Saccharide intermediates in the formation of the calicheamicin and esperamicin oligosaccharides

Intermediates useful in preparing the calicheamicin and esperamicin oligosaccharides, and particularly the sulfur- and O-substituted hydroxylamine-containing B ring of both oligosaccharides, are disclosed as are methods of making and using the same, including the preparation of chimeric antibiotics using the same.

DESCRIPTION 
TECHNICAL FIELD 
The present invention relates generally to the antibiotics calicheamicin 
and esperamicin, and more particularly to intermediates useful in the 
preparation of the calicheamicin and esperamicin oligosaccharide portions. 
BACKGROUND ART 
The calicheamicin [Lee et al., J. Am. Chem. Soc., 109:3466 (1987)] and 
esperamicin [Golik et al., J. Am. Chem. Soc., 109:3462 (1987)] families of 
antibiotics contain a complex bicyclic enediyne allylic trisulfide core 
structure linked through glycosyl bonds to an oligosaccharide chain. The 
oligosaccharide portions of each of those molecules contain a number of 
substituted sugar derivatives, and each of those oligosaccharide portions 
contains a tetrahydropyran ring that is substituted on the ring both with 
a sulfur atom and with the oxygen atom of a hydroxylamine group. 
The chemical structure of calicheamicin .gamma..sub.1.sup.I, which contains 
a more complex oligosaccharide group than an esperamicin, is illustrated 
in FIG. 1 herein. The saccharide unit lettered "B" is the before discussed 
sulfur- and O-hydroxylamine-substituted tetrahydropyran derivative. 
The saccharide rings of an esperamicin corresponding to rings "A" and "E" 
of FIG. 1 are substituted similarly to those depicted, except that the 
esperamicin ring corresponding to ring E includes an N-isopropyl rather 
than N-ethyl group. The corresponding "B" ring of an esperamicin contains 
an S-methyl rather than the S-(saccharide-substituted)-derivatized benzoyl 
group (C and D rings) shown in FIG. 1. The structures of esperamicin and 
some of its derivatives are illustrated in U.S. Pat. No. 4,837,206, whose 
disclosures are incorporated by reference. 
The enediyne-containing (aglycone or core) and carbohydrate portions of 
calicheamicin and esperamicin appear to carry out different roles in the 
biological activity of those molecules. Thus, the core portion appears to 
cleave DNA [Zein et al., Science, 240:1198 (1988)], whereas the 
oligosaccharide portion of calicheamicin appears to guide the drug to a 
double stranded DNA minor groove in which the drug anchors itself on the 
5' side of a TCCT sequence, and the core cleaves the DNA. Esperamicins are 
less sequence specific. [Zein et al., Science, 244:697 (1989)]. 
Studies of the effect on DNA cleavage of derivatization or removal of one 
or more of the D and E rings of calicheamicin (FIG. 1) indicate the 
following: removal of the E ring (amino sugar) provided a drug with the 
same DNA cleaving specificity as the parent, but having a DNA-cleaving 
efficiency 2 to 3 orders of magnitude less; acylation of the E ring amine 
maintained specificity but lowered efficiency; removal of the D ring 
(terminal rhamnose) maintained specificity, but lowered efficiency 50-100 
times; and removal of the D and E rings (terminal rhamnose and amino 
sugar) resulted in inhibition of cutting. [Zein et al., Science, 244:697 
(1989)]. 
Esperamicin lacks the C and D rings and includes a further complex 
saccharide structure linked to an additional core hydroxyl group. U.K. 
Patent Application 2,179,649A reports that acid hydrolysis of esperamicins 
led to cleavage of that second complex saccharide structure and a 
resulting esperamicin derivative referred to as BBM-1675C that was about 
as effective as the starting esperamicin BBM-1675A.sub.1 (esperamicin 
A.sub.1), and more so than esperamicin BBM-1675A.sub.2 (esperamicin 
A.sub.2) as an antitumor and antimicrobial agent. From the discussion in 
this U.K. application, the oligosaccharide portion of BBM-1675C contains 
rings analogous to the A, B and E rings of calicheamicin shown in FIG. 1. 
U.K. Patent Application 2,179,649A also disclosed that further hydrolysis 
of esperamicin BBM-1675C led to another esperamicin derivative named 
BBM-1675D that was also said to be about as effective as esperamicin 
BBM-1675A.sub.1, as an antitumor and antimicrobial agent. The data 
presented indicate that esperamicin BBM-1675D possessed only two 
saccharide rings; i.e. those corresponding to the A and E rings of FIG. 1 
herein. 
