CA1233816A - Derivatives of morpholinyl daunorubicin and morpholinyl doxorubicin, and analogues thereof - Google Patents

Abstract

Abstract of the Disclosure A group of new daunorubicin and doxorubicin derivatives having antitumor activity of the structure :

wherein R is -CO-CH3 or -CHOH-CH3, -CO-CH2OH or -CHOH-CH2OH;
hydroxy; a 1 to 3 carbon alkyl; a 1 to 3 carbon terminal hydroxyalkyl; 2 to 7 carbon organic acid-based esters and diesters of -CO-CH2OH, -CHOH-CH2OH, and -CHOH-CH3, and 13-ketimine derivatives of -CO-CH3 or -CO-CH2OH;
Y is usually methoxy (-OCH3) but can also be hydrogen, X is =O or -NH; R' and R" together are a hydrogen and a hydroxy (that is, either R' or R" is hydroxy with the other hydrogen, both are hydrogens or R' is methoxy and R" is hydrogen), and A is selected from cyano (-C?N) and hydrogen as set forth above. Z is selected from oxygen sulfur, wherein R''' is a 1 to 3 carbon alkyl, and -CH2- subject to the proviso that when Z is -CH2- or

Description

DERIVATIVES OF MORPHOLINYL DA~NORUBICI~ ~ND
MORPHOLINYL DOXORUBICIN AND ANALOGUES THEREOF.

This is a divisional of Canadian Patent Appli-cation 432,737 filed July 19, 1983.

Description The invention described herein was made in the course of work under Wational Cancer Institute Grant ~o.
CA25711 and CA32250 of the Departmen-t o Health and Human Services of the United States of America.
This invention is in the field of anthracyline chemistry. More particularly it concerns analogues of the anthracyclines doxorubicin and daunorubicin that are useful as anti~umor agentsO
Doxorubicin (adriamycin) described and claimed in US 3,590,028 F.Arcamone et al., is perhaps the most useful new anticancer drug in use at this time It (along with daunorubicin) is a principal agent in the treatment of an unsually wide number of solid tumors and leukemia. Regrett-ably, many patients with these tumors fail to respond and essentially no patients with some serious tumor types ~colon cancer, melanoma) respond. In addition, in some patients chronic treatment produces irreversible heart damage that can be fatal if continued. Thus~ there is great need for ~33~

analogues which ~ e a better rate of response, a wi~er s~e^trum of response, or red~ce~
cardiotoxicity. ~ore ef~ec~ive and less toxic agents are widely sought and are the fundamental object of this invention. The most active new analogues 50 ar, judging from screening results in a widely used test against rnouse leukemia P388 in a 3-dose treatment schedule (q4d 5,9,13), are two lipophilic derivatives (AD32 and N,N-dibenzyldaunorubicin) that required siynifican~ly higher doses, and that fail to interact with D~A in vitro although D~A is believed to be a primary biological target for the anthracycline series. Most N-alkyl derivatives have been active in the antitumor screen against mouse leuXemia P388, but are no~ significantly diferent fro~ doxorubicin or daunorubicin. A few such derivatives have been inactive, Much of the history and prior ar~ o doxoru-bicin and it~ anthracycline analogues is founcl in the ar-ticle "Adriamycin" by David W. Henry, A S Symposium Series, No~ 30, Cancer Chemotherapy, American Ch mical Society, pp. 15-57 ~1976) and in the book Doxorubicin by Federico ~rcamone, Academic Press, 1981. AD32 is disclosed in U.S. Patent No~ 4,035,566, dated July 12, 2S 1977.
5-Iminodaunorubicin is shown in United 5tates Pakent 4,109,076 which issued on August 22, 1978, to David W0 Henry and George 1.. Tong and which is assigned to the assignee of the present invention.
I'he doxorubicin equivalent is shown in '~Synthe$is and Preliminary Antitumor Evaluation of S-Iminodoxoru-bicin", J.~edicinal Chem., 24, 669 (1~81) by Edward M.
Acton ancl George L. Tong. S-Iminodaunorubicin retained activiey with red:ced sid- e'fects while ~ ~ 3 3 ~
1 5-iminodoxorubicin showed enhanced actlvity but required higher dosages.
3'-Deamino-3'-(4-morpholinyl)daunorubicin, disclosed in U.S. Patent No. 4,301,277 issued on Nov. 17, 1981 to Edward M. Acton and Carol W. Mosher and assigned to the assignee of the present invention, was active at one-fortieth the dose of doxorubicin but gave a substantially identical T/C value (166~ vs 160~ against P388). This -ompound and its preparation and properties are also disclosed in "Enhanced Antitumor Properties of 3'-(4-Morpholinyl) and 3l-(4-Methoxy-l-piperidinyl) Derivatives of 3~-Deaminodaunorubicin", J. Medicinal Chem., 25, pp. 18~24 (1982) by Carol W. Mosher, Helen Y.
Wu, Allan N. Fujiwara and Edward M. Acton A general reductive alkylation process for preparing new semi-synthetic anthracycline derivatives is described in "Adriamycin Analogs~ 3~ Synthesis of ~-Alkylated Anthracyclines with Enhanced Efficacy and Reduced Cardiotoxicity", J. Medicinal Chem., 22, pp. 912~
91~ (1979) by G.L. Tong, H. Y. WU9 T. H. Smith and D W.
Henry.
Statement of the Invention A group of new daunorubicin and doxorubicin derivatives has now been found. These compounds are represented by the General Formula I

~, ~33~

O OH

~ OH ~I) ~ N ~ A

wherein R is CO-CH3 or CHOH-CH3 in the case of daunorubicin derivatives or CO-CH2OH or CHOH-CH2OH in the case of doxorubicin derivatives, X is O or NH; and A is either a cyano group (C~) or a hydrogen, subject to the limitation that when X is O, A must be 2. cyano group. When A is hydrogén, these compounds can exist as acid additi~n salts, as well. These salts are an additional aspect o~ this invention.-In another, broader aspect the invent.ion provides derivatives of these compounds which derivatives have been formed by one or more modifications shown in the art to be effective with analoyous daunorubicin and doxorubicin materials.
Such modi~ications involve further chanyes in t.he Rgroup, removal of the 4 position methoxy, chan~es in the 4' carbon substituen-ts, changes in A and.
substitution in the morpholinyl ring. The compounds encompassed by these various derivatizations are .
generally classified as morpholinyl or analogues of morphoLinyl derivatives of daunorubicin and doxorubicin type materials and are represented by the General Formula II

.:

i .

~3~
. ~5_ o OH

O.'l (II) Y X OH O
R ' ~ J

wherein R is ~-CO-CH3 or -CHOH-CH3 in the case o simple daunorubicin deriva~ives, -CO-CH20H or -CHOH-CH20H in the case o~ simple doxorubicin derivatives; hydroxy; a l to 3 carbon alX~l, such as -CH2CH3; a l to 3 carbon terminal hydroxyal'.~yl su~h as .
-CH2-OrI or -CH2-CH2-OH; 2 to 7 carbon organic aeid-based esters and diesters of ~Co-CH20H, -C~OH-CH20H, and -C~OH-CH3 including for example acetate (-OAc), propionate ~-OPr), benzoate (~OBz) and glycolate (-O-Gl) esters such as -CO-CH2-OAc, -CO-CH2-OBz, -CO-CH2-OPr, -CO-CH2-OGl, -C~OAc)-CH2-OAc, -CH(OBz)-CH2-OBz, -CH(OAc)CH3, and -CH(OBz?CH3 or the liXe; a l to 6 earbon alkyl or aryl e-ther replaeement of one or more hydroxyls of -CO-CH20H, -CHOH-C~3, and -CHOH-CH~OH, such as -CH(OCH3)-CH3, -CO-C~20-CH3, CO CH20-C2H5~ -Co-cH2o-c6Hs or the liXe; and 13-ketimine derivatives of -CO-CH3 or -CO-CH~OH sueh as -C(NOH)-CH3, -C~NNHBz)CH3, -C(NOCE13)-CH3, -C(MOH)-CH20H, 20 -C(NOCH3)-CH20H, and -C(NNHB~)-CH20H or the liXe; Y is usually methoxy (-OC-~3~ but can also be hydrogen, X is =O or ~ , R' and R'' togeth~r are a hydro~en ~nd a hydroxy (that is, either R' or R'' is hydroxy with the ot'ner hydrogen), both are hydrogens or R' is methoxy and R'' is hydrogen, and A is selected from cyano (-C~N) and hydrogen as set forth above. When A is hydrogen, these compounds can exist as acid addition salts, as well. Z is selected from oxygen, sulfur, -C~- wherein R''' is a l to 3 carbon alkyl, OR'"
and -CH2- subject to the proviso that when Z is -C~2- or 7 r ~ i s -c ~N .
OR'"
Preparation of the compounds of the ~enera] formula II
. . . _ . .
The preparation of these compounds is charac-terized in that the known daunorubicin and doxorubicin and analogous thereof of structure :

O OH
~;IJ~
Y X OH
d R' ~ -wherein R, R', R " , X and Y have the above meanings, are reacted in a mixed aqueous polar organic medium with 20 a compound of the structure :

, . ... . . .

