The synthesis and biological activity of C2-fluorinated pyrrolo[2,1-c][1,4]benzodiazepines

Article abstract of DOI:10.1016/j.tetlet.2003.08.075

The novel C2-fluorinated pyrrolobenzodiazepines (1, 2 and 3) have been prepared from commercially available trans-hydroxyproline in good overall yield and were screened for in vitro cytotoxicity against a number of cancer cell lines. The 2R-fluoro isomer 2 exhibits an activity of 76 nM against the CH1 cell line.

Full text of DOI:10.1016/j.tetlet.2003.08.075

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 7809–7812
The synthesis and biological activity of C2-fluorinated
pyrrolo[2,1-c][1,4]benzodiazepines
Ian A. O’Neil,a,* Stephen Thompson,a S. Barret Kalindjianb and Terence C. Jenkinsc
aDepartment of Chemistry, University of Liverpool, Crown St, Liverpool L69 7ZD, UK
bJames Black Foundation, 68 Half Moon Lane, Dulwich, London SE24 9JE, UK
cYorkshire Cancer Research Laboratory of Drug Design, University of Bradford, West Yorkshire BD7 1DP, UK
Received 21 July 2003; revised 4 August 2003; accepted 14 August 2003
Abstract—The novel C2-fluorinated pyrrolobenzodiazepines (1, 2 and 3) have been prepared from commercially available
trans-hydroxyproline in good overall yield and were screened for in vitro cytotoxicity against a number of cancer cell lines. The
2R-fluoro isomer 2 exhibits an activity of 76 nM against the CH1 cell line.
© 2003 Published by Elsevier Ltd.
Within the scientific community, there is a considerable
amount of interest in the design and synthesis of small
molecules that can actively recognise and bind to specific
base-tract sequences of DNA. The pyrrolo[1,4]-
benzodiazepine (PBD) ring system is found in a number
ofantitumour agents belonging totheanthramycin family
of antibiotics. Members of this family include
anthramycin, porothramycin B and prothracarcin (Fig.
1). These agents exert their biological activity by cova-
lently binding to the C2-NH2 of the guanine base in
PuGPu sequences in the minor groove of DNA.1
with the vast majority bearing modifications to the
aromatic A-ring,1,2 with fewer reports of B-3 and C-ring
modifications.4 Dugave et al.5 have reported the synthesis
of Boc- and Fmoc-protected 4-fluoro- and 4,4%-difluoro-
prolinesfromthecommerciallyavailabletrans-4-hydroxy-
proline. Given our interest in defining structural activity
relationships for substituted PBDs, we envisaged these
compounds would be extremely useful in the synthesis of
the C2-fluoro-substituted PBDs 1, 2 and 3 using the
Staudinger/aza-Wittig methodology that we6 and others7
have previously reported. We now wish to report
the successful synthesis and biological activity of the
novel C2-fluoro-analogues 1, 2 and 3 using this method-
ology.
In order to probe their mode of action and biological
activity, several synthetic analogues have been prepared
Figure 1.
* Corresponding author. Tel./fax: 0044 (0)151 794 3485/3588; e-mail: ion@liv.ac.uk
0040-4039/$ - see front matter © 2003 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2003.08.075

7810
I. A. O’Neil et al. / Tetrahedron Letters 44 (2003) 7809–7812
Scheme 1. Reagents: (a) BOC2O, Et3N, DCM, 0°C to rt, 2 h, 95%; (b) (COCl)2, DMSO, Et3N, DCM, −78°C, 93%; (c) 5–7, DAST
(2.5 equiv.), DCM, −78°C to rt, o/n, 85%; (d) 6–8, DAST (5 equiv.), DCM, −78°C to rt, o/n, 90%.
Scheme 2. Reagents: (a) TrCl, Et3N, DCM, 0°C to rt, 2 h; (b) DEAD, DPPE, PhCO2H, toluene, rt, 2 h; (c) 5% KOH/MeOH,
2 h; (d) Boc2O, 10% aq. NaOH, THF:H2O (2:1), 0°C, o/n, 95%; (e) DEAD, PPh3, THF, 0°C to rt, o/n, 65%; (f) Amberlyst-15®,
MeOH, rt, o/n, 80%; (g) DAST, DCM, −78°C to rt, o/n, 81%.
Scheme 3. Reagents: (a) DIBAL-H, THF, −78°C to rt, o/n; (b) 4 M HCl in 1,4-dioxane, o/n; (c) (i) (COCl)2, DCM cat. DMF;
(ii) 13, 14 or 15, Et3N, DCM, −78°C to rt, o/n; (d) Dess–Martin periodinane, DCM, rt, o/n; (e) DPPE, THF, 2 h.

