Application of the Stille coupling reaction to the synthesis of C2-substituted endo-exo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs)

Article abstract of DOI:10.1016/j.bmcl.2004.08.002

The Stille coupling reaction has been employed to introduce novel vinyl, alkynyl and heterocyclic substituents to the C2-position of pyrrolo[2,1-c][1,4] benzodiazepines. Sodium borohydride reduction of the dilactam intermediates followed by N10-SEM deprotection has provided five novel C2-endo/exo-unsaturated analogues with significant cytotoxicity.

Full text of DOI:10.1016/j.bmcl.2004.08.002

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5041–5044
Application of the Stille coupling reaction to the synthesis
of C2-substituted endo–exo unsaturated
pyrrolo[2,1-c][1,4]benzodiazepines (PBDs)
Arnaud C. Tiberghien, David Hagan, Philip W. Howard* and David E. Thurston*
Cancer Research UK Gene Targeted Drug Design Research Group, Department of Pharmaceutical and Biological Chemistry,
The School of Pharmacy, 29–39 Brunswick Square, London WC1N 1AX, UK
Received 19April 2004; revised 2 August 2004; accepted 3 August 2004
Available online 1 September 2004
Abstract—The Stille coupling reaction has been used to introduce novel vinyl, alkynyl and heterocyclic substituents to the C2-posi-
tion of pyrrolo[2,1-c][1,4]benzodiazepine dilactams. Sodium borohydride reduction followed by N10-SEM deprotection has
provided five analogues (6b, 8a–d) that contain C2-endo/exo-unsaturation and novel C2-substituents. These analogues
have significant multilog cytotoxicity profiles in the NCI 60-Cell Line screen, and provide new SAR data for the PBD family.
Ó 2004 Elsevier Ltd. All rights reserved.
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a
family of potent sequence-selective DNA-interactive
antitumour antibiotics,1 which are being exploited as
components of a gene targeting strategy,2 and as antitu-
mour agents in the form of extended PBD dimers.3–5
SAR studies6–10 have shown that compounds with the
greatest cytotoxicity and DNA-binding affinity usually
possess endo–exo unsaturation in their C-rings com-
bined with conjugated planar C2-substituents, such as
the acrylamide side chain found in anthramycin (e.g.,
1, Fig. 1). However, the number of naturally occurring
molecules possessing this structural feature is limited.1
Therefore, as part of an ongoing investigation into
the chemistry and SAR of the PBD ring system, we
Ph
OH
OMe
H
O
O
N
H3C
9
H
N
H3C
H
10
N
N
CONH2
CONH2
O
O
1 (Anthramycin)
2
MeO
O
N
N
MeO
MeO
MeO
MeO
H
H
10
N
2
N
O
O
4
3
Figure 1.
Keywords: PBD; Antitumour agent; Anticancer agent; Stille coupling; DNA-binding; Cytotoxic.
*
Corresponding authors. Tel.: +44 (0)207 753 5932; fax: +44 (0)207 753 5935 (D.E.T.); e-mail: david.thurston@ulsop.ac.uk
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.002

