Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2001-09-19
2003-12-09
McKane, Joseph K. (Department: 1628)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C514S339000, C514S381000, C514S414000, C514S419000, C514S422000, C544S373000, C546S277100, C546S300000, C548S310700, C548S445000, C548S492000, C548S518000
Reexamination Certificate
active
06660742
ABSTRACT:
FIELD OF INVENTION
The present invention is directed to novel achiral seco-analogues of (+)-CC1065 and the duocarmycins and pharmaceutical compositions containing the said achiral analogues. The achiral analogues of (+)-CC1065 and the duocarmycins are useful as anticancer agents.
BACKGROUND OF THE INVENTION
One class of compounds that has received much attention recently is the DNA minor groove binders that exert their anticancer activity by alkylating specific sequences of DNA (1a). Minor groove interacting agents are more attractive than intercalating agents and major groove binders because the minor groove is typically only occupied by a spine of hydration and is therefore more accessible to anticancer agents. Additionally, covalent modifications in the minor groove are generally more cytotoxic to cells than their major groove alkylating counterparts, such as tallimustine versus L-phenylalanine mustard (1b). Examples of minor groove and AT sequence selective alkylating agents that have potent anticancer activity are (+)-CC-1065 (1c,d,e, 2) and the duocarmycins (2, 3). CC-1065 and the duocarmycins, exemplified by (+)-duocarmycin SA (or DUMSA) (2) and depicted in Table 1, belong to a group of natural products that have incredibly potent cytotoxic properties (ca. IC
50
values in the pM range against the growth of mouse L1210 leukemia cells in
Table 1. Structures and summaries of the biological properties of (+)-CC1065 and doucarmycin SA (DUMSA) culture). These compounds, which were isolated from the fermentation broth of
Streptomyces zelensis
(4) and Streptomryces sp. (5), respectively, derive their anticancer activity by reacting primarily with adenine-N3 groups in the minor groove of specific sequences of DNA. Confirmation of the adenine-N3 covalent reaction was achieved from isolation of an adenine-CC1065 adduct (6) and an adenine-duocarmycin SA adduct (7) from thermally cleaved DNA-drug adducts. (+)-CC1065 shows a preference for alkylating the adenine-N3 group which is flanked by 5′-A or 5′-T residues. The sequence preference for the three-base AT-rich alkylation site followed the order: 5′-AA
A
=5′-TT
A
>5′-TA
A
>5′-AT
A
(the alkylation site is denoted by the underlined base). In addition, this compound exhibited a strong preference for the fourth 5′-base to be an A or T residue, a weaker preference for the fifth 5′-base to be an A or T base, and a weak preference for the 3′ base preceding the alkylating site to be a purine. The consensus sequences 5′-PuNTT
A
-3′ and 5′-AAAA
A
-3′ for (+)-CC-1065 are suggested (6, 8). Although (+)-CC1065 and (+)-DUMSA generally have similar sequence selectivity, some subtle differences were observed, notably the lack of alkylation at 5′-GCAA
A
by CC1065 (2, 3, 7).
The mechanism by which CC1065 and the duocarmycins react with specific sequences of DNA is still a subject of intense debate (9). Two models for their sequence selective DNA alkylations have been proposed. The “noncovalent binding model” proposed by Boger's group (9) suggests that the forces stabilizing the DNA alkylation are a combination of stereoelectronically controlled covalent bond formation between the cyclopropylpyrroloindolone (CPI) subunit of CC1065 and adenine-N3 of DNA, as well as stabilizing noncovalent interactions derived from the hydrophobic and van der Waals contacts of the PDE-I subunits for CC1065, and the TMI (trimethoxyindole) unit of DUMSA (9). In the “alkylation site model” proposed by Hurley and coworkers (10), the covalent sequence specificty of the compounds, exemplified by (+)-CC1065, has been attributed to the unique conformational features of the consensus sequences, in which the sequences are flexible enough to adopt a transient bent conformation for recognition of the drug molecule. Further, the unique conformation provides an acidic proton on a phosphate which can activate the CPI system by general acid catalysis. In either case,
1
H-NMR studies showed that (+)-CC1065 effectively alkylated adenine-N3 of duplex 5′-GGCGGAGTT
A
GG-3′ (alkylation at the underlined A residue in the italicized sequence) and induced a bend of 17-22 ▭ at the TTA sequence (11). Similarly, a
1
H-NMR study of DUMSA with d-(GACTMTTGAC).d-(GTCATT
A
GTC) has also revealed that the drug undergoes significant conformational changes upon binding to the minor groove, which subsequently enhances its covalent reactivity (lid). Such conformational changes on the DNA has been postulated to entrap structures in the DNA that might be relevant to the biological control of gene expression and replication (12). A recent density functional and ab Initio study on the activation of the duocarmycin SA pharmacophore for DNA alkylation was reported (13). In the study the authors found that twisting of the indoline-amido-N bond (▭
2
) did not sufficiently explain the million-fold enhancement of DNA alkylation compared to solvolysis.
