U.S. patent application number 10/379049 was filed with the patent office on 2003-10-16 for pyrrolobenzodiazepines.
This patent application is currently assigned to Spirogen Limited. Invention is credited to Howard, Philip Wilson, Thurston, David Edwin.
Application Number | 20030195196 10/379049 |
Document ID | / |
Family ID | 10837953 |
Filed Date | 2003-10-16 |
United States Patent
Application |
20030195196 |
Kind Code |
A1 |
Thurston, David Edwin ; et
al. |
October 16, 2003 |
Pyrrolobenzodiazepines
Abstract
A compound with formula (I) 1 where R.sub.10 is a
therapeutically removable nitrogen protecting group; R.sub.2 and
R.sub.3 are independently selected from: H, R, OH, OR, .dbd.O,
.dbd.CH--R, .dbd.CH.sub.2, CH.sub.2--CO.sub.2R,
CH.sub.2--CO.sub.2H, CH.sub.2--SO.sub.2R, O--SO.sub.2--R,
CO.sub.2R, COR and CN; R.sub.6, R.sub.7 and R.sub.9 are
independently selected from H, R, OH, OR, halo, amino, nitro,
Me.sub.3Sn; X is S, O or NH; R.sub.11 is either H or R; and where
there is optionally a double bond between C1 and C2 or C2 and C3;
and R.sub.8 is selected from H, R, OH, OR, halo, amino, nitro,
Me.sub.3Sn, or R.sub.7 and R.sub.8 together form a group
--O--(CH.sub.2).sub.p--O--, where p is 1 or 2. Such compounds may
be used in methods of ADEPT, GDEPT, NPEPT or PDT.
Inventors: |
Thurston, David Edwin;
(Nottingham, GB) ; Howard, Philip Wilson;
(Nottingham, GB) |
Correspondence
Address: |
MICHAEL BEST & FRIEDRICH, LLP
ONE SOUTH PINCKNEY STREET
P O BOX 1806
MADISON
WI
53701
|
Assignee: |
Spirogen Limited
Isle of Wright
GB
|
Family ID: |
10837953 |
Appl. No.: |
10/379049 |
Filed: |
March 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10379049 |
Mar 4, 2003 |
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09763814 |
Feb 26, 2001 |
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6562806 |
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09763814 |
Feb 26, 2001 |
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PCT/GB99/02837 |
Aug 27, 1999 |
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Current U.S.
Class: |
514/220 ;
540/486; 540/496 |
Current CPC
Class: |
C07D 487/04 20130101;
A61P 31/12 20180101; A61P 33/00 20180101; Y02P 20/55 20151101; A61P
43/00 20180101; A61P 31/10 20180101; A61P 35/00 20180101 |
Class at
Publication: |
514/220 ;
540/486; 540/496 |
International
Class: |
A61K 031/551; C07D
487/04; C07F 007/22 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 27, 1998 |
GB |
9818731.3 |
Claims
1. A compound with the formula I: 36wherein: R.sub.10 is a
therapeutically removable nitrogen protecting group; R.sub.2 and
R.sub.3 are independently selected from: H, R, OH, OR, .dbd.O,
.dbd.CH--R, .dbd.CH.sub.2, CH.sub.2--CO.sub.2R,
CH.sub.2--CO.sub.2H, CH.sub.2--SO.sub.2R, O--SO.sub.2--R,
CO.sub.2R, COR and CN; R.sub.6, R.sub.7 and R.sub.9 are
independently selected from H, R, OH, OR, halo, amino, nitro,
Me.sub.3Sn; or R.sub.7 and R.sub.8 together form a group
--O--(CH.sub.2)--O--, where p is 1 or 2. X is S, O or NH; R.sub.11
is either H or R; where R is a lower alkyl group having 1 to 10
carbon atoms, or an aralkyl group, of up to 12 carbon atoms,
whereof the alkyl group optionally contains one or more
carbon-carbon double or triple bonds, which may form part of a
conjugated system, or an aryl group, of up to 12 carbon atoms; and
is optionally substituted by one or more halo, hydroxy, amino, or
nitro groups, and optionally contains one or more hetero atoms,
which may form part of, or be, a functional group; and where there
is optionally a double bond between C1 and C2 or C2 and C3; and
R.sub.8 is selected from H, R, OH, OR, halo, amino, nitro,
Me.sub.3Sn, where R is as defined above, or the compound is a dimer
with each monomer being the same or different and being of formula
I, where the R.sub.8 groups of the monomers form together a bridge
having the formula -T-R'-T- linking the monomers, where R' is an
alkylene chain containing from 3 to 12 carbon atoms, which chain
may be interrupted by one or more hetero atoms and/or aromatic
rings, and may contain one or more carbon-carbon double or triple
bonds, and each T is independently selected from O, S or N.
2. A compound according to claim 1, wherein R.sub.10 is of the
formula II: 37wherein n is 0 to 3, R.sup.(I) is H or R, and
R.sup.(II) is one or more optional substituents independently
selected from NO.sub.2, OR, or R, where R is as defined in claim 1,
and if two substituents R.sup.(II) are on adjacent atoms, they may
together be of the formula --O--(CH.sub.2).sub.m--O-- where m is 1
or 2.
3. A compound according to either claim 1 or claim 2, wherein
R.sub.10 is a photolabile protecting group.
4. A compound according to claim 3, wherein R.sub.10 is cleavable
by light with a wavelength of 250 to 550 nm.
5. A compound according to either claim 1 or claim 2 wherein
R.sub.10 is an enzyme labile group.
6. A compound according to claim 5, wherein R.sub.10 is a
nitroreductase labile group.
7. A compound according to claim 5, wherein R.sub.10 is a
penicillin V/G amidase labile group.
8. A compound according to claim 5, wherein R.sub.10 is a
L-.gamma.-glutamyl transpeptidase labile group.
9. A compound according to claim 5, wherein R.sub.10 is a
glutathione transferase labile group.
10. A compound according to claim 6, wherein R.sub.10 is of the
formula III: 38wherein n is 0 to 3, R.sup.(I) is H or R, and
R.sup.(III) is one or more optional substituents independently
selected from NO.sub.2O, OR, or R, where R is as defined in claim
1, and if two substituents R.sup.(III) are on adjacent atoms, they
may together be of the formula --O--(CH.sub.2).sub.m--O-- where m
is 1 or 2.
11. A compound according to claim 9, wherein R.sub.10 is of the
formula XI: 39wherein R is as defined in claim 1, and n is 0 to
3.
12. A compound according to claim 11, wherein R in the group
formula XI is a substituted or unsubstituted phenyl group.
13. A compound according to any one of the preceding claims,
wherein X is O.
14. A compound according to any one of the preceding claims,
wherein R.sub.11 is H.
15. A compound according to any one of the preceding claims,
wherein R.sub.6 and R.sub.9 are H.
16. A compound according to claim 15, wherein R.sub.7 and R.sub.8
are independently selected from H, OH, and OR, where R is as
defined in claim 1.
17. A compound according to any one of the preceding claims,
wherein the compound is a C8 dimer, wherein R.sub.6 and R.sub.9 are
H, and R.sub.7 is independently selected from H, OH, and OR, where
R is as defined in claim 1.
18. A compound according to any one of the preceding claims,
wherein the compound is a C8 dimer, and R' is
--O--(CH.sub.2).sub.P--O--, where p is from 1 to 12.
19. A method of preparing a compound of formula I as defined in any
one of claims 1 to 18 wherein XR.sub.11.noteq.OH from a compound of
formula Ia: 40wherein the substituents of the compound of formula
Ia are the same as for the compound of formula I to be
prepared.
20. A method of preparing a compound of formula Ia as defined in
claim 19 by the oxidation of a compound of formula IVa: 41wherein
the substituents of the compound of formula IVa are the same as for
the compound of formula Ia to be prepared.
21. A method according to claim 20, wherein the oxidation is a
Swern oxidation.
22. A method of preparing a compound of formula IVa as defined in
claim 20, by reacting a compound of formula Va: 42with a compound
of formula VI: Y--R.sub.10 (VI) wherein the substituents of the
compounds of formulae Va and VI are the same as for the compound of
formula IVa to be prepared, and Y is a halogen atom.
23. A method of preparing a compound of formula IVa according to
claim 20, wherein the compound of formula VI is a haloformate of
the formula Y-A, where A is a group of formula II as defined in
claim 2.
24. A method of making a compound of formula IVa as defined in
claim 20, by reacting a compound of formula VII: 43with a compound
of formula VI: Y--R.sub.10 (VI) to form a compound of formula VIII:
44and then reacting the compound of formula VIII with a compound of
formula IXa: 45via the formation of an acid chloride, wherein the
substituents for compounds of formulae VI, VII, VIII and IXa are
the same as for the compound of formula Va to be prepared, and
where Y is a halogen atom.
25. A method of preparing a compound of formula Ia as defined in
claim 19 by the unmasking of a compound of formula IVb: 46wherein
the substituents of the compound of formula IVb are the same as for
the compound of formula Ia to be prepared, Q is either O or S and
R.sup.(IV) is Me or Et, or together form --(CH.sub.2).sub.q-- where
q is 2 or 3.
26. A method according to claim 25, wherein Q is S, R.sup.(iv) is
Et and the unmasking is mercury-mediated unmasking.
27. A method according to claim 25, wherein Q is O, R.sup.(iv) is
Me and the unmasking is palladium-mediated unmasking.
28. A method of preparing a compound of formula IVb as defined in
claim 25, by reacting a compound of formula Vb: 47with a compound
of formula VI: Y--R.sub.10 (VI) wherein the substituents of the
compounds of formulae Vb and VI are the same as for the compound of
formula IVb to be prepared, and Y is a halogen atom.
29. A method of preparing a compound of formula IVb according to
claim 28, wherein the compound of formula VI is a haloformate of
the formula Y-A, where A is a group of formula II as defined in
claim 2.
30. A method of making a compound of formula IVb as defined in
claim 25, by reacting a compound of formula VII: 48with a compound
of formula VI: Y--R.sub.10 (VI) to form a compound of formula VIII:
49and then reacting the compound of formula VIII with a compound of
formula IXb: 50via the formation of an acid chloride, wherein the
substituents for compounds of formulae VI, VII, VIII and IXb are
the same as for the compound of formula Vb to be prepared, and
where Y is a halogen atom.
31. A method of making a compound of formula X: 51by the cleavage
of the therapeutically removable protecting group R.sub.10 of a
compound of formula I as defined in any one of claims 1 to 18,
wherein the substituent groups of the compound of formula X are the
same as the substituent groups of compound I used.
32. The use of a compound of formula I as defined in any one of
claims 5 to 8 or 10 in conjunction with an appropriate enzyme in
methods of ADEPT or GDEPT therapy.
33. The use of compounds of formula I as defined in any one of
claims 2 to 4 in conjunction with light of wavelengths between 250
and 550 nm in methods of PDT.
34. The use of compounds of formula I as defined in claim 9 or II
in methods of NPEPT.
35. A pharmaceutical composition comprising a compound of formula I
as defined in any one of claims 1 to 18 and a pharmaceutically
acceptable carrier or diluent.
36. The use of a compound of formula I as defined in any one of
claims 1 to 18 to prepare a medicament for the treatment of a
neoplastic disease.
37. The use of a compound of formula I as defined in any one of
claims 1 to 18 to prepare a medicament for the treatment of a
bacterial, viral or parasitic infection.
Description
[0001] The present invention relates to pyrrolobenzodiazepines
(PBDs), and is particularly concerned with the use of these
compounds as prodrugs for antibody-directed enzyme-prodrug therapy
(ADEPT), gene-directed enzyme-prodrug therapy (GDEPT), photodynamic
therapy (PDT) and naturally present enzyme-prodrug therapy
(NPEPT).
BACKGROUND OF THE INVENTION
[0002] Pyrrolobenzodiazepines (PBDs) have the ability to recognise
and bond to specific sequences of DNA; the most preferred sequence
is PuGPu (Purine-Guanine-Purine). The first PBD antitumour
antibiotic, anthramycin, was discovered in 1965 (Leimgruber et al.,
1965 J. Am. Chem. Soc., 87, 5793-5795; Leimgruber et al., 1965 J.
Am. Chem. Soc., 87, 5791-5793). Since then, a number of naturally
occurring PBDs have been reported, and over 10 synthetic routes
have been developed to a variety of analogues (Thurston et al.,
1994 Chem. Rev. 1994, 433-465). Family members include abbeymycin
(Hochlowski et al., 1987 J. Antibiotics, 40, 145-148), chicamycin
(Konishi et al., 1984 J. Antibiotics, 37, 200-206), DC-81 (Japanese
Patent 58-180 487; Thurston et al., 1990, Chem. Brit., 26, 767-772;
Bose et al., 1992 Tetrahedron, 48, 751-758), mazethramycin
(Kuminoto et al., 1980 J. Antibiotics, 33, 665-667), neothramycins
A and B (Takeuchi et al., 1976 J. Antibiotics, 29, 93-96),
porothramycin (Tsunakawa et al., 1988 J. Antibiotics, 41,
1366-1373), prothracarcin (Shimizu et al, 1982 J. Antibiotics, 29,
2492-2503; Langley and Thurston, 1987 J. Org. Chem., 52, 91-97),
sibanomicin (DC-102)(Hara et al., 1988 J. Antibiotics, 41, 702-704;
Itoh et al., 1988 J. Antibiotics, 41, 1281-1284), sibiromycin
(Leber et al., 1988 J. Am. Chem. Soc., 110, 2992-2993) and
tomamycin (Arima et al., 1972 J. Antibiotics, 25, 437-444). PBDs
are of the general structure: 2
[0003] They differ in the number, type and position of
substituents, in both their aromatic A rings and pyrrolo C rings,
and in the degree of saturation of the C ring. In the B-ring there
is either an imine (N.dbd.C), a carbinolamine (NH--CH(OH)) or a
carbinolamine methyl ether (NH--CH(OMe))at the N10-C11 position
which is the electrophilic centre responsible for alkylating DNA.
All of the known natural products have an (S)-configuration at the
chiral C11a position which provides them with a right-handed twist
when viewed from the C ring towards the A ring. This gives them the
appropriate three-dimensional shape for isohelicity with the minor
groove of B-form DNA, leading to a snug fit at the binding site
(Kohn, 1975 In Antibiotics III. Springer-Verlag, New York, pp.
3-11; Hurley and Needham-VanDevanter, 1986 Acc. Chem. Res., 19,
230-237). Their ability to form an adduct in the minor groove
enables them to interfere with DNA processing, hence their use as
antitumour agents.
[0004] The use of prodrugs represents a very valuable clinical
concept in cancer therapy. For example, a prodrug may be converted
into an antitumour agent under the influence of an enzyme that is
linked to a monoclonal antibody so that it can bind to a tumour
associated antigen. The combination of such a prodrug with such an
enzyme monoclonal antibody conjugate represents a very powerful
therapeutic strategy. This approach to cancer therapy, often
referred to as "antibody directed enzyme/prodrug therapy" (ADEPT)
is disclosed in WO88/07378.
[0005] A further therapeutic approach termed "virus-directed enzyme
prodrug therapy" (VDEPT) has been proposed as a method for treating
tumour cells in patients using prodrugs. Tumour cells are targeted
with a viral vector carrying a gene encoding an enzyme capable of
activating a prodrug The gene may be transcriptionally regulated by
tissue specific promoter or enhancer sequences. The viral vector
enters tumour cells and expresses the enzyme, thereby converting
the prodrug into the active drug within the tumour cells (Huber et
al., Proc. Natl. Acad. Sci. USA (1991) 88, 8039). Alternatively,
non-viral methods for the delivery of genes have been used Such
methods include calcium phosphate co-precipitation, microinjection,
liposomes, direct DNA uptake, and receptor-mediated DNA transfer.
These are reviewed in Morgan & French, Annu. Rev. Biochem.,
1993, 62;191. The term "GDEPT" (gene-directed enzyme prodrug
therapy) is used to include both viral and non-viral delivery
systems.
[0006] Photodynamic therapy (PDT) provides another method which
uses prodrugs to deliver desired drugs to specific sites in the
human body. Advances in the field of light delivery to internal
areas of the body allow delivery to organs and other areas without
the need for any extensive surgical procedures. The activation
process can be extremely site specific, as the direction of a laser
beam can be controlled with great precision, and the beam diameter
can be reduced far below that of a single cell, minimising any
possible damage to other neighbouring tissue from unwanted
activation of the drug. The high energy of ultra-violet light (e.g.
350 nm equivalent to 340 kJ/mol) is sufficient to break a range of
chemical bonds, since the bond energy spectrum of the majority of
organic molecules lies between 250 and 420 kJ/mol. For example,
there has been wide application of the photochemical deprotection
of amino acids, peptides and polysaccharides from their
o-nitrobenzyl carbamate, CBZ, and 4,5-dimethoxy-2-nitrobenzyl
carbamate forms at wavelengths longer than 350 nm.
[0007] A further class of prodrugs is those where the protecting
group is removed by an enzyme naturally present at the desired site
of action. These enzymes include dopa-decarboxylase,
L-.gamma.-glutamyl transpeptidase, and mixed function oxidases and
reductase (e.g. DT-diaphrase). This is method termed "naturally
present enzyme-prodrug therapy (NPEPT) in this application. One
enzyme of particular interest is glutathione transferase (GST),
which forms part of a major cellular defence mechanism based on the
use of the tripeptide, glutathione, as a scavenger of toxic
electrophiles. GST acts as a catalyst in the reaction between
glutathione and its target electrophiles. A consequence of this
defence mechanism is the inactivation of electrophilic therapeutic
agents Many human tumour cells exhibit elevated GST levels compared
to normal cells and the association of GST with resistance to DNA
alkylating agents has been demonstrated by Lewis et al.
(Carcinogenesis 1988, 9, 1283-1287), Kuzmich et al. (Journal of
Biochemistry 1992, 281, 219-224), and Tew et al.
(Glutathione-S-transferase and anti-cancer drug resistance, in
Mechanism of Drug Resistance in Neoplastic Cells; Wooley, P. V.,
Tew, K D, Eds.; Academic Press: Orlando, Fla., 1987; pp141-159).
Chemotherapeutic agents that take advantage of this intrinsic
property of cancer cells may prove highly useful in treating
refractory cancers.
[0008] Prodrugs which make use of this elevated GST level have been
made (Satyam et al., Med. Chem. 1996, 39, 1736-1747). They have a
glutathione molecule linked via a 2-sulphonylethyloxycarbonyl
linker to a phosphorodiamidate mustard. An alternative type of
prodrug has made use of the closely related
2-phenylsulphonylethyloxycarbonyl (Psec) group (Nicolaou et al.,
Science, 1992, 256, 1172-1178). Such prodrugs showed selectivity
between healthy human bone marrow cells and promeocytic and T cell
leukemia tumour lines.
DISCLOSURE OF THE INVENTION
[0009] A first aspect of the present invention provides a compound
with the formula I: 3
[0010] wherein:
[0011] R.sub.10 is a therapeutically removable nitrogen protecting
group;
[0012] R.sub.2 and R.sub.3 are independently selected from: H, R,
OH, OR, .dbd.O, .dbd.CH--R, .dbd.CH.sub.2, CH.sub.2--CO.sub.2R,
CH.sub.2--CO.sub.2H, CH.sub.2--SO.sub.2R, O--SO.sub.2--R,
CO.sub.2R, COR and CN;
[0013] R.sub.6, R.sub.7 and R.sub.9 are independently selected from
H, R, OH, OR, halo, amino, nitro, Me.sub.3Sn; or R.sub.7 and
R.sub.8 together form a group --O--(CH.sub.2).sub.P--O--, where p
is 1 or 2;
[0014] X is S, O or NH;
[0015] R.sub.11 is either H or R;
[0016] where R is a lower alkyl group having 1 to 10 carbon atoms,
or an aralkyl group (i.e. an alkyl group with one or more aryl
substituents), preferably of up to 12 carbon atoms, whereof the
alkyl group optionally contains one or more carbon-carbon double or
triple bonds, which may form part of a conjugated system, or an
aryl group, preferably of up to 12 carbon atoms; and is optionally
substituted by one or more halo, hydroxy, amino, or nitro groups,
and optionally contains one or more hetero atoms, which may form
part of, or be, a functional group;
[0017] and where there is optionally a double bond between C1 and
C2 or C2 and C3;
[0018] and R.sub.8 is selected from H, R, OH, OR, halo, amino,
nitro, Me.sub.3Sn, where R is as defined above, or the compound is
a dimer with each monomer being the same or different and being of
formula I, where the R.sub.8 groups of the monomers form together a
bridge having the formula -T-R'-T- linking the monomers, where R'
is an alkylene chain containing from 3 to 12 carbon atoms, which
chain may be interrupted by one or more hetero atoms and/or
aromatic rings, e.g. benzene or pyridine, and may contain one or
more carbon-carbon double or triple bonds, and each T is
independently selected from O, S or N.
[0019] If R is an aryl group, and contains a hetero atom, then R is
a heterocyclic group. If R is an alkyl chain, and contains a hetero
atom, the hetero atom may be located anywhere in the alkyl chain,
e.g. --O--C.sub.2H.sub.5, --CH.sub.2--S--CH.sub.3, or may form part
of, or be, a functional group, e.g. carbonyl, hydroxy.
[0020] R is preferably independently selected from a lower alkyl
group having 1 to 10 carbon atoms, or an aralkyl group, preferably
of up to 12 carbon atoms, or an aryl group, preferably of up to 12
carbon atoms, optionally substituted by one or more halo, hydroxy,
amino, or nitro groups. It is more preferred that R groups are
independently selected from a lower alkyl group having 1 to 10
carbon atoms optionally substituted by one or more halo, hydroxy,
amino, or nitro groups. It is particularly preferred that R groups
are unsubstituted straight or branched chain alkyl groups, having 1
to 10, preferably 1 to 6, and more preferably 1 to 4, carbon atoms,
e.g. methyl, ethyl, propyl, butyl.
