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Title:
COMPOSITION FOR DETERMINING THE LACTASE ACTIVITY
Document Type and Number:
WIPO Patent Application WO/2002/007778
Kind Code:
A1
Abstract:
The present invention relates to a composition for determining the lactase activity in a mammal. By means of the composition the lactase activity of a mammal, in particular a human being, can be determined in a manner that causes little discomfort. The composition according to the invention comprises lactose enriched with a first isotope (e.g. ?13¿C), and a source of an (Oligo)pentose or hexose enriched with a second isotope (e.g. ?2¿H). Establishing the isotope ratios after absorption via the intestine allows an extremely reliable determination of the lactase activity.

Inventors:
VONK ROELF JAN (NL)
Application Number:
PCT/NL2001/000569
Publication Date:
January 31, 2002
Filing Date:
July 23, 2001
Export Citation:
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Assignee:
STICHTING TECH WETENSCHAPP (NL)
VONK ROELF JAN (NL)
International Classes:
C07H3/04; C12Q1/34; (IPC1-7): A61K49/00; C07H3/04; C12N9/38
Other References:
R.J. VONK ET AL.: "Lactose (mal)digestion evaluated by the 13C-lactose digestion test", EUR. J. CLIN. INVEST., vol. 30, 2000, pages 140 - 146, XP000998498
R.J. VONK ET AL.: "The 13C/2H-glucose test for the determination of small intestinal lactase activity", EUR. J. CLIN. INVEST., vol. 31, 2001, pages 226 - 233, XP000998471
Attorney, Agent or Firm:
Altenburg, Bernardus Stephanus Franciscus (Octrooibureau Los En Stigter B.V. Weteringschans 96 XS Amsterdam, NL)
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Claims:
We claim:
1. A compound of the formula: AGENT wherein zz is 0 or 1, R^ is an acyl group derived from a Cj_ to C30 alkyl group, and R2 is hydrogen, an organic or inorganic cation , a carboxy protecting group, or a non toxic, metabolicallylabile esterforming group; and wherein the Cytotoxic Agent is a compound of the formula: .
2. A compound of claim 1, wherein R^ is i) Ci to C4 alkyl, Ci to C6 substituted alkyl, Ci to C4 alkoxy, Ci to C4 alkylthio, or a group of the formula: F3CSCHr CH2 NC \ :or NH2 or a protected amino and/or protected carboxy derivative thereof; ii) a group of the formula: wherein each of R5, Rβ , and R7 is, independently, hydrogen, halo, hydroxy, protected hydroxy, nitro, amino, protected amino, an amine salt, cyano, trifluoromethyl, aminomethyl, protected aminomethyl, N (methyl or ethylsulfonyDamion, Ci to C6 alkyl or Cl to C4 alkoxy, R4 is hydroxy, protected hydroxy, formyloxy, amino, protected amino, an amine salt, carboxy, a carboxylate salt, protected carboxy, phenyl carboxylate, (5indanyl)carboxylate, sulfonic acid, a sulfonate salt, azido, halo or Ci to Cζ alkyl; Rβ is Ci to C4, alkoxy; Z is oxygen or sulfur; n is 0, 1, 2, or 3; and m is 0 or 1; iii) a group of the formula: wherein R9 is a heterocyclic ring; Rio is hydroxy, protected hydroxy, formyloxy, amino, protected amino, an amine salt, carboxy, a carboxylate salt, protected carboxy, phenyl carboxylate, (5indanyl)carboxylate, sulfonic aacid, a sulfonate salt, azido, halo or Ci to Cβ alkyl; n is 0, i, 2 or 3; and Z is oxygen or sulfur.
3. A compound of claim 2, wherein R**_ is acetyl, propionyl, butanoyl, isopropionyl, pivaloyl, methoxymethyl, phenylacetyl, phenoxyacetyl, 2 (aminomethyl)phenylacetyl, 2phenyl2hydroxyacetyl, 2 phenyl2 (sodium sulfonato) acetyl, 2phenyl22 carboxyacetyl, 2 (4hydroxyphenyl) 2carboxyacetyl, 2 phenyl2aminoacetyl, 2 (4hydroxyphenyl) 2aminoacetal, 2 (3 (N (methylsulfonylamino) )phenyl) 2aminoacetyl, 2 phenyl2 (5indanyl carboxylate)acetyl, 2phenyl2 (phenyl carboxylate)acetyl, 2phenyl2azidoacetyl, 2 phenoxypropionyl, 2,5dimethoxybenzoyl, 2 (formyloxy) 2 phenylacetyl, 2 (2ethoxynaphth1yDacetyl, 2(naphthl yl) 2aminoacetyl, 2 (naphth2yl) 2aminoacetyl, 2 (2,5 dichlorophenylthio)acetyl, 2 (3,4 dichlorophenylthio)acetyl, 2 (1tetrazolyl)acetyl, 2 (N (3 ,5dichloropyrid4oxyl) )acetyl, 2 (2aminothiazol4 yDacetyl, 2 (2thienyl)acetyl, 2 (4pyridylthio)acetyl, 2 (Nmethyl4pyridiniumthio)acetyl, 2 (2amino4 phenylthiazol5yl)acetyl, 2 (3hydroxy4 carboxyisothiazol5ylthio)acetyl, 3phenyl5 methylisoxazolyl3formyl, 3 (2chlorophenyl) 5 methylisoxazolyl3formyl, 3 (2,5dichlorophenyl) 5 methylisoxazolyl3formyl, 3 (2fluoro5chlorophenyl) 5 methylisoxazolyl3formyl, 2 (2thienyl) 2aminoacetyl, 2 (2thienyl) 2 (sodium carboxylate)acetyl, 2(N(4 pyridinium) )acetyl, 2 (2benzothienyl)acetyl, 2(3 benzothienyl)acetyl, 2 (2benzofuryl)acetyl, and 2(3 benzofuryl)acetyl.
4. A compound of claim 3, wherein R*_ is 2 (thien2yl)acetyl and zz is 0.
5. A compound of claim 4, wherein the doxorubicin moiety is bound to the cephalosporin through the aminosaccharide nitorgen which is attched to the carbonyl of the 3 ' carbamato group of the cephalosporin.
6. A compound of claim 3, wherein Ri is 2 (thien2yl)acetyl and zz is 1.
7. A compound of claim 6, wherein the doxorubicin is bound to the cephalosporin through the aminosaccharide nitrogen which is attached to the carbonyl of the 3 'carbonato group of the cephalosporin.
8. A compound of claim 3, wherein Rj_ is phenoxyacetyl and zz is 1.
9. A compound of claim 8, wherein the doxorubicin is bound to the cephalosporin through the aminosaccharide nitrogen which is attached to the carbonyl of the 3 'carbonato group of the cephalosporin.
10. A method for the treatment of neoplastic diseases, which comprises: A) administering a therapeutically effective amount of the AntibodyEnzyme conjugate of the formula; Antibody (βLactamase Enzyme) wherein: the Enzyme reacts with a compound of claim 1, and causes the seperation of the Cytotoxic Agent from the cephalosporin; the Antibody complexes with antigens expressed by the target tissue. B) administering a therapeutically effective amount of the Substrate Cytotoxic Agent of claim 1 to the affected host, wherein the Substrate is the subtrate for the Enzyme.
11. A method of treatment of claim 10, wherein the Antibody complexes with careinoembryonic antigen.
12. A method of treatment of claim 11, wherein the Antibody complexes with the KS1/4 antigen.
13. A method of treatment of claim 12, wherein the Antibody complexes with the TAG72 antigen.
14. A method of treatment of claim 13, wherein the Antibody complexes with the MAT65 antigen.
15. A method of treatment of claim 14, wherein Ri is: NH2 or a protected amino and/or protected carboxy derivative thereof; ii) a group of the formula: wherein each of R5, Rβ , and R7 is, independently, hydrogen, halo, hydroxy, protected hydroxy, nitro, amino, protected amino, an amine salt, cyano, trifluoromethyl, aminomethyl, protected aminomethyl, N (methyl or ethylsulfonyl)amino, Ci to Cβ alkyl or Ci to C4 alkoxy, R4 is hydroxy, protected hydroxy, formyloxy, amino, protected amino, an amine salt, carboxy, a carboxylate salt, protected carboxy, phenyl carboxylate, (5indanyl) carboxylate, sulfonic acid, a sulfonate salt, azido, halo or Ci to Cβ alkyl; Rβ is Ci to C4, alkoxy; Z is oxygen or sulfur; n is 0, 1, 2, or 3; and m is 0 or 1; iii) a group of the formula: R9(CH2)n; wherein R9 is a heterocyclic ring; Rio is hydroxy, protected hydroxy, formyloxy, amino, protected amino, an amine salt, carboxy, a carboxylate salt, protected carboxy, phenyl carboxylate, (5indanyl) carboxylate, sulfonic acid, a sulfonate salt, azido, halo or Ci to Cβ alkyl; n is 0, 1, 2, or 3; and Z is oxygen or sulfur.
16. A method of treatment of claim 15, wherein Rj_ is acetyl, propionyl, butanoyl, isopropionyl, pivaloyl, methoxymethyl, phenylacetyl, phenoxyacetyl, 2 (aminomethyl)phenylacetyl, 2phenyl2hydroxyacetyl, 2 phenyl2 (sodium sulfonato)acetyl, 2phenyl22 carboxyacetyl, 2(4hydroxyphenyl) 2carboxyacetyl, 2 phenyl2aminoacetyl, 2 (4hydroxyphenyl) 2aminoacetal, 2 (3 (N(methylsulfonylamino) )phenyl) 2aminoacetyl, 2 phenyl2(5indanyl carboxylate)acetyl, 2phenyl2(phenyl carboxylate)acetyl, 2phenyl2azidoacetyl, 2 phenoxypropionyl, 2,5dimethoxybenzoyl, 2 (formyloxy) 2 phenylacetyl, 2 (2ethoxynaphth1yDacetyl, 2 (naphthl yl)2aminoacetyl, 2 (naphth2yl)2aminoacetyl, 2 (2,5 dichlorophenylthio)acetyl, 2(3, dichlorophenylthio)acetyl, 2 (1tetrazolyl)acetyl, 2(N (3,5dichloropyrid4oxyl) )acetyl, 2(2aminothiazol4 yDacetyl, 2 (2thienyl)acetyl, 2 (4pyridylthio)acetyl, 2* (Nmethyl4pyridiniumthio)acetyl, 2(2amino4 phenylthiazol5yl)acetyl, 2(3hydroxy4 carboxyisothiazol5ylthio)acetyl, 3phenyl5 methylisoxazolyl3formyl, 3(2chlorophenyl)5 methylisoxazolyl3formyl, 3 (2,5dichlorophenyl)5 methylisoxazolyl3formyl, 3(2fluoro5chlorophenyl)5 methylisoxazolyl3formyl, 2 (2thienyl)2aminoacetyl, 2 (2thienyl)2 (sodium carboxylate)acetyl, 2(N(4 pyridinium) )acetyl, 2 (2benzothienyl)acetyl, 2(3 benzothienyl)acetyl, 2 (2benzofuryl)acetyl, or 2(3 benzofuryl)acetyl.
17. A method of treatment of claim 16, wherein Rj_ of the cephalosporin Substrate is 2 (thien2yDacetyl and zz is 0.
18. A method of treatment of claim 17, wherein the Antibody is CEM231.6.7.
19. A method of treatment of claim 18, wherein the Cytotoxic Agent the doxorubicin is bound to the cephalosporin through the aminosaccharide nitrogen which is attached to the carbonyl of the 3 'carbonato group of the cephalosporin.
20. A method of treatment of claim 16, wherein R**L is 2 (thien2yl) acetyl and zz is 1.
21. A method of treatment of claim 20, wherein the Antibody is designated CEM231.6.7.
22. A method of treatment of claim 21, wherein the Antibody is designated CC49.
23. A method of treatment of claim 22, wherein the Antibody is designated ZCE025.
24. A method of treatment of claim 2, wherein doxorubicin is bonded through the aminosaccharide nitrogen to the carbonyl group of the 3 ' carbonato cephalosporin.
Description:
Title

Method for Delivery of Cytotoxic Agents and Components Thereof

Over the years, many cytotoxic drugs useful for treating neoplastic diseases have been developed. The chief drawback of all of the useful drugs has been the indiscriminant toxicity of these drugs to all cells of the patient, thus limiting their dosage and consequently the effectiveness of the drugs. The present invention, whereby a latent form of the Cytotoxic Agent is activated at the site of the neoplastic disease, offers a marked advantage in diminishing or possibly eliminating unwanted side effects of the Agent. As a further consequence of reduced deleterious side effects, higher concentrations of the drug at the tumor site may be brought about through administration of the Cytotoxic Agent at increased dosage.

One aspect of the present invention is a method for the treatment of neoplastic diseases, which comprises:

A) administering a therapeutically effective amount of an Antibody-Enzyme conjugate to the affected host; and then

B) administering a therapeutically effective amount of a Substrate - Cytotoxic Agent to the affected host, wherein the Substrate is a substrate for the Enzyme; wherein the Antibody-Enzyme conjugate and the Substrate - Cytotoxic Agent are as defined below in the Detailed Description.

Other aspects of the invention include the Antibody-Enzyme conjugates and the Substrate - Cytotoxic Agent compounds as defined below in the Detailed Description.

FIG.l depicts the biodistribution of the anti KS1/4 -- β-lactamase conjugate in nude mice as % injected dose in each organ divided by the organ weight.

FIG.2 depicts the same measurement of FIG.l expressed as % injected dose in each organ.

FIG.3 demonstrates the inhibition of T380 tumor growth in nude mice by the combination anti-CEA -- β- lactamase conjugate and the prodrug compound of Example 11. Therapy was administered in three courses over three weeks, each course consisting of 35μg conjugate followed after 72 hours by four daily doses of lmg prodrug/kg of body weight.

FIG.4 depicts a similar inhibitory effect of the same conjugate prodrug combination as in FIG.3 on T380 tumors, except that the prodrug is administered in four daily doses of 0.25mg of prodrug/kg of body weight.

FIG.5 compares the inhibition of LS174T tumor growth in nude mice by the combination of either anti-KSl/4 antibody --β-lactamase conjugate or anti-CEA -- β-lactamase conjugate and the prodrug of Example 11.

FIG.6 depicts the inhibitory effect of a combination of an anti-TAG72 -- β-lactamase conjugate and the prodrug of Example 11 on LS174T tumors in nude mice.

FIG.7 is a comparison of the effect of the dose of conjugate on the inhibition of T380 tumor growth by the combination of anti-CEA -- β-lactamase conjugate of this invention and the prodrug of Example 11.

One aspect of this invention is an Enzyme- Antibody conjugate of the formula

Antibody-Enzyme I

wherein : the Enzyme reacts with a compound of the formula

Substrate - Cytotoxic Agent II

which compound is described below, such that the Substrate is separated from the Cytotoxic Agent; and the Antibody complexes with the target tissue.

The Antibody of the present invention complexes with one or more antigens of the target cells. The target cells are a part of neoplastic tissues, whether benign or malignant. Examples of such target cells include psoriasis, squamous carcinoma cells, adenocarcinoma cells, small cell carcinoma cells, glyoma cells, melonoma cells, renal cell carcinoma cells, transitional cell carcinoma cells, sarcoma cells, cells of supporting tumor vasculature, and cells of lymphoid tumors such as leukemias and lymphomas.

The Antibody may be chosen from any class or subclass of immunoglobulin including IgG, IgA, IgM, IgE, and IgD. The species of origin is not critical so long as the Antibody complexes with target cells.

In the present state of the art, monoclonal antibodies are most preferred for use in the present invention. However, polyclonal antibodies are not excluded. A newer type of Antibody is the chimeric antibody, which is prepared in the laboratory by recombinant technology which permits expression of a modified DNA which encodes the antigen-binding region of any desired antibody, and also encodes any other desired amino acid sequences. Thus, chimeric antibodies of which one portion is derived from one species, and another

portion is derived from a different species, may be obtained and used in the present invention.

The origin and nature of the Antibody is not otherwise critical, so long as it targets the cell to be treated and is not, in itself, toxic to the patient. Those of ordinary skill can readily prepare enzyme conjugates with a candidate Antibody and evaluate them. How such antibodies are chosen will be discussed in detail below.

It will be understood that properly chosen fragments of antibodies have the same effect as the Antibody. Thus, in the practice of this invention, fragments of antibodies, such as F(ab')2 F(ab') and single domain antibodies (V H regions, or dAbs) and especially

F(ab') fragments, which recognize an antigen associated with the cell to be treated, may be just as useful as are intact antibodies. (dAbs are described by Ward E. , et al. , Nature. 341. p.544, 1989; Orlandi R. , et al.. Proc. Natl. Acad. Sci.. (USA) Jϋ . P-3833, 1989.)

A great number of antibodies are available to immunologists for use in the present invention, and further useful antibodies are being disclosed in every issue of the relevant journals. It is impossible, and entirely unnecessary, to give an exhaustive listing of antibodies which can be applied in the practice of this invention. Immunologists and chemists of ordinary skill are entirely able to choose antibodies from sources such as the catalogue of the American Type Culture Collection (ATCC) , Rockville, Maryland, U.S.A., and Linscott's Directory of Immunological and Biological Reagents, published by Linscott's Directory, 40 Glen Drive, Mill Valley, California, U.S.A., 94941. Thus, it is a simple matter for the artisan in the field to choose an antibody against virtually any antigen on the present target cells.

Alternatively, the necessary hybridomas for the production of such antibodies are obtainable through the ATCC or the Northern Regional Research Laboratories (NRRL)

of the U.S. Department of Agriculture, Peroia, Illinois, U.S.A. and other cell line collections.

A number of presently known antibodies are particularly interesting for use in the present invention. One preferred specific antibody is L/1C, produced by ATCC hybridoma HB9682. Another preferred is produced by ATCC hybridoma HB9620, which antibody (designated CEM231.6.7) complexes with a convenient carcinoembryonic antigen expressed by several types of tumor cells. Recently, chimeric antibody CEM231.6.7 has been described in U.S. Patent Application Nos. 07/165,856 and 07/272,577, filed March 3, 1988, and November 17, 1988, respectively. The Antibody is also discussed in Beidler C.B., et al.. J. Immunology. 141. pp.4053-4060, 1988. An Antibody which reacts with the MAT65 T-cell marker is the T-101 antibody. The T101 antibody is produced by ATCC hybridoma #CRL8023 and is described in U.S. Patent No. 4,675,386.

Another interesting antibody is KSl/4, first disclosed by Varki et al.. Cancer Research. 44, pp.681-686,

1984. A number of plasmids which comprise the coding sequences of the different regions of monoclonal antibody KSl/4 are now on deposit and can be obtained from the Northern Regional Research Laboratory. The plasmids can be used by those of ordinary skill to produce chimeric antibodies by recombinant means, which antibodies bind to a cell surface antigen found in high density on adenocarcinoma cells. The construction of such antibodies is discussed in detail in U.S. Patent Application No. 07/184,522, filed April 21, 1988. The following plasmids relate to KSl/4.

Plasmids pGKC2310, the coding sequence of the light chain, the signal peptide associated with the light chain, and the 5' and 3' untranslated regions; isolated from E.coli K12 MM294/pGKC2310, NRRL B-18356.

Plasmids pG2A52, the coding sequence of the heavy chain, the coding sequence of the signal peptide

associated with the heavy chain, and the 5' and 3 ' untranslated regions; isolated from E.coli K12

MM294/pG2A52, NRRL B-18357.

Plasmid CHKC2-6, the coding sequence of the light chain variable region, the coding sequence of the signal peptide associated with the light chain, and a sequence encoding the light chain constant region of a human IgG; isolated from E.coli K12 DH5/CHKC2-6, NRRL B-

18358.

Plasmid CHKC2-18, the coding sequence of a derivative light chain variable region, the coding sequence of the signal peptide associated with the light chain, and a sequence encoding the light chain constant region of a human IgG; isolated from E.coli K12 DH5/CHKC2-18, NRRL B- 18359.

Plasmid CH2A5, the coding sequence of the heavy chain variable region, the coding sequence of the signal peptide associated with the heavy chain, and a sequence encoding the heavy chain constant region of human igGl; isolated from E. coli K12 MM294/CH2A5, NRRL B-18360.

Plasmid CH2A5IG2, the coding sequence of the heavy chain variable region, the coding sequence of the signal peptide associated with the heavy chain, and a sequence which encodes the heavy chain constant region of human IgG2; isolated from E.coli K12 DH5/CH2A5IG2, NRRL B- 18361.

Plasmid CH2A5IG3, the coding sequence of the heavy chain variable region, the coding sequence of the signal peptide associated with the heavy chain, and a sequence encoding the heavy chain constant region of human IgG3; isolated from E.coli K12 DH5/CH2A5IG3, NRRL B-18362.

Plasmid CH2A5IG4, the coding sequence of the heavy chain variable region, the coding sequence of the signal peptide associated with the heavy chain, and a sequence encoding the heavy chain constant region of human IgG4; isolated from E.coli K12 DH5/CH2AIG4, NRRL B-18363.

