Modification of the antibiotic olivomycin

The aureolic acid family of antitumor antibiotics includes a group of
clinically active agents such as olivomycin I (olivomycin A), mithramycin,
and also chromomycin A3 and durhamycin.1 The antibiotics of
the aureolic acid family interact with the DNA minor groove in high-
GC-content regions in a nonintercalative way and with the requirement
for Mg2+ ions.

The antitumor antibiotic olivomycin I was discovered at the Gause
Institute of New Antibiotics, Moscow.3 Comparative study of the
antitumor action of olivomycin I and chromomycin A3 in in vivo
experiments on murine lymphosarcoma LY01 revealed that the chemotherapeutic
index (LD50/DIT50) of olivomycin I is more favorable
(2.35) than that of chromomycin A3 (0.99). A similar study on the
inhibitory effect of olivomycin I, chromomycin A3 and mithramycin
against transplantable murine leukemia La showed that a similar
antitumor effect (an increase in the lifespan of mice by 25%) can be
achieved at lower doses of olivomycin I than those for the other
aureolic acid antibiotics studied. Clinical investigations of mithramycin
and olivomycin I showed that these antibiotics give favorable results
in treatment of testicular tumors. It was shown that these antibiotics
exhibit side effects such as gastrointestinal, hepatic, renal and bone
marrow toxicities. The major clinically limiting toxicity of mithramycin
was a hemorrhagic diathesis associated with a precipitous thrombocytopenia.
It is of considerable interest that hemorrhagic diathesis
was not observed after administration of olivomycin I.

As olivomycin I possesses the best chemotherapeutic index among
the aureolic acid antibiotics, it can be considered as the best scaffold
for the development of novel semisynthetic aureolic acid analogs with
increased therapeutic indices and lower toxicity compared with the
parent antibiotic.
Here we describe chemical modifications of olivomycin I at the
2ў-keto group of the side chain of the aglycone moiety. The
antiproliferative and topoisomerase I (Topo-I)-poisoning activities
of the novel derivatives (2–7) were tested. One of the novel derivatives
showed pronounced antitumor activity in in vivo experiments on mice
bearing lymphocyte leukemia P-388, together with lower toxicity to
animals compared with the parent olivomycin I.

RESULTS

Chemistry
We developed a novel method of chemical modification of olivomycin
I (1) based on the introduction of a carboxyl group into the molecule
of the antibiotic. Reaction of olivomycin I (1) with carboxymethoxylamine
gave the key intermediate, 2ў-carboxymethoxime-olivomycin
I (2) (Scheme 1), which was further reacted with different amines in
the presence of PyBOP to give the corresponding amides 3–7. The
resulting compounds were purified by column chromatography on
silica gel.


Biological testing

The cytotoxicity of the compounds in comparison with the parent
olivomycin I was tested. Cells were incubated with drugs for 48–72 h
to ensure the completion of late events in cell death. Table 1 shows
the comparative potencies of these compounds against the
wild-type murine leukemia L1210 cells, the human leukemia cell
line K562 and the human malignant T-lymphocyte Molt4/C8 and
CEM cells.
All novel derivatives (2–7) caused cell death at higher concentrations
than olivomycin I. Remarkably, amides with the bulky
hydrophobic substituents (adamantyl- 5; tert-butyl-, 6) showed antiproliferative
activity that was at an IC50 of only one order of
magnitude higher (for L1210: 0.19 and 0.20 mM, correspondingly)
than that of olivomycin I, but at a markedly lower IC50 than that of
2ў-carboxymethoxime-olivomycin I (2) or the amides with small or
hydrophilic substituents 3, 4 and 7 (IC50 for L1210: 6.5–20 mM).
To identify tentative intracellular targets important for cytotoxicity
of olivomycin I and its novel derivatives (2–7), we tested these
compounds for their ability to modulate Topo-I activity in vitro.
Olivomycin I (1) and all novel derivatives (2–7) were potent Topo-I
inhibitors at all concentrations investigated (0.5–20 mM) (data not
shown). Figure 1 shows the results of electrophoretic analysis of the
relaxation products of the Topo-I-dependent supercoiled DNA relaxation
in the absence and presence of the antibiotics olivomycin I and 5.
In the absence of antibiotics (track of Topo-I), the reaction led to a set
of topoisomers and the disappearance of the supercoiled form of
DNA. This effect was revealed by the presence of residual amounts of
rapidly migrating topoisomers. Olivomycin I (1) inhibited Topo-I
activity at all concentrations investigated (0.5–20 mM); compound 5
was less active but still a potent Topo-I inhibitor—the rapidly
migrating topoisomers were observed on the track starting at the
compound concentration of 2.5 mM.

When 5 was i.p. injected to mice with leukemia P-388 72 h after i.p.
implantation of tumor, 62% increase of lifespan (ILS) at the dose
of 50mg kg was achieved.

Compound 5 did not show any cumulative toxic effects at the
quintuple doses of 5 and 10mg kg (25 and 50mg kg 1 total dose,
respectively), whereas all of the mice that received 1 (5 mg kg 1 daily)
had drug-related toxic death after the third injection (data no shown).
All mice that received daily doses of 5 and 10mg kg 1 had tumorrelated
death. The ILS was 43% compared with the group of untreated
mice (Figure 2). Quintuple injections of 5 at 20mg kg 1 per dose
(100mg kg total dose) resulted in drug-related death of 15% of the
mice.