Even though (+)-CC1065 has potent cytotoxic properties, its usefulness as an anticancer drug is hampered by its limiting toxicity of delayed lethality in mice at therapeutic doses (4b). Interestingly, DUMSA is devoid of this toxic side effect (2a), and it is the most solvolytically stable and the most potent in this class of compounds (2, 14). Facilitated by an extensive array of analogues of CC1065 synthesized by Upjohn scientists (1a, b, 4c), structure-activity relationships have been sharply defined (10b, 11), and the ethano bridges (see
FIG. 1
) were found to be responsible for the undesired delayed lethality. These ethano groups enter into favorable van der Waals and hydrophobic interactions with adenine-H2 atoms on the floor of the minor groove that stabilizes the drug-DNA complex (11a, b). It has been suggested that the delayed lethality of (+)-CC1065 and its CDPI
2
analogue (see Table 2) is a consequence of their inability to “reversibly” alkylate DNA (7, 15). The strong noncovalent binding of these compounds due to the ethano groups could either prevent the reverse reaction after alkylation has occurred (7, 15a), or keeps the drug bound so that it realkylates the same site (15b). In agreement with this suggestion, CC1065 analogues, such as adozelesin (15b) and DUMSA (15a, 16), which lack the ethano bridges, are devoid of the delayed toxicity problem, yet they exhibit potent anticancer activity.
Adozelesin (1b, 4c, 17, patent 1), carzelesin (18), bizelesin (19, patent 2), and KW2189 (20, patent 3) are examples of analogues of (+)-CC1065 and duocarmycins that are presently undergoing clinical trials for the treatment of cancer. A phase I clinical trial of adozelesin has shown a partial response on a melanoma patient (17e). Carzelesin is a prodrug that upon carbamate hydrolysis provides the seco-prodrug U76073, which readily cyclizes to the corresponding cyclopropane containing CPI “drug” U76074 (18). It is worthy to note that carzelesin has a higher in-vivo anticancer activity against L1210 leukemia than adozelesin (150±8% ILS, 2/6 survival versus 90±11%, 0/6 survival) (18). This outcome is likely to be due to the lower toxicity of carzelesin, and consequently it can be administered at a higher dose (400 &mgr;g/kg vs 100 &mgr;g/kg for adozelesin) (18). Bizelesin is a DNA interstrand crosslinking agent incorporating two seco-CPI alkylating units and is approximately 20-30 times more potent than (+)-CC1065 itself (IC
50
(L1210)=1 pM vs 20 pM) (19). KW2189, a semisynthetic duocarmycin B2 derivative, which possesses improved anticancer activity, water solubility, and stability, has been selected for clinical evaluation in Japan (20). Its mechanism of activation also involves hydrolysis of the carbamate group to release a seco-prodrug, which cyclizes to produce the actual cyclopropane containing drug, or DU-86.
The biological properties of these clinically studied compounds indicate that seco-prodrugs are as active as their cyclopropane c
McKane Joseph K.
Rothwell Figg Ernst & Manbeck P.C.
Saeed Kamal
Taiho Pharmaceutical Co. Ltd.
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