Design and synthesis of novel pyrrolobenzodiazepine (PBD) prodrugs for ADEPT and GDEPT

Article abstract of DOI:10.1016/S0960-894X(00)00404-2

Three N10-(4-nitrobenzyl)carbamate-protected PBD prodrugs (9a, 9b and 15) have been synthesized and evaluated for potential use in nitroreductase-based ADEPT and GDEPT therapies. An approximately 100-fold activation was observed for the DC-81 prodrug 9a.

Full text of DOI:10.1016/S0960-894X(00)00404-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2083±2086
Design and Synthesis of Novel Pyrrolobenzodiazepine (PBD)
Prodrugs for ADEPT and GDEPT
Marina J. Sagnou,a Philip W. Howard,a,y Stephen J. Gregson,a,y
Ebun Eno-Amooquaye,b Philip J. Burkeb and David E. Thurstona,*,y
aCRC Gene Targeting Drug Design Research Group, School of Pharmacy and Biomedical Sciences,
University of Portsmouth, St. Michael's Building, White Swan Road, Hants PO1 2DT, UK
bDepartment of Medical Oncology, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK
Received 19 May 2000; revised 3 July 2000; accepted 7 July 2000
AbstractÐThree N10-(4-nitrobenzyl)carbamate-protected PBD prodrugs (9a, 9b and 15) have been synthesized and evaluated for
potential use in nitroreductase-based ADEPT and GDEPT therapies. An approximately 100-fold activation was observed for the
DC-81 prodrug 9a. # 2000 Elsevier Science Ltd. All rights reserved.
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) belong
to a group of antitumour antibiotics that exert their
biological activity through sequence-selective covalent
bonding via their electrophilic N10-C11 imine function-
ality to the N2-position of guanine within the minor
groove of DNA.1 More recently, members of the PBD
family such as DC-81 (1) and tomaymycin (2) have been
used as templates for the design of PBD dimer analogues
such as DSB-120 (3a)2 5 and SJG-136 (3b)6 which are
among the most potent DNA interstrand cross-linking
agents known to date (Fig. 1).
approach but relies on the selective delivery to the
tumour site of a gene coding for the relevant enzyme.7
Nitroreductase is an enzyme used in ADEPT and
GDEPT that requires either NADH or NADPH to
reduce aromatic nitro groups to hydroxylamines,8
a
process which can induce self-immolation of a 4-nitro-
benzylcarbamate functionality. This observation led to
the design and synthesis of the N10-(4-nitrobenzyl) car-
bamate-protected prodrugs of benzyl DC-81, benzyl
tomaymycin and DSB-120 reported here (i.e. 9a, 9b and
15). These molecules are unable to interact with DNA
and do not exert their cytotoxic e€ect until the N10-
protecting groups are removed to give 10a, 10b and 16,
respectively.
The ability to deliver such agents selectively to a tumour
site could lead to an e€ective treatment for solid
tumours. Antibody-directed enzyme prodrug therapy
(ADEPT) is a two-stage therapeutic approach with the
objective of selectively generating a cytotoxic agent
from its non-toxic prodrug form at the tumour site.7 A
tumour-speci®c monoclonal antibody±enzyme con-
jugate is administered and time allowed for localization
at the tumour site followed by subsequent clearance of
the conjugate from healthy tissues. This is followed by
administration of a non-toxic prodrug which is con-
verted enzymatically to the active agent only at the
tumour site thus avoiding collateral toxicity. Gene
directed enzyme prodrug therapy (GDEPT) is a related
The N10-protected PBD molecules were synthesized as
shown in Schemes 1 and 2 using a modi®ed Fukuyama
approach9 involving TPAP oxidation to cyclize appro-
priately substituted pro-N10-4-(nitrobenzyl)carbamate-
protected amino alcohols of type 8a, 8b and 14. This
route was found to have a number of advantages over
previous approaches to PBD synthesis.10 In the case of
the monomer prodrugs (9a and 9b), the previously
reported11 nitrobenzoic acid fragment (4) was coupled
to the appropriate C-ring fragments 5a or 5b using
oxalyl chloride in the presence of potassium carbonate or
TEA to provide intermediates 6a and 6b (Scheme 1). The
tomaymycin C-ring (5b) was produced by a variation of
a method reported by Gregson.12 Compound 6a could
be conveniently reduced to the amino alcohol 7a using
Raney Ni and hydrazine hydrate, however, 6c (prepared
*Corresponding author. Tel.: +44-0115-8466076; fax: +44-0115-951-
3114; e-mail: david.thurston@nottingham.ac.uk
yPresent address: Cancer Research Laboratories, School of Pharma-
ceutical Sciences, University of Nottingham, University Park,
Nottingham, NG7 2RD, UK.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00404-2