Antibody 5E9C11, produced by an ATCC hybridoma, HB21, recognizes transferrin receptor, which is expressed by many tumors. An Antibody called CC49, available from the National Cancer Institute (NCI) , recognizes the TAG-72 antigen expressed by both breast and colon carcinoma (Muraro R., et al. Cancer Research. 48. pp. 588-4596,

1988) . Also Antibody ZCE025 is suitable for use in this invention. The antibody was first described by Jean-Pierre Mach and his group (e.g., Mach J.P., et al.. Int. J. Cancer. 33. pp.643-649, 1984; and Patt Y. , et al. , Cancer Bull.. 40. pp.218-221, 1988). Another useful Antibody, reactive with an antigen present on many carcinomas, designated BRE3 and is useful in the practice of this invention (Peterson J.A., et al.. Hybridoma, £, pp.221-235, 1990) . Yet another useful antibody for this invention also recognizes the KS1/4 antigen and is designated 007B. The antibody is available from Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285.

The Enzyme for the present conjugate is one that recognizes a Substrate, and which will also recognize that Substrate when it is bound to the present Cytotoxic Agent. Furthermore, the Enzyme must retain its activity once conjugated to the present Antibody.

As with the Antibody, it is impossible, and unnecessary to list all of the enzymes for use in the instant invention. Many suitable examples are available commercially, such as from Sigma Chemical Company, St. Louis, Missouri; Boehringer Mannheim Company, Indianapolis, Indiana; Cal Biochem, San Diego, California, and the like. Various methods for isolating and purifying the enzymes can be found in such reference works as Methods In Enzvmolocrv and the like. One skilled in the art will also realize that fragments of enzymes that retain the whole enzymes catalytic activity and specificity (or any catalytic agent, such as catalytic antibodies, with comparable activity and specificity) are suitable for use in the present invention.

Enzymes that can be used in this invention catalyze one of two broad classes of reactions. The first class of reaction is one in which the Substrate - Cytotoxic Agent compound is cleaved into its two component parts. The activity of the Enzyme toward the Substrate must be substantially unimpaired by the presence of the Cytotoxic Agent. A preferred Enzyme for this type of reaction is one that is practically insensitive to the presence of the Cytotoxic Agent, regardless of the structure of the Agent. Such an Enzyme would allow that particular conjugate to be used for a variety Substrate - Cytotoxic Agent combinations. Examples of such (insensitive) Enzymes include β-galactosidase, the various isoenzymes of alkaline phosphatase, certain carboxypeptidases (such as that of Pseudomonas s . ) . D-aminopeptidase (Asano Y., et al. , J. Biol. Chem.. 264. pp.14233-14239, 1989; Asano Y. , et al. Biochem. Biophvs. Res. Comm.. 162, pp.470-474, 1989; Asano Y., et al.. Biochem.. 31. pp.2316-2328, 1992), pyroglutamate aminopeptidase, and various β-lactamases.

These Enzymes recognize galactose residues, phosphate residues, certain simple amino acids, 1-pyroglutamate, d- amino acid amides and cephalosporin compounds, respectively, as Substrates, and separate these Substrates from any other molecules to which they are bound. Inherent in these desired properties for the Enzymes is the trait that the Enzyme reacts only with one particular functional group, i.e., the functional group that links the Substrate and Cytotoxic Agent, such that the Cytotoxic Agent can be substituted with any number of different functional groups that are unaffected by the Enzyme, β-lactamase is a prime example of an Enzyme with such properties. The penicillins, cephalosporins, cephalosporin εulfoxides, 1- oxadethiacephalosporins, and 1-carbadethiacephalosporins that are its Substrates are composed of a number of functional groups that are untouched by the Enzyme. The beta-lactamase simply reacts with the β-lactam ring of

these substrate compounds. As a result of this reaction the 3 ' - substituent of the Substrate will often cleave from the rest of the cephalosporin nucleus.

The second class of reactions catalyzed by the present Enzymes are anabolic ones, in other words, reactions where the Enzyme converts the precursor into a Cytotoxic Agent. Thus, with this type of Enzyme, the precursor would serve as first the Substrate and then the Cytotoxic Agent. Examples of such combination Substrate- Enzyme compounds are the trichothecenes, saxitonin, or amanitin.

Regardless of the class of reaction it catalyzes, the purpose of the Enzyme is the same - to generate the Cytotoxic Agent in vivo (and in high concentration in the loci of the Antibody-Enzyme conjugate) after the Cytotoxic Agent has been administered to the patient masked in the form of a Substrate - Cytotoxic Agent compound. Enzymes that can be used for the instant invention include the various β-lactamases, regardless of the species of microorganism the Enzyme is isolated from, L-pyroglutamate aminopeptidase, β-galactosidase, D-amino peptidase, the various isoenzymes of alkaline phosphatase, various carboxypeptidases such as Pseudomonas so. carboxypeptidase G2, and the like.

Enzymes that are most useful are those that can be inhibited specifically by substances that are both not normally found in the body and that do not inhibit other enzymes found in the body. Especially useful examples of this class of Enzyme are the β-lactamases, which can be inhibited by various β-lactamase resistant penicillins and cephalosporins, such as cloxacillin. Such penicillins and cephalosporins often have the advantage that they are already approved for use in humans. Furthermore, being molecules of relatively low molecular weight, they would be expected to rapidly penetrate organs where the

prodrug/Antibody-Conjugate drug is causing undesired effects.

Methods for conjugating the Enzyme and Antibody are well known in the art. See, for example, a listing of the protein-protein coupling reagents commercially available from Pierce Chemical Company. The various functional groups employed for enzyme-antibody bonding are generally not determined, and furthermore it is not important for the purposes of this invention. All that is required is that the Antibody retain a substantial part of the immunoreactivity of its unconjugated state. Similarly, all that is required of the conjugated Enzyme is that it retain a substantial portion of its activity from its unconjugated state. Finally, both the conjugate and a newly formed Substrate-Cytotoxic Agent must demonstrate practical pharmacokinetics in the affected host. Reagents that can be used in conjugating the Enzyme to the Antibody include SMCC, sulfo-SMCC, SPDP, 2-iminothiolane, MBS, Sulfo-MBS, SMPB, Sulfo-SMPB, SIAB, sulfo-SIAB, GMBS, EMCS, N,N' -bis(3-maleimidopropionyl) -2-hydroxy-l,3- propanediamine, N-succinylbromoacetate (Sigma Chemical Company), BMME (Boehringer Mannheim) and the like. Depending on the particular functional groups available for reaction on both the Antibody and the Enzyme, the Antibody fragment chosen, and the reactivity of residues in or near the enzyme active site and antibody binding site, these reagents may be used in combination. Also, reagents that crosslink between two amino groups can be used, such as DMA or DSS (Pierce Chemical Company) . Certain enzymes and antibodies may have amino, sulfhydryl, or hydroxyl groups in their active sites or binding sites, respectively. Thus, these reactive functional groups can first be masked by protecting groups such as citraconic anhydride, DTNB, and DNFB (according to procedures referenced in the Pierce Chemical Company Catalog and Schmuel and Shaltiel, Biochem. and Bioohvs Res. Comm.. 29. pp.178-183, 1967) before they

are conjugated, with for example, the reagents listed above. The Pierce Chemical Company Catalog lists references describing the use of many of the reagents above. The use of any remaining reagents may be found in the scientific literature.

It is also desirable, but not essential, that the Enzyme not be found in humans, and also that the Substrate not be found in humans. One skilled in the art could readily see that if the Enzyme or the Substrate is found in the human body, the chance for the Cytotoxic Agent to be released in an area other than the location of the target tissue is increased.

Although any combination of Antibody and Enzyme as described above is sufficient for the present invention, the preferred conjugates are ones that are as homogenous as possible given the conjugation technology. Unlike whole antibody, F(ab') fragments may be derivitized regiospecifically through the thiol residues found near the carboxy terminus and away from the antigen binding site, and are preferred in the invention. Other methods are available for preparing conjugates that are more homogeneous. One such method is disclosed by Drs. Offord and Rose in European Patent Applicatios Nos. 243 929, 359 428, and 360 433, and in Fisch I., Kϋnzig G., Rose K. , and Offord R. , Site-Specific Modification of a Fragment of a Chimeric Monoclonal Antibody Using Reverse Proteolysis, Bioconi . Chem.. 3 , pp.147-153, 1992. Another method involves cloning and fusion of genes encoding the functional portions of the Enzyme and Antibody molecules; fusing these genes together, and expressing the fusion protein in an appropriate host cell. Such fusion proteins have been prepared. (European Patent Application No. 392 745 AZ) . Furthermore, the conjugate should not be metabolized in degratory organs such as the liver and the spleen, an event that could prevent sufficient conjugate from accumulating at the target tissue. Yet, on the other

hand, it is desirable that the conjugate be rapidly cleared from the bloodstream after the conjugate has had the opportunity to localize to the target tissue. Both of these pharmacokinetic properties reduce the chance that Cytotoxic Agent will be generated at a site other than the target tissue. These pharmacokinetic properties are facilitated by conjugate with a low molecular weight, a property supplied by Antibody fragments, and particularly F(ab') fragments. Along the same line of thought, Enzymes with a low molecular weight (e.g., 50,000 or less) are preferred for the same reason as the Antibody fragments. Preferred embodiments of the present Antibody- Enzyme conjugate include:

A. A conjugate as described above, wherein additionally the Enzyme is β-lactamase, β-galactosidase, pyroglutamate aminopeptidase, or D-aminopeptidase;

B. A conjugate of A, wherein additionally the Enzyme is β-lactamase;

C. A conjugate of B, wherein additionally the Antibody complexes with antigens expressed solely, or in supernatural abundance, by malignant tumor cells;

D. A conjugate of C, wherein additionally the Antibody complexes with carcinoembryonic antigen;

E. A conjugate of C, wherein additionally the Antibody complexes with the KSl/4 antigen;

F. A conjugate of C, wherein additionally the Antibody complexes with the TAG-72 antigen;

G. A conjugate of C, which complexes with the MAT-65 antigen;

H. A conjugate of D, wherein additionally the Antibody is designated ZCE025;

I. A conjugate of D, wherein additionally the Antibody is designated CEM231.6.7;

J. A conjugate of C, wherein additionally the Antibody is designated T101;

K. A conjugate of C, wherein additionally the Antibody is designated CC49;

L. A conjugate of C, wherein additionally the Antibody is designated 007B;

M. A conjugate of C, D, E, F, G, H, I, J, K, or L, wherein additionally the Antibody is a F(ab') fragment and the Enzyme is a β-lactamase taken from

Enterobacter cloacae.

A further aspect of this invention are compounds of the Formula II:

Substrate - Cytotoxic Agent II

wherein: the Substrate is the Substrate for an Enzyme, or active fragment thereof, wherein reaction of said Enzyme with the Substrate - Cytotoxic Drug causes the chemical separation of the Substrate from the Cytotoxic Agent; and the Substrate and the Cytotoxic Agent are bonded together through an ether, thioether, ester, amide, amino, hydrazido, carbonate, carbamate, thiocarbamate, azacarbamate, thioester, thioamide, or thiocarbonate group formed from the appropiate reactive group on the Substrate and a hydroxy, thioether, amine, amido, hydrazido, carboxy, or carbamato group on the Cytotoxic Agent. Alternatively, the Substrate may be a precursor for a Cytotoxic Agent.

The term "Cytotoxic Agent" means compounds that are useful in the treatment of neoplasms, whether benign or malignant. Such drugs include, in general, alkylating agents, antiproliferative agents, tubulin-binding agents, cytotoxins in general, and the like. Preferred classes of such compounds are the nitrogen mustard agents, anti- folates, nucleoside analogs, the vinca alkaloids, the anthracyclines, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, the podophyophyllotoxins, the sulfonylureas (as described in

European Patent Publication No. 222,475, published May 20, 1987), and low-molecular-weight toxins such as the trichothecenes and the colchicines. Particularly useful members of those classes include, for example, doxorubicin, daunorubicin, aminopterin, methotrexate, taxol, methopterin, dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide, melphalan, vinblastine, vincristine, desacetylvinblastine hydrazide, leurosidine, vindesine, leurosine, trichothecene, desacetylcolchicine and the like. It will be understood that unimportant chemical modifications may be made by the ordinarily skilled chemist to the preferred and generally described compounds in order to make.reactions of them more convenient.

It will be understood that preferred Substrate - Cytotoxic Agent compounds are prepared from the preferred Cytotoxic Agent. The preferred embodiments of the compound of Formula II are listed below. In the following list, each new preferred embodiment carries all of the definitions and limitations of the preceeding embodiments to which it refers. Also, it will be understood that reference to an Enzyme in the present specification also refers to an active fragment thereof. Thus, preferred embodiments of this aspect of the invention include:

A. A compound of Formula II, wherein the Substrate is a substrate for a β-lactamase, L-pyroglutamate aminopeptidase, β-galactosidase, or D-aminopeptidase enzymes; (For example, the Substrate for a β-lactamase enzyme could be a penicillin, a penem, a carbapenem, cephalosporin, a cephalosporin sulfoxide, 1- carbadethiacephalosporin, or 1-oxadethiacephlosporin; )

B. A compound of embodiment A, wherein the Substrate is: i) a cephalosporin of the formula;

AGENT

wherein zz is 0 or 1, R*^ is an acyl group derived from a C- j _ to C30 alkyl group, R2 is hydrogen, an organic or inorganic cation, a carboxy-protecting group, or a non-toxic, metabolically-labile ester-forming group; ii) L-pyroglutamate; iii) β-D-galactose; or iv) D-amino acid, especially D-alanine C. A compound of embodiment B, wherein the Substrate is a cephalosporin of the formula:

AGENT

A compound of embodiment C, wherein the R^

i) Cl to C4 alkyl, Cl to C6 substituted alkyl, Ci to C4 alkoxy, Ci to C4 alkylthio, or a group of the formula:

F 3 C-S-CH ~

CH 2

NC ; or

or a protected amino and/or protected carboxy derivative thereof; ii) a group of the formula:

wherein each of R5, Rζ, and R7 is, independently, hydrogen, halo, hydroxy, protected hydroxy, nitro, amino, protected amino, an amine salt, cyano, trifluoromethyl, aminomethyl, protected aminomethyl, N- (methyl- or ethyl-sulfonyl)amion, Ci to Cδ alkyl or Ci to C4 alkoxy, R4 is hydroxy, protected hydroxy, formyloxy, amion, protected amino, an amine salt,

carboxy, a carboxylate salt, protected carboxy, phenyl carboxylate, (5-indanyl)-carboxylate, sulfonic acid, a sulfonate salt, azido, halo or Ci to Cβ alkyl; Rβ is Ci to C4, alkoxy; Z is oxygen or sulfur; n is 0, 1, 2, or 3; and m is 0 or 1; iii) a group of the formula:

R 9 -(CH 2 ) n -;

•■ -10

wherein R9 is a heterocyclic ring; Rio is hydroxy, protected hydroxy, formyloxy, amino, protected amino, an amine salt, carboxy, a carboxylate salt, protected carboxy, phenyl carboxylate, (5-indanyl)carboxylate, sulfonic aacid, a sulfonate salt, azido, halo or Ci to Cβ alkyl; n is 0, 1, 2, or 3; and Z is oxygen or sulfur.

E. A compound of embodiment D, wherein the Cytotoxic Agent is of the formulae:

wherein Rn is hydrogen or hydroxy;

wherein R12 is amino or hydroxy;

Rl3 is hydrogen or methyl;

Rl4 is hydrogen, fluoro, chloro, bromo, iodo;

Rl5 is hydroxy or a moiety which completes a salt of the carboxylic acid;

wherein Ri6 is hydrogen or methyl;

wherein R17 is amino, C1-C3 alkylamino, di-(Cι-C3* alkyl)amino, or C4-C6 polymethylene amino;

(VIII )

wherein one of the Ri8 moieties is a bond and the others are hydrogen;

wherein R19 is hydrogen or methyl; R20 is methyl or thienyl;

wherein R21 is H, CH3 or CHO; when R23 and R24 are taken singly, R24 is H, and one of R22 and R23 is ethyl and the other is H or OH; when R23 and R24 are taken together with the carbons to which they are attached, they form an oxirane ring in which case R22 is ethyl; R25 is hydrogen, (C1-C3 alkyl) -CO-, or chloro-substituted (C1-C3 alkyl) -CO; p is 0 or 1;

R28 i a bond, -(C2-C4 alkyl) -X, or a group that requires that p is 1 and which is in turn bonded to a carbonyloxy group;

X is -0-, -S-, or -NH-;

wherein R27 is a base of one of the formulae:

wherein R28 is hydrogen, methyl, bromo, fluoro, chloro, or iodo ;

R29 is -OR18 or -NHRis;

R30 is hydrogen, bromo, chloro, or iodo;

wherein A is -0-, -NCH3-, -CH2-, -CH2CH2-, or -CH2O- D is -CH2- or -0-; R31 is hydrogen or halo; R32 is halo or trifluoromethyl;

5-fluorouracil or desacetylcolchicine.

In the above preferred formulae, compounds of Formula III represent the anthrocyclines of compounds; Formula IV represents the methotrexate group of compounds; Formula V represents the mitomycins; Formula VI represents the bleomycins; Formula VII represents melphalan; Formula VIII represents 6-mercaptopurine; Formula IX represents cytosine arabinoside; Formula X represents the podophyllotoxins; Formula XI represents the vinca drugs; Formula XII represents the difluoronucleosides and Formula XIII and Formula XIV represents the sulfunoylurea compounds.

F. A compound of embodiment E, wherein R- ] _ is acetyl, propionyl, isopropionyl, pivaloyl, butanoyl, methoxymethylacetyl, phenylacetyl, phenoxyacetyl, 2- (aminomethyl)phenylacetyl, 2-phenyl-2-hydroxyacetyl, 2- phenyl-2- (sodium sulfonato) -acetyl, 2-phenyl-2-2- carboxyacetyl, 2- (4-hydroxyphenyl) -2-carboxyacetyl, 2- phenyl-2-aminoacetyl, 2- (4-hydroxyphenyl) -2-aminoacetal, 2- (3- (N- (methylsulfonylamino) )phenyl) -2-aminoacetyl, 2- phenyl-2- (5-indanyl carboxylate)acetyl, 2-phenyl-2- (phenyl carboxylate)acetyl, 2-phenyl-2-azidoacetyl, 2- phenoxypropionyl, 2,5-dimethoxybenzoyl, 2 - (formyloxy) -2 - phenylacetyl, 2- (2-ethoxynaphth-l-yl)acetyl, 2- (naphth-l- yl) -2-aminoacetyl, 2- (naphth-2-yl) -2-aminoacetyl, 2- (2, 5- dichlorophenylthio)acetyl, 2- (3, 4- dichlorophenylthio)acetyl, 2- (1-tetrazolyl)acetyl, 2-(N- (3, 5-dichloropyrid-4-oxyl) )acetyl, 2- (2-aminothiazol-4- yl)acetyl, 2- (2-thienyl)acetyl, 2- (4-pyridylthio)acetyl, 2- (N-methyl-4-pyridiniumthio)acetyl, 2- (2-amino-4- phenylthiazol-5-yDacetyl, 2- (3-hydroxy-4- carboxyisothiazol-5-ylthio)acetyl, 3-phenyl-5- methylisoxazolyl-3-formyl, 3- (2-chlorophenyl) -5- methylisoxazolyl-3-formyl, 3- (2, 5-dichlorophenyl) -5- methylisoxazolyl-3-formyl, 3- (2-fluoro-5-chlorophenyl) -5- methylisoxazolyl-3-formyl, 2- (2-thienyl) -2-aminoacetyl, 2- (2-thienyl) -2- (sodium carboxylate)acetyl, 2-(N-(4- pyridinium) )acetyl, 2- (2-benzothienyl)acetyl, 2-(3- benzothienyl)acetyl, 2- (2-benzofuryl)acetyl, and 2-(3- benzofury1)acetyl;

G. A compound of embodiment F, wherein the

Cytotoxic Agent is methotrexate, 5-fluorouracil, desacetylvinblastine aminoethanethiol, a desacetylvinblastine hydrazidocarboxy moiety, a 7-

(carboxyamino)desacetyl colchicine moiety, or doxorubicin; H. A compound of embodiment G, wherein R- ] _ is

2- (thien-2-yl)acetyl and zz is zero;

I. A compound of embodiment H, wherein the Cytotoxic Agent is methotrexate;

J. A compound of embodiment I, wherein methotrexate is bonded through its carboxylate group to the cephalosporin Substrate;

K. A compound of embodiment H, wherein the Cytotoxic drug is 5-fluorouracil;

L. A compound of embodiment K, wherein the 5- fluorouracil is bonded through its N-l nitrogen to the cephalosporin Substrate;

M. A compound of embodiment H, wherein the Cytotoxic Agent is desacetylvinblastine aminoethanethiol;

N. A compound of embodiment M, wherein the desacetylvinblastine aminoethanethiol is bonded through the thiol group of its aminoethanethiol moiety to the cephalosporin moiety; and

0. A compound of embodiment H, wherein the Cytotoxic Agent is a desacetylvinblastine hydrazidocarboxy moiety;

P. A compound of embodiment 0, wherein the desacetylvinblastine moiety is bonded through an oxygen atom of the hydrazidocarboxy group to the cephalosporin

Substrate;

Q. A compound of embodiment G, wherein R-*_ is

2- (thien-2-yDacetyl and zz is 1;

R. A compound of embodiment Q, wherein the Cytotoxic Agent is a 7- (carboxyamino)desacetylcolchicine moiety;

S. A compound of embodiment R, wherein the 7- (carboxyamino)desacetylcolchicine moiety is bonded through an oxygen atom of the 7-(carboxyamino) group to the cephalosporin Substrate;

T. A compound of embodiment Q, wherein the Cytotoxic Agent is desacetylvinblastine aminoethanethiol;

U. A compound of embodiment T, wherein the Cytotoxic Agent is desacetylvinblastine aminoethanethiol;

V. A compound of embodiment Q, wherein the Cytotoxic Agent is the desacetylvinblastine hydrazidocarboxy moiety;

W. A compound of embodiment V, wherein the desacetylvinblastine hydrazidocarboxy moiety is bonded to an oxygen atom of the hydrazidocarboxy group to the cephalosporin moiety;

X. A compound of embodiment Q, wherein the Cytotoxic Agent is doxorubicin;

Y. A compound of embodiment X, wherein the doxorubicin moiety is bonded through the aminosaccharide nitrogen to the carbonyl carbon of the 3-(carbonyl- orymethylene) group of the cephalosporin sulfoxide moiety; Z. A compound of embodiment G, wherein R- j _ is phenoxyacetyl and zz is 1;

AA. A compound of embodiment Z, wherein the Cytotoxic Agent is the desacetylvinblastine hydrazidocarboxy moiety; and

BB. A compound of embodiment AA, wherein the desacetylvinblastine hydrazidocarboxy moiety is bonded through an oxygen atom of the aminocarboxy group to the cephalosporin Substrate.

