Design and synthesis of a novel water soluble benzotetrazepinone

Article abstract of DOI:10.1016/S0960-894X(00)00461-3

In order to confer water solubility to the benzotetrazepinone ring system, the synthesis of 12 was undertaken. The design and synthesis of 12 were based upon previously established structural requirements for the stability of the 1,2,3,5-tetrazepin-4-one ring system. Tetrazepinone 12 was extremely water soluble and was 10-fold more potent than its imidazo-1,2,3,5-tetrazin-4-one counterpart 1a, against the human MCF-7 breast cancer cell line. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00461-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2325±2327
Design and Synthesis of a Novel Water Soluble
Benzotetrazepinone
Beatrice Dumont-Hornebeck, Yi Ning Strube, Daniela Vasilescu and
Bertrand Jacques Jean-Claude*
Cancer Drug Research Laboratory, Division of Medical Oncology, Department of Medicine, McGill University/Royal Victoria Hospital,
687 Pine Avenue West, Rm M719, Montreal, Quebec, Canada H3A1A1
Received 9 March 2000; accepted 7 August 2000
AbstractÐIn order to confer water solubility to the benzotetrazepinone ring system, the synthesis of 12 was undertaken. The design
and synthesis of 12 were based upon previously established structural requirements for the stability of the 1,2,3,5-tetrazepin-4-one
ring system. Tetrazepinone 12 was extremely water soluble and was 10-fold more potent than its imidazo-1,2,3,5-tetrazin-4-one
counterpart 1a, against the human MCF-7 breast cancer cell line. # 2000 Elsevier Science Ltd. All rights reserved.
Tetrazepinones of type 2a are a novel class of anti-
tumour agents which are more potent than temozolo-
mide 1a (a strong alkylating agent) against alkylating-
agent-resistant Mer+ tumour cells.1,2 In contrast to 1a,
Maxam±Gilbert assays show that tetrazepinones are
weak alkylating agents that create barely detectable levels
of guanine-N7 alkylation in DNA.3 Alkaline sucrose
density±gradient sedimentation studies indicate that
despite their weak alkylating activity, tetrazepinones
induce signi®cant levels of single strand breaks in
human tumour cells. All the results obtained thus far
suggest that tetrazepinones may damage DNA by a
novel mechanism.
the chemistry of this novel class of heterocycles,4 9 the
marked instability of the 1,2,3,5-tetrazepin-4-one moi-
ety precluded the synthesis of compounds containing
functional group at the 3- and 5-positions: bulky sub-
stituents at these positions destabilize the 1,2,3,5-tetra-
zepin-4-one ring, whereas electron-withdrawing groups
at the aromatic ring stabilize it.5 In addition, tetra-
zepinones are rapidly converted to their corresponding
2-diazoniumphenyl urea precursors at acidic pH.7,9
Therefore, benzoic acid-fused tetrazepinones like 2b,
which could be used for coupling with a water soluble
amine, cannot be synthesized. In contrast, imidazote-
trazinone 1b is stable enough to be converted to its cor-
responding acyl chloride for further coupling with
amine-containing biomolecules.10,11 To circumvent the
problems associated with the instability of the tetra-
zepinone ring system, we designed compound 12, which
contains all the structural requirements for a stable
molecule. The 3- and 5-positions are substituted with
methyl groups and the water soluble 4-aminoethylmor-
pholine moiety is connected to the aromatic moiety via
an electron-withdrawing carboxamido (in order to pre-
serve the electron-withdrawing character of the benzene
ring). Ring closure to the 1,2,3,5-tetrazepinone ring was
planned to be performed at the last step of the total
synthesis.
Studies designed to mimic the interactions of tetra-
zepinones with nucleosides under physiological conditions
have been extremely limited by the poor water solubility
of the existing compounds. During the development of
The preparation of 12 proceeded according to Scheme 1.
