A 15N NMR investigation of a series of benzotriazinones and related antitumour heterocycles

Article abstract of DOI:10.1002/mrc.994

A series of 3-substituted 1,2,3-benzotriazin-4-ones, 1 and 2, were synthesized by standard methods and the ¹⁵N NMR spectra were recorded. All spectra were obtained using the natural abundance of the nitrogen-15 isotope. The chemical shifts appear in the normal range for N-1, N-2 and N-3 of the triazine ring, and also correlate with the chemical shifts in the spectra of the imidazolotriazinone, 4, and the imidazolotetrazinone, 5. Significantly, the spectra of 1a, 2 and 4, recorded with full NOE, show inversion of the singlet assigned to N-3, demonstrating that these compounds exist in the tautomeric form shown. The structure of the 4-iminobenzotriazinone (3) was confirmed by this ¹⁵N NMR analysis. The spectrum shows a signal for the NH-bearing imino-nitrogen atom, which is an inverted singlet in the NOE spectrum, whereas the signal from the N-3 atom of 3 is not inverted in the NOE spectrum. Copyright

Full text of DOI:10.1002/mrc.994

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2002; 40: 300–302
Spectral Assignments and Reference Data
A 15N NMR investigation of a series of
benzotriazinones and related antitumour
heterocycles
O
N
H
O
N
N
N
N
N
O
Cl
H
R
N
N
N3
H
N2
N1
4
Keith Vaughan,1∗ Derry E. V. Wilman,2
Richard T. Wheelhouse2 and Malcolm F. G. Stevens3
2
1
R
NH
1
2
a) H
O
Saint Mary’s University, Halifax, Nova Scotia, Canada B3H 3C3
Institute of Cancer Research, CRC Laboratory, 15 Cotswold Road,
CH2CONH2
b) CH3
N
CH3
N5 N3
c) CH2Ph
d) CH2CH2Cl
e) CH2CONH2
f) OH
N
N7
H2NOC
Belmont, Sutton, Surrey, SM2 5NG, UK
N
N2
3
N1
Cancer Research Laboratories, Department of Pharmaceutical Sci-
3
ences, University of Nottingham, Nottingham NG7 2RD, UK
5
g) OCH2Ph
Received 12 July 2001; revised 6 November 2001; accepted 6 November 2001
Scheme 1
A series of 3-substituted 1,2,3-benzotriazin-4-ones, 1 and
2, were synthesized by standard methods and the 15N
NMR spectra were recorded. All spectra were obtained
using the natural abundance of the nitrogen-15 isotope.
The chemical shifts appear in the normal range for N-1, N-
2 and N-3 of the triazine ring, and also correlate with the
chemical shifts in the spectra of the imidazolotriazinone,
4, and the imidazolotetrazinone, 5. Significantly, the
spectra of 1a, 2 and 4, recorded with full NOE, show
inversion of the singlet assigned to N-3, demonstrating
that these compounds exist in the tautomeric form shown.
The structure of the 4-iminobenzotriazinone (3) was
confirmed by this 15N NMR analysis. The spectrum
shows a signal for the NH-bearing imino-nitrogen atom,
which is an inverted singlet in the NOE spectrum,
whereas the signal from the N-3 atom of 3 is not inverted
in the NOE spectrum. Copyright  2002 John Wiley &
Sons, Ltd.
synthesis of 15N isotopically labelled temozolomide allowed the
assignment of all the 15N resonances of the compound. In the
present work, we recorded the 15N NMR spectra of a series of
1,2,3-benzotriazinones (1a–g and 2), the imino-1,2,3-benzotriazine
(3) and 2-azahypoxanthine (4), and assigned the chemical shifts to
the respective nitrogen atoms by comparison with the chemical shifts
observed for temozolomide (5).
EXPERIMENTAL
Sample preparation
1,2,3,-Benzotriazin-4(3H)-one (1a)
This was prepared by diazotization of anthranilamide (6 X D R D H)
according to the method of Heller3 (Scheme 2).