CC. A compound of embodiments I through BB, wherein R2 is hydrogen, a sodium cation, or a potassium cation.

It will be understood that the compounds of Formula II may also exist as solvates and hydrates. Thus, these compounds may crystallize with, for example, waters of hydration, or one, or a number of, or any fraction thereof of molecules of the mother liquor solvent. The solvates and hydrates are included within the scope of this invention.

Similarly, the compounds of Formula II can exist as pharmaceutically-acceptable salts. The compounds include salts of both the acidic functions, such as carboxy and sulfonate groups, and the basic functional groups, such

as amino groups. Such salts include the organic and inorganic cations discussed below, plus the salts formed from acid-base reactions of basic groups with acids such as hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, d-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids.

As used in the above preferred embodiments, the term "acyl group derived from a C- j _ to C3 Q carboxylic acid" respresented by R- j _ refers to the acyl moieties which have been bonded to the C-6 amino group of penicillins, the C-7 amino group of cephalosporins, 1-sulfoxide cephalosporin, 1-oxadethiacephalosporins or 1-carbacephalosporins and the C-3 amino of monocyclic β-lactams (such as the azthreonam series) . The "acyl group derived from a C- j _ to C3 Q carboxylic acid" can be optionally interrupted by heteroatoms. Examples of such acyl groups can be found in references such as "Cephalosporins and Penicillins, Chemistry and Biology" edited by Edwin W. Flynn, Academic Press, New York, 1972 and "Chemistry and Biology of β- lactam Antibiotics" edited by Robert B. Morin and Marvin

Gorman, Vols. 1,2, and 3, Academic Press, New York, 1982. Examples of acyl groups at R- j _ can also be found in Yoshioka M. , et al.. U.S. Patent No. 4,478,997, issued

October 23, 1984; Belleau B.R., et al.. U.S. Patent No.

4,172,199, issued October 23, 1979: Kamiya T., et al. , U.S.

Patent No. 4,472,300, issued September 18, 1984,

(especially columns 25 through 36) all of which are herein incorporated by reference. Additional examples of "acyl groups derived from a C- j _ to C3 Q carboxylic acid" can be found in Koster et al.. U.S. Patent No. 4,478,749, issued

October 23, 1984.

The term "organic or inorganic cation" refers to counter-ions for the carboxylate anion of a carboxylate

salt. The counter-ions are chosen from the alkali and alkaline earth metals, such as lithium, sodium, potassium, barium and calcium; ammonium; and organic cations such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis (2-hydroxyethyl)ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium and like cations. Other cations encompassed by the above term include the protonated form of procaine, quinine and N-methylglucosamine, and the protonated forms of basic amino acids such as glycine, ornithine, histidine, phenylglycine, lysine and arginine. Furthermore, any zwitterionic form of the instant compounds formed by a carboxylic acid and an amino group is referred to by this term.

The term "carboxy-protecting group" as used in the specification refers to one of the ester derivatives of the carboxylic acid group commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound. Examples of such carboxylic acid protecting groups include 2-nitrobenzyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4- dimethoxybenzyl, 2, -dimethoxybenzyl, 2,4,6- trimethoxybenzyl, 2, 4, 6-trimethylbenzyl, pentamethylbenzyl, 3, 4-methylenedioxybenzyl, benzhydryl, 4,4'- dimethoxybenzhydryl, 2,2 ' , 4, 4 ' -tetramethoxybenzhydryl, t- butyl, t-amyl, trityl, 4-methoxytrityl, 4,4'- dimethoxytrityl, 4, 4 ' , 4 ' ' -trimethoxytrityl, 2-phenylprop-2- yl, trimethylsilyl, t-butyldimethylsilyl, phenacyl, 2,2,2- trichloroethyl, β-(trimethylsilyl) ethyl, β-(di(n- butyDmethylsilyl)ethyl, p-toluenesulfonylethyl, 4- nitrobenzylsulfonylethyl, allyl, cinnamyl, 1- (trimethylsilylmethyl)prop-l-en-3-yl, and like moieties. The species of carboxyl-protecting group employed is not critical so long as the derivatized carboxylic acid is stable to the condition of subsequent reaction(s) on other positions of the cephalosporin molecule and can be removed at the appropriate point without disrupting the remainder

of the molecule. A preferred carboxylic acid protecting group is the allyl group. Similar carboxy-protecting groups used in the cephalosporin, penicillin and peptide arts can also be used to protect carboxy group substituents of the instant cephalosporin substrate. Further examples of these groups are found in Haslam E. , "Protective Groups in Organic Chemistry", McOmie H.G.W., Ed., Plenum Press,

New York, N.Y., 1973, Chapter 5, and Greene T.W.,

"Protective Groups in Organic Synthesis", John Wiley and

Sons, New York, N.Y., 1981, Chapter 5. A related term is

"protected carboxy", which refers to a carboxy group substituted with one of the above carboxy-protecting groups.

The term "non-toxic, metabollically-labile, ester-forming group" refers to those biologically active ester forms which induce increased blood levels and prolong the efficacy of the corresponding non-esterified forms of the compounds. Such ester groups include the lower alkoxymethyl groups, for example, methoxymethyl, ethoxymethyl, iso-propoxymethyl and the like; the α-(C-^to

C4) alkoxyethyl groups, for example, methoxyethyl, ethoxyethyl, propoxyethyl, iso-propoxyethyl, and the like; the 2-oxo-l,3-dioxolen-4-ylmethyl groups, such as 5-methyl-

2-oxo-l,3-dioxolen-4-ylmethyl, 5-phenyl-2-oxo-1, 3-dioxolen- 4-ylmethyl, and the like; the C- j _ to C3 alkylthiomethyl groups, for example methylthiomethyl, ethylthiomethyl, iso- propylthiomethyl, and the like; the acyloxymethyl groups, for example, pivaloyloxymethyl, α-acetoxymethyl, and the like; the ethoxycarbonyl-1-methyl group; the α-acyloxy-α- substituted methyl groups, for example α-acetoxyethyl; the 3-phthalidyl or 5, 6-dimethylphthalidyl groups; the 1- (C- j _ to C4 alkyloxycarbonyloxy) eth-l-yl groups such as the 1- (ethoxycarbonyloxy)eth-l-yl group; and the 1-(C*^ to C4 alkylaminocarbonyloxy) eth-l-yl groups such as the 1- (methylaminocarbonyloxy)eth-l-yl group.

Several terms used in conjunction with embodiment D above should be defined. Thus, the term "C- ] _ to Cg alkyl" denotes such radicals as methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, amyl, tert-amyl, hexyl and the like.

The term "C** j _ to Cg substituted alkyl" denotes the above C- j _ to Cg alkyl groups that are substituted by one or two halogen, hydroxy, protected hydroxy, amino, protected amino, C** j _ to C- acyloxy, nitro, carboxy, protected carboxy, carbamoyl, carbamoyloxy, cyano, methylsulfonylamino or C- j _ to C4 alkoxy groups. The substituted alkyl groups may be substituted once or twice with the same or with different substituents.

The term "C* j _ to C4 alkoxy" as used herein denotes groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy and like groups.

The term "protected amino" as employed in the above definition has reference to an amino group substituted with one of the commonly employed amino blocking groups such as the tert-butoxycarbonyl group (t- BOC), the benzyloxycarbonyl group, the 4- methoxybenzyloxycarbonyl group, the 2,2,2- trichloroethoxycarbonyl group, the trimethylsilyl group, and like amino protecting groups. The nature of such amino protecting groups is not critical so long as the protected amino functionality is stable under the reaction conditions described hereinafter.

The term "protected hydroxy" has reference to any group stable under the reaction conditions of the subsequent step in the synthesis of the cephalosporin substrate compounds, but readily cleavable thereafter. Such groups include the formyloxy group, the chloroacetoxy group, the benzhydryloxy group, the trityloxy group, the trimethylsilyl group, and the like.

In the foregoing definitions, hydroxy, amino, and carboxy protecting groups are not exhaustively defined.

The function of such groups is to protect the reactive functional groups during the preparation of the desired products and then to be removed without disrupting the remainder of the molecule. Many such protecting groups are well known in the art and the use of other groups equally applicable to the process and compounds of the present invention, such as those described in McOmie J.F.W.,

"Protective Groups in Organic Chemistry", Plenum Press,

1973, and Greene T.W., "Protective Groups in Organic

Chemistry", John Wiley and Sons, New York, N.Y. , 1981, will be recognized as suitable. Thus, there is no novelty or inventiveness asserted with regard to the "protecting groups" alluded to in this specification.

The term "heterocyclic ring", when used in conjunction with the term "acyl group derived from a C- j _ to

C3 Q carboxylic acid", refers to the rings in Durckheimer W. , et al.. U.S. Patent No. 4,278,793. Particular examples are unsubstituted or substituted rings such as tetrazolyl, oxazolyl, isoxazolyl, thienyl, furyl, thiazolyl, thiadiazolyl, isothiazolyl, pyridyl, 4-pyridonyl, pyrididyl, benzthienyl, benzfuryl or indolyl. Of course, these examples can be substituted with the substituents discussed in the Durckheimer et al.. '793 patent.

A third aspect of this invention is a method of treatment for the treatment of neoplastic diseases which comprises:

1) administering to the affected host a therapeutically effective amount of the Antibody-Enzyme conjugate compound of Formula 1; then

2) administering to the affected host a therapeutically effective amount of the Substrate - Cytotoxic Agent of Formula II above.

It will be understood that the term "therapeutically-effective" , as it relates to the Substrate - Cytotoxic Agent will be an amount sufficient to cause some target neoplastic cell death, and may further refer to

the use of this drug to keep a neoplastic disease in remission, or, in general, in an amount sufficient for prophylaxis.

In general, the term "therapeutically effective" means at least the same molar equivalent amount of the Substrate - Cytotoxic Agent be given for the Cytotoxic Agent alone for the particular purpose at hand. Larger amounts of the Substrate -Cytotoxic Agent may be given in the instant invention because the toxicity of the Cytoxic Agent is initially masked by the attached Substrate but subsequently unmasked upon reaction with the Antibody- Enzyme conjugate at its point of delivery, thus relieving the patient of the side effects normally associated with that amount of Cytotoxic Agent alone.

A therapeutically effective dose of the Antibody-Enzyme conjugate is the amount that delivers between 1 through 500, and preferrably, 1 through 50, milligrams of Antibody (or fragments thereof) to the affected host.

Preferred embodiments for the present method are listed below. As with the compound aspects of the invention, the embodiments listed below contain the limitations and definitions of the embodiment it refers to. Examples of the preferred embodiments of the present method include:

A. A method wherein the Substrate is a substrate for a β-lactamase, pyroglutamate aminopeptidase, β-galactosidase, or D-aminopeptidase, and the Antibody- Enzyme conjugate compound wherein the Enzyme is a β- lactamase, pyroglutamate aminopeptidase, β-galactosidase, or D-aminopeptidase;

B. A method of A, wherein the Enzyme is a β- lactamase;

C. A method of B, wherein the Antibody complexes with antigens that are present solely, or are in supernatural abundance, on malignant tumor cells;

D. A method of embodiment C, wherein the Substrate is a cephalosporin of the formula:

AGENT

wherein zz is 0 or 1; R- ] _ is an acyl group derived from a C^ to C30 alkyl group, R2 is hydrogen, an organic or inorganic cation, a carboxy-protecting group, or a non-toxic, metabolically-labile ester-forming group; as those terms are defined regarding preferred embodiment B of the Substrate-Cytotoxic Agent aspect of this invention;

E. A method of embodiment D, wherein the Antibody complexes with carcinoembryonic antigen;

F. A method of embodiment D, wherein the Antibody complexes with the KSl/4 antigen;

G. A method of embodiment D, wherein the Antibody complexes with the TAG-72 antigen;

H. A method of embodiment D, wherein the

Antibody complexes with the MAT 65 antigen;

I. A method of embodiment E, wherein R^ is the same as R- j _ in the preferred embodiment D of the compounds of Formula II;

J. A method of embodiment I, wherein the

Cytotoxic Agent is a compound of the formula listed in the preferred embodiment E of the compounds of Formula II;

K. A method of embodiment J, wherein the R-*_ is the same as for preferred embodiment F of the compounds of Formula II;

L. A method of embodiment K, wherein the

Cytotoxic Agent is methotrexate, 5-fluorouracil, desacetylvinblastine aminoethanethiol, a desacetylvinblastine aminoacarboxy moiety, a 7-

(carboxyamino)desacetylcolchicine moiety; or doxorubicin;

M. A method of embodiment L, wherein R2 of the cephalosporin Substrate is 2- (thien-2-yl)acetyl and zz is 0;

N. A method of embodiment M, wherein the Antibody is designated CEM 231.6.7;

0. A method of embodiment N, wherein the Cytotoxic Agent is methotrexate;

P. A method of embodiment 0, wherein the methotrexate is bonded through a carboxy group to the cephalosporin Substrate;

Q. A method of embodiment N, wherein the Cytotoxic Agent is 5-fluorouracil;

R. A method of embodiment Q, wherein 5- fluorouracil is bonded through its N-l nitrogen to the cephalosporin Substrate;

S. A method of embodiment N, wherein the Cytotoxic Agent is desacetylvinblastine aminoethanethiol;

T. A method of embodiment S, wherein the desacetylvinblastine aminoethanethiol is bonded through the thiol group of the aminoethanethiol moiety to the cephalosporin moiety;

U. A method of treatment of embodiment N, wherein the Cytotoxic Agent is a desacetylvinblastine aminocarboxy moiety.

V. A method of treatment of embodiment U, wherein the desacetylvinblastine hydrazidocarboxy moiety is bonded through an oxygen atom of the hydrazidocarboxy group to the cephalosporin Substrate.

W. A method of treatment of embodiment L, wherein R- j _ is 2- (thien-2-yl) acetyl and zz is 1.

AA. A method of treatment of embodiment X, wherein the Cytotoxic Agent is a 7- (carboxyamino)desacetylcolchicine moiety.

BB.' A method of treatment of embodiment AA, wherein the Cytotoxic Agent is 7- (carboxyamino)desacetylcolchicine moiety.

CC. A method of treatment of embodiment X, wherein the Cytotoxic Agent is desacetylvinblastine aminoethanethiol.

DD. A method of treatment of embodiment CC, wherein the desacetylvinblastine aminoethanethiol is bonded through the thiol group to the cephalosporin Substrate.

EE. A method of treatment of embodiment X, wherein the Cytotoxic Agent is a desacetylvinblastine hydrazidocarboxy moiety.

FF. A method of treatment of embodiment EE, wherein the desacetylvinblastine hydrazidocarboxy moiety is bonded through an oxygen atom of the hydrazidocarboxy group to the cephalosporin Substrate.

GG. A method of treatment of embodiment W, wherein the Cytotoxic Agent is doxorubicin.

HH. A method of treatment of embodiment GG, wherein doxorubicin is bonded through nitrogen of the aminosaccharide moiety to the cephalosporin Substrate.

II. A method of treatment of embodiment L, wherein R- j _ is (phenoxy)acetyl and zz is 1.

JJ. A method of treatment of embodiment II, wherein the Cytotoxic Agent is a desacetylvinblastine hydrazidocarboxy moiety.

KK. A method of treatment of embodiment JJ, wherein the desacetylvinblastine hydrazidocarboxy moiety is bonded through an oxygen atom of the hydrazidocarboxy group to the cephalosporin .Substrate.

LL. A method of embodiments N through KK; wherein the Antibody is a F(ab') fragment.

The Substrate - Cytotoxic Agent compounds of the instant invention can be synthesized by a variety of methods that are known. Thus, a nucleophilic group of the

Substrate could be used to displace a leaving group at an electrophilic center of the Cytotoxic Agent under S n l or

S n 2 conditions, or vice versa. Carboxy, amino, thiol or hydroxy groups on the Substrate and the Cytotoxic Agent that would interfere with the reaction of desired functional groups could be masked with protecting groups before the reaction, then deprotected after the reaction.

The use of suitable protecting groups is described, among other places, in the works of McOmie and Greene set forth above. All that is required of the instant Cytotoxic Agent compounds is that the Cytotoxic Agent be a potent antineoplastic agent after chemical seperation from the

Substrate as is effected by the Enzyme, and that the derivatization of the Substrate with Cytotoxic Agent does not significantly decrease the reactivity of the Substrate with Enzyme. It is preferred that the Substrate -

Cytotoxic Agent compound mask the toxicity of the Cytotoxic

Agents or that the cytotoxicity be no more than 70% of the cytotoxicity of the unsubstituted Cytotoxic Agent.

For the purposes of the present method, it is desirable that Enzyme have a reasonable catalytic rate. In light of the meager concentrations of Substrate (in the form of the Substrate - Cytotoxic Agent compounds) the

Enzyme will encounter, it is especially important that the enzyme-substrate combination have a high ratio of k cat /K m .

Thus, it is desirable to choose an Enzyme that has a k cat /K m ratio of at least 1 x 10° M -1 S ~ . This same ratio is desirable in the Enzyme once it is conjugated to the Antibody. Similarly, it is preferred for the Antibody to have high affinity for the antigen on the target neoplastic cell, preferably about 1 x 10° (liter/mole) or above. It is also desirable that the conjugated Antibody retain about

80% of its unconjugated activity, thus desirably exhibiting a 60% immunoreactivity once conjugated.

Conjugating an Enzyme to an Antibody is accomplished by methods standard in the protein chemistry art. Conjugates could also be formed by Offord and Rose methodology described above, or by gene fusion to produce fusion protein (European Patent Application No. 392 745, published October 17, 1990; Neuberger M.S., et al.. Nature. 312. p.604, 1984); Seehaus T., et al.. Gene. 114. p.l, 1992). Heterobifunctional and homobifunctional reagents can be used. Thus, the present conjugates can be formed using MBS, Sulfo-MBS, SMCC, Sulfo-SMCC, SIAB, SPDP, Sulfo- SMPB, DIDS, DFDNB, BMH, (which are commercially available) and the like. Purification of the resulting conjugate may be accomplished by appropriate, well-known chromatographic methods such as gel permeation or ion exchange. Thus, nothing is novel in the instant invention in the way the Enzyme and the Antibody are conjugated. It is preferred that the stoichiometry and other conditions of the conjugation reaction (and purification) be adjusted such that a 1:1 Antibobdy : Enzyme conjugate results, although conjugates with disproportionately larger amounts of either moiety in the conjugate are encompassed in the present invention.

After choosing the Antibody and Enzyme, one skilled in the art must decide whether to use the whole Antibody or a fragment thereof. The methods for making fragments, using pepsin or papain, for instance, are elaborated in M. Brennan et al.. Science. 229. pp.81-83, 1985, and M. Glennie e_£. al. , i. Immunology. 139, pp.2367- 2375, 1987, and will not be discussed here, other than to mention that F(ab') is the preferred form for the Antibody of this invention.

After isolating an Enzyme with the criteria detailed above, conjugating it to the appropriate Antibody as described above, and purifying the conjugate, it is

useful to analyze the conjugate by gel electrophoresis.

Alternatively, high pressure liquid chromatography (HPLC) may be used to analyze the conjugate.

The activity of the Enzyme of the conjugate is again measured, this time employing Substrate bound to a molecule that imparts to the Substrate (or the molecule itself) a measurable spectrophotometric change upon cleavage to measure the k cat and the K M This measurement will demostrate whether or not the Enzyme has retained its activity through the conjugation step. The next parameter to be measured is the immunoreactivity of the conjugate in vitro. This measurement is usually made by first radioactively labeling the conjugate (with, e.g., 125 I) coating a solid surface (e.g., small plastic beads or microtiter plate wells) with the target antigen. This measurement will detect if the Antibody portion of the conjugate has been unacceptably altered in the conjugation step. The first step toward seeing if the components of the instant method function in the presence of the target antigen comes when the k cat and the K M of the conjugate are measured with the Substrate - Cytotoxic Agent. (That free Cytotoxic Agent is released by the Enzyme can be shown chromatσgraphically. ) The conjugate is then studied to assess if it will localize to tumors in vivo. This is typically accomplished by injecting radiolabeled conjugate into tumor-bearing mice, which tumors express the target antigen, then sacrificing the mice and detecting the amount of radioactivity present in the tumor(s) and the various organs. This study will demonstrate that the conjugate localizes to the tumor, or is unacceptably targeting an undiseased organ, or furthermore is metabolized owing to its status as a foreign protein.