- 6~ ~
f~

z 2 CHO

or a suitable precursor thereof, and in which Z is selec-ted from oxygen, sulfur, -CH2-, -C~I- , and R''' has the above OR'~
meaning, the reaction being carried out in the presence of a cyanoborohydride salt, such as an alkali metal cyanoboro-hydride, and tha-t the desired compounds are isolated and pwrified in a manner knwon "per se". These compounds are related to t]1e established anticancer drugs daunorubicin and doxorubicin (adriarnycin), are prepared from -them by chemical syn1:hesis and derivati~ation technigues and are active agents against cancer. They appear to combine two advantageous and sought-after properties - high antitumor efficacy and low dose requirements. Thus, they offer the promise of high effectiveness with reduced dose-related side effects such as cardiotoxicity as compared with materials disclosed heretofore.
In other aspects, this invention provides phar~
maceutical preparations con-taining these new derivatives as well as a method for treating mammalian cancer by ad-ministering such preparations to a mammal in need of such treatment.
In another aspect the present invention provides a process for the preparation of a compound having the structure E:

- 6b - ~
~o3~

O OH

~ / R

~ Q I
~CH3 y E
HO ~
` ~ ~ CN
~J
wherein R is selected from -CO-CH3, -CHOH-CH3 -CO-C~I20~
and -CHOlI-CH20H, characterized in that a compound of the structure o OH

¦ OH

~ro~/

~J
HOI
NH2.HCl wherein R represents a -CO--CH3 or a -CO-CII20H group, is xeacted în a mixed aqueous polar organic medium with an excess tl8 molar equivalent) of 2,2'-oxydiacetaldehyde of the structure:

~3~
- 6c ~ CIIO

prepared "in situ" by cleavage of 1,4-anhydroerythritol in aqueous solution with sodium perioda-te at room temperature the reaction being carried out at room temperature in the presence of two molar eqllivalents with respect to the star-ting material of an alkali metal cyanoborohydride such as sodium or potassium c~anoborohydride, and tha-t the raw reaction product, obtained as a mixture of basic morpholino derivatives of the staxting materials together with their 13- dihydro analogues and of the corresponding neutral 3l--cyano-morpholino analogues, is separated into basic and neutral fractions, and the neutral fractions are submitted to a cromatographic separation on a silica gel column using as eluent a CH2C12-CH30H system (9:1 v/v) with an increasing amount of CH30H to obtair. in the f.irst eluate 3''-cyano morpholino derivatives of formula I (R = -CO-CH3 or -CO-CH20H) and in the su~sequent elution the corresponding 3''-cyano-13-dihydroderivatives of formula I (R = -OEIOH-CH3 or -CHOH-CII20H), which are eventually purifi.ed by methods known per se.
In another aspect the presen-t invention provides a process ~or the preparation of a compound having the structure G:

- 6~ - ~
~3~

~ OH

~ I
1~/
HO ~

wherein R is selected from CO-CH3, CHOH-CH3, CO-CH20H and CHOH-CH2011 and A i5 selected from CN and H, characterized in tha- a compound having the structure:
OH

H3CO O OH o O
ï

N~A
o ~3~
- 6e - S

wherein R has -the mcanings cJiven above and after the hydroxy group of the -C0-CH20M group, if present, has been suitably blocked by a mild acid labile protecting group such as a p-methoxy-tri-tyl group to give a 14-ester, which is eventually purified by chromatography on a silica gel column using as eluent system a mixture of CH2C12-CEI30H
(99:1 V/V wi-th an increasing amount of.CH30H till 90:10 v/v), said compoundis reacted at a temperature at from 0C to 3C with an excess of alcoholic ammonia and, upon slitting off, i~ necessary, said mild acid labile protective group by trea-tment with acetic acid or cold trifluoroacet.ic acid. at room temperature, that the de-sired compour,ds are obtained as the relevant free bases which, after a chromatographic purification on a silica gel column, using as eluent system CHC13-CH30H (9:1 v/v), are isolated as such as compounds of structure E wherein A is CN and c;ptionally are transformed by treatment with 0.1 N hydrochloric ac.id into their relevant hydrochlorides being compounds of structure E wherein A is ~I.

D~tailed Description of_the Invention The present invention provides morpholinyl deri-vatives of irninodaunorubicln and iminodoxorubicin and the pharmaceutically acceptable salts thereof as well as cyanomorpholinyl derivatives oE daunorubicin, ~;~3~

doxo~ubicin, iminodaunorubicin and iminodoxorubicin.
These compounds are ~isted in Table I.

Table I
Compounds o the Invention 5 X A R C~.f~
NH H CO-CH3 3'-deamino-3'-(4''-morpho-linyl~-5-iminodaunorubicin an~
~harmaceutically acceptable salts thereof 10 NH H CHOH-CH3 3'-deamino-3'-~4''-morpho-linyl)-13-dihydro-5-imino-daunorubiein and pha~maceutieally acce~table salts thereof 15 NR H CO-CH2OH 3'-deamino-3'-(4''-morpho-.
linyl)-5-iminodoxorubie.in and pharmaceutically ac.ceptable salts thereof NH H CHOH-CH2OH 3'-de~ino-3'-(4''-morpho-. linyl~-13-dihydro-5-imi.~odoxo-rubicin and pharmaceuti,_ally ~ aeeeptable salts thereo~
O CN CO-CH3 3'-deamino-3'-(3''-eyan~-4''-morpholinyl)daunorubiein 25 O CN CHOH-CH3 3'-deamino~ 3''-eyano-4"-morpholinyl)-13-dihydro~
daunorubiein O CN CO-CH2oH 3'-deamino-3'-(3''-eyano-4''-morpholinyl)doxorubicin , Table I
Comp~unds of the Invention (con-inued) X A R Compound N~me o CN CHOH-CH20H 3'-deamino-3'-(3''-cyano-4''-morpholin~ 13-dihydrodoxoru-bicin NH CN CO-CH3 3'-deamino-3'-(3''-cyano-4''-morpholinyl)-5-iminodaunorubi-ein NH C~ CHOH-CH3 3'-deamino-3'-(3''-cyano-4''-morpholinyl)-13-dihydro-5-iminodaunorubicin NH CN CO-CH20H 3'-deamino-3' -(3''-cyano-4''-morpholinyl)-5-iminodoxorubiein NrI CN C~OH-C~2OH 3'-deamino-3'-(3''-e~ano-4''-morpholi~yl)-13-dihydro-5-iminodoxorubiein Five of these materials are preferred because of their exeellent aetivity a~ ant.i-tumor agents. These are 3'-deamino-3'--~3''-eyano-4''-morpholinyl)-.
doxorubiein;
3'-deamino-3'-~3''-eyano-4''-morpholinyl)-13-dihydrodoxorubicin;
3'-deamino~3'-t3''-eyano~4''-morpholinyl)daunorubicin;
3'-deamino-3'-~3''-eyano-4''-morpholihyl~-13-dihydrodaunorubiein: and 3'-deamino~3'-~4''-morpholinyl)-5-iminodoxorubiein.

~3~

The first of these five materials is the most prererred material.
The first four compounds or the invention listed in Table I can be the free bases shown in Table I or ~h~y can be pharmaceuti^ally-acceptable acid addition salts of these bases. The acid addition salts offer the advantage of being soluble in water and aqueous mixed solvents such as water-alkanols or water-alXandiols. Examples of these mixed solvents are water-propylene ~lycol, water-ethanol, water-e~hylene glycol, saline, various other aqueous injec~able media, and the li~e. The free bases are soluble in less polar organic solvents such as ch~oLo~o~m methylene chloride, mixed chloroform-methanol solvents, and the like. They may also beused as suspensions.
The salts are the acid addition products of the free bases with a pharmaceutically acceptable acid. A ~Ipharmaceutically-acceptable~ acid i5 one which is nontoxic and generally employed in pharma~
ceutical products. Examples of these acids are inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acids, and organic acids such as the carboxylic acids, e.g. acetic, glycolic, maleic, malic, hydroxymaleic, tartaric, citric, and salicylic acids and the organosulfonic acids, e.g.
methanesulfonic and p-toluenesulfonic acid. M~xtures of two or more acids may be used as may mixtures of one ox more free base plus one or more acid addition salt. For reasons of simplicity and ready solubility, the hydrochloric acid and hydrobromic acid addition salts are preferred.
As previously noted, these compounds can also be present as derivatives. These derivatives are . .

for,ned so as to increase ~he solubiliLy o~ the co~pounds or 50 as to vary other physical properties of the cornpounds.

Preparation o~ some preferred compounds of the invention .
These compounds can be prepared by the ~ollowing general route-:
First, commercially available daunorubicin or doxorubicin ~as an acid addition salt) is caused to react ~mder reductive alXylation conditions with 2,2'-10 oxydiaceta~ldehyde O=CH CH=O
CH2-0-CH2 This alkylation yields a mixed product containing four principal components.
In the case of daunorubicin these components are;
3'-deamino-3'-(4''-morpholinyl)daunorubicin, 3'-deamino-3'-(4''-morpholinyl3~13-dihydrodaunorubicin, 3'-deamino-3'-(3''-cyano-4''-morpholinyl~daunorubicin, and 3'-deamino-3'-(3''-cyano-4''-morp~olinyl)-13-dihydrodaunorubicin.
In the case of doxorubicin the reaction product contains, 3'-deamino-3'-(4''-morpholinyl)dQxorubicin, 3'-deamino-3'-(4''-morpholinyl)-13-dihydrodoxorubicin, 3'-deamino-3`-~3''-cyano-4''-morpholinyl3do20rubicin, and 3'-deamino-3'~(3''-cyano-4''-morpholinyl)-13-dihydrodoxorubicin.

2,2'-oxydiacetaldehyde c2n be formed by acid hydrolysis of 2,2'-oxydiacetaldehyde bis(dieth~l acetall .

