removal reaction of protective groups of protected carboxy groups for R.sup.p and R.sup.q and or amino protective group for R.sub.1.sup.r, details ofwhich are explained as follows:
Process 4-1: Elimination of an amino protective group for R.sub.1.sup.r
The elimination of an amino protective group for R.sub.1.sup.r is carried out substantially in the same manner as that of Process 2, and accordingly the detailed explanation for Process 2 as made hereinabove is to be referred to.
Process 4-2: Removal of carboxy protective group of protected carboxy for R.sub.1.sup.p and R.sub.1.sup.q.
The reaction for removal of protective group of the protected carboxy group is carried out by a conventional method such as hydrolysis and reduction or the like, details of which are explained in the following.
(i) For hydrolysis which refers to the same meaning as solvolysis including, for example, acidolysis, alcoholysis, aminolysis, hydroxinolysis, etc.:
Hydrolysis is preferably carried out in the presence of an acid or base.
Suitable acid includes an inorganic acid (e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, etc.), an organic acid (e.g. formic acid, acetic acid, trifluoroacetic acid, propionic acid, benzensulfonic acid, p-toluenesulfonic acid, etc.), anacidic ion-exchange resin and the like.
Suitable base includes an inorganic base such as alkali or alkaline earth metal hydroxide or the corresponding carbonate or bicarbonate (e.g. sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, sodiumbicarbonate, calcium hydroxide, magnesium hydroxide, etc.), ammonium hydroxide or the like; an organic base such as an alkoxide or phenoxide of the above metal (e.g. sodium ethoxide, sodium methoxide, lithium phenoxide, etc.), an amine such as mono-, di-or trialkylamine (e.g. methylamine, ethylamine, propylamine, isopropylamine, butylamine, N,N-dimethyl-1,3-propanediamine, trimethylamine, triethylamine, etc.), unsubstituted, mono- or disubstituted arylamine (e.g. aniline, N-methylaniline,N,N-dimethylaniline, etc.), a heterocyclic base (e.g. pyrrolidine, morpholine, N-methylmorpholine, N-methylpiperdine, N,N-dimethylpiperazine, pyridine, etc.), hydrazines (e.g. hydrazine, methylhydrazine, ethylhydrazine, etc.) or the like; a basicion-exchange resin or the like.
The hydrolysis is preferably conducted under somewhat milder conditions such as cooling or warming and usually in a solvent which does not have adverse influence to the reaction, e.g. water, a hydrophilic solvent such as alcohol (e.g. methanol,ethanol, propanol, etc.), acetone, N,N-dimethylformamide, tetrahydrofuran, dioxane, dimethylsulfoxide, etc. or a mixture thereof, and other hydrophobic solvent such as benzene diethylether, etc. may also be used as a solvent. A liquid abovementionedacid or base can also be used as solvent.
(ii) For reduction:
Reduction, including chemical reduction and catalytic reduction, is carried out in a conventional manner.
Suitable reducing agents to be used in chemical reduction are a metal (e.g. tin, zinc, iron, etc.), or a combination of such metal and/or metallic compound (e.g. chromium chloride, chromium acetate, etc.) and an organic or inorganic acid (e.g.formic acid, acetic acid, propionic acid, trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid, hydrobromic acid, etc.).
Suitable catalysts to be used in catalytic reduction are conventional ones such as platinum catalysts (e.g. platinum plate, spongy platinum platinum black, colloidal platinum, platinum oxide or platinum wire, etc.), palladium catalysts (e.g.spongy palladium, palladium black, palladium oxide, palladium on carbon, colloidal palladium, palladium or barium sulfate, palladium or barium carbonate, etc.), nickel catalysts (e.g. reduced nickel, nickel oxide, Rabey nickel, etc.), cobalt catalysts(e.g. reduced cobalt, Raney cobalt, etc.), iron ctalysts (e.g. reduced iron, Raney iron, etc.), copper catalysts (e.g. reduced copper, Raney copper, Ullman copper, etc.) or the like.
The reduction is usually carried out in a solvent. A suitable solvent to be used may be, e.g. water, alcohol (e.g. methanol, ethanol, propanol, etc.) and other conventional organic solvent or a mixture thereof. Additionally, the afore-mentionedliquid acids to be used in chemical reduction can also be used as solvent. Further, a suitable solvent to be used in catalytic reduction may be, e.g. the above-mentioned solvent, and other conventional solvent, such as diethyl ether, dioxane,tetrahydrofuran, etc. or a mixture thereof.
The reaction is preferably carried out under somewhat milder conditions such as cooling or warming.
Among these methods for removal of protective groups, preferred one and appropriate combination methods are to be selected according to kinds of the protective groups of carboxy group and amino protective group to be removed off.
It is noted that this process includes the following cases of removal of protective groups and amino protective group, that is, one case that all of the carboxy protective groups for R.sub.1.sup.p and R.sub.1.sup.q and the amino protective groupfor R.sub.1.sup.r in the Compound (Ic) are simultaneously removed by a method to be employed to the reaction, and the other case that the carboxy protective groups and the amino protective group are sequentially and stepwise removed by a method which isappropriately selected according to the kinds of the protective group to be removed.
Process 4-3: Removal of hydrazino group
A protected carbazoyl of the formula: --CONHNHY wherein Y is an amino protective group can be removed by subjecting the Compound (Ic) at first to the reaction of Process 4-1 for eliminating amino protective group (i.e. R.sub.1.sup.r) to give--CO.NHNH.sub.2 group and then subjecting the reaction product to the reaction of this step to give --COOH group, and particular of this reaction step is as follows.
The reaction of this step is carried out in a conventional manner by treating the Compound (Ic) with a conventional oxidizing agent which is capable of oxidizing a group of the formula: --CONHNH.sub.2 to form into a group of the formula: --COOHand accordingly preferred example of such an oxidizing agents may be halogen such as iodine, bromine etc., perhalogenic acid such as periodic acid or its salt (e.g. sodium salt, potassium salt, etc.), perchloric acid, etc., N-haloimide such asN-bromosuccinimide, etc., lead tetraacetate, hydrogen peroxide or its salt (e.g. nickel peroxide, etc.), metal oxide such as mercuric oxide, manganese dioxide, nickel peroxide, etc., cupric compound (e.g. cupric acetate, cupric sulfate, etc.) and thelike.
This reaction is usually carried out in a solvent such as water, acetic acid, methanol, ethanol, tetrahydrofuran, dioxane and the like and a mixture thereof, which should be appropriately selected in accordance with the kind of oxidizing agent tobe used.
This reaction is usually carried out under ice-cooling at at ambient temperature, or under reflux.
As to Process 4 for Elimination of protective groups (i.e. Process 4-1, Process 4-2 and Process 4-3), it is to be noted that, in case that acyl for R.sup.1 has one or more protective groups for hydroxy, amino and (or) carboxy, such protectivegroups also may be simultaneously removed and such a case is included within the scope of this process.
(2) Fermentation: A strain of Streptmyces.fwdarw.Compound (Ie)
The compound (Ie) (hereinafter referred to as FR-900156 substance) can be produced by fermentation of a FR-900156 substance producing strain belonging to the genus Streptomyces in a nutrient medium, details of which are explained in thefollowing.
The microorganism which can be used for the production of the FR-900156 substance is a strain belonging to the genus Streptomyces, among which a strain of Streptomyces olivaceogriseus and Streptomyces violaceus have been newly isolated from asoil sample as a suitable strain of a FR-900156 substance-producing strain belonging to the genus Streptomyces.
it is to be understood that, for the production of the FR-900156 substance, this invention is not limited to the use of the particular oganism as described herein, which is given for illustrative purpose only. This invention also includes theuse of any mutants which are capable of producing the FR-900156 substance, including natural mutants which are produced by natural mutation of the organisms as well as artificial mutants which can be produced from the described organism by conventionalmeans, such as X-rays, ultra-violet radiation, nitrogen mustard oils and the like.
I. Re. Streptomyces olivaceogriseus nov. sp.C-353;
Streptomyces olivaceogriseus nov. sp.C-353 has been isolated from a soil sample collected at Kochi Prefecture, Japan and deposited with and added to a permanent stock culture collection of the America Type Culture Collection under the numberATCC 31427.
Streptomyces olivaceogriseus nov. sp. C-353 (ATCC 31427) has the following morphological, cultural and physiological characteristics.
(1) Morphological characteristics:
Microscopic observations were made on cultures which were grown from 10 to 14 days on sucrose-nitrate agar, glycerin-asparagine agar, yeast-malt extract agar, oatmeal agar, starch-inorganic salts agar and Bennett agar. Sphorophore morphology wasobserved on undisturbed plates cultures.