Once it is determined that the conjugate localized to the tumor, it is necessary to demonstrate that the Substrate -Cytotoxic Agent will be cleaved by the localized conjugate. Thus, target-antigen expressing cells

are suspended and incubated either with conjugate or, for a control, Enzyme only. The cells so treated are resuspended in another amount of buffer, then incubated with a Substrate that is coupled with a compound such that the pair absorb at different wavelengths in the visible spectrum before and after cleavage. The supernatant of these suspensions are then measured to determine if the conjugate-containing suspensions demonstrate a time and concentration dependence on the amount of conjugate added. Thus, if the control samples are exhibiting such a dependence, some condition other than the conjugate is causing the cleavage. Assuming that the components of the present invention demonstrate in the preceding test that localized conjugate is indeed the agent causing the Substrate - Cytoxic Agent cleavage, the serum kinetics of the conjugate in a test animal such as mice should be measured to determine if and when the Substrate - Cytotoxic Agent can be administered to the affected host after administration of the conjugate. It is desirable that the conjugate demonstrate a short serum half-life, so that the Substrate - Cytotoxic Agent molecule can be administered within a reasonable time (e.g., 72 hours) after administration of the conjugate. Once the serum half-life of the conjugate has been found acceptable, the Enzyme activity of the conjugate localized in vivo in test animals, such as the mice, is determined. The conjugate is administered to the target-tumor-bearing mice, and after sufficient time has passed for the conjugate to localize, the mice are sacrificed and their tumors are excised. The tumor is minced and incubated with a chromogenic substrate. The supernatants of these suspensions are measured for signs of enzymatic activity.

If the components of the present method have successfully overcome all of these hurdles, the in vitro cytotoxicity of the components is measured.

Target cells are then suspended and incubated with conjugate. The treated cells are then resuspended and incubated with Substrate - Cytotoxic Agent. Cell growth is monitored by the uptake of radiolabeled nutrients subsequently added to the culture media. The final step in evaluating the present method is to evaluate the efficacy of the components in a nude mouse tumor model study.

A further aspect of this invention is the pharmaceutical compositions of the Antibody-Enzyme conjugate of Formula I and the Substrate - Cytotoxoic Agent compounds of Formula II. In particular, these pharmaceutical compositions are useful for the treatment of neoplastic diseases of the present invention and comprise a suitable vehicle and a therapeutically effective amount of either the conjugates of Formula I or the compounds of Formula II.

With regard to compositions for oral administration (e.g., tablets and capsules), the term "suitable vehicle" means common excipients such as binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidine (Povidone) , methylcellulose, ethylcellulose, sodium carboxymethyl- cellulose, hydroxypropylmethylcellulose, surcrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium, phosphate, sodium chloride and alginic acid; disintegrators such as croscarmellose sodium, microcrystalline cellulose, corn starch, sodium starch glycolate, alginic acid and mutable wetting agents such as sodium lauryl sulfate; and lubricants such as magnesium stearate and other metallic stearates, stearic acid, silicone fluid, talc, waxes oils and colloidal silica. Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the like can also be used. It may be desirable to add a coloring agent to make the dosage form more aesthetically pleasing in appearance or to help

identify the product. The tablets may also be coated by methods well known in the art.

The pharmaceutical compositions of the present invention may also be in the form of oral liquid preparations, which may be either a) aqueous or oily suspensions, solutions, emulsions or syrups; or b) a dry powder to be reconstituted with water or another suitable vehicle before use. When used in conjunction with such oral liquid preparations, the term "suitable vehicle" means conventional additives such as suspending agents, for example, sorbitol, syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible oils, for example almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; and preservatives such as methyl or propyl p-hydroxybenzoates or sorbic acid.

The pharmaceutical composition can also be for intravenous (IV) use. Specifically, a water soluble form of the conjugate or Substrate - Cytotoxic Agent can be dissolved in one of the commonly used intravenous fluids and administrated by infusion. When used in conjunction with compositions for IV use, the term "suitable vehicle" means such fluids as physiological saline, Ringer's solution or 5% dextrose solution.

For intramuscular preparations a sterile formulation of a suitable salt form of the conjugate or Substrate - Cytotoxic Agent compound (for example, the hydrochloride salt or sodium salt) can be formulated with a "suitable vehicle". Examples of such sterile formulations are a suitable salt form either dissolved in a pharmaceutical diluent (e.g., Water-for-Injection, physiological saline, 5% glucose) or suspended in an aqueous base or a pharmaceutically acceptable oil base (e.g., an ester of a long chain fatty acid such as ethyl oleate) .

Topical compositions can be formulated with "suitable vehicles" such as hydrophobic or hydrophilic bases. Such bases include ointments, creams or lotions.

Veterinary pharmaceutical compositions of the conjugates and the Substrate - Cytotoxic Agent compounds may be administered in the feed or the drinking water of farm animals. Alternatively, the conjugate or Substrate - Cytotoxic Agent compounds can be formulated as intramammary preparations with "suitable vehicles" such as long-or quick-release bases.

The Antibody-Enzyme conjugate of Formula I and the Substrate - Cytotoxic Agent compounds of Formula II can also be formulated in unit dosage form in sterile vials sterile plastic pouches containing a port with a septum, or sterile, hermetically sealed ampoules. The conjugate or compound (or the corresponding pharmaceutically-acceptable salt) may be a dry powder or in crystalline or lyophylized form. The amount of the conjugate or Substrate - Cytotoxic Agent compound per unit dosage may vary from about 5 milligrams to about 10 grams.

The following Experiments and Procedures are provided to further illustrate the present invention. Abbreviations used below have the meanings commonly associated with them in the relevant art unless otherwise noted. The n.m.r. spectra were taken on General Electric G.E.-300 instrument. The infrared spectra were obtained on a Perkin-Elmer 281 instrument. Ultraviolet spectra were obtained on a Cary 118 instrument (except for Example 7 and Procedures 6 through 13, where the U.V. spectra were obtained on a Hewlett-Packard 8451A instrument.) Fast atom bombardment mass spectra were obtained on a VG ZAB-3 instrument.

Procedure 1 Allyl 7-β-(2- (Thien-2-yl)Ace amido)

-3-Acetoxymethylene-3-Cephem- -Carboxyl te

Sodium 7-β-(2- ( hien-2-yl)acetamido) -3-

(acetoxymethylene) -3-cephem-4-carboxylate (11.Og) was dissolved in a 1:1 mixture of DMF and water. Allyl bromide

(3.6g) was added and the reaction mixture was stirred for

48 hrs. at room temperature under a nitrogen atmosphere.

The reaction mixture was diluted with ethyl acetate and then 0.1N hydrochloric acid. The organic layer was separated and dried over magnesium sulfate, filtered, and concentrated in vacuo to give an orange oil. The oil was flash chromatographed on a 3 inch column with a 100-200 mesh activated silica support eluted with ethyl acetate.

White crystals formed from some of the product-containing fractions on standing. These crystals were collected by filtration then recrystallized from 1:1 chloroform:ether and dried to give 5.5g of the title product. NMR: (300MHz,CDCl 3 ) : 5 7.25 (d, 1,J=7) ; 7.0(m,2);

6.27 (br.d,l,J=9) , 6.0-5.8 (m, 1) ; 5.83 (dd, 1,J=5, 9) ; 5.4- 5.2(m,2); 5.06(1/2 of ABq,1,J=14) ; 4.96 (d, 1,J=5) ; 4.81(1/2 of ABq,l,J=14) ; 4.74 (br.d,2,J=6) ; 3.85(s,2); 3.55(1/2 of ABq,l,J=18); 3.33(1/2 of ABq, 1,J=18) ; 2.06(s,3) .

Procedure 2

Allyl 7β-(2- (Thien-2-yl)Acetamido) -3- ( Iodo ethyl) -3-Cephem-4-Carboxylate

Allyl 7β-(2-thien-2-yl)acetamido) -3- acetoxymethyl-3-cephem-4-carboxylate (5.01g, 11.5mmol) was dissolved in methylene chloride (55ml) . TMSI (3.27ml) was added and the resultant solution was stirred at room temperature for 70 min. The solution was diluted with ethyl acetate, then washed sequentially with ice cold 10% sodium thiosulfate, saturated aqueous sodium bicarbonate solution, and brine. The solution was dried over sodium sulfate and concentrated in vacuo to give 4.6g, 79% yield

of a yellow-orange tinted foam of the title product: NMR: (300MHz,CDCl3) : δ 7.25 (d, 1, J=7 ) ; 7.0(m,2) ; 6.42 (d, 1, J=9) ;

6.0-5.85(m,l) ; 5.78 (dd, 1, J=5 , ) ; 5.4-5.2 (m, 2 ) ;

4.95(d,l,J=5) ; 4.74 (d, 2 , J=6) ; 4.38 (ABq, 2 , J=10) ; 3.85(s,2);

3.72(1/2 of ABq,l,J=18); 3.44(1/2 of ABq,l,J=18) .

Example 1

Allyl 7β-(2- ( Thien-2-yl ) Acetamido ) -3- ( (Methotrexate -Gamma -Ester) Methylene) -3-Cephem-

4 -Ester

Allyl 7β-(2- (Thien-2-yl) acetamido) -3- (iodomethyl) -3-cephem-4-carboxylate (4.6g, 9.lmmol) , methotrexate-gamma-ester (4.6g, 9.lmmol, Sigma) , DMF (50ml), and sodium bicarbonate (1.54g, 18.25mmol) were combined. The solution was stirred at room temperature for 18 hours then added dropwise to a solution of acetic acid (4ml) in water (400ml) . The resultant precipatate was collected and dried in vacuo to yield approximately 7g of powder. The powder was flash chromatographed over a silica gel column eluted with a solution of 15% methanol : 2% acetic acid in chloroform, to give approximately 2.lg of yellow-orange solid. The solid was flash chromatographed on silica gel eluted with a gradient of 10% methanol : 1% acetic acid in chloroform to 2% acetic acid in chloroform then by a 15% methanol and 2% acetic acid in chloroform to give 1.06g (R f =0.5 in 2% HOAc, 15% MeOH in CHCl 3 ) of the title product: IR: (KBr) : 1782, 1730, 1607, 1562, 1513 cm "1 . FABMS: Calc'd. for C 37 H3 8 N 10 O 9 S 2 Na=853.2162.

Found-=853.2106. UV: (EtOH) lambda max =370 (ε=3290) ;

297 (ε=12000) ; 260(8=13300) . NMR: (300MHz,DMSO-d ) : δ

9.42(bd,l,J=8) ; 9.13 (d, 1,J=9) ; 8.52(s,l), 7.45-6.7 (m, 5) ; 6.6(bs,2); 5.88 (m, 1,allyl CH) ; 5.39 (dd, 1,J=5, 8) ; 5.35- 4.55(m,7) with 4.7(s,2) superimposed; 4.16(m,l) ; 3.70(s,2); 3.50(ABq,2,J=10) ; 3.2 (s, 3); 2.1(m,2); 1.9 (m, 2) .

Example 2

7β-(2- (Thien-2-yl)Acetamido) -3- ( (Methotrexate- Gamma-Ester)Methylene) -3-Cephem-4-Carboxylic Acid

Palladium (II) acetate (3mg, 0.013mmol) and triphenylphosphine (14mg, 0.053mmol) were combined in ethyl acetate (0.5ml). Allyl 7β-(2- (thien-2-yl)acetamido) -3-

( (methotrexate ester)methylene)-3-cephem-4-carboxylate (130mg, 0.15mmol) was suspended in a mixture of 1:1:1 ethyl acetate : methanol : acetic acid followed by the addition of triethylsilane (0.1ml). The resultant reaction mixture was stirred for 16 hours then concentrated in vacuo. The concentrate was diluted with water (15ml) and the pH of the solution was adjusted to 7 by the addition of aHC03. The resultant --.jlution was layered with ethyl acetate and the mixture was stirred for 1 hour. The mixture was filtered, the phases were separated and the aqueous phase was extracted with additional ethyl acetate then freeze dried.

The freeze-dried solid was subjected to medium pressure liquid chromatography on a C- j _g reverse phase column. The column was first equilibrated with a solution of 15% acetonitrile in water. Then the freeze dried material was loaded on the column and eluted first with 30% acetonitrile : 1% acetic acid in water (600ml); then 40% acetonitrile : 1% acetic acid in water. Fractions containing the peak eluting at 4.8 min (Waters C- j _g μbondpak analytical column, eluted with 30% acetonitrile, 1% HOAc, water, 2ml/min) were combined and freeze dried to give the title product: NMR: (300MHz,DMSO-dg) : δ 9.0(d,1,J=9) ;

8.52 (s,l); 7.7(d,2,J=10) ; 7.64 (br.s,2) ; 7.30(m,l); 6.88(m,2); 6.75 (d,2,J=10) ; 6.55 (br.s,2) ; 5.42(dd,l,J=4.5,9) ; 4.94 (d,1,J=ll.6,1/2 of ABq) ; 4.89(d,l,J=4.5); 4.80(d,1,J=ll.5,1/2 of ABq) ; 4.72(s,2); 4.06(m,l); 3.60(s,2); 3.15(s,3); 2.1-1.7 (m,4) . IR: (KBr) 1763, 1757, 1606, 1559 cm -1 . FABMS: Found=791.2037

Example 3

Allyl 7β-(2- (Thien-2-yl)Acetamido) -3-( (N-l- ( 5-Fluorouracil )yl)Methylene) -3-Cephem-4-

Carboxylate

Allyl 7β-(2- (thien-2-yl)acetamido) -3- (acetoxymethylene) -3-cephem-4-carboxylate (l.Og) was dissolved in methylene chloride (5ml) then trimethylsilyl iodide (0.65ml, 5mmol) was added to the solution. The dark brown solution was stirred at room temperature for 90 minutes under a nitrogen atmosphere. The reaction mixture was diluted with ethyl acetate, then washed sequentially with cold IN sodium thiosulfate, saturated aqueous sodium bicarbonate solution, and brine. The layers were separated and the organic layer was dried over sodium sulfate and concentrated in vacuo.

5-Fluorouracil (0.30g, 2.3mmol) was dissolved in dry DMF. Triethylamine (0.40ml, 2.3mmol) then the 3- (iodomethylene) cephem compound from above, dissolved in DMF, was added to the 5-fluorouracil solution. The resultant solution was stirred at room temperature for 2 hours then concentrated in vacuo. The concentrate was diluted with cold IN HCl and ethyl acetate. The ethyl acetate layer was separated and dried over magnesium sulfate and concentrated in vacuo to give 0.70g of a brown solid. The brown solid was flash chromatagraphed over silica gel eluted with 5% methanol in methylene chloride. NMR: (300MHz,CDCl 3 ) , 10.10 (br.s, 1) , 7.81 (d, 1,J=5) ;

7.20(m,l); 7.05(d,l,J=7) ; 6.92(m,2), 6.0-5.8 (m, 2) ; 5.40-

5.25(m,2,); 4.95 (d, 1,J=5) ; 4.85 (d, 1,J=9) ; 4.75 (d,2,J=5) ;

4.41(d,l,J=9) ; 3.80(s,2); 3.45 (d, 1,J=12) ; 3.30 (d, 1,J=12) .

IR: (chloroform) : 1792, 1720, 1704, 1671, 1507 cm-1; UV: (EtOH) ; lambda maχ : 272 (ε=14, 100) , 238 (ε=12, 900) . FABMS:

M+l = 507.

Example 4

7-β-(2- (Thien-2-yl)Acetamido) -3-((N-l(5- Fluorouracil)yl) ethylene) -3-Cephem-4-Carboxylic

Acid

Tetrakis[triphenylphosphine] palladium [0] (8mg, O.OOδmmol), and triphenylphosphine (3mg, O.Olmmol) were combined in a 1:1 mixture of methylene chloride and ethyl acetate (2ml). Allyl 7-β-(2- (Thien-2-yl)acetamido)-3-( (N-

1(5-fluorouracil)yl)methylene)-3-cephem-4-carboxylate

(0.112g, 0.221mmol) was also dissolved in a 1:1 mixture of methylene chloride and ethyl acetate and the resultant brown solution was added to the catalyst-containing solution at room temperature. To this solution was added sodium 2-ethylhexanoate (0.040g, 0.243mmol). The resultant solution was stirred for 1.5 hours and the precipitate was isolated by first adding acetone to reaction mixture and transferring the mixture to a centrifuge tube. After centrifugation, the supernatant was discarded and the beige precipitate washed with ether and dried in a vacuum descicator. While drying, the beige solid turned brown.

The brown solid was dissolved in water, washed with ether, and freeze-dried to give 85mg of off-white solid. The solid was subjected to reverse-phase HPLC (Waters

Associates C-18 column) eluted with 15% acetonitrile/1% acetic acid/water. The product-containing fractions were combined and freeze-dried to give 15mg of the title product: NMR: (DMSO-dg,300MHz) : δ 9.08 (d, 1,J=8.3) ;

8.24(bd,l,J=5.8) ; 7.30(m,l); 6.88(m,2); 5.59 (dd,1,J=4.9 and

3.4); 4.97 (d,l,J=4.9) ; 4.72 (d,1,J=14.6) ; 4.23 (d,1,J=14.6) ;

3.68 (s,2); 3.50 (d,1,J=14.6) ; (other doublet obscured by water in DMSO at 3.3 δ) . IR: (CHCl 3 ) 1782, 1695, 1666 cm "1 .

UV: (ethanol) : lambda maχ =271nm(ε=10,800) , 237 (ε=10,200) . FABMS: Found=467.0489.

Procedure 3

N-t-Butoxycarbonyl-2-Aminoethanethiol

2-Aminoethanethiol hydrochloride (9.9g, O.Oδmol) was suspended in acetonitrile (approximately 25ml) under a nitrogen atmosphere, di (Isopropyl)amine (14.9ml) was added as the mixture was kept at 0°C during the addition. After 10 minutes at 0°, di (t-butyDdicarbonate (20.0ml) was added and the resultant mixture was stirred at 0° for 15 minutes then at room temperature for another 45 minutes. The reaction mixture was concentrated in vacuo and a 40:60 mixture of ethyl acetate:hexane was added. The white solid thus formed was removed by filtration. The filtrate was flash chromagraphed over a 7 inch silica gel column eluted with a 40:60 mixture of ethyl acetate:hexane. The product- containing fractions were combined and concentrated in vacuo to give 15g of a colorless oil of the title product. NMR: (300 MHz,CDCl 3 ) : 4.92 (bs, 1,NH) ; 3.28 (q,2,J=6.3 ) ;

2.62 (q,2,J=6.9) ; 1.42(s,9); 1.33 (t, 1,J=8.5) .

Procedure 4

7-β-(2- (Thien-2-yl)Acetamido) -3-( (N-(t- Butoxycarbonyl mino) -2-Ethylsulfide) Methylene) -3- Cephem-4-Carboxylic Acid

Sodium 7-β-(2-(Thien-2-yl)acetamido) -3- (acetoxymethylene) -3-cephem-4-carboxylate (2.0g, 5.05mmol) was stirred in distilled water, followed by the addition of potassium iodide (8g) and N-t-butoxycarbonyl-2- aminoethanethiol (0.87g) . The mixture was heated slowly to 65°C and held at this temperature for 2 hours, then at 70°C for an additional 2 hours. The reaction mixture was cooled and poured into a mixture of ice water and ethyl acetate with stirring. IN Hydrochloric acid was added to the mixture to adjust the pH to 2. The ethyl acetate layer was collected, dried over magnesium sulfate, filtered and concentrated in vacuo to give 2g of a white solid of the

title product. NMR: (300 MHz,DMSO-dg) : δ 9.10 (d,NH,J=9) ;

7.30(m,l); 6.88(m,2); 5.59(m,l); 5.05 (d,1,J=5) ; 3.71(s,2); 3.60(m,4); 3.26 (bs,2); 3.02 (m, 2); 2.45 (m, 2, overlapping with DMSO signal) .

Procedure 5

Allyl 7-β-(2- ( hien-2-yl)Acetamido) -3-(((l-(t- Butoxycarbony1amino) -2-Ethylsulfide)Methylene) -3-

Cephem-4-Carboxylate

7-β-(2- (Thien-2-yl)acetamido) -3- ( ( (1- (t- butoxycarbonylamino) -2-ethylsulfide)methylene) -3-cephem-4- carboxylate (0.50g, 0.975mmol), allyl bromide (0.25ml, 2.92mmol) and sodium bicarbonate (0.4g) were combined in 5ml DMF and stirred overnight at room temperature under a nitrogen atmosphere. The reaction mixture was diluted with ethyl acetate and 0.1 N hydrochloric acid. The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated in vacuo to give 0.5g of a yellow oil. The oil was flash chromatographed over 6 inches of silica gel eluted with 1:1 ethyl acetate:hexane and the product-containing fractions were combined and concentrated in vacuo to give 200mg of the title product. NMR: (CDC1 3 ,300 MHz) : δ 7.20 (m,1) ; 6.92(m,2); 5.90(m,l);

5.75(dd,l,J=5,9) ; 5.30(m,2); 4.98(d,1,J=5) ; 4.68(d,2); 3.80(s,2); 3.55(q,2,J=12) ; 3.4-3.1(m,4) ; 2.6(t,2,J=5) ; 1.40(s,9). IR: (KBr) 1771, 1756, 1714, 1680, 1670, 1531 cm-1. FDMS: M+l = 554.