[0021] Alternatively, R.sub.6, R.sub.7, R.sub.8, and R.sub.9 may
preferably be independently selected from R groups with the
following structural characteristics:
[0022] (i) an optionally substituted phenyl group;
[0023] (ii) an optionally substituted ethenyl group;
[0024] (iii) an ethenyl group conjugated to an electron sink.
[0025] The term `electron sink` means a moiety covalently attached
to a compound which is capable of reducing electron density in
other parts of the compound. Examples of electron sinks include
cyano, carbonyl and ester groups.
[0026] The term `therapeutically removable nitrogen protecting
group` means any group which can protect the 10-nitrogen, but which
is removable under therapeutic conditions in vivo, that is,
removable under conditions which occur or can be caused to occur in
vivo and are medically acceptable generally by elimination to
produce a N10-C11 imine group or an equivalent, capable of
interacting with DNA. The removal of the protecting group should
leave the rest of the structure of the PBD unaffected.
[0027] Suitable removal techniques include applying light, e.g.
with a wavelength of 250 to 400, or 550 nm, changing the ambient
pH, or cleavage by the action of an enzyme. One particularly
suitable enzyme is nitroreductase, although other suitable enzymes
include penicillin V/G amidase, .beta.-lactamase, phosphatase,
L-.gamma.-glutamyl transpeptidase, and .alpha.-galactosidase. The
action of some of these enzymes is described in Jungheim, L. N. and
Shepherd, T. A., Design of Antitumour Prodrugs: Substrates for
Antibody Targeted Enzymes, Am. Chem. Soc. Chem. Rev., 1994, 94: 6,
1553-1566. Another particularly suitable enzyme is glutathionare
transferase, as discussed above.
[0028] One possible group is R.sub.10 of the formula II: 4
[0029] wherein n is 0 to 3, R.sup.(I) is H or R, and R.sup.(II) is
one or more optional substituents independently selected from
NO.sub.2, OR, or R, where R is as defined in any of the definitions
above; and if two substituents R.sup.(II) are on adjacent atoms,
they may together be of the formula --O--(CH.sub.2).sub.m--O--,
where m is 1 or 2. R.sup.(II) is preferably NO.sub.2.
[0030] If the therapeutically removable group R.sub.10 is one which
is susceptible to nitroreductase, it may be of the formula III:
5
[0031] wherein n is 0 to 3, R.sup.(I) is H or R, and R.sup.(III) is
one or more optional substituents independently selected from
NO.sub.2, OR or R, where R is as defined in any of the definitions
above, and if two substituents R.sup.(III) are on adjacent atoms,
they may together be of the formula --O--(CH.sub.2).sub.m--O--,
where m is 1 or 2.
[0032] Another possible therapeutically removable nitrogen
protecting group, R.sub.10, is of the formula XI: 6
[0033] where R is as defined in any of the definitions above and n
is 0 to 3, preferably 0. For this formula, R is most preferably a
phenyl group, substituted or unsubstituted. This protecting group
may be removable by the action of glutathione transferase (GST),
which is present at high levels in many human tumour cells (see
above).
[0034] It is preferred in compounds of formula I that X is O and,
independently, that R.sub.11 is H.
[0035] If there is a double bond in the C ring, it is preferably
between C2 and C3.
[0036] Additionally, it is preferred that R.sub.6 and R.sub.9 are
H, and further preferred that R.sub.7 and R.sub.8 are independently
selected from H, OH, and OR. It is further preferred that R.sub.2
and R.sub.3 are H.
[0037] If the compound of formula I is a dimer, the dimer bridge
may be of the formula --O--(CH.sub.2).sub.P--O--, where p is from 1
to 12, preferably 3 to 9. Further, R.sub.6 and R.sub.9 are
preferably H, and R.sub.7 is preferably independently selected from
H, OH, and OR.
[0038] A second aspect of the present invention provides a method
of preparing a compound of formula I as described in the first
aspect of the invention wherein XR.sub.11.noteq.OH, from a
corresponding compound Ia which is a compound of formula I in which
XR.sub.11.dbd.OH. A product in which XR.sub.11 is OR may be
prepared by direct etherification of compound Ia. A product in
which X is S may be prepared by treatment of compound Ia with
R.sub.11SH and a catalyst (generally a Lewis acid such as
Al.sub.2O.sub.3) A product in which X is NH may be prepared by
treatment of compound Ia with an amine R.sub.11NH and a catalyst
(generally a Lewis acid such as Al.sub.2O.sub.3).
[0039] A third aspect of the present invention provides a method of
preparing a compound of formula Ia as described in the second
aspect of the invention, by the oxidation of a compound of formula
IVa: 7
[0040] wherein the substituents of the compound of formula IVa are
the same as for the compound of formula Ia to be prepared. (For
preparation of dimeric compounds, the monomers linked through C8 by
-T-R'-T- are both of formula IVa. Similar comments apply to other
intermediates in dimer synthesis.) The preferred oxidation method
is Swern oxidation.
[0041] A fourth aspect of the present invention provides a method
of preparing a compound of formula IVa as described in the third
aspect of the invention, by reacting a compound of formula Va:
8
[0042] with a compound of formula VI:
Y--R.sub.10 (VI)
[0043] wherein the substituents of the compounds of formulae Va and
VI are the same as for the compound of formula IVa to be prepared,
and Y is a halogen atom.
[0044] If the therapeutically removable nitrogen protecting group
is to be of formula II, then it is preferred that the compound of
formula VI is a haloformate of the formula VIa: 9
[0045] wherein the substituents are as defined for the group of
formula II, and Y is a halogen atom.
[0046] If the therapeutically removable nitrogen group is to be of
formula XI, then it is preferred that the compound of formula VI is
a haloformate of the formula VIb: 10
[0047] where Y is a halogen atom, and R and n are as defined for
formula XI.
[0048] A fifth aspect of the present invention provides an
alternative synthesis of a compound of formula IVa as described in
the third aspect of the invention, by reacting a compound of
formula VII: 11
[0049] with a compound of formula VI:
Y--R.sub.10 (VI)
[0050] to form a compound of formula VIII: 12
[0051] and then reacting the compound of formula VIII with a
compound of formula IXa: 13
[0052] (e.g. by means of (COCl).sub.2), wherein the substituents
for compounds of formulae VI, VII, VIII and IXa are the same as for
the compound of formula IVa to be prepared, where Y is a halogen
atom.
[0053] A sixth aspect of the present invention provides a method of
preparing a compound of formula Ia as described in the second
aspect of the invention, by the unmasking of a compound of formula
IVb: 14
[0054] wherein the substituents of the compound of formula IVb are
the same as for the compound of formula Ia to be prepared, and Q is
either S or O and R.sup.(IV) are independently selected from Me or
Et or may together form --(CH.sub.2).sub.q-- where q is 2 or 3.
(For preparation of dimeric compounds, the monomers linked through
C8 by -T-R'-T- are both of formula IVb. Similar comments apply to
other intermediates in dimer synthesis.) The preferred unmasking
method when Q=S is mercury-mediated unmasking. Unmasking when Q=O
is preferably carried out by the use of acid conditions, e.g. TFA,
methanol and water or palladium catalysis.
[0055] A seventh aspect of the present invention provides a method
of preparing a compound of formula IVb as described in the sixth
aspect of the invention, by reacting a compound of formula Vb:
15
[0056] with a compound of formula VI:
Y--R.sub.10 (VI)
[0057] wherein the substituents of the compounds of formulae Vb and
VI are the same as for the compound of formula IVb to be prepared,
and Y is a halogen atom.
[0058] If the therapeutically removable nitrogen protecting group
is to be of formula II, then it is preferred that the compound of
formula VI is a haloformate of the formula VIa: 16
[0059] wherein the substituents are as defined for the group of
formula II, and Y is a halogen atom.
[0060] An eighth aspect of the present invention provides an
alternative synthesis of a compound of formula IVb as described in
the second aspect of the invention, by reacting a compound of
formula VII: 17
[0061] with a compound of formula VI:
Y--R.sub.10 (VI)
[0062] to form a compound of formula VIII: 18
[0063] and then reacting the compound of formula VIII with a
compound of formula IXb: 19
[0064] (e.g. by means of (COCl).sub.2), wherein the substituents
for compounds of formulae VI, VII, VIII and IXb are the same as for
the compound of formula IVb to be prepared, where Y is a halogen
atom.
[0065] A ninth aspect of the present invention provides a method of
making a compound of formula X: 20
[0066] by cleavage of the therapeutically removable protecting
group R.sub.10 of a compound of formula I as described in the first
aspect of the invention, wherein the substituent groups of the
compound of formula X are the same as the substituent groups of the
compound I used.
[0067] A tenth aspect of the present invention provides a use of a
compound of formula I, wherein the therapeutically removable
nitrogen protecting group (R.sub.10)is enzyme labile, in
conjunction with an appropriate enzyme in methods of ADEPT or GDEPT
therapy. If the enzyme labile group is susceptible to
nitroreductase, then compounds of formula I, may be used in
conjunction with nitroreductase enzymes (for example, those
isolated from E. coli) in methods of ADEPT and GDEPT therapy.
[0068] An eleventh aspect of the present invention provides a use
of a compound of formula I, wherein the therapeutically removable
nitrogen protecting group (R.sub.10) is photolabile, in conjunction
with light of wavelengths between 250 and 400 or 550 nm in methods
of PDT.
[0069] A twelfth aspect of the invention provides a use for a
compound of formula I, where the therapeutically removable nitrogen
protecting group (R.sub.10) is labile by conditions occurring
naturally at specific localised sites in the patient in therapy.
Suitable compounds of formula I may be those susceptible to a
nitroreductase enzyme when used to treat hypoxic tumour cells, or
those susceptible to enzymes which are naturally occurring at
specific localised sites, such as glutathione transferase.
[0070] The drug produced by the cleavage of the therapeutically
removable nitrogen protecting group, in either the tenth or
eleventh or twelfth aspect of the invention, may be used for
treating cancers or other site-specific diseases where a local
increase of toxicity is beneficial to the patient. Cancers that may
be treated are solid cancers including ovarian, colonic cancer,
renal, breast and bowel CNS, melanoma, as well as leukemias. Such
drugs may also be suitable for treating bacterial, viral or
parasitic infections by exploiting a unique enzyme produced at the
site of the infection which is not natural to the host, or by
exploiting an elevation in the amount of an enzyme which does
naturally occur in the host.
[0071] A thirteenth aspect of the present invention is a
pharmaceutical composition comprising a compound of formula I as
described in the first aspect of the invention. Pharmaceutical
compositions according to the present invention, and for use in
accordance with the present invention, may comprise, in addition to
the active ingredient, i.e. a compound of formula I, a
pharmaceutically acceptable excipient, carrier, buffer, stabiliser
or other materials well known to those skilled in the art. Such
materials should be non-toxic and should not interfere with the
efficacy of the active ingredient. The precise nature of the
carrier or other material will depend on the route of
administration, which may be oral, or by injection, e.g. cutaneous,
subcutaneous or intravenous.
[0072] Pharmaceutical compositions for oral administration may be
in tablet, capsule, powder or liquid form. A tablet may comprise a
solid carrier or an adjuvant. Liquid pharmaceutical compositions
generally comprise a liquid carrier such as water, petroleum,
animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included. Capsules may comprise a solid
carrier such as gelatin.
[0073] For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active ingredient will be
in the form of a parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and stability. Those
of relevant skill in the art are well able to prepare suitable
solutions using, for example, isotonic vehicles such as Sodium
Chloride Injection, Ringer's Solution, or Lactated Ringer's
Injection. Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
[0074] A fourteenth aspect of the present invention provides the
use of a compound of formula I as described in the first aspect of
the invention, to prepare a medicament for the treatment of
neoplastic disease or other site-specific diseases where a local
increase of toxicity is beneficial to the patient. The compound of
formula I may be provided together with a pharmaceutically
acceptable carrier or diluent. The preparation of a medicament is
described in relation to the thirteenth aspect of the
invention.
[0075] Aspects of the invention will now be further described with
reference to the accompanying drawings in which:
[0076] FIG. 1 is a synthesis scheme according to the present
invention;
[0077] FIGS. 2a & 2b are a synthesis scheme for dimers
according to the present invention;
[0078] FIG. 3 is a synthesis scheme showing an alternative
cyclisation, for use in the present invention;
[0079] FIG. 4 is a graph illustrating the cytotoxicity results for
prodrug compound 7 (see example 1), both before and after
activation with nitroreductase/NADH in SW1116 cells and LS174T
cells;
[0080] FIG. 5 is a graph illustrating the percentage of compound 11
(see example 3) cleaved by UVA (365 nm) exposure over a 3 hour time
course;
[0081] FIG. 6 is a graph illustrating the in vitro cytotoxicity
(IC.sub.50; .mu.M) of benzyl DC-81 and compound 11, with varying
irradiation times, against human chronic myeloid leukaemia K562
cells in DMF at 1 mM initial drug concentration;
[0082] FIG. 7 is a graph illustrating the in vitro cytotoxicity
(IC.sub.50; .mu.M) of benzyl DC-81 and compound 11, with varying
irradiation times, against human chronic myeloid leukaemia K562
cells in DMF at 10 mM initial drug concentration;
[0083] FIG. 8 is a graph illustrating the in vitro cytotoxicity
(IC.sub.50; .mu.M) of benzyl DC81 and compound 11, with varying
irradiation times, against human chronic myeloid leukaemia K562
cells in methanol at a 1 mM initial drug concentration;
[0084] FIG. 9 is a graph illustrating the in vitro cytotoxicity
(IC.sub.50; .mu.M) of DSB-120 and compound 32, with varying
irradiation times, against human chronic myeloid leukaemia K562
cells in methanol at a 1 mM initial drug concentration.
PREFERRED SYNTHETIC STRATEGIES
[0085] A key step in a preferred route to compounds of formula I is
a cyclisation to produce the B-ring, involving generation of an
aldehyde (or functional equivalent thereof) at what will be the
11-position, and attack thereon by the 10-nitrogen: 21
[0086] The "masked aldehyde", --CPQ, may be an acetal or thioacetal
(e.g. P=Q=SEt or OMe), which may be cyclic, in which case the
cyclisation involves unmasking. Alternatively, the masked aldehyde
may be an aldehyde precursor, such as an alcohol, --CHOH, in which
case the reaction involves oxidation, e.g. by means of TPAP or DMSO
(Swern oxidation).
[0087] The masked aldehyde compound can be produced by condensing a
corresponding 2-substituted pyrrolidine with a 2-nitrobenzoic acid:
22
[0088] The nitro group can then be reduced to --NH.sub.2 and
protected by reaction with a suitable agent, e.g. a chloroformate,
which provides the therapeutically removable nitrogen protecting
group R.sub.10 in the compound of formula I.
[0089] A process involving the oxidation-cyclization procedure is
illustrated in FIG. 1 (an alternative type of cyclisation will be
described later with reference to FIG. 3). If R.sub.11 is other
than hydrogen, the compound of formula I, may be prepared by direct
etherification of the alcohol Ia. If X.dbd.s, and not O, the
alcohol Ia, or the OR derivative, can be treated with H.sub.2S, and
a catalyst such as Al.sub.2O.sub.3, or by the addition of a thiol,
e.g. EtSH. If X is NH then treatment of the alcohol Ia with the
appropriate amine yields the desired compound of formula I.
[0090] Exposure of the alcohol (IVa) (in which the pro-10-nitrogen
is generally protected as an amide carbamate) to
tetrapropylammonium perruthenate (TPAP)/N-methylmorpholine N-oxide
(NMO) over A4 sieves results in oxidation accompanied by
spontaneous B-ring closure to afford the desired product. The
TPAP/NMO oxidation procedure is found to be particularly convenient
for small scale reactions while the use of DMSO-based oxidation
methods, particularly Swern oxidation, proves superior for larger
scale work (e.g. >1 g).
[0091] The uncyclized alcohol (IVa) may be prepared by the addition
of a nitrogen protection reagent of formula VI, which is preferably
a chloroformate or acid chloride, to the amino alcohol (Va),
generally in solution, generally in the presence of a base such as
pyridine (preferably 2 equivalents) at a moderate temperature (e.g.
at 0.degree. C.). Under these conditions little or no O-acylation
is usually observed.
[0092] The key amino alcohol (Va) may be prepared by reduction of
the corresponding nitro compound (XIa), by choosing a method which
will leave the rest of the molecule intact. Treatment of XIa with
tin (II) chloride in a suitable solvent, e.g. refluxing methanol,
generally affords, after the removal of the tin salts, the desired
product in high yield.
[0093] Exposure of XIa to hydrazine/Raney nickel (or hydrogentation
with a catalyst) avoids the production of tin salts and may result
in a higher yield of Va, although this method is less compatible
with the range of possible C and A-ring substituents. For instance,
if there is C-ring unsaturation (either in the ring itself, or at
R.sub.2 or R.sub.3), this technique may be unsuitable.
[0094] The nitro compound of formula XIa may be prepared by
coupling the appropriate o-nitrobenzoyl chloride to a compound of
formula IXa, e.g. in the presence of K.sub.2CO.sub.3 at -25.degree.
C. under a N.sub.2 atmosphere. The o-nitrobenzoyl chloride is
synthesised from the o-nitro benzoic acid of formula XII--many of
these are commercially available, and the synthesis of some
examples has been reported by Althuis (Althuis, T. H. and Hess, H
-J, 1977, Synthesis and Identification of the Major Metabolites of
Prazosin Formed in Dog and Rat, Journal of Medicinal Chemistry 20,
1: 146-266) Compounds of formula IXa can be readily prepared, for
example by olefination of the ketone derived from L-trans-hydroxy
proline. The ketone intermediate can also be exploited by
conversion to the enol triflate for use in palladium mediated
coupling reactions such as the Heck, Stille and Suzuki
reactions.
[0095] Dimer Synthesis (FIG. 2)
[0096] PBD dimers may be synthesized using the strategy developed
for the synthesis of the protected PBD monomers (FIG. 2a). FIG. 2
also shows a synthesis route where the dimer linkage is of the
formula --O--(CH.sub.2).sub.n--O--. The step of dimer formation is
normally carried out to form a bis(nitro acid) XII' (FIG. 2b).
[0097] The bis(nitro acid) XII' may be obtained by nitrating (e.g.
using 70% nitric acid) the bis(carboxylic acid). This can be
synthesised by alkylation of two equivalents of the relevant
benzoic acid with the appropriate diiodoalkane under basic
conditions (Route 2a). Many benzoic acids are commercially
available and others can be synthesised by conventional
methods.
[0098] An alternative synthesis of the bis(nitro acid) involves
oxidation of the bis(nitro aldehyde), e.g. with potassium
permanganate. This can be obtained in turn by direct nitration of
the bis(aldehyde), e.g. with 70% HNO.sub.3. Finally, the
bis(aldehyde) can be obtained via Mitsunobu etherification of two
equivalents of the benzoic aldehyde with the appropriate alkanediol
(Route 2b).
[0099] Alternative Cyclisation (FIG. 3)
[0100] In FIGS. 1 and 2, the final or penultimate step is an
oxidative cyclisation. An alternative, using thioacetal coupling
unmasking, is shown in FIG. 3 (which shows it applied to a dimer,
with a dimer linkage of formula --O--(CH.sub.2).sub.n--O--).
Mercury-mediated unmasking causes cyclisation to the desired
compound (Ia').
[0101] The thioacetal compound may be prepared as shown in FIG. 3:
the thioacetal protected C-ring [prepared via a literature method:
Langley, D. R. & Thurston, D. E., J. Organic Chemistry, 52,
91-97 (1987)] is coupled to the bis(nitro carboxylic acid) core
using a literature procedure. The resulting nitro compound cannot
be reduced by hydrogenation, because of the presence of the
thioacetal group, so the tin(II) chloride method is used to afford
the bis(amine). This is then N-protected, e.g., by reaction with a
chloroformate or acid chloride, such as
p-nitrobenzylchloroformate.
[0102] An alternative to thioacetal coupling is the use of acetal
coupling. The method is the same as that illustrated in FIG. 3, but
with the thioacetal group replaced by an acetal group (e.g.
--CH(OMe).sub.2). Acid or palladium-mediated unmasking is the
preferred method of unmasking to cause cyclisation to the desired
compound of formula Ia or I'a.
[0103] GDEPT
[0104] Vector Systems
[0105] In general, the vector for use in GDEPT therapies may be any
suitable DNA or RNA vectors.
[0106] Suitable non-viral vectors include cationic liposomes and
polymers. Suitable viral vectors include those which are based upon
a retrovirus. Such vectors are widely available in the art. Huber
et al. (ibid) report the use of amphotropic retroviruses for the
transformation of hepatoma, breast, colon or skin cells. Culver et
al. (Science (1992) 256; 1550-1552) also describe the use of
retroviral vectors in GDEPT. Such vectors or vectors derived from
them may also be used. Other retroviruses may also be used to make
vectors suitable for use in the present invention. Such
retroviruses include Rous sarcoma virus (RSV).
[0107] Englehardt et al. (Nature Genetics (1993) 4; 27-34) describe
the use of adenovirus-based vectors in the delivery of the cystic
fibrosis transmembrane conductance product (CFTR) into cells, and
such adenovirus-based vectors may also be used. Vectors utilising
adenovirus promoter and other control sequences may be of use in
delivering a system according to the invention to cells in the
lung, and hence useful in treating lung tumours.
[0108] Other vector systems including vectors based on the Molony
murine leukaemia virus are known (Ram, Z et al., Cancer Research
(1993) 53; 83-88; Dalton & Treisman, Cell (1992) 68; 597-612).
These vectors contain the Murine Leukaemia virus (MLV) enhancer
cloned upstream at a .beta.-globin minimal promoter. The
.beta.-globin 5' untranslated region up to the initiation codon ATG
is supplied to direct efficient translation of the enzyme.
[0109] Suitable promoters which may be used in vectors described
above, include MLV, CMV, RSV and adenovirus promoters. Preferred
adenovirus promoters are the adenovirus early gene promoters.
