Synthesis of a novel C2/C2′-aryl-substituted pyrrolo[2,1-c][1,4]benzodiazepine dimer prodrug with improved water solubility and reduced DNA reaction rate

Article abstract of DOI:10.1016/j.bmcl.2009.09.012

A prodrug form (17) of a novel C2/C2′-aryl-substituted pyrrolobenzodiazepine (PBD) dimer (16) has been synthesized by introducing sodium bisulfite groups to the C11/C11′-positions of the parent bis-imine. The prodrug form is highly water soluble, stable in aqueous conditions, and the rate of DNA cross-link formation is much slower compared to the parent bis-imine.

Full text of DOI:10.1016/j.bmcl.2009.09.012

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6463–6466
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of a novel C2/C20-aryl-substituted pyrrolo[2,1-c][1,4]benzodiazepine
dimer prodrug with improved water solubility and reduced DNA reaction rate
a
a
a
a
Philip W. Howard a, , Zhizhi Chen , Stephen J. Gregson , Luke A. Masterson , Arnaud C. Tiberghien ,
*
Nectaroula Cooper b, Min Fang a, Marissa J. Coffils a, Sarah Klee a, John A. Hartley a, David E. Thurston a,b,
*
a Spirogen Ltd, The School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK
b Gene Targeted Drug Design Research Group, School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK
a r t i c l e i n f o
a b s t r a c t
A prodrug form (17) of a novel C2/C20-aryl-substituted pyrrolobenzodiazepine (PBD) dimer (16) has been
synthesized by introducing sodium bisulfite groups to the C11/C110-positions of the parent bis-imine. The
prodrug form is highly water soluble, stable in aqueous conditions, and the rate of DNA cross-link forma-
tion is much slower compared to the parent bis-imine.
Article history:
Received 27 July 2009
Revised 2 September 2009
Accepted 4 September 2009
Available online 9 September 2009
Ó 2009 Published by Elsevier Ltd.
Keywords:
Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs)
Suzuki coupling
Palladium (0)
Triflate
Cross-linking
Interstrand
DNA
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of
naturally occurring antitumour antibiotics produced by Streptomy-
ces species.1–4 The PBD monomers (e.g., natural products: anthra-
mycin, porothramycin and sibiromycin;1 synthetic agents:
DRH-417;5 Fig. 1) exert their biological activity by binding
sequence-selectively to Pu-G-Pu motifs within the minor groove
of DNA followed by covalent bond formation between their C11-
position and the C2–NH2 functionality of a guanine base. The bio-
logical activity of these molecules can be potentiated by joining
two PBD units together through their C8/C80-positions via a flexible
alkylene linker (e.g., DSB-120 and SJG-136; Fig. 1).6,7 The PBD di-
mers are known to form sequence-selective DNA lesions such as
the palindromic 50-Pu-GATC-Py-30 interstrand cross-link8–10 which
is thought to be mainly responsible for their biological activity. One
example of a PBD dimer, SG2000 (SJG-136, 6),11–13 has recently
completed Phase I clinical trials in the oncology area and is about
to enter Phase II.
the application of Suzuki coupling chemistry (e.g., DRH-417, 4),
and have established that C2-unsaturation enhances potency.18
We have applied this finding to the synthesis of a PBD dimer series
and report one example here, the C2/C20-aryl-substituted dimer
SG2202 (16, Scheme 1). Although this molecule had significantly
greater in vitro cytotoxicity compared to the C2-unsubstituted par-
ent, DSB-1206 (5, Fig. 1), the additional C2/C20-aryl substituents
lowered the water solubility to a level that became problematic
for in vivo evaluation. Therefore, we converted 16 to the C11/
C110-bisulfite adduct SG2285 (17)19 based on methodologies re-
ported for the synthesis of C11-bisulfite adducts of PBD mono-
mers.20,21 Importantly, we found that we could control the
stereochemistry of the C11/C110-bisulfite substituents through
the reaction conditions, and the product formed was highly water
soluble and stable in aqueous conditions thus allowing full in vitro
and in vivo evaluation. Crucially, the bis-bisulfite 17 was found to
be significantly slower at cross-linking DNA in vitro compared to
the parent dimer 16, suggesting that it is behaving as a prodrug
form of the parent PBD dimer. It was also found to have significant
antitumour activity across a range of in vivo models.