1. Type of branching of spore-forming hyphae: Monopodial branching.
2. Form of spore-forming hyphae: Retinaculiaperti (closed spirals, loops).
3. Numbers of spores: 5-20 spores.
4. Surface appearance and size of spores. Smooth 0.6-1.2.times.1.0-1.9 micron.
5. Existence of zoospores: Not observed.
6. Existence of sporangium: Not observed.
7. Formation of spores: At aerial mycelium.
8. Fragmentation of substrate mycelium: Not observed.
(2) Cultural characteristics:
The following observations were made on slant cultures which were grown on various media at 30.degree. C. for 10-14 days.
______________________________________ Aerial mass Reverse side Soluble Medium color of colony Pigment ______________________________________ Sucrose-nitrate none or pale yellow, none agar very thin, small colonies powdery Glucose-aspara- light gray to pale yellow none gine agar greenish to pale gray, yellowish brown powdery small colonies Glycerin-aspara thin, powdery pale yellowish none or gine agar light gray brown, trace small colonies Starch-inorganic olive gray, grayishyellow none salts agar powdery brown, small colonies Tyrosine agar light olive yellowish brown, light gray, thin small colonies brown powdery Nutrient agar none pale yellow, none flat Yeast-malt greenish yellowish brown, none extractagar gray, wrinkled powdery colonies Oatmeal agar light gray to colorless, none greenish small colonies gray, powdery Peptone-yeast none colorless to light iron agar pale yellow, brown slightly wrinkled Glucose-peptone white, colorless topale brown gelatin stab thin powdery yellow, wrinkled colonies Milk white, colorless, none very thin surface ring powdery growth ______________________________________
(3) Biological and physiological properties:
1. Temperature requirements (on Bennett agar slants)--growth from 15.degree. C. to 40.degree. C. optimum 28.degree. C.
2. Hydrolysis of starch (on starch-inorganic salts agar)--hydrolyzed weakly.
3. Liquefaction of gelatin (on glucose-peptone gelatin stab)--negative.
4. Action on milk--no coagulation, no peptonization.
5. Production of melanin (on tyrosine agar, peptone-yeast iron agar and tryptone-yeast broth)--positive.
6. Utilization of various carbon compounds (on Pridham-Gottlieb basal agar medium)
______________________________________ L-Arabinose - D-Xylose .+-. D-Glucose + D-Fructose + D-Galactose + Sucrose + Glycerin + Inositol + Lactose + L-Rhamnose - Maltose + Raffinose - D-Mannitol + D-Mannose + Salicin - ______________________________________ Symbols: +, good utilization; .+-., doubtful utilization; -, no utilization
7. Cell wall pattern I (LL-diaminopimelic acid).
As a result of looking up the strain possessing the characteristics mentioned above by referring to the literature, namely "Bergey's Manual of Determinative Bacteriology" eighth edition (1975), and "The International Streptomyces Project Reports"written by E. B. Shirling and D. Gottlieb Cf. International Journal of Systematic Bacteriology Vol. 18, pages 69 and 279 (1968), Vol. 19, pages 391 (1969) and Vol. 22, pages 265 (1972), Streptomyces eurythermus and Streptomyces galbus (Okami) have beendetected as species having relatively analogous characteristics to those of the strain ATCC 31427.
The strain ATCC 31427, however, is different from these analogous species in the following:
Streptomyces eurythermus (Okami):
A strain of the species can assimilate arabinose and can not assimilate inositol. Assimilation of rhamnose and raffinose by a strain of the species is indefinite. Loops are not formed. Straights or flexous mycelium are sometimes observed.
Streptomyces galbus:
Open-spirals are generally formed. A strain of the species produces a soluble yellow - yellow green pigment, and can not assimilate surcrose.
In view of the result of the observation, the strain ATCC 31427 can be judged to be a new species belonging to the genus Streptomyces and this has been designated as Streptomyces olivaceogriseus nov. sp. C-353.
II. Re. Streptomyces violaceus No. 6724
Streptomyces violaceus No. 6724 was isolated from a soil sample collected at Ishigaki island, at Okinawa Prefecture, Japan, and deposited with and added to a permanent stock culture collection of the American Type Culture Collection under thenumber ATCC 31481.
Streptomyces violaceus No. 6724 has following morphological, cultural and physiological characteristics.
(1) Morphological characteristics
Microscopic observations were made on cultures which were grown on sucrose-nitrate agar, glycerin-asparagine agar, starch-inorganic salts agar, yeast-malt asparagine agar, starch-inorganic salts agar, yeast-malt extract agar and oatmeal agar at30.degree. C. for 10 to 14 days.
1. Type of branching of spore-forming hyphae: Monopodial branching.
2. Form of spore-forming hyphae: Spirales.
3. Numbers of spores: 10-50.
4. Surface appearance and size of spore: Spiny, 0.3-0.7.times.0.6-1.1.mu..
5. Existence of zoospore and sporangium: Not observed.
6. Formation of spores: At aerial mycelium.
7. Fragmentation of substrate mycelium: Not observed.
(2) Cultural characteristics
The following observations were made on cultures which were grown on various madia at 30.degree. C. for 10 to 14 days.
______________________________________ Aerial mass Reverse side Soluble Medium color of colony Pigment ______________________________________ Sucrose-nitrate purplish white, small colonies purple agar powdery Glucose-aspara- purplishwhite, yellowish red, pink gine agar powdery small colonies Glycerin-aspara- pinkish white- Yellowish red, pink- gine agar pink, powdery small colonies, red slightly wrinkled Starch-inorganic whitish red, yellowish red, red salts agarshort cottony slightly wrinkled Tyrosine agar none red, wrinkled none or colonies trace Nutrient agar none or very colorless-purple reddish thin powdery flat purple Yeast-malt pink-purplish reddish brown- reddish extract agar pink,purplish brown, purple short cottony wrinkled colonies Oatmeal agar pink-purplish colorless- pink- pink, powdery purplish pink, purple small colonies Peptone-yeast none colorless, brown iron agar wrinkled colonies Glucose-peptone white,thin red, wrinkled faint gelatin stab powdery colonies brown Milk thin powdery red growth on reddish surface yellow ______________________________________
Reverse mycelium pigment is pH indicator, changing from red to violet (purple) with addition of 0.05 N NaOH or from violet to red (pink) with addition of 0.05 N HCl. Soluble pigment is also pH sensitive, showing the same changes noted forreverse mycelium pigment.
(3) Biological and physiological properties
1. Temperature requirements (on Bennett agar slants)--growth from 15.degree. C. to 40.degree. C. (optimum 28.degree. C.).
2. Liquefaction of gelatin (on glucose-peptone gelatin stab)--negative.
3. Hydrolysis of starch (on starch-inorganic salts agar)--positive.
4. Action on milk--coagulation, no peptonization.
5. Production of melanoid pigment (on tyrosine agar, peptone-yeast iron agar and tryptone-yeast extract both)--positive, very weak or not on tyrosine agar.
6. Utilization of various carbon compounds (on Pridham-Gottlieb basal agar medium)
______________________________________ L-Arabinose + D-Xylose + L-Rhamnose + D-Glucose + D-Fructose + D-Mannose + D-Galactose + Sucrose + Lactose + Maltose + Raffinopse + Inulin .+-. Cellulose - Chitin - Glycerin + D-Mannitol + Salicin + Inositol + Na-Acetate - Na-Citrate + Na-Succinate + ______________________________________ Symbols: + good utilization .+-. doubtful utilization - no utilization
The FR-900156 substance of this invention is produced when the FR-900156 substance-producing strain belonging to the genus Streptomyces (e.g. Streptomyces olivacegriseus nov. sp.C-353 and Streptomyces violaceus, etc.) is grown in a nutrientmedium containing sources of assimilable carbon and nitrogen under aerobic conditions (e.g. shaking culture, submerged culture, etc.).
The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, fructose, glycerin, starch and the like. Other sources which may be included are lactose, arabinose, xylose, dextrin, molasses and the like.
The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cottonseed meal, soybean meal, corn steep liquor, dried yeast, wheat germ, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammoniumnitrate, ammonium sulphate, ammonium phosphate, etc.), urea, amino acid and the like.
The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form because less pure materials, which contain traces of growth factors and considerable quantities of mineral nutrients, are alsosuitable for use. When desired, there may be added to the medium mineral salts such as calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, magnesium salts, copper slats and the like. If necessary, especially when the culturemedium foams seriously a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.