Example 5

Allyl 7 -β- ( ( 2 -Thien-2 -yl ) Acetamido ) - 3 - ( ( ( 1 -

( Desacetylvi blast ine ) amino ) -2 -

Eethyl sul f ide ) Methylene ) - 3 -Cephem- 4 -Carboxylate

Allyl 7-β- ( (2 -thien-2-yl ) acetamido ) -3- ( ( ( 1- (t- butoxycarbonylamino) -2 -ethylsulf ide ) methylene) -3 -cephem-4- carboxylate ( 0 .46g, 0 . 823mmol ) was dissolved in methylene

chloride (5ml) and cooled to 0°C under a nitrogen atmosphere. Trifluoroacetic acid (neat, 2ml) was added and the resultant mixture was stirred for 1 hour. The reaction mixture was concentrated in vacuo, diluted with toluene and concentrated in vacuo twice, then diluted with CHCI3 and concentrated in vacuo twice to give the deprotected amino compound.

The deprotected amino compound was dissolved in methylene chloride (3ml) and the solution was cooled to 0°C. N-Methylmorpholine (0.3ml) was added to the mixture followed by the slow addition of a methylene chloride solution (5ml) of desacetylvinblastine azide (preparation described in U.S. Patent No. 4,203,898, Example 5, Column 20) (0.47g, 0.543mmol) . The resultant mixture was stirred at 0°C for 30 minutes, the reaction mixture was concentrated to 1/2 of its original volume then stirred overnight at room temperature. The reaction mixture was diluted with water, the layers were separated and the organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The concentrate was flash chromatgraphed over silica gel eluted with 4% methanol in methylene chloride. The product-containing fractions were combined and concentrated to give lOOmg of the title product. NMR: (CDCI3) : δ (mixture of Δ-2 and Δ-3 iεomerε)

9.68(s,l); 9.58(s,l); 8.60 (d, 1,J=9) ; 8.05(s,l);

7.55(d,l,J=9) ; 7.25-7.10 (m, 6) ; 7.0(m,2); 6.90(s,l);

6.60(s,l); 6.10(s,l); 6.0-5.6 (m, 5) , 5.60(s,l); 5.40-

5.3 (m,2); 4.8-4.6 (m,3) ; 3.86 (s,2); 3.80 (s,3); 3.75(s,2);

3.62 (s,3); 2.80 (s,3); 0.90 (two overlapping triplets, 6); remainder of protons are a mixture of multiplets from 4.0- 1.0. UV: (ethanol) lambda maχ 265 nm (Absorbance=0.6609) ,

214 (Absorbance=1.8358) . FABMS: M+l = 1190.4720.

Example 6

7-β-(2- (Thien-2-yl)Acetamido) -3- ( ( (1-

(Desacetylvinblastine)Amino) -2- Ethylsulfide)Methylene) -3-Cephem-4-Carboxylic Acid

Under a nitrogen atmosphere, tetrakis[triphenylphosphine] palladium [0] (5.8mg) and triphenylphosphine (1.8mg) were dissolved in 1:1 ethyl acetate:hexane. Allyl 7-β-(2- (thien-2-yl)acetamido)-3-

( ( (1- (desaceylvinblastine)amino)-2-ethylsulfide)methylene) - 3-cephem-4-carboxylate (0.20g, 0.168mmol) was added to the solution followed by the addition of triethylsilane (40.3μl, d=0.728). The reaction mixture was stirred at room temperature. After 1 hour, glacial acetic acid (2 drops) was added and the mixture was stirred for an additional hour. Ether was added to the reaction mixture to precipitate the product, and the reaction mixture was centrifuged to collect the resultant beige precipitate

(85mg) . The precipitate was chromatographed by reverse phase HPLC using a Waters C- j _g column eluted at lOml/min with 30% acetonitrile, 0.2% formic acid in water, monitored at 254nm. The product-containing fractions were combined and concentrated to give 30mg of white solid title product. NMR: (DMSO-dg,300 MHz) : δ 9.30(s,l); 9.0 (d,1,J=9) ;

8.45(m,l); 8.17(br.s,l) ; 7.9-7.6 (m,3) ; 7.31(m,2); 7.20(d,l,J=9) ; 7.0-6.8(m,4) ; 6.39(s,l); 6.14(ε,l); 5.7- 5.5 (m,2); 5.40(dd,l,J=5,9) ; 4.92 (d,1,J=5) ; 3.90 (s,l); 3.70(s,2); 3.67(s,3); 3.49(s,3); 2.67(s,3); 0.74 (t,3,J=7) ;

0.68 (t,3,J=7) ; remainder of protons are several multiplets from δ 4.9-1.1. IR: (KBr) 1768, 1764, 1648, 1630, 1616 cm- 1; UV: (ethanol) lambda maχ =267nm (ε=22,900), 215(ε=53,800) ; FABMS: 1150.44435.

Example 7 Synthesis of Antibody (F(ab')) Enzyme Conjugate

A solution of Sulfo-SMCC (Pierce Chemical

Company) was prepared in isotonic phosphate buffered saline, pH 7.2 (PBS) at a concentration of approximately

35mM. The true concentration of the solution was determined by measuring the absorbance at 260nm of an aliquot before and after hydrolysis with 0.3M Na2Cθ3, using a molar extinction coefficient of 8,000. β-lactamase ("β-L") from the P99 strain of

Enterobacter cloacae (available from Sigma Chemical Company) was purified according to the method of Cartwright and Waley, (Biochem. J.. 221. pp.505-512, 1984), then dialyzed against 50mM sodium borate, lOOmM sodium chloride, pH 8.0 (BBS) and stored. A volume of the solution containing 20mg of the protein was concentrated to about lOmg/ml with a Centiprep™ concentrator (Amicon Corporation, Danvers, Masssachusetts) after addition of Nonidet P-40 (Sigma Chemical Company) to 0.01%, and its exact concentration measured by absorbance at 280nm (extinction lmg/mL= 1.4, Cartwright and Waley, Biochem. J. , 2£ ^ pp.5329-5337 (1984)).

A volume of Sulfo-SMCC solution containing 2.0 molar equivalents (~35μD was added to the β-L solution, and the pH was adjusted to 8.0 with 0.3M Na 2 Cθ3. The unstirred solution was left at room temperature for 1 hour. The reaction mixture was then eluted through a 50mL P6DG (Bio-Rad Laboratories, Richmond, California) column with 50mM ammonium citrate, ImM DTPA, lOOmM NaCl, pH 6.2.

The protein containing peak (monitored at 280nm) was collected in a single fraction, and its concentration determined by measurement of the Absorbance at 280nm of an aliquot of the solution. The maleimide content of the protein solution was determined by reaction of an aliquot with a twofold excess of cysteine (10 min., room temperature) , and detection of the residual cysteine with

DTNB: molar extinction coefficient at 412nm of released thio-nitro-benzoic acid= 13,600. Under the stated reaction conditions, an average molar ratio of -0.9 maleimide/β-L is obtained. The derivatized protein was used within two hours.

F(ab') fragments of the anti-carcinoembryonic antigen antibody, CEM231, were prepared by pepsin digestion followed by cysteine reduction according methods known to the art and described by Breman, et al. , and Glennie, e_t al. cited above. The ratio of free thiol/F(ab") was determined by measuring the Absorbance at 280nm of an aliquant of the protein solution (extinction of a lmg/mL solution=1.4) , and quantitating the thiol concentration with DTNB. Typical average thiol/F(ab') ratios are in the range of 2.0 to 2.5.

Molar equivalents of maleimide derivatized β-L and CEM231 F(ab') at a concentration of 1 to 5mg/mL each were reacted at room temperature, unstirred, for 1 hour in 50mM ammonium citrate, ImM DTPA, lOOmM NaCl, pH 6.2. At the end of the reaction period, excess N-ethyl maleimide was added to stop the reaction.

The reaction mixture was then applied directly to a 500mL Sephadex G-150 Superfine column and eluted with BBS buffer. Product fractions were those determined by PAGE to contain pure material of molecular weight -90,000. Pooled product fractions were then concentrated to -1.5mg/mL, sterile filtered, and stored in sterile septum capped vials.

Procedure 6

Determination of β—lactamase Enzymatic Activity

Part A below demonstrates Enzymatic activity was quantified using either cephalothin (Sigma Chemical Company) or PADAC (Cal-Biochem) as substrate, in a Hewlett-

Packard 8451A Spectrophotometer equipped with a stirred thermostatable cell holder, monitored at 264 or 570nm, respectively. In a typical assay, substrate, at an assay concentration sufficient to give an absorbance of 0.5-1.0 at the wavelength of interest, (e.g., 50μM) , was added to a stirred, referenced solution of -5nM P-99 E. cloacae β- lactamase Enzyme ("β-L") in an appropriate buffer (PBS,

HEPES, etc.) at an appropriate pH (generally 7.2) equilibrated at 37°C. (The β-L Enzyme (also referred to as

"pencillinase") can be obtained commercially (e.g., Sigma Chemical Company) and from procedures in the literature.) The change in absorbance was then determined by the spectrophotometer at intervals of 5 or more seconds.

Part B demonstrates the determination of enyzmatic activity of β—L-CEM 231 conjugate on the compound of Example 11.

Part A Determination of K M and v maχ for β-L Enzyme and

Conjugates

A stock solution of cephalothin was prepared by dissolving 5.5mg in l.OmL lOmM sodium phosphate, 150mM sodium chloride, pH 7.2 (PBS): 13mM. A stock solution of P-99 E. cloacae β-lactamase ("β-L") enzyme was determined by A2 Q to be 1.3μM.β-L solution, 7.7μL, was added to a cuvette containing stirred, temperature equilibrated PBS, such that a final volume of 2.0mL would be obtained, and the spectrophotometer was referenced. Substrate stock solution was then added to obtain assay concentrations between 5 and 130μM. The spectrophotometer was instructed to wait 5 seconds for initial substrate mixing, then record the absorbance at 264nm every 10 seconds for 2 minutes.

The results were converted to change in absorbance from the initial value, and plotted using the RS/3 program (BBN Software Products Company, Cambridge, Massachusetts) . Inspection of the graphs showed that the initial rates were identical above 50μM Substrate

concentration, and that initial rates could be estimated from times prior to 25 seconds. (In subsequent determinations absorbance readings were collected every 5 seconds.) The Δ-OD values after 20 seconds of reaction were converted to Δ- [substrate] /sec. and were correlated with Substrate concentration in a double reciprocal plot: 1/V χ£ 1/[S] ( wherein V = delta [substrate] /second) , from which K M and V maχ were determined with their associated error limits. K cat was calculated based on the relation that: k cat = V maχ /[E] (The Substrate concentrations were calculated from the 264nm absorbance at the 5 second time point using a molar extinction coefficient of 8,540 for cephalothin. )

Similar experiments were performed on two separate lots of β-L-CEM231 conjugate. The values for K M were 4.8+/-0.1, 4.4+/-0.3, and 5.2+/-0.4μM; and for k cat

54+/-0.7, 35+/-1.4, and 49+/-2.8 sec "1 , for β-L and the two lots of conjugate, respectively.

Part B

1.5ml of a PBS buffer solution (pH 7.4) in stirred cell was incubated at 37°C with different concentrations of the compound of Example 11. The reaction was started in each case by addition of β-Lactasmase-CEM

231 conjugate at a concentration of O.llnM and allowed to incubate for 90 seconds. Samples were quenched by adding

0.5ml of the reaction mixture to 0.5ml of a solution composed of 34% acetonitrile and 200mM KH2PO4 buffer adjusted to pH 3 with H3PO4.

The decrease in concentration of the cephalosporin Prodrug was determined by averaging duplicate HPLC injections of the quenched sample on a 0.46 x 15cm C- 18 reversed phase column monitored at 266nm. The flow rate was 1ml per minute, run isocratically with the 34% acetonitrile buffer described above.

Data Collected

Minutes μM Prodrug μM/min 1/C 1/V

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

: 'ccaat /K m = 11.1 ± 42 sec "1 μM -1 for the compound of Example 11

Procedure 7

Demonstration of Immunoreactivity of β-lactamase Conjugated CEM231

Iodination: 10μg conjugate at lμg/μL was treated with 200uCi 125 ι in the presence of Biorad Enzymobeads (BioRad Laboratories) and 25μL 1% β-D-glucose at room temperature for 20 minutes in 0.2M sodium phosphate, pH 7.1. The mixture was quenched with 50μL of a solution of PBS containing 0.1% sodium azide and 0.35mg/mL sodium metabisulfite, pH 7.2. Carrier was added in the

form of 100μL of PBS containing 0.1% sodium azide, 0.1% gelatin, and 1% sodium iodide.

The mixture was purified on a 5mL Sephadex G-25 column equilibrated with PBS, 0.1% sodium azide, 0.1% gelatin solution, and eluted with PBS containing 0.1% sodium azide, pH 7.5, into tubes containing 100μL of a 0.1% gelatin solution in 0.5mL fractions. The fractions were counted for radioactivity in an ISODATA 20/20 Series Gamma Counter (RIA Data Systems, Rolling Meadows, Illinois) , and the first peak of radioactivity was the labeled conjugate.

Immunoreactivity: Polystyrene beads were prepared with carcinoembryonic antigen (CEA) or with irrelevant protein (for non-specific binding control) by soaking the beads in a solution of the desired protein at a concentration of 150ng/bead in 10% horse serum. Iodinated conjugate was diluted to about 200cpm/μL in 10% horse serum. Three each antigen positive and antigen negative beads were placed in individual gamma counting tubes, washed, and treated with 200μL labeled conjugate. The beads were incubated at 37°C overnight. Total counts were measured in each tube; supernatants were transferred to tubes containing a second bead of the same type. Finally, all beads were washed, and the fraction of counts on each bead was determined. Immunoreactivity was calculated as: A + + B + (l-A + ), and Non-specific Binding was calculated as: A_ + B_(l-A_) , where A + and B + are the fractions bound on the antigen positive beads in the first and second rounds, respectively, and A_ and B_are the fractions bound on the antigen negative beads in the first and second rounds, respectively.

Immunoreactivity for the β-L-CEM231 conjugate was found to be about 80%, which compares well with values obtained for unmodified F(ab') fragments of CEM231. Non-

specific binding had also not increased by conjugation with β-lactamase.

Procedure 8

Demonstration of Release of Substituents from the 3 ' -Methylene Group of Cephalosporins onTreatment with β-lactamase

Release of substituents (Cytotoxic Agents) from the 3 ' position of cephalosporins can be demonstrated by chromatographic (especially HPLC) analysis of the reaction mixture, or by spectrophotometric assay of the released functional group. (If the detection method is not instantaneous, the Enzyme can be stopped at the desired time by addition of an inhibitor such as cloxacillin (Sigma Chemical Company) ) at its effective concentration: 5μM for cloxacillin. )

For instance, a model system for the present method employs the expulsion of aminoethanethiol from 7-β-

(2-(thien-2-yl)acetamido) -3- ( (2-aminoethyl-l- sulfide)methylene)-3-cephem-4-carboxylic acid. A typical enzyme assay was performed (following the 264nm absorbance), but in 50mM Tris, lOOmM NaCl buffer, pH 8.0. Quantitation of the release of thiol was achieved in a subsequent assay by including 250mM DTNB (dithionitrobenzoic acid) in the reaction mixture, and observing the change in absorbance at 412nm. Using molar extinction coefficients of 8,540 for the 264nm absorbance, and 13,600 for the 412nm absorbance, the rates of the two reactions can be compared (reaction of thiol with DTNB is instantaneous under these conditions) . The graphs of the results showed that thiol is released as a result of β- lactamase catalyzed cleavage of the cephalosporin, but that the release rate is slower than the catalytic rate.

To demonstrate the expulsion of 5-fluorouracil from 7-β-(2- (thien-2-yl)acetamido) -3- ( (N-l (5-

fluorouraciDyl)methylene)-3-cephem-4-carboxylic acid (ceph-5-FU) , a stock solution of the cephalosporin was prepared (lmg of cephalosporin in 2ml of 20% N,N- dimethylacetamide in pH 7.0 HEPES buffer, final concentration of 500μg/ml) . A stock solution of P-99 E. cloacae β-lactamase Enzyme was prepared. To the stock solution of the cephalosporin Substrate (90μl) was added stock β-lactamase solution (lOμl) . The reaction was monitered by HPLC (on a Waters Associates C^ μbondapak column) eluted with 25% acetonitrile/ 1% acetic acid in water (monitered at 254nm absorbance) . The retention time for 5-fluorouracil was 1.9 minutes and was 5.3 minutes for the cephalosporin - (5-fluorouracil) compound. Approximately 30 seconds after mixing a 5μl aliqout of the reaction mixture was injected into the HPLC. The HPLC trace indicated that the cephalosporin had been consumed, and a new compound with a retention time of 4.3 minutes appeared, along with a small amount of 5-fluorouracil with a retention time of 1.9 minutes. Twenty-one minutes after mixing the two stock solutions, the compound eluting at 4.3 minutes was diminished and a large amount of compound eluting at 1.9 minutes was observed. Thirty-six minutes after mixing the stock solutions the amount of compound eluting at 4.3 minutes was further diminished, and the amount of compound eluting at 1.9 minutes was correspondingly larger by intergration of the peak areas.

Thus, the Enzyme caused conversion of the cephalosporin - (5-fluorouracil) Substrate - Cytotoxic Agent to the Cytotoxic Agent.

Procedure 9_

Tumor Localization of lodinated β-L-CEM231 Conjugate

Conjugate was labeled with 12 5ι as described for the immunoreactivity assay. Twelve nude mice in which LS174T tumor cells had been injected subcutaneously and

allowed to reach a size of about 0.4g were injected in the tail vein with 50μg of the labeled conjugate in 100μL BBS.

At 4 and 24 hours 6 mice were sacrificed, dissected, and the weight and amount of radioactivity in each organ was quantitated. The results are reported as % of injected dose/gm tissue.

Resμlt-s:

Blood Bone Heart Kidney Liver Lung Muscle Skin Spleen Tumor GI

4 HOUR

15.8 2.4 4.3 7.4 3.7 8.1 1.3 3.8 4.2 13.9 3.1

24 HOUR

1.5 0.2 0.4 0.7 0.6 1.0 0.1 0.8 0.4 7.5 0.7

These results show that the conjugate does accumulate in tumor preferentially, and that it shows no untoward accumulation in critical organs. It also shows a rapid clearance from the serum which is desirable from the standpoint of conversion of Substrate - Cytotoxic Agent to Cytotoxic Agent specifically at the target. The results are comparable to those seen for murine hybrid antibodies of the same approximate molecular weight, and show no unusual metabolic treatment of the bacterial protein.

Procedure 10

Enzymatic Assay of Conjugate Bound to Antigen

Expressing Cells

In order to show that β-lactamase activity could be bound to tumor cells via the prepared β-L-CEM231 conjugate, CEA expressing LS174T cells and CEA negative MOLT4 cells were prepared as single cell suspensions in Autopow media (GIBCO, Grand Island, New York) . Both suspensions were at a density of 2xl0 7 cells/mL; LS174T

were 23% viable, MOLT4 80% viable. Suspensions were maintained on ice until use.

Cells (lmL) were incubated for either 1 or 10 minutes with 10 or 50pmol of conjugate or unconjugated β- lactamase. Cells were then centrifuged, the supernatant discarded, re-suspended in buffer, centrifuged again, then suspended in l.OmL of buffer containing PADAC (Cal-Biochem) at a known concentration (determined spectrophotometrically) around 30μM. After incubating 10 minutes with Substrate, the cells were centrifuged, and the concentration of PADAC in the supernatant determined. Results are reported as % of original absorbance remaining after incubation at the PADAC peak absorbance wavelength of 570nm.

Results:

Conjugate Quantity Binding Time %OD 570 Remaining pmol/ml min after incubation

LS174T Cells

46 23 95 87 62 36

94 93

These results show that β-lactamase activity can be bound to antigen positive cells in a specific manner, that the cells have no such activity in the absence of the conjugate, that binding does not impede the catalytic activity of the Enzyme, and that the binding is time and concentration dependent.

Procedure 11

PADAC Assay of Serum Kinetics of β-L-CEM231

Con-iuσate β-L-CEM231 conjugate (50μg) in 50mM sodium borate, lOOmM NaCl (BBS) was injected into the tail vein of each of 18 tumor- free nude mice. Three mice each at 1, 24, 48, 72, 96, and 120 hours after injection were bled, and their sera pooled and frozen. The serum samples were assayed for β—L activity using the standard assay conditions. Appropriate quantities of serum were diluted to 2.OmL with PBS, and the time of the assay was varied as necessary to detect activity. PADAC (20_L of an EtOH solution prepared by dissolving 2.4mg/mL) was used as substrate to avoid interference from serum proteins.

Linear portions of the absorbance y^. time plots were determined by inspection and those points were fitted by linear regression (RS/3, BBN Software Products Company). The slopes of these lines were compared to the slope obtained for a known amount of conjugate under identical conditions, and corrected for dilution, to calculate the concentration of conjugate present in the serum.

Results: μL Serum in Assay μg β-LCEM231/mL Serum

10 20

100 1.0

50 0.33

200 0.16

600 0.06 600 0.06

These results show that no β-lactamase activity exists in normal serum of these mice, and that the serum level drops rapidly, so that Substrate - Cytotoxic Agent treatment could begin relatively soon (48 hours) after injection of conjugate (in nude mice) . Similar experiments would show when sufficient serum clearance had occurred in

humans. Comparison of the enzymatically determined level of conjugate with the level determined by radiodetection of iodinated conjugate, (measured at 4 and 24 hours after injection) , shows that enzyme is not inactivated or inhibited in the serum. (See Procedure 9).

