Synthesis of novel C2-aryl pyrrolobenzodiazepines (PBDs) as potential antitumour agents

Article abstract of DOI:10.1039/b205136b

Three novel C2-aryl substituted pyrrolobenzodiazepines (PBDs) have been synthesised and evaluated in a number of cell lines revealing selective cytotoxicity at the sub-nanomolar level towards melanoma and ovarian cancer cell lines.

Full text of DOI:10.1039/b205136b

Synthesis of novel C2-aryl pyrrolobenzodiazepines (PBDs) as potential
antitumour agents
Nectaroula Cooper, David R. Hagan, Arnaud Tiberghien, Temitope Ademefun, Charles S. Matthews†,
Philip W. Howard* and David E. Thurston*
CRUK Gene Targeted Drug Design Research Group, School of Pharmacy, University of London, 29/39
Brunswick Square, London, UK WC1N 1AX. E-mail: crukgtddrg@ulsop.ac.uk; Fax: 0207 753 5935;
Tel: 0207 753 5932
Received (in Cambridge, UK) 30th May 2002, Accepted 3rd July 2002
First published as an Advance Article on the web 15th July 2002
Three novel C2-aryl substituted pyrrolobenzodiazepines
(PBDs) have been synthesised and evaluated in a number of
cell lines revealing selective cytotoxicity at the sub-nano-
molar level towards melanoma and ovarian cancer cell
lines.
Although the initial synthetic steps are based on the classical
approach of Leimgruber et al.,3 a novel use of an N10-
hemiaminal protecting group is employed. Furthermore, to-
wards the latter stages of the synthesis, the Suzuki reaction is
used for the first time on a PBD skeleton to introduce the C2-
aryl substituents.
Anthramycin, the first example of a series of PBD antitumour
antibiotics, was isolated from Streptomyces refuineus in 1965
by Leimgruber et al.1 Many other PBD natural products have
since been found in Streptomyces species, and a large number of
synthetic analogues have been investigated and reported.2 The
PBDs exert their biological activity by binding covalently to the
N2-position of guanine within the minor groove of DNA in a
sequence-selective manner, preferring to interact with purine-
guanine-purine sequences.
Using a new synthetic approach, we report here the synthesis
of a series of C2-aryl C2/C3-unsaturated PBDs that represent a
structural sub-class not observed in nature. Compared to PBD
monomers with other substitution patterns, these C2-aryl
analogues have remarkably selective cytotoxicity at the sub-
nanomolar level towards melanoma and ovarian cell lines.
The synthetic route starts from commercially available
6-nitroveratric acid (1) and the C-ring building block 2 to
provide the target molecules 13–15 in nine steps (Scheme 1).
The pre-formed C-ring4 was coupled to 1 to provide the A–C
ring backbone 3. For the next step involving reduction of the
nitro group, several methods were investigated including the
sodium dithionite method originally employed by Leimgruber
et al.3 Catalytic hydrogenation was selected as the preferred
method. On a scale of greater than 1 g, it was found that, on
formation of the amine spontaneous cyclisation occurred to give
4, thus eliminating the need for the two-day acid-treatment step
employed by Leimgruber. This spontaneous cyclisation was not
encountered using other reduction methods. Following cyclisa-
tion, the C2 alcohol was protected as a TBDMS ether.
The PBD dilactam 5 was next treated with SEM-Cl under
strongly basic conditions to provide the SEM version of the N10
hemiaminal shown by Mori et al.5 to promote C11-lactam
reduction. Having successfully derivatized the N10 position, the
C2-TBDMS ether was selectively cleaved with TBAF at room
temperature without affecting the orthogonal N10-SEM pro-
tecting group. Swern oxidation furnished the C2 ketone 8,
Scheme 1 Reagents and conditions: i, (COCl)2, DMF, TEA, anhydrous DCM, RT, 57%; ii, 10% Pd/C, EtOH, H2, 75%; iii, TBDMS-Cl, imidazole, anhydrous
DMF, RT, 95%; iv, SEM–Cl, NaH, anhydrous DMF, 0 °C, 77%; v, TBAF, THF, RT, 70%; vi, (COCl)2, anhydrous DMSO, anhydrous DCM, TEA, N2, 255
°C, 49%; vii, anhydrous pyridine, anhydrous DCM, anhydrous triflic anhydride, RT, 60%; viii, benzeneboronic acid, benzene, EtOH, water, N2, Na2CO3,
Pd(PPh3)4, RT, 78%; ix, methylbenzeneboronic acid, benzene, EtOH, water, N2, Na2CO3, Pd(PPh3)4, RT, 66%; x, methoxybenzeneboronic acid, benzene,
EtOH, water, N2, Na2CO3, Pd(PPh3)4, RT, 90%; xi, NaBH4, anhydrous EtOH, anhydrous THF, wet silica gel, N2, RT, 13:‡ 74%, 14:§ 38%, 15:¶ 72%; Note:
The C2-unsubstituted PBD 16 shown for comparative purposes was not synthesised using this route.
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This journal is © The Royal Society of Chemistry 2002