As in the case of the preferred methods used for the production of other antibiotics in massive amounts, submerged aerobic cultural conditions are preferred for the production of the FR-900156 substance in massive amounts. For the production insmall amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferably to use the vegetative form of the organism for inoculation in the production tanks in order toavoid growth lag in the process of production of the FR-900156 substance. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of theorganism and culture them and then to transfer the cultured vegetative inoculum aseptically to large tanks. The medium, in which the vegative inoculum is produced, is substantially the same as or different from the medium utilized for the production ofthe FR-900156 substance.
Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or bythe passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.
The fermentation is usually conducted at a temperature between about 20.degree. C. and 40.degree. C., preferably 30.degree. C., for a period of about 50 hours to 100 hours.
The FR-900156 substance can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known antibiotics.
In general, most of the FR-900156 substance produced are found in the cultured broth, and accordingly the FR-900156 substance can be separated from the filtrate, which is obtained by filtering of centrifuging the culture broth, by a conventionalmethod such as concentration under reduced pressure, lyophilization, pH adjustment, treatment with a resin (e.g. anion or cation exchange resin, non-ionic absorption resin, etc.), treatment with an adsorbent (e.g. activated charcoal, silicic acid, silicagel, cellulose, alumina, etc.), crystallization, recrystallization and the like.
The FR-900156 substance thus produced in the culture broth can be isolated in the free form, i.e., FR-900156 substance per se and when the solution or its concentrate containing the FR-900156 substance is treated with a base, i.e. with aninorganic base such as an alkali metal compound (e.g. sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, etc.), an alkaline earth metal compound (e.g. calcium hydroxide, magnesium hydroxide, etc.), ammonia and the like, with anorganic base (e.g. ethanolamine, triethylamine, dicyclohexylamine, etc.); or with an acid i.e. with an inorganic acid (e.g. hydrochloric acid, sulfuric acid, phosphoric acid, etc.); or with an organic acid (e.g. formic acid, acetic acid,p-toluenesulfonic acid, citric acid, tartaric acid etc.) during operation of the processes, e.g. extraction, isolation or purification processes, the FR-900156 substance may be transformed into and isolated in the form of the corresponding salts thereof. Alternatively, thus prepared salts of the FR-900156 substance can easily be converted to the free form, i.e. FR-900156 substance per se in a conventional manner.
Further, the FR-900156 substance obtained in the free form may be converted to the corresponding salts thereof with a base or an acid as mentioned above in a conventional manner.
Accordingly, it is to be understood that this invention includes within the scope thereof the FR-900156 substance as well as salts thereof as mentioned above.
The FR-900156 substance possesses the following physical and chemical properties (The following data are those of the product obtained by Example for fermentation (3)):
(1) Form and color: White powder.
(2) Nature of substance: Amphoteric.
(3) Color reaction: Positive; each reaction with ninhydrin, potassium permanganate and sulfuric acid. Negative; Dragendorff reaction and Ehrlich reaction.
(4) Solubility: Soluble; water. Sparingly soluble; methanol. Insoluble; ethanol, acetone, ethyl acetate, benzene, hexane, chloroform.
(5) MP; 143-148 (dec.).
(6) Specific rotation [.alpha.].sub.D.sup.25 =-27.1 (c=0.4 in water).
(7) Ultraviolet absorption spectrum: end absorption.
(8) Infrared absorption spectrum (KBr): 1050, 1130, 1235, 1340, 1400, 1450, 1535, 1660, 1735, 2950, 2980, 3080, 3350 cm.sup.-1.
(9) Elementary analysis: Qualitative analysis revealed that the FR-900156 substance comprises the following elements: Carbon, Hydrogen, Nitrogen and Oxygen.
(10) Thin layer chromatography:
______________________________________ Developing Stationary phase Solvent Rf Value ______________________________________ Eastman cellulose sheeet*.sup.1 BuOH, Acetic acid 0.35 Water(4:1:2) Silicagel sheet Merck*.sup.2 60%i-propanol-water 0.65 ______________________________________ Note *.sup.1 Trade name, made by Eastman Kodak Co. *.sup.2 Trade name, made by Merck & Co.
(11) Molecular weight: Mass spectrometory (Field desorption method): M=519 (base peak: M+1=520).
(12) Nuclear magnetic resonance absorption spectrum: As shown in the Figure of accompanying drawing. (Solvent: D.sub.2 O, Reference: TMSP).
(13) Amino acid analysis:
______________________________________ Molar ratio ______________________________________ Glycine 1.00 Glutamic acid 1.06 Alanine 1.04 .alpha., .epsilon.-Diaminopimelic acid 1.03 (Molar ratio is expressed as assuming Glycine = 1.00) ______________________________________
(14) Molecular formula: C.sub.20 H.sub.33 N.sub.5 O.sub.11.
Further, physical and chemical properties of more purified sample of FR-900156 substance (i.e. a product obtained by Example for fermentation (4)) was measured, as a result of which MP of said sample is 147.degree.-153.degree. C. (dec.) andothers properties thereof were the same as the above.
From analysis of the above physicochemical properties and the further investigation for elucidation of chemical structure, the chemical structure of the FR-900156 substance has been elucidated as follows: ##STR20##
Preparation of Starting Compounds (II) and (III)
The starting Compounds (II) and (III) each include new compound and can be prepared by methods as described below. Among the Compound (III), a new compound is represented by the following formula: ##STR21## wherein R.sup.1, R.sub.a.sup.2,R.sub.b.sup.2, R.sup.q and R.sup.r are each as defined above, provided that when R.sup.1 is hydrogen or t-butoxycarbonyl and R.sub.b.sup.2 is hydrogen, the R.sub.a.sup.2 is mono- or di-carboxy lower alkyl having 1 and 3 to 6 carbon atoms,.alpha.-carboxyethyl or ar(carboxy)-lower alkyl whose aryl moiety may be substituted by hydroxy.
Detailed explanation of preparation of the starting Compounds (II) and (III) are made in the following. ##STR22##
In the above formula,
R.sub.2.sup.r is an amino protective group,
R.sub.a.sup.2' is mono or di-protected carboxy-lower alkyl or ar(protected carboxy)lower alkyl whose aryl moiety may be substituted by hydroxy,
R.sub.3.sup.q is carboxy or protected carboxy excepting protected carbazoyl, and
R.sub.c.sup.1, R.sub.b.sup.1, R.sup.p, R.sub.1.sup.r, R.sub.1.sup.q, R.sub.b.sup.2, Y, m and n are each ad defined above.
Particulars of the definitions for R.sub.2.sup.r, and R.sub.a.sup.2' and preferred example thereof are the same as those for R.sub.1.sup.r and R.sub.a.sup.2, respectively.
(1) Process 1.sup.s : Compound (V-I)+Compound (IV).fwdarw.Compound (III-1)
This process relates to a method for preparing Compound (III-1) by reacting Compound (V-1) or its salt with a Compound (IV) or its salt.
The starting Compound (V-1) includes a known one described in e.g. Biochemistry, vol. 9, pages 823-831, 1971, and new one which can be prepared according to the method as described in said literature.
This reaction is carried out in substantially the same manner as that of Process 1 as explained hereinabove.
(2) Process 2.sup.s : Compound (III-1).fwdarw.Compound (III-2)
This process relates to a method for preparing Compound (III-2) by subjecting Compound (III-1) to elimination reaction of both of amino protective groups for R.sub.1.sup.r and R.sub.2.sup.r.
This process is applied to case that both amino protective groups for R.sub.1.sup.r and R.sub.2.sup.r are ones which can be removed by a method to be employed in this process.
This reaction is carried out by conventional methods such as catalytic reduction method, liquid ammoniaalkalimetal method, acid method, zinc-acid method, base method, hydrazine method and the like. Among these methods, preferred one is selectedaccording to kind of the amino protective groups for R.sub.1.sup.r and R.sub.2.sup.r of Compound (III-1).
This reaction is carried out in substantially the same manner as that of Process 2 as explained hereinabove.
(3) Process 3.sup.s : Compound (III-1).fwdarw.Compound (III-3)
This process relates to a method for preparing Compound (III-3) or its salt by removing selectively an amino protective group for R.sub.2.sup.r of Compound (III-1) or its salt.
This process is applied in case that the amino protective group for R.sub.2.sup.r reveals a different chemical property from that for R.sub.1.sup.r against each kind of the removal methods as mentioned in the foregoing Process 2 and canselectively be removable by a method to be employed.
This reaction is carried out by conventional methods as mentioned in explanation for Process 2. Among these methods, preferred one is selected according to kinds of the amino protective group for R.sub.2.sup.r. Each of such a method and thekinds of such amino protective group are to be referred to explanations in Process 2.
(4) Process 4.sup.s : Compound (III-1).fwdarw.Compound (III-4)
This process relates to a method for preparing Compound (III-4) or its salt by removing selectively an amino protective group for R.sub.1.sup.r of Compound (III-1) or its salt.