Many of the most potent, naturally occurring PBD monomers
such as anthramycin (1),14 porothramycin (2)15,16 and sibiromycin
(3)17 have endo/exo-unsaturation at their C2-position. We have
developed synthetic PBD monomers that retain this motif through
To prepare dimers 16 and 17, the known 2-nitrobenzoic acid di-
mer core11 7 was converted to its acid chloride with oxalyl chloride
and added as a solid to methyl 4-hydroxyprolinate hydrochloride
in the presence of TEA to afford a quantitative yield of the bis-ester
8. Reduction and spontaneous B-ring cyclisation to afford the tetr-
alactam 9 was achieved with Raney nickel and hydrazine (CAU-
* Corresponding authors. Tel.: +44 0207 753 5934 (PWH); +44 0207 753 5931
(DET); fax: +44 0207 753 5964.
E-mail addresses: philip.howard@spirogen.com (P.W. Howard), david.thurston@
pharmacy.ac.uk (D.E. Thurston).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.09.012

6464
P. W. Howard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6463–6466
OH
OH
H
N
OR2
OMe
H
H
N
Me
O
H
9
R1
11a
N
10
11
A
B
1
R3
R4
C
3
N
N
Me
O
2
O
Me
MeHN
O
O
HO
OH
1
2
3 Sibiromycin
Anthramycin Methyl Ether, R1 = Me; R2,R3,R4 = H
Porothramycin B, R1 = H; R2 = Me; R3,R4 = Me
N
N
N
8
O
MeO
MeO
O
8'
H
H
H
10'
10
11'
11
2
2'
5
2
5'
N
N
N
OMe
MeO
X
X
O
O
O
OMe
5 DSB-120, X = H,H
4
DRH-417
6
SJG-136 (SG2000), X = CH2
Figure 1. Structures of PBD monomers and dimers.
TION PYROPHORIC!) in 85% yield. Alternative reducing conditions
(Pd/C, H2 or H-cube) gave the uncyclized bis-amine, although sub-
sequent treatment with hydrazine furnished the desired tetralac-
tam 9, thus offering a less pyrophoric approach to cyclisation in
comparable yield (81%). The 2/20-hydroxy groups were then pro-
tected as silyl ethers (10) under standard conditions in order to
allow introduction of the SEM group (n-butyllithium, SEM-Cl) at
the N10/N100 positions (11). The silyl ethers could be selectively
removed in the presence of the SEM group to afford the N10/
N100-protected tetralactam 12 (78% yield over three steps). The
newly formed C2/C20-secondary alcohol was oxidized under Swern
conditions to give 13 in 56% yield, but a modified version of the
method of Fey et al.22 (TEMPO, NaOCl and KBr in DCM) afforded
the same product in 75% yield on a 20 g scale. Triflation repre-
sented one of the most important steps in the synthesis, as the
resulting bis-triflate intermediate 14 could be used to prepare a
diverse set of C2/C20-aryl substituted PBD dimers. Our original tri-
flation conditions (pyridine, triflic anhydride) yielded 14 in repro-
ducible but moderate yield (39%). However, replacing pyridine
with the more-hindered 2,6-lutidine and performing the reaction
at lower temperature raised the triflation yield to a more accept-
able 70%. Next, Suzuki coupling with 4-methoxybenzeneboronic
O
NO2
O
OMe
O2N
O
O
b
MeO
HO
O2N
O
O
NO2
a
N
N
OMe MeO
HO2C
OMe
MeO
CO2H
OH
O
O
8
7
SEM
O
SEM
N
R1
N
R1
N
O
H
O
H
O
H
N
H
O
O
O
O
f
g
N
O
N
N
N
OMe
MeO
OMe
MeO
O
OR2
O
R2O
O
O
O
13
9
R1, R2, = H
c
10
11
12
R
1 = H; R2 = TBS
d
e
R1 = SEM; R2 = TBS
R1 = SEM; R2 = H
SEM
N
SEM
N
SEM
N
SEM
N
O
H
O
H
O
H
O
H
O
OMe
O
O
O
h
N
N
MeO
N
N
OMe
MeO
OTf
TfO
O
O
O
O
OMe
MeO
15
14
H
N
H
N
NaO3S
H
SO3Na
H
N
N
O
O
O
O
H
H
i
j
N
N
N
OMe
N
MeO
OMe
MeO
O
O
O
O
OMe
MeO
OMe
MeO
16 (SG2202)
17 (SG2285)
Scheme 1. Synthesis of the C2/C20-bis-aryl PBD dimer SG2202 (16) and the novel C11/C110-bis-bisulfite prodrug SG2285 (17). Reagents and conditions: (a) (i) (COCl)2, cat.