Procedure 12 β-lactamase Activity Bound to Antigen Expressing Tumor Tissue In Vivo

Nude mice implanted with LS174T or MOLT4 cells which had been allowed to develop into 0.5 to 1.Ogm tumors were injected in the tail vein with 50μg of β-L-CEM231 conjugate diluted in 100μL BBS. Twenty-four hours after injection the tumors were excised and cut into -lOOmg chunks.

The chunks were minced, then incubated 10 minutes with a lmL PBS solution to which had been added 10μL of a stock solution made by dissolving lmg of PADAC in 400μL 100% EtOH. Exact concentrations of PADAC in the assay solutions were determined spectrophotometrically ε 570 nm =48,000). The samples were then centrifuged (10 minutes, lOOOxg) , and the supernatants examined spectrophotometrically. Because the supernatants were very cloudy, quantitation was impossible, but the color change from purple to orange was clearly visible only in the LS174T tumor sample. The result was recorded photographically. Identical positive results were obtained in sections of LS174T tumors from two different nude mice, and negative results from sections of MOLT4 tumors from two different nude mice.

Procedure 13

In Vitro Cytotoxicity Determination

Target cells (antigen positive or antigen negative) are resuspended in 75% leucine deficient EBSS-MEM

(GIBCO) + 10% dialyzed fetal bovine serum (FBS) + gentamicin (GIBCO) at 200,000 cells/ml. Aliquots of this cell suspension, 0.2ml, are seeded into 96 well plates and incubated overnight at 37°C, 5% CO2. To the supernatants is added the antibody-enzyme conjugate to a final concentration of 25μg/ml (unconjugated enzyme, unconjugated antibody, no treatment) . The supernatants are removed after 1 hour incubation, and the cells rinsed once with media. The media is then replaced with the same media, but containing a Substrate - Cytotoxic Agent at concentrations between 0.001 and lOμg/ml. The cells are incubated with the Substrate - Cytotoxic Agent compound for a period of time ranging from 3 to 48 hours at 37°C, 5% CO2. Media containing 4μCi of ^H-leucine are added per well. The cells are incubated 24 hours with the labeled leucine, then harvested. Uptake of labeled leucine is determined by liquid scintillation counting. (All samples are run in triplicate.) Results are reported as the Substrate -

Cytotoxic Agent concentration at which leucine incorporation is reduced to 50% of the control value (ID 50 ).

As an example of the above general procedure, the cytotoxicity of 7-β-(2- (thien-2-yl)acetamido)-3-((( (1-

(desacetylvinblastine)amino) -2-ethylsulfide)methylene)-3- cephem -4-carboxylic acid ("COMPOUND") towards CEA - bearing LS174T (ATCC) tumor cells that were either treated or untreated with β-L-CEM231 conjugate ("conjugate") was measured. Thus, the appropiate wells were exposed to the β-L-CEM231 conjugate at 25μg/ml for 1 hour at 37°C, 5% C0 2 .

The cells were then rinsed and resuspended in media containing the counpound. At the 24 hour time point, the

appropriate wells were washed and 0.2ml of fresh media was added to complete the 48 hour incubation. The rest of the experiment was carried using the above standard procedure.

The results of this experiment are as follows: COMPOUND treatment time: cells treated with—: 6 Hours 48 Hours

1) COMPOUND and conjugate: 0.265 0.001

2) COMPOUND alone: 2.154 <0.001

3) Cytotoxic Agent 2 alone: 0.260 <0.001

1 values are ID^Q (μg/ml)

2 Desacetylvinblastine aminoethanethiol

These data show that the COMPOUND is much less toxic to the tumor cells than the Cytotoxic Agent from which it is derived, but that when the cells are first treated with the conjugate, the toxicity of the COMPOUND is dramatically increased.

Procedure 14 t-Butyl 7-β-(2- (Phenoxy)Acetamido) -3- ( (p- Nitrophenylcarbonato)Methylene-3-Cephem-4-

Carboxylate t-Butyl 7-β-(2- (phenoxyacetamido) -3- (hydroxymethylene) -3-cephem-4-carboxylate (0.50g, 1.15 mmol) was dissolved in dry tetrahydrofuran (5ml). The resultant solution was cooled to 0°C under nitrogen. To the cooled solution was added (p-nitrophenyDchloroformate (0.35g, 1.72mmol) then dimethylaminopyridine (DMAP, 2mg) and finally 2,6-lutidine (0.20ml) (slowly) . The resultant mixture, exhibiting a heavy precipitate, was allowed to stand overnight in an ice bath. Additional THF was added and the precipitate was broken up with a spatula. The mixture was filtered then flash chromatographed over silica

gel eluted with a 1:1 mixture of methylene chloride and ethyl acetate to yield 0.40g of the title product: NMR: (CDCl 3 , DMSO-dg 300 MHZ); δ 8.01 (d,2,J=8.9) ;

7.75(d,l,J=10.2) ; 7.25-7.00 (m,3) ; 6.80-6.62 (m, 4) ;

5.86(dd,l,J=4.9,10.2); 5.28 (d,1,J=13.3) ; 4.67 (d, 1,J=13.3) ;

4.56(d,l,J=4.6); 4.33(s,2); 3.65 (d,1,J=18.6) ; 3.32(d,l,J=18.5) ; 1.30(s,9); IR: (CHCI3) 3020, 1807, 1771,

1725, 1696 cm "1 ; UV: (EtOH) , lambda maχ 392(ε=1100), 268(6=13,500) ; FABMS: (M+H)602, 546, 363.

Examp1e 8

t-Butyl 7-β-(2- (Phenoxy)Acetamido) -3-

( (Desacetylvin lastinehydrazido) Carbonyloxymethylen e) -3-Cephem-4-Carboxylate

The free base of desacetylvinblastine hydrazide sulfate was made by dissolving the sulfate salt (900mg) in aqueous sodium bicarbonate solution then extracting the solution with methylene chloride. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to give a white solid. The free base was dissolved in dry pyridine (6ml) and added to t-butyl 7-β-(2-

(phenoxy)acetamido) -3- (0- (p-nitrophenyDcarbonato) methylene) -3-cephem-4-carboxylate (0.40g, 0.666mmol) . The addition flask was washed with another portion (2ml) of pyridine and added to the reaction mixture. A small amount of N,N-Diisopropylethylamine (two drops) was added to the reaction mixture and the resultant mixture was stirred overnight at room temperature under nitrogen. The reaction mixture was diluted with ethyl acetate then concentrated in vacuo. The concentrate was flash chromatographed over silica gel eluted with 90/10 methylene chloride/methanol to give 0.54g of the title product: NMR: (CDCI3 , 300mHz) : δ

10.0(s,l); 8.70(s,l); 8.02(s,l); 7.93 (d,1,J=12) ; 7.50(d,l,J=12) , 7.38-6.90(m,10) ; 6.10(s) overlapping with 6.12(m,l); 5.80 (d, 1,J=12) ; 5.65 (d,1,J=12) ; 4.38(S,2);

4.02 (d,l,J=12) ; 3.75(s,3); 3.58(s,3); 2.8(brs,3);

1.52 (s, 9); 0.90 (brs, 6) remainder are mutiplets ranging from 4.0-l.lppm.; IR: (CHCI3) 1802, 1721, 1696, 1515 cm "1 ; UV:

(EtOH) , lambda maχ =267(ε=23,800) , 214 (ε=53 , 000) ; FABMS: Calc'd. (M+H)=1231.53853. Found=1231.54230.

Example 9

7 -β-(2- (Phenoxy) Acetamido) -3-

( ( De sac e ty 1 vinbl as tinehydr azido) Carbonyloxymethylen e) -3-Cephem-4-Carboxylic Acid Trifluoroacetic Acid

Salt t-Butyl 7-β-(2- (phenoxy)acetamido)-3- ( (desacetylvinblastinehydrazido)carbonyloxymethylene) -3- cephem-4-carboxylate (123mg, 0.lmmol) was dissolved in methylene chloride (3ml) . The solution was cooled to 0_C and triethylsilane (1ml) then trifluoroacetic acid (1ml) was added. The resultant solution was stirred for 30 minutes. Additional identical portions of triethylsilane and trifluoroacetic acid were added. The ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The mixture was concentrated in vacuo. diluted with cold acetonitrile (3 volumes) , and again concentrated in vacuo. Carbon tetrachloride was added and the mixture was again concentrated in vacuo to yield 120mg of the title product: NMR: (DMSO-dg,300MHz) : δ

9.76(br.ε,l); 9.46(br.s,1) ; 9.41(br.s,1) ; 8.14(d,1,J=9.3) ;

7.47(d,l,J=7.9Hz) ; 7.35-7.21(m,4) ; 7.18-6.80(m, 6) ;

6.70(s,l); 6.30(s,l); 6.00(dd,1,J=4.8,9.5) ; 5.75(s,2);

5.15 (d,l,J=12) ; 4.92 (d,1,J=4.8) ; 4.6(s. overlapping broad H 2 0 peak) ; 3.72(s,3), 3.53(s,3); 2.79 (br.s,3) ;

0.82 (t,3,J=7) ; 0.70 (t,3,J=7) ; remainder are multiplets ranging from 4.0-l.lppm; IR: (KBr) 1788, 1720, 1685, 1677 cm "1 ; UV: (EtOH), lambda maχ =311 (ε=3800) , 268(ε=18,500) ,

215(ε=45,100) ; FABMS: Calc'd. (M+H)=1175.47593.

Found=1175.47600.

Procedure 15

Benzhydryl 7-β—(2- (Thien-2-yl ) acetamido) -3- (Hydroxymethyl) -2-Cephem-4-Carboxylate

7-β-(2- (Thien-2-yl)acetamido) -3- (Hydroxymethyl) -

2-cephem-4-carboxylic acid (5.0g, 14.lmmol), obtained as described in Kukolja S., J. Med Chem. , p.1114, 1970, was dissolved in a 1/1 mixture of acetone and acetonitrile. To this solution was added in a dropwise fashion an acetonitrile solution of diphenyldiazomethane (2.7g, 13.9mmol) . The resultant reaction mixture was stirred at room temperature for 1 hour then concentrated in vacuo.

Acetonitrile was added to the concentrate and the resultant biege solid (4.4g), the title product, was collected by filtration. NMR: (CDCl 3 ^ 300MHz) , 7.40-7.23 (m, 11) ; 7.02-

6.95 (m,2); 6.88 (s,l); 6.45 (d,1,J=7.6) ; 6.25(s,l);

5.61(dd,l,J=5,8) ; 5.20 (d, 1,J=5) ; 5.15(s,l); 4.10 (q,2,J=8 3.82 (s,2); IR: (CHCI3) 1778 cm -1 ; 1743, 1683; EA: Calc'd

C=62.29, H=4.64, N=5.38. Found: C=62.07, H=4.73, N=5.51

Procedure 16

Benzhydryl 7-β-(2- (Thien-2-yl )Acetamido) -3-

(Hydroxy ethylene) -3-Cephem-1-β-Sulfoxide-4-

Carboxylate

Benzyhydryl 7-β-(2- (Thien-2-yl)acetamido) -3-

(hydroxymethylene) -2-cephem-4-carboxylate (2.0g, 3.85mmol) was dissolved in a 4/1 mixture of methylene chloride/isopropanol (125ml) and the solution was cooled to

-10_C. Metachloroperbenzoic acid (1.21g) was dissolved in isopropanol (25ml) and added to the previous cooled solution in a dropwise fashion over a period of 30 minutes.

The resultant reaction mixture was taken to dryness in vacuo. Benzene was added and the mixture was taken to dryness in vacuo. Ethyl Acetate was added to the solid, and the tan solid was collected by filtration and dried to yield 1.5g of the title product: NMR: (300MHz,CDCI3 +

DMSO-dg) : δ 7.4-7.0 (m, 11) ; 6.80(m,2) ; 6.70(s,l);

5.82 (dd,l,J=4.6,9.4) ; 4.42 (d, 1, J=4.7 ) ; 4.37 (d, 1, J=15.0) ;

4.18(d,l,J=15.0) ; 3.84 (d, 1, J=19.2) ; 3.67(s,2),

3.22 (d,l,overlaps with H 2 0) ; (OH and NH not identified);

UV: (EtOH), lambda maχ =258nm(ε=6540) ; IR: (KBr) 1654 cm "1 ,

1721, 1755, 1773, 1785; EA: Theory C=60.43, H=4.51, N=5.22. Found C 60.57, H 4.37, N 5.01; FABMS: M+H = 537.

Procedure 17

Benzhydryl 7-β-(2- ( hien-2-yl)Acetamido-3- (O- (p-

Nitrophenyl ) Carbonato)Methylene) -3-Cephem-1-β- Sulfoxide-4-Carboxylate , Benzhydryl 7-β-2- (thien-2-yl) acetamido-3- (hydroxymethyl) -3-cephem-l-β-sulfoxide-4-carboxylate (1.0g, 1.87mmol) was partially dissoved in dry THF (20ml) and the mixture was cooled to 0°C under nitrogen. First (p- nitrophenyDchloroformate (0.49g, 2.43mmol), then dimethylaminopyridine (5mg) , and finally dry 2,6 lutidine (0.28ml) was added to the cooled solution. The resultant solution was stirred for 30 minutes at 0°C then for 1 hour at room temperature. The mixture was filtered, the filtered solid was washed with ethyl acetate, and the filtrate was concentrated in vacuo. The resultant solid was flash chromatographed over silica gel eluted with a 60/40 mixture of methylene chloride / ethyl acetate to yield 0.32g of the colorless title product: NMR: (300MHz,DMSO-dg) : δ 8.49 (d, 1,J=8.3Hz) ; 8.27 (d, 2,J=9.2) ;

7.5-7.2 (m, 13) ; 6.92(m,3); 5.94 (dd,1,J=4.9 , 8.2) ; 5.25(d,l,J=13.0) ; 4, 93 (d, 1,J=4.4) ; 4.85 (d, 1,J=13.0) ; 4.04(d,l,J=18.7) ; 3.88 (d,1,J=15.3) ; 3.78 (d,1,J=15.3) ; 3.66(d,l,J=18.7) ; UV: (EtOH) 265nm(ε=3570) ; IR: (KBr) 1720 cm "1 , 1765, 1658; EA: Theory C=58.20, H=3.88, N=5.99. Found: C=58.48, H=4.02, N=5.90.

Example 10

Benzhydryl 7-β-(2- (Thien-2-yl)Acetamido) -3-

( (Desacetylvinblastinehydrazido)Carbonyloxy)

Methylene) -3-Cephem-1-β-Sulfoxide-4-Carboxylate

The free base of desacetylvinblastinehydrazide sulfate (0.50g) was made as described above in Example 8. The free base was dissolved in dry pyridine (10 ml) and added to benzhydryl 7-β-(2- (thien-2-yl)acetamido) -3- (0- (p-nitrophenyl)carbonato)methylene) -3-cephem-l-β- sulfoxide (0.32g, 0.456mmol). The mixture was allowed to stir at room temperature under nitrogen after N,N- diisopropylethylamine (2 drops) was added. The resultant reaction mixture was stirred overnight at room temperature under nitrogen, then diluted with ethyl acetate and concentrated in vacuo. The solid thus obtained was flash chromatographed over silica gel eluted with a 90/10 mixture of methylene chloride/methanol to yield 0.34g of an off- white solid that was the title product: NMR: (CDC1 3 ,300MHz) : δ 9.91(s,l) ; 8.66(br.s,1) ; 8.01(s,l); 7.53-

6.86(m,23); 6.55(s,l); 6.08(s,l); 6.05(dd,1,J=4.7,9.8) ; 5.8-5.6(m,2) ; 5.3-5.2 (m,1) ; 4.85 (br.s,1) ; 4.47 (d,1,J=4.4) ; 3.83 (s,2); 3.74 (s,3); 3.57 (s,3); 0.87 (t, 6,J=7.2) remainder are buried multiplets ranging from 4.1-l.lppm.; UV: (EtOH), lambda maχ =268nm(ε=25200) ; IR: (CHCI3) 1690cm "1 , 1730, 1803; FABMS: Calc'd. for (M+H) C 71 H 79 Ng0 14 S 2 =1331.51569.

Found=1331.51224.

Example 11

7 -β- ( 2 - ( hi en- 2 -yl ) Acetamido ) - 3 -

( ( Desacetylvinblast inehydrazido ) carbonyloxy) methylene ) -3 -Cephem- 1-β-Sul f oxide- 4 -Carboxyl ic

Acid, Trif luoroacetic Acid Salt

Benzhydryl 7-β- (2- ( Thien- 2 -yl) acetamido) -3 -

( (desacetylvinblastinehydrazido) carbonyloxy) ethylene) -3 - cephem-l-β-sulfoxide-4-carboxylate ( 105mg, 0.00789mmol ) was

dissolved in methylene chloride (3ml) . The solution was cooled to 0°C under nitrogen, then triethylsilane (0.5ml) followed by trifluoroacetic acid (0.5ml) was added. After 15 minutes the solution was diluted with acetonitrile and concentrated in vacuo twice. The solid was dissolved first in chloroform then in diethyl ether, each time followed by concentration in vacuo. The resultant solid was dried under vaccuum at room temperature to yield lOOmg of the title product: NMR: (300MHz,DMSOdg) : δ 9.76(s,l);

9.46(s,l); 9.40(s,l); 8.43 (d,1,J=8.1) ; 7.47 (d,1,J=8.0) ,

7.4-6.9(m,16) ; 6.68(s,l); 6.28(s,l); 5.80 (dd,1,J=5.8) ; overlapping with 5.75 (s,l) ; 5.16 (d, 1,J=12.7) ; 4.85(m,l);

4.64(d,l,J=13.2) ; 3.88(s,2); 3.78(s,2); 3.71(s,3);

3.53(s,3); 2.78(br.s,3) ; 0.80(t,3,J=7) ; 0.70(t,3,J=7) ; remainder are multiplets ranging from 4.3-1.0ppm.; UV: (EtOH), lambda maχ =266nm (ε=19,300) , 214 (ε=49,800) ; FABMS: Found=1165.43290.

Example 12

Benzhydryl 7-β-(2- (Thien-2-y1)Acetamido) -3-

( ( (Desacetylvinblastinehydrazido) carbonyloxy)Methyl ene) -3-Cephem-4-Carboxylate

Benzhydryl 7-β-(2- (thien-2-yl)acetamido)-3-

( ( (desacetylvinblastinehydrazido)carbonyloxy)methylene)-3- cephem-l-β-sulfoxide-4-carboxylate (0.24g, 0.180mmol) and stannous chloride (0.10g, 0.451mmol) were dissolved in dimethylformamide (DMF, 3ml) . The solution was cooled with an ice bath and acetyl chloride (0.32ml) was added slowly with a syringe. The resultant reaction mixture was stirred for two minutes with the ice bath in place then for twenty minutes at room temperature. The reaction mixture was poured into cold water (20ml) . The pH of the aqueous layer was taken to 8 with cold aqueous sodium bicarbonate solution. The resultant mixture was extracted with ethyl

acetate, and the organic layer was dried over sodium sulfate, filtered, and taken to dryness in vacuo. The solid was flash chromatographed over silica gel eluted with a 90/10 mixture of methylene chloride/methanol to yield 47mg of the title product: NMR: (300MHz,CDCl 3 ) : δ

10.30(s,l); 9.90(s,l); 9.20(s,l); 8.70(s,l);

8.02 (d,2,J=8Hz) ; 7.6-6.8 (m,25) ; 6.58 (br.s, 1) ; 6.05(s,l);

5.83 (dd,l,J=5,8) ; 5.8-5.7 (m, 2) ; 5.12 (d,1.,J=13) ;

4.92 (d,l,J=5) ; 4.10 (d, 1,J=8) ; 3.78(s,2); 3.72{s,3);

3.55(s,3); 2.77(s,3); 0.88 (t, 6, J=7) ; Remainder are buried multiplets ranging from 4.0-l.lppm.; FABMS: Calc'd. for C 71 H 79 N 8 0 13 S 2 =1315.52077. Found=1315.51781.

Example 13

7-β- (2- (Thien-2-yl) Acetamido) -3-

( ( (Desacetylvinblastinehydrazido) Carbonyloxy) Methyl ene) -3-Cephem-4-Carboxylic Acid, Trifluoroacetic

Acid Salt

Benzhydryl 7-β-(thien-2-yl)acetamido) -3- ( ( (Desacetylvinblastinhydrazido) carbonyloxy)methylene) -3- cephem-4-carboxylate (46mg, 0.0035mmol) was dissolved in methylene chloride (2ml) . The solution was cooled to 0_C under nitrogen, then triethylsilane (0.5ml) followed by trifluoroacetic acid (0.5ml) were added. The resultant reaction mixture was stirred for 30 minutes under nitrogen a 0°C, cold acetonitrile was added and the solution was taken to dryness in vacuo. The procedure with acetonitrile was repeated, then the solid was taken up in diethyl ether and taken to dryness in vacuo to leave an off-white solid

(42mg after drying) of the title product: NMR:

(300MHz,DMSO-dg) : δ 9.78(8,1); 9.46(8,1); 9.35(s,l);

9.10(d,l,J=8) ; 7.47(d,l,J=8) ; 7.4-6.8 (m, 16) ; 6.68{s,l); 6.29(s,l); 5.75(8,1); 5.65 (dd, 1,J=5, 8) ; 5.2-4.9 (m,2) ; 3.85(s,2), 3.72(s,3); 3.50(s,3); 2.75 (br.s, 3) ; 0.80 (t, 3 ,J=7) ; 0.65 (t, 3,J=7) remainder are mutiplets

ranging from 4.2-1.0ppm; FABMS: Calc'd. for C 58 Hg9N 8 0;ι_3S2 = 1149.44252. Found 1149.44007.