This process is applied to case that the amino protective group for R.sub.1.sup.r and the amino protective group of the protected carbazoyl for R.sub.1.sup.q reveals mutually the same chemical property and they reveal a different chemicalproperty from that for R.sub.2.sup.r against each kind of the removal methods as mentioned in the foregoing Process 2 and they can selectively be removable by a method to be employed.
(5) Process 5.sup.s : Compound (III-4).fwdarw.Compound (III-5)
This process relates to a method for preparing Compound (III-5) or its salt by removing an amino protective group for R.sub.2.sup.r or its salt.
This reaction is carried out in substantially the same manner as that of Process 3.sup.s as explained hereinabove.
(6) Process 6.sup.s : Compound (III-5).fwdarw.Compound (III-6)
This process relates to a method for preparing Compound (III-6) by reacting Compound (III-5) with an amino protective agent.
The amino protecting agent includes organic carboxylic, sulfonic, sulfinic, phosphoric and carbonic acids, aldehyde and ketone compounds which are composed of the same protective group as explained for the amino protective group for R.sup.r,R.sub.1.sup.r and Y and their reactive derivatives.
The reactive derivatives include conventional ones such as halides, activated amides, activated esters, acid azide and the like.
When a free acid form of such an amino protecting agent is employed, the reaction is preferably carried out in the presence of a condensing agent conventionally used in the field of amino acid and peptide chemistry.
This reaction is carried out in water or an aqueous organic solvent in the presence of a metal compound such as cupric chloride, cupric sulfate cupric carbonate, cupric acetate or the like.
(7) Process 7.sup.s : Compound (III-5).fwdarw.Compound (III-7)
This process relates to a method for preparing Compound (III-7) by reacting Compound (III-5) with an amino protecting agent.
The amino protecting agent to be used in this reaction is the same as one illustrated in Process 6.sup.s.
This reaction is carried out in a conventional solvent, preferably within the range of pH 6-9, more preferably at pH of around neutrality under ice-cooling to at ambient temperature.
(8) Process 8.sup.s : Compound (III-6).fwdarw.Compound (III-10)
This process relates to a method for preparing Compound (III-10) by reacting Compound (III-6) with an amino protecting agent.
The amino protecting agent is the same as that of Process 6.sup.s and the reaction condition is the same as those of Process 3 as explained hereinabove.
(9) Process 9.sup.s : Compound (VI).fwdarw.Compound (VI-1)
This process relates to a method for preparing Compound (VI-1) by reacting Compound (VI) with an amino protecting agent.
In this reaction, it is necessary that such an amino protecting group to be introduced into one of two amino groups of the Compound (VI) be different from the amino protecting group for Y in case of obtaining stereospecific compound.
The amino protecting agent to be used is the same as the one illustrated in Process 6.sup.s.
This reaction is carried out in substantially the same manner as that of Process 6.sup.s.
(10) Process 10.sup.s : Compound (VI).fwdarw.Compound (VI-2)
This process relates to a method for preparing Compound (VI-2) by reacting Compound (VI) with an amino protecting agent.
In this reaction, such an amino protective group to be introduced into the amino group may be the same as or different from that for Y.
Preferred examples of an amino protecting agent are the same as that of Process 6.sup.s.
This reaction is carried out in water or an aqueous organic solvent and preferably within the range of pH 6-9, more preferably at pH of around neutrality under ice-cooling to at ambient temperature.
(11) Process 11.sup.s : Compound (VI-1).fwdarw.Compound (VI-3)
This process relates to a method for preparing Compound (VI-3) by reacting Compound (VI-1) with an amino protective agent.
In this reaction, it is necessary that such an amino protective group to be introduced into the amino group is different from that for R.sub.2.sup.r of Compound (VI-1) in case of obtaining sterospecific compound, but may be the same as that forY.
This reaction is preferably carried out at neutral-alkaline condition, and other reaction conditions are the same as that of Process 3 as explained hereinabove.
(12) Process 12.sup.s : Compound (VI-2).fwdarw.Compound (VI-3)
This process relates to a method for preparing Compound (VI-3) by reacting Compound (VI-2) with an amino protecting agent.
In this reaction, it is necessary that such an amino protective group to be introduced into the amino group (VI-3) is different from that for R.sub.1.sup.r and Y of Compound (VI-2) in case of obtaining sterospecific compound.
Reaction conditions are the same as those of Process 11.sup.s.
(13) Process 13.sup.s : Compound (VI-3) .fwdarw.Compound (III-8)
This process relates to a method for preparing Compound (III-8) by reacting Compound (VI-3) with a compound of the formula (IV): ##STR23## or its salt.
This reaction is carried out in substantially the same manner as that of Process 1 as explained hereinabove.
(14) Process 14.sup.s : Compound (VI-3).fwdarw.Compound (VI-4)
This process relates to a method for preparing Compound (VI-4) by removing selectively an amino protective group for Y of Compound (VI-3).
This process is applied to case that the amino protective group for Y reveals a different chemical property from that for R.sub.1.sup.r and R.sub.2.sup.r against each kind of the removal methods as mentioned in the foregoing Process 2 and canselectively be removed by a method to be employed.
This reaction is carried out in substantially the same manner as that of Process 2 as explained hereinabove.
(15) Process 15.sup.s : Compound (VI-4).fwdarw.Compound (III-11)
This process relates to a method for preparing Compound (III-11) by reacting Compound (VI-4) or its salt with a compound of the formula: ##STR24## or its salt.
This reaction is carried out in substantially the same manner as that of Process 1.sup.s.
(16) Process 16.sup.s : Compound (III-11).fwdarw.Compound (III-5)
This process relates to a method of preparing Compound (III-5) by removing both of amino protective groups for R.sub.1.sup.r and R.sub.2.sup.r of Compound (III-11).
This reaction is carried out in substantially the same manner as that of Process 2.sup.s.
(17) Process 17.sup.s : Compound (VI-5).fwdarw.Compound (VI-6)
This process relates to a method for preparing Compound (VI-6) by reacting Compound (VI-5) with an esterifying agent.
An esterifying agent to be used in this reaction may include a conventional one such as an alcohol (e.g. methanol, ethanol, propanol, benzylalcohol, etc.) or its reactive equivalent (e.g. halide, sulfonate, sulfate, diazo compound etc.) and thelike.
This reaction is usually carried out in the presence of a base as illustrated in the aforementioned Process 2, in a conventional solvent.
(18) Process 18.sup.s : Compound (VI-6).fwdarw.Compound (VI-4)
This process relates to a method for preparing Compound (VI-4) by removing selectively an amino protective group for Y of Compound (VI-6).
This reaction is carried out in substantially the same manner as that of Process 14.sup.s.
(19) Process 19.sup.s : Compound (III-12).fwdarw.Compound (III-13)
This process relates to a method for preparing Compound (III-13) by reacting Compound (III-12) with an esterifying agent.
An esterifying agent to be used in this process is the same as that of Process 17.sup.s.
This reaction is carried out in substantially the same manner as that of Process 17.sup.s.
(20) Process 20.sup.s : Compound (III-13).fwdarw.Compound (III-5)
This process relates to a method for preparing Compound (III-5) by removing both of amino protective groups for R.sub.2.sup.r and R.sub.1.sup.r of Compound (III-13).
This reaction is carried out in substantially the same manner as that of Process 16.sup.s.
(21) Process 21.sup.s : Compound (V-2).fwdarw.Compound (VI-7)
This process relates to a method for preparing Compound (VI-7) by reacting Compound (V-2) with a halogenating agent.
A halogenating agent to be used in this reaction is to be such a halogenating agent as being capable of halogenating a carboxy group to provide the corresponding acid halide and may include a conventional one such as phosphorus trihalide (e.g.phosphorus trichloride, etc.), phosphorus pentahalide (e.g. phosphorus pentachloride, etc.), thionyl halide (e.g. thionyl chloride, etc.), phosgene and the like.
In this reaction, the followings are to be noted: That is, in case that a compound (V-2), wherein both of R.sup.r and R.sup.r are a carbonic acyl group (e.g. alkoxycarbonyl, etc.), is employed as a starting compound, all of the halogenating agentas exemplified above can be used, and the reaction is conducted by reacting Compound (V-2) with the halogenating agent under warming or heating. On the other hand, in case that a compound (V-2) wherein both of R.sup.r and R.sup.r are hydrogen, isemployed as a starting compound, phosgene only is used as a halogenating agent, and the reaction is carried out by reacting Compound (V-2) with phosgene under cooling to warming.
(22) Process 22.sup.s : Compound (VI-7).fwdarw.Compound (VI-8)
This process relates to a method for preparing Compound (VI-8) by reacting Compound (VI-7) or its salt with a compound of the formula: H.sub.2 NNHY or its salt.
