Synthesis and isolation of nitro-β-carbolines obtained by nitration of commercial β-carboline alkaloids

Article abstract of DOI:10.1002/jhet.5570380510

Nitration of commercial full aromatic β-carboline alkaloids nor-harmane (1), harmane (2), harmine (3), harmol (4), and the 7-acetylated derivative of harmol (5) is described. Advantages and disadvantages of different nitration reagents which involve acidic conditions (HNO₃/H⁺) and neutral conditions (Cu(NO₃)₂; ceric ammonium nitrate) are discussed. A complete ¹H and ¹³C-nmr characterization including ms and also uv absorption spectra in neutral and acid media is presented. A detailed ei-ms and ld-tof-ms study is enclosed because the nitro-β-carbolines constitute a new family of β-carboline-like chromophores with potential use as matrix in uv-maldi-tof-ms.

Full text of DOI:10.1002/jhet.5570380510

Sep-Oct 2001
Synthesis and Isolation of Nitro-β-carbolines Obtained by Nitration of
Commercial β-Carboline Alkaloids
1071
María A. Ponce and Rosa Erra-Balsells*
Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Pabellón II, 3° Ciudad Universitaria, 1428, Buenos Aires, Argentina
Received October 20, 2000
In Memorial of Dr. Eduardo G. Gros, the Teacher, the Sensei.
Nitration of commercial full aromatic β-carboline alkaloids nor-harmane (1), harmane (2), harmine (3),
harmol (4), and the 7-acetylated derivative of harmol (5) is described. Advantages and disadvantages of dif-
+
ferent nitration reagents which involve acidic conditions (HNO /H ) and neutral conditions (Cu(NO ) ;
3
3 2
1
13
ceric ammonium nitrate) are discussed. A complete H and C-nmr characterization including ms and also
uv absorption spectra in neutral and acid media is presented. A detailed ei-ms and ld-tof-ms study is
enclosed because the nitro-β-carbolines constitute a new family of β-carboline-like chromophores with
potential use as matrix in uv-maldi-tof-ms.
J. Heterocyclic Chem., 38, 1071 (2001).
Introduction.
are used as matrix in polysaccharide analysis, in the nega-
tive ion mode [32-34,39].
The commercially available β-carboline alkaloids [1]
nor-harmane, harmane, harmine, harmol, harmaline and
harmalol have been studied mainly because of their bio-
logical and pharmacological properties and also because of
their photochemical behaviour. They are present in many
plant families [2], human urine [3], human lenses [4] and
cigarette and tobacco smoke [5]. They are known to be
hallucinogens [6], to inhibit the monoamine oxidase
The simultaneous presence, in the aromatic rings of the
molecule, of a group that can be deprotonated, such as NH,
and a basic group such as pyridinic nitrogen together with
the nature and properties of the low energy electronic
excited states cause the special property mentioned above
[32-39].
Up to now we have studied for commercially available
β-carbolines the photochemical behavior, the effect of the
medium on the properties of the ground and the electronic
excited states (intermolecular interactions) and their poten-
tial use as matrix in uv-maldi-tof-ms [12,22-27,32-39]. In
order to study the effect of different substituents present in
the β-carboline moiety (intramolecular interactions) on its
photochemistry and particularly on its efficiency to work
as matrix in uv-maldi-tof-ms analysis of macromolecules,
we decided to prepare β-carboline derivatives.
To begin with, preparation of nitro-β-carbolines was
selected because the nitro group as a substituent induces
strong modification of the acid-base properties and of
the nature of the electronically excited states of aromatic
compounds.
Although the synthesis of nitro-β-carbolines has been
attempted by different authors, the full characterization of
these compounds from the usual point of view has not been
reported yet. For example, the first preparations of a nitro-
harmine were carried out by Fritzsche in 1848 and in 1853
[40,41] who described as product of nitration of harmaline
(3,4-dihydro-harmine) with concentrated nitric acid a
mononitrated-harmine. This nitration reaction was subse-
quently carried out by Konovalova et al., [42] in 1935 who
informed for the mononitro-harmine a mp 204-205°. The
position of the nitro group was assumed by these authors to
be para to indole nitrogen (C-6). Siddiqui et al., in 1973,
[43] stated that the nitration of harmine proceeds in a very
complicated manner and that after considerable experi-
mentation it was possible to obtain mononitro-harmine
(
MAO) [7,8] and to have photosensitizing activity towards
a variety of systems, including bacteria, fungi, viruses and
insects [9,10]. They have been reported to produce singlet
oxygen and super oxide radical anion with varying effi-
ciency [11] and to yield under uv irradiation symmetric
and asymmetric β-carboline dimers [12]. It is also known
that they form charge transfer complexes with flavins [13],
the prosthetic group of MAO, and they bind selectively
with DNA [14]. Because of their emission properties they
have been proposed as fluorescence standards [15,16].
Particular interest has been shown in the study of the acid-
base properties of the β-carbolines in aqueous and in
organic media, in the ground and in the electronic excited
states [17-28]. Electronic excitation results in significant
charge-density changes due to the presence of two differ-
ent types of nitrogen atoms in their structure (pyridinic and
pyrrolic); the pK and the excited state pK* values have
been determined [16-29] together with some kinetic para-
meter [29,30]. Besides, fluorescence quantum yields
and/or life times have been determined in water at different
pH values [28,31]. Recently, they have been described as
efficient matrices (photosensitizers) in matrix-assisted
ultraviolet laser desorption/ionization time-of-flight mass
spectrometry (uv-maldi-tof-ms) for proteins [32-34], syn-
thetic polymers (polyesters [35], polysilesquioxanes
[
36,37], polyamides [38]) and carbohydrates [32,33].
Specially, the sulfated oligosaccharide uv-maldi-tof-ms
analysis has being highly improved [32,39] since commer-
cially available β-carbolines and particularly nor-harmane

