From distamycin to brostallicin : a case history

1
From distamycin to brostallicin a case history

• Paolo Cozzi
• investigational drug from Pharmacia Co.

2
List of items

• A short account of cancer epidemiology
• DNA minor groove binders distamycin and
  tallimustine
• a-Halogenoacrylic derivatives of distamycin
• a new class of cytotoxics
• A new drug in development brostallicin
• The role of GSH in brostallicin mechanism of
  action
• Synthetic chemistry of distamycin-like cytotoxics

3
The burden of cancer

• The estimated worldwide incidence of cancer is
  about 10 M
• roughly half of which in developped countries.
• The incidence and mortality in the USA and Italy
  are respectively about
• 1.3 M / 550 K and 250 K / 80 K.

4
The burden of cancer

• In spite of progress in diagnosis, surgery and
  therapy,
• the cancer mortality, even somewhat declined in
  very last years, remains high, with some
  exceptions for specific tumors.
• In the USA the cancer deaths 2000 vs 1985 were
• 160 K vs 120 K for lung, 110 K vs 60 K for colon,
  
• 41 K vs 38 K for breast, 32 K vs 25 K for
  prostate,
• 24 K vs 19 K for urinary tract, 14 K vs 12 K for
  ovary.

5
The burden of cancer

• The classical five-year survival rate for the
  average of most common tumors is about 60, but
  is as low as 14 for lung cancer.
• The five-year survival rate is increasing,
• however this reflects more a progress in early
  diagnosis, e.g. the large scale mammografy, PSA,
  Pap test practices, than a real therapeutic
  control.

6
Success stories against cancer

• A dramatic decrease of mortality took place in
  cancers
• in which early diagnosis and full organ surgery
• are possible or a big drop of incidence occurred.
• In the USA the mortality 1995 vs 1950 dropped
• 67 for uterus, 76 for cervix, 80 for stomach.
• (both mortality and incidence for lung increased
  about 250
• in the same year range)

7
The medical need

• Although many chemotherapeutic agents
• are available, the medical need is largely unmet
  because of
• Lack of selectivity, leading to toxicity
• Metastatic spreading on sites other than the
  primary tumor
• Heterogenicity of the disease about 100 types
  of cancer
• Intrinsic or acquired resistance (multi-drug
  resistance)

8
Distribution by therapeutic classes of the total
340 B foreseen prescription drugs sales in
2002

• Cardiovascular 25
• CNS 16
• Anti-infective 14
• Gastro-intestinal/metabolic 12
• Respiratory 8
• Oncology/immunosuppressive 6

Source S.S. Cheng, Decision Resources Inc.
Spectrum 2002
9
New approaches

• A number of innovative approaches are in
  development,
• focused on molecular targets of tumor cell e.g.
• oncogene encoded signal transducers proteins like
  Ras,
• cell cycle targets as cyclin-dependent kinases,
• telomerase, histone deacetylases, etc.
• These approaches met first success with tyrosine
  kinase inhibitor imatinib (gleevec) recently
  approved for chronic myelogenous leukemia, while
  gefitinib (iressa) is the promising forerunner of
  EGFR inhibitors.

10
New approaches

• As far as the approach of angiogenesis
  inhibition,
• focused molecular targets have been e.g.
• matrix metalloproteinases, endothelial tyrosine
  kinases, aVb3 and more recently aVb5
  transmembrane integrins.
• However it will take some years to fully define
  the role of new approaches, therefore cytotoxics
  will continue to represent a chief part of the
  cancer therapy in the near future,
• also in combination with innovative agents.

11
Minor groove binders

• The mode of action of many antitumor agents
  involves DNA damage, either by direct DNA
  interaction or by interaction with DNA-binding
  proteins as topoisomerases.
• DNA minor groove binders (MGBs) are a class of
  cytotoxics which was hypothesised to provide
  significant improvement in cancer therapy due to
  high affinity and selectivity of interaction with
  DNA.