~33.~s~ ~ ~

(Et-0)2-cH CH~(o-Et)2 by the method of Field, et al's Belgian Patent 655,436 or by cleavage of 1,4- anhydroerythritol HO-C~ - 7~-OH
CH2-O~CH2 by the method of Barry, et al, Carbohydrate Research, 7, 299 (1968) and Greenberg, et al, Carbohydrate Research, 35, 195 (197~).
The reductive alkylation can be carried out using an excess of the dialdehyde in a mixed aqueous -polar organic medium, such as water - acetonitrile, generally at a pH of about 7 in the presence of a reducing agent such as an alkali metal cyanoborohydride e.g~ sodium or potassium cyanoborohydride. This is a relatively facile reaction which can usually be completed in an hour or less at room temperature. The reductive alkylation is illustratecl in the Examples and is al30 shown in previously United States Patent 4,301,277 and J. Medicinal Chem., 25, pp. 18-24 ~1982)~
The work-up of the mixed reaction product may be carried out by any method that effect3 the desired isolation and separation. Acid extraction oE the reaction product is effective to separate the ~cid-extractable non-cyano-substituted materials from the acid-insoluble cyano-substitu-ted materials. The resulting paris of materials can then be separated into individual compounds by various chromatography methods such as preparative scale layer chromatography, column chromatography, or preparative scale high performance liquid chromatography.
The 5-imino compounds can be easily and directly prepared from the isolated 5-oxo compounds using the ~2~

1 method disclosed in the above J. Medicinal Chem, 24, pp.669 (1981) article. In this method the 5-oxo materials are contacted with an excess of alcoholic ammonia at low to moderate temperatures such as Erom -25C to ~25C for from about 0.5 to about 100 hours~ In the case of 3'-deamino-3'-(4''-morpholinyl)doxorubicin and 3'-deamino-3'-(3''-cyano-4l~-morpholinyl)doxorubicin, it is necessary to block the hydroxyl on the 14 carbon before the ammonia treatment Any mild acid-labile protecting group can be L0 used. Because of its wide use in pharmaceutical chemistry, methoxy-trityl is a preferred protecting groupO The ~rityl functionality can be introduced by treating 3'-deamino-3'-(4''-morpholinyl)doxorubicin or 3'-deamino-3'-(3 " -cyano-4''-morpholinyl)doxorubicin with excess p-anisyl chlorodiphenylmethane at room temperature or the like. After the reaction with ammonia is complete, the 14-hydroxyl can be regenerated by contact with acid such as acetic acid or cold aqueous trifluoroacetic acid.
Derivatives _nd Analogues In addition, the present inven-ion p~ovides derivatives and analo~ues of the foregoing 12 primary compounds. As shown in General ~ormula II, these derivatives can include one or more of the following modifications of the primary compounds.
a. one or more of any hydroxyls present in R
can be present as esters of 2 to 7 carbon organic acidst including alkanoic acids, oxyalkanoic acids, hydroxyalkanoic acids, and benzoic acid. This modification can give rise to R groups such as shown in Table II.

Table II
Es~er R's Acid Ester R

Acetic acid ~CO-CH2-O-COCH3 -C~1(OCOC~3~ C~2-O-CoC~3 -CH~OCOCH3)-CH2OH
-CH(OCOCH3)-C.~13 Propionic acid -Co-C~2-o-coc2Hs -CH(OCOC2Hs)-cH2-O-(-oc2H5 -C~(OCOC2H5)-cH3 Glycolic acid CO-C~2-O~COC~12OH
-CH(OCOCH2O~)-CH~-O-OCH2OY
. -CH~OCOCH2OH~-CH3 Benzoic acid -ro-CH2 o-COC6H5 -C~Ococ6Hs)-cH2-~coc~Hs -CH(OCOC6H5)-~H3 More complex acids such as HOOC-CH(OC2H5)2 -co-cH2-o-cocH(oc2Hs~2 etc.
, Such esters o~ doxorubicin described [Arcamone, et al, J. Medicinal Chem., l7, 335~1974);
Maral, et al,Belgian Patent 848,219 (May lOt l971)~
can ~e readily conver-ted by -the herein described reduc-tive alXylation method to the corresponding ester derivatives of compounds o~ this invention.
b. One or more of any hydroxyls present in R
CaQ be present as ethers - particularly 1 to 6 carbon alkyl ethers or about 6 or 7 carbon aryl ethers.
Representative "ether" R units are shown in Table III.

Table III
Ether R's Methyl ether -CO-CH2-OCH3 -CH(OH)-cH2~ocH3 Ethyl ether -Co-cH~-oc2H5 --CH ( OH )--CH2--oC2H5 Butyl ether CO CH20C4H9 Phenyl ether -Co-cH2-o-c6Hs -c~(OH)-c~2-oc6H5 Such 14-ethers of doxorubicin have been described [Masi/ et al, Il Farmaco, Ed. Sci., 34, 907 -(1979)] and can be used as starting materials in the reductive alkylation method of this invention.
c. The substikuents o~ the 4' carbon in the "sugar" ring can be modified. The 4'-0-methyl deriva-tives (in the sugar uni~) of doxorubicin and daunoru-bicin are readily obtained ~Cassinelli, J. Medicinal Chem., 2~, 121 (1979)~ and converted to compounds of ~. .
this invention. Other known structural changes at the 4'- position of the sugar unit are the 4'-deoxy ~no OH) and 4'-epi ~OE~ up) derivatives of doxorubicin and daunorubicin (Suarato, et al, Carbohydrate Res~ 98, cl (1981)) which show promising pharmaceutical proper-ties. These compounds are reaaily conver-ted by the xeductive alXylation process to the corresponding compounds o~ this invention.
d. ~he 4-demethoxy analogues o~ doxorubicin and daunorubicin (no CH30 in A-ring of the aglycone) are readily obtained [Arcamone, et al, Cancer Test .
.

~23~.hP~;

Rpts., 60, 829 (1~76); Arcamone, et al, Cerman Patent 2,652,391 (May 26, 1977)] and converted to compounds of this invention.
o e. Carbonyl groups (-C-)in the R units o~
daunorubicin and doxorubicin can be readily converted to (-C-) groups by the common methods for converting ketones to oximes, hydrazones, and c)ther Xetimines.
Table IV lists representative ketimi.ne R's .
Table IV
. Ketlmine R's -C(NOH)-CH2OH
-C(NOH)-C~3 --C~NOC~3 )--CH2~
-C(NOCH3)~CH3 -C(NNHCOC6H5~-CH2OH
-C(~Hcoc6Hs)-c~3 --C ( N~HCONH2 ) CH20H
-C(NNHCONH2)-CH3 and ~he l.iXe.

These 13-ketimine R materials can be readily converted to compounds of this inver..tion by the above described xeductive alkylation. . .
. f. ~he R unit can be simplified to eliminate the carbonyl and give rise to the simple hydroxyl R
units shown in Table V.

~ 33~
~16-Ta~le V
Simplified R's --0~ .
-CH~O~
-C2~OH

These daunorubicin and doxorubicin materials are shown irl Penco et al, German Patent 27 57 057 ~uly 7, 197~ and Penco et al, J._Antibiotics, 30:764 (1977) and care converted to compounds of this invention by reductive alkylation.
g. 2-Cyanopiperidino (Z = CH2~ arld 2-Cyano-4-methoxypiperidino (Z - CHOCH3) (Formula II). Th~
piperidino clerivatives of daunorubicin and doxorubicin have been described in U.S. patents.
' .
~,N~ U.S. 4~02,967 ¦ ~ May 13, 19~0 \/ .

U.S. ~,3l4,054 ~eb. 2, 1982 The correspondiny 2-cyano-1-piperidinyl derivatives can be synthesized by converting the above compounds with meta-chloroperbenzoic acid in dichlorome-thane soluti.on to the ~-oxides, and rearrangement of the N-oxides with trifluoroacetic anhydride in the presence of cyanide ion (Polonovski-Potier-~lusson).

~3~

~N ~ N~CN

Z= -CH2- or CHOCH3 When the reductive alkylation procedure of this invention is carried out on daunorubicin, except that 2,2'-thiobisacetaldehyde O=CHCH2SUCH2CH=O; Carboh~drate Res., 110, ]95(1982)) is used in place of 2~2'--oxybisacetaldehyde, and the pH is weakly acidic (pH 6 instead of pH 7.2), the thiomorpholino derivative (A=~) (Formula I) of daunor~abicin is obtained. This produc~ is as active V mouse leukemia P388 ~T/C = 169~) as doxorubicin (T/C - 160%), although a higher dose is required (50 mg~kg instead of 8 mg/kg). Similarly, the thiomorpholino derivative oE doxorubicin is formed when doxorubicin is used in this reaction.
The neutral product fraction from these reactions, which remains in the organic layer after extraction with aqueous acid to remove the thiomorpholino derivative as the water soluble acid salt, contains the coresponding 3-cyano-1-thio-4-morpholinyl derivatives (A=CN) of daunorubicin and doxorubicin.
This invention will be further shown by the following examples~ These are presented to illustrate the invention and are not to be construe~ as limiting the invention's scope.