Strong mammalian promoters may also be suitable. An example of such
a promoter is the EF-1.alpha. promoter which may be obtained by
reference to Mizushima and Nagata ((1990), Nucl. Acids Res. 18;
5322). Variants of such promoters retaining substantially similar
transcriptional activities may also be used.
[0110] Other suitable promoters include tissue specific promoters,
and promoters activated by small molecules, hypoxia or X-rays.
[0111] If nitroreductase is the enzyme of choice for the activation
of compounds of formula I, then preferably the enzyme is a
non-mammalian nitroreductase such as a bacterial nitroreductase. An
E. coli nitroreductase as disclosed in WO93/08288 is particularly
preferred. The enzyme may be modified by standard recombinant DNA
techniques, e.g. by cloning the enzyme, determining its gene
sequence and altering the gene sequence by methods such as
truncation, substitution, deletion or insertion of sequences for
example by site-directed mutagenesis. Reference may be made to
"Molecular Cloning" by Sambrook et al. (1989, Cold Spring Harbor)
for discussion of standard recombinant DNA techniques. The
modification made may be any which still leaves the enzyme with the
ability to reduce the nitro group in suitable compounds of formula
I, but alters other properties of the enzyme, for example its rate
of reaction or selectivity.
[0112] In addition, small truncations in the N- and/or C-terminal
sequence may occur as a result of the manipulations required to
produce a vector in which a nucleic acid sequence encoding the
enzyme is linked to the various other vector sequences.
[0113] For information on the use of penicillin V/G amidase, and
.beta.-lactamase in GDEPT, see Jungheim, L. N. and Shepherd, T. A,
Design of Antitumour Prodrugs: Substrates for Antibody Targeted
Enzymes, Am. Chem. Soc. Chem. Rev., 1994, Vol 94, No. 6,
1553-1566.
[0114] ADEPT
[0115] For applications in ADEPT systems, an antibody directed
against a tumour specific marker is linked to the relevant enzyme,
which may be modified as described above The antibody may be
monoclonal or polyclonal. For the purposes of the present
invention, the term "antibody", unless specified to the contrary,
includes fragments of whole antibodies which retain their binding
activity for a tumour target antigen. Such fragments include Fv,
F(ab') and F(ab').sub.2 fragments, as well as single chain
antibodies. Furthermore, the antibodies and fragments thereof may
be humanised antibodies, e.g. as described in EP-A-239400.
[0116] The antibodies may be produced by conventional hybridoma
techniques or, in the case of modified antibodies or fragments, by
recombinant DNA technology, e.g. by the expression in a suitable
host vector of a DNA construct encoding the modified antibody or
fragment operably linked to a promoter. Suitable host cells include
bacterial (eg. E. coli), yeast, insect and mammalian cells. When
the antibody is produced by such recombinant techniques the enzyme
may be produced by linking a nucleic acid sequence encoding the
enzyme (optionally modified as described above) to the 3' or 5' end
of the sequence of the construct encoding the antibody or fragment
thereof.
[0117] PDT
[0118] The activation process in PDT can be highly site specific.
The direction of a laser beam can be controlled with great
precision, and the beam diameter can be reduced to a width far
below that of a single cell. Therefore, it can act upon a very
limited area, minimising damage to neighbouring tissue.
[0119] Ultra-violet light is sufficient to break a range of
chemical bonds since the energy spectrum for bond breakage for the
majority of organic molecules lies between 250 and 420 kJ/mol, and,
for example, 350 nm is equivalent to 340 kJ/mol. For example, a
broad range of light-mediated deprotection reactions have been
demonstrated including the photochemical deprotection of amino
acids, peptides and polysaccharides from their CBZ and
o-nitrobenzyl and 4,5-dimethoxy-2-nitrobenzyl carbamate forms at
wavelengths longer than 350 nm, (Pillai, R. V. N., Photoremovable
protecting groups in organic chemistry, Synthesis (1980), 1-26),
(Bayley, H., Gasparro, F. and Edelson, R., Photoactivatible drugs,
TIPS (1987) 8, 138-143, (Star, W. M., Light delivery and light
dosimetry for photodynamic therapy, Laser in Medical Science (1990)
5, 107-113. On the other hand, highly reactive and thus cytotoxic
species can also result from relatively low energy activations. For
example, a reactive excited state of molecular oxygen, the singlet
state, differs in only 90 kJ/mol from its ground triplet state.
However, this enables sufficient concentrations of the toxic
species to be formed by those sensitisers which absorb at
wavelengths longer than 600 nm, (Carruth, J. A. S., Clinical
applications for photodynamic therapy, J Photochem Photobiol (1991)
9, 396-397).
[0120] The main limitation of this approach arises from the physics
of light itself and its interaction with human tissue. The ability
of light to penetrate tissue has been found to be
wavelength-dependent. Penetrating ability increases with increasing
wavelength but limitations arise due to light scattering and
reflection. In biological tissues the scattering coefficient, for
example of red light, is much greater than the absorption
coefficient, (Carruth, J. A. S., Clinical applications for
photodynamic therapy, J Photochem Photobiol (1991) 9, 396-397),
(Kennedy, J. C. And Pottier, R. H, Endogenous protoporphyrin IX, a
clinical useful photosensitiser for photodynamic therapy, J
Photochem Photobiol (1992) 14, 275-292). As a result, photons
entering the tissue are scattered several times before they are
either absorbed or diffused. Although this might be expected to
increase the energy delivered to certain areas, internal reflection
results in an exponential decrease of energy flux with increasing
distance from the tissue-air interface. These limitations have been
partially overcome in the treatment of relatively bulky tumours or
when deeper penetration is necessary by the use of multiple
interstitial optical fibres.
[0121] Several tumour types have been identified as potential
targets for PDT. They include head and neck tumours, carcinomas of
the bronchus, malignant brain tumours, superficial tumours of the
bladder and vascular disease, which have all shown promising
responses in the clinic, (Regula, J., Mac Roberts, A. J, Gorchein,
A., Buonaccorsi, Thorpe, S. M., Spencer, G. M., Hartfield, A. R. W.
and Bown, S. G., Photosensitisation and photodynamic therapy of
oesophageal, duodenal and colorectal tumours using 5-aminoleavulic
acid induced protoporphyrin IX-a pilot study, Gut (1995) 36,
67-75).
[0122] The technique of PDT as discussed above can be used in
combination with appropriate compounds of formula I when the
therapeutically removable nitrogen protecting group is photolabile.
The preferred wavelength of UV light used is 250 to 400 or 550
nm.
[0123] Applications of the Invention
[0124] Compounds of the invention can be used in vitro or in vivo
for a range of applications. For example, a number of vector
systems for tie expression of nitroreductase in a cell have been
developed. The further development of such systems (e.g. the
development of promoters suitable for specific cell types) requires
suitable candidate prodrugs capable of killing cells when activated
by nitroreductase. Prodrug compounds of formula I susceptible to
nitroreductase may be used in such model systems.
[0125] The model systems may be in vitro model systems or in vivo
xenograft model systems comprising for example human tumour cells
implanted in nude mice. Compounds of formula I susceptible to
different enzymes may be used in similar systems which have been
appropriately modified.
[0126] Compounds of formula I which are not activatable by an
enzyme may be tested in vitro with other suitable forms of
activation against panels of different tumour cell types to
determine efficacy against such tumour cells The efficacy of
compounds of the invention against a range of tumour cell types may
be used as points of reference for the development of further
antitumour compounds. Compounds of formula I may also be tested in
combination with additional anti-cancer compounds to determine
potential combination drug systems, for example combinations which
are synergistic.
[0127] Compounds of formula I may also be used in a method of
treatment of the human or animal body. Such treatment includes a
method of treating the growth of neoplastic cells in a patient with
neoplastic disease which comprises administering to a patient in
need of treatment compounds of formula I as part of an ADEPT, GDEPT
or PDT system or treatment with compounds of formula I alone, where
neoplastic diseases include leukaemia and solid tumours such as
ovarian, colonic, lung, renal, breast, bowel, CNS and melanomas.
The treatment can also be the treatment of other site-specific
diseases where local increase in toxicity is beneficial to the
patient.
[0128] It will be understood that where treatment of tumours is
concerned, treatment includes any measure taken by the physician to
alleviate the effect of the tumour on a patient. Thus, although
complete remission of the tumour is a desirable goal, effective
treatment will also include any measures capable of achieving
partial remission of the tumour as well as a slowing down in the
rate of growth of a tumour including its metastases. Such measures
can be effective in prolonging and/or enhancing the quality of life
and relieving the symptoms of the disease.
[0129] Therapies
[0130] Methods of ADEPT and GDEPT will now be described with
reference to nitroreductase, although other enzymes as previously
described could be substituted with appropriate modifications to
the methods described.
[0131] The basis of PDT has been described above, but the
information on the administration of products below also applies to
this type of therapy. This information is also relevant to
therapies where the prodrug is activated by conditions naturally
occurring within the body (e.g. hypoxia, elevated level of GST--see
discussion of NPEPT above).
[0132] ADEPT Therapy
[0133] The antibody/enzyme conjugate for ADEPT can be administered
simultaneously but it is often found preferable, in clinical
practice, to administer the enzyme/antibody conjugate before the
prodrug, e.g. up to 72 hours or even 1 week before, in order to
give the enzyme/antibody conjugate an opportunity to localise in
the region of the tumour target. By operating in this way, when the
prodrug is administered, conversion of the prodrug to the cytotoxic
agent tends to be confined to the regions where the enzyme/agent
conjugate is localised, i.e. the region of the target tumour. In
this way, the premature release of the compound produced by the
action of the nitroreductase on the prodrugs of the present
invention is minimised.
[0134] In ADEPT the degree of localisation of the enzyme/agent
conjugate (in terms of the ratio of localized to freely circulating
active conjugate) can be further enhanced using the clearance
and/or inactivation systems described in WO89/10140. This involves,
usually following administration of the conjugate and before
administration of the prodrug, the administration of a component (a
"second component") which is able to bind to part of the conjugate
so as to inactivate the enzyme in the blood and/or accelerate the
clearance of the conjugate from the blood. Such a component may
include an antibody to the enzyme component of the system which is
capable of inactivating the enzyme.
[0135] The second component may be linked to a macromolecule such
as dextran, a liposome, albumin, macroglobulin or a blood group o
erythrocyte so that the second component is restrained from leaving
the vascular compartment. In addition, or as an alternative, the
second component may include a sufficient number of covalently
bound galactose residues, or residues of other sugars such as
lactose or mannose, so that it can bind the conjugate in plasma but
be removed together with the conjugate from plasma by receptors for
galactose or other sugars in the liver. The second component should
be designed for use and administered such that it will not, to any
appreciable extent, enter the extravascular space of the tumour
where it could inactivate localised conjugate prior to and during
administration of the prodrug.
[0136] In ADEPT systems, the dose of the prodrug and conjugate will
ultimately be at the discretion of the physician, who will take
into account such factors as the age, weight and condition of the
patient. Suitable doses of prodrug and conjugate are given in
Bagshawe et al. Antibody, Immunoconjugates, and
Radiopharmaceuticals (1991), 4, 915-922. A suitable dose of
conjugate may be from 500 to 200,000 enzyme units/m.sup.2 (e.g.
20,000 enzyme units/m.sup.2) and a suitable dose of prodrug may be
from about 0.1 to 200 mg/Kg, preferably from about 10 to about 100
mg/Kg per patient per day.
[0137] In order to secure maximum concentration of the conjugate at
the site of desired treatment, it is normally desirable to space
apart administration of the two components by at least 4 hours. The
exact regime will be influenced by various factors including the
nature of the tumour to be targeted and the nature of the prodrug,
but usually there will be an adequate concentration of the
conjugate at the site of desired treatment within 48 hours.
[0138] The ADEPT system when used with nitroreductase also
preferably comprises a suitable co-factor for the enzyme. Suitable
co-factors may include a riboside or ribotide of nicotinic acid or
nicotinamide.
[0139] The antibody/enzyme conjugate may be administered by any
suitable route usually used in ADEPT therapy This includes
parenteral administration of the antibody in a manner and in
formulations similar to that described below.
[0140] GDEPT Therapy
[0141] For use of the vectors in therapy, the vectors will usually
be packaged into viral particles and the particles delivered to the
site of the tumour, as described in for example Ram et al. (supra).
The viral particles may be modified to include an antibody,
fragment thereof (including a single chain) or tumour-directed
ligand to enhance targeting of the tumour. Alternatively the
vectors may be packaged into liposomes. The liposomes may be
targeted to a particular tumour. This can be achieved by attaching
a tumour-directed antibody to the liposome. Viral particles may
also be incorporated into liposomes. The particles may be delivered
to the tumour by any suitable means at the disposal of the
physician. Preferably, the viral particles will be capable of
selectively infecting the tumour cells. By "selectively infecting"
it is meant that the viral particles will primarily infect tumour
cells and that the proportion of non-tumour cells infected is such
that the damage to non-tumour cells by administration of a prodrug
will be acceptably low, given the nature of the disease being
treated. Ultimately, this will be determined by the physician.
[0142] One suitable route of administration is by injection of the
particles in a sterile solution. Viruses, for example isolated from
packaging cell lines, may also be administered by regional
perfusion or direct intratumoral injection, or direct injection
into a body cavity (intracaviterial administration), for example by
intra-peritoneal injection.
[0143] The exact dosage regime for GDEPT will, of course, need to
be determined by individual clinicians for individual patients and
this, in turn, will be controlled by the exact nature of the
prodrug and the cytotoxic agent to be released from the prodrug.
However, some general guidance can be given. Chemotherapy of this
type will normally involve parenteral administration of modified
virus, and administration by the intravenous route is frequently
found to be the most practical.
[0144] In GDEPT systems the amount of virus or other vector
delivered will be such as to provide a similar cellular
concentration of enzyme as in the ADEPT system mentioned above.
Typically, the vector will be administered to the patient and then
the uptake of the vector by transfected or infected (in the case of
viral vectors) cells monitored, for example by recovery and
analysis of a biopsy sample of targeted tissue. This may be
determined by clinical trials which involve administering a range
of trial doses to a patient and measuring the degree of infection
or transfection of a target cell or tumour. The amount of prodrug
required will be similar to or greater than that for ADEPT
systems.
[0145] In using a GDEPT system the prodrug will usually be
administered following administration of the vector encoding an
enzyme. Suitable doses of prodrug are from about 0.1 to 200 mg/Kg,
preferably from about 10 to about 100 mg/Kg per patient per
day.
[0146] Administration of Prodrugs
[0147] While it is possible for the compounds of formula I to be
administered alone, it is preferable to present them as
pharmaceutical formulations, for use with any of the above methods.
The formulations comprise the compounds, together with one or more
acceptable carriers thereof and optionally other therapeutic
ingredients, or diluents. The carrier or carriers must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the
recipients thereof, for example, liposomes. Suitable liposomes
include, for example, those comprising the positively charged lipid
(N[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA), those
comprising dioleoylphosphatidylethanolamine (DOPE), and those
comprising
3.beta.[N-(n'N'-dimethylaminoethane)-carbamoyl]cholesterol
(DC-Chol).
[0148] Formulations suitable for parenteral or intramuscular
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats,
bacteriocidal Antibiotics and solutes which render the formulation
isotonic with the blood of the intended recipient; and aqueous and
non-aqueous sterile suspensions which may include suspending agents
and thickening agents, and liposomes or other microparticulate
systems which are designed to target the compound to blood
components or one or more organs. The formulations may be presented
in unit-dose or multi-dose containers, for example sealed ampoules
and vials, and may be stored in a freeze-dried (lyophilized)
condition requiring only the addition of a sterile liquid carrier,
for example Water for Injection, immediately prior to use.
Injection solutions and suspensions may be prepared
extemporaneously from sterile powders, granules and tablets of the
kind previously described.
[0149] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations may include other
agents conventional in the art having regard to the type of
formulation in question. Of the possible formulations, sterile
pyrogen-free aqueous and non-aqueous solutions are preferred.
[0150] The doses may be administered sequentially, eg. at hourly,
daily, weekly or monthly intervals, or in response to a specific
need of a patient. Preferred routes of administration are oral
delivery and injection, typically parenteral or intramuscular
injection or intratumoural injection. For methods of PDT dermal or
topical administration may be preferred, e.g. subcutaneous
injection or creams and ointments, and such methods of
administration are well known.
[0151] The exact dosage regime will, of course, need to be
determined by individual clinicians for individual patients and
this, in turn, will be controlled by the exact nature of the
compound of formula I, but some general guidance can be given
Typical dosage ranges generally will be those described above which
may be administered in single or multiple doses. Other doses may be
used according to the condition of the patient and other factors at
the discretion of the physician.
EXAMPLES
[0152] Embodiments of the present invention will now be described
in detail by way of example.
[0153] General Experimental Methods
[0154] Melting points (mp) were determined on a Gallenkamp P1384
digital melting point apparatus and are uncorrected. Infrared (IR)
spectra were recorded using a Perkin-Elmer 297 spectrophotometer.
.sup.1H- and .sup.13C-NMR spectra were recorded on a Jeol GSX 270
MHZ FT-NMR spectrometer operating at 20.degree. C. .+-.1.degree. C.
Chemical shifts are reported in parts per million (.delta.)
downfield from tetramethylsilane (TMS). Spin multiplicities are
described as: s (singlet), bs (broad singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quartet), p (pentuplet) or m
(multiplet). Mass spectra (MS) were recorded using a Jeol JMS-DX
303 GC Mass Spectrometer (EI mode: 70 eV, source 117-147.degree.
C.). Accurate molecular masses (HRMS) were determined by peak
matching using perfluorokerosene (PFK) as an internal mass marker,
and FAB mass spectra were obtained from a
glycerol/thioglycerol/trifluoroacetic acid (1:1:0.1) matrix with a
source temperature of 180.degree. C. Optical rotations at the Na-D
line were obtained at ambient temperature using a Perkin-Elmer 141
Polarimeter. Flash chromatography was performed using Aldrich flash
chromatography "Silica Gel-60" (E. Merck, 230-400 mesh). Thin-layer
chromatography (TLC) was performed using GF.sub.254 silica gel
(with fluorescent indicator) on glass plates. All solvents and
reagents, unless otherwise stated, were supplied by the Aldrich
Chemical Company Ltd. and were used as supplied without further
purification. Anhydrous solvents were prepared by distillation
under a dry nitrogen atmosphere in the presence of an appropriate
drying agent, and were stored over 4 .ANG. molecular sieves or
sodium wire. Petroleum ether refers to the fraction boiling at
60-80.degree. C.
Example 1
[0155] Synthesis of a nitroreductase-activated benzyl DC-81 Prodrug
for ADEPT (7). 23
[0156] Synthesis of 4-Benzyloxy-3-methoxybenzoic Acid (2)
[0157] A solution of benzyl chloride (24.6 ml, 209 mmol, 1.1 eq.)
in THF (100 ml) was added dropwise at 0.degree. C. over 15 min, to
a mechanically stirred solution of 4-hydroxy-3-methoxybenzoic acid
(vanillic acid, 1) (30 g, 179 mmol) in THF (90 ml) and 2.0M aq.
NaOH (225 ml). The mixture was allowed to warm to room temperature
and then heated under reflux for 48 hours. After cooling, the
mixture was washed with hexane (2.times.100 ml) and the THF was
removed in vacuo. The remaining aqueous phase was acidified to pH 1
with conc. HCl. The resulting precipitate was collected by
filtration, washed with water and dried to afford
4-benzyloxy-3-methoxybenzoic acid (2) as a pale amorphous
solid.
[0158] Yield (after recrystallisation from EtOAc) 31 g (67%); mp
171-172 C; IR (cm.sup.-1) 3700-3200, 2820-3000, 2210, 2140, 1670,
1600, 1580, 1510, 1450, 1430, 1410, 1380, 1340, 1300, 1265, 1220,
1180, 1130-1110, 1030, 1010; .sup.1H NMR (CDCl.sub.3+DMSO-d.sub.6)
.delta.7.60 (d, J=2 Hz, 1H), 7.55 (d, J=2 Hz , 1H), 7.30-7.44 (m,
5H), 6.90 (d, J=8.4 Hz, 1H), 5.19 (s, 2H), 3.91 (s, 3H); .sup.13C
NMR (CDCl.sub.3+DMSO-d.sub.6) .delta.168.3, 151.7, 148.8, 136.3,
128.5, 128.0, 127.2, 123.7, 123.5, 112.5, 112.2, 70.6, 55.9; MS
(EI) (m/z, relative intensity) 258 (M.sup.+, 20), 91 (100), 79 (3),
65 (10), 51 (3); EI-HRMS m/z 258.0949 (calc'd f.degree. r
C.sub.15H.sub.14O.sub.4 m/z 258.0892).
[0159] Synthesis of 4-Benzyloxy-5-methoxy-2-nitrobenzoic Acid
(3)
[0160] Method A: A freshly prepared mixture of SnCl.sub.4 (5 g,
19.5 mmol) and fuming nitric acid (1.67 g, 26.5 mmol) was added
dropwise over 5 minutes to a mechanically stirred solution of 2
(4.35 g, 17 mmol) in DCM at -25.degree. C. (dry ice/carbon
tetrachloride). The mixture was maintained at the same temperature
for a further 15 min, quenched with water (150 ml), and allowed to
warm to room temperature After the organic layer was separated, the
aqueous layer was extracted with EtOAc (2.times.75 ml). The
combined organic phase was dried (MgSO.sub.4) and evaporated in
vacuo to afford a light brown gum which was recrystallised to form
3 as pale yellow needles. Yield=4.16 g (82%)
[0161] Method B: 4-benzyloxy-3-methoxybenzoic acid (8.5 g, 32.9
mmol) was added in small portions over 30 minutes to stirred
solution of 70% nitric acid (100 ml). When addition was complete
the reaction mixture was allowed to warm to 15.degree. C. and
maintained at that temperature for a further 30 min. The reaction
mixture was then poured onto ice and the resultant precipitate was
collected by filtration, washed with ice-cold water and dried to
afford the nitrated product 3 as a yellow powder.
