Preparation and NMR characterization of new substituted benzo[a]phenazines

Article abstract of DOI:10.1002/(SICI)1097-458X(199807)36:7<529::AID-OMR326>3.0.CO;2-K

Benzo[a]phenazines were prepared by condensation of β-lapachone with 1,2-phenylenediamme and 4-chloro-1,2-phenylenediamine. The latter diamine gave two regioisomers that could be separated and unambiguously identified by means of their ¹H and ¹³C NMR spectra with the aid of 2D NMR experiments, mainly HETCOR and COLOC.

Full text of DOI:10.1002/(SICI)1097-458X(199807)36:7<529::AID-OMR326>3.0.CO;2-K

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 36, 529È532 (1998)
Note
Preparation and NMR characterization of new substituted
benzo[a]phenazines
Violeta Benedetti-Doctorovich,* Natalia Escola and Gerardo Burton
Departamento de Qu•mica Orga nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Pabellon 2,
Ciudad Universitaria, 1428 Buenos Aires, Argentina
Received 23 December 1997; accepted 16 February 1998
ABSTRACT: Benzo[a]phenazines were prepared by condensation of b-lapachone with 1,2-phenylenediamine and
4-chloro-1,2-phenylenediamine. The latter diamine gave two regioisomers that could be separated and unam-
biguously identiÐed by means of their 1H and 13C NMR spectra with the aid of 2D NMR experiments, mainly
HETCOR and COLOC. ( 1998 John Wiley & Sons, Ltd.
KEYWORDS: NMR; 1H NMR; 13C NMR; b-lapachone; benzo[a]phenazines
INTRODUCTION
RESULTS AND DISCUSSION
Chemistry
Benzo[a]phenazine derivatives are efficient DNA inter-
calating ligands which have shown to possess antitumor
activity when evaluated in vitro and in vivo against leu-
kemia and solid tumor models.1 On the other hand, b-
lapachone (2,2-dimethyl-3,4,5,6-tetrahydro-2H-oxine-5,
6-dione, 1) is a naturally occurring o-naphthoquinone
with antineoplastic activity.2 These structural features
may be combined to give benzo[a]phenazine analogs of
b-lapachone (1) by condensation of the o-quinone with
1,2-phenylenediamines. We report here the synthesis
and NMR spectral assignment of derivatives 2, 3 and 4.
Regiochemistry of the chlorine-substituted isomers 2
and 3 could be unambiguously established by means of
2D NMR spectroscopy, mostly COLOC and
HETCOR. The complete assignment of the 13C NMR
spectrum of b-lapachone is also given.
Reaction of b-lapachone with phenylenediamines in
reÑuxing methanol gave the corresponding bright
yellow benzo[a]phenazines. When 4-chloro-1,2-pheny-
lenediamine was used, the two possible isomers were
obtained. The preparation of other benzo[a]phenazines
monosubstituted at C-11 or C-12 has been reported3,4
but no assignment of the NMR spectra has been made.
The new compounds were characterized by 1H and 13C
NMR and mass spectrometry.
b-Lapachone (1)
The complete assignment of the benzo[a]phenazine
spectra required the unambiguous assignment of the 1H
and 13C NMR spectra of b-lapachone. Table 1 shows
the spectral data and assignments, which di†er from
those reported previously.5 The aromatic hydrogen
resonances were assigned based on their multiplicities
and by comparison with the spectrum of 8,9-dimethoxy-
b-lapachone described by Sha†ner-Sabba et al.6 In this
compound, H-7 next to the carbonyl resonates down-
Ðeld compared with H-10, next to the ether group.
Hence the double doublets at 8.06 and 7.82 ppm were
assigned to H-7 and H-10, respectively. Selective
decoupling of these hydrogens allowed us to distinguish
between H-8 and H-9. Assignment of H-3 and H-4 was
straightforward based on their chemical shifts. These
assignments were corroborated by means of HÈC long-
range correlations from the COLOC experiment (see
below). Methyne and methylene 13C NMR signals were
then assigned by their one-bond HÈC correlations in a
* Correspondence to: V. Benedetti-Doctorovich, Departamento de
Qu•mica Organica, Facultad de Ciencias Exactas y Naturales, Uni-
versidad de Buenos Aires, Pabellon 2, Ciudad Universitaria, 1428
Buenos Aires, Argentina.
Contract/grant sponsor: CONICET.
( 1998 John Wiley & Sons, Ltd.
CCC 0749-1581/98/070529È04 $17.50