~3~ 3 1 Example 1 Preparation, isolation and identi~ication of 3'-deamino-3'-(3''-cyano-4'-morpholinyl~daunorubicin A. Following the method for preparing -3'-deamino-3'-morpholinodaunorubicin hydrobromide shown in Mosher, et al, J. ~edicinal Chem., 25, pp. 18-24 (1982) a crude reaction product containing 3'-deamino-3'-(4''-morpholinyl)daunorubicin, 3'-deamino-3'(4''-morpholinyl)-13-dihydrodaunorubicin, 3'-deamino-3'-(3''-cyano-4''-morpholinyl)daunorubicin, and 3'-deamino-3'-(3''-cyano-4''-morpholinyl)-13-dihydrodaunorubicin was prepared. The crude material was extracted with CHC13 as noted therein to remove the four primary products into the CHC13 phase. Exhaustive extraction of the CHC13 phase ~ith O.OlN HCl removed the two basic morpholino products, as described in the reEerenceO The neutral product-rich CHC13 was washed with NaHC03 solution~ dried and evaporated. Samples were dissolved in 4:1 CHC13.CH30H and placed on a Waters Radial-Pak C-18 high performance liquid ! 20 chromatography column, and eluted with 2 mL/minute of a 65:35 0.05M pH4 citrate buffer~CH3CN eluent. One material (usually 19-24~ of total) eluted at 6.1-6.8 minutes while another material ~usually 26-27~) eluted at 11.9 minutes~ Detection was by UV at 254 nm. As will be shown, the 6~1-6~8 minute material was 3'-deamino-3i(3 "-cyano-4''-morpholinyl)-13-dihydrodaunorubicin and the 11.9 minute material was 3'-deamino-3'-(3''-cyano-4''-morpholinyl)daunorubicin.
B. On a larger scale, 5.41 g of the solid byproduct was dissolved in 500 mL o~ 9:1 CHC13-CH30H.
This solution was washed with O.OlN HCl (3 x 100 mL), H20 (1 x 100 mL) and dilute NaHC03 (1 x 100 ~L). The organic phase was dried, evaporated to dryness and the residue was vacuum dried at 0.1 mm and room temperature to give S.10 g of gla5sy residue.
The aqueous phase W25 retained, also.
C. The glassy resldue of ~art B, 5.09 g, was dissolved in S0 mL cf 4:1 CH2C12-CH30~ The solution was stirred wh;lc 30 mL of CH3CN was added dropwise. The turbid solution which resulted was evaporated to dryness to afford a semisolid residue which was triturated with 200 mL of CH3CN in the darX.
The insoluble solid was collected and triturated a second time with 100 mL of CH3CN. Th~ liquid phases of the two triturations contained the desired pro-duct. They were evaporated to give 2.23 g of semi~
lS solid residue.
D. The semisolid residue of Part C was dissolved in 5 mL of CH~C12 and applied on a 3.1 c~
o.d. x 59 cm column of ~2Cl-washed 200-325 mesh Mallinckrodt Silic AR C~'-7 silica gel. The column was 20 eluted with CH2C12 (500 mL) and then CH2C12-CH3OH
(99:1, 1500 mL; 98:2, 1()00 mL; 97:3, 1500 mL, 90:10, 500 mL). After collect:Lon (10 mL fractions, monitored by TLC) o 2550 mL o initial eluate, a 190 mL rac-tion was evaporated to l~ield O.48 g o product. The primary component was determined by comparative high performance li~uid chromatography and thin layer chromatography to be identical to a material later proven to be 3'-de~ino-3'-~3''-cyano-4''-morpho-linyl)daunorubicin.
E. A 0.35 g sample o~ the material o Part D was further purified first in the dark on ive 2 mm x 20 x 20 cm silica gel plates with -two CH2C12-CH30~ 29:1 de~elopments. A center band containing 65% of the applied weight o~ sample was CUt out, 21uted, filtered and evaporate~l ~o dryness.
F. The product of Part E, together wi-th other equiv~lently purified materials recov~red ~rom chromatography flanking zones, (0.18 g~ was given a 5 final purification on a 1.1 cm o.d. x 27 cm column Oc 200-400 mesh silica gel. The column was eluted with CH2C12-EtOAc (80:20, 30 mL, 60:~0, 30 mL; 40:60, 30 mL; 20:80, 30 mL) and then E~OAc ~175 mL). A~t~r collection (2 mL fract~ons monitored by T~C) of 1~ 162 mL ol initial eluate, an 88 mL fraction was col-lec-ted and evaporated to afford 0.15 g of product.
Elemental analysis of this pure material verified the structure to be 3'-deamino-3'-(3''-cyano-4''-m~rpholinyl)daunorubicin as did 360 MHz N~R, UV, IR and m~ss spectroscopy.
The occurrence of this product as ~ dia-stereoisomeric mixture was indicated by HPLC and 360 MHz NMR analysis. HPLC analysis on a Waters Radial-Pak C-18 column with 0.05M pH4 citrate buffer-CH3C~ (60:40) at 2 mL/minute c~owed two closely spacedpeaXs (at g.6 minutes and 10.2 minutes) in the ra~io 53:44. The 360 MHz ~MR spec-trum of this material exhibited two resonances for the 6-OH, 11-OH, 1 H, 2-H, 3-H, l'-H, 7-H, 9-OH, lOA-H, 14-H3 and 6-H3 protons.
360 MHz ~MR CDC1~ f 13.99, 13.98 (2s, 6-OFI~, 13.2S, 13.24 (2s, ll-OH), 8.02, 8.00 ~2d, l-H), 7.79, 7.77 (2t, 2-H), 7.40, 7.38 (2d, 3-H), 5.59, 5.56 (2d, l'-H), 5.29, 5.26 (2bs, 7-H), 4047*, 4.34* (~s, 9-OH), 4.08 (s, OCH3), 3.97-4.07 (m, 2'l B-H, S'-H), 3.92 (t, J~12Hæ 3"-H), 3.74 (m, 2"A-H), 6"B-H), 3.68 ~bs, 4'H),

3.58 (t, J=12Hz, 6"A-H), 3.20 (d, J=19Hæ, lOB-H), 2.91, 2.gO (2d, J-19Hz, lOA-H) 2.75-2.95 ~m, 3'-H;, 2.68 (m, 5"-H2), 2.43, 2.42 (2s, 14-H3), 2.35 (m, 8B-H), 2.13 (m, 8~-~1), 1.70-~.t~ (m, 2'-H2), 1.86 ~s, 4'-OH, H20), 1.37, 1.36 ~2d, J=6.4Hz), 6-E~3) *exchangeable with D2O

Mass Spectrum:
5 Cas the trimethylsilyl (TMS) derivatives], mje 910 [M(T~S~4], 895 CM(TMS)~ Me], 883 [M(TMS)4-HC~], 838 [M(~S)3~, 823 tM(TMS)3-Me], 811 ~M(TMS)3-HC~, MS at 70 ev. showed a base peaX (HCN~ at m/e 27.

.
Example 2 Isolation of 3'-deamino 3'-~3''-cyano-4''-morpholinyl)-13-dihydrodaunorubicin In Example 1, Part D, a mixture of 3'-deamino-3'-(3''-cyano-4''-morpholinyl)daunorubicin and 3'-deamino-3'-(3''-cyano-4'l-morpholinyl)-13-dihydro-daunorubicin was chromatographed and a series o~~ractions were taken. After collect:ion of 3460 mL of eluent a 430 mL fraction containing 12.5~ o the ini-tially charged material as essentiall~ a single com-pound was collected and ~vaporated. The com~ound of this ~raction was determined by ~PL(' to be identical to a material previously charac-terized by ~R and mass spectroscopy to be 3'-deamino-3'-(3l'-cyano-4''-mor p~olinyl)-13-dihydxodaunorubicin. 1'his material could be puri~ied essentially ~y the me~hods shown in E~ample 1, Parts E and F to yield essentially ~ure 3'-de~lino-3'-(3''-cyano-4''-morpholinyl)-13-dihydrodau-norubicin.
.

1 Examole 3 IsolatiQn of 3'-deamino-3'-(4'l-morpholinyl)d~unorubicin and 3'-deamino 3'-(4''-morpholinyl)-13-dihydrodaunorubicin In Example 1~ Parts A and B, the O.OlN HCl phase containiny 3'-deamino-3'-(4''-morpholinyl)daunorubicin and 3'-deamino-3'-(4''morpholinyl~-13-dihydrodaunorubicin was isolated. This aqueous phase contained about 40% oE the charged material. This aqueous phase was then worked up using the method shown in the previously ~. Medicinal Chem., 25, reference to yield 3l-deamino-31_(41l_ __ morpholinyl)claunorubicin and 3'-deamino-3'-(4''~
morpholinyl)-13-dihydrodaunorubicin as separate isolated compoundsO

Example 4 .

Preparation and isolation of 3'-deamino-3'-(3l'-cyano-4''-morpholinyl)doxorubicin and 3'-deamino-3'-(3''-cyano-4l'-morpholinyl)-13-dihydrodoxorubicin.