I. A. O’Neil et al. / Tetrahedron Letters 44 (2003) 7809–7812
Table 1. In vitro cytotoxicity for fluorine-substituted C-ring modified PBDs
7811
Table 2. Effect upon thermal denaturation of calf thymus DNA by C-ring modified PBDs
DTm/°C after incubationa
Entry
PBD
0 h
4 h
18 h
48 h
1
2
3
4
5
1
2
3
23
24
0.41+/−0.06
0.57+/−0.07
0.37+/−0.07
0.35+/−0.09
0.31+/−0.10
0.63+/−0.07
0.82+/−0.08
0.54+/−0.06
0.51+/−0.09
0.49+/−0.13
0.85+/−0.07
1.03+/−0.10
0.78+/−0.09
0.74+/−0.10
0.65+/−0.14
0.98+/−0.11b
1.21+/−0.12c
0.86+/−0.10
0.88+/−0.13
0.72+/−0.11
a Determined with either a Varian-Carey 100 or Varian-Carey 1E spectrophotometer fitted with a Peltier temperature controller, following
incubation at 37.0+/−0.1°C within an external water bath for the times shown. Values represent the mean+/−s.e.m. from at least three
determinations.
b Value of 0.99+/−0.12°C after 72 h incubation.
c Value of 1.25+/−0.11°C after 72 h incubation.