This reaction is preferably carried out in the presence of a weak acid such as acetic acid, oxalic acid or the like. This reaction is carried out in a conventional solvent under ice-cooling to at ambient temperature.
(23) Process 23.sup.s : Compound (VI-8).fwdarw.Compound (VI-9)
This process relates to a method for preparing Compound (VI-9) by reacting Compound (VI-8) with a conventional leucineaminopeptidase.
This reaction is preferably carried out in a conventional solvent, e.g. water, a hydrophilic solvent such as alcohol (e.g. methanol, ethanol, etc.),acetone or the like or a mixture thereof within the range of pH 7 to 10, preferably pH 8 to 9, at20.degree. to 40.degree. C., preferably 37.degree. C.
(24) Process 24.sup.s : Compound (VI-9).fwdarw.Compound (VI-10)
This process relates to a method for preparing Compound (VI-10) with an amino protecting agent.
An amino protecting agent to be used are the same as that of Process 6.sup.s.
This reaction is carried out in substantially the same manner as that of Process 3 as explained hereinabove.
(25) Process 25.sup.s : Compound (VI-10).fwdarw.Compound (VI-11)
This process relates to a method for preparing Compound (VI-11) by reacting Compound (VI-10) with a halogenating agent.
A halogenating agent is the same as that of Process 21.sup.s.
This reaction is carried out in substantially the same manner as that of Process 21.sup.s. In this respect, it is to be noted that, in case that a compound (VI-10), wherein R.sub.2.sup.r is a carbonic acyl (e.g. alkoxycarbonyl), is employed as astarting compound, all of the halogenating agent as exemplified in the explanation for Process 21.sup.s can be explained.
(26) Process 26.sup.s : Compound (VI-11).fwdarw.Compound (VI-12)
This process relates to a method for preparing compound (VI-12) by subjecting compound (VI-11) to hydrolysis.
This reaction is carried out in substantially the same manner as that of Process 4-2 as explained hereinabove.
(27) Process 27.sup.s : Compound (III-9).fwdarw.Compound (III-9')
This process relates to a method for preparing compound (III-9') by reacting compound (III-9) with an esterifying agent.
An esterifying agent to be used is the same as that of Process 17.sup.s.
This reaction is carried out in substantially the same manner as that of Process 17.sup.s.
(28) Process 28.sup.s : Compound (III-3).fwdarw.Compound (III-14)
This process relates to a method for preparing compound (III-14) by reacting compound (III-3) with an amino protecting agent.
An amino protecting agent to be used is the same as that of Process 6.sup.s.
This reaction is carried out in substantially the same manner as that of Process 8.sup.s.
(29) Process 29.sup.s : Compound (III-14).fwdarw.Compound (III-4)
This process relates to a method for preparing compound (III-4) by removing selectively an amino protective group for R.sub.1.sup.r.
This reaction is carried out in substantially the smae manner as that of Process 4.sup.s.
(30) Process 30.sup.s : Compound (III-4).fwdarw.Compound (III-15)
This process relates to a method for preparing Compound (III-15) by reacting compound (III-4) with an amino protecting agent.
An amino protecting agent to be used is the same as that of Process 6.sup.s.
This reaction is carried out in substantially the same manner as that of Process 8.sup.s.
(31) Process 31.sup.s : Compound (III-15).fwdarw.Compound (III-6)
This process relates to a method for preparing compound (III-6) by removing selectively an amino protective group for R.sub.2.sup.r.
This reaction is carried out in substantially the same manner as that of Process 3.sup.s.
(32) Process 32.sup.s : Compound (VI-11).fwdarw.Compound (III-16)
This process relates to a method for preparing compound (III-16) by reacting compound (VI-11) with a Compound of the formula: ##STR25## or its salt.
This reaction is carried out in substantially the same manner as that of Process 22.sup.s.
(33) Process 33.sup.s : ##STR26##
This process relates to a method for preparing Compound (II) by reacting Compound (II-1) with an acylating agent.
The starting Compound (II-1) includes known one (e.g. Journal of Med. Chemistry, vol. 9, 971 (1966) and new one, and said new compound can be prepared according to the method described in such literature.
The acylating agent to be used in this reaction is the same as illustrated in the aformentioned Process 3.
The acylation is carried out in substantially the same manner as that of Process 3 as explained hereinabove.
(34) Process 34.sup.s : Compound (II-2)+Compound (II-3) .fwdarw.Compound (II)
This process relates to a method for preparing Compound (II) or its salt by reacting Compound (II-2) or its salt with Compound (II-3) or its salt.
This reaction is carried out in substantially the same manner as that of Process 1 as explained hereinabove.
(35) Process 35.sup.s : Compound (II-4)+Compound (II-5) .fwdarw.Compound (II)
This process relates to a method for preparing Compound (II) or its salt by reacting Compound (II-4) or its salt with Compound (II-5) or its salt.
This reaction is carried out in substantially the same manner as that of Process 1 as explained hereinabove.
As to the object Compound (I) and starting Compounds (II) and (III) which are prepared according to the aforementioned Processes, it is to be noted that each of said compounds includes one or more stereoisomers which is due to the asymmetriccarbon atoms in their molecule and all of such isomers are included within the scope of this invention.
The new peptide (I) and its pharmaceutically acceptable salts of this invention have been found to possess enhancing activities of immune response (i.e., enhancing activities of cellular immunity and humoral antibody production) andreticuloendotherial system, mitogenic activity; inducing activity of interferon, protective efficacy in experimental infection and anticancer activity.
Accordingly, the new peptide (I) and its pharmaceutically acceptable salts are useful for the therapeutic treatment of infectious diseases caused by pathogenic microorganism, especially gram-negative bacteria and gram-positive bacteria and funge,and of cancer in animals.
Further, Compounds (II) and (III) are useful as intermediate for preparing Compound (I) having biologically active properties as mentioned above.
For the purpose of showing pharmaceutical utility of the new peptide (I), pharmacological test data thereof are illustrated in the following.
1. BLOOD STREAM CLEARANCE OF CARBON
Reagents
1. Carbon suspension. Rotoring drawing ink (170 mg carbon/ml.) was diluted to 1/5 of the original concentration in saline containing 1% gelatin.
2. 0.1% aqueous sodium carbonate solution.
Procedure
Mice (DDY male 5-6 W) were injected via the tail vein with a dose of 0.01 ml/g body weight of carbon. Blood samples were taken by means of a pointed capillary pippet calibrated to hold a 50 .mu.l and previously washed in heparin. This wasplunged into the retrooribital venous sinus at the nasal angle of the eye. The samples were removed at 3 and to 6 min.
The blood was immediately discharged into 3.0 ml. of the sodium carbonate solution. This hemolyszed the blood and allowed the quantitation of carbon. The samples were then read in a spectrophotometer at 660 nm, the log concentration beingobtained from a previously determined standard curve. The clearance value K may be determined by plotting log carbon concentration against time according to the following relationship; ##EQU1## in which T.sub.1 and T.sub.2 represent the time in min whenthe sample were withdrawn and C.sub.1 and C.sub.2 represent the concentrations of carbon in the blood at the time T.sub.1 and T.sub.2, respectively.