12
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13
Distamycin A

• Distamycin A, an antibiotic mainly active against
  viruses,
• binds reversibly to DNA minor groove with high
  affinity and high selectivity for TA-rich
  sequences

14
TA sequence recognition in DNA minor groove
15
Distamycin bound to the minor groove
DST bound to DNA dodecamer G/C green, AT blue,
sugar-phosphate backbone yellow, DST red.
16
Distamycin A and its benzoyl nitrogen mustard
tallimustine
L1210 murine leukemia IC50 Distamycin A 10.0
mM / BAM 65.0 mM
Tallimustine 68.5 nM
(Cisplatin 90 nM)
17
Tallimustine - DNA interaction

• Tallimustine is a mild sequence-specific
  alkylator at T4GA sequences of the minor groove.
• Distamycin-driven DNA binding, by increasing drug
  concentration near DNA target, increases the
  potency, limiting the drug loss by reaction with
  other targets.

tallimustine
melphalan
L1210 IC50 - tallimustine 68.5 nM melphalan
980.5 nM
18
Mustardsfrom a chemical weapon to anticancer
drugs
Sulfur mustard
Nitrogen mustard
First DNA alkylating drug
19
DNA major groove alkylators nitrogen mustards
20
Alkylation mechanism of classical nitrogen
mustards
21
Preferential nucleotide alkylation (G-N7)of
classical nitrogen mustards
mustard
deoxyguanosine
a strand
base on the a strand intrastrand crossliking
base on the b strand interstrand crossliking
22
Distamycin A and its benzoyl nitrogen mustard
tallimustine
L1210 murine leukemia IC50 Distamycin A 10.0
mM / BAM 65.0 mM
Tallimustine 68.5 nM
23
Interaction of tallimustine with DNA duplex
tridecamer containing the T4GA consensus sequence.
24
The different functional moieties of tallimustine
25
Alkylation mechanism of classical nitrogen
mustards
higher the electronic availability of the
nitrogen higher the reactivity, i.e. the
alkylating power, of the mustard
26
Alkylating power of phenyl mustards
27
Tallimustine as a model

• Tallimustine was the first distamycin-derived
  cytotoxic clinically developed, however it showed
  a severe myelotoxicity and was discontinued.
• Nevertheless tallimustine has represented a model
  for the design of new potent agents by combining
  a moiety of
• mild chemical reactivity with a binding-frame
  acting as
• a DNA sequence-selective vector.

28
New distamycin-derived mustards

• Key points of the followed rationale
• fine tuning of alkylating power
• isosterical replacement of pyrrole rings
• modification of strong basic amidino moiety
• many potent compounds were obtained and SAR
  defined
  however
• the overall activity profile was not dramatically
  improved

29
In vitro activity vs L1210 of tallimustine and
close analogs
30
Alkylating power of phenyl mustards
31
Distamycin nitrogen half-mustard and sulfur
mustard
L1210 IC50 PNU-160366 4.0 nM / PNU-193821 0.9
nM tallimustine 68.5 nM // (cisplatin 90 nM)
These compounds show the possibility of achieving
potent activity by tethering one-arm mustards to
a distamycin frame, while classical nitrogen
half-mustards and sulfur mustards are not
cytotoxic, due to the impossibility of DNA
crosslinking.
32
A new class of cytotoxic minor groove binders
a-bromoacrylic distamycin-like derivatives
33
PNU-151807 the lead of distamycin-derived
?-halogenoacrylamides
PNU-151807 IC50 (L1210 ) 6.3 nM
Tallimustine IC50 (L1210)
68.5 nM PNU-151807 was found to bind to DNA
minor groove, but unreactive in classical in
vitro DNA alkylation assays,
at variance with tallimustine.
34
The role of the halogen on the activity of
?-halogenoacrylamido distamycin derivatives In
vitro and in vivo activity ( L1210 leukemia )
in vitro
i n vivo
R
IC
ng/mL
OD mg/kg T/C
50
Br
4.7 1.0
1,56
200
Cl
2.7 1.0
1,56
133
F
gt500
nd
nd
H
gt2500
nd
nd
35
Putative mechanism of nucleophilic attack
to ?-halogenoacrylic moiety
36
Reactivity of ?-bromoacrylamido-pyrrolecarboxyanil
ide
No reaction
N
No reaction
H
CH
COCH
/ H
O
N
3
3
2
N
O
pH 10
H
N
N
r.t. 48h or 60C 48h
imidazole (1eq)
O
DMF r.t. 72h or 80C 8h
Diethylamine (4 eq.)
Br
EtOAc, r.t., 1 week
H
N
N
O
N
H
N
H
N
N
Isobutyl amine (4 eq.)
imidazole (2 eq)
O
O
H
r.t. 12 h or 70C
N
K
CO
2 eq.60 C
N
N
2
3
O
H
N
O
H
N
Adenine (1.2 eq)
N
Adenine (1.2 eq)
DMF, r.t. 12h
O
K
CO
1eq.
N
H
2
3
2
DMF, r.t. 12h
N
N
N
N
No reaction
H
N
O
H
N
N
O
37
Reactivity of ?-fluoro-acrylamido-pyrrolecarboxyan
ilide
?-chloro-acrylamido-pyrrolecarboxyanilide reacts
like ?-bromo analog with lower yields and in
harder conditions
38
PNU-151807 as a lead of a new class