7812
I. A. O’Neil et al. / Tetrahedron Letters 44 (2003) 7809–7812
Key to the strategy was the synthesis of BOC-protected
cis and trans 4-fluoro- 7 and 9 and 4,4%-difluoroproline
methyl esters 8. The syntheses of 7 and 8 were achieved
in 83 and 78% yield, respectively, from trans-4-hydroxy-
proline methyl ester hydrochloride 4, using similar
methodology to Dugave5 (Scheme 1).
Nevertheless, both the 2S-F 1 and 2-F2 3 PBDs are
190- and 75-fold, respectively, more active than the
unsubstituted PBD 23 against the CH1 cell line (Table
1, entries 1 and 3 versus entry 4), indicating the pres-
ence of considerably favourable stereoelectronic effects.
It is of note that the 2R-F isomer 2 is more active
against the CH1 cell line than the naturally occuring
PBD DC-81 24.
Attempts to synthesise N-BOC-trans-4-fluoroproline
methyl ester 9 employing the conditions reported by
Dugave proved to be inconsistent in our hands. Purifi-
cation of the crude N-trityl-cis-4-hydroxyproline 10 by
column chromatography gave low yields of the desired
product (Scheme 2). These results were attributed to the
acidity of the silica gel employed during purification,
resulting in the removal of the trityl protecting group.
Use of base-treated silica gel and basic alumina also
failed to give any of the desired product, therefore an
alternative strategy was sought. BOC-protection of
trans-4-hydroxyproline 11 followed by an intramolecu-
lar Mitsunobu reaction and ring-opening of the resul-
tant lactone with Amberlyst-15® in methanol afforded
the desired N-BOC-cis-4-hydroxyproline methyl ester
12 in 50% yield over the three steps. Treatment of 12
with 2.5 equivalents of DAST afforded the desired
trans-4-fluoro analogue 9 in 81% yield.
As can be seen in Table 2, the fluorinated PBDs 1, 2
and 3 significantly increase the thermal stability of the
calf thymus DNA duplex. Longer DNA-PBD contact
times lead to increased thermal stabilisation, a feature
commonly associated with PBDs. In addition, in all
cases the differential effects upon the low-T and high-T
portions of the melting curves suggest that the guanine
tracts (the higher melting temperature events) are selec-
tively modified. Again, it is noteworthy that 1, 2 and 3
all exhibit greater DNA binding affinity than the natu-
rally occuring PBD DC-81 24.
In summary, the replacement of a hydrogen with a
fluorine atom in the C2-position of the PBD ring
system leads to a significant increase in cytotoxicity.
Reduction of esters 7, 9 and 8 with DIBAL-H followed
by removal of the BOC-protecting group by treatment
with 4 M HCl in 1,4-dioxane afforded the cis-4-fluoro-
13, trans-4-fluoro-14 and 4,4%-difluoroprolinol 15
hydrochlorides in 82, 78 and 78% yields, respectively
(Scheme 3). Coupling of 13, 14 and 15 with 2-azidoben-
zoic acid (16) via its acid chloride furnished azido-alco-
hols 17, 18 and 19 in 74, 66 and 81% yields,
respectively. Oxidation of alcohols 17, 18 and 19 with
Dess–Martin periodinane afforded the corresponding
aldehydes 20, 21 and 22 in 81, 80 and 64% yields,
respectively. Aldehydes 20, 21 and 22 underwent a
Staudinger/aza-Wittig cyclisation upon treatment with
DPPE in THF, to give the desired fluoro-substituted
PBDs 1, 2 and 3 in 71, 80 and 62% yields, respectively.
Acknowledgements
We would like to thank the EPSRC for a case award to
S.T. and the James Black Foundation for their contin-
ued financial support of this work. We thank Yorkshire
Cancer Research for programme support (to T.C.J.).
References
1. (a) Thurston, D. E.; Bose, D. S. Chem. Rev. 1994, 94, 433;
(b) Thurston, D. E.; Thompson, A. S. Chem. Br. 1990, 26,
767.
2. Kamal, A.; Rao, M. V.; Reddy, B. S. P. Khimiya
Geterosiklicheskikh Soedineii 1998, 12, 1588.
3. O’Neil, I. A.; Murray, C. L.; Potter, A. J.; Kalindjian, S.
B. Tetrahedron Lett. 1997, 38, 3609.
4. (a) Reddy, B. S. P.; Damayanthi, Y.; Lown, J. W. Synlett
1999, 7, 1112; (b) Gregson, S. J.; Howard, P. W.; Jenkins,
T. C.; Kelland, L. R.; Thurston, D. E. Chem. Commun.
1999, 797.
The C-ring fluorinated PBDs 1, 2 and 3 were screened
for in vitro cytotoxicity (IC50) against the A27808 and
CH1 human ovarian carcinoma cell lines as well as the
L1210 mouse leukaemia cell line and for DNA-binding
affinity by means of the stabilisation given to the
duplex form calf thymus DNA towards thermal denat-
uration.9 The data in Tables 1 and 2 indicate that the
rank order of DNA reactivity and stabilising efficiency
for the C-ring fluorinated PBDs is:-
5. Demange, L.; Menez, A.; Dugave, C. Tetrahedron Lett.
1998, 39, 1169.
6. O%Neil, I. A.; Thompson, S.; Murray, C. L.; Kalindjian, S.
B. Tetrahedron Lett. 1998, 39, 7787.
7. (a) Molina, P.; Diaz, I.; Tarraga, A. Tetrahedron 1995, 51,
5617; (b) Eguchi, S.; Yamashita, K.; Matsushita, Y.;
Kakehi, A. J. Org. Chem. 1995, 60, 4006; (c) Kamal, A.;
Reddy, B. S. P.; Reddy, B. S. N. Tetrahedron Lett. 1996,
37, 6803.
8. Smallie, M.; Kelland, L. R.; Thurston, D. E.; Souhami, R.
L.; Hartley, J. A. Br. J. Cancer 1994, 70, 48.
9. McConnaughie, A. W.; Jenkins, T. C. J. Med. Chem.
1995, 38, 3488.
2R-F 2 >2S-F 1 >2-F2 3
These results suggest that the 2R-F isomer offers the
better presentation of the fluorine group whilst the
PBD is bound, leading to a 550-fold increase in activity
against the CH1 cell line when compared to the unsub-
stituted PBD 23 (Table 1, entry 2 versus entry 4). Initial
molecular modelling studies indicate that the 2R orien-
tation is favoured as it enables superior alignment of
the PBD within the minor groove without wall clash.