Procedure 18

Allyl 7-β-(2- (Thien-2-yl)Acetamido) -3-((l-(t-

Butoxycarbony1amino) -2-ethylsulfide) Methylene) -3 -

Cephem-1-β-Sulfoxide-4-Carboxylate

Allyl 7-β-(thien-2-yl)acetamido) -3- ( (1- (t- butoxycarbonylamino) -2-ethylsulfide)methylene) -3-cephem-4- carboxylate (l.Og, 1.81mmol) was dissolved in methylene chloride (20ml) and the solution was cooled to -78_C under nitrogen. Meta-chloroperbenzoic acid (0.37g) was dissolved in methylene chloride (20ml) and the solution was added dropwise to the cold cephalosporin solution. The reaction mixture was stirred under nitrogen for 15 minutes, then warmed to -10_C using an ice/brine bath. After 15 minutes, the reaction mixture taken to dryness in vacuo. The solid was flash chromatographed over silica gel eluted with a 2% methanol in methylene chloride solution to give 0.60g of the title product: NMR: (DMSO-dg, 300MHz) : δ

8.40(d,l,J=8.3Hz) ; 7.32(m,l); 6.90(m,2); 6.80 (t, 1,J=4.8) ;

6.0-5.8(m,l) ; 5.78 (dd, 1,J=4.8, 8.3) ; 5.34 (d, 1,J=17.2) ;

5.21(d,l,J=10.5) ; 4.91 (d, 1,J=4.4) ; 4.71 (d, 1,J=5.4) ; 4.0- 3. (m, 6); 3.0(m,2); 2.5 (m,2), 1.32 (s, 9); 13 C (DMSO-dg,

270MHz) 28.19; 30.68; 32.70; 35.67; 46.42; 58.05; 66.06;

66.54; 77.69; 118.76; 122.93; 123.10; 125.04; 126.44;

126.66; 131.78; 136.78; 155.50; 160.65; 164.12; 170.00; UV: (EtOH); 271(ε=8,330) , 234(ε=ll,000); IR: (CHCI3) 1801,

1728, 1700, 1696cm "1 ; FABMS: M+l=570; EA: Calc'd C 50.60, H 5.48, N 7.38. Found: C 50.83, H 5.55, N 7.32.

Example 14

Allyl 7-β-(2- ( hien-2-yl)Acetamido) -3- ( ( (1-

Desacetylvinblastine)Amino) -2-

Ethylsulfide)Methylene) -3-Cephem-1-β-Sulfo ide-4-

Carboxylate

The procedure of Example 5 was repeated, substituting the cephalosporin-1-β-sulfoxide starting material of Procedure 18 (0.34g, 0.598mmol) for the cephalosporin sulfide of Example 5. Also, the following amounts of other reagents were used:

Desacetylvinblastine hydrazide (0.46g, 0.60mmol);

N-Methylmorpholine (0.20ml, l.δmmol);

Sodium Nitrite (0.083g, 1.2mmol) .

For the current, the reaction was stirred overnight at room temperature under nitrogen, instead of 30 minutes at 0°C in

Example 5. The product was flash chromatographed over silica gel eluted with a gradient of 95/5 to 90/10 methylene chloride/methanol, to yield 0.30g of the title product as a yellow solid: NMR: (CDCI3, 300MHz) : δ

9.48(s,l); 8.01(8,1); 7.5-6.9(m,12) ; 6.52(s,l); 6.02(s,l); 5.95 (dd,l,J=4.2, 9.5Hz) ; overlapping with 5.92(m,l,); 5.70(8,2); 5.35(d,l,J=17.1); 5.25 (d,1,J=7.5) ; 4.73 (br.ε,2) ; 4.62(d,l,J=4.2) ; 3.71(s,2); 3.67(s,3); 3.66(s,3); 3.55(8,3); 2.73(s,3); 0.88 (t,3,J=7) ; overlapping with

0.85 (t,3, J=7); remainder are various multiplets ranging from 4.1-l.lppm.; UV (EtOH), lambda maχ = 268(ε=22,600) ,

214(ε=57,000) ; IR: (CHCI3) 1799, 1730, 1669, 1615cm "1 ;

FABMS: Calc'd. for C 2 H 7 gN 7 0 12 S3 = 1206.47139. Measured mass = 1206.47138.

Example 15

7-β-(2- (Thien-2-yl) acetamido) -3- ( ( (1-

(Desacetylvinblastine) amino) -2-

Ethylsulfide)methylene) -3-Cephem-1-β-Sulfoxide-4-

Carboxylic Acid

Allyl 7-β-(2- (thien-2-yl)acetamido) -3- ( ( (1-

(Desacetylvinblastine) amino) -2-ethylsulfide)methylene) -3- cephem-l-β-sulfoxide-4-carboxylate (0.30g, 0.249mmol) was dissolved in methylene chloride (3ml) . The solution was added to a methylene chloride (1ml) solution of tetrakis[triphenylphosphine] palladium (0) (11.4mg,

0.0099mmol) and triphenylphosphine (2.6mg, 0.0099mmol) .

The resultant reaction mixture was stirred at room temperature under nitrogen for 10 minutes then triethylsilane (4 drops) and glacial acetic acid (2 drops) were added. The reaction mixture was then stirred overnight at room temperature under nitrogen. An identical portion of the palladium, phosphine, silane and acetic acid reagents were added to the reaction mixture and the mixture was stirred for another 3 hours. Diethyl ether was added and the resultant solid was collected using a centrifuge.

The collected off-white solid was dried under vacuum at room temperature to yield 183mg of the title product. A portion (lOOmg) of this material was purified by HPLC (C- j _g

50 x 350 column, 1000 psi, 50 ml/min, eluted with 15% acetonitrile/ 1% formic acid /84% water for 10 minutes, a linear gradient over 18 minutes to 30% acetonitrile/ 69% water/ 1% formic acid until completion) . NMR: (DMSO-dg,

300MHz): δ9.40(s,l); 8.32 (d,1,J=8Hz) ; 8.0-7.6 (m,4) ; 7.4-

7.2(m,4); 7.0-6.8(m,4) ; 6.39(s,l); 6.15(8,1); 5.75-

5.50(m,3); 4.81 (d, 1,J=4) ; 3.75(s,2); 3.65(8,3); 3.50(s,3);

2.62(s,3); 0.75(t,3,J=7) ; 0.65 (t,3,J=7) ; remainder are multiplets ranging from 4.0-l.lρpm; UV: (EtOH), lambda maχ =

266(ε=25,600) , 215ws (ε=59,200) ; IR: (CHCI3) 1792, 1623, 1616, 1602cm "1 ; FABMS: Calc'd. for C 5 9H 72 N 7 0 12 S3 -

1166.44009. Found = 1166.43436.

Procedure 19

Benzhydryl 7-β-(2- (Thien-2-yl)acetamido) -3- ( ( (0- (p-Nitrophenyl)carbonato)methylene) -2-Cephem-

4-Carboxylate

Benzhydryl 7-β-(2- (thien-2-yl)acetamido)-3- (hydroxymethyl)-2-cephem-4-carboxylate (3.0g, 5.77mmol) was dissolved in dry tetrahydrofuran (THF, 5ml) . The solution was cooled to 0_C under nitrogen then (p- nitrophenyl)chloroformate (1.74g, 8.65mmol) and dimethylaminopyridine (2mg) were added. To this reaction mixture dry 2,6-lutidine (l.OmL, 8.65mmol) was added in a dropwise fashion then the reaction was stirred overnight under nitrogen gradually allowing to warm to 23°C. The reaction mixture was gravity-filtered, and the filtrate was concentrated in vacuo. The concentrate was flash chromotagraphed over silica gel eluted with 95/5 methylene chloride/ethyl acetate. The chromatography yielded 2.9g of a white foam of the title product: NRM: (CDC1 3 ,300MHz) : δ

8.28(d,2,J=9.D; 7.42-7.28(m,13) ; 7.05-7.00(m,2) ;

6.92(s,l); 6.55(s,l); 6.35(d,1,J=8.7) ;

5.65 (dd,l,J=4.0,8.7Hz) ; 5.24 (d,1,J=4.0) ; 5.21(S,1); 4.82(d,l,J=12.4) ; 4.72 (d,1,J=12.5) ; 3.88(s,2); IR: (CHCI3)

1777, 1749, 1686, 1528cm "1 ; UV: (EtOH), lambda maχ =

244(6=16,800); FABMS: Calc'd. (M+H)=686.12669.

Found=686 . 12451 .

Example 16

Benzhydryl 7 -β- ( 2 - ( Thien-2 -yl ) Acetamido ) - 3 -

( ( Desacetylcol chic ine - 7 -

Amino ) Carbonyloxy ) Methylene ) - 3 -Cephem- 4 -Carboxylate

Desacetylcolchicine (70mg, 0 .196mmol ) was made according to the procedure described in Raffauf , J . Amer .

Chem. Soc . . p .5292 , 1953 . The desacetylcolchicine thus prepared was dissolved in dry pyridine under nitrogen at

room temperature and the solution was added to benzhydryl 7-β-(2- (thien-2-yl)acetamido) -3- ( (O- (p- nitrophenyl) carbonato)methylene) -2-cephem-4-carboxylate (130mg, 0.196mmol), followed by the addition of N,N- diisopropylethylamine (1 drop) . The reaction mixture was stirred overnight at room temperature under nitrogen. The reaction mixture was diluted with ethyl acetate then concentrated in vacuo. The dilution/concentration was repeated and the concentrate was flash chromatographed over silica gel eluted with 10% methanol in methylene chloride.

The chromatography yielded 0.17g of a yellow oil of the title products: NMR: (CDCl3,300 MHz), 1:1 mixture of δ 2 : δ 3 ); 8.78(br.s,l); 8.10 (d, 1,J=6) ; 7.6-7.1 (m, 13) ; 7.0- 6.8(m,4); 6.52(8,1); 6.38(s,l); 5.8 (dd,1,J=4, 8) ; 5.7(d,l,J=6); 5.55(dd,l,J=4,8); 5.30 (d, 1,J=6) ; 5.16(d,l,J=4) ; 5.10(8,1); 4.95 (d, 1,J=12) ; 4.90 (d, 1,J=4) ; 4.65(d,l,J=12); 4.4-4.2 (m, 1) ; 3.92 (s,3); 3.90(ε,3); 3.88(8,3); 3.60(s,3); 3.42 (d, 1,J=12) ; 3.23 (d, 1,J=12) ; 2.58- 2.45(m,l); 2.40-2.18 (m,2) ; UV: (EtOH) 341(8=81,600), 219(8=214,000), 202(6=311,000); FABMS: (M+H)904.

Example 17

Benzyhydryl 7-β-(2- (Thien-2-yl)Acetamido) -3-

( ( (Desacetylcolchicine-7 -

Amino) Carbonyloxy) ethylene) -3-Cephem-1-β-Sulfoxide-

4-Carboxylate Benzhydryl 7-β-(2- (thien-2-yl) acetamido) -3-

( ( (desacetylcolchicine-7-amino) carbonyloxy)methylene) -2,3 - cephem-4-carboxylate (0.17g, 0.188mmol) was dissolved in methylene chloride and the solution was cooled to 0_C under nitrogen. Meta-Chloroperbenzoic acid (0.038g, 0.188mmol) was dissolved in methylene chloride (2ml) and this solution was slowly added to the cephalosporin solution. The reaction mixture was stirred at 0_C under nitrogen for one hour. The reaction mixture was taken to dryness in vacuo then the solid was flash chromatographed over silica gel

eluted with a 90/10 methylene chloride/methanol mixture to yield 70mg of the title product: NMR: (CDC1 3 ,300 MHz): δ

8.08(d,l, =6.2Hz) ; 7.48-7.10 (m, 13 ) ;

7.0-6.8(m,4) ; 6.51(8,1), 6.0(m,l); 5.15 (d, 1,J=13) ;

4.62(d,l,J=13) ; 4.45 (d, 1,J=4) ; 4.32(m,l); 3.95(s,2);

3.92(s,3); 3.90(s,3); 3.82(s,3); 3.60(s,3); overlapping with 3.6 (d,l,J=13) ; 3.15 (d,1,J=13) ; 2.50-2.18 (m,3) ; 1.78 (m,l); UV: (EtOH), lambda maχ =346 (ε=14, 500) ; FABMS:

Calc'd. (M+H)=920.25228. Found=920.25448.

Example 18

7-β-(2- ( hien-2-yl)Acetamido) -3-

( ( (Desacetylcolchicine-7 -

Amino) Carbonyloxy)Methylene) -3-Cephem-1-β-Sulfoxide-

4-Carboxylic Acid

Benzhydryl 7-β-(2- (thien-2-yl) acetamido) -3-

( ( (desacetylcolchicine-7-amino)carbonyloxy)methylene) -3- cephem-l-β-sulfoxide-4-carboxylate (70mg) was dissolved in methylene chloride (3ml) and cooled to 0°C under nitrogen. To this cooled solution was added triethylsilane (1ml) then trifluoroacetic acid (1ml) . The resultant reaction mixture was stirred at 0°C under nitrogen for 30 minutes. The reaction mixture was diluted with cold acetonitrile and taken to dryness in vacuo. Cold acetonitrile was added to the residue and the resultant solution was again taken to dryness in vacuo. The residue was taken up in diethyl ether and the solution was taken to dryness in vacuo giving a yellow solid. After drying, the preceeding procedure afforded 70 mg of the title product: NMR: (DMSO- dg,300MHz); 8.37 (d,1,J=8.4) ; 8.13 (d,1,7.9) ;

8.06(d,l,J=9.1); 7.38-6.80 (m, 6) ; 6.70(s,l); 5.74(dd,l,J=4,8) ; 5.07 (d, 1,J=13.2) ; 4.82 (d,1,J=4) ; 4.42(d,l,J=13.2) ; 4.05 (m,l); 3.88 (s,2); 3.82 (s, 3); 3.77(s,3); 3.73(s,3); 3.46(s,3); with 2 buried protons 2.58-2.40 (m,l); 2.20-1.90 (m,2) ; 1.83-1.70 (m, 1) ; IR: (KBr)

1788, 1721, 1693, 1592cm "1 ; UV: (EtOH), lambda maχ =345 (8=14,200) , 240(8=33,000); FABMS: Calc'd.

(M+H)=754.17403. Found=754.17598.

Procedure 20

Preparation of Allyl 7-β-(2- (Thien-2-yl)Acetamido) - 3- (Hydroxymethyl) -2-Cephem-4-Carboxylate

7-β-(2-(thien-2-yl)acetamido)-3- (hydroxymethyl)-

2-cephem-4-carboxylic acid (17.5g, 49.5mmoles) dissolved in

250ml DMF and 150ml dioxane. To this solution was added sodium bicarbonate (4.5g, 53.6mmoles)followed by allyl bromide (5.9ml, 68.3mmoles). The resulting mixture was heated to reflux for 1 hour. The reaction mixture cooled to room temperature and partitioned between ethyl acetate

(500ml) and brine (500ml) . The organic phase was washed with brine and water 6 to 7 times (400ml) , then dried

(sodium sulfate) and concentrated in vacuo to give 10.2g of a dark brown oil of the title product. Crude yield: 52%.

This crude material was used in the next step without further purification. An analytical sample was obtained for analysis by dissolving 0.84g of the crude oil in 5ml

1:1 ethyl acetate/methylene chloride and adding hexanes

(50ml) until a yellow solid formed. After filtration, the filtrate, on standing, yielded the title compound as a white solid (0.04g). IR: (CHCI3): 1779, 1750, 1684, 1510 cm " ; EA: C 17 H 18 N2θ 5 S2: Calc'd. C=51.76, H=4.50, N=7.10. Found: C=51.50, H=4.56, N=6.93. UV: (EtOH) 6max = 234(6=14,384), 201(6=15,766); NMR: (30θMHz,CDCl 3 ) : δ

7.27(m,l); 7.00(m,2); 6.38 (d,1,J=9) ; 6.28(s,l); 5.9(m,l); 5.65(d of d,l,J=4,9) ; 5.3(m,3); 5.08(s,l); 4.66 (d,2,J=6) ; 4.20(q,2,J=13) ; 3.85(s,2) .

Procedure 21

Preparation of Allyl 7-β-(2- (Thien-2-yl)Acetamido) -

3- ( (p-Nitrophenylcarbonato) Methylene-3-Cephem-4-

Carboxylate and the Corresponding 2-Cephem Isomer

Allyl 7-β-(2- (thien-2-yl)acetamido) -3- (hydroxymethyl) -2-cephem-4-carboxylate (10.2g, 25.8mmol) was dissolved in 50ml THF and the solution was cooled in an ice-water bath. Lutidine (4.8ml, 41.3mmol) was added followed by p-nitrophenyl-chloroformate (8.50g, 41.3mmol) and dimethylaminopyridine (0.10g, 0.8mmol). The reaction mixture was allowed to warm to room temperature and stirred 0.5 hours. The reaction was filtered to collect the off- white precipitate, and the filtrate concentrated in vacuo. The resultant residue was purified by flash chromatography with 5% ethyl acetate/95% methylene chloride to afford the title compound as a dark yellow gum (5.66g, 39% yield) . This material was predominantly the β-2 isomer. It was used in the next step without further purification. An analytical sample was obtained by flash chromatography (5% ethyl ace ate/CH2Cl2) of 0.28g of the yellow gum to afford

0.08g of the β-2 isomer as a clear colorless oil. IR: (CHC1 3 ) : 1779, 1730, 1685, 1529, 1510 cm "1 : EA: C 24 H 21 N 3°9 S 2 : Calc'd. C=51.51, H=3.78, N=7.51, S=11.46. Found: C=51.72, H=3.88, N=7.27, S=11.22. UV: (EtOH) : εmax = 239(8=17,365), 201(8=24,226); NMR: (300MHz,CDCI3) : ε max =

8.28(d,2,J=9) ; 7.38 (d, 2,J=9) ; 7.25(m,l); 7.01(m,2); 6.56(s,l); 6.32(m,l,J=8) ; 5.91(m,l); 5.68 (d of d,1,J=4,9) ; 5.33(m,3); 5.09(s,l); .92 (d, 1,J=12) ; 4.77 (d,1,J=12) ; 4.68(d,2,J=7) ; 3.86(s,2) .

Example 19

Preparation of Allyl 7-β-(2- (Thien-2-yl )

Acetamido) -3- ( (p-Nitrophenylcarbonato)

Methylene-3-Cephem-1-β-Sulfoxide-4-Carboxylate

A mixture of allyl 7-β-(2- (thien-2- yl)acetamido) -3- ( (p-nitrophenylcarbonato) methylene-3- cephem-4-carboxylate (5.66g, lO.lmmoles) and the corresponding β-2 isomer was taken up in 225ml CH2CI2 and the resultant solution was cooled in an ice-water bath. To this solution was added a solution of meta-chloroperbenzoic acid (3.23g, 55%, 10.3mmoles dissolved separately in 50ml CH2CI2). The perbenzoic acid solution was added in dropwise fashion over 15 minutes. The combined solutions at room temperature another 10 minutes. The reaction mixture concentrated in vacuo to a brown oil and triturated with methanol to afford 4.24g, 74% yield of the title compounds as a light tan solid. IR: (CHCI3): 1808, 1772,

1733, 1689, 1530 cm "1 MS: (FAB) : M+=576. EA: C 24 H 21 N 3°10 S 2 : Calc'd. C=50.08, H=3.68, N=7.30. Found: C=50.02, H=3.63, N=7.04. UV: (EtOH): λmax=400 (8=688) , 332(8=2,394), 325(8=2,353), 270(8=17,094), 239(8=14,438), 201(8=28,842). NMR: (300MHz,DMSO-d ) : λ = 8.48 (d, 1,J=8) ;

8.29 (d,2,J=9) ; 7.52 (d, 2,J=9) ; 7.33(m,l); 6.92(m,2); 5.9(m,2); 5.2-5.41 (m,3 ) ; 4.91 (d, 1,J=4) ; 4.87 (d, 1,J=13) ; 4.74(d,2,J=5) ; 4.04 (d, 1,J=18) ; 3.82 (AB q, 2,J=15) ; 3.66(d,l,J=18) .

Example 20

Preparation of Allyl 7-β-(2- (Thien-2-yl)Acetamido) - 3-0-[3'-N-ureido] Doxorubicin]Methylene-3-Cephem-1-β- Sulfoxide-4-Carboxylate

Doxorubicin-HCl (0.87g, 1.5mmoles) was slurried in (5.0ml, Aldrich Sure-Seal) DMF. Disopropylethylamine (0.3ml, dried over KOH pellets, 0.25mmoles) was added to the slurry, then allyl-7-β-(2- (thien-2-yl)acetamido) -3- ( (p-

nitrophenylcarbonato) methylene-3-cephem-l-β-sulfoxide-4- carboxylate (0.87g, 1.51mmoles) was added to the resultant mixture and the reaction was shielded from light and stirred for 2.5 hours. Ethyl ether was added to the reaction to precipitate 1.29g of a red solid. This material may be used without purification in Example 21.