• In vitro / in vivo activity superior to
  tallimustine
• Better cytotoxicity /myelotoxicity ratio
• Unusual / unknown mechanism of action
• Non traditional alkylating moiety

39
The effect of the length of distamycin-like frame
on the activity of ?-halogenoacrylamido
derivatives
40
The length of distamycin-like frame and the
activity

• The classical explanation for the correlation
  cytotoxicity / length
• is a tighter DNA binding, arising from the
  multiplicity of pyrrole-carboxamide / DNA
  interactions, mainly by hydrogen bonding.
• However, using Caco-2 cells, we found in our
  series a progressive increase of permeability
  from one to four pyrrole units derivatives.
• This may represent an additional explanation for
  high cytotoxicity of derivatives with three or
  more pyrrole units, suggesting also the existance
  of an active transport mechanism for these
  compounds.

41
Caco-2 cells permeability for abromoacrylic
oligopyrroles.  
 
aApparent permeability, method of P. Artusson b
ACD calculated LogD at pH 7.4
42
In vitro and in vivo activity of ?-bromoacrylic
distamycin-like derivatives modified at the
amidino moiety.
43
Examples of isosteric a-bromoacrylic analogs
PG. Baraldi et Al., Bioorg. Med. Chem. Lett. 6,
1247 (1996) PG. Baraldi et Al., Bioorg. Med.
Chem. 7,251 (1999)
44
Representative ?-bromoacrylic derivatives of
isosteric imidazole / pyrazole analogs of
PNU-151807
45
PNU -166196 / INN brostallicin
?-bromoacrylamido-tetrapyrrole guanidino
derivative undergoing Phase II clinical trials
46

• Brostallicin activity profile
• cytotoxicity on tumor cells resistant to
  camptothecin
• and alkylating agents
• broad antitumor activity
• dramatically reduced myelotoxicity on human
• hematopoietic progenitor cells
• well tolerated in patients (Phase I data)

47
Brostallicin myelotoxicity The
in vitro myelotoxicity for human hematopoietic
progenitor cells is radically reduced compared
to other MGB
In vitro
MYELOTOXICITY
CYTOTOXICITY
Cytotoxicity/
Human CFU-GM
Tumor Cells
COMPOUND
Myelotoxicity
IC
ng/ml
IC
ng/ml
50
50
Ratio
2377
29.3
81.1
BROSTALLICIN
290
330
0.9
TALLIMUSTINE
0.15
0.04
3.7
CARZELESIN
0.18
0.17
1
BIZELESIN
0.17
0.04
4.2
ADOZELESIN

• The myelotoxicity of brostallicin examined in
  mice, rats
• and monkeys is mild and
  reversible.

48

• Brostallicin Mechanism
• Brostallicin is 20 fold more active than
  tallimustine
• in inducing apoptosis (in hu. ovarian A2780
  cells).
• Brostallicin, as the parent PNU-151807,
  interacts
• reversibly with AT-rich regions of the
  minor groove
• but appears unreactive in DNA alkylation
  assays.