EXAMINATION OF EFFECT ON THE NEW PEPTIDE ON CARBON CLEARANCE
The aqueous solution of the test compound as given was administered subcutaneously to mice. Twenty four hours later, blood stream clearance of carbon was measured. K value obtained with treated mice was compared with that of control mice. Thetest results are shown in Table 1 to 3. ##STR27##
TABLE 1 __________________________________________________________________________ Reference Dose compound R.sup.1 R.sub.b.sup.1 n R.sub.a.sup.2 *1 *2 *3 *4 kg)(mg/ ##STR28## __________________________________________________________________________ ##STR29## CH.sub.3 1 ##STR30## L D L D 1 0.1 1.3 1.0 1.3 0.9 " " " ##STR31## " " " " 1 0.1 1.6 1.1 1.3 0.9 " " " ##STR32## " " " " 1 0.1 2.8 2.6 2.3 1.2 H -- 0 CH.sub.2COOH -- " " " 100 1.8 -- CH.sub.3 CO " " " " " " " 1 1.5 2.4 1 1.2 1.0 ##STR33## " " " " " " " 0.1 1 0.1 0.8 1.8 1.9 0.8 1.8 1.9 ##STR34## " " " " " " " 100 1 1.7 1.5 -- 1.8 H " " ##STR35## " " " " 100 1 2.7 1.1 -- 1.8 ##STR36## " " " " "" " 1 0.1 1.6 1.7 1.8 1.7 CH.sub.3 (CH.sub.2).sub.5 CO " " " L D L D 1 1.9 1.8 0.1 2.5 1.7 CH.sub.3 (CH.sub.2).sub.10 CO " " " " " " " 1 1.7 1.8 0.1 2.1 1.9 CH.sub.3 (CH.sub.2).sub.16 CO CH.sub.3 1 CH.sub.2 COOH " " " " 1 1.5 1.3 0.1 1.2-- ##STR37## " 2 " " " " " 1 0.1 1.3 1.5 1.3 0.9 CH.sub.3 CO " " " " " " " 100 4.2 -- 1 4.7 4.5 ##STR38## H 1 " " " " " 100 1 2.0 0.9 -- 2.0 " CH.sub.2 OH " " " " " " 100 2.3 -- 1 1.1 2.0 ##STR39## " " " " " " 100 1 2.3 1.7 -- 2.0 "##STR40## " " " " " " 100 1 2.9 1.6 -- 2.3 ##STR41## CH.sub.3 " " " " " " 1 0.1 1.4 1.8 1.8 1.9 ##STR42## " " " " " " " 1 0.1 2.0 1.6 1.8 1.2 ##STR43## " " " " " " " 100 1 2.2 1.5 -- 1.8 ##STR44## " " " " " " " 1 0.1 1.2 1.6 1.8 1.7 ##STR45## " " " " " " 1 0.1 2.0 1.3 1.8 1.9 ##STR46## " " ##STR47## " " " " 1 0.1 1.8 1.5 1.8 1.7 " " " ##STR48## " " " " 1 0.1 1.8 1.2 1.8 1.9 HOCH.sub.2 CO CH.sub.3 1 CH.sub.2 COOH " " " " 100 2.5 -- 1 0.9 2.1 ##STR49## " " " " " " " 10.1 2.3 1.5 2.4 1.4 ##STR50## " " " " " " " 100 1 2.0 1.8 -- 2.0 ##STR51## " " " " " " " 100 1 1.6 1.9 -- 2.2 ##STR52## " " " " " " " 100 1 2.1 1.9 -- 2.2 ##STR53## " " " " " " " 100 1 1.6 2.2 -- 1.7 ##STR54## " " " " " " " 100 1 1.9 0.7 -- 2.0 (CH.sub.3).sub.3 COO " " " " " " " 100 2.9 -- 1 2.3 1.8 ##STR55## " " " " " " " 100 1 2.1 2.0 -- 2.2 CH.sub.3 (CH.sub.2).sub.10 CO " " " " " " " 1 1.7 1.3 0.1 1.6 0.9 ##STR56## " " " " " " " 1 0.1 1.6 1.2 1.3 0.9 ##STR57## " " " " "" " 1 0.1 3.0 1.7 3.6 1.6 ##STR58## " " " " " " " 1 0.1 2.1 1.0 1.6 1.1 H CH.sup.3 1 CH.sub.2 COOH " " " " 100 2.1 -- 1 0.8 2.1 ##STR59## " " " " " " " 100 1 1.0 0.7 -- 1.0 " " " " " " " L 10 4.3 5.0 1 1.2 4.5 " " " " " L " D 100 2.8 -- 1 1.4 3.6 ##STR60## " " " " D " " 100 1 2.6 2.4 -- 2.2 ##STR61## " " " D " " " 1 0.1 2.2 1.9 1.8 1.2 CH.sub.3 CH.sub.2 CO " " " L " " " 100 2.0 -- 1 1.6 2.1 CH.sub.3 CO " " " " " " " 100 2.0 -- 1 1.1 2.2 CH.sub.3 (CH.sub.2).sub.5 CO " " "" " " " 1 2.3 2.0 0.1 1.4 1.1 ##STR62## " " " " " " " 100 1 2.0 2.4 -- 1.7 ##STR63## " " " " " " " 100 1 2.3 2.0 -- 2.6 ##STR64## " " " " " " " 1 0.1 2.5 1.3 2.0 1.1 ##STR65## " " " " " " " 100 1 1.5 1.3 -- 1.4 ##STR66## " " " " " " " 1001 1.5 1.3 -- 1.4 __________________________________________________________________________ Note: ##STR67## - R.sub.b.sup.1, is CH.sub.3, n is 1, R.sub.a.sup.2 is CH.sub.2 COOH, *1 is L, *2 is D, *3 is L and *4 is D.
TABLE ______________________________________ Dose Drug (mg/mouse) K.sub.treated /K.sub.control ______________________________________ Krestin 10 1.1 1 1.0 Levamisol 10 -- (death) 1 0.5 Tuftsin 400 1.4 125 1.0 FR-900156 substance 0.252.9 0.06 1.5 Control (saline) 1.0 ______________________________________ ##STR68##
TABLE 3 ______________________________________ Reference Compound Test Compound Dose FR-900156 *1 *2 *3 *4 *5 (mg/kg) K.sub.treated /K.sub.control substance ______________________________________ D L L L D 100 2.0 -- 1 0.9 1.9 D D L LD 100 1.7 -- 1 1.6 D L D L L 100 2.7 -- 1 0.9 2.5 L L D L D 100 2.6 -- 1 2.4 2.2 D D D L D 1 2.2 1.8 0.1 1.9 1.2 ______________________________________
2. ENHANCING ACTIVITIES OF CELLULAR IMMUNITY AND HUMORAL ANTIBODY PRODUCTION
Guinea pigs (groups of five) were given 0.1 ml of FIA (Freund's Incomplete Adjuvant) emulsion containing 500 .mu.g of ovalbumin in both posterior footpads. Control groups received antigen in FIA only, whereas the test groups received the antigenwith the following test compounds in FIA. The animals were skin-tested on day 14 and bled on day 16.
(1) Test Compound (1) ##STR69##
TABLE 4 __________________________________________________________________________ Humoral hemag- Cellular gluti- immunity immunity *1 nation hemolysin Dose skin reaction titer titer (.mu.g/ (mm diameter, (M .+-. S.E. (M .+-. S.E. R.sup.1 R.sub.b.sup.1 n R.sub.a.sup.2 site) M .+-. S.E.) log.sub.2) *2 log.sub.2) __________________________________________________________________________ *2 H CH.sub.3 1 CH.sub.2 COOH 1 1.8 .+-. 0.7 *3 11.9 .+-. 0.5 *3 5.6 .+-. 0.5 10 8.9.+-. 0.9 *3 12.3 .+-. 0.2 *3 6.6 .+-. 0.3 100 10.8 .+-. 3.3 *3 12.0 .+-. 0.3 *3 6.7 .+-. 0.5 CH.sub.3 CH.sub.2 CO " " " 1 2.0 .+-. 2.0 12.3 .+-. 0.2 *3 7.3 .+-. 0.4 *3 10 8.6 .+-. 1.0 *3 11.4 .+-. 0.5 *3 7.8 .+-. 0.3 *3 100 3.1 .+-. 1.3 *3 9.7 .+-. 0.4 7.2 .+-. 0.3 *3 HOCH.sub.2 CO " " " 1 10.4 .+-. 2.4 *3 11.8 .+-. 0.5 *3 7.4 .+-. 0.6 *3 10 14.6 .+-. 1.1 *3#2 12.4 .+-. 0.5 *3 7.3 .+-. 0.5 *3 100 6.6 .+-. 1.1 *3 10.8 .+-. 0.2 *3 6.0 .+-. 0.2 Control " " " 0 0 9.4 .+-. 0.4 5.4.+-. 0.5 1 6.7 .+-. 0.8 *3 8.2 .+-. 0.1 *3 10 10.2 .+-. 1.6 *3 7.6 .+-. 0.5 *3 ##STR70## " " " 1 8.7 .+-. 