Acid 3 was treated with aqueous methylamine in a
pressure vessel kept at 160 ꢀC to give 4 which was con-
verted to the corresponding acyl chloride 5 by treatment
with thionyl chloride. Esteri®cation with trimethylsilyl
*Corresponding author: Tel.: +1-514-842-1231, ext. 5841; fax: +1-
514-842-1475; e-mail: bertrand@med.mcgill.ca
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00461-3

2326
B. Dumont-Hornebeck et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2325±2327
Scheme 1. (a) aq CH3NH2 (40%), sealed (160ꢀC), 70%; (b) SOCl2 (neat) re¯ux, 100%; (c) 2-(trimethylsilyl)ethanol (1.1 equiv), pyridine (1 equiv), 72%;
(d) H2/Pd±C, methanol, 90%; (e) K2CO3, PhCH2OCOCl; (f) CH3NCO in CH2Cl2, 35%; (g) tetrabutylammonium ¯uoride (1M in THF), 82%; (h) SOCl2
(neat), re¯ux, 100%; (i) K2CO3, 4-(aminoethyl)morpholine, 98%; (j) H2/Pd±C, methanol, 90%; (k) NaNO2/H+ and K2CO3, 51%; Z=carbobenzyloxy.
ethanol and hydrogenation gave 6, which was treated
with benzylchloroformate to provide a mixture of 7 and
8 in a 2:1 ratio. Isomers 7 and 8 were extremely dicult
to separate by columnn chromatography. Therefore, the
mixture was treated with methyl isocyanate to provide
the corresponding ureas. Separation of the mixture on
silica gel provided the desired urea 9. After removal of
the trimethylsilylethyl group by treatment with tetra-
butyl ammonium ¯uoride (1.5 equiv) in tetrahydrofuran,
the resulting acid was re¯uxed in thionyl chloride for
30 min to give acyl chloride 10 in quantitative yield.
Coupling of 10 with 4-(2-aminoethyl)morpholine pro-
vided the corresponding amide 11a which was converted
to amine 11b by catalytic hydrogenation. The latter
amine was diazotized with 15N NaNO2 and cyclized at
pH 9 to give the desired tetrazepinone 12.
CDCl3 and as a singlet at 3.1 ppm in D2O. In 15N
NMR, N2 appeared at 69.16 ppm in H2O (Fig. 1a). The
peak assignment was based on literature values.4,9 These
data provide the ®rst evidence for a ring-closed tetra-
zepinone structure in aqueous medium.
The CI mass spectrum showed a minor MH+ 29 (15NN)
peak and a large MH+ MeNCO peak. The molecular
ion could not be detected by mass spectrometry using
chemical ionization, however the characteristic MH+
N2 and MH+ MeNCO peak were observed.4,5
Tetrazepinone 12 was tested against the resistant MCF-7
breast tumour cell line using the sulforhodamine B
assay.13 As shown in Figure 1b, 12 (referred to as
MOPH) was 10-fold more potent than temozolomide
against the Mer+ breast tumour cell line MCF-7 [IC50
(temozolomide): 800 mM, IC50 (12): 80 mM].
The structure of 12 was con®rmed by 1H and 15N NMR
spectroscopy, chemical ionization (CI) mass spectro-
metry and elemental analysis.12 Tetrazepinone 12 was
The limitations imposed by the instability of the 1,2,3,5-
tetrazepinone ring to the synthesis of conjugated structures
can be circumvented by carefully designing a conjugated
1
extremely soluble in water. In H NMR, the 3-methyl
group appeared as a doublet at 3.4 (3J 15NH=2.8 Hz) in
Figure 1. (a) 15N NMR spectrum of 12 in D2O; (b) dose±response curve of MCF-7 cells exposed to temozolomide and 12 for 72 h.

B. Dumont-Hornebeck et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2325±2327
2327
2-aminoaromatic urea, which can be cyclized by diazo-
tation at the last step of the total synthesis. The avail-
ability of this ®rst water soluble tetrazepinone will
stimulate further studies on the interactions of tetra-
zepinones with water soluble biomolecules and thereby
signi®cantly improve our understanding of the mechan-
ism of action of this novel class of cytotoxic agents.
8. Jean-Claude, B. J.; Britten, J. F.; Just, G. Acta Crystallogr.
1993, C49, 1070.