530
V. BENEDETTI-DOCTOROVICH, N. ESCOLA AND G. BURTON
Table 1. 1H and 13C NMR spectral data [d (ppm) and J (Hz)] for b-
lapachone (1) in deuteriochloroform
H
C
HÈC long-range correlationsa
2
3
4
4a
5
6
6a
7
8
9
È
79.1
31.4
16.0
H-4 (3J), CH (2J)
3
CH (3J)
3
1.86 (t, J \ 6.6)
2.58 (t, J \ 6.6)
H-3 (2J)
È
È
È
È
112.5
178.3
179.6
130.0b
128.3
130.5
134.6c
123.9
132.4b
161.8
26.6
H-3 (3J), H-4 (2J)
H-4 (3J)
H-7 (3J)
H-10 (3J), H-8 (3J)
H-8 (2J)
8.06 (dd, J \ 1.4, 7.5)
7.50 (dt, J \ 1.2, 7.4)
7.64 (dt, J \ 1.4, 7.6)
7.82 (dd, J \ 1.0, 7.7)
È
H-10 (3J)
H-7 (3J)
10
H-8 (3J)
10a
10b
CH
H-9 (3J), H-7 (3J)
H-10 (3J), H-4 (3J)
È
1.47 (s)
3
a From COLOC experiment.
b,c Pairs of assignments corrected from Ref. 5.
HETCOR experiment. Carbonyl C-5 was distinguished
from C-6 by the long-range correlations with H-4 and
H-7, respectively, observed in the COLOC experiment.
The chemical shift of C-10b (161.8 ppm) is indicative of
a quaternary sp2 carbon bonded to an oxygen. The
expected long-range correlations with H-4 and H-10
were also observed; the latter conÐrmed the assignment
of the aromatic methynes described above. C-4a was
easily assigned by its sp2 chemical shift (112.5 ppm) and
the long-range correlation with H-3. To distinguish
C-6a from C-10a, the long-range correlations with the
pairs H-8/H-10 and H-7/H-9, respectively, were conclu-
sive.
Chlorine-substituted benzo[a]phenazines 2 and 3
Prior to the assignment of the NMR spectra of 2 and 3,
both regioisomers had to be unambiguously identiÐed.
In these compounds the chlorinated aromatic ring is
isolated from the rest of the molecule by the hetero-
cyclic pyrazine, hence no long range HÈC correlations
Table 2. 1H and 13C NMR spectral data [d (ppm) and J (Hz)] for benzophenazines 2 and 3 in deuteriochloroform
2
3
HÈC long-range
correlationsa
HÈC long-range
correlationsa
H
C
H
C
1
2
3
4a
4b
5
6
7
3.29 (t, J \ 6.6)
18.1
32.3
76.1
È
3.30 (t, J \ 6.7)
18.1
32.3
76.1
È
2.07 (t, J \ 6.6)
CH (3J)
2.07 (t, J \ 6.7)
CH (3J)
3
3
È
È
È
CH (2J), H-1 (3J)
È
È
È
H-1 (3J), H-2 (2J), CH (2J)
H-5 (3J), H-1 (3J)
3
3
152.2
129.2
122.1
129.5
127.6
124.9
130.3
139.9
142.5
127.3
135.0
128.8
130.6
138.4
144.8
109.3
26.8
H-1 (3J), H-5 (3J)
151.7
129.3
122.0
129.7
127.6
125.1
130.2
140.3
140.8
129.8
130.2
133.3
128.0
140.0
144.3
109.4
26.7
H-8 (3J)
H-8 (3J)
8.31 (m)
7.77 (m)
7.75 (m)
9.29 (m)
È
È
È
È
8.32 (m)
7.78 (m)
7.75 (m)
9.29 (m)
È
H-8 (3J), H-5 (2J)
H-5 (3J)
8
È
8a
8b
9a
10
11
12
È
È
È
H-7 (3J), H-5 (3J)
È
È
È
H-7 (3J), H-5 (3J)
H-8 (3J)
H-8 (3J)
H-13 (3J)
H-13 (3J), H-11 (3J)
È
8.23 (d, J \ 2.3)
È
8.14 (d, J\ 9)
È
H-13 (3J)
7.72 (dd, J\ 2.3, 9)
H-13 (3J)
7.68 (dd, J \ 2.3, 9)
È
È
H-13 (2J), H-10 (3J)
13
8.22 (d, J \ 9)
È
H-12 (3J), H-10 (3J)
H-1 (3J)
H-1 (2J), H-2 (3J)
È
8.29 (d, J\ 2.3)
È
13a
14a
14b
CH
È
È
È
È
È
È
H-10 (3J)
H-1 (3J)
H-1 (2J), H-2 (3J)
È
1.53 (s)
1.53 (s)
3
a From COLOC experiment.
( 1998 John Wiley & Sons, Ltd.
MAGNETIC RESONANCE IN CHEMISTRY, VOL. 36, 529È532 (1998)