DMF, DCM, rt, 16 h, (ii) (2S,4R)-methyl-4-hydroxyprolinate hydrochloride, TEA, ꢀ40 °C, 100%; (b) Raney Ni (CAUTION!), N2H4ꢁH2O, MeOH, , 85%, or (i) 10% Pd–C, H2, DMF,
D
8 h, 45 psi, (ii) N2H4ꢁH2O, EtOH,
D
, 50 min, 81%; (c) TBS-Cl, imidazole, DMF, rt, 3 h, 99%; (d) n-BuLi, SEM-Cl, THF, ꢀ30 °C, 100%; (e) TBAF, THF, rt, 1 h, 79%; (f) TEMPO, NaOCl,
KBr, DCM, 0 °C, 1 h, 75%; (g) Tf2O, 2,6-lutidine, DCM, ꢀ40 °C, 1 h, 70%; (h) 4-methoxyphenylboronic acid, Pd(PPh3)4, Na2CO3, H2O, EtOH, toluene, rt, 3 h, 87%; (i) (i) LiBH4, THF,
EtOH, 15–25 °C, 1 h, (ii) silica gel, EtOH, DCM, H2O, 72 h, 94%; (j) NaHSO3, IPA, H2O, rt, 45 min, 82% (11S,11S0-diastereomer), or NaHSO3, DCM, H2O, rt, 25 h, 62% (11S,11R0-
diastereomer).

P. W. Howard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6463–6466
6465
acid proceeded smoothly at room temperature to give the
C2/C20-aryl-substituted PBD dimer 15 in 87% yield. The presence
of the N10/N100-SEM protecting groups allowed regioselective
reduction of the C11/C110-carbonyl functionalities in the presence
of the C5/C50-carbonyl groups. Previously, sodium tetraborohy-
dride has been employed as a reducing agent for this step,2 how-
ever we found that the extended reaction times required for this
reagent could led to in situ removal of the SEM groups followed
by reduction of the nascent PBD N10–C11/N100–C110 imines to
secondary amine functionalities. However, use of the more power-
ful lithium tetraborohydride reduced the C11/C110-carbonyl
groups sufficiently rapidly so that premature N10-deprotection
did not occur.23 Subsequent treatment with silica gel afforded
the free bis-imine 16 in 94% yield on a 12 g scale.
DNA.24 A longer incubation time was required to reach the peak
of cross-linking for the bis-bisulfite 17 (18 h) compared to the par-
ent N10–C11 bis-imine 16 (2 h). An XL50 value of 0.9 nM was
achieved for 17, indicating that it is a highly efficient cross-linking
agent. Further investigations showed that while 17 alone is stable
in phosphate buffer for at least six days, in the presence of double-
stranded oligonucleotides containing Pu-GATC-Py motifs (e.g., du-
plex 50-TATAGATCTATA-30; drug/DNA molar ratio 4:1; 18 °C) in the
same buffer it reacts within hours to form interstrand cross-linked
adducts (e.g, 10.8%, 70.1% and 92.5% cross-linked in 4, 24 and 48 h,
respectively).25 Assuming that 17 does not react directly with DNA
(which would require ejection of the relatively bulky C11/C110-
bisulfite groups within the minor groove), this suggests that in
aqueous solution 17 may exist in equilibrium with a small amount
of the N10–C11/N100–C110 bis-imine form (i.e., 16), and it is this
species that reacts with DNA, thus pulling the equilibrium towards
16 as the latter is consumed by DNA adduct formation.
Compound 16 was then converted to the bis-C11/C110-bisulfite
adduct SG2285 (17) by treatment with sodium bisulfite. Initially,
reaction in a two-phase DCM/water system afforded a 2:1 mixture
of two diastereomers that could be separated by mass-directed
preparative HPLC. 500 MHz NMR revealed the major and minor
components to be the 11S,11R0 and 11S,11S0 isomers, respectively.