Table 1 Cytotoxicity of compounds 13–15
GI50ad/nM
TGIbd/nM
LC50cd/nM
Cell line
16e
15
14
13
16e
15
14
13
16e
15
14
13
MALME3M
(Melanoma)
SKMEL28
(Melanoma)
SKOV3 (Ovarian) 3490
MCF7 (Breast)
HCT116 (Colon)
H460 (Lung)
2275
3175
0.73
2.4
11.09
4850
1.67
4.71
21.63 53715
6.69
61.02
84.92
82.89
> 10
> 10
3.32
0.03
2.89
3.83
4.09
5.95
0.04
4.47
2.31
3.51
9.9
0.05
17.24
3.83
9.14
9720
NA
> 100000
> 100000
> 100000
6.74
6.22
9.27
8.01
8.2
12.37
4.67
99.51
7.59
43.17 23305
25.87 > 100000
1001.63 > 100000
9.55 > 100000
1707.32 > 100000
17.25
> 10
> 10
9.17
9.68
71.36
> 10
> 10
9.04
9.97
2490
2730
2925
82.54
5984.73
8.03
a Dose required to inhibit cell growth by 50% compared to PBD-free controls after incubation for 48 h at 37 °C; b Dose required for complete inhibition of
cell growth compared to PBD-free controls after incubation for 48 h at 37 °C; c Dose required to kill 50% of cells compared to PBD-free controls after
incubation for 48 h at 37 °C; d The MTT assay was used to measure cytotoxicity, and PBDs were dissolved in DMSO/culture medium prior to addition to
the culture medium; e Data for compound 16 were obtained from the NCI’s 60 cell line panel.
which was converted to the C2–C3 enol triflate 9 using
trifluoromethanesulfonic anhydride in the presence of pyr-
idine.6
tumour cell lines and to elucidate the precise mechanism of
action. Compound 15 has been selected for in vivo studies and
these results will be reported elsewhere.
Three trial Suzuki reactions were performed on the enoltri-
flate 9. The reactions with methoxybenzeneboronic (x) me-
thylbenzeneboronic (ix) and benzeneboronic acids (viii) in
benzene proceeded at room temperature.7 This observation
coupled with the diverse array of commercially available
arylboronic acids suggested the possibility of generating
libraries of analogues through parallel combinatorial synthetic
methodologies.
Reduction of the dilactam in the presence of the N10-SEM
group was achieved using sodium borohydride in anhydrous
EtOH/THF. The resulting unstable N10-SEM protected carbi-
nolamines underwent spontaneous cleavage in the presence of
wet silica gel to afford the required C2-aryl PBDs 13, 14 and 15
in their imine forms without the need for treatment with
additional reagents such as TBAF.
Compound 15 was evaluated in the standard NCI 60-cell line
screen and was shown to have nanomolar potency (at the LC50
level) against six melanoma (MALME-3M, M14, SK-MEL-2,
SK-MEL-5, UACC-257, UACC-62), two non-small cell lung
(NCI-H460, NCI-H522), one CNS (SF-539) and two colon