1H AND 13C NMR OF BENZO[a]PHENAZINES
531
Table 3. 1H and 13C NMR spectral data [d (ppm) and J (Hz)] for benzophe-
nazine 4 in deuteriochloroform
H
C
HÈC long-range correlationsa
1
2
3
4a
4b
5
6
7
3.34 (t, J \ 6.5)
18.4
32.5
76.1
È
2.07 (t, J \ 6.5)
È
CH (3J)
3
H-2 (2J), H-1 (3J), CH (2J)
3
È
È
151.6
129.4
122.2
129.5
127.6
125.1
130.6
140.2
142.6
128.8
129.4
128.0
129.6
140.2
144.3
109.7
26.9
H-5 (3J)
H-6 (3J)
8.33 (m)
7.76 (m)
7.78 (m)
9.34 (m)
È
H-7 (3J)
H-8 (3J)
È
8
H-6 (3J)
8a
8b
9a
10
11
12
H-7 (3J), H-5 (3J)
H-8 (3J)
H-11 (3J), H-13 (3J)
H-12 (3J)
È
È
8.24 (m)
7.78 (m)
7.78 (m)
8.30 (m)
È
È
È
1.53 (s)
H-13 (3J)
H-10 (3J)
13
H-11 (3J)
13a
14a
14b
CH
H-10 (3J), H-12 (3J)
H-1 (3J)
H-2 (3J), H-1 (2J)
È
3
a From COLOC experiment.
are available to correlate those ring nuclei (H or C) to
known b-lapachone related signals. Furthermore,
molecular modeling calculations (AM1, AMPAC 5.01)
predicted a distance of [4 Ó between H-8 and H-10,
thus precluding the use of a NOESY experiment to
assign the latter hydrogen.
H-6, H-7, H-11 and H-12 are heavily overlapped, ren-
dering an unresolved multiplet at 7.75 ppm (Table 3).
Among the signals at ca. 8.3 ppm, H-5 was assigned to
the resonance at 8.33 ppm by HÈC correlation and
comparison with 2 and 3. H-13 and H-10 were at 8.30
and 8.24 ppm; in this case, again we considered that
H-10 in the concave side is shielded compared with
H-13. The latter assignment was conÐrmed by long-
range HÈC correlations in the COLOC experiment.
The 13C NMR spectrum of 4 showed only 18 signals
for the 20 carbons (Table 3). A quaternary carbon could
be found by a QUAT experiment at 129.4 ppm. From
long-range HÈC correlations it could be found that
C-11 and C-4b overlapped at this chemical shift value
(Fig. 1) and that the signal at 142.6 ppm corresponded
to the resonance of C-9a. Signals at 144.3 and 140.2
ppm were assigned to C-14a and C-8b, respectively, by
comparison with 2 and 3 and conÐrmed by the
observed long-range HÈC correlations. Besides the
correlation for C-8b (H-8), the signal at 140.2 ppm
showed cross peaks at 8.24 ppm (H-10) and at 7.78 ppm
(H-12). These correspond to the expected three-bond
correlations for C-13a, which must then overlap with
C-8b.
In benzo[a]phenazine systems it has been established
that the hydrogen inside the concave side of the mol-
ecule, H-8, is downÐeld compared with H-5 on the
convex side. On the other hand, on the benzene ring
next to the heterocycle, H-10 is upÐeld compared with
H-13 on the convex side.7 These di†erences were used
to distinguish between the regioisomers. The isolated
hydrogen in both isomers (i.e H-10 in 2 and H-13 in 3)
can be easily identiÐed from its multiplicity and small
coupling constant (Table 2). Further, the regioisomer
with this H at higher Ðeld must be 2 (H-10 at 8.23 ppm)
and the other one 3 (H-13 at 8.29 ppm). Concomitantly,
H-13 in 2 resonated at lower Ðeld than H-10 in 3 (8.22
vs. 8.14 ppm). This is in agreement with the substitution
pattern of each regioisomer.
The other 1H NMR signals of these compounds
could be identiÐed by comparison with b-lapachone.
13C NMR signals were assigned from the HÈC corre-
lations obtained from HETCOR (one-bond) and
COLOC (two- and three-bond) experiments; all data
were in agreement with the assignments shown in Table
2.
EXPERIMENTAL
1H and 13C NMR spectra were measured at 200.13 and
50.32 MHz, respectively, in a Bruker AC-200 NMR
spectrometer in deuteriochloroform (with tetra-
methylsilane as internal standard) using standard
Bruker software. All spectra were recorded at 303 K.
Typical conditions for 13C NMR spectra were 20 000
Benzo[a]phenazine 4
The assignment of the 1H NMR spectrum of this com-
pound was complicated by the fact that the signals of
( 1998 John Wiley & Sons, Ltd.
MAGNETIC RESONANCE IN CHEMISTRY, VOL. 36, 529È532 (1998)