[0162] Yield (after recrystallisation from EtOAc/hexane) 7.8 g
(78%); mp 182-185.degree. C.; IR (cm.sup.-1) 3400-3200, 2820-2930,
1670, 1600, 1580, 1550, 1510, 1450, 1410, 1400, 1370, 1350, 1330,
1265, 1210, 1180, 1160, 1055, 1005; .sup.1H-NMR
(CDCl.sub.3+DMSO-d.sub.6) .delta.7.36-7.45 (m, 6H), 7.16 (s, 1H),
5.19 (s, 2H), 3.95 (s, 3H); .sup.13C-NMR (CDCl.sub.3+DMSO-d.sub.6)
.delta.167.3, 152.7, 149.0, 140.9, 135.2, 128.4, 127.6, 127.3,
111.1, 71.2, 56.5; MS (EI) (m/z, relative intensity) 303 (M.sup.+,
64), 286 (36), 273 (5), 259 (5), 181 (7), 123 (8), 105 (13), 91
(100), 77 (8), 65 (63), 51 (23); EI-HRMS m/z 303.0824 (calc'd for
C.sub.15H.sub.13NO.sub.6 m/z 303.0743).
[0163] Synthesis of (2S)-N-(4-benzyloxy-5-methoxy-2-nitrobenzoyl)
pyrrolidine-methanol (4)
[0164] A catalytic amount of DMF (3 drops) was added to a solution
of 4-benzyloxy-5-methoxy-2-nitrobenzoic acid (3) (3.5 g, 11.55
mmol) and oxalyl chloride (1.75 g, 13.56 mmol, 1.2 eq) in dry
acetonitrile (30 ml). The solution was allowed to stir under
nitrogen overnight. The resulting acid chloride solution was then
added dropwise over 30 minutes to a stirred suspension of
pyrrolidinemethanol (1.168 g, 11.56 mmol, 1 eq) and K.sub.2CO.sub.3
(3.675 g, 26.63 mmol, 2.3 eq) in acetonitrile (80 ml) at
-25.degree. C. under a nitrogen atmosphere. The reaction mixture
was stirred at the same temperature for a further 1 hour and then
allowed to return to room temperature and quenched with water (200
ml). The solution was then extracted with chloroform (4.times.100
ml) and the combined organic phase was washed with 1M HCl
(2.times.50 ml), water (2.times.75 ml), brine (2.times.50 ml) and
water (100 ml), dried (MgSO.sub.4) and the solvent was evaporated
in vacuo to afford a light yellow oil. The product was further
purified by means of flash chromatography (5% MeOH/CHCl.sub.3) to
afford (4) as a pale yellow oil which slowly crystallised on
standing.
[0165] Yield 3.92 g (88%); [.alpha.].sup.20.sub.D: 62.3.degree. (c
0.45, CHCl.sub.3); IR (cm.sup.-1) 3500-3250, 2860, 2910, 1600,
1580, 1520, 1455, 1430, 1370, 1330, 1275, 1220, 1210, 1185, 1150,
1110, 1060, 1025, 1000; .sup.1H-NMR (CDCl.sub.3) .delta.7.76 (s,
1H), 7.34-7.47 (m, 5H), 6.83 (s, 1H), 5.21 (s, 2H), 4.38-4.40 (bs,
1H), 3.98 (s, 3H), 3.80-3.95 (m, 2H), 3.15-3.20 (m, 2H), 2.17-2.21
(m, 2H), 1.69-1.89 (m, 2H); .sup.13C-NMR (CDCl.sub.3) .delta.155.0,
148.1, 136.9, 135.2, 128.8, 128.5, 127.9, 127.6, 109.2, 109.1,
71.3, 66.1, 61.5, 56.8, 49.5, 28.4, 24.4; MS (EI) (m/z, relative
intensity) 386 (M.sup.+, 4), 368 (6), 355 (39), 286 (90), 121 (4),
91 (100), 65 (4); EI-HRMS m/z 386.1531 (calc'd for
C.sub.20H.sub.22N.sub.2O.sub.6 m/z 386.1478).
[0166] Synthesis of (2S)-N-(2-amino-4-benzyloxy-5-methoxybenzoyl)
pyrrolidine-methanol (5)
[0167] Method A: A solution of the nitro compound (4) (1.4 g, 3.62
mmol) and SnCl.sub.2.2H.sub.2O (4.58 g, 20.08 g, 5.5 eq) in
methanol (70 ml) was heated at reflux for 45 min. The solvent was
removed by evaporation in vacuo and the resulting brown oil was
diluted with EtOAc (150 ml), treated with sat. aq. NaHCO.sub.3 (150
ml) and allowed to stir under N.sub.2 overnight. The resulting
suspension was filtered through Celite, the organic phase was then
separated and washed with brine (2.times.100 ml), dried
(MgSO.sub.4) and the excess solvent was finally evaporated in
vacuo. The residual light brown oil was further purified by column
chromatography (5% MeOH/CHCl.sub.3) to afford the amine (5) as a
bright yellow oil. Yield =0.82 g (62%).
[0168] Method B: Hydrazine hydrate (4.53 g, 90.67 ml, 5 eq) was
added dropwise to a solution of the nitro compound 4 (7.0 g, 18.13
mmol) in dry methanol (20 ml) and a catalytic amount of Raney Ni
(0.544 g) over antibumping granules whilst a gently reflux was
maintained. The mixture was heated at reflux for a further 15
minutes when TLC (5% MeOH/CHCl.sub.3) indicated that the reaction
had gone to completion. The Ni catalyst was then removed by
filtration through Celite and the solvent removed by evaporation in
vacuo. The product was further purified by flash chromatography (5%
MeOH/CHCl.sub.3) to afford the amine 5 as a bright yellow unstable
oil which required storage at low temperature.
[0169] Yield 5.2 g (81%); [.alpha.].sup.20.sub.D: -15.4.degree. (c
0.13, CHCl.sub.3); .sup.1H-NMR (CDCl) .delta.7.28-7.42 (m, 5H),
6.76 (s, 1H), 6.26 (s, 1H), 5.09 (s, 2H), 4.37-4.42 (bs, 1H), 3.78
(s, 3H), 3.58-3.77 (m, 4H), 2.04-2.10 (m, 2H), 1.69-1.92 (m, 2H);
.sup.13C-NMR (CDCl.sub.3) .delta.171.8, 151.0, 141.2, 136.5, 128.6,
127.6, 127.1, 112.9, 102.9, 70.6, 66.8, 60.9, 57.1, 51.0, 28.5,
24.9; IR (cm.sup.-1) 3500-3100, 2950, 1680, 1620, 1590, 1510, 1450,
1425, 1400, 1330, 1260, 1230, 1170, 1165, 1130, 1100, 1080, 1055,
1030; MS (EI) (m/z, relative intensity) 356 (M.sup.+, 100), 256
(68), 237 (14), 226 (4), 164 (6), 138 (15), 100 (8), 91 (96), 84
(22), 65 (8); EI-HRMS m/z 356.1785 (calc'd for
C.sub.20H.sub.24N.sub.2O.sub.4 m/z 356.1736).
[0170] Synthesis of
(2S)-N-[2-(p-nitrobenzyloxy)carboxamido-4-benzyloxy-5--
methoxybenzoyl]pyrrolidinemethanol (6)
[0171] A solution of 4-nitrobenzyl chloroformate (0.6 g, 2.8 mmol,
1 eq) in dry DCM (15 ml) was added dropwise over 20 minutes to a
solution of the amine (5) (1 g, 2.8 mmol) and pyridine (0.44 g, 5.6
mmol, 2 eq) in dry DCM (20 ml) at 0.degree. C. under N.sub.2. After
the addition was complete the solution was allowed to stir under
for a further 1.5 hours. The reaction mixture was then washed with
sat. aq. CuSO.sub.4 (2.times.100 ml), water (150 ml), brine (100
ml), dried (MgSO.sub.4) and the excess solvent removed under
vacuum. The resulting oil was subjected to flash chromatography (3%
MeOH/CHCl.sub.3) to afford the protected amine (6) as a pale yellow
oil.
[0172] Yield 1.16 g (77%); IR (cm.sup.-1) 3500-3100, 2950, 1680,
1620, 1590, 1510, 1450, 1425, 1400, 1330, 1260, 1230, 1170, 1165,
1130, 1100, 1080, 1055, 1030; .sup.1H-NMR (CDCl.sub.3, rotamers)
.delta.9.04 (s, 1H), 8.20 (d, J=2 Hz, 2H), 7.81 (s, 1H), 7.28-7.55
(m, 5H), 6.86 (s, 1H), 5.14-5.24 (bs, 4H), 4.39 (bs, 1H), 3.82 (s,
3H), 3.55-3.89 (m, 4H), 2.04-2.14 (m, 2H), 1.70-1.87 (m, 2H);
.sup.13C-NMR (CDCl.sub.3) .delta.170.7, 153.2, 150.3, 147.5, 143.6,
136.1, 128.7-127.7, 123.7, 111.5, 106.0, 70.6, 65.2, 60.9, 56.5,
51.7, 28.2, 25.1; MS (FAB) (m/z, relative intensity) 535 (MH.sup.+,
4), 435 (2), 356 (3), 256 (9), 192 (2), 185 (4), 166 (3), 136 (13),
120 (5), 102 (26), 91 (100), 84 (5).
[0173] Synthesis of
(11aS)-8-benzyloxy-7-methoxy-1,2,3,10,11,11a-hexahydro-
-11-hydroxy-10-(p-nitrobenzyloxy) carboxy-5H-pyrrolo[2,
1-c][1,4]-benzodiazepine-5-one (7)
[0174] A solution of the carbamate 6 (0.4 g, 0.75 mmol), NMO (0.131
g, 1.1.2 mmol, 1.5 eq) over 4 .ANG. molecular sieve (0.375 g) in a
solvent mixture of dry DCM:CH.sub.3CN (9:3 ml) was allowed to stir
at room temperature under nitrogen for 15 min. A portion of TPAP
(13 mg) was then added and the solution was allowed to stir for a
further 2 hours. TLC (2% MeOH/CHCl.sub.3) indicated that reaction
incomplete and a further amount of NMO (65 mg, 0.845 mmol, 0.75 eq)
and TPAP (6.5 mg) was added. After 15 min, TLC indicated a complete
loss of starting material. The molecular sieve was removed by
filtration through Celite and the solvent was removed under reduced
pressure. The black residue was purified by column chromatography
(1% MeOH/CHCl.sub.3 followed by EtOAc:Petroleum ether, 95:5) to
afford the cyclised final product 7.
[0175] Yield 0.124 (31%); IR (cm.sup.-1) 3600--3200, 2820-3000,
1710, 1600, 1510, 1450, 1430, 1400, 1370, 1350, 1305, 1270, 1220,
1120-1100, 1050, 1030, 1010; .sup.1H-NMR (CDCl.sub.3, rotamers)
.delta.8.10 (d, J=8.43 Hz, 2H), 7.27-7.53 (m, 6H), 7.20 (d, J=8.4
Hz, 2H), 6.70 (s, 1H), 5.62 (d, J=10.2 Hz , 1H), 5.07 (d, J=10.7
Hz, 4H), 3.94 (s, 3H), 3.52-3.72 (m, 3H), 2.04-2.10 (m, 4H);
.sup.13C-NMR (CDCl.sub.3) .delta.166.83, 149.26, 142.85, 136.03,
128.79-127.17, 126.38, 114.58, 112.53, 111.00, 105.92, 86.23,
71.12, 66.21, 59.90, 56.21, 47.32, 46.42, 28.68, 23.05; MS (FAB)
(m/z, relative intensity) 535 (M.sup.++2, 2), 353 (6), 337 (10),
286 (5), 256 (3), 241 (2), 228 (2), 192 (3), 136 (7), 91 (100);
EI-HRMS m/z 533.1813 (calc'd for C.sub.28H.sub.27N.sub.3O.sub.8 m/z
533.1798).
Example 2
[0176] Synthesis of a Benzyl DC-81 Prodrug for the PGA ADEPT (9)
24
[0177] Synthesis of (2S)-N-[2-phenyl
acetamido-4-benzyloxy-5-methoxybenzoy- l]pyrrolidine methanol
(8)
[0178] A catalytic amount DMF(4 drops) was added to a solution of
phenyl acetic acid (0.42 g, 3.3 mmol, 1.2 eq) and oxalyl chloride
(0.51 g, 3.96 mmol, 1.4 eq) in dry acetonitrile (10 ml) and the
reaction mixture was allowed to stir overnight under nitrogen. The
resulting acid chloride was added dropwise over 20 minutes to a
mixture of the amine (5) (1 g, 2.8 mmol) and K.sub.2CO.sub.3 (0.97
g, 7.02 mmol, 2.5 eq) in dry acetonitrile (60 ml) at -25.degree. C.
under N.sub.2. The reaction mixture was allowed to stir for a
further 2 hours at -25.degree. C. and then allowed to warm to room
temperature. The reaction mixture was diluted with water (200 ml)
and it was extracted with CHCl.sub.3 (4.times.100 ml). The combined
organic phase was washed with 1M HCl (2.times.50 ml), water
(2.times.75 ml) and brine (100 ml) and dried over MgSO.sub.4. The
solvent was removed under vacuum and the residual oil was further
purified by flash chromatography (5% MeOH/CHCl.sub.3) to afford the
phenylacetamide (8) as a pale oil.
[0179] Yield 0.91 g (68%); IR (cm.sup.-1) 3400, 2093, 1660, 1613,
1519, 1495, 1454, 1435, 1401, 1345, 1262, 1218, 1175, 1101, 1028,
1003, 969; .sup.1H-NMR (CDCl.sub.3, rotamers) .delta.9.27 (s, 1H),
7.90 (s, 1H), 7.26-7.48 (m, 10H), 6.77 (s, 1H), 5.11 (s, 2H),
4.19-4.29 (bs, 1H), 3.78 (s, 3H), 3.65 (s, 2H), 3.35-3.60 (m, 4H),
1.81-2.08 (m, 2H), 1.62-1.81 (m, 2H); .sup.13C-NMR (CDCl.sub.3)
.delta.169.8, 149.9, 145.0, 136.2, 134.5, 129.3-127.3, 110.9,
107.8, 70.6, 66.2, 61.2, 56.5, 51.2, 44.8, 28.3, 24.9; MS (EI)
(m/z, relative intensity) 474 (M.sup.+, 7), 374 (44), 284 (7), 256
(9), 228 (3), 166 (9), 105 (14), 102 (25), 91 (100), 84 (5);
EI-HRMS m/z 474.2142 (calc'd for C.sub.28H.sub.30N.sub.2O.sub.5 m/z
474.2155)
[0180] Synthesis of
(11aS)-8-benzyloxy-7-methoxy-1,2,3,11,11a-pentahydro-1-
1-hydroxy-10-phenylacetyl-5H-pyrrolo[2,1-c][1,4]-benzodiazepine-5-one
(9)
[0181] A solution of the phenylacetamide (8) (0.5 g, 1.05 mmol) and
NMO (0.184 g, 1.57 mmol, 1.5 eq) over molecular sieve (0.525 g) in
a solvent system consisting of dry DCM and CH.sub.3CN (9:3 ml) was
allowed to stir for 15 minutes at room temperature under nitrogen.
TPAP (19 mg, 5% molar) was then added and the mixture was stirred
for 1 hour when TLC (5% MeOH/CHCl.sub.3) indicated complete
consumption of starting material. The reaction mixture was filtered
and evaporated in vacuo. The product was further purified by flash
chromatography (2% MeOH/CHCl.sub.3) to afford the protected PBD 9
as an opaque oil.
[0182] Yield 0.15 g (30%); [.alpha.].sup.20.sub.D: +1325.degree. (c
0.04, CHCl.sub.3); IR (cm.sup.-1) 3400, 2956, 2927, 2094, 1631,
1553, 1514, 1454, 1433, 1407, 1379, 1352, 1278, 1219, 1202, 1181,
1136, 1067, 1009, 752; .sup.1H-NMR (CDCl.sub.3, rotamers)
.delta.7.19-7.38 (m, 11H), 6.47 (s, 1H), 5.76 (d, J=10.1 Hz, 1H),
4.87 (d, J=12.1 Hz, 2H), 3.95 (s, 3H), 3.42-3.67 (m, 3H), 2.62 (s,
2H), 1.98-2.11 (m, 4H); .sup.13C-NMR (CDCl.sub.3) .delta.173.56,
166.54, 149.74, 135.81, 134.84, 130.94-126.56, 114.77, 111.45,
84.38, 70.60, 59.87, 56.28, 46.41, 28.54, 22.67; MS (EI) (m/z,
relative intensity) 472 (M.sup.+, 40), 374 (25), 352 (15), 326 (6),
284 (7), 136 (5), 91 (100), 70 (17); EI-HRMS m/z 472.1944 (calc'd
for C.sub.28H.sub.28N.sub.2O.sub.5 m/z 472.1998).
Example 3
[0183] Synthesis of a photolabile Benzyl DC-81 Prodrug (11) 25
[0184] Synthesis of
(2S)-N-[2-(2'-nitro-4',5'-dimethoxybenzyloxy)carboxami-
do-4-benzyloxy-5-methoxybenzoyl]pyrrolidinemethanol (10)
[0185] A solution of 4,5-dimethoxy-2-nitrobenzyl chloroformate
(NVOC--Cl) (0.77 g, 2.8 mmol, 1 eq) in dry DCM (10 ml) was added
dropwise over 15 minutes to a solution of the amine (5) (1 g, 2.8
mmol) and pyridine (0.44 9, 5.6 mmol, 2 eq) in dry DCM at 0.degree.
C. under N.sub.2. The reaction mixture was then stirred at
0.degree. C. for a further 2.5 hours when TLC (3% MeOH/CHCl.sub.3)
indicated completion of the reaction. The solution was then washed
with aq. sat. CuSO.sub.4 (2.times.75 ml), water (2.times.100 ml),
brine (150 ml) and dried over MgSO.sub.4. The excess solvent was
removed under reduced pressure to give the crude product. Further
purification by column chromatography (5% MeOH/CHCl.sub.3) afforded
10 as a yellow oil.
[0186] Yield 1.45 g (87%); [.alpha.].sup.20.sub.D: -175.5.degree.
(c 0.225, CHCl.sub.3); IR (cm.sup.-1) 4330, 4253, 3428, 2924, 2854,
1711, 1626, 1513, 1463, 1377, 1266, 1216, 1173, 761, 722;
.sup.1H-NMR (CDCl.sub.3, rotamers) .delta.8.90 (s, 1H), 7.82 (s,
1H), 7.73 (s, 1H), 7.26-7.47 (m, 6H), 6.85 (s, 1H), 5.58 (d, J=15
Hz, 2H), 5.16 (s, 2H), 4.29-4.39 (bs, 1H), 3.95-3.99 (s, 6H), 3.84
(s, 3H), 3.49-3.72 (m, 4H), 1.86-2.17 (m, 2H), 1.72-1.86 (m, 2H);
.sup.13C-NMR (CDCl.sub.3) .delta.153.7, 153.2, 150.4, 148.1, 144.5,
139.6, 136.1, 128.5, 128.1, 127.8, 127.6, 111.5, 110.8, 109.9,
108.1, 70.7, 66.4, 63.7, 62.7, 61.0, 56.7-56.4, 51.6, 28.3; MS
(FAB) (m/z, relative intensity) 596 (M.sup.++1, 16), 356 (6), 256
(19), 196 (95), 181 (21), 166 (20), 149 (14), 102 (32), 91 (100),
87 (10), 73 (44), 61 (22), 57 (31).
[0187] Synthesis of
(11aS)-8-benzyloxy-7-methoxy-1,2,3,10,11,11a-hexahydro-
-11-hydroxy-10-(2'-nitro-4',5'-dimethoxybenzyloxy)
carboxy-5H-pyrrolo[2,1-- c][1,4]-benzodiazepine-5-one-(11)
[0188] A solution of the NVOC protected amine, 10 (0.4 g, 0.67
mmol) and NMO (0.236 g, 20.17 mmol, 3 eq) over molecular sieve
(0.335 g) in a mixture of dry DCM:CH.sub.3CN (9:3 ml) was allowed
stir for 15 minutes at room temperature under N.sub.2. TPAP (23 mg,
10% molar) was then added to the reaction mixture and it was
allowed to stir for a further 2 hours when the reaction was found
to be complete by TLC (2% MeOH/CHCl.sub.3). The molecular sieve was
removed by filtration through Celite and the resulting solution was
evaporated in vacuo. The resulting dark oil was subjected to flash
chromatography (1% MeOH/CHCl.sub.3) to give the photolabile
protected PBD (11).
[0189] Yield 0.21 g (49%); [.alpha.].sup.20.sub.D: 212.5.degree. (c
0.08, CHCl.sub.3); IR (cm.sup.-1) 4329, 4258, 3405, 2925, 2361,
1713, 1620, 1602, 1579, 1514, 1463, 1377, 1342, 1277, 1219, 1103,
1065, 1012, 968, 870, 791, 722; .sup.1H-NMR (CDCl.sub.3, rotamers)
.delta.7.66-7.71 (s, 1H), 7.11-7.39 (m, 6H), 6.80-6.92 (s, 1H),
6.32 (s, 1H), 5.14-5.67 (m, 5H), 3.85-4.15 (m, 9H), 3.49-3.67 (m,
3H), 2.01-2.15 (m, 4H); .sup.13C-NMR (CDCl.sub.3) .delta.170.41,
166.73, 194.97, 149.03, 135.92, 128.67-126.30, 124.52, 114.64,
112.24, 110.87, 109.82, 108.92, 107.97, 86.02, 71.51, 66.38, 65.03,
60.40, 56.48-56.19, 46.67, 28.70, 26.41, 23.65, 21.05; IR
(cm.sup.-1) 3600-3100, 3020, 2400, 2105, 1765, 1640, 1525, 1430,
1385, 1350, 1310, 1280, 1215, 1170, 1110, 1045; MS (FAB) (m/z,
relative intensity) 594 (MH.sup.+, 9), 353 (5), 337 (4), 282 (6),
256 (4), 241 (8), 196 (100), 180 (15), 166 (16), 151 (13), 136 (6),
123 (5), 105 (17), 91 (98); EI-HRMS m/z 593.2010 (calc'd for
C.sub.30H.sub.31N.sub.3O.sub.10 m/z 593.2042).