49
Interaction of brostallicin with DNA duplex
tridecamer 5-d(ACCTTTTTGATGT)-3 3-d(TGGAAAAACTA
CA)-5
50

• Brostallicin Mechanism
• About the apparent lack of DNA alkylation,
  
• we speculated that an intracellular reactive
  nucleophile
• e.g. GSH, could perform a Michael-type attack
• followed by a further reaction leading to
  alkylation of
• DNA nucleophilic functions.
• Thus brostallicin might alkylate DNA
• only in the presence of GSH.

51
GSH reactivity

• GSH is the most abundant non-protein thiol
  present in concentrations up to 5 mM in cells,
• (cytosol, nucleus, mitochondria)
• GSH acts as an antioxidant (one electron
  transfer) scavenging Reactive Oxygen Species
  (ROS)
• GSH reacts also as a nucleophile (two electron
  transfer) forming hydrophilic conjugates and
  leading to xenobiotics detoxification.

52
Putative role of GSH in interaction between
DNA and ?-bromoacrylic derivatives
Nu GS / OH / other
53
Interaction with plasmidic DNA
(Agarose gel electrophoresis after incubation
with plasmid containing sequences
T4GA) Change of the plasmidic DNA from the
supercoiled form to the circular
form. Brostallicin no change
PNU-151807 no change Tallimustine change
Distamycin no change Brostallicin
GSH change PNU-151807 GSH change ??
?-fluoroacrylic analogs GSH no change
54
Cytotoxicity of brostallicin, PNU-151807 and
tallimustine against L1210 and L1210/L-PAM
murine leukemia cells.
IC
ng/mL ( 48 h treatment )
50
L1210
L1210/L-PAM
Brostallicin
1.62
0.49
PNU-151807
0.86
0.26
Tallimustine
22.5
27.4
L1210/L-PAM murine leukemia cells present
a three-fold increase of GSH levels.
55
Cytotoxicity and apoptotic effect on A2780 cells
pretreated with BSO
Dose
Growth
Apoptosis
Treatment
inhibition
()
()
1
?
50
65
g/
mL
Brostallicin a
3
?
71
71
g/
mL
1
Brostallicin BSO 0.1 mM a
?

22
8
g/
mL
3
?
31
17
g/
mL
b
5
0
BSO
0.1
mM
a) Cells incubated with the comp. for 48 h.
Apoptosis determined by morphological
examination. b) Cells exposed to BSO for 24 h
before and during treatment.
56
The role of GSH/GST on brostallicin efficacy in
vivo

• Brostallicin shows increased antitumor efficay
• in tumors overexpressing GSH-S-transferase-p
• (GST-p), developed in nude mice after
  implantation of A2780 clones transfected with
  human GST-p genes.

57
The role of GSH and GSH-S-transferases (GSTs)

• GSH thiol (pKa 9) is protonated under
  physiological conditions, however GSTs activate
  GSH thiol to thiolate enhancing its
  nucleophilicity

58

• Summary of brostallicin mechanism
• Interacts with AT-rich regions of DNA minor
  groove.
• Shows evidence of DNA alkylation in vitro only
  in the
• presence of GSH.
• Shows increased cytotoxicity on tumor cells
  which
• overexpress GSH/GST and decreased
  cytotoxicity when
• GSH synthesis is inhibited.
• Shows increased efficacy in tumors
  overexpressing GST-p
• in mice.

59
GSH role in cancer High levels of GSH/GST are
one of the main causes of resistance to
different anticancer drugs, such as alkylating
agents and anthracyclines. There is increasing
evidence that a number of human tumors display
increased levels of GSH and GST-p isozyme in
respect to normal tissues.


60

• Conclusions
• a-bromoacrylamido-pyrrole oligomers are a new
  class of
• MGB with potent antitumor activity
• they appear unreactive in classical DNA
  alkylation assays
• in vitro, but may be activated in vivo by
  GSH, leading to
• covalent DNA interaction
• brostallicin shows high potency and low
  myelotoxicity
• and additionally
• might have a particular role for the treatment
  of tumors
• overexpressing GSH-GST-p levels.