Thus, the art has recognized the importance of the oligosaccharide portions 
of the calicheamicin and esperamicin antibiotics, and has recognized that 
the saccharide rings in the calicheamicin group can affect the activity of 
the drug. The results disclosed in Zein et al., Science, 244:697 (1989) 
and those in U.K. Patent Application 2,179,649A indicate a possible 
conflict as to the effect of the individual saccharide portions on 
efficacy, although different assay methods were used. 
It would be important therefore to be able to prepare an oligosaccharide 
portion of a calicheamicin or the corresponding portion of an esperamicin 
and derivatives thereof so that the specificities of those materials can 
be further studied and fine-tuned. It would also be of importance to link 
a calicheamicin or esperamicin oligosaccharide or a derivative or analog 
thereof to another known DNA cleaving chemical to create a synthetic, 
chimeric antibiotic. 
The present invention describes the synthesis of key intermediates useful 
in the preparation of a calicheamicin or esperamicin oligosaccharide 
portion, an oligosaccharide portion derivative or analog, as well as the 
synthesis of chimeric antibiotics containing such an oligosaccharide. 
BRIEF SUMMARY OF THE INVENTION 
The present invention contemplates intermediate compounds useful in the 
preparation of the oligosaccharide portions of calicheamicin and 
esperamicin, as well as analogs and derivatives thereof and a method of 
preparing the same. 
One contemplated intermediate compound corresponds in structure to that of 
the formula below, 
##STR1## 
wherein R is a moiety selected from the group consisting of hydrogen, 
C.sub.1 -C.sub.5 alkyl, phenyl and m-chlorophenyl; and 
R.sup.2 is a moiety selected from the group consisting of tri-C.sub.1 
-C.sub.6 alkylsilyl, di-C.sub.1 -C.sub.6 alkylphenylsilyl, and C.sub.1 
-C.sub.6 alkyldiphenylsilyl. 
In preferred practice, R is a substituted phenyl moiety such as 
m-chlorophenyl, and R.sup.2 is a tri-C.sub.1 -C.sub.6 alkylsilyl group 
such as t-butyldimethylsilyl or triethylsilyl. This preference for R.sup.2 
holds for all of the compounds in which such a group is present. 
Another contemplated compound corresponds in structure to that shown in the 
formula below, 
##STR2## 
where R.sup.1 is hydrogen or COR, and R is a moiety selected from the 
group consisting of hydrogen, C.sub.1 -C.sub.5 alkyl, phenyl and 
m-chlorophenyl; 
R.sup.2 is a moiety selected from the group consisting of tri-C.sub.1 
-C.sub.6 alkylsilyl, di-C.sub.1 -C.sub.6 alkylphenylsilyl, and C.sub.1 
-C.sub.6 alkyldiphenylsilyl; and 
--W is (i) an N-hydroxy cyclic imido group, --ON=R.sup.4 in which R.sup.4 
has 4 to about 8 carbon atoms or (ii) an O,N-di-substituted oxime group, 
--ON=R.sup.5, whose R.sup.5 carbon-containing portion is derived from a 
group consisting of a C.sub.1 -C.sub.6 alkyl ketone or aldehyde, and a 
tetrahydropyranone derivative with the proviso that R.sup.1 is COR when 
--W is --ON=R.sup.4. 
In one preferred embodiment, --W is --ON=R.sup.4, and --ON=R.sup.4 together 
constitute the residuum of an N-hydroxy cyclic imido group having 4 to 
about 8 carbon atoms as are present in an N-hydroxysuccinimido or 
N-hydroxyphthalimido moiety, and R.sup.1 is COR. 
In another preferred embodiment, --W is --ON=R.sup.5, and --ON=R.sup.5 
together constitute an O,N-di-substituted oxime whose carbon-containing 
R.sup.5 portion is derived from a group consisting of a C.sub.1 -C.sub.6 
ketone or aldehyde and a tetrahydropyranone derivative. R.sup.5 is most 
preferably derived from a tetrahydropyran-4-one that can also contain a 
tetrahydropyran substituent. 
A compound whose structure corresponds to that of the formula shown below 
constitutes yet another contemplated compound of this invention. 
##STR3## 
wherein R.sup.2 is a moiety selected from the group consisting of 
tri-C.sub.1 -C.sub.6 alkylsilyl, di-C.sub.1 -C.sub.6 alkylphenylsilyl, and 
C.sub.1 -C.sub.6 alkyldiphenylsilyl; 
--ON=R.sup.5 is an O,N-disubstituted oxime group in which R.sup.5 is 
derived from a group consisting of a C.sub.1 -C.sub.6 alkyl ketone or 
aldehyde, and a tetrahydropyranone derivative; and 
R.sup.6 is selected from the group consisting of hydrogen, C.sub.1 -C.sub.6 
alkyl, benzoyl, m-chlorobenzoyl, C.sub.1 -C.sub.6 alkyl oxycarbonyl and 
N-carbonyl imidazyl. 