A. In a reaction analogous to tha~ shown in the J Medicinal Chem., 25 article, to a stirred solution of 6.25 g t60,0 mmol) of 1,4-anhydro-erythritol, OH OH
. ~ ~
~ o ~
, in 75 mL of H2O cooled in a water bath al: 15-20C was added 6.42 g (30.0 mmol) oE
sodium metaperiodate. The resulting clear solution was stirred at room temperature for 17 hours~ The solution pH
was adjusted from 4.0 to 7.3 with NaHCO3 .

and -then diluted with s-tirring wi~h 75 mL of CH3CN. A
precipita'e formed. The mixture was s-tirred and 0.126 g (2.0 mmol) of NaBH3C~ in 5 mL of 1:1 (vol) C~3C~-H20 was added. To this mixture was then added 1.16 g (2.0 mmol) o doxorubicin hydrochloride in 30 mL of 1:1 CH3CN-H20. After 10 minutes, the reaction mixture was diLuted with 50 mL of dilute ~aHC03 and extrActed thrice with 50 mL portions of CHC13. This crude extract contained 3'-deamino-3'-(4"-morpholinyl)doxoru~)icin, 3'-de~mino-3'-(4''-morpholinyl)-13-dihydrodoxorubicin, 3'-deamino-3'-(3''-cyano-4''-morpholinyl)doxorubicin and 37-deamino-3'-(3''-cyano-4''-morphclinyl)-13-dihydrodoxoru-bicin. Combined extrac~s were extracted with 0.1 N
15 acetic acid (5 x 25 mL) and then with H20 and washed with dilute ~a2HC03 and saturated aqueous NaCl. The acidic aqueous phase wa; retained. The chloroform phase was dried o~er ~a~,04, filtered through Celite diatomaceous earth and concentra-ted to yield a residue. This residue ~as dissolved in 25 mL of CHC13 and solvent re-evaporat~d under vacuum at room temper-ature. This afforded 0.518 g (40%) of a dark red foamed glass.
B. A sample ~f the foamed glass of Part A
was dissolved i.n CH3CN ~nd injected into a Waters Radial-Pa~ C-18 high performance liquid chromatography column and eluted with pH 4.0 0.05 M citrate bu~er -CH3CN 55:45 at 2 mL/minute. The elution of compounds was detec~ted at 254 nm. At 2.3 minutes a material identified as 3'-deamino-3'-~3''-cyano-4''-morpho-linyl~-13-dihydrodoxorubicin came of in 13~ yield ~based on in~ection rnixture) and a-t 3.8 minutes, 3'-deamino-3'-(3''-cyano-4''-morpholinyl)doxorubicin came off in 69% yield. Other similar pooled products were obtained and separated by HPLC.

~33~
-2~-C. The isolation of 3'-Deamino-3'-(3''-cyano-4''-morpholinyl)doxorubicin and 3'-De~mino-3'-(3''-cyano-4''-morpholinyl)-13-di'nydoxorubicin was subsequently repeated as follows:
~ 0.424 g sample of the foamed glass oE Part A ~s dissolved in 1.5 mL of CH2C12 and applied on a 1.5 x 35.5 cm column of CH2C12-washed 200-400 mesh Bio--Sil A Silica gel. Th2 column was eluted with C~I2C12 (50 mL) and then CEI~C12-CH3OH (99:1, 150 ~L:
9~3:2, 150 mL, 97:3, 300 mL; 95:5, 100 mL and 90:10, 300 mL). After collec-tion of 445 mL of initial eluate, an 80 mL fraction was evaporated to yield 0.217 g o~ p~oduct. This material was com~ined with 0.039 y ~f purified product from an earlier prepara-tion and the mixture was dissolved in 2 mL of CH2C12, diluted with 10 mL of CH30H and evaporated to dry-ness. Trituration of this residue with 5 mL of CH30H
afforded O.218 g of 3'-declmino-3'-t3''-cyano-4''-morpholin-~l)doxorublcin.
HPLC and 400 .~Hz ~MR analysis indicated this product wzs a diastereoisomeric mixture. HPLC
an21ysis on a Waters Radial-Pak C-18 column with 0.05 M pH 4 citrate buffer-CH3CN ~65:35~ at 2 mL/minute showed two clo~ely spaced peaks (at 14.4 minutes and 15.7 minutes~ in the ratio 5B:39. The 400 MHz spectrum of this materi.al exhibited two ' resonances for the l-H, ~-H, 3~H, 1'-~, 7-H, 14-H2, 9-OH, OCH3, 10A-H and 6'-H3 protons.
' 400 MHz NMR CDC13 ~ 14.02 ~s, 6-OH), 13.26 (s, ll-OH), 8.05, 8.04, (2d, l-H), 7~80, 7.79 (2t, 2-H), 7.41, 7.40 (2d, 3-H~, 5.61, 5.S7 (2d, l'-H), ~.34, 5.3C~ (2m, 7-H), 4.79, 4.78 (2s, 14-H~, 4,54, 4.42 (2s, 9-OH), 4.11, 4.10 (2s, OCH3), 4.05 ~m, 5'-H), 3.97 ~m, 2"B-H, ,3"-H, 6"B-H), 3~71 ~m, 4'-H, 6"A-H), ~25-.

3.S8 (t, 2"A~H), 3.30 (d, J = 19 Hz, lOB-H), 3.07, 3.06 (2d, lOA-~), 3.03 (m, 3'-H), 2.69 (m, 5"-H2), 2.38 (m, 8B-H), 2.22 (m, 8A-H), 1.84 (m, 2'-H2~, 1.61 (s, H20), 1.40, 1.39 (2d, J = 6.5 Hæ, 6'-H3).
~V-Vis (CH30H) max 234 nm ( 40,100), 252 (27,700), 289 (9,~20), ~78 (13,000), 49~ (12,900), 530 (7,l90). Mass spectrum [as the trirlethylsilyl ~TMS) derivative], m/e 899 M(T~S)4-HCN.
C H N
Calcd for C32H34N2l2 2H2 56.97 5.68 4.15 Found 57.0l 5.37 4.17 Further elution of the abc~ve column ~ave 0.041 g of 3'~deamino-3'-(3''-cyano-4i'-morpholin~l)-13-dihydrodoxorubicin. UV-Vis (CH3~)H) max 234 nm (~
37,400), 252 (28,000), 2~9 (g,390), ~75 ~12,7~0~, ~96 (12,800), 530 (7,410). Mass spectrum ~as the trimethylsilyl (TMS) derivative~, m~'e 985 M~TMS)5-CH3, 973 M(TMS)5-HCN
C H H
Calcd for C32H36N212 1 5H2 - 5.89 4.. 20 Found 57~35 5.94 3.82 Example 5 Isolation of 3'~deamino-3'~(4''-morpholinyl) aO
rubicin and 3'~deamino-3' (4''-morpholi~yl~-13-dihydrodoxorubicin A. The acidic aqueous phase obtained inPart A o~ ~xample 4 was basified with NaHC03 and extracted with CHC13. The CHC13 phase was washed with saturated NaCl, dried over Na2S0~. filtered through Celite , concentrated and dried to give 0.828 ~ of a red foam which by HPLC, 90 MHz ~IR, 300 MHz NMR, UV-Vis spectroscopy and mass spectroscopy was ~hown to . ' ~ ~ .
.' . ................ ', '. .,, .,,~.

30~

cont~in two primary components, 3'-deamino-3'-~4"-morp~olinyl)doxorubicin and 3'-deamino-3'-(.''-morpholinyl)-13-dihydrodo~orubicin.
3. A pool of 0.98 g of the foamed glass S such as was prepared in Part A was made up. rFhis ma~erial was dissolved in 3 mL of CH2C12 and chromato-graphed on a 2.2 x 33 cm column oF silica gel. r~he column was eluted wit'n CH2C12 (50 mL~ and then CH2C12-CH30H (99:1, 300 mL; 98:2, 300 mL; 97:3, 900 mL
and 90:10, 700 mL~. After collecting an initial 1170 mL o~ eluent, a 490 mL fraction was isolated and evaporated to give a residue. This residu~ contained 45% of the charged sample and by HPLC was seen to be essentially pure (99~) 3'-aeamino-3'~(4''-morpho-linyl)doxorubicin.
90 MHz ~MR CDC13 ~ 13.88 (s, 6-OH), 1-;.07 (s, ll-OH), 7090 (d, J - 8 Hz, l-H), 7.72 ~t, J =
8 Hz, 2-H~, 7.38 ~d, J = 8 Hz, 3-H), 5.51 tbs, ]'-H), 5.20 (bs, 7-~), d.75 (S, 14-H2), 4.68 ~S, 9-OH), 4.07 (s, OCH3), 3;98 (m, 5'-H), 3.67 (m, 4'-H, 2"-H2, 6"-H2), 3.0g (d, 3 = 19 Hz, lOB-H), 2.83 (d, J = lC~ Hz, lOA-H), 2.80-3.20 (m, 3'-H), 2.50-3.00 (bs, 4'-OH, 14 OH), 1~95-2;65 (m, 8-~2~ 3 -H2~ 5 ~H2), 1 H~, 1.3~ (d, ~ = 6.5 Hz, 6'-H3)~ Mass spectrum ~as the trimeth~lsilyl ~TMS) derivatives], m/e 973 ~MS)5, 901 M(r~MS)~.
The free base was suspended in H20 and acidi~ied to pH 4.4 with O.lN HCl. rFne r~sultant solution w~s lyophilized and the produc-t was dissolved in CH30H and precipitated with 10 volumes of ether to affor~ the hydrochloride.

~ ;33~$~ i C H Cl- ~1 lcd _orC31E~35N012 HCl 2H~O 54.27 5~88 5.17 2 04 Found 54.08 5.35 4.78 2.00 UV-Vis (CH30H) max 234 nm (E 39,000), 252 (26,300), 290 (8,990), 480 (12,600~, 4~5 ~12,500), 530 (6,700).

A 190 mL fraction was taken Eollowed by a 160 mL frac-tion. This latter fraction was evaporated and found to contain 19.5~6 of the charg2d material as 97~6 pure 3'-deamino-3'-(4''-morpholinyl)-13-dihydrodoxorubicin.