5042
A. C. Tiberghien et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5041–5044
have investigated the application of Stille coupling to
the synthesis of C-ring endo–exo unsaturated C2-substi-
tuted PBDs. This work complements the recent
study10 of the use of the Heck reaction to achieve similar
unsaturation/substitution patterns in the C-rings of
PBDs.
The coupling products (7a–e) and 6a were subjected to
sodium borohydride reduction to transiently afford the
SEM-protected carbinolamine intermediates, which
converted to the PBD imines 8a–d and 6b upon exposure
to moist silica gel (Scheme 2). Although sufficient quan-
tities of these final products were obtained for biological
evaluation, the yields for the final reduction/deprotec-
tion steps were disappointing (on average ca. 25%).
The yields were particularly poor for the C2-alkynic
compounds, where significant collateral reduction of
the triple bond was observed. For example, reduction
of the acetylene intermediate 7e afforded mainly the
2-vinyl PBD (8a), while reduction of the phenylethynyl
intermediate 7c afforded a mixture of the anticipated
PBD (8c) along with styryl and phenethyl by-products.
The Stille coupling reaction has been employed to great
effect in modern organic chemistry.11–13 In 1989, Pena
and Stille reported the application of Stille coupling to
a total synthesis of the naturally occurring PBD, anthra-
mycin14 (1). Initially they followed the strategy of Mori
et al.,15 which relies on N10-MOM protection to allow
subsequent reduction of the dilactam C11-carbonyl.
However, this was unsuccessful and the target anthra-
mycin (1) was eventually obtained by returning to Leim-
gruberÕs original O9/N10-benzal protecting group
strategy (e.g., 2, Fig. 1).16 Intriguingly, these workers
also described a number of C2-alkynic and vinylic
N10-MOM-protected PBD precursors prepared via
Stille coupling (e.g., 3, Fig. 1), but did not report their
conversion to the N10-C11-imine containing final prod-
ucts. We report here the use of Stille coupling to produce
five novel PBDs (6b and 8a–d, Scheme 2), and explore
the limitations of the final, critical, reduction/deprotec-
tion step.
All novel PBDs were evaluated in the NCI 60-Cell Line
screen. The mean GI50 data (Table 1) indicate that all
five PBDs (6b, 8a–d) are significantly cytotoxic with
most GI50 values in the sub-micromolar range. Com-
pound 4 (GI50 = 2.39lM), a C-ring saturated/unsubsti-
tuted PBD20 but with an A-ring identically substituted
to 6b and 8a–d, is included in Table 1 for comparison.
Interestingly, introduction of a 2,3-endo double bond
as in 6b results in a modest (i.e., 2-fold) increase in cyto-
toxicity. However, inclusion of a C2-vinyl substituent in
addition to the double bond (i.e., 8a) increases the activ-
ity by an order of magnitude (i.e., to 0.123lM). Activity
is further potentiated by up to 10-fold by incorporation
of heteroaryl groups such as thiophenyl or furyl (e.g.,
0.023 and <0.01lM for 8b and 8d,21 respectively). The
results for the heteroaryl compounds 8b and 8d are thus
similar in potency to the reported C2-aryl substituted
PBDs.17 Finally, isolating the aryl moiety from the
C-ring with an alkyne functionality (e.g., 8c) maintains
the significant cytotoxicity of the directly conjugated
C2-aryl17 and C2-heteroaryl PBDs. These results are
consistent with the hypothesis that the presence of pla-
nar C2-substituents enhance the fit of the PBD structure
in the DNA minor groove.22
The key enol triflate (5)17 was coupled to a number of
commercially available organotin reagents under Stille
conditions18 to provide 7a–e (Scheme 1). Typically, yields
of between 45% and 81% were obtained, with the best
from the 2-furanyl coupling reaction (81% for 7d19),
and the lowest from the unsubstituted acetylene coupling
(45% for 7e). In the latter case, the modest yield was
probably due to side reactions occurring at the unpro-
tected terminal acetylenic position. In general, the reac-
tions proceeded to completion within 2.5h, although
the 2-thiophenyl tin reagent was less reactive, taking 5h
to completely consume the triflate. In marked contrast
to the other tin reagents, and previous results obtained
from Suzuki reactions,17 tributylphenyltin failed to
afford any coupled product (see Table 1). An attempt
to convert the enol triflate 5 to the more versatile trimeth-
ylstannyl alkene failed to afford the expected product,
but yielded instead the simple unsubstituted endo-2,3-
unsaturated product 6a in 41% yield (Scheme 1).
In summary, this study demonstrates that Stille coupling
is complementary to the Suzuki reaction in being able to
introduce heteroaromatic moieties to the C2 position of
the PBD structure. However, it also demonstrates that,
although C2-vinylic and acetylenic moieties can be effi-
SEM
O
SEM
SEM
N
O
N
O
MeO
MeO
H
N
MeO
MeO
MeO
MeO
Sn2Me6 /Pd[PPh3]4
THF/ reflux
H
H
RSnBu3 /Pd[PPh3]4
THF/ reflux
N
2
N
N
3
OTf
R
O
O
O
6a
5
7a-e
SEM = 2-(Trimethylsilyl)ethoxymethyl
7b: R=
7c: R=
7d: R=
S
O
H
Ph
7e: R=
7a: R=
Scheme 1.

A. C. Tiberghien et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5041–5044
5043
Table 1. Summary of tributyl tin coupling partners, yields of Stille coupling reactions and cytotoxicities of final products (6b, 8a–d) compared to the
C-ring saturated/unsubstituted PBD 4
Tributyl tin
coupling partner
Stille coupling
yield for 7a–e (%)
Final PBD products (6b, 8a–d) and 4
a
Structures
Number
Cytotoxicity: Mean GI50 (lM)
N
MeO
H
H
—
—
4
2.39
N
N
N
N
MeO
O
N
MeO
MeO
Sn2Me6
Bu3Sn
40 (see Scheme 1)
6b
8a
1.28
O
N
MeO
MeO
H
72
0.123
O
N
MeO
MeO
H
66
8b
0.023
<0.01
<0.01
0.123
Bu3Sn
S
O
S
N
MeO
MeO
H
Bu3Sn
Ph
67
8c
N
O
Ph
N
MeO
MeO
H
8119
8d21
N
Bu3Sn
O
O
O
N
MeO
MeO
H
Bu3Sn
H
45
8a
N
O
N
MeO
MeO
H
No coupling observed
Ref. 17
<0.01
N
Bu3Sn
O
a Mean GI50: Mean of concentrations of PBD that inhibits 50% of cell growth (measured using the Sulforhodamine B (SRB) assay) across each of the
60 cell lines of the NCI panel after incubation for 48h at 37°C. For details see: www.dtp.nci.nih.gov/branches/btb/ivclsp.html.
SEM
N
SEM
N
O
OH
MeO
MeO
MeO
MeO
N
H
MeO
MeO
H
NaBH4
H
Silica gel
N
EtOH/Water
EtOH/Water
N
N
R
R
R
O
O
O
6a (R=H)
7a-e
6b (R=H)
8a-d
N10-SEM Carbinolamine Intermediates
Scheme 2.
ciently introduced through Stille coupling, they are
vulnerable to reduction later in the synthetic pathway.
These observations suggest that reducible C2-substitu-
ents should be introduced to a N10-protected PBD carb-
inolamine intermediate (i.e., one that does not require a
final reduction step) rather than to a N10-protected di-
lactam precursor such as 5. The five novel compounds
prepared (6b, 8a–d) all possess potent cytotoxicity, and
their SAR indicate the importance of planar C2-substit-
uents in potentiating biological activity.