KEYWORDS: NMR; 15N NMR; benzotriazinones; antitumour
6-Chloro-1,2,3-benzotriazin-4(3H)-one (2)
6-Chloroanthranilamide (6, X D Cl, R D H) (Aldrich Chemical)
was diazotized in 2 M hydrochloric acid with sodium nitrite and
neutralized to afford 6-chloro-1,2,3-benzotriazin-4(3H)-one (2).
drugs
INTRODUCTION
The antitumour drug temozolomide (5) (Scheme 1) is of considerable
current interest and is now used widely for the treatment of
malignant glioma.1 A previous study2 reported a multinuclear NMR
study of temozolomide and the related drug mitozolomide. The
3-Methyl-1,2,3-benzotriazin-4-one (1b) and related compounds
Methyl anthranilate (9) was diazotized and the resulting diazonium
salt coupled with methylamine according to the method of LeBlanc
and Vaughan,4 to afford the 1-aryl-3-methyltriazene (10 R D Me)
(Scheme 3), which was cyclized by heating in ethanolic solution to
afford the triazinone (1b).
The same method was used to prepare 3-(2-chloroethyl-)1,2,3-
benzotriazin-4-one (1d), 3-benzyl-1,2,3-benzotriazin-4-one (1c)4 and
3-carbamoylmethyl-1,2,3-benzotriazin-4-one (1e). Full details of the
characterization of 1e are reported elsewhere.5
ŁCorrespondence to: K. Vaughan, Saint Mary’s University, Halifax, Nova
Scotia, Canada B3H 3C3. E-mail: keith.vaughan@stmarys.ca
Contract/grant sponsor: Natural Sciences and Engineering Research Council
of Canada (NSERC).
Contract/grant sponsor: Saint Mary’s University Senate Research Fund.
O
R
N
X
X
CONHR
NH2
HNO2
0˚C
N
N
1
6
CN
i) HNO2, 0˚C
3
ii) NH2CH2CONH2
NH2
7
CONH2
N
i) HNO2, 0˚C
4
ii) NH3 , pH 10-11
N
H
NH2
8
Scheme 2
DOI: 10.1002/mrc.994
Copyright  2002 John Wiley & Sons, Ltd.

301
Spectral Assignments and Reference Data
Table 1. 15N chemical shift data (υ, ppm) for a series of
3-alkyl-1,2,3-benzotriazinones (fully 1H decoupled, without NOE)
CO2Me
NH2
CO2Me
N
i) HNO2, 0˚C
ii) RNH2
R
N
N
H
Compound
N-1a
N-2
N-3
Other
9
10
1a
1b
1c
1d
1e
1f
1g
2
ꢀ16.5
ꢀ16.0
ꢀ13.5
ꢀ14.0
ꢀ14.7
ꢀ23.8
ꢀ17.1
ꢀ18.8
ꢀ35.3
29.3
32.8
32.4
31.8
33.1
24.7
25.2
30.0
22.4
ꢀ152.5b
ꢀ153.8
ꢀ143.8
ꢀ150.3
ꢀ151.3
ꢀ113.4
ꢀ104.1
ꢀ152.5d
ꢀ142.0
—
—
—
O
N
R
N
N
O
N
ꢀ274.6c
N
N
R
R = CH3, CH2Ph,
CH2CONH2
N
N
N
X
H
R′
12
11
Scheme 3
e
4
—
aSee Scheme 1 for atom notation.
3-Hydroxy-1,2,3-benzotriazin-4-one (1f) and the 3-benzoyloxy
compound (1g)
ꢀ152.6 ppm, inverted singlet when 1H coupled with full NOE.
b
c
ꢀ274.7 ppm, inverted triplet when 1H coupled with full NOE.
Methyl anthranilate was treated with hydroxylamine in the presence
of sodium hydroxide to afford the o-aminobenzhydroxamic acid (6
X D H, R D OH), which was diazotized in hydrochloric acid with
sodium nitrite according to the method of Ahern et al.6 to afford
3-hydroxy-1,2,3-benzotriazin-4-one (1f).
ꢀ152.5 ppm, inverted doublet when 1H coupled with full NOE.
d
eN atoms of imidazole ring not observed.