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M. J. Sagnou et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2083±2086
Figure 1. Structures of representative pyrrolobenzodiazepines.
Scheme 1. (i) (COCl)2, DMF, CH3CN, 24 h then 5a or 5b, K2CO3, CH3CN, 25 ꢁC, 88% for 6a, 66% for 6b; (ii) TBAF, THF, 0 ꢁC, quant. for 6c;
(iii) Raney Ni, NH2NH2, MeOH, Á, 81% for 7a,; Sn(II)Cl2, MeOH, Á, 66% for 7b; (iv) 4-nitrobenzyl chloroformate, pyridine, CH2Cl2, 0 ꢁC, 77%
for 8a, 76% for 8b; (v) TPAP, NMO, 4 A molecular sieves, CH2Cl2:CH3CN (3:1), 31% for 9a, 45% for 9b.
Scheme 2. (i) (COCl)2, DMF, CH3CN:THF (1:1), 24 h then 5a, K2CO3, CH3CN, 25 ꢁC, 65%; (ii) Raney Ni, NH2NH2, MeOH, Á, 70%; (iii) 4-
nitrobenzyl chloroformate, pyridine, CH2Cl2, 0 ꢁC, 81%; (iv) TPAP, NMO, 4 A molecular sieves, CH2Cl2:CH3CN (3:1), 42% (PNZ=p-nitro-
benzyloxycarbonyl).
from 6b) which contains unsaturation had to be reduced
to 7b with tin(II) chloride which gave a slightly lower
yield. The amino alcohols 7a and 7b were treated with
4-nitrobenzyl chloroformate and pyridine to a€ord the
pro-N10-protected intermediates (8a, 8b) which were
cyclized with TPAP/NMO to give 9a13 and an E/Z
mixture of 9b.
protected (14) and oxidized to a€ord the bis(N10,N100-
[4-nitrobenzylcarbamate]) PBD dimer prodrug 15.14
Prodrug 9a was established to be non-toxic in the
human adenocarcinoma cell-line LS174T (IC50=
>500 mM), whereas in the presence of nitroreductase
and NADH co-factor the IC50 was found to be between
5.0 and 6.0 mM (Fig. 2 and Table 1), suggesting an acti-
vation factor of ꢀ100-fold or greater. This appears to
represent only partial release of the parent C8-O-benzyl-
DC-81 (10a), as an authentic sample of 10a was shown
Similarly, the previously reported nitro dimer core4 (11)
was coupled to pyrrolidinemethanol to give the bis(nitro
amide) 12 (Scheme 2). This was reduced (13), bis-N-