1
072
M. A. Ponce and R. Erra-Balsells
Vol. 38
1
that decomposed at about 260° and whose H-nmr spec-
trum allowed to fix the position of the nitro group at C-6. It
by using an specific synthetic route. Thus, the higher-melting
nitro derivative was shown to be 6-nitroharmane (2a) and that
with the lower-melting point was supposed presumably to be
the 8-nitroharmane (2b). This paper includes the description
of the ir spectrum of each compound. Besides, there are some
papers which describe that by treatment of harmane with
nitric acid the 6-nitroharmane was prepared in order to study
several properties by using the uv spectra [31,45-47]. Among
them, only in references 45 and 46 are the mp's of the pre-
pared nitro-harmane reported as proof of characterization. As
part of a study of structure–inhibitory activity relationship of
derivatives of β-carbolines, 6-nitroharmane was prepared by
treatment of harmane with concentrated nitric acid and was
characterized by mp and the m/z value of the molecular ion
is interesting to point that this is the only nitro-β-carboline
Scheme 1, compound 3a) whose H-nmr spectrum has
been described in the literature. On the contrary, for the
-nitro-harmine (Scheme 1, compound 3b), obtained by
oxidation of 8-nitro-harmaline with cromic acid, only the
1
(
8
mp was reported in 1935 [42], and for 6,8-dinitro-harmine
(3c) its preparation has never been reported.
Scheme 1
+
obtained by ei-ms (M , 227) [48].
Finally, in the case of nor-harmane (1), after the descrip-
tion of Saxena of a mononitro derivative that he presumed to
be the 6-nitronor-harmane (1a), obtained by treatment of 1
with concentrated nitric acid [49], several studies have been
published. Some of these studies only include the descrip-
tion of the uv of 1a [50,51] because of its acid-base proper-
ties. For others, the only proof of identity given for the nitro
derivative presumed to be 1a is that the method used for its
preparation is the treatment of 1 with concentrated nitric
acid [52,53]. In these cases, the authors do not take into
account the possible formation of 8-nitronor-harmane (1b).
Here we report full details of various attempts for nitra-
tion of the commercial full aromatic β-carboline alkaloids
R1
H
R7
H
Compound R1
R6
R7
R8
Compound
1
1a
H
H
NO2
H
H
H
H
H
H
NO2
H
NO2
H
1b
2
3
Me
H
2a
Me NO2
Me
2b
H
Me MeO
Me AcO
3a
Me NO2 MeO
Me
Me NO2 MeO NO2
Me NO2 AcO
Me AcO NO2
3b
H
MeO NO2
3c
5
5a
H
5b
H
(
Scheme 1) nor-harmane (1), harmane (2), harmine (3),
harmol (4) and O-acetyl harmol (5) together with a com-
plete characterization of the nitro-β-carbolines obtained.
These compounds constitute an interesting family of β-
carboline-like chromophores with potential use as uv-
maldi matrix. Thus, a detailed ei-ms and ld-tof-ms study
(
ultraviolet laser desorption/ionization time-of-flight mass
R1 R7
R1
HCO
R6
H
R7
R8
H
spectrometry) is enclosed. In order to compare the ms data
obtained for nitro-β-carbolines with those of nitro-car-
bazoles, the 1-, 3-, 1,6-di- and 3,6-dinitrocarbazoles 6a-d
were also prepared (Scheme 1) and the corresponding ei-
and ld-tof- mass spectra were measured.
4
Me HO
4d
HO
Results and Discussion.
Nitration Procedures.
Nitration of nor-harmane (1) and harmane (2) as clean
6
R1
NO2
H
NO2
H
R3
H
NO2
H
R6
+
reactions were conducted with HNO /H [54,55] with high
3
6
a
H
H
NO2
NO2
yields (1a (64%), 1b (30%), 2a (66%) and 2b (32%)),
recovering in low percentage the corresponding β-carbo-
line. However, this acidic oxidative condition results in
lower yields for nitro-harmine derivatives (3a (10%), 3b
6b
6
c
6d
NO2
(5%)) because the following side reactions take place: oxi-
About the nitro derivatives of harmane (Scheme 1, com-
pounds 2a and 2b), Snyder et al., in 1948 [44] described the
preparation, by using concentrated nitric acid, of two isomeric
nitro-harmanes with the proof of the structure of one of them
dation of harmine (3), a total oxidation/dimerization of
harmol (4), and the removal of the acetyl group from
acetyl-harmol (5). Thus, a variety of oxidized products are
generated that are unstable and difficult to purify.