cancer lines (COLO 205, HCC-2998).
Notes and references
† Cytotoxicity studies were carried out at the Cancer Research Laboratories,
University of Nottingham, University Park, Nottingham, UK NG7 2RD.
‡ 1H (250 MHz, CDCl3) NMR: d 7.91 (d, 1H, J 5 Hz, H-11), 7.53 (s, 1H,
H-9), 7.52 (s, 1H, H-3), 7.43–7.11 (m, 5H, Ar-H), 6.84 (s, 1H, H-6), 4.44
(ddd, 1H, J 2.5, 5, 10 Hz, H-11a), 3.98 & 3.95 (2s, 6H, 7,8-MeO), 3.60 (ddd,
1H, J 2.5, 12.5, 17.5 Hz, H-1), 3.42 (ddd, 1H, J 2.5, 5, 17.5 Hz, H-1).
§ 1H (250 MHz, CDCl3) NMR: d 7.90 (d, 1H, J 3.9 Hz, H-11), 7.53 (s, 1H,
H-9), 7.47 (br s, 1H, H-3), 7.31 (d, 2H, J 8.15 Hz, Ar-Tolyl), 7.18 (d, 2H,
J 8.04 Hz, Ar-Tolyl), 6.84 (s, 1H, H-6), 4.42 (ddd, 1H, J 4.1, 5.2, 11.4 Hz,
H-11a), 3.98 & 3.95 (2s, 6H, 7,8-MeO), 3.59 (ddd, 1H, J 1.9, 11.5, 16.3 Hz,
H-1), 3.39 (ddd, 1H, J 1.9, 5.3, 16.6 Hz, H-1), 2.36 (s, 3H, Ar-Me).
¶ 1H (250 MHz, CDCl3) NMR: d 7.91 (d, 1H, J 2.5 Hz, H-11),7.53 (s, 1H,
H-3), 7.43–7.30 (m, 3H, H-6 & Ar-Phenyl), 6.94-6.84 (m, 3H, H-9 & Ar-
Phenyl), 4.46–4.38 (ddd, 1H, J 2.5, 5, 12.5 Hz, H-11a), 3.98 & 3.95 (2s, 6H,
7,8-MeO), 3.81 (s, 3H, Ar–MeO), 3.60 (ddd, 1H, J 2.5, 12.5, 17.5 Hz, H-1)
3.41 (ddd, 1H, J 2.5, 5.0, 17.5 Hz).
1 W. Leimgruber, V. Stefanovic, F. Schenker, A. Karr and J. Berger, J. Am.
Chem. Soc., 1965, 87, 5791.
Compounds 13, 14 and 15 were subsequently tested in a more
focused 6-cell line panel and the results are shown in Table 1.
Data for the C-ring unsubstituted analogue 16 have been
included for comparative purposes. All analogues exhibit
nanomolar potency at the GI50 level against the two melanoma
lines, and picomolar potency against SKOV3, an intrinsically
cisplatin-resistant ovarian cancer cell line.
Efforts are now underway to carry out a Structure Activity
Relationship (SAR) study through the synthesis of further C2-
aryl analogues in order to maximise the cytotoxicity in key
2 D. E. Thurston, Advances in the study of Pyrrolo[2,1-c][1,4]benzodiaze-
pine (PBD) Antitumour Antibiotics, in Molecular Aspects of Anticancer
Drug–DNA Interactions, ed. S. Neidle and M. Waring, Macmillan Press
Ltd, London, vol. 54–88, 1993.
3 W. Leimgruber and A. D. Batcho, J. Am. Chem. Soc., 1968, 90, 5641.
4 M. A. Williams and H. Rapoport, J. Org. Chem., 1994, 59, 3616.
5 M. Mori, Y. Uozumi, M. Kimura and Y. Ban, Tetrahedron, 1986, 42,
3793.
6 M. R. Pena and J. K. Stille, J. Am, Chem. Soc., 1989, 111, 5417.
7 N. Yasuda, L. Xavier, D. L. Rieger, Y. Li, A. E. DeCamp and U.-H.
Dolling, Tetrahedron Lett., 1993, 34, 3211.
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