1.8 *3 8.9 .+-. 0.2 *3 10 7.7 .+-. 2.4 *3 8.7 .+-. 0.2 *3 Control " " " 0 0 6.8 .+-. 0.3 ##STR71## " " " 1 11.5 .+-. 2.1 *3#1 10.8 .+-.0.3 *3 7.1 .+-. 0.4 *3 10 11.1 .+-. 0.7 *3#2 12.3 .+-. 0.5 *3 8.5 .+-. 0 *3 100 1.1 .+-. 1.1 9.1 .+-. 0.6 4.30 .+-. 6 *3 Control 0 0 9.2 .+-. 0.3 5.9 .+-. 0.1 ##STR72## H " " 1 10.0 .+-. 0.3 *3 5.9 .+-. 0.3 10 9.6 .+-. 0.3 6.9 .+-. 0.5 *3 100 10.0 .+-. 0.4 7.0 .+-. 0.4 *3 ##STR73## " " 1 10.6 .+-. 0.3 *3 6.9 .+-. 0.3 *3 10 9.9 .+-. 1.0 *3 7.0 .+-. 0.4 *3 100 10.5 .+-. 0.4 *3 6.3 .+-. 0.5 *3 " HOCH.sub.2 " " 1 10.1 .+-. 1.1 *3#2 10.5 .+-. 0.2 *3 7.8 .+-. 0.3 *3 10 10.7 .+-.1.3 *3 9.8 .+-. 0.4 7.3 .+-. 0.3 *3 100 5.4 .+-. 2.2 9.6 .+-. 0.4 7.1 .+-. 0.2 *3 Control 0 0.9 .+-. 0.9 8.5 .+-. 0.5 5.5 .+-. 0.3 ##STR74## CH.sub.3 " " 1 5.0 .+-. 2.2 8.5 .+-. 0.2 *3 10 7.1 .+-. 0.9 *3 7.9 .+-. 0.4 *3 ##STR75## " " " 15.4 .+-. 2.2 *3 7.6 .+-. 0.4 *3 10 12.6 .+-. 0.9 *3 8.4 .+-. 0.2 *3 Control 0 0 5.8 .+-. 0.3 CH.sub. 3 (CH.sub.2).sub.5 CO " " " 1 10.0 .+-. 1.2 *3 9.5 .+-. 0.4 *3 10 7.5 .+-. 2.2 *3 9.2 .+-. 0.6 *3 ##STR76## " " " 1 5.5 .+-. 1.4 *3 8.7 .+-.0.5 *3 10 8.5 .+-. 1.0 *3 7.4 .+-. 0.6 *3 Control 0 0 6.6 .+-. 0.6 ##STR77## " " " 1 10.2 .+-. 1.1 *3 8.3 .+-. 0.3 *3 10 6.3 .+-. 1.3 *3 7.7 .+-. 0.3 *3 CH.sub.3 CO " 2 " 1 9.0 .+-. 1.0 *3 7.7 .+-. 0.5 D 10 8.1 .+-. 2.5 *3 8.4 .+-. 0.5 *3 L ##STR78## CH.sub.3 1 " 1 8.0 .+-. 1.1 *3 8.8 .+-. 0.3 *3 10 11.9 .+-. 2.5 *3 8.3 .+-. 0.3 *3 Control 0 0 7.1 .+-. 0.1 ##STR79## " " " 0.1 11.2 .+-. 1.1 *3 10.4 .+-. 0.3 *3 1 7.6 .+-. 2.2 *3 10.2 .+-. 0.3 *3 10 6.4 .+-. 2.5 9.3 .+-. 0.2 *3 ##STR80## " " " 0.1 6.4 .+-. 2.7 9.9 .+-. 0.5 *3 1 10.2 .+-. 2.6 *3 10.6 .+-. 0.3 *3 10 8.6 .+-. 1.8 *3 10.6 .+-. 0.3 *3 ##STR81## " " " 0.1 10.5 .+-. 2.9 *3 9.1 .+-. 0.4 1 9.3 .+-. 0.7 *3 9.1 .+-. 0.4 10 4.2 .+-. 1.3 9.8 .+-. 0.2 Control 00.9 .+-. 0.9 8.5 .+-. 0.4 CH.sub.3 CO -- 0 " 1 6.4 .+-. 2.1 *3 8.8 .+-. 0.3 10 8.7 .+-. 1.0 *3 9.1 .+-. 0.4 ##STR82## CH.sub.3 1 ##STR83## 1 13.7 .+-. 1.6 *3 11.1 .+-. 0.5 10 10.8 .+-. 0.9 *3 10.7 .+-. 0.3 __________________________________________________________________________ (2) Test Compound (2) ##STR84##
TABLE 5 __________________________________________________________________________ Humoral Hemag- Cellular gluti- immunity immunity*1 nation hemolysin Dose skin reaction titer titer Test Compound (.mu.g/ (mm diameter, (M .+-. S.E. (M .+-. S.E. *1 *2 *3 *4 *5 site) M .+-. S.E.) log.sub.2)*2 log.sub.2)*2 __________________________________________________________________________ D L D L D 0 0 9.1 .+-. 0.19 4.5 .+-. 0.45 0.1 8.2 .+-. 2.8*3 9.9 .+-. 0.10*3 6.4 .+-. 0.76 114.5 .+-. 2.1*3 11.5 .+-. 0.61*3 7.7 .+-. 0.64*3 10 11.0 .+-. 1.3*3 12.2 .+-. 0.56*3 7.1 .+-. 0.58 100 4.2 .+-. 1.7*3 10.0 .+-. 1.01 5.9 .+-. 0.89 D L L L D 0 0.9 .+-. 0.9 8.5 .+-. 0.4 -- 1 2.4 .+-. 1.5 10.4 .+-. 0.5*3 -- 10 7.9 .+-. 2.1*3 11.0 .+-. 0*3 -- D L D 0 0 9.2 .+-. 0.3 5.9 .+-. 0.1 DL 1 4.3 .+-. 1.8*3 10.2 .+-. 1.1*3 6.5 .+-. 0.4 10 9.0 .+-. 3.2*3 10.4 .+-. 0.3*3 7.5 .+-. 1.2*3 100 6.4 .+-. 1.7*3 10.9 .+-. 0.2*3 6.7 .+-. 1.2*3 L L D L D 0 0 6.8 .+-. 0.3 -- 1 6.7 .+-.0.8*3 8.2 .+-. 0.1*3 -- 10 10.2 .+-. 1.6*3 7.6 .+-. 0.5*3 -- __________________________________________________________________________ Note: *1 The skin test was performed on the back by intradermal injection of 5 .mu.g of antigen dissolved in 0.1ml of saline. Skin reaction of the test site was measured at 48 hours. *2 Antibody estimation was carried out as follows: Ovalbumincoated sheep red blood cells were prepared by chromium chloride. Antibody titer was expressed as the reciprocal of thehighest dilution of serum evoking threshold hemogglutination and hemolysin. The results were converted to log.sub.2 unit. *3 Significance was calculated by Student's ttest; P < 0.05 #1 2/5 of animal, Central necrosis #2 1/5 of animal, Centralnecrosis
3. MITOGENIC ACTIVITIES FOR MOUSE SPLEEN CELLS
(Materials and Methods)
(1) Animal: Mice used for this experiment were male BALB/C strain, aged 13 weeks (Test 1) or female BALB/C strain, aged 9 weeks (Test 2).
(2) Tissure Culture Medium: The tissue culture medium employed was a complete medium designated Roswell Park Memorial Institute (RRMI)-1640. All media employed contained 100 units/ml of penicillin G and 100 .mu.g/ml of streptomycin sulfate and10% fetal calf serum.
(3) Cell Preparation: Spleens were removed under sterile conditions, and washed with Hanks solution and then teased in the tissue culture medium. The cells were suspended in the tissue culture medium to contain 8.times.10.sup.6 cells/ml.
(4) Culture Conditions: Into each hole of Microtest II tissue culture plate (8.times.12 hole) (maker: Falcon Plastics Co.) were poured 0.1 ml of the above cells suspension and 0.1 ml of the prescribed concentrate of the test compound as describedbelow and then the cultures were incubated in tirplicate at 37.degree. C. in a humidified atmosphere (95% air, 5% CO.sub.2) for 48 hours.
The control culture contained 0.1 ml of the culture medium instead of the medium containing the test compound.
(5) (3H) Thymidine uptake: In all tests, 20 .mu.l of 10 micro-curine (.mu.Ci)/ml of tritiated thymidine (3H-thymidine) was added to each hole for the final 24 hours of culture. After the culture was completed, the resultant cells were filteredwith a filter paper, Whatman (GF83 and washed successively with saline and with 5% trichloracetic acid. The filter paper was dried and placed in a scintillator (toluene 1 l, containing 0.1 g of p-bis(5-phenyloxazoyl)benzene and 4 g of2,5-diphenyloxazoyl), and 3H-thymidine incorporated into DNA was measured.