M. J. Sagnou et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2083±2086
2085
Table 1. Cytotoxicity of PBD prodrugs before and after activation with nitroreductase/NADH in the LS174T cell line
Prodrug
IC50 (mM)a of prodrug
in LS174T cells
IC50 (mM)a in presence of
enzyme and NADH
Activation
factor
IC50 (mM)a of
parent PBD
9a
>500
86.2
215.3
5.0±6.0
6.4
13.7
100 (or>)
13.5
15.7
0.008
0.001±0.01
0.0005
9bb
15
aDose of prodrug required to inhibit cell growth by 50% compared to drug-free controls.
bEvaluated as mixture of E/Z isomers.
authentic parent molecules suggests that only partial
deprotection may be occurring. This could be due to a
number of factors including sub-optimal activity of the
enzyme or detrimental steric and/or electronic interac-
tions between structural features of the prodrugs and
the active site of the enzyme. Alternatively, the parent
PBDs 10a, 10b and 16 may have enhanced cellular
penetration in LS174T cells under the conditions of the
experiment. Nevertheless, these results establish the
principle that PBDs can be modi®ed for use as prodrugs
in ADEPT- or GDEPT-type therapies, and that an
activation factor of at least 100-fold can be achieved.
Based on this, the design and synthesis of a second
generation of PBD prodrugs with potentially higher
activation factors is currently underway.
Acknowledgements
The Cancer Research Campaign (UK; Programme
Grant SP1938/0401 to D.E.T.) and the Bodossaki
Foundation (Athens, Greece; Fellowship to M.J.S.) are
thanked for ®nancial support of this work.
Figure 2. Cytotoxicity results for prodrug 9a before and after activa-
tion with nitroreductase/NADH in LS174T cells. 2000 cells/well were
treated with di€erent concentrations of prodrug in the presence and
absence of enzyme for 1 h. Cells were then washed and incubated for 3
days (-*- without enzyme; -~- with enzyme).
References and Notes
1. Thurston, D. E. In Molecular Aspects of Anticancer Drug±
DNA Interactions; Neidle, S.; Waring M. J., Eds.; Macmillan:
London, 1993; pp 54±88.
to have an IC50 of 0.008 mM in the same cell line. Simi-
larly, a 13.5-fold activation was observed for the benzyl
tomaymycin prodrug E/Z mixture 9b in LS174T cells
after addition of the enzyme, again suggesting incom-
plete activation given that the IC50 of authentic benzyl-
tomaymycin (10b) is 0.001±0.01 mM in the same cell line.
However, it is possible that either the pure E or Z ver-
sion of 9b may have a higher activation value than the
mixture, and this will be addressed in future studies. A
similar result was observed for the dimer prodrug 15
which was relatively non-toxic towards LS174T cells
(IC50=215.3 mM) but produced an IC50 value of
13.7 mM after the addition of enzyme, representing a
>15-fold activation. The IC50 of authentic DSB-120 in
the same cell line was found to be 0.0005 mM, suggesting
a less ecient activation compared to the monomer
analogue 9a.
2. Bose, D. S.; Thompson, A. S.; Ching, J. S.; Hartley, J. A.;
Berardini, M. D.; Jenkins, T. C.; Neidle, S.; Hurley, L. H.;
Thurston, D. E. J. Am. Chem. Soc. 1992, 114, 4939.
3. Bose, D. S.; Thompson, A. S.; Smellie, M.; Berardini, M.
D.; Hartley, J. A.; Jenkins, T. C.; Neidle, S.; Thurston, D. E.
J. Chem. Soc., Chem. Commun. 1992, 20, 1518.
4. Thurston, D. E.; Bose, D. S.; Thompson, A. S.; Howard, P.
W.; Leoni, A.; Croker, S. J.; Jenkins, T. C.; Neidle, S.; Hart-
ley, J. A.; Hurley, L. H. J. Org. Chem. 1996, 61, 8141.
5. Walton, M. I.; Goddard, P.; Kelland, L. R.; Thurston, D.
E. Cancer Chemother. Pharmacol. 1996, 38, 431.
6. Gregson, S. J.; Howard, P. W.; Jenkins, T. C.; Kelland, L.
R.; Thurston, D. E. J. Chem. Soc., Chem. Commun. 1999, 797.
7. Denny, W. A.; Wilson, W. R. Journal of Pharmacy and
Pharmacology 1998, 50, 387.
8. Mauger, A. B.; Burke, P. J.; Somani, H. H.; Friedlos, F.;
Knox, R. J. J. Med. Chem. 1994, 37, 3452.
9. Fukuyama, T.; Liu, G.; Linton, S. D.; Lin, S. C.; Nishino,
H. Tetrahedron Lett. 1993, 34, 2577.
10. Thurston, D. E.; Bose, D. S. Chem. Rev. 1994, 94, 433.
11. Althuis, T. H.; Hess, H. J. J. Med. Chem. 1977, 20, 146.
12. Gregson, S. J., PhD Thesis, University of Portsmouth,
1998.
13. For 9a: [a]2d0: 62.3 (c=0.4497, CHCl3); 1H NMR (270
MHz, CDCl3) (rotamers): d=8.11 (d, 2Harom, J=8.43 Hz),
These results provide evidence that N10-protected ana-
logues of PBD monomers and dimers have potential use
as prodrugs in ADEPT- and GDEPT-type therapies.
However, the ®nding that activation results in a lower
cytotoxicity than equivalent concentrations of the

2086
M. J. Sagnou et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2083±2086
7.27±7.53 (m, 6Harom), 7.21 (d, 2Harom, J=8.4 Hz), 6.70 (s,
1Harom), 5.62 (d, 1H, J=10.2 Hz), 5.07 (d, 4H, J=10.7 Hz),
3.94 (s, 3H), 3.52±3.72 (m, 3H), 2.04±2.10 (m, 4H); 13C NMR
(68.7 MHz, CDCl3): d=166.8, 149.3, 142.9, 136.0, 128.8±
127.2, 126.4, 114.6, 112.5, 111.0, 105.9, 86.2, 71.1, 66.2, 59.9,
56.2, 47.3, 46.4, 28.7, 23.1; IR (neat): n (cm 1): 3600±3200,
2820±3000, 1710, 1600, 1510, 1450, 1430, 1400, 1370, 1350,
1305, 1270, 1220, 1120±1100, 1050, 1030, 1010; MS (FAB): (m/z,
relative intensity)=535 (M+Á+2, 2), 353 (6), 337 (10), 286 (5),
256 (3), 241 (2), 228 (2), 192 (3), 136 (7), 91 (100); HRMS:
calcd 533.1798; found 533.1813.
(d, 4H, J=8.8 Hz), 7.74 (s, 2H), 7.47 (d, 4H, J=8.8 Hz), 6.71
(s, 2H), 5.65 (d, 2H, J=10 Hz), 5.26 (s, 4H), 4.29 (t, 4H,
J=6 Hz), 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); 13C
NMR (68.7 MHz, CDCl3): d=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; IR (neat): n (cm 1): 3500±
3000, 2933, 2253, 1728, 1599, 1523, 1465, 1431, 1409, 1348, 1270,
1206, 1174, 1111, 1060; MS (FAB): (m/z, relative intensity)=925
(M+Á 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).
1
14. For 15: H NMR (270 MHz, CDCl3) (rotamers): d=8.18