The diastereomeric ratio was found to be very sensitive to reaction
and work-up conditions. Performing the reaction in a miscible
aqueous solvent system such as isopropanol/water (2:1) for
45 min followed by flash freezing of the reaction mixture and lyo-
philisation afforded almost exclusively the 11S,11S0 diastereomer
(96.4:3.6; 11S,11S0:11S,110R). Conversely, performing the reaction
in an immiscible mixture of DCM and water (1:1) over 25 h, fol-
lowed by separation and lyophilisation of the aqueous phase, affor-
ded a (9:1) mixture in favour of the 11S,110R diastereomer. Unlike
the parent bis-imine 16 which was poorly soluble in water, the
N10/N100-bisulfite adducts of 17 were highly soluble (approxi-
mately 11 mg/ml).
In summary, the first example of a C2/C20-aryl PBD dimer (16) is
reported that can be converted into a stable, highly water soluble
form (17) by conversion to C11/C110-bisulfite diastereomers.
Importantly, 17 appears to behave as a prodrug in that the rate
of reaction with DNA is significantly delayed compared to the par-
ent N10–C11-bis-imine (16), although a similar level of cross-link-
ing is eventually achieved by both. 16 and 17 are cytotoxic at
picomolar concentrations in a range of tumour cell lines, clearly
demonstrating the potency-enhancing effect of the C2-aryl motif.
Preliminary in vivo data show that 17 has significant antitumour
activity across a wide range of human tumour xenograft models,
and these data will be reported elsewhere. Finally, it is noteworthy
that the versatile, late-stage, enol triflate intermediate 14 can be
produced on a large scale and used to prepare a diverse set of
C2/C20-aryl PBD dimers.
Dimers 16 and 17 were evaluated in a panel of ten human tu-
mour cell lines as shown in Table 1, with both exhibiting picomolar
activity across the panel (continuous drug exposure, Alamar Blue
assay), with the bis-bisulfite 17 only slightly less potent than the
parent imine (16). Interestingly, the ratio of C11/C110 diastereo-
mers did not appear to influence the cytotoxicity of dimer 17. Both
16 and 17 were more potent than the structurally-equivalent
monomeric PBD 4 in the K562 leukemia cell line (360-fold for 16
and 250-fold for the bisulfite 17) due to their ability to cross-link
DNA. Compounds 16 and 17 were also significantly more potent
than the C2-exo-unsaturated PBD dimer SG2000 (SJG-136, 6) in
the K562 leukemia cell line (86-fold for 16 and 40-fold for 17).
The ability of 17 to cross-link naked DNA was investigated using
a gel-based interstrand cross-linking assay with pUC18 plasmid
Acknowledgement
Dr. Emma Sharp is thanked for her assistance with the prepara-
tion of the Letter.
References and notes
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Table 1
Cytotoxicity data (GI50) for PBD dimers 16 and 17 across a panel of human tumour cell
lines (Alamar Blue assay, continuous exposure)
Cell line
Cell type
SG2202 (16)a
mean GI50 (nM)
SG2285 (17)a
mean GI50 (nM)
K562b
CCRF-CEM
RPMI8226
A549
DU145
LNCaP-FGC
A2780
MCF7
LOXIMVI
LS174T
CML
ALL
Myeloma
NSCL carcinoma
Prostate carcinoma
Prostate carcinoma
Ovarian carcinoma
Breast carcinoma
Melanoma
0.013
0.0185
0.0014
0.0278
0.0194
0.0064
0.0007
0.0005
0.031
10. Narayanaswamy, M.; Griffiths, W. J.; Howard, P. W.; Thurston, D. E. Anal.
Biochem. 2008, 374, 173.
0.0001
0.0051
0.033
0.0154
0.0027
0.0024
0.0186
0.0225
0.0007
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C.; Kelland, L. R.; Thurston, D. E. J. Med. Chem. 2001, 44, 737.
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0.0535
0.0043
Colon
a
GI50 = concentration required to reduce growth by 50% following continuous
drug exposure using Alamar Blue.
Comparative GI50 value for SJG-136 (6) in K562 under identical
conditions = 0.419 nM.
b
16. Langlois, N.; Rojas-Rousseau, A.; Gaspard, C.; Werner, G. H.; Darro, F.; Kiss, R. J.
Med. Chem. 2001, 44, 3754.