Sep-Oct 2001
Nitro-β-carbolines Obtained by Nitration of Commercial β-Carboline Alkaloids
1073
Acetyl nitrate [54,55] (in situ generation from nitric aid
and acetic anhydride) in tetrahydrofuran at 0° also gave the
dimmer of harmol, dimeric 1-formyl-harmol (4d), from
both harmol (4) and its acetyl derivative (5).
On the contrary, efficient and clean nitration of harmol,
as acetyl harmol (5), was obtained by treatment with cop-
per(II) nitrate/acetic anhydride [56,57] at room tempera-
ture for 48 hours (yields: 5a 9%; 5b 33%) although harmol
Cerium ammonium nitrate (CAN) in acetonitrile has been
recently described as an efficient reagent for nitration, which
does not require acid catalysis [58]. Thus, mononitro car-
bazoles have been successfully prepared with this reagent.
This shows that CAN is a mild nitration reagent because
polynitro carbazoles were not obtained [58]. In our experi-
ments with CAN and β-carbolines we improved the work-up
of the reaction in order to eliminate excess cerium in the
reaction mixture avoiding the formation of β-carboline-Ce
complexes. Thus, from harmane and harmine the mononitro
derivatives 2a, 2b, 3a and 3b were obtained in very low yield
(17%, 3%, 12% and 4% respectively), together with dimeric
products. From nor-harmane, which is not specially activated
for electrophilic substitution, the conversion to mononitro
derivatives is very low (< 5%) and nor-harmane remains
almost unchanged. The fact that in nor-harmane only one
(4) itself was completely oxidized even at low tempera-
ture. Analogous treatment of nor-harmane (1) and harmane
(
2) gave the same reaction pattern as treatment with
+
HNO /H , but in lower yield (yields: 1a 32%; 1b 18%; 2a
3
5
5%; 2b 5%). For harmine, the reaction was cleaner than
+
that with HNO /H . After this treatment, harmine was
3
recovered unchanged in high percentage and the yield of
the nitro derivatives 3a, 3b and 3c was low (yields: 12%,
+
4% and 2% respectively).
ring is activated for the attack by NO2 while in carbazole
Table 1
Static Charge Distribution for β-Carbolines
Compound
Method [a]
Atom number
1-C
2-N
3-C
4-C
5-C
6-C
7-C
8-C
1
AM1
PM3
ZINDO/S
AM1
PM3
AM1
AM1
AM1
AM1
AM1
PM3
ZINDO/S
AM1
PM3
AM1
AM1
AM1
AM1
AM1
PM3
ZINDO/S
AM1
PM3
AM1
AM1
AM1
AM1
PM3
ZINDO/S
AM1
PM3
-0.088
-0.061
0.112
-0.120
-0.065
-0.380
-0.071
0.583
-0.113
-0.068
-0.114
-0.068
-0.123
-0.064
-0.397