Example 4
[0190] Synthesis of a Benzyl tomaymycin Prodrug for nitroreductase
ADEPT (147) 2627
[0191] Synthesis of (2s,
4S)-N-(Allyloxycarbonyl)-4-hydroxypyrrolidine-2-c- arboxylic acid
(13)
[0192] A solution of allyl chloroformate (33.17 g, 275 mmol, 1.2
eq) in THF (30 ml) was added dropwise over 30 minutes to a
Suspension of trans-4-hydroxy proline (30 g, 229 mmol) in THF (150
ml) and water (150 ml) at pH 9 (adjusted with 4M NaOH) and at
0.degree. C. The reaction mixture was allowed to stir at 0.degree.
C. and pH 9 for a further 1 hour. The aqueous layer was separated
and saturated with NaCl, washed with EtOAc (4.times.100 ml) and the
pH of the aqueous phase was adjusted to 2 with conc. HCl. The
resulting oil was extracted with EtOAc (3.times.100 ml). The
combined organic phase was dried (MgSO.sub.4) and evaporated in
vacuo to afford the Alloc protected hydroxy proline (13), as a
thick transparent oil which was used for the next stage without
further purification.
[0193] Yield 41 g (84%); [.alpha.].sup.20.sub.D: -236.degree. (c
0.25, CHCl.sub.3); IR (cm.sup.-1) 3429, 2522, 2340, 2258, 2130,
1688, 1437, 1415, 1345, 1277, 1222, 1177, 1133, 1085, 1047, 1025,
994, 827, 768; .sup.1H-NMR (CDCl.sub.3, rotamers) .delta.5.80-5.99
(m, 1H), 5.13-5.34 (m, 2H), 4.52-4.63 (m, 2H), 4.37-4.44 (m, 2H),
3.53-3.60 (m, 2H), 2.00-2.36 (m, 2H); .sup.13C-NMR (CDCl.sub.3)
.delta.174.7, 174.5, 154.8, 154.5, 132.8, 132.7, 117.1, 116.7,
69.2, 68.5, 65.7, 65.6, 57.9, 57.6, 54.9, 54.4, 38.9, 38.2; MS (EI)
(m/z, relative intensity) 215 (M.sup.+, 14), 170 (100), 130 (95),
126 (34), 108 (35), 68 (51), 56 (21); EI-HRMS m/z 215.0759 (calc'd
for C.sub.9H.sub.13NO.sub.5 m/z 215.0743).
[0194] Synthesis of
(2S)-N-(Allyloxycarbonyl)-4-oxopyrrolidine-2-carboxyli- c Acid
(14)
[0195] The alloc protected hydroxy proline (13) (18 g, 83.72 mmol)
was dissolved in acetone (1260 ml). The Jone's reagent
[CrO.sub.3:26.6 g/H.sub.2SO.sub.4:21.3 ml and the solution was made
up to 100 ml with water] (87 ml) was added over 10 min. The
resulting mixture was stirred for a further 45 minutes when the
excess oxidant was quenched with methanol (15 ml). The green
chromium salts were removed by filtration through Celite and the
filtrate was diluted in CHCl.sub.3 (1000 ml). The combined organic
phase was washed with brine several times (5.times.500 ml) and the
solvent was evaporated under reduced pressure, to yield the ketone
14 which was used without any purification.
[0196] Yield 14.68 (82%); IR (cm.sup.-1) 3471, 3020, 2932, 2610,
2041, 1761, 1691, 1649, 1547, 1411, 1343, 1266, 1195, 1164, 1128,
972, 939, 877, 759; .sup.1H-NMR (CDCl.sub.3, rotamers) .delta.9.20
(bs, 1H), 5.86-6.00 (m, 1H), 5.21-5.35 (m, 2H), 4.87-4.91 (m, 2H),
4.87-4.91 (m, 1H), 3.87-3.99 (s, 2H), 2.68-2.95 (m, 2H);
.sup.13C-NMR (CDCl.sub.3) .delta.207.6, 207.1, 175.8, 175.3, 155.3,
154.2, 131.9, 118.4, 118.2, 67.0, 66.8, 55.8, 52.5, 52.3, 41.0,
40.3; MS (EI) (m/z, relative intensity)=213 (M.sup.+, 19), 168
(81), 152 (8), 128 (100), 112 (15), 100 (37), 96 (36), 68 (14), 58
(13), 56 (37); EI-HRMS m/z 213.0641 (calc'd for
C.sub.9H.sub.11NO.sub.5 m/z 213.0637).
[0197] Synthesis of
(2S)-(E,Z)-N-(Allyloxycarbonyl)-4-ethylidenepyrrolidin-
e-2-carboxylic Acid (15)
[0198] A mixture of NaH (60% suspension) (4 g, 100 mmol, 4 eq) and
ethyl triphenylphosphonium bromide (37 g, 100 mmol, 4 eq) in
freshly distilled THF (250 ml) was refluxed for 4 hours under
nitrogen. A solution of the ketone 14 (5.325 g, 25 mmol) in freshly
distilled THF (50 ml) was added dropwise over 40 minutes and the
reaction mixture was refluxed overnight under nitrogen. It was then
cooled and poured into a 5% w/v aq. solution of NaHCO.sub.3 (500
ml) and stirred for 10 min. The mixture was then washed with EtOAc
(2.times.250 ml). The aqueous layer was acidified to pH 3 with dil.
HCl (vigorous effervescence was observed). The emulsion was
extracted with EtOAc (4.times.200 ml) and the combined organic
phase was washed with brine (250 ml) and dried (MgSO.sub.4). The
solvent was then evaporated in vacuo to give an orange oil which
was used in the next stage without further purification. Crude
yield 3.81 g (68%)
[0199] Synthesis of
Methyl-(2S)-(E,Z)-N-(Allyloxycarbonyl)-4-ethylidenepyr-
rolidine-2-carboxylate (16)
[0200] Oxalyl chloride and DMF (4 drops) was added to a solution of
the Wittig product 15 (3.5 g, 16 mmol) in dry toluene (15 ml) and
it was stirred overnight at room temperature and under N.sub.2.
Freshly distilled methanol (20 ml) was then added and the mixture
was stirred for a further 4 hours when TLC (EtOAc:Petroleum ether,
1:1) revealed complete loss of starting material. The solvent was
then evaporated in vacuo. The resulting oil was dissolved in EtOAc
(60 ml) and the solution was washed with sat. aq. NaHCO.sub.3
(4.times.25 ml), brine (50 ml), dried (MgSO.sub.4) and the solvent
was removed under reduced pressure to afford the esterified Wittig
product (16).
[0201] Yield 3.48 g (94%); [.alpha.].sup.20.sub.D: -377.degree. (c
0.045, CHCl.sub.3); IR (cm.sup.-1) 4214, 3452, 3019, 2955, 2862,
2400, 1746, 1704, 1649, 1437, 1410, 1343, 1310, 1274, 1215, 1179,
1120, 1072, 1029, 991, 932, 755; .sup.1H-NMR (CDCl.sub.3, rotamers)
.delta.5.83-6.00 (m, 1H), 5.16-5.48 (m, 3H), 4.56-4.68 (m, 3H),
4.05-4.14 (s, 2H), 3.87-3.99 (m, 3H), 2.53-2.97 (m, 2H), 1.60-1.63
(m, 38); .sup.13C-NMR (CDCl.sub.3) (E/Z isomers) .delta.172.9,
15498, 134.1, 134.0, 133.1, 133.0, 132.8, 132.7, 118.2, 118.1,
117.8, 117.7, 117.5, 117.4, 117.1, 66.1, 66.0, 65.9, 58.8, 58.6,
58.4, 52.3, 51.1, 50.6, 48.3, 47.7, 36.6, 35.7, 32.6, 31.7, 14.7,
14.6, 14.4; MS (EI) (m/z, relative intensity) 239 (M.sup.+, 4), 180
(70), 154 (100), 136 (55), 94 (33), 80 (10), 67 (19), 59 (8);
EI-HRMS m/z 239.1030 (calc'd for C.sub.12H.sub.17NO.sub.4 m/z
239.1158).
[0202] Synthesis of
(2S)-(E,Z)-N-(Allyloxycarbonyl)-4-ethylidenepyrrolidin- emethanol
(17)
[0203] A solution of the Alloc protected C-Ring ester (16) (2.5 g,
10.46 mmol) was dissolved in freshly distilled THF (50 ml) and
stirred under nitrogen at 0.degree. C. Lithium borohydride (0.4 g,
17.95 mmol, 1.7 eq) was added slowly and in small portions to that
solution (effervescence was observed). The mixture was allowed to
warm to room temperature and stirred for a further 4 hours when the
reaction was complete as it was indicated by TLC (EtOAc:Petroleum
ether, 1:1). It was then cooled back down to 0.degree. C. and the
reaction was stopped by the addition of water (40 ml) dropwise over
15 min. The mixture was neutralised with 2M HCl (40 ml) which was
added very slowly and vigorous effervescence occurred. It was then
extracted with EtOAc (3.times.100 ml), the combined organic phase
was washed with water (150 ml) and brine (100 ml), dried
(MgSO.sub.4) and the solvent was removed under reduced pressure to
afford an oil which was further purified by flash chromatography
(EtOAc:Petroleum ether, 60:40) to give the pure product 17.
[0204] Yield 1.57 g (71%); IR (cm.sup.-1) 3425, 2947, 2864, 2085,
1678, 1547, 1536, 1411, 1349, 1308, 1236, 1203, 1114, 1047, 975,
931, 883, 851, 769; .sup.1H-NMR (CDCl.sub.3, rotamers)
.delta.5.87-6.01 (m, 1H), 5.19-5.40 (m, 3H), 4.59-4.61 (s, 2H),
3.64-4.15 (m, 5H), 2.37-2.80 (m, 2H), 1.62-1.64 (m, 3H);
.sup.13C-NMR (CDCl.sub.3) (E/Z isomers) .delta.156.3, 154.5, 133.1,
132.8, 131.8, 131.7, 130.9, 130.7, 128.7, 128.6, 125.5,
117.5-115.9, 66.1, 65.9, 65.7, 64.6, 60.4, 59.9, 57.9, 57.3, 51.6,
51.3, 48.3, 48.1, 34.7, 34.4, 34.2, 34.1, 30.6, 30.3, 29.9, 29.5,
25.6, 14.5, 14.4, 14.2.
[0205] Synthesis of
(2S)-(E,Z)-N-(Allyloxycarbonyl)-4-ethylidene-o-(tert-b-
utyldimethylsilyl)-pyrrolidinemethanol (18)
[0206] Tert-butyldimethylsilyl chloride (1.11 g, 7.37 mmol, 1.2 eq)
was added to a solution of the hydroxy compound 17 (1.3 g, 6.36
mmol) and imidazole (1.05 g, 15.41 mmol, 2.5 eq) in dry DMF (3 ml)
and the reaction mixture was allowed to stir overnight under
N.sub.2. The reaction mixture was then saturated with water (200
ml) and extracted with EtOAc (3.times.100 ml). The organic layer
was washed with water (100 ml), brine (150 ml), dried (MgSO.sub.4)
and excess solvent was evaporated in vacuo to give a light brown
oil which was subjected to flash chromatography (EtOAc:Petroleum
ether, 50:50) to afford the pure silyl ether (18).
[0207] Yield 1.54 g (75%); IR (cm.sup.-1) 3416, 2093, 1642, 1406,
1253, 1189, 1104, 775; .sup.1H-NMR (CDCl.sub.3, rotamers)
.delta.5.89-5.95 (m, 1H), 5.18-5.34 (m, 3H),4.62 (s, 2H), 3.53-4.08
(m, 5H), 2.49-2.70 (m, 2H), 1.43-1.59 (m, 3H), 0.91 (bs, 9H),
0.02-0.003 (bs, 6H); .sup.13C-NMR (CDCl.sub.3) (E/Z isomers)
.delta.162.5, 154.4, 136.2, 135.1, 133.1, 130.9, 128.8, 117.4,
117.1, 116.7, 116.1, 65.9, 65.7, 65.5, 63.9, 63.6, 63.1, 58.6,
58.2, 58.1, 51.2, 48.3, 48.1, 36.5, 34.6, 33.9, 31.8, 31.4, 30.4,
30.3, 29.7, 29. 2, 29.1, 22.7, 18.1, 18.0, 14.5, 14.4, 14.1, -3.6,
-5.4; MS (EI) (m/z, relative intensity) 325 (M.sup.+, 6), 310 (3),
268 (100), 256 (6), 240 (18), 224 (7), 182 (22), 180 (38), 168 (10
), 154 (5), 136 (24), 115 (8), 94 (6), 75 (13), 73 (21), 57 (17);
EI-HRMS m/z 325.2003 [(M-isobutyl) m/z 268.1233] (calc'd for
C.sub.17H.sub.31NO.sub.3 m/z 325.2073 [(M-isobutyl) m/z
268.1369).
[0208] Synthesis of
(2S)-(E,Z)-4-ethylidene-O-(tert-butyldimethylsilyl)-py-
rrolidinemethanol (19)
[0209] Tributyltinhydride (1.49 g, 5.1 mmol, 1.1 eq) was added to a
solution of 18 (1.5 g, 4.62 mmol) in DCM (30 ml) in the presence of
water (0.48 g, 27.11 mmol, 6 eq) and a catalytic amount of
Pd(PPh.sub.3).sub.2Cl.sub.2 (0.132 g, 0.189 mmol, 4% molar) and the
reaction mixture was allowed to stir for 5 minutes at room
temperature. The reaction mixture was diluted with DCM (40 ml),
dried (MgSO.sub.4) and excess solvent removed under vacuum. The
pure amine 19 was isolated by flash chromatography (EtOAc:Petroleum
ether, 50:50) to afford a light brown oil.
[0210] Yield 0.67 g (60%); IR (cm.sup.-1) 4329, 4257, 3426, 2923,
2728, 2672, 2360, 2341, 2035, 1777, 1713, 1641, 1512, 1463, 1377,
1302, 1242, 1169, 1019, 890, 760, 721; .sup.1H-NMR (CDCl.sub.3)
.delta.5.33-5.37 (m, 1H), 4.06 (s, 1H), 3.28-3.75 (m, 4H),
2.17-2.47 (m, 2H), 1.54-1.63 (m, 3H), 0.89 (bs, 9H), 0.06-0.007
(bs, 6H).
[0211] Synthesis of
(2S)-(E,Z)-N-(4-benzyloxy-5-methoxy-2-nitrobenzoyl)-2--
ethylidene-O-(tert-butyldimethylsilyl)-pyrrolidinemethanol (20)
[0212] A catalytic amount of DMF (4 drops) was added to a solution
of 4-benzyloxy-5-methoxy-2-nitrobenzoic acid (3) (2.5 g, 8.25 mmol)
and oxalyl chloride (1.25 g, 9.84 mmol, 1.2 eq) in dry THF (10 ml)
was allowed to stir overnight at room temperature The resulting
chloride was added dropwise over 20 minutes to a solution of the
deprotected C-ring 19 (1.98 g, 8.25 mmol, 1 eq), TEA (1.75 g, 17.35
mmol, 2.1 eq) and water (0.6 ml) at 0.degree. C., under nitrogen.
The reaction mixture was allowed to stir for a further 2.5 hours,
at which point TLC (EtOAc:Petroleum ether, 40:60) indicated that
the reaction was complete. The organic solvent was then removed
under reduced pressure and the residue was partitioned between
EtOAc (150 ml) and water (150 ml) and the layer separated. The
aqueous layer was washed with EtOAc (100 ml) and the combined
organic phase was washed with sat. NH.sub.4Cl (100 ml), brine (150
ml), dried (MgSO.sub.4) and evaporated in vacuo to afford, after
flash chromatography (EtOAc:Petroleum ether, 70:30), the coupled
product 20.
[0213] Yield 2.85 g (66%); [.alpha.].sup.2O.sub.D: -11.7.degree. (c
0.305, CHCl.sub.3); TR (cm.sup.-1) 4328, 3424, 2960, 2855, 2359,
2332, 2084, 1709, 1641, 1548, 1526, 1462, 1377, 1278, 1215, 1107,
1058, 836, 761, 722; .sup.1H-NMR (CDCl.sub.3) .delta.7.37-7.47 (m,
6H), 6.77 (s, 1H), 5.33-5.37 (m, 1H), 5.22 (s, 2H), 4.43-4.59 (bs,
1H), 3.95 (s, 3H), 3.59-3.86 (m, 4H), 2.47-2.75 (m, 2H), 1.60-1.67
(m, 3H), 0.89 (bs, 9H), 0.087-0.09 (bs, 6H); .sup.13C-NMR
(CDCl.sub.3) (E/Z isomers) .delta.154.8, 135.3, 128.9, 127.6,
109.2, 71.3, 58.0, 57.6, 56.6, 30.3, 29.7, 25.7, 14.6, 14.4, -5.4,
-5.6; MS (CI) (m/z, relative intensity) 527 (MH.sup.+, 25), 418
(82), 388 (6), 359 (9), 328 (7), 304 (25), 279 (45), 258 (13), 240
(35), 219 (11), 184 (10), 161 (51), 147 (21), 133 (41), 113 (21),
107 (100), 91 (25), 81 (8), 73 (40); EI-HRMS m/z 526.2642 (calc'd
for C.sub.28H.sub.38N.sub.2O.sub.6Si m/z 526.2499).
[0214] Synthesis of
(2S)-(E,Z)-N-(4-benzyloxy-5-methoxy-2-nitrobenzoyl)-2-- ethylidene
pyrro-lidine methanol (21)
[0215] A solution of TBAF (1M solution in THF, 1.75 ml, 1.75 mmol,
1.2 eq) was added dropwise over 15 minutes at 0.degree. C. under
N.sub.2 to a solution of the TBDMS protected intermediate 20 (0.75
g, 1.42 mmol) in dry THF (30 ml). The reaction mixture was allowed
to warm to room temperature and to stir for a further 30 minutes
when TLC (EtOAc) indicated complete consumption of the starting
material. The reaction mixture was diluted with sat. aq. NH.sub.4Cl
(150 ml) and extracted with EtOAc (3.times.100 ml). The organic
phase was washed with brine (150 ml), dried (MgSO.sub.4) and
evaporated under reduced pressure to afford the deprotected product
21 which was used for the next step without further
purification.
[0216] Yield 0.64 g (109%, some TBDMSF associated with the product;
Yield.sub.max 0.57 g); IR (cm.sup.-1) 4214, 3406, 3020, 2958, 2925,
2854, 2400, 2085, 1,631, 1581, 1523, 1463, 1453, 1433, 1378, 1335,
1278, 1215, 1053, 870, 754; .sup.1H-NMR (CDCl.sub.3)
.delta.7.37-7.47 (m, 6H), 6.77 (s, 1H), 5.33-5.37 (m, 1H), 5.22 (s,
2H), 4.43-4.59 (bs, 1H), 3.95 (s, 3H), 3.59-3.86 (m, 4H), 2.47-2.75
(m, 2H), 1.60-1.67 (m, 3H), 0.89 (bs, 9H), 0.087-0.009 (bs,
6H).
[0217]
(2S)-(E,Z)-N-(2-amino-4-benzyloxy-5-methoxybenzoyl)-2-ethylidenepyr-
rolidine methanol (22)
[0218] A solution of the nitro compound 21 (0.64 g, 1.38 mmol,
assumes quantitative yield in previous step) in MeOH (30 ml) and
SnCl.sub.2.2H.sub.2O (1.58 g, 7 mmol, 5 eq) was heated to reflux
for 40 minutes when TLC (5% MeOH/CHCl.sub.3) indicated the reaction
was complete. The excess solvent was evaporated in vacuo and the
residual oil was partitioned between EtOAc (100 ml) and sat. aq.
NaHCO.sub.3 (100 ml) and allowed to stir overnight under N.sub.2.
The organic layer was then separated and washed with brine (150
ml), dried (MgSO.sub.4) and evaporated in vacuo. The residue was
purified by flash chromatography (5% MeOH/CHCl.sub.3) to give the
pure amine intermediate 22 as a bright yellow oil.
[0219] Yield 0.32 g (61%); IR (cm.sup.-1) 4329, 4257, 3427, 2923,
2727, 2360, 2341, 2036, 1712, 1624, 1513, 1463, 1377, 1264, 1215,
1172, 1120, 1002, 872, 761, 722; .sup.1H-NMR (CDCl.sub.3)
.delta.7.35-7.51 (m, 5H), 6.87 (s, 1H), 6.27 (s, 1H), 5.28-5.42 (m,
1H), 5.17 (s, 2H), 4.43-4.59 (bs, 1H), 3.84 (s, 3H), 3.59-3.78 (m,
4H), 2.25-2.75 (m, 2H), 1.60 (d, J=6.8 Hz, 3H); .sup.13C-NMR
(CDCl.sub.3) (E/Z isomers) .delta.150.9, 141.5, 136.6, 134.5,
128.8, 127.9, 127.1, 117.7, 117.3, 112.6, 103.0, 70.6, 66.1, 60.4,
59.8, 57.2, 34.3, 30.0, 29.4, 21.0, 14.6, 14.3, 13.6.