6466
P. W. Howard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6463–6466
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4.17–4.11 (m, 3H, CH2O ‘S’ and H-11 ‘R’), 4.06 (t, 2H, J = 6.4 Hz, CH2O ‘R’), 3.94
(d, 1H, J = 10.3 Hz, H-11 ‘S’), 3.76 (s, 3H, OMe ‘S’), 3.75 (s, 3H, OMe ‘S’), 3.72 (s,
3H, OMe ‘R’), 3.66 (s, 3H, OMe ‘R’), 3.66–3.62 (m, 1H, H-1 ‘R’), 3.52–3.45 (m, 1H,
H-1 ‘S’), 3.29–3.16 (m, 2H, H-1 ‘S’ and ‘R’), 2.28–2.18 (m, 2H, CH2) 13C NMR
(100 MHz, DMSO-d6) d 163.9, 158.2, 151.3, 143.1, 140.1, 126.8, 126.0, 122.5,
122.2, 115.4, 114.1, 112.7, 106.5, 78.4, 65.1, 56.7, 56.0, 55.1, 35.4, 28.6; IR (ATR,
neat) 3370 (weak), 2935 (weak), 1605, 1572, 1512, 1450, 1434, 1383, 1241,
19. Analytical data for the two diastereomers of 17: 11S/11S0: ½a D21
ꢂ
= +53 (c 0.12,
DMSO); 1H NMR (500 MHz, DMSO-d6) d 7.41 (s, 2H, H-3), 7.38 (d, 4H, J = 8.7 Hz,
H-20 C2-aryl), 7.05 (s, 2H, H-6), 6.91 (d, 4H, J = 8.8 Hz, H-30 C2-aryl), 6.51 (s, 2H,
H-9), 5.26 (s, 2H, NH), 4.29 (td, 1H, J = 10.3, 3.6 Hz, H-11aS), 4.18–4.11 (m, 4H,
CH2O), 3.92 (d, 1H, J = 10.4 Hz, H-11), 3.75 (s, 6H, OMe), 3.71 (s, 6H, OMe),
2.28–2.18 (m, 2H, CH2); note: H-1 signals obscured by residual buffer. 13C NMR
(100 MHz, DMSO-d6) d 163.9, 158.2, 151.3, 143.1, 140.1, 126.8, 126.0, 122.6,
122.1, 115.5, 114.1, 112.7, 106.6, 78.4, 65.1, 56.7, 56.0, 55.1, 35.5, 28.6; IR (ATR,
neat) 3386 (weak), 2935 (weak), 1604, 1572, 1510, 1494, 1450, 1433, 1383,
1246, 1202, 1179, 1146, 1109, 1028, 970, 867, 822, 786, 758 cmꢀ1; 11S/11R0:
1200, 1178, 1143, 1107, 1028, 975, 872, 823, 786, 755 cmꢀ1
.
20. Kaneko, T.; Wong, H.; Doyle, T. W.; Rose, W. C.; Bradner, W. T. J. Med. Chem.
1985, 28, 388.
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8154.
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Evaluate the DNA-Interaction of Pyrrolobenzodiazepines (PBDs) with
Oligonucleotides, University of London, 2008, p 182.
½ ꢂ
a 2D2 = +291 (c 0.32, DMSO); 1H NMR (500 MHz, DMSO-d6) d: 7.41 (s, 1H, H-3
‘S’), 7.37 (d, 2H, J = 8.7 Hz, 20-H C2-aryl, ‘S’), 7.35 (s, 1H, H-3, ‘R’), 7.32 (s, 1H, H-
6, ‘R’), 7.29 (d, 2H, J = 8.6 Hz, H-20 C2-aryl, ‘R’), 7.05 (s, 1H, H-6 ‘S’), 7.03 (d, 1H
J = 6.4 Hz, NH ‘R’), 6.91 (d, 2H, J = 8.9 Hz, H-30 C2-aryl), 6.89 (d, 2H, J = 8.5 Hz, H-
30 C2-aryl), 6.52 (s, 1H, 9-H ‘R’), 6.51 (s, 1H, 9-H ‘S’), 5.25 (s, 1H, NH ‘S’), 4.42
(dd, 1H, J = 12.4, 6.2 Hz, H-11aS ‘R’), 4.29 (td, 1H, J = 10.3, 3.5 Hz, H-11aS ‘S’),