5044
A. C. Tiberghien et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5041–5044
15. Mori, M.; Uozumi, Y.; Kimura, M.; Ban, Y. Tetrahedron
1986, 42, 3793–3806.
Acknowledgements
16. Leimgruber, W.; Batcho, A. D.; Czajkowski, R. C. J. Am.
Chem. Soc. 1968, 90, 5641–5643.
17. Cooper, N.; Hagan, D. R.; Tiberghien, A.; Ademefun, T.;
Matthews, C. S.; Howard, P. W.; Thurston, D. E. Chem.
Commun. 2002, 1764–1765.
This work was supported by Cancer Research UK
through Programme Grant C180/A1060 (Previously
SP1938/0402) to D.E.T. and P.W.H.
18. General Stille coupling procedure: The enol triflate 5
(700mg scale) was heated at reflux in dry THF (under
nitrogen) in the presence of Pd(PPh3)4 (0.05equiv), lithium
chloride (3equiv) and the appropriate tin reagent
(1.3equiv) for 2.5–5h. Removal of THF afforded the
crude product, which was partitioned between dichloro-
methane and 10% aqueous ammonium hydroxide. The
separated organic phase was washed with water and brine,
and dried over magnesium sulfate. Excess dichlorometh-
ane was removed by rotary evaporation and the residue
purified by flash column chromatography (silica gel,
gradient elution: ethyl acetate/hexane) to give the final
product.
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1
19. 7d: H NMR (400MHz) (CDCl3): d 7.35 (s, 2H), 7.27 (s,
1H), 7.23 (s, 1H), 6.37 (dd, J1 = 1.7Hz, J2 = 3.2Hz, 1H),
6.26 (d, J1 = 3.2Hz, 1H), 5.52 (d, J = 10Hz, 1H), 4.67 (d,
J = 10Hz, 1H), 4.58 (dd, J1 = 3.3Hz, J2 = 10.6Hz, 1H),
3.91 (s, 3H), 3.90 (s, 3H), 3.86–3.74 (m, 2H), 3.69–3.63 (m,
1H), 3.05 (ddd, J1 = 2.0Hz, J2 = 10.6Hz, J3 = 15.8Hz,
1H), 0.95 (m, 2H), 0.00 (s, 9H); 13C NMR (100MHz)
(CDCl3): d 169.5, 163.0, 153.4, 150.6, 148.7, 143.7, 135.0,
122.6, 122.4, 117.7, 112.7, 108.6, 107.2, 79.7, 68.51, 58.75,
57.5, 31.8, 19.7, 0.0; IR (cmꢀ1, thin film): 2952, 1689, 1640,
1606, 1518, 1453, 1430, 1358, 1276, 1248, 1209, 1102, 1069,
1010, 858, 836, 787, 757; MS (ES+): 471.2 (M+Å+H, 100%),
19
353.1 (65%); ½aꢁD +175 (c 0.02, CHCl3).
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1H), 6.44 (dd, J1 = 1.7Hz, J2 = 3.2Hz, 1H), 6.28 (d,
J = 3.2Hz, 1H), 4.41 (m, 1H), 3.98 (s, 3H), 3.96 (s, 3H),
3.50 (m, 1H), 3.35 (ddd, J1 = 1.6Hz, J2 = 5.2Hz,
J3 = 16.2Hz, 1H); 13C NMR (100MHz) (CDCl3): d
163.8, 162.8, 153.4, 150.7, 149.3, 143.9, 141.8, 124.3,
120.5, 115.8, 113.1, 112.9, 111.3, 108.2, 57.7, 57.6, 55.2,
36.0; IR (cmꢀ1): 3117, 2936, 1626, 1601, 1559, 1508, 1450,
1426, 1377, 1344, 1265, 1215, 1102, 1072, 1039, 1007, 956,
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Å
20
914, 882, 778, 730; MS (ES+): 325.1 (M+ +H, 100%); ½aꢁD
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