[0220] Synthesis of
(2S)-(E,Z)-N-[2-(p-nitrobenzyloxy)carboxamido-4-benzyl-
oxy-5-methoxy benzoyl]-2-ethylidenepyrrolidine methanol (23)
[0221] The freshly made amine 22 (0.5 g, 1.31 mmol) and dry
pyridine (0.207 g, 2.62 mmol, 2 eq) was dissolved in dry DCM (30
ml) at 0.degree. C. under N.sub.2. A solution of 4-nitrobenzyl
chloroformate (0.282 g, 1.31 mmol, 1 eq) was added to the amine
solution dropwise over 20 min. The reaction mixture was allowed to
stir under the same conditions for 2 hours at which time TLC (5%
MeOH/CHCl.sub.3) revealed complete consumption of the starting
material. The mixture was then dissolved in DCM (100 ml) and washed
with sat. aq. CuSO.sub.4 (2.times.75 ml), water (2.times.100 ml),
brine (150 ml) and dried (MgSO.sub.4). The organic solvent was
evaporated in vacuo and after purification by flash chromatography
(3% MeOH/CHCl.sub.3) the p-nitrobenzyl carbamate was isolated.
[0222] Yield 0.56 g (76.3%); [.alpha.].sup.20.sub.D: +204.60 (c
0.22, CHCl.sub.3); IR (cm.sup.-1) 4329, 4257, 3426, 2924, 2853,
2093, 1710, 1628, 1524, 1462, 1406, 1377, 13460, 1269, 1219, 1175,
1118, 722; .sup.1H-NMR (CDCl.sub.3, rotamers) .delta.8.81 (bs, 1H),
8.19 (d, J=8.6 Hz, 2H), 7.80 (s, 1H), 7.31-7.61 (m, 7H), 6.85 (s,
1H), 5.29-5.35 (m, 1H), 5.24 (s, 2H), 5.15 (s, 2H), 4.08-4.13 (bs,
1H), 3.83 (s, 3H), 3.66-3.86 (m, 4H), 2.39-2.75 (m, 2H), 1.51 (d,
J=6.6 Hz, 3H); .sup.13C-NMR (CDCl.sub.3) (E/Z isomers)
.delta.153.2, 150.3, 147.6, 143.6, 136.1, 134.1, 128.7, 128.4,
128.1, 127.7, 126.9, 123.7, 117.8, 111.2, 70.7, 65.2, 63.8, 56.6,
29.7, 14.6, 14.4; MS (FAB) (m/z, relative intensity) 562 (MH.sup.+,
15), 279 (77), 256 (15), 240 (35), 231 (37), 136 (13), 128 (25),
106 (14), 91 (100), 73 (11), 57 (11); EI-HRMS m/z 561.2150 (calc'd
for C.sub.30H.sub.31N.sub.3O.sub.8 m/z 561.2111).
[0223] Synthesis of
(11aS)-8-benzyloxy-7-methoxy-1,3,10,11,11a-tetrahydro--
2-ethylidene-11-hydroxy-10-(p-nitrobenzyloxy)carboxy-5H-pyrrolo[2,1-c][1,4-
]-benzodiazepine-5-one (24)
[0224] A solution of the uncyclised carbamate 23 (0.3 g, 0.535
mmol), NMO (94 mg, 0.803 mmol, 1.5 eq) and mol. sieve (0.267 g) in
a mixture of dry (DCM:CH.sub.3CN, 9:3 ml) was allowed to stir at
room temperature under N.sub.2 for 15 min. TPAP (9.4 mg, 0.026
mmol, 5% molar) was then added and the mixture was allowed to stir
under the same conditions for a further 2.5 hours. The reaction
mixture was filtered through Celite and the filtrate evaporated in
vacuo. The residue was subjected to flash chromatography (1%
MeOH/CHCl.sub.3) to obtain the target compound (24).
Example 5
[0225] Synthesis of PBD Dimer Prodrugs
[0226] Example 5(a)-Synthesis of
1,1'-[(Propane-1,3-diyl)dioxy]bis[[2-(4-n-
itrobenzyloxycarboxamido)]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-11-hyd-
roxy-5H-pyrrolo [2-c][1,4]benzodiazepine -5-one (30) 28
[0227] Synthesis of 1',3'-Bis(4-carboxy-2-methoxyphenoxy)propane
(25)
[0228] A solution of diiodopropane (4.2 g, 14.2 mmol) was dissolved
in THF (30 ml) was added dropwise to a vigorously stirred solution
of vanillic acid (1) (4-hydroxy-3-methoxybenzoic acid) (5 g, 29.8
mmol, 2.1 eq) and aq. 0.5 M NaOH (70 ml) in THF (50 ml). The
reaction mixture was allowed to reflux for 48 hours and the organic
solvent was removed under reduced pressure. The remaining aqueous
phase was washed with Petroleum ether 40-60 (3.times.100 ml) and it
was then acidified with conc. HCl to pH 2 until no further
precipitation was observed, The precipitate was collected
filtration, washed with water, dried to afford the dimer acid (25)
as white crystals.
[0229] Yield 7.63 g (68%); .sup.1H-NMR (CDCl.sub.3+DMSO-d.sub.6)
.delta.7.64 (dd, J.sub.1=8.3 Hz, J.sub.2=8.3 Hz, 2H), 7.54 (s, 2H),
6.96 (d, J=8.4 Hz, 2H), 4.29 (t, J=6.1 Hz, 4H) 3.88 (s, 6H), 2.38
(t, J=6.2 Hz, 2H); .sup.13C-NMR (CDCl.sub.3+DMSO-d.sub.6)
.delta.173.1, 168.1, 151.9, 148.6, 123.5, 112.4, 111.45, 65.21,
55.8, 28.8; IR (cm.sup.-1)=3600-3100, 2925, 2855, 1713, 1680, 1597,
1516, 1459, 1377, 1344, 1309, 1275, 1223, 1178, 1133, 1045, 1021;
MS (EI) (m/z, relative intensity) 376 (M.sup.+, 34), 208 (31), 168
(43), 152 (15), 101 (100), 69 (87); EI-HRMS m/z 376.1136 (calc'd
for C.sub.19H.sub.20O.sub.8 m/z 376.1158)
[0230] Synthesis of
1',3'-Bis(4-carboxy-2-methoxy-5-nitrophenoxy)propane (26)
[0231] The dimer acid 25 (5 g, 13.3 mmol) was added slowly in small
portions over 30 minutes to a stirred solution of 70% HNO.sub.3 (50
ml) at 0.degree. C. After the addition was complete the reaction
mixture was allowed to warm to 15.degree. C. and stirring continued
for a further 2 hours. The reaction mixture was poured onto ice
causing precipitation of the product. The yellow precipitate was
collected by filtration, washed with cold water and dried to afford
the nitrated dimer acid core (26) as a pale yellow solid.
[0232] Yield 4.52 g (73%); mp. 241-246.degree. C.; IR (cm.sup.-1)
3500-3200, 2924, 1713, 1604, 1582, 1523, 1459, 1377, 1282, 1218,
1189, 1053; .sup.1H-NMR (CDCl.sub.3+DMSO-d.sub.6) .delta.7.44 (s,
2H), 7.16 (s, 2H), 4.32 (t, J=6.5 Hz, 4H), 3.94 (s, 6H), 2.44 (t,
J=6.4 Hz, 2H); .sup.13C-NMR (CDCl.sub.3+DMSO-d.sub.6) .delta.172.3,
167.11, 152.5, 149.2, 141.1, 132.1, 128.6, 122.7, 111.2, 108.3,
65.2, 56.4, 33.9; MS (EI) (m/z, relative intensity) 467 (MH.sup.+,
1), 436 (5), 423 (10), 376 (2), 256 (6), 210 (4), 183 (10), 164
(10), 153 (3), 91 (9), 77 (4), 51 (13), 44 (100); EI-HRMS m/z
466.0876 (calc'd for C.sub.19H.sub.18N.sub.2O- .sub.12
466.0858).
[0233] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[(2-nitro-5-methoxy--
1,4-phenylene) carbon yl]bis[pyrrolidinemethanol](27)
[0234] A solution of the nitrated dimer core 26 (1 g, 2.14 mmol) in
a mixture of dry CH.sub.3CN/THF (30:5 ml) was treated overnight
with oxalyl chloride (0.64 g, 5.14 mmol, 2.4 eq) and DMF (5 drops).
The resulting acid chloride was then added dropwise over 30 minutes
to a suspension of pyrrolidinemethanol (0.432 g, 4.28 mmol, 2 eq)
and K.sub.2CO.sub.3 (1.2 g, 8.56 mmol, 4 eq) in dry acetonitrile
(80 ml) at -25.degree. C., under nitrogen. The resulting mixture
was allowed to stir at -25.degree. C. for a further 1.5 hour when
the reaction mixture was diluted with water (100 ml). The solution
was then extracted with chloroform (4.times.100 ml). The combined
organic phase was washed with aq. 1M HCl (2.times.75 ml), water
(2.times.75 ml), brine (100 ml), dried (MgSO.sub.4) and evaporated
in vacuo. The resulting orange oil was purified by column
chromatography (10% MeOH/CHCl.sub.3) to afford the pure product 27
as a yellow viscous oil.
[0235] Yield 0.88 g (65%); [.alpha.].sup.20.sub.D: +246.1.degree.
(c 0.067, CHCl.sub.3); IR (cm.sup.-1) 4329, 4257, 3370, 2916, 2728,
1787, 1713, 1614, 1574, 1513, 1462, 1377, 1337, 1274, 1215, 1058,
868, 823, 749, 723; .sup.1H-NMR (CDCl.sub.3) .delta.7.73 (s, 2H),
6.80 (s, 2H), 4.31-4.36 (m, 6H), 3.94 (s, 6H), 3.74-3.87 (m, 4H),
3.17 (t, J=6 Hz, 4H), 2.44 (t, J=5.9 Hz), 2.12-2.22 (m, 4H),
1.70-1.92 (m, 4H); .sup.13C-NMR (CDCl.sub.3) .delta.154.8, 148.3,
137.0, 128.0, 109.1, 108.4, 66.0, 65.6, 61.4, 56.7, 49.5, 28.6,
24.4; MS (FAB) (m/z, relative intensity) 633 (M.sup.++1, 52), 449
(12), 236 (11), 219 (17), 206 (12), 196 (15), 191 (22), 178 (20),
166 (20), 151 (34), 135 (23), 128 (11), 122 (33), 115 (11), 102
(47), 91 (100), 84 (45).
[0236] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[(2-amino-5-methoxy--
1,4-phenylene) carbonyl]bis[pyrrolidinemethanol](28)
[0237] Hydrazine hydrate (0.87 g, 1.74 mmol, 10 eq) was added
dropwise to a solution of the nitro intermediate (27) (1 g, 1.58
mmol) over Raney Nickel (0.25 g) in gently refluxing methanol (20
ml). (Anti-bumping granules were used to ensure even boiling). The
reaction mixture was heated at reflux for a further 15 minutes at
which time TLC (10% MeOH/CHCl.sub.3) indicated that the reaction
had gone to completion. The catalyst was then removed by filtration
(causation! pyrophoric Ni) and the filtrate was evaporated in vacuo
to give a dark brown oil which was purified by flash chromatography
(7.5% MeOH/CHCl.sub.3) to afford the coupled dimer amine (28) as-a
bright yellow oil.
[0238] Yield=0.632 g (70%); IR (cm.sup.-1) 3100-3600, 2925, 1611,
1460, 1377, 1275, 1216, 1175; .sup.1H-NMR (CDCl.sub.3) .delta.6.74
(s, 2H), 6.33 (s, 2H), 4.31-4.42 (m, 2H), 4.19-4.23 (m, 4H), 3.76
(s, 6H), 3.50-3.66 (m, 8H), 2.27-2.40 (m, 2H), 2.02-2.15 (m, 4H),
1.71-1.87 (m, 4H); .sup.13C-NMR (CDCl.sub.3) .delta.151.1, 141.0,
112.8, 102.3, 65.1, 60.8, 57.1, 50.9, 28.7, 28.4; MS (FAB) (m/z,
relative intensity) 574 (M.sup.++2, 44), 503 (3), 473 (30), 389
(11), 371 (31), 219 (14), 206 (50), 198 (13), 192 (27), 180 (30),
166 (18), 149 (28), 137 (23), 128 (17), 102 (43), 93 (68), 84 (57),
70 (33), 57 (35).
[0239] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(4-nitrobenzylox-
ycarboxamido)-5-methoxy-1,4-phenylene]carbonyl]bis-pyrrolidinemethanol
(29)
[0240] A solution of 4-nitrobenzyl chloroformate (0.347 g, 1.61
mmol, 2.3 eq) in dry DCM (10 ml) was added dropwise over 20 min,
under nitrogen to a freshly prepared solution of dimer amine 28(0.4
g, 0.7 mmol) in dry DCM (15 ml) and pyridine (0.193 g, 2.45 mmol,
3.5 eq) at 0.degree. C. The resulting reaction mixture was allowed
to stir for a further 1.5 hours at 0.degree. C. at which time TLC
(10% MeOH/CHCl.sub.3) indicated complete consumption of starting
material. The reaction mixture was diluted with chloroform (50 ml)
and washed with a saturated aqueous solution of CuSO.sub.4
(2.times.50 ml), water (2.times.50 ml), brine (100 ml) and dried
(MgSO.sub.4). The organic solvent was removed in vacuo and the pure
dimer carbamate 29 was obtained after flash chromatography (7%
MeOH/CHCl.sub.3) as a bright yellow foam.
[0241] Yield 0.53 g (81%); IR (cm.sup.-1) 3424, 2088, 1641, 1502,
1247; .sup.1H-NMR (CDCl.sub.3) .delta.9.02 (bs, 2H), 8.21 (d, J=8.8
Hz, 4H), 7.72 (s, 2H), 7.56 (d, J=8.8 Hz, 4H), 6.82 (s, 2H), 5.26
(s, 4,H), 4.28-4.38 (m, 2H), 4.26 (t, J=6 Hz, 4H), 3.79 (s, 6H),
3.42-3.79 (m, 8H), 2.27-2.42 (m, 2H), 2.11-2.18 (m, 4H), 1.71-1.88
(m, 4H); .sup.13C-NMR (CDCl.sub.3) .delta.170.7, 153.2, 150.6,
147.6, 143.6, 128.5, 126.9, 123.9, 111.5, 105.9, 65.3, 63.8, 61.0,
56.6, 51.6, 30.3, 28.4; MS (FAB) (m/z, relative intensity) 932
(M.sup.++2, 3), 753 (5), 551 (5), 249 (7), 232 (13), 222 (8), 206
(23), 192 (32), 179 (27), 166 (46), 149 (28), 136 (66), 120 (22),
106 (45), 102 (100), 91 (80), 84 (48), 73 (71), 57 (47).
[0242] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(4-nitrobenzylox-
ycarboxamido)]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-11-hydroxy-5H-pyrr-
olo[1,2-c][1,4]benzodiazepine-5-one (30)
[0243] TPAP (13.4 mg, 0.114 mmol, 0.3 eq) was added in one portion
to a solution of the bis carbamate 29 (0.35 g, 0.37 mmol) and NMO
(0.134 g, 1.14 mmol, 3.1 eq) in dry DCM/CH.sub.3CN (9:3 ml) which
has been allowed to stir over molecular sieve (0.350 g) for 15
minutes under N.sub.2 and at room temperature. Progress of the
reaction was followed by TLC (7% MeOH/CHCl.sub.3). After 2 hours
reaction was still incomplete requiring the addition of a further
amount of NMO (67 mg, 0.55 mmol, 1.5 eq) and TPAP (6.7 mg, 0.05
mmol, 0.15 eq). After stirring for a further 30 minutes TLC
revealed the complete consumption of starting material. The
reaction mixture was filtered through Celite and the filtrate
evaporated in vacuo. The resulting black residue was subjected to
column chromatography (1.5% MeOH/CHCl.sub.3) to afford the product
as an opaque pale yellow oil.
[0244] Yield 0.143 (42%); IR (cm.sup.-1) 3500-3000, 2933, 2253,
1728, 1599, 1523, 1465, 1431, 1409, 1348, 1270, 1206, 1174, 1111,
1060; .sup.1H-NMR (CDCl.sub.3, rotamers) .delta.8.18 (d, J=8.8 Hz,
4H), 7.74 (s, 2H), 7.48 (d, J=8.8 Hz, 4H), 6.71 (s, 2H), 5.65 (d,
J=10 Hz, 2H), 5.26 (s, 4H), 4.29 (t, J=6 Hz, 4H), 4.07-4.16 (m,
2H), 3.83 (s, 6H), 3.42-3.75 (m, 8H), 2.25-2.32 (m, 2H), 2.10-2.22
(m, 4H), 1.68-1.75 (m, 4H); .sup.13C-NMR (CDCl.sub.3) .delta.166.9,
153.2, 147.6, 143.8, 142.8, 128.2, 123.9, 113.9, 110.9, 108.3,
86.2, 69.1, 66.3, 65.6, 60.0, 56.4, 46.4, 29.7, 28.7, 23.1, 14.8;
MS (FAB) (m/z, relative intensity) 925 (M.sup.+-1, 1), 889 (5), 711
(6), 501 (3), 286 (10), 252 (7), 213 (15), 192 (32), 197 (11), 185
(22), 181 (42), 165 (15), 149 (47), 131 (18), 119 (16), 105 (29),
91 (96), 73 (100), 57 (54).
Example 5(b)
[0245] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(2,3-dimethoxy-5-
-nitrobenzyloxy
carboxamido)]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-11--
hydroxy-5H-pyrrolo[1,2-c][1,4]benzodiazepine-5-one (32) 29
[0246] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(2,3-dimethoxy-5-
-nitrobenzyloxy
carboxamido)-5-methoxy-1,4-phenylene]carbonyl]bis-pyrrolid-
inemethanol (31)
[0247] A solution of 2-nitro-4,5-dimethoxybenzyl chloroformate
(0.443 g, 1.61 mmol, 2.3 eq) in dry DCM (10 ml) was added dropwise
over 20 minutes to a stirred solution of the amino alcohol 28 (0.4
g, 0.7 mmol) and pyridine (0.193 g, 2.54 mmol, 3.5 eq) in dry DCM
(15 ml) at 0.degree. C., under N.sub.2. TLC (10% MeOH/CHCl.sub.3)
revealed the reaction had gone to completion after 2 hours. The
reaction mixture was then diluted with chloroform (50 ml) and
washed with saturated aqueous solution of CuSO.sub.4 (2.times.50
ml), water (2.times.50 ml), brine (100 ml) and dried (MgSO.sub.4).
Removal of the solvent under reduced pressure gave a dark yellow
oil which was further purified by flash chromatography (7%
MeOH/CHCl.sub.3) to afford the pure product as a yellow foam.
[0248] Yield 0.564 (77%); [.alpha.].sup.20.sub.D: -43.3.degree. (c
0.485, CHCl.sub.3); IR (cm.sup.-1) 4214, 3416, 3020, 2973, 2940,
2613, 2400, 2254, 2075, 1727, 1618, 1585, 1522, 1465, 1438, 1408,
1331, 1278, 1216, 1174, 1118, 1071, 1030, 987, 909, 874, 850, 754;
.sup.1H-NMR (CDCl.sub.3, rotamers) .delta.8.93 (bs, 2H), 7.71 (s,
2H), 7.69 (s, 2H), 7.10 (s, 2H), 6.82 (s, 2H), 5.60 (m, 4H), 4.34
(m, 2H), 4.26 (t, J=6 Hz, 4H), 3.99 (s, 12H), 3.94 (s, 6H),
3.51-3.79 (m, 8H), 2.26-2.36 (m, 2H), 2.05-2.17 (m, 4H), 1.80-1.88
(m, 4H); .sup.13C-NMR (CDCl.sub.3) .delta.170.6, 153.7, 150.45,
148.1, 139.5, 132.9, 127.8, 111.2, 110.4, 109.9, 108.1, 65.4, 63.8,
62.4, 60.8, 56.4, 53.5, 51.4, 50.6, 28.2, 25.0, 21.0, 14.2; MS
(FAB) (m/z, relative intensity) 1053 (M.sup.++2, 7), 814 (6), 416
(3), 306 (13), 292 (5), 280 (27), 246 (46), 197 (81), 186 (38), 180
(25), 166 (37), 151 (19), 102 (23), 93 (100), 84 (10), 75 (37), 57
(42).
[0249] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(2,3-dimethoxy-5-
-nitrobenzyloxy carboxamido)]bis[(11aS)
-7-methoxy-1,2,3,11a-tetrahydro-11-
-hydroxy-5H-pyrrolo[1,2-c][1,4]benzodiazepine-5-one (32)
[0250] NMO (0.138 g, 1.16 mmol, 3.1 eq) and molecular sieve (350
mg) was added to a stirred solution of the NVOC dimer carbamate 31
(0.4 g, 0.38 mmol) in dry DCM and acetonitrile (9:3 ml) under
nitrogen. This mixture was allowed to stir for 15 minutes before
the addition of TPAP (13.8 mg, 0.116 mmol, 0.3 eq). Further
stirring of the reaction mixture for 2 hours at room temperature
was followed by the addition of an additional amount of TPAP (6.9
mg, 0.058 mmol, 0.15 eq) and NMO (69 mg, 1.58 mmol, 1.5 eq) to
drive the reaction to completion [TLC (5% MeOH/CHCl.sub.3)] after a
further 45 minute period of vigorous stirring. The organic solvent
was evaporated in vacuo to afford a black residue which was further
purified by column chromatography (1% MeOH/CHCl.sub.3) to afford
the pure final product as a dark yellow oil.