An analytical sample was obtained by passing a portion of the red solid (130mg) through a chromatotron, eluting with 6% methanol/94% CH2CI2 on a 1 micron plate to afford the desired product as 29mg of a red solid. Fractions were analyzed on a C-18 reverse phase column (Waters C 18 _bondpack) eluted with 35%/35%/30% MeOH/acetonit^ite/H 0

+ 0.5% ammonium acetate. The desired product has retention time © 3.6 min (flow rate = 2 ml/min) . The ti' le product was obtained in 90% purity, when analyzed by t e above HPLC conditions. IR: (CHCI3): 1806, 1724, 1700, 1610, 15£2,

1509 cm "1 ; EA: Calc'd: C=55.15, H=4.63, N=4.2 Found: C=55.07, H=4.51, N=4.52. UV: (EtOH): εmax=532 \l=A,27 ) , 496(8=7,857), 480(8=7,839); 349 (ε=l,467) , 251(8=21,091), 234(8=30,361); NMR: (300 MHz,DMSO dg): δ 13.9, (s,l);

13.2, (s,l); 8.35, (d,l,J=8); 7.84, (m=2); 7.56, (m,l); 7.31, (d,l,J=4) ; 6.90, (m,3); 5.9-5.75, (m,1) overlapping with 5.76, dd,l,J=5,9) ; 5.32, (m,l); 5.16, (m,2); 5.02, (d,1,J=13) ; 4.8, (m,2); 4.65, (m,2); 4.53, (bs,2); 4.44, (d,1,J=13) ; 4.10, (d,l,J=5) ; 3.92, (s,3); 3.88-3.70, (m,4) ; 3.62, (m,l); 3.5-3.1, (m,2) overlapping with 3.28, s,2); 2.83, (AB q,2,J=18); 2.2-2.0, (m,2) ; 1.76, (m,l); 1.40, (m,l); 1.08(d,3,J=6) .

Example 21

Preparation of 7-β-(2- (Thien-2-yl)Acetamido) -3-0-

[3 ' -N-ureido]Doxorubicin]Methylene-3-Cephem-1-β- Sulfoxide-4-Carboxylic Acid

Procedure A Palladium acetate (3.5mg, .016mmol) and triphenylphosphine (22mg, .084mmol) were dissolved in ethyl acetate (0.1ml) and the mixture was sonicated approximately 2 minutes. Allyl-7-β-(2- (thien-2-yl)acetamido)-3-0-[3 ' -N- ureido] oxorubicin]methylene-3-cephem-l-β-sulfoxide-4- carboxylate (O.llg, O.llmmol) was dissolved separately in a solvent mixture consisting of 5.5ml EtOAc, 2.5ml MeOH, 3ml CH 2 Cl2 and 80 microliters acetic acid. This solution was added to the palladium solution by pipette followed by the addition of triethylsilane (0.02ml, 0.13mmoles). The reaction mixture was protected from light and stirred at room temperature for 4 hours. The reaction mixture was concentrated to a red oil (0.25g). A portion of this crude oil (54mg) was dissolved in DMSO (1.5ml) and injected onto a 22 x 250mm RSIL Phenyl preparative HPLC column (Alltech

Associates) and eluted (flow rate of 8ml/min, detector @ 480nm) with a mixture of 35% CH 3 CN/0.05% TFA in water for

12 minutes, then with a mixture of 45% CH3CN/0.05% TFA in water for 18 minutes, then with a mixture of 60% CH3CN/0.05% TFA in water after 30 minutes running time.

Product-containing fractions were lyophilized to afford the title product as 26mg of a red solid. IR: 1780 cm "1 . MS: (ESI): 962.1(MNa + ). MS: HRMS: (FAB): Calc'd for c 42 H 41 N 3°18 S 2 Li (MLi +) =946.1987. Found=946.1955. NMR: (DMSO-dg): δ l4.0,(s,l); 13.2,(8,1); 8.33, (d,1,J=8) ;

7.91, (d,2,J=4) ; 7.64, ( ,1,J=5) ; 7.35, (m,l); 6.91, (m,3); 5.71, (dd,l,J=5,8) ; 5.43, (s,l); 5.19, (m,l); 5.05, (m,1); 4.92, (m,l); 4.82, (m,2); 4.70, (d,1,J=5) ; .55, (d,2,J=6) overlapping with 4.46, (m,l) ; 4.12, (d,1,J=9) ; 3.97, (s,3);

3.80, (ABq,2,J=15; HOD obstructs the spectrum from 3.4-3.0); 2.96, (m,2); 2.12, (m,2); 1.82, (m,l); 1.43, (m,l); 1.09, (d,3,J=6) .

Procedure B Palladium acetate (0.04g, O.lδmmol) and triphenylphosphine (0.2g, 0.76mmol) were combined and slurried in 1ml of ethyl acetate. This mixture was sonicated one minute to produce a white precipitate. Ethyl acetate (50ml), methanol (25ml) and acetic acid (0.75ml) were added to this precipitate. Allyl-7-β-(2- (thien-2- yl)acetamido)-3-0-[3 '-N-ureido]doxorubicin]methylene-3- cephem-l-β-sulfoxide-4-carboxylate (1.36g, 1.39 mmol) prepared in identical fashion to Example 20, was added along with 125ml of 10% MeOH / 90% CH 2 C1 2 to improve solubility. Triethylsilane (0.35ml, 2.20mmol) was added. After 4 hours, the reaction was only partially complete. Palladium acetate, triphenyl phosphine and triethylsilane were added in the same portions as before and the reaction was stirred overnight. The reaction mixture was concentrated in vacuo to afford the title compound as 1.4g of a red solid. This material was purified in small aliquots using reverse-phase HPLC. Two 25x100mm columns (Waters μbondapak C-18 column) were connected in series and eluted with 30% acetonitrile / 70% water + 0.1% TFA. The flow rate was 8ml/min and the output was monitored at 480nm. After 25 minutes the mobile phase was changed to 40% acetonitrile / 60% water + 0.1% TFA. The title compound eluted at about 55 minutes under these conditions. Fractions of >95% analytical purity were lyophilized to afford the title compound. 76mg of crude material gave 24mg of .95% puree title compound.

Procedure 22

Rat Hematoloσv Protocol

The purpose of the following procedure was to compare the toxicity of the Substrate-Cytotoxic Agent to the toxicity of Cytotoxic Agent alone.

1. Day 0: 25 male Fischer 344 rats (@100g) are pre- bled (eyebleed) and injected i.v. as follows:

GROUP A: 5 rats + vehicle control: 0.5ml, lOmM HEPES, 150 NaCl, pH7.1 buffer containing 10% ethanol.

GROUP B: 5 rats + lmg/kg 4-desacetylvinblastine -3- carboxhydrazide (desacetylvmblastmehydrazide) . Dissolved lmg desacetylvmblastmehydrazide in 0.5ml absolute ethanol and added 4.5ml HEPES-NaCl buffer. Injected each rat with 0.5ml.

GROUP C: 5 rats + lmg/kg LY262758 (7-β-2- (phenoxyacetamido)-3-

( ( (desacetylvinblastinehydrazido)carbonyloxy)methylene) -3- cephem-l-β-sulfoxide-4-carboxybiacid, trifluoroacetic acid salt) (vinca content, 58% vinca) . Dissolved 2mg as above and injected 0.5ml.

GROUP D: 5 rats + lmg/kg LY266494, (7-β-(2- (thien-2- yl)acetamido-3-

( ( (desacetylvinblastinehydrazido)carbonyloxy)methylene) -3- cephem-4-carboxylic acid, hydrochloride salt (55.8% vinca). Dissolved 2mg and injected 0.5 ml.

GROUP E: 5 rats + lmg/kg compound of Example 17 7-β-(2-

(thien-2-yl)acetamido-3- ( ( (desacetylvinblastinehydrazido) carbonyloxy)methylene)-3-cephem-l-β-sulfoxide-4-carboxylic

acid, trifluoroacetic acid salt. Dissolved 2mg and injected 0.5ml.

All animals were inden ifed by ear punch and were bled on days 3 and 7 post treatment for white blood cell (WBC) determination. The raw data are on the following page. The desacetylvinblastinehydrazide-treated animals exhibited a significant WBC suppression on day 3 compared to all other groups. The WBC values of the groups were indistinguishable at day 7 (and lower than the pre- bleed which may have been due to using a different instrument for the WBC determination) .

Rat Hematoloσv Data

Values x 1000 = WBC' s

Group Day 0 Day 3 Day 7

14.2 6.0

16.4 9.3 13.7 6.8

10.3 10.1 13.1

13.5 8.1 2.2 2.0

** 5.0

4.4 2.8

9.0 19.8 5.4 **

10.6 **

7.4 9.2

2.9 9.2

13.3 14.0

14.6 13.0 15.0 12.5 16.3 9.0

14.7 7.7

14.8 11.2 1.1 2.7

**Sample clotted--could not determine WBC count.

Procedure 23

In Vivo Nude Mouse Model for Evaluation of Prodrug/Antibody-Enzyme Conjugate System.

The following procedure was carried out to determine the dose at which toxicity for the Prodrug is first observed. Also, the maximum dose of Prodrug for which anti-tumor activity is not observed is also determined.

1. DAY 0: Implant 30 Nude Mice (CHARLES RIVER, 25G) WITH 1X10 7 LS174T Tumor Cells (ATCC) subcutaneously;

2. The stock Prodrug treatment solution was prepared as follows:

7-β-(2- (thien-2-yl)acetamido-3- ( ( (desacetylvinblastine hydrazido) carbonyloxy)methylene)-3-cephem-l-β-sulfoxide-4-

carboxylic acid, trifluoroacetic acid salt, was dissolved in water. Sodium chloride solution (9%) was added to give an isotonic (0.9%) saline solution containing Prodrug at a

2mg/ml concentration. The resultant solution was filter sterilized prior to use, and Prodrug was injected as soon as possible after formulation.

GROUP A: Inject with 0.25ml saline per mouse

GROUP B: Placed 1.7M1 stock solution in vial, injecte

0.25M1 per mouse

GROUP C: 0.625M1 stock solution + 0.775ml saline; injected 0.2ml GROUP D: 0.31M1 stock solution + 1.09ml saline; injected 0.2ml GROUP E: 0.15M1 stock solution + 1.25ml saline; injected 0.2ml GROUP F: 0.075 Ml stock solution + 1.325 ml Saline; injected 0.2ml

3. Days 3,4,5,6 the nude mice were treated I.V. as follows:

GROUP A 5 mice injected with 0.25ml saline

GROUP B 5 mice injected with 20mg/kg ceph-vinca PRODRUG GROUP C 5 mice injected with 10MG/KG PRODRUG

GROUP D 5 mice injected with 5MG/KG PRODRUG GROUP E 5 mice injected with 2.5MG/KG PRODRUG GROUP F 5 mice injected with 1.25MG/KG PRODRUG

4. The tumors were measured on days 14,21,2 post implantion.

5. The results were as follows:

The Group B dosage was toxic. The Group C dosage, while not toxic, showed that Prodrug alone had anti-tumor activity at that level. It is speculated from these results that a maximum effective dose would be somewhere in the range of 1 to 8mg/kg.

Procedure 24

Labeling and Biodistribution Studies

The biodistribution of the anti-KSl/4(i.e.007B) — β-lactamase conjugate was measured by labeling the conjugate with 1- L 1 X 1- L Indium. The conjugate was first reacted with isothiocyanatobenzyl DTPA, then labeled according to established procedures (Meares C, et al.. Anal. Biochem.. 142, pp.68-78, 1984). The labeled conjugate (20μg and lOμCi per animal) was injected into the tail vein of nude mice with LS174T tumors. The animals were separated into groups of six, and groups were sacrificed at 4, 24, 48, 72 and 120 hours after conjugate injection. Procedures for sacrificing, specimen collection, counting and data reduction have been described previously (Halpern S.E., e_t al. , Can. Res.. 43. pp.5347-5355, 1983.). Tumors were implanted by subcutaneous inoculation of 1x10' cells in the flank of female athymic nude mice (Charles River Breeding

Laboratories, Wilmington, Massachusetts) . The LS174T tumors implanted by these methods reached a mean size of 0.2gm in nine days. The results of these studieε are set forth in FIG.l and 2. They show that conjugate localizes at the tumor and that it clears rapidly (i.e., within 72 hours) from the serum.

Procedure 25

The Growth of T380 Colon Tumors in Nude Mice Following ADC Treatment.

T380 Tumors were implanted on day 0 by subcutaneous inoculation of a 0.1ml tumor slurry in the flank of female athymic nude mice. In 14 days the tumors were established and had reached a size of approximately 0.3gm as determined by caliper measurements and calculated from the formula V=0.5xLxW , where L and W are the longest dimension and its perpendicular measurement, respectively. All injections were made in the tail vein. Animal weightε were also measured as an indication of toxicity in the study depicted in FIG.3,4.

Treatment groups of 8 mice each were injected with either the anti-CEA (i.e., CEM231) -- β-lactamase (35μg) conjugate, the irrelevant-antibody (i.e., CHA255) -- β-lactamase (35μg) conjugate, or saline on days 14, 21 and 28. These injections were followed by four daily injections of lmg/Kg (FIG.3) or 0.25mg/Kg (FIG.4) of the prodrug compound of Example 11 beginning 72 hours after conjugate injection. In the control arms of the experiment, the prodrug injections were substituted with either saline, or 0.6mg/kg (FIG.3) or 0.15mg/kg (FIG.4) desacetylvmblastmehydrazide, the molar equivalent of the prodrug doses.

The data points in FIG.3,4 represent the following results for this Procedure: filled symbols represent experimental (tumor specific conjugate/prodrug) arms; and the open symbols, represent controls. Other

symbols on the figure are to be interpretted as follows: open circles: saline/saline; open triangles: saline/prodrug; open squares: saline/drug; open diamonds: irrelevant antibody -- β-lactamase conjugate/prodrug; open arrows: conjugate or saline injection; filled arrows: first of 4 days of either the prodrug, drug, or saline injections. With the experimental treatment, the tumors regressed and animals showed long-term disease-free survival. The control arms showed only tumor growth inhibition, no regressions, and long-term disease stabilization.

Procedure 26

Growth of LS174T Colon Tumors in Nude Mice Following ADC Treatment.

LS174T tumors were implanted as in Procedure 24 and measured as in Procedure 25. Animals (ten in the saline control group; five in the treatment groups) were treated with 35μg each of either the anti-KSl/4(007B) -- β- lactamase conjugate, the anti-CEA(CEM231) -- β-lactamase conjugate, the irrelevant antibody(CHA255) -- β-lactamase conjugate, or saline on days 9, 16 and 23. These injections were followed after 96 hours by a single injection of either the prodrug (the compound of Example 11) (4mg/Kg) , the drug

(desacetylvmblastmehydrazide) (2.3mg/Kg), or saline. Tumor volumes were measured and are depicted in FIG.5. As with T380, tumors shown in FIG.3, tumor regressions were observed only in the tumor-specific treatment groups, not in any of the control groups.

The data points in FIG.5 represent the following results for this Procedure: filled circles: anti-KSl/4 -- β-lactamase conjugate/prodrug; filled squares: anti-CEA -- β-lactamase conjugate/prodrug; open circles: saline/saline; open triangles: saline/prodrug; open squares: saline/drug; open diamonds: irrelevant antibody

-- B-lactamase conjugate/prodrug; open arrows: conjugate or saline injection; filled arrows: prodrug, drug, or saline injections. The control arms of the experiments were also run using anti-KSl/4 -- β-lactamase conjugate/drug, anti-CEA -- β-lactamase conjugate/drug and irrelevant antibody -- β-lactamase conjugate/drug to test whether a non-specific synergy between conjugate and drug led to enhanced efficacy. No difference from saline/drug was observed (data not shown in the FIG.).

Animal weights were measured. Significant weight loss was measured in the animals receiving free drug at this dosage, and animal death has resulted from this drug dose and schedule in other experiments. Less pronounced weight loss was measured in the group receiving the anti-CEA -- β-lactamase conjugate/prodrug, but none in the group receiving the anti-KSl/4 -- β-lactamase conjugate (data not shown in the FIG. ) .

Procedure 27

Growth of LS174T Tumors Following ADC Treatment.

The procedure here is identical to that of Procedure 26 except that the tumors were allowed to reach a larger size before treatment was started, and that the conjugate used is anti-TAG-72 (CC49) -- β-lactamase. Even with large tumors and TAG-72 as target antigens, tumor regressions resulted from, tumor specific treatment, but not from any control treatmen .

The data points in FIG.6 represent the following results for this procedure: filled circles: anti-TAG-72 — β—lactamase conjugate/prodrug; open circles: saline/saline; open triangles: saline/prodrug; open squares: saline/drug; open diamonds: irrelevant antibody (CHA 255) -- B -lactamase conjugate/prodrug; Open arrows: conjugate or saline injection; filled arrows: prodrug, drug, or saline injections.

Procedure 28

Dependence of Tumor Growth Inhibition on Conjugate

Dose.

The procedure followed here was generally that of Procedure 25, with the following exceptions. Mice received either 35μg (filled squares), 14_g (filled circles), or 3.5μg (filled triangles) of the anti- CEA(CEM231) — β-lactamase conjugate on days 14, 21, and 28 followed after 72 hours by prodrug at lmg/Kg/day for four days. A fourth experimental group received 35μg of the anti-CEA -- β-lactamase conjugate on day 14 only (filled diamonds) followed by prodrug on the same schedule as the other mice. The data points in FIG.5 represent the following results for this procedure: open circle: saline/saline; open squares: saline/drug; open arrows: conjugate or saline injection; filled arrows: first of 4 days of prodrug, drug, or saline injections. The results show that maximal efficecy may be duration, not magnitude, of therapeutic effect depended on conjugate dose. The results also show that conjugate in each course of therpy contributes to the duration of response.

Procedure 29

Preparation of Benzhydryl-7-β-(2- (Thien-2-yl)

Acetamido) -3- (0- (p-Nitrophenyl)Carbanato)

Methylene) -2-Cephem-4-Carboxylate

To a 0°C solution of benzhydryl 7-β-(2- (thien-2- yl)acetamido-3-hydroxymethyl-2-cephem-4-carboxylate (3.Og, 5.77mmol) in dry THF (5mL) was added dimethylaminopyridine (2mg) followed by p-nitrophenylchloroformate (1.74g, 8.65mmol). Dry 2,6-lutidine (l.OmL, 8.65mmol) was added dropwise and the resulting solution was allowed to warm to room temperature overnight. Some insoluble material was removed by filtration and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography

(5% EtOAc in CH 2 C1 2 ) to give 2.9g, 74%, of the title compound as a white foam. NMR: (CDC1 3 ) δ 8.28 (d,2,J=9.1) ;

7.42-7.28(m,13) ; 7.05-7.0(m,2) ; 6.92(s,l); 6.55(s,l);

6.35(d,l,J=8.7); 5.65(dd,1,J=4,8.7) ; 5.24 (d,1,J=4) ;

5.21(8,1); 4.82(d,l,J=12) ; 4.72(d,1,J=12) ; 3.88(s,2). 13 C NMR: (CDCI3) δ 169.99, 165.90, 164.17, 155.26, 152.04,

145.46, 138.76, 138.69, 134.59, 128.84, 128.77, 128.60,

128.47, 127.90, 127.55, 127.14, 126.79, 126.07, 125.30,

124.64, 121.72, 117.93, 79.49, 69.90, 60.45, 53.2, 50.14, 37.07. IR: (CHCI3) 1777, 1749, 1686, 1528 cm "1 . UV:

(EtOH) λ maχ =244(ε=16,800) . FABMS: Calc'd. for

C 34 H 27 N 3°9 S 2 : C=59 . 55 ; H=3 . 97 ; N=6 . 13 . Found : C=59 . 34 ;

H=4 . 05 ; N=5 . 93 .

Procedure 30

Preparation of Benzhydryl 7-β-(2- (Thien-2- yl)Acetamido-3- (0- (p-Nitrophenyl)

Carbonato) ethylene) -3-Cephem-1-β-Sulfoxide-4-

Carboxylate

To a 0°C solution of benzhydryl-7-β-(2- (thien-2- yl)acetamido)-3- (O- (p-nitrophenyl)carbanato)methylene) -2 - cephem-1-4-carboxylate (2.5g, 3.65mmol) in CH2Cl2(40mL) was added a solution of 55% meta-chloroperoxybenzoic acid (l.lg, 3.65mmol eq. ) in CH 2 C1 2 (lOmL) . After 1 hour at 0°C

TLC (20% EtOAc in CH 2 Cl 2 ) indicated the starting material had been consumed. The solvent was removed in vacuo and the residue subjected to flash chromatography (20% EtOAc in CH2CI2 to give 2.2g, 86%, of the title compound as a white solid. NMR: (DMSO-dg): δ 8.49 (d,1,J=8) ; 8.27(d,2,J=9) ;

7.5-7.2 (m,13); 6.92 (m,3); 5.94(dd,1,J=4.5,8) ; 5.25(d,l,J=13) ; 4.93 (d,1,J=4.5) ; 4.85 (d,1,J=13) ; 4.04(d,l,J=18.7) ; 3.88 (d,1,J=15.3) ; 3.78(d,1,J=15.3) ; 3.66(d,l,J=18.7) . IR: (KBr) 1765, 1720, 1658 cm -1 . UV: ( EtOH ) ε maχ =265(ε=3570) . Anal. Calc'd for C34H 2 7N3θ 10 s 2 : C=58.20; H=3.88 N=5.99. Found: C=58.48; H=4.08; N=5.90.