61
Possible moral of the story

• The most fruitful basis for the discovery of a
  new drug is to start with an old drug. (Prof.
  James Black)
• Distamycin
• Tallimustine
• PNU-151807
• Brostallicin

62
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63
Some synthetic chemistryondistamycin-like
compounds
64
Synthesis of brostallicin
65
Alternative syntheses of brostallicin from
distamycin
66
Synthesis of brostallicin from distamycin via
Curtius rearrangement
I. Beria, M. Nesi, Tetrahedron Letters, 2002,
43,7323
67
Synthesis of brostallicin from distamycin via
selective amide hydrolysis
I. Beria, M. Nesi, Tetrahedron Letters, 2002,
43,7323
68
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69
Reactivity of ?-fluoro-acrylamido-pyrrolecarboxyan
ilide
70
a-fluoroacrylic moiety does not react with
nucleophiles as corresponding bromo-, chloro-
moieties and acrylic moiety

No reaction

71
a-fluoroacrylic moiety does not react with
nucleophiles as corresponding bromo- or chloro-
moieties and acrylic moiety do
72
a-fluoroacrylic moiety does not react with
nucleophiles as corresponding bromo- or chloro-
moieties and acrylic moiety do
73
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74
The general synthesis of distamycin derivatives
coupling of appropriate acid with
desformyl-distamycin
75
Synthesis of a-halogenoacrylic derivatives of
distamycin a representative example
76
Synthesis of mustards in which the amidino moiety
is replaced by an amide group
77
A general method of synthesis of distamycin-like
derivatives with modified amidino groups
78
Alkylation mechanism of classical nitrogen
mustards
79
Synthesis of mustards in which the amidino moiety
is replaced by cyano group
80
The conversion of distamycin amidine into cyano
group is performed by all five-membered
anhydrides. Acetic and glutaric anhydrides gave
N-acylation.
81
Putative mechanism of amidine-nitrile
conversion.Succinimide formation was detected by
HPLC.Tetrahedron Letters 39 (1998) 3551
82
Aknowledgement Medicinal Chemistry Nicola
Mongelli, Italo Beria, Marina Caldarelli Pharmacia
, Global Chemistry PierGiovanni Baraldi, Romeo
Romagnoli Dept.of Pharmaceutical Chemistry,
Ferrara University Pharmacology Cristina
Geroni Pharmacia, Discovery Research Oncology
Biochemistry Molecular Pharmacology Enzio
Ragg, Stefania Mazzini DI.S.M.A., Faculty of
Agriculture, Milan University Maurizio
DIncalci, Massimo Broggini Department of
Oncology, Mario Negri Institute, Milan.
83
Back-ups
84
The agents derived from antibiotic CC-1065
85
Ecteinascidin 743 (ET743) alkylating central G
of DNA minor groove triplets
C-21 electrophilic centre
marine-derived antibiotic / Pharma Mar - BMS
86
Lexitropsins

• Lexitropsins are oligopeptides in which the
  typical pyrrole units of distamycin are replaced
  by imidazole, thiazole or furan rings leading to
  to preferential GC recognition.

dista protruding proton
lexi donor sp2 N
Tri-imidazole lexitropsin
87
Synthesis of nitro-pyrrole-carboxylic acid
88
Techniques to study in vitro drug-DNA interactions

• DNase footprinting a ligand bound to a specific
  DNA sequence interferes with the digestion by
  endonuclease Dnase I. The digestion pattern
  allows the identification of a protected region
  (footprint).
• The technique is used in combination with
  Maxam-Gilbert method of DNA sequencing, that
  cleaves chemically DNA at specific bases, giving
  fragments resolved by electrophoresis.
• Taq polymerase stop assay the enzyme
  synthesizes a new strand on a DNA template, until
  it encounters the site of covalent binding, where
  it stops.

89
Putative GSH-brostallicin adduct alkylating DNA
The synthesis of this adduct was performed but
its identification is not absolutely sure
The attempted synthesis of this adduct failed