[0251] Yield 0.162 (39%); [.alpha.].sup.20.sub.D-121.5.degree. (c
1.07, CHCl.sub.3);IR (cm.sup.-1) 4329, 4258, 3426, 2926, 2854,
2728, 2360, 2341, 2046, 1712, 1620, 1583, 1523, 1464, 1408, 1330,
1278, 1219, 1172, 1149, 1071, 1030, 987, 871, 796, 759; .sup.1H-NMR
(CDCl.sub.3, rotamers) .delta.7.71 (s, 2H) , 7 62 (s, 2H), 7.10 (s,
2H), 6.43 (s, 2H), 5.48-5.59 (m, 6H), 4.29-4.36 (m, 2H), 4.24-4.28
(t, J=6 Hz, 4H), 3.95 (s, 12H), 3.88 (s, 6H), 3.47-3.53 (m, 8H),
2.32-2.43 (m, 2H), 2.07-2.17 (m, 4H), 1.67-1.92 (m, 4H);
.sup.13C-NMR (CDCl.sub.3) .delta.170.4, 153.8, 150.2, 148.9, 139.6,
130.9, 128.8, 127.6, 111.5, 110.6, 108.1, 86.0, 65.4, 65.0, 63.8,
60.9, 56.4, 46.4, 31.9, 28.2, 25.9, 22.7, 14.1.
Example 5(c)
[0252] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(phenylacetamide-
)]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-11-hydroxy-5H-pyrrolo[1,2-c][1-
,4]benzodiazepine-5-one (34) 30
[0253] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(phenylacetamide-
)-5-methoxy-1,4-phenylene]carbonyl]bis-pyrrolidinemethanol [33]
[0254] A catalytic quantity of DMF (4 drops) was added to a stirred
solution of phenyl acetic acid (0.2 g, 1.47 mmol, 2.1 eq) and
oxalyl chloride (0.224 g, 1.76 g, 2.5 eq) in dry acetonitrile (10
ml) and the reaction mixture was allowed to stir overnight at room
temperature under N.sub.2. The solution of the resulting acid
chloride was added dropwise over 30 minutes to a stirred solution
of the amine 28 (0.4 g, 0.7 mmol) in dry acetonitrile (40 ml) over
K.sub.2CO.sub.3 (0.406 g, 2.94 mmol, 4.2 eq) at -25.degree. C.,
under N.sub.2 and the reaction mixture was allowed to stir for a
further 1.5 hours. The reaction mixture was diluted in chloroform
(50 ml) and washed with 1M HCl (2.times.50 ml), water (2.times.75
ml), brine (100 ml), dried (MgSO.sub.4). Excess solvent was
evaporated in vacuo to give a dark yellow oil which, after flash
chromatography (10% MeOH/CHCl.sub.3), afforded the pure
phenylacetamide protected dimer amino alcohol as a pale yellow
oil.
[0255] Yield 0.344 (64%); .sup.1H-NMR (CDCl.sub.3, rotamers)
.delta.9.31 (bs, 2H), 7.59 (s, 2H), 7.35 (s, 10H), 6.68 (s, 2H),
4.20-4.28 (m, 6H), 4.14-4.18 (m, 4H), 3.71 (s, 6H), 3.24-3.56 (m,
8H), 2.27-2.39 (m, 2H), 2.02-2.17 (m, 4H), 1.64-1.81 (m, 4H);
.sup.13C-NMR (CDCl.sub.3) .delta.169.7, 150.1, 134.7, 130.2, 129.4,
128.9, 127.3, 110.5, 108.2, 65.3, 61.1, 56.3, 50.8, 44.7, 29.7,
28.3, 24.7.
[0256] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(phenylacetamide-
)]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-11-hydroxy-5H-pyrrolo[1,2-c][1-
,4]benzodiazepine-5-one (34)
[0257] NMO (0.141 g, 1.2 mmol, 3.1 eq) and mol. sieve (350 mg) was
added to a stirred solution of the phenylacetamide protected dimer
carbamate 33(0.3 g, 0.38 mmol) in dry DCM and acetonitrile (9:3 ml)
under nitrogen. This mixture was allowed to stir for 20 minutes
before the addition of TPAP (14.1 mg, 0.12 mmol, 0.31 eq). Further
stirring of the reaction mixture for 1.5 hours at room temperature
was followed by the addition of an additional amount of TPAP (7 mg,
0.06 mmol, 0.15 eq) and NMO (70 mg, 0.6 mmol, 1.5 eq) to drive the
reaction to completion [TLC (5% MeOH/CHCl.sub.3)] after a further
45 minute period of vigorous stirring. The organic solvent was
evaporated in vacuo to afford a black residue which was further
purified by column chromatography (1% MeOH/CHCl.sub.3) to afford
the pure final product as a dark yellow oil.
[0258] Yield 0.138 g (47.5%); [.alpha.].sup.20.sub.D+1320 (c 0.265,
CHCl.sub.3); IR (cm.sup.-1) 3412, 2959, 2924, 2854, 2094, 1642,
1462, 1377; .sup.1H-NMR (CDCl.sub.3, rotamers) .delta.7.28-7.39 (s,
10H), 7.15 (s, 2H), 6.46 (s, 2H), 5.75-5.78 (d, J=10 Hz, 2H),
4.10-4.35 (m, 10H), 3.89 (s, 6H), 3.35-3.73 (m, 8H), 2.27-2.32 (m,
2H), 1.68-2.05 (m, 8H); .sup.13C-NMR (CDCl.sub.3) .delta.169.9,
153.2, 147.6, 143.8, 142.8, 128.2, 123.9, 113.9, 110.9, 108.3,
86.5, 69.1, 66.3, 65.6, 60.0, 56.4, 46.4, 29.7, 28.7, 23.1,
14.8.
Example 5(d)
[0259] Alternative Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(4-n-
itrobenzyloxycarboxamido)]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-11-hyd-
roxy-5H-pyrrolo[2-c][1,4]benzodiazepine-5-one (30) 31
[0260] Synthesis of
1,1'-[[(propane-1,3-diyl)dioxy]bis[(2-nitro-5-methoxy1-
,4-phenylene)
carbonyl]]bis[pyrrolidine-2-carboxaldehydediethyldithioaceta- l)
(35)
[0261] 3-4 drops of DMF was added to a stirred suspension of the
dimer nitro acid 26 (0.25 g, 1.01 mmol) and oxalyl chloride (0.3 g,
2.33 mmol, 2.3 eq) in dry THF volume and the reaction mixture was
allowed to stir overnight under nitrogen. The resulting acid
chloride was added dropwise, over 20 min, to a stirred solution of
(2S)-pyrrolidine-2-carbaldehyde diethyl thioacetal (0.416 g, 2.02
mmol, 2 eq) and triethylamine (0.41 g, 4.04 mmol, 4 eq) at
0.degree. C., under N.sub.2. The reaction mixture was allowed to
warm to room temperature and stirring was continued for a further
1.5 hours. Excess THF was then removed and the residue was diluted
with water (5 ml) and extracted with EtOAc (15 ml). The pH of the
aqueous phase was adjusted to pH 3 with two drops of conc. HCl and
subsequently extracted with EtOAc (3.times.10 ml). The combined
organic phase was washed with water (2.times.10 ml), brine (15 ml),
dried (MgSO.sub.4) and the organic solvent was evaporated in vacuo
to afford a dark red oil. Purification by flash chromatography
(EtOAc:Petroleum ether 40-60, 1:1) afforded the pure product
35.
[0262] Yield 0.558 g (66%); .sup.1H-NMR (CDCl.sub.3) .delta.7.72
(s, 2H), 6.83 (s, 2H), 4.89 (d, J=4.0 Hz, 2H), 4.64-4.70 (m, 2H),
4.30-4.40 (m, 4H), 3.91 (s, 6H), 3.15-3.30 (m, 4H), 2.59-2.76 (m,
8H), 1.70-2.38 (m, 8H), 1.30-1.38 (m, 12H).
[0263] Synthesis of
1,1'-[[(propane-1,3-diyl)dioxy]bis[(2-amino-5-methoxy1-
,4-phenylene)
carbonyl]]bis[pyrrolidine-2-carboxaldehydediethyldithioaceta-
l](36)
[0264] A solution of the nitro thioacetal 35 (0.4 g, 0.47 mmol) and
SnCl.sub.22H.sub.2O (1.4 g, 6.22 mmol, 13.2 eq) in methanol (7 ml)
was heated at reflux for 40 minutes at which time TLC
(EtOAc:Petroleum ether 40-60, 1:1) indicated the reaction was
complete. After the reaction mixture was allowed to return to room
temperature the pH was adjusted to pH 8 by the addition of sat. aq.
NaHCO.sub.3: The resulting suspension was diluted with EtOAc (100
ml) and it was allowed to stir overnight under nitrogen. The
organic phase was separated and washed with brine (50 ml), dried
(MgSO.sub.4) and the solvent was removed under reduced pressure to
give a dark yellow gum which was further purified by flash
chromatography (EtOAc:Petroleum ether 40-60, 3:7) to afford the
pure dimer amino thioacetal as a bright yellow oil.
[0265] Yield 0.281 (56%); .sup.1H-NMR (CDCl.sub.3) .delta.6.82 (s,
2H), 6.29 (s, 2H), 5.30 (d, J=4.0 Hz, 2H), 4.69-4.87 (m, 2H),
4.08-4.21 (m, 4H), 3.76 (s, 6H), 3.60-3.66 (m, 4H), 2.65-2.73 (m,
8H), 1.80-2.35 (m, 8H), 1.22-1.31 (m, 12H).
[0266] Synthesis of
1,1'-[[(propane-1,3-diyl)dioxy]bis[(2-(4-nitrobenzylox-
ycarbonylamine-5-methoxyl,4-phenylene)
carbonyl]]bis[pyrrolidine-2-carboxa-
ldehydediethyldithioacetal](37)
[0267] A solution of 4-nitrobenzyl chloroformate (0.100 g, 0.46
mmol, 2 eq) in dry DCM (10 ml) was added dropwise to a solution of
the bis amine 36 (0.18 g, 0.23 mmol) and pyridine (0.101 g, 1.28
mmol, 5.5 eq) in dry DCM (15 ml) over 20 minutes at 0.degree. C.,
under nitrogen. The reaction solution was allowed to stir at
0.degree. C. for a further 1.5 hours (TLC: 7% MeOH/CHCl.sub.3),
after which time it was allowed to warm to room temperature and
diluted with chloroform (50 ml). The organic phase was washed with
sat. aq. CuSO.sub.4 (2.times.50 ml), water (75 ml), brine (75 ml),
dried (MgSO.sub.4) and the excess solvent evaporated in vacuo to
give a dark yellow oil. After purification by flash chromatography
(7% MeOH/CHCl.sub.3) the pure product was obtained as a dark yellow
oil.
[0268] Yield 0.179 (68%); [.alpha.].sup.20.sub.D+100.degree. (c
0.232 CHCl.sub.3); IR (cm.sup.-1) 4214, 3426, 3020, 2400, 2085,
1658, 1609, 1525, 1466, 1452, 1408, 1348, 1268, 1216, 1174, 1112,
1052, 1015, 908, 753; .sup.1H-NMR (CDCl.sub.3, rotomers)
.delta.9.18 (bs, 2H), 8.19-8.24 (d, J=8.8 Hz, 4H), 6.82 (s, 2H),
6.29 (s, 2H), 5.30 (d, J=4.0 Hz, 2H), 4.69-4.87 (m, 2H), 4.08-4.21
(m, 4H), 3.76 (s, 6H), 3.60-3.66 (m, 4H), 2.65-2.73 (m, 8H),
1.80-2.35 (m, 8H), 1.22-1.31 (m, 12H).
[0269] Synthesis of
1,1'-[[(Propane-1,3-diyl)dioxy]bis[[2-(4-nitrobenzylox-
ycarboxamido)]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-11-hydroxy-5H-pyrr-
olo[1,2-c][1,4]benzodiazepine-5-one (30)
[0270] Mercury chloride (1.6 g, 5.88 mmol, 4.5 eq) was added to a
slowly stirred solution of the N-protected amino thioacetal 37(0.15
g, 0.13 mmol) and CaCO.sub.3 (65 mg, 0.65 mmol, 5 eq) in
acetonitrile/water (4:1, 5 ml) and stirring continued at room
temperature for 24 hours. The reaction mixture was diluted with
EtOAc (20 ml) and filtered through Celite. The filtrate was washed
with sat. aq. NaHCO.sub.3 (2.times.15 ml), brine (20 ml), dried
(MgSO.sub.4) and evaporated under reduced pressure to afford a
yellow oil which was further purified by column chromatography (0
to 2% MeOH/CHCl.sub.3) to afford the dimer prodrug compound 30
(Yield=67.3 mg (56%))
Example 5(e)
[0271] Alternative Synthesis of Intermediate (26) in Examples 5(a)
to 5(d) 32
[0272] Synthesis of 1',3'-Bis(4-carbonyl-2-methoxyphenoxy)propane
(39)
[0273] A solution of diethyl azodicarboxylate (4.57 g, 26 3 mmol, 1
eq) in dry THF (15 ml) was added dropwise over 15 minutes to a
solution of vanillin (38) (10.4 g, 68.3 mmol, 2.6 eq),
1,3-propanediol (2 g, 26.3 mmol, 1 eq) and triphenylphosphine (6.87
g, 26.3 mmol, 1 eq) in dry THF (50 ml) under nitrogen. The reaction
mixture was allowed to stir overnight and diluted with chloroform
(70 ml), washed with 1M NaOH (2.times.75 ml). The organic solvent
was removed by rotary evaporation and the residue was triturated
with toluene (150 ml) for 24 hours. Triphenylphosphine oxide was
removed by filtration and the filtrate was washed with NaOH (100
ml), dried (MgSO.sub.4), evaporated in vacuo and the remaining
opaque oil was purified by flash chromatography (100% CHCl.sub.3)
to afford a white solid.
[0274] Yield 6.27 (70%); .sup.1H-NMR (CDCl.sub.3+DMSO-d.sub.6)
.delta.9.84 (s, 2H), 7.62-7.66 (d, J=8.3 Hz, 2H), 7.38 (s, 2H),
7.02-7.05 (d, J=8.4 Hz, 2H), 4.24-4.28 (t, J=6.1 Hz, 4H), 3.91 (s,
6H), 2.05-2.12 (t, J=6.2 Hz, 2H); .sup.13C-NMR
(CDCl.sub.3+DMSO-d.sub.6) .delta.190.8, 154.0, 149.6, 131.9, 129.7,
128.5, 126.7, 11.5, 109.1, 66.6, 61.4, 58.8, 55.9, 31.8, 14.5; MS
(FAB) (m/z, relative intensity) 344 (M.sup.+, 2), 210 (54), 152
(100), 123 (9), 109 (14), 81 (11), 65 (10), 51 (11).
[0275] Synthesis of
1',3'-Bis(4-carbonyl-2-methoxy-5-nitrophenoxy)propane (40)
[0276] The dimer aldehyde (39) (5 g, 14.5 mmol) was added in small
portions over a period of 30 minutes to HNO.sub.3 (100 ml, 70%) at
0.degree. C. The resulting suspension was stirred for a further 30
minutes at 15.degree. C. when it was poured onto ice-water and the
bright yellow precipitate was collected by filtration, washed with
cold water and dried to afford the nitrated intermediate.
[0277] Yield 4.63 g (74%); .sup.1H-NMR (CDCl.sub.3+DMSO-d.sub.6)
.delta.10.41 (s, 2H), 7.67 (s, 2H), 7.37 (s, 2H), 4.41-4.45 (t,
J=6.1 Hz, 4H), 3.99 (s, 6H), 2.87-2.92 (t, J=6.2 Hz, 2H);
.sup.13C-NMR (CDCl.sub.3+DMSO-d.sub.6) .delta.187.7, 172.2, 153.4,
151.5, 143.6, 132.0, 128.6, 125.6, 110.3, 109.9, 108.4, 65.8, 56.6,
33.8; MS (EI) (m/z, relative intensity) 434 (M.sup.+, 1), 277 (33),
269 (37), 214 (13), 197 (21), 167 (100), 152 (21), 122 (20), 111
(15), 96 (10), 79 (13), 72 (32).
[0278] Synthesis of 1',3'-Bis
(4-carboxy-2-methoxy-5-nitrophenoxy)propane (26)
[0279] Hot aqueous KMnO.sub.4 (10% w/v, 200 ml) was added dropwise
to a solution of the aldehyde (40) (4 g, 9.21 mmol) in acetone (300
ml) over 15 min. The resulting mixture was stirred for 40 minutes
and the insoluble material was removed by filtration through
celite. The pad was washed with hot water and the combined filtrate
concentrated in vacuo. The remaining aqueous phase was acidified
with conc. HCl to afford a pale yellow precipitate which was
collected by filtration, washed with water and dried to give the
dimer acid (26) (Yield 3.08 g (71.8%)).
Example 6
[0280] Synthesis of a Psec-protected PBD (52) and Related
Ptec-Protected PBD (55a/b) (for Comparison) 33
[0281] Synthesis Secondary Amine 45
[0282] Lithium tetrahydroborate (2.6 g, 0.12 mol) was added
portionwise to a solution of N-carbobenzyloxy-L-proline methyl
ester 41 (21 g, 0.08 mmol) in THF (500 ml) at 0.degree. C. The
reaction mixture was allowed to stir at room temperature for 48
hours. The solution was then cooled to 0.degree. C. and ice water
(150 ml) was added to quench excess lithium tetrahydroborate. The
resulting suspension was adjusted to pH 4.0 with aqueous HCl (1.0
N) and extracted with Et.sub.2O (250 ml). The organic phase was
separated and washed with H.sub.2O (3.times.100 ml), brine
(2.times.100 ml), dried (MgSO.sub.4) and concentrated to give
alcohol 42 as a pale yellow oil (18.6 g, 99%). .sup.1HNMR (270 MHz,
CDCl.sub.3) d 2.1-1.77 (m, 4H); 3 76-3.35 (m, 4H); 4.1-3.77 (m,
1H); 5.14 (2.times.s, 2H); 7.38-7.28 (m, 5H). CIMS 236
(M.sup.+)
[0283] A solution of triethylamine (32 ml, 0.23 mol) and
SO.sub.3.pyridine complex (37 g, 0.23 mol) in DMSO (210 ml) was
added to a solution of alcohol 42 (18 g, 0.077 mol) in
CH.sub.2Cl.sub.2 (250 ml) at -10.degree. C., under a nitrogen
atmosphere. The reaction mixture was allowed to warm to room
temperature and stirred for 30 minutes and then poured into ice
water, (200 ml) and extracted with Et.sub.2O. The organic phase was
washed with aqueous HCl (1.0 N, 3.times.150 ml), H.sub.2O
(3.times.150 ml), brine (2.times.150 ml), dried (MgSO.sub.4) and
concentrated to give a yellow oil. The crude material was purified
by flash column chromatography (silica gel, EtOAc) to give aldehyde
43 as a colourless oil (12.6 g, 71%). .sup.1H NMR (270 MHz,
CDCl.sub.3) .delta.2.16-1.8 (m, 4H); 3.66-3.5 (m, 2H); 4.33-4.17
(m, 1H); 5.22-5.13 (m, 2H); 7.37-7.3 (m, 5H); 9.59 (2.times.s, 1H).
CIMS 234 (M.sup.++1).
[0284] Thionyl chloride (5.5 ml) was added to a solution of
aldehyde 43 (11 g, 0.047 mol) and trimethyl orthoformate (36 ml,
0.33 mol) in MeOH (55 ml) at 0.degree. C. The reaction mixture was
heated at 60.degree. C. for 2 hours. The solution was allowed to
cool to room temperature, and treated with excess solid
Na.sub.2CO.sub.3 and diluted with Et.sub.2O (60 ml). The suspension
was filtered to remove insoluble inorganics and resultant filtrate
was concentrated in vacuo and then redissolved in EtOAc. The
organic solution was washed with saturated aqueous NaHCO.sub.3
(3.times.50 ml), brine (2.times.50 ml), dried (MgSO.sub.4) and
concentrated to give acetal 44 as a yellow liquid (12.5 g, 95%).
.sup.1H NMR (270 MHz, CDCl.sub.3) .delta.2.16-1.7 (m, 4H);
3.64-3.33 (m, 8H); 4.02-3.91 (br. m, 1H); 4.4 and 4.6 (2.times.br.
s, 1H); 5.17-5.1 (m, 2H); 7.47-7.28 (m, 5H).
[0285] A solution of acetal 44 (5.8 g, 0.02 mol) in EtOH (50 ml)
was allowed to stir for 16 hours at room temperature over Raney
nickel (0.2 g), in order to remove the trace amounts of sulphur
impurities prior to hydrogenation. Nickel was removed by filtration
through Celite.
[0286] 10% Palladium on carbon (580 mg) was added to the alcoholic
solution which was subjected to hydrogenation under pressure (c 50
psi). After 16 h, the reaction mixture was filtered through Celite
and the pad was washed with EtOAc, the combined organic solutions
were concentrated to give secondary amine 45 as a pale green liquid
(2.9 g, 100%). .sup.1H NMR (270 MHz, CDCl.sub.3) .delta.1.93-1.59
(m, 4H); 3.1-2.92 (m, 2H); 3.4-3.3 (d, J=6.9 Hz, 1H); 3.41
(2.times.s, 6H); 3.53 (br.s, 1H); 4.2 (d, J=6.8 Hz, 1H).
[0287] Synthesis of Amine 48
[0288] A solution of acetal 45 (1 g, 6.9 mmol), 4,5-dimethoxy
2-nitrobenzoic acid 46 (1.6 g, 6.9 mmol), TBTU (2.2 g, 6.9 mmol)
and DIPEA (1.2 ml, 6.9 mmol) in DMF (30 ml) were stirred at room
temperature for 16 hours. The reaction mixture was concentrated in
vacuo and the crude material was extracted with EtOAc and washed
with saturated aqueous NaHCO, (3.times.30 ml), HCl (1.0 N,
3.times.30 ml), H.sub.2O (3.times.30 ml), brine (3.times.30 ml),
dried (MgSO.sub.4) and concentrated to give a yellow semi solid.