Reaction of 1f with benzoyl chloride in pyridine afforded 3-
DISCUSSION
benzoyloxy-1,2,3-benzotriazin-4-one (1g).6
Table 1 gives the 15N chemical shifts of the benzotriazinones (1a–g
and 2) and of azahypoxanthine (4). The chemical shift of N-1 is in
the range ꢀ13 to ꢀ19 ppm for all of the triazinones, except for 1f
at ꢀ23.8, compared with the chemical shift of ca ꢀ35 ppm in the
imidazolo-fused compounds (4 and 5). The chemical shift of N-2 is in
the range 20–33 ppm for all compounds and N-3 is in the range ꢀ142
to ꢀ153 ppm for most compounds of the series, with the exception
of temozolomide, with an upfield shift to ꢀ180 ppm, and the N-oxy
compounds (1f and 1g), which show a downfield shift to ꢀ104 to
ꢀ113 ppm. Compounds 1e, 3 and 5 exhibit a signal a ca ꢀ275 ppm
arising from the amide N-atom; this signal has been unambiguously
assigned to the amide nitrogen by full 1H NOE, which shows the
signal as an inverted triplet. Compound 5 shows the expected signals
for the imidazole nitrogens at ꢀ105.6 and ꢀ199.8 ppm. Surprisingly,
the N-atoms of the imidazole ring in azahypoxanthine (4) are not
observed, possibly owing to a combination of signal broadening and
the effect of exchange of the hydrogen atom between both nitrogen
sites of the imidazole ring.
3-(Carbamoylmethyl)-4-imino-1,2,3-benzotriazine (3) (Scheme 2)
Anthranilonitrile (7) was diazotized in hydrochloric acid with
sodium nitrite to afford the diazonium salt, which was coupled
with glycinamide. The resulting intermediate triazene cyclized
spontaneously to give the 4-iminotriazine (3). Full details of the
characterization of 3 are described elsewhere.5
2-Azahypoxanthine (4) (Scheme 2)
5-Aminoimidazole-4-carboxamide (8) (0.50 g) was dissolved in 1 M
°
hydrochloric acid (6.0 ml), cooled to 0 C and then added slowly to
a well-stirred ice-cold solution of sodium nitrite (2.0 g) in water
(15 ml). The mixture was stirred for 10 min and then treated
with concentrated ammonia to pH 10–11. The resulting clear
brown solution was stirred for a few minutes until precipitation
was complete. The product was filtered and dried to afford 2-
°
azahypoxanthine (4) (0.31 g, 57%), m.p.>250 C.
When the spectra of 1a, 2 and 4 are recorded with 1H coupled
NOE, the signal from N-3 is inverted in each case, showing that
a hydrogen atom is bonded to N-3. This observation confirms that
these molecules do indeed exist in the tautomeric form shown
(e.g. 1a), rather than the alternative tautomer with the hydrogen
atom located at N-1 as in structure 11 (Scheme 3). The spectra of
2, with and without NOE, are shown in Fig. 1 to illustrate these
observations. The appearance of a doublet for the N-3 nitrogen in
the spectrum of 2 with NOE [Fig. 1(b)] allows the measurement of
the coupling constant 1J(N,H), which is found to be 99 š 5 Hz in this
case. This value is very close to previously reported 1J(N,H) coupling
constants.2
Temozolomide (5)
This was obtained from Aston Molecules (Birmingham, UK).
Spectroscopy techniques
15N NMR spectra were recorded in DMSO solution on a Bruker
AC250 spectrometer (at the Institute of Cancer Research, Sutton)
at 25.36 MHz using a multinuclear 10 mm probe. Solutions were
prepared by dissolving 1 mmol of each compound in dimethyl
sulphoxide (2.4 ml) containing 30% (w/w) DMSO-d6 with 30 mg of
chromium acetylacetonate. A concentric capillary tube containing
nitromethane was used as external reference. Spectra were recorded
at 305 K. All spectra were obtained with the natural abundance of the
nitrogen-15 isotope; no isotope enrichment was necessary to allow
unequivocal assignment of all signals.
The value of these 15N chemical shift assignments is evident in
the analysis of the 15N NMR spectrum of 3-(carbamoylmethyl)-4-
imino-1,2,3-benzotriazine (3). In an independent study,5 a series of
1-aryl-3-carbamoylmethyltriazenes, ArN N—NH—CH2CONH2,
were prepared and characterized. As part of this study, the
diazotization of anthranilonitrile (7) was carried out and the resulting
diazonium salt was coupled with glycinamide, NH2CH2CONH2,
to afford an unstable triazene, which undergoes spontaneous
cyclization. The cyclization product was assigned structure 3 on
the basis of IR and 1H and 13C NMR spectroscopic evidence, but
the structure was not unequivocal. The 15N NMR spectrum of 3
removed the ambiguity from the structural assignment; the 15N
chemical shifts of 3 are shown in Table 2. Chemical shifts for N-1,
N-2 and N-3 are all consistent with the previous observations, and
the amide nitrogen at ꢀ275.05 ppm shows the appropriate inversion
with NOE. The crucial evidence is the signal at ꢀ177 ppm, which
The microprogram INVGATE.AU (standard Bruker software)