532
V. BENEDETTI-DOCTOROVICH, N. ESCOLA AND G. BURTON
sequence (COLOC in the Bruker operating software).
The spectra were acquired with 4K ] 256 data points
and a data acquisition of 128 scans ] 128 increments in
the t dimension. Spectral widths of 700È900 Hz for F
1
1
and 6500È7500 Hz for F were used. The relaxation
2
delay was 1 s, the refocusing delay was 15È25 ms and
the corresponding polarization delay was twice this
value. Data were processed using shifted squared sine-
bell functions in both dimensions.
Melting points were determined with a FisherÈJohns
apparatus and are uncorrected. b-Lapachone (m.p. 155È
156 ¡C) was isolated from heartwood of T abebuia
avellanedae8 and also prepared by acid cyclization of
lapachol9 isolated from the same source. 4-Chloro-o-
phenylenediamine was obtained by reduction of the 4-
chloro-2-nitroaniline with Zn in 20% NaOH ethanolic
solution.10
11-Chloro-3,3-dimethyl-2,3-dihydro-1H-benzo[a]oxino[2,3-c]phenazine
(2) and 12-chloro-3,3-dimethyl-2,3-dihydro-1H-benzo[a]oxino[2,3-
c]phenazine (3). b-Lapachone (0.3 g) and 4-chloro-o-phenylenediamine
(0.21 g) were dissolved in methanol (10 ml). The mixture was stirred
under reÑux for 3 h, giving a yellow precipitate. Evaporation of the
solvent followed by Ñash chromatography of the residue eluting with
tolueneÈhexane (4 : 5) a†orded 2 (120 mg, 42%), m.p. 161È162 ¡C,
EIMS m/z 348 (M`, 40), 333 (8), 305 (100), 293 (42), and 3 (80 mg,
28%), m.p. 168.5È169 ¡C, EIMS m/z 348 (M`, 21), 333 (4), 305 (51),
293 (23), 41 (100).
3,3-Dimethyl-2,3-dihydro-1H-benzo[a]oxino[2,3-c]phenazine (4). b-
Lapachone (1.00 g) and o-phenylenediamine (0.55 g) were dissolved in
methanol (15 ml). The mixture was stirred under reÑux for 3 h, giving
a yellow precipitate. Evaporation of the solvent followed by Ñash
chromatography of the residue eluting with hexaneÈethyl acetate (8 : 2)
a†orded 4 (1.27 g, 98%), m.p. 133.5 ¡C (lit.11 130.5È133.5 ¡C), EIMS
m/z 314 (M`, 44), 299 (7), 285 (7), 271 (100).
Acknowledgements
Figure 1. Expansions of the COLOC spectrum of
4
We thank CONICET (Argentina) for Ðnancial support of this work.
showing C–H correlations through 2J and 3J
.
CH
CH
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MAGNETIC RESONANCE IN CHEMISTRY, VOL. 36, 529È532 (1998)