The crude material was purified by flash column chromatography
(silica gel, 2:1 EtOAc petroleum ether 40-60) to give the nitro
compound 47 as a pale cream solid (1.28 g, 51%). .sup.1H NMR (270
MHz, CDCl.sub.3) .delta.2.26-1.67 (m, 4H); 3.19-3.06 (m, 2H); 3.59
and 3.57 (2.times.s, 6H); 3.98 (s, 6H); 4.45-4.4 (m, 1H); 4.95-4.94
(d, J=2.6 Hz, 1H); 6.76 (s, 1H); 7.71 (s, 1H); CIMS 355
(M.sup.++1).
[0289] 10% Palladium on carbon (130 mg) was added to a solution of
the nitro compound 47 (1.28 g, 3.6 mmol) in EtOH (50 ml), which was
subjected to hydrogenation under pressure (c. 50 psi). After 20
hours, the reaction mixture was filtered through Celite and the pad
was washed with EtOAc, the combined organic solutions were
concentrated to give secondary amine 48 as a pale oil (1.26 g,
98%). .sup.1H NMR (270 MHz, CDCl.sub.3) .delta.2.18-1.65 (m, 4H);
3.57-3.47 (m, 8H); 3.85 and 3.8 (2.times.s, 6H); 4.42-4.38 (m, 1H);
4.74-4.7 (m, 1H); 6.3 (s, 1H); 6.77 (s, 1H); .sup.13C NMR (68.7
MHz, CDCl.sub.3) .delta.24.03, 25.07, 50.56, 55.8, 56.24, 56.86,
57.65, 58.82, 101.07, 105.01, 111.75, 112.52, 141.07, 151.81,
169.76; EIMS 324 (M.sup.+).
[0290] Synthesis of Psec-Protected PBD 52
[0291] Pyridine (55 .mu.l, 0.67 mmol) was added to a solution of
2-(phenylsulfonyl)ethanol 49 (375 mg, 2.01 mmol) and triphosgene
(200 mg, 0.67 mmol) in CH.sub.2Cl.sub.2 (10 ml) at 0.degree. C. The
reaction mixture was stirred at room temperature for 16 hours.
Pyridine (150 .mu.l, 1.85 mmol) and crude chloroformate 50 were
added to a solution of amine 48 (0.5 g, 1.5 mmol) in
CH.sub.2Cl.sub.2 (20 ml) at 0.degree. C. The reaction mixture was
allowed to stir at room temperature for 3 hours. Then the solution
was concentrated in vacuo and extracted with CH.sub.2Cl.sub.2. The
organic phase was washed with 10% citric acid (2.times.20 ml),
H.sub.2O (20 ml), brine (20 ml), dried (MgSO.sub.4) and
concentrated to give a yellow oil. The crude material was purified
by flash column chromatography (silica gel, EtOAc) to give
carbamate 51, as a colourless oil (770 mg, 93%).
[0292] .sup.1H NMR (270 MHz, CDCl.sub.3) .delta.2.17-1.72 (m, 6H),
3.55-3.34 (m, 10H), 3.92 and 3.84 (2.times.s, 6H), 4.5-4.4 (m, 2H),
4.75 (br.s, 1H), 6.82 (s, 1H), 7.97-7.56 (m, 6H), 8.89 (br.s, 1H);
.sup.13C NMR (68.7 MHz, CDCl.sub.3) .delta.14.19, 21.04, 23.88,
55.31, 56.12, 56.31, 56.39, 57.52, 58.25, 58.94, 60.38, 103.91,
104.74, 111.15, 127.97, 128.09, 129.43, 133.98, 134.05, 139.29,
143.75, 151.09, 152.72, 168.83; FABMS 536 (M.sup.+).
[0293] Trans-bis(acetonitrile)palladium (II) chloride (107 mg, 0.41
mmol) was added to a solution of carbamate 51 in anhydrous acetone
(14 ml). The reaction mixture was stirred at room temperature for
16 hours. The solution was concentrated in vacuo to give a brown
foam. The crude material was purified by flash column
chromatography (silica gel, EtOAc) to give methyl ether 52, as an
orange oil (690 mg, 85%).
[0294] .sup.1H NMR (270 MHz, CDCl.sub.3) .delta.2.12-1.78 (m, 6H),
3.22-3.17 (m, 1H), 3.7-3.4 (m, 7H), 3.98 and 3.94 (2.times.s, 6H),
4.69-4.65 (m, 1H), 5.46-5.43 (d, J=9.3 Hz, 1H), 7.03 (s, 1H), 7.22
(s, 1H), 7.87-7.53 (m, 5H); .sup.13C NMR (68.7 MHz, CDCl.sub.3)
.delta.14.19, 21.06, 23.21, 28.98, 46.33, 54.74, 56.15, 56.39,
56.45, 58.58, 60.09, 60.39, 93.33, 110.18, 113.32, 126.24, 128.07,
128.27, 129.49, 134.13, 138.59, 148.77, 151.15, 155.65, 167.36;
EIMS 504 (M.sup.+).
[0295] Synthesis of Ptec-PBD 55a/b 34
[0296] Pyridine (65 .mu.l, 0.77 mmol) was added to a solution of
2-(phenylthio)ethanol 53 (355 mg, 2.3 mmol) and triphosgene (230
mg, 0.77 mmol) in CH.sub.2Cl.sub.2 (10 ml) at 0.degree. C. The
reaction mixture was stirred at room temperature for 16 hours.
Pyridine (150 .mu.l, 1.85 mmol) and crude chloroformate 54 were
added to a solution of amine 47 (0.5 g, 1.5 mmol) in
CH.sub.2Cl.sub.2 (20 ml) at 0.degree. C. The reaction mixture was
allowed to stir at room temperature for 3 hours. Then the solution
was concentrated in vacuo and extracted with CH.sub.2Cl.sub.2. The
organic phase was washed with HCl (1.0 N. 2.times.20 ml), H.sub.2O
(20 ml), brine (20 ml), dried (MgSO.sub.4) and concentrated to give
a brown oil. The crude material was purified by flash column
chromatography (silica gel, 2:1 EtOAc: petroleum ether 40-60) to
give a mixture of carbinolamine 55a and methyl ether 55b, as a
yellow gum (510 mg, 66%).
[0297] .sup.1H NMR (270 MHz, CDCl.sub.3) .delta.2.1-1.99 (m, 4H);
3.15-2.98 (m, 2H), 3.71-3.44 (m, 4H), 3.93 and 3.89 (2.times.s,
6H), 4.38-4.07 (m, 1H), 5.45-5.41 (d, J=9.3 Hz, 1H), 5.65-5.61 (d,
J=9.5 Hz, 1H), 6.76 (s, 1H), 7.29-7.19 (m, 6H); .sup.13C NMR (68.7
MHz, CDCl.sub.3) .delta.23.05, 23.21, 28.7, 28.98, 29.36, 29.68,
32.72, 32.99, 46.36, 56.13, 56.19, 56.59, 59.9, 60.13, 63.91,
64.19, 86.07, 93.37, 110.36, 112.55, 112.85, 125.67, 126.69, 128.3,
129.15, 129.72, 129.97, 134.85, 148.36, 150.83,. 156.02, 167.05;
EIMS 458 (M.sup.+, 55a), 472 (M.sup.+, 55b).
Example 7
[0298] Nitroreductase-Activation of benzyl DC-81 Prodrug (Compound
7)
[0299] Compound 7, synthesized according to example 1, was
evaluated in two different cell lines, namely SW1116 and LS174T
Both SW1116 and LS174T are human adenocarcinoma colonic cell lines,
which were grown at Charing Cross hospital. The cells, at a
concentration of 2500 cells/ml, were plated in 96-well microtitre
plates, and were incubated at 37.degree. C. for 1 hour with
different concentrations of the prodrug, in the presence or absence
of E. coli nitroreductase-monoclonal antibody conjugate in
phosphate buffer saline (available from Sigma), and NADH (the
co-factor necessary for enzyme function) in DMSO. The cells were
then washed and incubated for a further 3 days at 37.degree. C. At
the end of this period, the cells were fixed (TCA) and stained. The
concentration of the remaining viable cells adhering to the plates
was quantified by a sulforhodamine B (SRB) dye. The control sets of
cells treated with the prodrug alone were used in order to evaluate
the cytotoxicity of the compound prior to enzymatic activation.
[0300] The results are illustrated in FIG. 4. The prodrug alone was
found to be essentially non-toxic in SW1116 cells even at
concentrations of up to 500 .mu.M. A slight toxicity was observed
in the LS174T cell line at concentrations higher than 100 .mu.M. In
the presence of the enzyme and co-factor, the IC.sub.50 of compound
7 was established as 1-5 .mu.M in both cell lines.
[0301] The parent drug, benzyl DC-81 (Thurston et al, 1990, Chem.
Brit., 26, 767-772), was also evaluated in the same cell lines
under the same conditions in order to establish the extent of
activation achieved by the prodrug/enzyme system. A difference was
observed between the IC.sub.50 value of the parent drug
(IC.sub.50=0.008 .mu.M) and the nitroreductase-activated prodrug
(IC.sub.50=1-5 .mu.M) of 20-100 fold.
Example 8
[0302] Nitroreductase-Activation of DSB-120 Prodrug (Compound
30)
[0303] Compound 30, see examples 5(a) and 5(e), was evaluated in
the LS174T cell line, under the same conditions as were used in
example 6. The IC.sub.50 value was found to be 215.3 .mu.M (average
of two measurements) which reduced to 13.7 .mu.M after the addition
of the enzyme and co-factor, representing a 15-16 fold activation
factor. The cytotoxicity (IC.sub.50) of the parent agent DSB-120 in
this cell line was found to be 0.0005 .mu.M, indicating less
efficient activation of the dimer prodrug compared to compound
7.
Example 9
[0304] Nitroreductase-Activation of benzyl tomaymycin Prodrug
(Compound 24)
[0305] Compound 24, see example 4, was examined in the same cell
lines as example 6, and under the same conditions. It exhibited an
IC.sub.50 value of 86.2 .mu.M (average of two experiments) which,
after addition of the enzyme conjugate and NADH, dropped to 6.4
.mu.M, indicating an activation factor of approximately 13.5. In
this case, the parent benzyl tomaymycin was unavailable for
evaluation as a control. However, assuming that the addition of a
benzyl group to the C8-position of the A-ring of tomaymycin does
not change the cytotoxicity of the compound significantly (as is
the case for DC-81/C8-benzyl DC-81), the cytotoxicity of C8-benzyl
tomaymycin should be in the order of 0.01-0.00 .mu.M.
Example 10
[0306] Light-Activation of the benzyl DC-81 Prodrug (Compound
11)
[0307] This experiment involved the irradiation of compound 11, see
example 3, in DMF at a concentration of 1 mM using a Stratagene UVA
Crosslinker(365 nm). Small aliquots (100 .mu.L) were removed at 30
minutes, 1 hour, 2 hours and 3 hours and the cleavage of the
photolabile NVOC group was monitored by HPLC, using a Waters C4,
300 angstrom reversed phase column and a mobile phase of 50%.
methanol/50% water. Compound 11 has a retention time of 11.25
minutes and upon radiation produced a new peak with a retention
time of 8.58 minutes. Authentic benzyl DC-81 has an identical
retention time of 8.58 minutes. The time course of the deprotection
process is shown in FIG. 5. Complete conversion is achieved by 2
hours of irradiation at 365nm under the conditions employed.
[0308] An MTT assay was used to evaluate the ability of aliquots of
the activated analogues, which were removed at time intervals, to
inhibit the growth of chronic human histiocytic leukaemia K562
cells in culture. Following treatment of cells with a range of drug
doses, cells were transferred to 96-well microtitre plates,
10.sup.4 cells per well, 8 wells per sample. Plates were incubated
at 37.degree. C. in a humidified atmosphere containing 5% CO.sub.2.
The assay is based on the ability of viable cells to reduce a
yellow soluble tetrazolium salt
3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT), to
an insoluble purple formazan precipitate. Following incubation of
the plates for 4 days (to allow control cells to increase 10-fold
in number), 20 .mu.L of a 5 mg/mL solution of MTT in
phosphate-buffered saline was added to each well and the plates
were incubated for a further 5 hours. The plates were then
centrifuged for 5 minutes at 300 g and the bulk of the medium was
removed from the cell pellet. DMSO (200 .mu.L) was added to each
well, and the samples agitated to ensure complete mixing. The
optical density was then measured at a wavelength of 550 nm on a
Titertek Multiscan ELISA plate reader and expressed as a percentage
of the control optical density. For each curve, an IC.sub.50 value
was read as the dose required to reduce the final optical density
to 50% of the control value.
[0309] The PBD prodrug compound 11 investigated was itself shown to
have negligible cytotoxicity (IC.sub.50=47.5 .mu.M; FIG. 6).
Following irradiation, significantly increased cytotoxicity was
observed (FIG. 6) with an IC.sub.50 value of 0.6 .mu.M achieved
after 1 hour of irradiation. The IC.sub.50 value of authentic
benzyl DC-81 under similar conditions was 0.5 .mu.M.
1 Irradiation Time IC.sub.50 Compound (hours).sup.+ (.mu.M) benzyl
DC-81* 0 0.5 11 0 47.5 0.5 0.98 1.0 0.60 2.0 0.60 *comparative
example .sup.+irradiation was in DMF at 1 mM initial drug
concentration
[0310] The efficiency of photoinduced cleavage was reduced at the
higher concentration of 10 mM of prodrug in DMF, when 2 hours
irradiation gave an IC.sub.50value of 1.75 .mu.M, see FIG. 7.
2 Irradiation Time IC.sub.50 Compound (hours).sup.+ (.mu.M) benzyl
DC-81* 0 0.5 11 0 48.5 0.5 4.33 1.0 2.17 2.0 1.75 *comparative
example .sup.+irradiation was in DMF at 10 mM initial drug
concentration
[0311] It was thought that the high concentration of prodrug might
be preventing efficient absorption of UV light. Furthermore,
changing the solvent from DMF to methanol resulted in a less
efficient conversion, see FIG. 8.
3 Irradiation Time IC.sub.50 Compound (hours).sup.+ (.mu.M) benzyl
DC-81* 0 0.5 11 0 47.5 0.5 4.5 1.0 4.0 2.0 3.2 *comparative example
.sup.+irradiation was in DMF at 1 mM initial drug concentration
Example 11
[0312] Light-activation of the dimer DSB-120 Prodrug (Compound
32)
[0313] The dimer derivative 32, see example 5(b), was found to be
slightly cytotoxic before UV irradiation with an IC.sub.50 of 4.5
.mu.M. Incubation of a solution of compound 32 with cells following
2 hours irradiation reduced the IC.sub.50 to 1.25 .mu.M (FIG. 9).
This was further reduced to 0.85 .mu.M following 5 hours
irradiation. The parent PBD dimer DSB-120 gave an IC.sub.50 of 0.55
.mu.M in the same cell line, see FIG. 9.
4 Irradiation Time IC.sub.50 Compound (hours).sup.+ (.mu.M)
DBS-120* 0 0.55 32 0 4.50 0.5 1.90 1.0 1.75 2.0 1.25 5.0 0.85
*Comparative example .sup.+irradiation was in DMF at 1 mM initial
drug concentration
[0314] For these experiments, the HPLC data (not shown) suggested
that almost complete conversion of the prodrug 32 (retention time
5.18 minutes) to a product with retention time=3.64 minutes
occurred by 2 hours irradiation, with complete conversion by 5
hours. Authentic DSB-120 eluted with a retention time of 3.64
minutes.
Example 12
[0315] Comparison of Biological Activity of Psec and Ptec Protected
PBDs (Compounds 52 and 55)
[0316] Compounds 52 (UP 2073) and the comparative compound 55b
(UP2090) were evaluated for their cytotoxic activity in ovarian
cell lines by Dr Lloyd R. Kelland's group at The Institute of
Cancer Research, Sutton, UK. The five cell lines investigated were
SKOV-3, A2780/A2780cisR and CH1/CH1cisR (cisR denotes that the cell
line is resistant to cisplatin).
[0317] Single viable cells were seeded in growth medium (160 .mu.L)
in 96-well microtitre plates and allowed to attach overnight. The
test compounds were then dissolved in DMSO (to give 20 mM drug
concentrations) immediately prior to adding the cells in
quadruplicate wells The final drug concentrations in the wells
ranged from 100 .mu.M to 2.5 nM as follows:100, 25, 10, 2.5, 1
.mu.M, 250, 100, 25, 10, 2.5 nM (drugs were diluted in growth
medium and then 40 .mu.L added to the existing well volume of 160
.mu.L to give final concentrations as above). After 96 hours, the
medium was removed and the remaining cells fixed by exposure to
10%. trichloroacetic acid on ice for 30 minutes. The wells were
then washed 3-4 times with tap water, air dried overnight and
treated with 100 .mu.L of sulphorhodamine B (0.41) dissolved in 1%
acetic acid. Staining was allowed to continue for 10-15 minutes,
then the wells were washed 3-4 times with 1% acetic acid, air dried
and then added to Tris base (100 .mu.L of 10 mM). Plates were then
shaken and absorbance readings at 540 nM were determined using a
plate reader. The IC.sub.50 values were calculated from plots of
concentration versus percentage absorbance (compared with 8
untreated wells).
[0318] The assay was also carried out using compound 56 (UP2025):
35
[0319] which is the unprotected version of compound 52.
[0320] The results are shown below:
5 UP2090 UP2073 UP2025 Cell Lines IC.sub.50 (.mu.M) IC.sub.50
(.mu.M) IC.sub.50 (.mu.M) A2780 >25 0.48 0.064 A2780cisR >25
0.49 0.155 RF N/A 1 2.4 CH1 >25 0.4 0.082 CH1cisR >25 0.47
0.11 RF N/A 1.2 1.3 SKOV3 >25 0.56 1.7
[0321] RF is the resistance factor, which is the cytotoxicity of
the compound in the cisplatin resistant cell line divided by the
cytotoxicity in the normal cell line.
[0322] The Ptec protected PBD (55b), UP2090, was essentially
inactive in against the ovarian cell lines, as expected as the Ptec
protecting group lacks the acidic protons thought to be required to
trigger the fragmentation of the protecting group when exposed to
GST. However, the Psec-protected prodrug (52) UP2073 was found to
be at least 50 times more toxic than the Ptec control. The compound
was particularly active against the SKOV3 cell line; this is
noteworthy as this cell line is intrinsically resistant to
electrophilic cytotoxic agents due to the presence of high levels
of glutathione/glutathione transferase. Interestingly, the Psec
prodrug UP2073 was actually more active in this cell line than the
PBD it was based upon UP2025. Without wishing to be bound by
theory, it is possible that the prodrug is protected from
glutathione and other biological nucleophiles until it is
deprotected close to the site of action.
[0323] UP2073 also underwent screening carried out by The National
Cancer Institute (NCI), Bethesda, Md. USA. The NCI has available an
in vitro cytotoxicity screen which consists of approximately 60
human tumour cell lines against which compounds are tested at a
minimum of five concentrations each differing 10-fold. A 48 hour
continuous exposure protocol is used, where cell viability or
growth is estimated with an SRB protein assay.
[0324] Method
[0325] The test compound was evaluated against approximately 60
human tumour cell lines. The NCI screening procedures were
described in detail by Monks and co-workers (Monks, A et al.,
Journal of the National Cancer Institute, 1991, 83, 757). Briefly,
cell suspensions were diluted according to the particular cell type
and the expected target cell density (5000-40,000 cells per well
based on cell growth characteristics), and added by pipette (100
.mu.L) into 96-well microtitre plates. The cells were allowed a
preincubation period of 24 hours at 37.degree. C. for stabilisation
Dilutions at twice the intended test concentration were added at
time zero in 100 .mu.L aliquots to the wells. The test compounds
were evaluated at five 10-fold dilutions (10.sup.-4, 10.sup.-5,
10.sup.-6, 10.sup.-7 and 10.sup.-8 .mu.M). The test compounds were
incubated for 48 hours in 5% CO.sub.2 atmosphere and 100% humidity.
The cells were then assayed using the sulphorhodamine B assay. A
plate reader was used to read the optical densities and a
microcomputer processed the readings into LC.sub.50 values, which
is the dosage required to kill half of the cells IC.sub.50 values,
the dosage required to inhibit the growth of half the cells, was
also measured.
[0326] UP2073 did well in the screening showing activity against
cell lines in the lung, colon, CNS, melanoma, renal and breast
tumour cell line panels. Interestingly, analysis of the LC.sub.50
data across 53 cell lines suggested some correlation to glutathione
transferase activity. Selected results are shown below:
6 Cell Lines IC.sub.50 (.mu.M) IC.sub.50 (.mu.M) Lung, NCI-H552
0.11 1.38 Colon, Colo 205 0.19 0.71 CNS, SNB-75 0.72 9.00 Melanoma,
SK-MEL-5 0.29 6.78 Renal, RXF 393 0.24 2.97 Breast, MDA-MB-231 0.45
4.33
[0327] Key to Figures
[0328] FIG. 4 : SW1116-(.box-solid.) Compound 7; (.circle-solid.)
Compound 7+enzyme+NADH LS174T-(.tangle-solidup.) Compound 7;
(.tangle-soliddn.) Compound 7+enzyme+NADH
[0329] FIG. 6 & 7: (.box-solid.) Benzyl DC-81; (.quadrature.)
Compound 11; (.circle-solid.) Compound 11+UVA 30 mins;
(.largecircle.) Compound 11+UVA 1 h; (.tangle-solidup.) Compound
11+UVA 2 h.
[0330] FIG. 8: (.box-solid.) Benzyl DC-81; (.quadrature.) Compound
11; (.circle-solid.) Compound 11+UVA 30 mins; (.largecircle.)
Compound 11+UVA 1 h; (.tangle-solidup.) Compound 11+UVA 2 h
[0331] FIG. 9: (.box-solid.) DSB-120; (.quadrature.) Compound 32;
(.circle-solid.) Compound 32+UVA 30 mins; (.largecircle.) Compound
32+UVA 1 h; (.tangle-solidup.) Compound 32+UVA 2 h; (.DELTA.)
Compound 32+UVA 5 h
* * * * *