300 MHz N~IR CDC13 ~ 13.98, 13.96 (2s, 6-OH), 13.34, 13~32 (2s, ll--OH), 8.03 ~a, l-H), 7.79 (t, 2--H),7.40 (d, 3--H),5.~6 (bs, l'--H), 5.29 (bs~ 7--E~),

4.64, 4.59 (2s, 9-OH), 4.09 (s, OCH3), 4.03 (m, 5'~
3.~2-4.05 (rn, 4'-H, 13-H), 3.68 (m; 2"-H2, 6"-H2, 15 14B-H), 3.54 (bs, 14A-H), 3.30 (m, 10B-H), 2.g8 ~bs, OH), 2.87 (bs, OH), 2.77 (m, 10A-H, 3'-H), 2-30-2.70 (m~ 8Es-H, 3"-H2, 5"-H2), 1-99 (m, 8A-E), 1.78 ~m, 2~-H2), 1.41 ~d, 6'-H3). Mass spectrum ~as the tximethylsilyl (IMS) derivativel. m/e 975 M(TMS)5.

20 Example 6 Preparation of 3'-deamino-3'-(4'' rnorpholinyl)-5-iminodoxorubicin A. To a solution of 0.396 g of 3'-deamino-3'-(4''-morpholinyl)doxorubicin prepared as showrl in 25 Example 5 in 5 mL of dry pyridine was added 0.990 g of ~-anisyl chlorodiphenylmethane. The mixture was allowed to react at room temperature in the darlc for about two days. The solution was cooled in ice water and 0.5 m~ of CH30H was added. The mixture was .

stirred ror 2 hr and added to 50 mL of dilute NaHCO~
and extracte~ with CH2C12. The extrac~s were concen-trated to give a gummy residue which was dissolved in toluene, concentrated, dissolved in

5 CH2C12 and precipitated by slowly adding 35~-60 petroleum ether. This precipitate was recovered, re-dissolved in CH2Cl~ and precipitated with 2:1 petroleum ether:diethyl ether to afford 14-0-p-anisyldiphenyl methyl-3'-deamino-3'-(4''-morpho-linyl~doxorubicin~ (IIII, as an amorphous solid in 94% yield.

~ ~ ~ CO-CH20-C ~ ~ OC~l3 ~01 ~CH3 \

HOV

. ~0~
This material was ident:Lfied by 90 MHz ~R in CDC13.
B. A solution of 0.532 g of 14-0-p-anisyldiphenyl methyl-3'-deamino-3'-(4'.'-morpho-linyl~doxorubicin in 10 mL of CH2C12 was added to 30 mL of CH30H saturated with ammonia at 0C. The-mixture w~s stirred at 0C for an hour and then allowed to stand at 3C for 27 hours. Solven-t in the reaction product was evaporated. The residue was dissolved in 4:1 CH2C12-CH30H and concentrated. This was repeated twice and -the solid was dissolved in CH2C12, filtered through Celite , concentra-ted, dissolved in 1:2 CH2C12-CrI30'H and again concentrated and dried to yield 0.52 g (97%~ of a violet residue.
C~ The residue of Part B was dissolved in 2 mL of CH2C12 and applied on a 1.5 x 40 cm silica ~el column and eluted with CH2C12 (50 mL3 and then CH2C12-CH30H (99:1, 150 mL; 98:2, 150 mL; 97:3, 500 mL; 95:5, 100 mL; 93:7, 100 mL; and 90:10, 200 mL)O
Following 565 mL of eluent, a 335 mL fraction was separated, filtered and evaporated to contain 59.9~ of the applied sample as a single material. This mater-ial was identified as 14-O-p-anisyldïphenylmethyl-3 deamino-3'-(4 "-morpholinyl)-5-iminodoxorubicin by 90 MHz ~.
D. A 0.341 g sample of the product o~ Step C was dissolved in 20 mL of 80~ acetic acid and the soluti~n was s-tirred in the dark for seven hours. The solution was then diluted with 50 mL of wa~er and extracted three times with CHC13. The aqueous phase contained the desired product and was lyophilized in the dark to give 0.2~4 g o~ solid. The solid was dissolved in 0.1 N acetic acid. The solution was washed with CHC13 basified with NaHC03 and extracte~
with CHC13- The desired product went into the organic phase which was washed, dried, filtered and concen-trated to give a residue. This residue was dissolved in CHC13-CH30H (1:10) concentrated and dried to yive 0.228 ~ o 3'-deamino-3'-(4''~morpholinyll-5-imino~
doxorubicin. The material's identit~ was veriied by 300 MHz ~MR and elemental analysis.

~. ,~ , .

3~p ~

Example 7 Preparation of acid addition salt The free ~se product of Example 6 was sus--pended in 20 mL o water. ~he mixture was stirred and 5 3.2 mL of 0.1 N HCl was slowly added to give-a pH of 4.5. The suspended solid gradually dissolved. l'he solution was lyophili~.ed in the dark to give the acid addition salt 3'-deamino-3'-~4''-morpnolinyl)-5-iminodoxorubicin hydrochloride in 97~% purity by HPLC
10 analysis.
C HCl-- ~
alcd for C~31H36N2ll HCl2H20 54~35 ~.0~ 5.17 4 og Found 54.20 5.96 4.33 4.03 Example 8 Preparation and isolation of 3'-deamino~3'-(4''-15 morpholinyl~~5-imino-13-dihydrodoxorubicin A. A solution of 0.186 g of 3'-deamino-3'-(4''-morpholinyl)-13-dihydrodoxorubicin prepared as in Example 5 in 6 mL of 1:1 CH2C12-CH30H was added tc>
20 mL of CH30H saturated wi-th aumnonia at O~C. The 20 mixture was stirred for an hour and then stored at 3C
for about 27 hours, concentrated and the residual product dissolved in CH2C12-CH30H (4:1) and concentra-tea thrice to completely remove ammonia. The resulting residue was purified by preparative thin 25 layer chromatography on 2 mm x 20 cm x 20 cm silica gel plates using CHC13-CH30H 9:1 development. Bands containing essentially pure 3'-deamino-3'-~4''-morpholinyl~5~imino-13-dihydrodoxorubicin were separated, eluted and the eluent dried to afford 30 0.139 g o the free base product as identified by 300 MHz NMR.

:~2~

B. The free base oE Part A was converted to the hydrochloride by the method o F Example 7. By HPLC
ana~ysis, ~he hydrochlori~e was 97-98% pure.

C H Cl- N
Calcd ror C31H38N2ll HCl 2H2 5 Found 54.17 5.95 4.8~ 3.87 Example 9 Preparation of 3'-deamino-3'-(3''-cyano-4''-morpholinyl)-5-iminodaunorubicin A solution of 0.031 g of 3'-deamino-3'-(3''-cyano-4''-morpholinyl)daunorubicin in 1.0 mL of CH2C12 was added to 5 mL of ammonia-saturated methanol a~ -o3c. The mixture was stirred for 30 minutes ana then stored at 3C for 45 hours. The product of this reaction was evaporated to dryness to give a residue which was dissoIved in 5 mL of 19:1 CHC13-CH30H and concen~ra~ed. This step ~as repeat d. This r~sidue was dissolved in CHC13-CH30H and applied to a 2 mm 20 cm x 20 cm silica gel plate and developed using 9:1 C~C13-CH30H. The major band was eluted and analyzed. Massspectroscopy verified the compound to be 3'-deamino-3'-(3''-cyano-4''-morpholinyl)-5~iminodaunorubicin.
.

Example 10 The preparation of Example 6 was repeated u.sing 3'-deamino-3'-(3''-cyano~4''-morpholinyl)doxo-rubicin prepared as in Example 4 as feed material in place of 3'-deamino-3'-(4''-morpholinyl)doxorubicin.
l~e blocking-amination-deblocXing isolation sequence of ~xample 6 is used to yield 3'-deamino-3' (3''-cyano-4''-morp~olinyl)-5-iminodoxorubicin as the final product.

In more detail, this prepara~ion was as follows:
A. To a solution o~ 0.241 9 of 3'-aeamino-3'-(3l'-cyano-4''-morpholinyl)doxoru~icin prepared as shown in Example 4 in 4 mL of dry pyridine was added 0.587 g o~ p-anisylchlorodiphenylmeth~ne. ~ne solution was stirred at room temperature in the dark ~or 44 hr. The reaction mixture was cooled, diluted with 0.5 rnL of CH30H, stirred at room temperature for 3 hr and then added to 50 mL of dilute ~aHCO3 and extracted with CH2C12. The extracts were concentrated, the resi~ue was dissolved in 3 mL of CH2C12 and precipitated by slowly adding 40 mL of diethyl ether to afford 0.333 g (97%) of 14-O-p-anisyldiphenylmethyl-3'-~5 deamino-3'-(3''-cyano-4''-morpholinyl)doxorubicin.
90 MH~ ~R CDC13 ~ 13.84 (s, 6-OH), 12.99 ~sl ll-OFI), 7.82 ~d, l-H), 6.70-7.75 (m, 2-H, 3-H, trityl-aryl), 5.42 ~bs, l'-H~, 5.08 (~s, 7-~), 4.45 ~bs, 2, 14-H2), 4.19 (s, 9-OH), 4.00 (s, OCH3), 3.79 (s, OCH3), 3.30-4.15 ~m, 4'-H, 5'-H, 2"-H2, 3"-H, 6" H2), 1.60-3.10 (m, 2'-H2, 8-H2, 5"-H2, 10-H~, 3'-H), 1 13 (d, ~ -~I3)-B. A solution of 0.369 g of 14-O-p-anisyldiphenylmethyl-3'-deamino-3'-~3'`-c~ano-4'' morpholinyl)doxorubicin in 8 mL of CH2C12 was added ~o 25 mL of CH30H saturated with ammonia at 0C. The mixture was stirred at 0C for an hour and then allowed to stand at 3C or 26 hr. The reaction mix.ture was evaporated, to completely remove ammonia and yield 0.376 g o~ a violet residue.
C. The residue of Part B in 1.5 mL of ~I2C12 was applied on a 1.5 x 28 cm (200-400 mesh) silica geL
column and eluted with CH2C12 (50 mL) and then CH2C12-CH30H (99:1, 200 mL: 98:2, 300 mL; 97:3, 100 mL; 95:5, 100 mL; and 90:10, 200 mL3. After collec~ion of 360 mL