(6) Stimulation Index ##EQU2##
(7) Test Compound (1) ##STR85##
TABLE 6 __________________________________________________________________________ (1) Test 1 Concen- 3H-thymidine tration uptake net Stimulation R.sup.1 R.sup.2 (.mu.g/ml) cpm:av .+-. S.E. Index __________________________________________________________________________ CH.sub.3 (CH.sub.2).sub.5 CO CH.sub.3 100 2,728 .+-. 73 5.2 10 2,660 .+-. 45 5.0 ##STR86## 1 2,899 .+-. 126 5.5 100 2,174 .+-. 142 4.1 10 1,547 .+-. 79 2.9 1 1,103.+-. 76 2.1 ##STR87## CH.sub.3 100 2,234 .+-. 170 4.2 10 2,046 .+-. 138 3.9 1 1,378 .+-. 112 2.6 ##STR88## CH.sub.3 100 2,312 .+-. 158 4.4 10 2,388 .+-. 97 4.5 1 1,775 .+-. 94 3.4 Control -- -- 528 .+-. 28 1.0 ##STR89## CH.sub.3 100 2,306.+-. 54 5.5 10 1,788 .+-. 314 4.3 1 1,928 .+-. 87 4.6 HOCH.sub.2 CO CH.sub.3 100 2,605 .+-. 117 6.2 10 1,725 .+-. 86 4.1 1 1,120 .+-. 55 2.7 CH.sub.3 CH.sub.2 CO CH.sub.3 100 2,023 .+-. 142 4.9 10 1,474 .+-. 44 3.5 1 732 .+-. 28 1.8 ##STR90## H 100 1,541 .+-. 73 3.7 10 1,119 .+-. 63 2.7 1 937 .+-. 30 2.2 ##STR91## ##STR92## 100 1,675 .+-. 22 4.0 10 1,679 .+-. 98 4.0 1 1,019 2.4 ##STR93## HOCH.sub.2 100 1,541 .+-. 41 3.7 10 1,468 .+-. 12 3.5 1 927 .+-. 33 2.2 ##STR94## CH.sub.3 100 1,635 .+-. 192 3.9 10 1,573 .+-. 69 3.8 1 1,001 .+-. 73 2.4 Control -- -- 417 .+-. 13 1.0 ##STR95## CH.sub.3 100 1,979 .+-. 174 5.0 10 2,343 .+-. 22 5.9 1 1,469 .+-. 224 3.7 ##STR96## CH.sub.3 100 1,537 .+-. 113 3.9 10 1,788 .+-. 30 4.5 1 1,144 .+-. 142 2.9 ##STR97## CH.sub.3 100 1,627 .+-. 38 4.1 10 1,837 .+-. 138 4.6 1 1,338 .+-. 74 3.4 ##STR98## CH.sub.3 100 1,528 .+-. 122 3.8 10 1,225 .+-. 42 3.1 1 866 .+-. 37 2.2 ##STR99## CH.sub.3 100 1,588.+-. 167 4.0 10 1,469 .+-. 87 3.7 1 1,065 .+-. 89 2.7 Control -- 399 .+-. 51 1.0 __________________________________________________________________________
TABLE 7 ______________________________________ Concen- 3H-thymidine Stimu- tration uptake net lation R.sup.1 R.sup.2 (.mu.g/ml) cpm:av .+-. S.E. Index ______________________________________ CH.sub.3 CO CH.sub.3 100 1,920 .+-. 135 4.2 10 1,067 .+-. 63 2.4 1 785 .+-. 33 1.7 ##STR100## CH.sub.3 100 2,772 .+-. 215 6.1 10 2,326 .+-. 140 5.1 1 1,713 .+-. 102 3.8 ##STR101## CH.sub.3 100 2,979 .+-. 199 6.6 10 2,177 .+-. 66 4.8 1 1,477 .+-. 46 3.3 ##STR102## CH.sub.3 1002,186 .+-. 164 4.8 10 1,370 .+-. 58 3.0 1 756 .+-. 42 1.7 Control -- 452 .+-. 30 1.0 ##STR103## CH.sub.3 100 4,227 .+-. 217 4.2 10 3,664 .+-. 122 3.7 1 2,809 .+-. 83 2.8 CH.sub.3 (CH.sub.2).sub.10 CO CH.sub.3 100 4,027 .+-. 87 4.0 103,963 .+-. 198 4.0 1 4,210 .+-. 255 4.2 ##STR104## CH.sub.3 100 3,967 .+-. 348 4.0 10 2,933 .+-. 126 2.9 1 2,134 .+-. 32 2.1 Control -- 1,000 .+-. 94 1.0 ______________________________________
(8) Test Compound (2) ##STR105##
TABLE 8 ______________________________________ Concent- 3H--thymidine Stimu- Test Compound ration uptake net lation *1 *2 *3 *4 *5 Test (.mu.g/ml) cpm: av .+-. S.E. Index ______________________________________ D L D L D 1 100 2,028 .+-.89 4.9 10 1,486 .+-. 120 3.6 1 835 .+-. 64 2.0 0 417 .+-. 13 1.0 2 100 3,666 .+-. 42 4.1 10 3,223 .+-. 402 3.6 1 2,741 .+-. 319 3.0 0 901 .+-. 105 1.0 D L D DL 1 100 1,284 .+-. 131 3.1 10 1,414 .+-. 38 3.4 1 996 .+-. 35 2.4 0 417.+-. 13 1.0 L L D L D 2 100 2,772 .+-. 215 6.1 10 2,326 .+-. 140 5.1 1 1,713 .+-. 102 3.8 0 452 .+-. 30 1.0 D L D L L 1 100 1,271 .+-. 29 3.2 10 1,003 .+-. 50 2.5 1 742 .+-. 61 1.9 0 399 .+-. 51 1.0 ______________________________________
4. PROTECTIVE EFFICACY IN EXPERMENTAL INFECTION IN MICE
(1) In determining the protective efficacy against experimental infections in mice, the test compound was dissolved in and diluted with sterile saline to provide prescribed concentrations of drug.
Male ICR-strain mice, aged 4 weeks were used in groups of ten mice. E. coli 22 was cultivated overnight at 37.degree. C. on trypticase soy agar and then were suspended in a sterile saline to obtain microbial cell concentration of9.0.times.10.sup.7 CFU/ml. Mice were inoculated intraperitoneally with 0.2 ml of the suspension. Each of the test drugs was given intraperitoneally in various doses to a group of ten mice 24 hours before challenge.
Survival percent were found from the number of the surviving animals after four days of injection. Results are shown in Table 9.
(1) Test Compound (1): ##STR106##
TABLE 9 ______________________________________ Survival (%) Dose Dose 10 1 R.sup.1 R.sup.2 mg/kg mg/kg ______________________________________ CH.sub.3 (CH.sub.2).sub.5 CO CH.sub.3 60 70 (CH.sub.3).sub.3 CCO CH.sub.3 70 70 CH.sub.3(CH.sub.2).sub.10 CO CH.sub.3 50 60 CH.sub.3 88.9 100 ##STR107## CH.sub.3 100 88.9 ##STR108## CH.sub.3 100 88.9 ##STR109## CH.sub.3 87.5 75.0 ##STR110## CH.sub.3 30 50 ##STR111## CH.sub.3 20 60 ##STR112## CH.sub.3 50 30 ##STR113## CH.sub.3 70 70 ##STR114## CH.sub.3 50 50 ##STR115## CH.sub.3 50 50 ##STR116## CH.sub.3 80 60 ##STR117## ##STR118## 60 60 ##STR119## CH.sub.3 40 70 HOCH.sub.2 CO CH.sub.3 75 100 CH.sub.3 CH.sub.2 CO CH.sub.3 100 87.5 ##STR120## ##STR121## 87.5 100 ##STR122##HOCH.sub.2 87.5 87.5 ##STR123## H 100 100 ##STR124## CH.sub.3 87.5 62.5 ##STR125## CH.sub.3 100 87.5 ##STR126## CH.sub.3 100 87.5 CH.sub.3 CO CH.sub.3 62.5 25.0 ______________________________________
(2) Test Compound (2) ##STR127##
TABLE 10 __________________________________________________________________________ Survival (%) Dose Dose R.sup.1 R.sub.b.sup.1 n R.sub.a.sup.1 *1 *2 *3 *4 10 mg/kg 1 mg/kg __________________________________________________________________________ CH.sub.3 1 CH.sub.2 COOH L D L D 75.0 50.0 ##STR128## " " " " " " " 87.5 75.0 ##STR129## " " " " " " " 75.0 87.5 ##STR130## " " " " " " " 75.0 87.5 ##STR131## " " " " " " "87.5 62.5 ##STR132## " " ##STR133## " " " " 87.5 87.5 ##STR134## " " ##STR135## " " " " 70.0 70.0 " " " ##STR136## " " " " 90.0 80.0 " " " ##STR137## " " " " 60 60 H -- 0 CH.sub.2 COOH -- " " " 66.6 22.2 CH.sub.3 CO " " " " " " " 100 88.9 ##STR138## " " " " " " " 87.5 75.0 ##STR139## -- 0 CH.sub.2 COOH -- " " " 87.5 50.0 H " " ##STR140## " " " " 75.0 87.5 ##STR141## " " " " " " " 62.5 50.0 CH.sub.3 (CH.sub.2).sub.5 CO " " " " " " " 62.5 62. | | | |