9. Jean-Claude, B. J.; Williams, C. I. Magn. Res. Chem. 1998,
36, 87.
10. Clark, S. A.; Deans, B.; Stevens, M. F. G.; Tisdale, M. J.;
Wheelhouse, R. T.; Denny, B. J.; Hartley, J. A. J. Med. Chem.
1995, 38, 1493.
11. Horspool, K. R.; Stevens, M. F. G.; Newton, C. G.; Lunt,
E.; Walsh, R. J. A.; Pedgrift, B. L.; Baig, G. U.; Lavelle, F. J.
Med. Chem. 1990, 33, 1393.
Acknowledgements
12. Experimental data for 12: Urea 11b (95 mg, 0.2 mmol) was
dissolved in 2.4 N HCl (10 mL) and a solution of 27% NaNO2
(1 mL) [50% enriched with 15N NaNO2] was added dropwise
at 0 ꢀC. After 30 min, the solution was extracted three times
with 25 mL portions of CH2Cl2. The aqueous layer was sepa-
rated and neutralized with saturated sodium bicarbonate, after
which it was further re-extracted three times with 25 mL por-
tions of CH2Cl2. The organic layer was removed and dried
over anhydrous potassium carbonate and evaporated to give
12 (50 mg, 51%) as a pale brown powder: mp 125±130 ꢀC
(e€ervescence); dH (CDCl3) 7.95 (1H, d, J=2 Hz, Ar), 7.93
(1H, dd, J=8.6 Hz, J=2, Ar), 7.26 (d, 1H, J=8.6, Ar), 7.05
(1H, br s, NHCO), 3.78 (4H, m, (CH2)2), 3.59 (2H, br q,
NCH2CH2NHCO), 3.42 (3H, d, 3J 15NH=2.8 Hz, 15NNCH3),
3.31 (3H, s, N(CO)CH3), 2.64 (2H, t, J=4.5 Hz, NCH2CH2
NHCO), 2.5 (4H, m, morpholine CH2N); dN (CDCl3): 69.16.
dC (CDCl3): 165.25, 159.02, 140.82, 130.42, 130.07, 125.63,
125.57, 119.98, 119.76, 66.58, 57.01, 53.29, 35.90, 35.07. CIMS
(NH3) m/z 319 (MH+ 15NN, 3.3%), 291 (MH+ CH3NCO,
100). Anal. calcd for C16H22N6O3: C, 55.5; H, 6.4; N, 24.3.
Found: C, 55.27; H, 6.33; N, 24.5.
We thank the National Cancer Institute of Canada
(NCIC) and the Fonds pour la formation des Chercheurs
et l'Aide a la Recherche (FCAR) for ®nancial support.
References and Notes
1. Jean-Claude, B. J.; Mustafa, A.; Watson, A. J.; Damian,
Z.; Vasilescu, D.; Chan, T. H.; Leyland-Jones, B. J. Pharma-
col. Exp. Ther. 1999, 288, 467.
2. Jean-Claude, B. J.; Mustafa, A.; De Marte, J.; Yen, R.; Chan,
T. H.; Leyland-Jones, B. Can. J. Infect. Dis. 1997, 6, 465C.
3. Jean-Claude, B. J.; Mustafa, A.; Damian, Z.; De Marte, J.;
Yen, R.; Chan, T. H.; Leyland-Jones, B. Br. J. Cancer 1997,
76, 467.
4. Jean-Claude, B. J.; Just, G. J. Chem. Soc., Perkin Trans. 1
1991, 2525.
5. Jean-Claude, B. J.; Just, G. Heterocycles 1998, 48, 1347.
6. Williams, C. I.; Whitehead, M. A.; Jean-Claude, B. J. J.
Mol. Sruct. (Theochem.) 1997, 392, 27.
13. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.;
McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Ken-
ney, H. S.; Boyd, M. R. J. Natl. Cancer Inst. 1991, 82, 1107.
7. Williams, C. I.; Whitehead, M. A.; Jean-Claude, B. J. J.
Org. Chem. 1997, 62, 7006.