of init~al eluate, a 125 mL fraction was evaporated to yield 0.2~3 g of 14-O-p-anisyldiphenylmethyl-3'-deamin~-3'-(3''-cyano-4''-morpholinyl)-5-iminodoxorubicin.
D. A 0.158 g sample of .~e residue Oc Part C
was cooled to 0C and dissolved in 8 mL of ice cold 50 trifluoroacetic acid. The solution was stirred at 0~C
for 2 min and then poured into 100 mL of ice water.
The aqueous mixture was extracted wi.th CHCI3 ~4 x 10 mL) and the combined extract:s were washed with dilute NaHC03 and H2O, dried over Nz~2SO4, filtered through Celite and evaporated. Th~! residue was dissolved in 3 mL of CHC13-CH30H (4:1~; the s~lution was stirred and 25 mL of ether was cldded dr~pwise. T~e resulting precipitate was collected to af~ord 0.093 g of 3'-deamino-3-(3''-cyano-4''-m~rph~linyl) 5- -iminodoxorubicin.
HPLC and 300 MHz ~R anal~sis indicated this material was a diastereoisomeric mi~t~re. HPLC
analysis on a Waters Radial-Pak C-l~ column with 0.05M
pH 4 citr~te buffer-CH3OH (40:60) s~owed peaks a~
18.4 min and 25.0 min in the ratio ~9:31. The 30Q MHz spectrum of this product exhibited two resonances for the l-H, 2-H, 3-H, l'-H, 7-H, 14-H2, 9-OH, OCH3, 10A-H, and 6' H3 protons.
300 ~Hz NMR CDC13 ~ 15.61 (s, ll-OH), t3.74 (d, 6-OH), 9.27 (d, NH), 8.21, 8.19 (2d, l-H), 7.73, ?.72 ~2t, 2-H), 7.33, 7.32 (2d, 3~H), 5.77, 5.72 (2d, l'-H), 5.41, 5.38 (2m, 7-H), 4.79, 4.77 (2s, 14-H2), 4.72, 4.66 (2s, 9-OH), 4.15, 4.14 (2s, OCH3), 4.04 (m, 5'-H), 3.97 (m, 3"-H, 2"B-H), 3.75 (m, 6"-H2, 4'-H), 3.59 (m, 2"A H~, 3.23 (d, 10B-H), 3.03 (m, 10A~H, 3'-H), 2.72 (m, 5"-H2), 2.33 (m, 8B-H), 2.14 (m, 8A-~), 1:8S (m, 2'-H2~, 1.38, 1.37 (2d, 6'-H3).

~3~
-3~-UV-Vis (CH30E-~) max 221 nm (~ 31,000), 252 (32,900), 307 (7,110), 520 sh (9.110~, 551 (1~,400), 592 (20,700). DCI-~S m/e 638 (~ -~ H), 611 t.~5 ~ ~-HC~) C H N
5 Calcd for C32H35N3ll H20 58...... .62 5.69 6.41 Found 58.79 5.47 6.30 Example 11 The preparation of Example 8 is repeated four times each time using an equivalent molar amount o~ a different starting material in place of 3'-deamino-3'-(4''-morpholinyl~-13-dihydrodoxorubicin. In the ~irst repeat, 3'-deamino-31-~3''-cyano-4''-morpholinyl)-13-dihydrodoxorubicin is used as feed mater;.al to give 3'-deamino-3'-(3''~cyano-4''-morpholinvl)-5-imino-13-.dihydrodoxorubicin as ~inal product. In the secondrepeat, 3'-deamino-3'-(4''-morpholinyl)daunorubicin is used as feed material to give 3'-deamino-3'~(4''- .
morpholinyl)-5-iminodaunorubicin as final product. In the third repeat 3'-deamino-3'-(4''-morpholinyl)-13-dihydrodaunorubicin is used as feed mater.ial to yive3'-deamino-3'-(4''-morpholinyl)-5-imino-13-dihydro-daunorubicin as final product. In the forth repeat 3'-deamino-3i-(3''-cyano-4''-morpholinyl)-13-dihydrodaunorubicin is used as feed material to gi.ve 3'-deamino-3'-(3''-cyano-4''-morpholinyl)--5-imino-13-dihydrodaunorubicin as final product.

, , Exam~le 1~

The preparations of Examples 1 11 are repeated using in place of aoxorubicin or daunorubicin as starting materials the range of derivatized doxorubicins and daunorubicins described and prepared as described herein in the section denominated "Derivatives and Analogues'`. These repeats give rise to the co~responding derivatives and analogues of the compounds ~f the invention.

The co~pounds o~ this invention have utility as mammalian antitumor agents. This activity is evidenced by in vivo and in vitro studies. In one in vivo test, conducted in accordance with the protocol described in Cancer Chemotherapy Reports, ~ational lS Canc~r Institute, _, No. 2, Part 3, September, 1972, healthy mice were inoculated i.p. with Lymphocyte Leu~2mia P-388 ascitic fluid. The inocuLated mice were then treat~d on days 5, 9 and 13 of the suceeeding period with various amounts o compounds o~ the invention. As comparisons, other mice were untreated and additi~nal mice were treated with daunorubicin, or doxorubicin; 3'-deamino-3'-(4''-morpholinyl)daunoru-bicin or 3'-deamino-3'-(4''-morpholinyl)-13-dihydrodaunorubicin of U.S. Patent 4,301,277 or 3'-de~mino-3'-(4-methoxy-1-piperidinyl~daunorubicin or its 13~dihydro equivalent shown in U.S. Patent ~,314,054.
The average survival time of the various treated mice was determined and compared with that of the mice inoculated with the leukemia ascitic fluid but given no treatment with the test compounds. Presente~
in the following Table A are the data so obtained. The data are presented as ~ T/C values which are the Y3t~

survival time of the treated mice divided by the survival time of the controls multipliea by 100. Also given in Table A are the dosage levels of the various compounds which were observed to produce the best survival time improvements.

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8 E ~ ~ v E I ~ '~
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t~ ~t ~ I o I O~) U~ t i In ~ u-~
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Tab1e ~ (~ontin~) Activity vs Le~kemia Leukemia P-388 in Mice ~-1210 Cells Optimum Inhibition Survi~al Dose Oc Synthesis S ~ime lq4a 5,9,13) ED50"~M
Co~pouna ~SC ~o. % T~C mg~kg DNA R~
3'-de~mino-3'-(3''-~y~no-4''-morpholinyl) daunorubicin 33Z,304 197 0.¢ 0.012 0.002 l0 3~-deamino-3'-~3''-cyano-4 "-morpholin~
13-dih~drodau~o-rubicin 332,305 143 0.1 .. 0.019 1l~002 3~-~eamino-3~ -(3~- 357,704 187 0.07S 0.003 0.0005 cyano-¢''-mor?holinyl)-doxorubicin 3'-deamino-3'-(3 " - 360,291 150 0.2 0.021 l).0030 cyano-4''-morpholinyl)-13-dihydrodo~orubicin 3'-deamino-3'-~4''-355,465 161 S0 . ~100 ~.
~orpholinyl~ -13-aihya 5-iminoaoxorubicin For Comparison:
.
daunoru~icin 82,1~1 130 8 0.66 0.33 aoxorubicin 123,127 160 8 1.5 ~.58 . -* NSC No. = National Service Center of US
Nati.onal Cancer Institute registration number.
.
,, .

. ~ ~ 3J~

Tabl~ ~ (continued) Act.ivity vs Leuk~mia Leukemi~
P-388 in Mice L-1~10 Cells Optimum Inhibition Survival Dose of Synthesis Time (q4d.~9,13) ED50'~M
NSC No. ~ T/C mq/kq DNA RNA
3'-deamino-3'--~4''-morpholinyl~
d~unorubicin 327,~51 166 0.2 0.76 0.10 10 3' deamu~o-3'-~4 "-m~rpholinyl)--13-dihydrodau;lo~lbicin 327,450 132 0,2 2.2 0.67 3'-de~mino-3'-(4 "-methoxy-'l''-piperidinyll- - . -daunorubicin 334,353 199 6.25 0.63 0.12 15 3'-deamuno-3'~
(4-methoxy-1-]?iperidinyl)-1 3-dihydrodauno- : , rubicin 334,354 199 12.5 0.58 O.OB
These resul~s show that compounds of this inven~ion have good to superior in vivo antitumor activity at low optimum dosages. Compound NSC 357704 showed an optimum dose level about l/lSOth that re~uired with the parent compound. Other materials of the invention show optimu]n dose levels far lower than daunorubicin and doxor~b:icin. This gives promise o providing an active anti-~umor agent with substantially decreased cardiotoxic:ity.
In vitro tests of 3'-de~nino-3'-(3''-cyano-4'' morpholinyl~daunorubicin and 3'~deamino-3'-(3''-cyano-*''-morpholinyl)-13-dihydrodaunorubicin also showed the increased activity in this class o~ com-pounds. When these materials were tested as inhibi-tors of DNA and RNA synthesis in L 1210 Cells by the .