R.sup.6 is most preferably hydrogen or m-chlorobenzoyl, and R.sup.2 and 
R.sup.5 are preferably as discussed above. 
Also contemplated is a compound whose structure corresponds to that shown 
below in Formula VII. 
##STR4## 
wherein R.sup.8 is hydrogen or a selectively removable nitrogen atom 
protecting group such as FMOC, t-BOC, CBZ or NVOC as is used for 
protecting .alpha.-amino groups during peptide synthesis reactions; 
R.sup.9 is C.sub.1 -C.sub.6 alkyl, with methyl, ethyl and iso-propyl being 
particularly preferred; 
R.sup.10 is hydrogen or a previously defined R.sup.2 group; 
Z is O or an oximino group that is the reaction product of the compound 
where Z=O with hydroxylamine or with an O-substituted hydroxylamine having 
up to 7 carbon atoms in the substituent such as O-benzylhydroxylamine or 
O-methylhydroxylamine; and 
X is selected from the group consisting of o-nitrobenzyloxy, benzyloxy, 
halo such as chloro, bromo or preferably fluoro, hydroxyl, and 
trichloroacetimido [OC(NH)CCl.sub.3 ]. 
A disaccharide compound of Formula VII is useful as an intermediate and in 
forming a chimeric antibiotic with an aglycone as is shown in FIG. 14. 
Exemplary compounds whose structures correspond to Formula VII include 
Compounds 148, and 160-167. 
A compound whose structure corresponds to that of Formula VIII, shown 
below, constitutes another embodiment of this invention. 
##STR5## 
In the above formula, X is a leaving group, as can be selected from the 
group consisting of o-nitrobenzoxy, benzoxy, halo such as chloro, bromo, 
or preferably fluoro, and trichloractimido, and R.sup.8, R.sup.9 and 
R.sup.10 (R.sup.8-10) are as previously described, with R.sup.8 preferably 
being FMOC. A compound of Formula VIII is particularly useful as an 
intermediate in preparing calicheamicin .gamma..sub.1.sup.I and chimeras 
with DNA-cleaving compounds as are discussed hereinafter. 
A chimer of Formula IX is also contemplated, wherein Z is a reacted 
DNA-cleaving compound discussed or shown herein, and R.sup.8-10 are as 
discussed above, and in which R.sup.8 is preferably FMOC. 
##STR6## 
A compound of Formula X is also a contemplated chimer of the invention, 
where Z is a reacted DNA-cleaving compound other than calicheamicinone 
when R.sup.9 is ethyl, and R.sup.9 is defined above. The wavy line to the 
nitrogen of the hydroxylamine indicates that both of the .alpha.- and 
.beta.-epimers are contemplated. 
##STR7## 
A molecule that corresponds in structure to Formula XI, below, is also 
contemplated herein, wherein R.sup.12 is --OR.sup.2, o-nitrobenzyloxy, 
benzyloxy, halo such as chloro, bromo or more preferably fluoro or 
trichloroacetimido. 
##STR8## 
A molecule of Formula XI corresponds to the smaller esperamicin saccharide 
and is useful in preparing chimers as discussed elsewhere herein. 
A method of forming (preparing) a compound whose structure corresponds to 
that of Formula A is also contemplated. 
##STR9## 
wherein R.sup.2 is a moiety selected from the group consisting of 
tri-C.sub.1 -C.sub.6 alkylsilyl, di-C.sub.1 -C.sub.6 alkylphenylsilyl, and 
C.sub.1 -C.sub.6 alkyldiphenylsilyl; and 
--ON=R.sup.5 is an O,N-disubstituted oxime group in which R.sup.5 is 
derived from a group consisting of a C.sub.1 -C.sub.6 alkyl ketone or 
aldehyde and a tetrahydropyranone derivative; 
comprising the steps of 
a) heating the compound whose structure corresponds to that of Formula B 
##STR10## 
wherein R.sup.2 and R.sup.5 are defined above, in a liquid medium for a 
time period and at a temperature sufficient for that compound to rearrange 
to form a compound whose structure corresponds to that of Formula A. 
The N-carbonylimidazyl group of a compound whose structure corresponds to 
that of Formula A is preferably replaced with hydrogen. It is also 
preferred that the compound of Formula A and the compound having hydrogen 
in place of N-carbonylimidazyl be recovered.