was used to acquire NOE-suppressed proton-decoupled 15N spectra
by inverse-gated heteronuclear decoupling. Confirmation of proton
attachment was obtained by heteronuclear gated decoupling with the
GATEDEC.AU microprogram which provided 1H coupled spectra
with full NOE. The parameters used were pulse width 14 µs (45 ),
decoupler power 18 H and 90 pulse for composite pulse decoupling
163 µs. The spectral width was 20 kHz and with 16 K data points gave
an acquisition time of 0.41 s and a digital resolution of 2.44 Hz per
point. Typically, a total of 32 000 scans were acquired for unlabelled
compounds; the FID was exponentially multiplied (line broadening
5 Hz) before Fourier transformation.
°
°
Copyright  2002 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2002; 40: 300–302

302
Spectral Assignments and Reference Data
Table 3. 15N chemical shift data
(υ, ppm) for 8-carbamoyl-3-
methylimidazolo [5,1-d]-1,2,3,5-
tetrazine-4-one (5)
Atoma
υ
b)
N-1
ꢀ34.0
20.0
N-2
N-3
ꢀ180.4
ꢀ199.8
ꢀ105.6
ꢀ274.9b
N-5
N-7
Amide N
aSee Scheme 1 for atom notation.
bInverted triplet when 1H cou-
pled with full NOE.
a)
significant effect. The change from N-methyl in 1b to N-benzyl in 1c
is more significant, causing a 10 ppm downfield shift. Introducing
the chlorine substituent in the benzene ring as in 2 causes almost no
change in any of the 15N resonances when compared with those of
1a; only N-1, the closest nitrogen to the halogen, is shifted marginally
by 2.4 ppm.
100
50
0
−50 −100 −150 −200 −250
ppm
The 15N resonances assigned to N-1, N-2 and N-3 in the
benzotriazines have chemical shifts with a similar order to the
chemical shifts found in the open-chain analogues, the 1-aryl-3,3-
dialkyltriazenes (12) (Scheme 3), for which typical values are N-1,
ꢀ13 to ꢀ35 ppm, N-2, 68–75 ppm and N-3, ꢀ170 to ꢀ220 ppm.8 The
most important deviation in comparing the cyclic and open-chain
variants is in the chemical shift of N-2, which differs by ca 40 ppm.
The most likely explanation for this difference is in the differing
geometry of the N-1 N-2 double bond; the cyclic benzotriazines are
locked into the cis geometry, whereas the 1-aryl-3,3-dialkyltriazenes
are known to prefer the trans geometry in the solid state.9
Figure 1. 15N NMR spectra of 6-chloro-1,2,3-benzotriazin-4(3H)-one
(2) in DMSO, (a) without NOE and (b) with NOE.
Table 2. 15N chemical shift data (υ, ppm) for
3-carbamoylmethyl-4-imino- 1,2,3-
benzotriazinone (3), with NOE
Atom
υ
N-1
ꢀ29.0
31.0
Acknowledgements
N-2
This work was supported by the Natural Sciences and Engineering
Research Council of Canada (NSERC) and by the Saint Mary’s
University Senate Research Fund. We are grateful to the Institute
of Cancer Research, Sutton, UK for access to the Bruker AC250
NMR spectrometer, to Aston Molecules for providing a sample
of temozolomide and to the Cancer Research Laboratories at
Nottingham University, UK, for laboratory resources.
N-3
ꢀ168.4
Amide NH2
ꢀ275.2
(inverted triplet)
ꢀ177.8
Imino NH
(inverted broad doublet)
is assigned to the imino-nitrogen atom at position 4 in the triazine
ring. This nitrogen atom is shown to carry a hydrogen atom by the
observed inversion with NOE.
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(i.e. shifts to lower field) with increasing electron withdrawal by
attached groups. The chemical shift data reported in this paper are
consistent with this general principle. For example, compare the
chemical shift of N-3 in 1b and 1f; the electron-withdrawing effect of
the oxygen atom in 1f, compared with the methyl carbon in 1b, causes
a large downfield shift of ca 40 ppm. On the other hand, changing
the substituent from a methyl group in 1b to a chloroethyl group
in 1d causes only a marginal downfield shift of 3.5 ppm because
the halogen atom is too far removed from the nitrogen to have a
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Copyright  2002 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2002; 40: 300–302