method described in G. Tong, W.W. Lee, D.R. Black and D.W. Henry J. Medicinal Chem, 19 3g5 (1976) they , were ac-tive a~ doses tha~ were as much as 600 times lower than the doses of daunorubici'n, doxorubicin, or the previous analogues. They were also observed to be much more inhibitory toward RNA synthesis than toward DNA sysnthesis (ED 50 Ratio DNA/RNA = 10 to 11). It has been sul3gested by S.T. Crooke, et al, Mol.
Ph~rmacol., 14, 290 (1978) that,such a ratio indicates Class TI anthracyclines having improved therapeutic properties. These data are shown in Table A.
The data in I'able A, showing increased antitumor p~tency with the morpholino structure and further increase in efficacy with the cyanomorpholino structure, typify activity with this class of co~pounds.
Additlonal tests were run to verify the' biological activity of compounds o this invention.
These were in vivo tests in mice against P-3~8 and L1210 leukemia ancl B-16 ~elanoma carried out essen-t'ially by the method of the above-noted Cancer y Report~, 197~. Various dose schedules and I.P., I.V. and oral rou-tes of administration were,- ' tested.- These results are given in Table B.

n n ~n u~~nIn ~n ~_N O t~ N N ~1 No o OO ~L ~ 3 ~
oo _ o ~ oo o r~) N N ~ C~
m ~ ~ ~ ~ -~ ~ ~ ~
Ln Ln r~Ln Ln ~ r~Ln ~`1 N \ DN O ~DLD t`J
~O ' O - ~O O O r O O O O O--~ O o O
ro ~ --~o -n~ o o o ~n o o o D ~ .~ o co ~ '~
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_~ ~ ~ ~ ~ ~ a~
L~7 Ln Ln ~I Ln Ln L
_ ~ O ~ O ~D o o ~
V ~ . O . o o o -- O -- O -- -- ^ ^
~ ~r ~ ~ ~ -- ~r Ln O
E ~D Ln ~ CD ~ N 1` -- t~ t~ ~ t~
_ n '- o . .~ _ ~
r~ ~ O O ~ O
d~ Ln C~ Lo O t~l o ~ ~ ~0 tD ~o E I t~) E ~ E El e E
O l ~r Ln ~n Ln a ~ ~ ~
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n ~

. ~ C
C ~ Ln L~ ~ O C X D
In . O . o ~;l U o o 1 '` Ln . - I x, .1 ~ n ~.
Ln ~ LO o ~ ~ O
~ -- ~ Ln ~ O
-_ ~ ~ Ln to r ~
doxorubicin ~ N A ` ~ ~ S ~ O
_ . ~ S
E o ~ ~ ~, ! ! ! e o Ln Ln ~ ~ ~r ~ ~
daunorubicin~ ~ 3 _~
a O O O O
E~
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o~ :zi ., ~ J ~ ~ Ln ~ .o '1 ~7 ~ ~
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u z .~ a ~ ,~ a ~ .~ a 3~

The compounds of this inve~tion, including the salts thereof can be administered by any available route, including oral and parenteral (intravenous, intraperitoneal, subcutaneous, and intramuscular) administration. Parenteral administration, especially intravenous administration, has historically been the mode of choice and this is preferred. The doc;ing regimen and amount administered is sufficient to ameleriorate the leukemia or other type of cancer against which the compounds hereof are eEfective. For example, in the treatment of lower test anima]s, a dosage o~ a compound of the present invention within the range from about 0.0010 mg/kg to about 25 mg~kg per day should be sufficent to amelerioriate leukemia.
The upper dosage limit is that imposed by toxic side erfects and can be determined by trial and error for the animal to be treated. In general, the dosage with compounds o~ this invention will be lower than ~e.g.
1/20 to 1/200 times3 that required with the parent compounds. Dosing regimens of one dose every 2 to 7 days are effective ~hile shorter in-tervals, say one day or less, between dosings may be used as well.
To facilitate administration, the compounds of this invention, including the salts thereof, can be provided in pharmaceutical composition form, and par-ticularly in unit dosage form. While the compo-unds can be adminis-tered per se, it is more common to administer them in conjunction with a pharmaceutically acceptable carrier which dilutes the compound and acilitates handling. The term "pharmaceutically acceptable" means that the carrier (as well as the resulting composition) is sterile and nontoxic.
For oral dosage, the carrier or diluent can be solid, semisolid, or liquid, and can serve as a ~o~
-4~-vehicle, excipient, or medium or the agent as described in p~a~macology texts. For parenteral administr~tion, the compound is dissolved or suspended in a suitable inj~ctable li~uid medium as is Xnown in the art.
In the preparation of these dosage focms, one can use the art accepted techniques for for:nula-ting water-soluble pharmaceutical agents (in the case of salts) and water-insoluble agents ~in the case of 10 the free bases). For example, injectable materials can be formulated as follows.

Formulation A:
Sterile Suspension in Aqueous Vehicle for Injection Mg lS Compound of Examples 1, 2, 3, 4~ 5, 6, ~ or 10 as a suspendable powder 3 Sodium citrate 5,7 Sodium carboxy~ethylcellulose (low viscosity grade) ~.0 20 Methyl para-hydroxybenzoate 1.5 Propyl para-hydroxybenzoate 0.2 Water for injection to 1.0 mL

.
Formulation A':
Sterile Suspension in 25 A~ueous Vehicle for In~ection "3'-Deamino-3'-(3''-cyano-41'-morpholi.nyl3 doxorubicin of Example 4 0.5 Sodium citra-te 5,7 Sodium carboxymethylcellulose 30 ~low viscosity grade) 2.0 Methyl para-hydroxybenzoate 1.5 Propyl para-hydroxybenzoate 0.2 Water for injection to 1.0 mL

,. , , ~

7~ ~ e ~

Formulation B:
Sterile Solution in Aqueous Carrier System for Injection ~g Compound of Example 7 5 Sodium citrate 5.7 Sodium carbox~me-thylcellulose (low viscosity grade) 2.0 Methyl para-hydroxybenzoate 1,5 Propyl para-hydroxybenzoate 0.2 10 Water for injection to 1.0 mL

Similarly, one could formulate tablets for oral administration as follo~s.

Formulation C:
Tablet Formulation M~
15 Compound of Example 7 5.0 Lactose 91 Cornstarch ~dried) 51.5 ~elatin . 2.5 Magnesium stearate 1,0 The compound of Example 7 is powdered and passed through a mesh sieve ancl well mixed with the lactose and 30 mg o the cornstarch, both passed through a sieve.
The mixed powders are massed with a ~arm gelatin solution, prepared by stirring -the gelatin in water and heating to form a 10~ w/w solution. The mass is granulated by passing through a sieve, and the moist granules dried at 40C.
~he dried granules are re-granulated by 3U passing through a sieve and the balance o~ the starch ~ 2 ~ .L ~

and the magnesium stearate is aclded and thoroughly mixed.
The granules are compresse~ to produce tablets each weighing 150 mg.
Capsules could be formulated as follows.

Formulation D:
Capsule Formulation Mg Compound of Example 8 10 Lactose 190 10 Formulation D': .

Capsule Formulation Mg 3'-Deamino-3'-(3''-cyano 4''-morp'nolinyl~) doxorubicin of Example 4(C) 15 Lactose lgg - Compound o-E Example 8 c~r 4(C) and lactose are passed through a sieve and the powders well mixed together.
beore filling into hard gelatin capsules of suitable size, 50 that each capsule contains 200 mg of mixed powders.

Claims (6)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the preparation of a compound having the structure G:

(G) wherein R is selected from CO-CH3, CHOH-CH3,CO-CH2OH
and CHOH-CH2OH and A is selected from CN and H,charac-terized in that a compound having the structure:


1. Claim 1 continued....
   
   wherein R has the meanings given above and after the hydroxy group of the -CO-CH2OH group, if present, has been suitably blocked by a mild acid labile protecting group to give a 14-ester which is purified by chromato-graphy on a silica gel column using as eluent system a mixture of CH2Cl2-CH3OH (99: lv/v with an increas-ing amount of CH3OH till 90:10 v/v said compound is reacted at a temperature at from 0°C to 3°C with an excess of alcoholic ammonia and, upon splitting off, if necessary, of said mild acid labile protective group by treatment with acetic acid or cold trifluoroacetic acid at room temperature, the desired compounds are obtained as their relevant free bases which, after a chromato-graphic purification on a silica gel column, using as eluent system CHCl3-CH3OH (9:1 v/v), are isolated as compounds of the structure G wherein A is CN or are transformed into their relevant hydrochlorides to be compounds of the structure G wherein A is H by treat-ment with 0.1N hydrochloric acid.
2. 2. The process of claim 1 wherein said mild acid labile protecting group is a p-methoxy-trityl group.
   
   3. A process for the preparation of a compound having the structure G:
   
   Claim 3 continued....
   
   (G) wherein R is selected from CO-CH3, CHOH-CH3, CO-CH2OH
   and CHOH-CH2OH and A is selected from CN and H, charac-terized in that a compound having the structure:
   
   
3. Claim 3 continued in which R has the above meaning and after the 14-hydroxy-group, if present, has been suitably blocked by any mild acid labile protecting group, said compound is reacted with an excess of alcoholic ammonia at a tem-perature between -25°C and +25°C and, upon splitting off said mild acid labile protecting group, the desired compounds are isolated and purified.
4. 4. A compound having the structure G as defined in claim 1 wherein R and A are as defined in claim 1 when produced by the process of claim 1 or an obvious chemical equivalent.
5. 5. A compound having the structure G as defined in claim 1 wherein R and A are as defined in claim 1 when produced by the process of claim 2 or an obvious chemical equivalent.
6. 6. A compound having the structure G as defined in claim 1 wherein R and A are as defined in claim 1 when produced by the process of claim 3 or an obvious chemical equivalent.