-0.079
0.556
-0.117
-0.069
-0.118
-0.072
-0.067
-0.068
-0.408
-0.089
0.528
-0.117
-0.063
-0.134
-0.125
-0.068
-0.410
-0.089
0.531
-0.103
-0.089
0.116
-0.127
-0.096
-0.036
-0.100
0.010
-0.122
-0.096
-0.124
-0.094
-0.133
-0.105
-0.046
-0.101
0.004
-0.128
-0.099
-0.131
-0.099
-0.111
-0.111
-0.046
-0.116
-0.017
-0.130
-0.110
-0.134
-0.138
-0.112
-0.044
-0.115
-0.016
-0.131
-0.130
-0.044
-0.110
-0.019
-0.131
-0.132
-0.069
-0.041
-0.010
-0.041
-0.017
0.004
-0.020
-0.018
-0.001
-0.069
-0.041
-0.011
-0.043
-0.017
0.004
-0.020
-0.018
-0.001
-0.002
-0.006
-0.010
0.001
-0.161
-0.127
-0.037
-0.140
-0.094
-0.178
-0.154
-0.185
-0.155
-0.162
-0.127
-0.037
-0.140
-0.095
-0.179
-0.154
-0.186
-0.155
-0.208
-0.166
-0.051
-0.222
-0.140
-0.187
-0.241
-0.163
-0.245
-0.169
-0.055
-0.228
-0.142
-0.179
-0.173
-0.050
-0.160
-0.146
-0.202
-0.202
-0.095
-0.074
-0.013
-0.045
-0.022
-0.032
-0.001
-0.025
0.014
-0.096
-0.074
-0.014
-0.047
-0.023
-0.032
-0.001
-0.025
0.014
0.119
-0.120
0.159
0.175
0.184
0.152
0.218
0.191
0.115
0.131
0.154
0.173
0.194
0.128
-0.12-
0.158
0.197
0.183
0.202
0.160
-0.157
-0.108
-0.040
-0.151
-0.097
-0.178
-0.156
-0.181
-0.165
-0.157
-0.109
-0.041
-0.152
-0.099
-0.178
-0.156
-0.183
-0.162
-0.156
-0.198
-0.046
-0.204
-0.217
-0.201
-0.465
-0.161
-0.193
-0.213
-0.033
-0.202
-0.229
-0.239
-0.243
-0.045
-0.254
-0.199
-0.270
-0.199
1H
-0.045
-0.232
-0.081
-0.035
-0.075
-0.024
-0.025
-0.034
0.153
-0.041
-0.267
-0.096
-0.031
-0.096
-0.037
-0.096
-0.083
0.117
-0.041
-0.259
-0.089
-0.030
-0.089
-0.034
-0.080
-0.079
0.117
-0.031
-0.239
-0.087
-0.072
-0.067
-0.093
-0.078
0.110
-0.031
-0.239
-0.096
-0.094
0.116
1
1
1
1
2
a
aH
b
bH
2H
0.034
-0.175
-0.017
-0.001
-0.011
-0.001
-0.032
-0.031
0.160
2
2
2
2
3
a
aH
b
bH
3H
0.033
-0.158
-0.017
-0.017
0.015
-0.023
-0.030
0.155
0.036
-0.159
0.026
0.027
0.160
0.043
-0.158
-0.015
-0.006
0.019
0.006
0.056
0.015
-0.029
-0.002
-0.009
0.003
3a
3b
3c
4
4H
0.024
5
AM1
PM3
ZINDO/S
AM1
PM3
-0.121
-0.122
-0.405
-0.090
0.527
-0.038
-0.040
-0.011
-0.017
-0.015
0.028
5H
0.032
-0.238
-0.084
-0.085
5
5
a
AM1
AM1
-0.118
-0.117
b
-0.009
[a] Single Point calculations were performed using semiempirical methods (AM1; PM3; ZINDO/S) after geometry optimization at HF/6-31G level

1
074
M. A. Ponce and R. Erra-Balsells
Vol. 38
both rings are equally activated explains why CAN, a mild
nitration reagent, is more effective for mononitration of car-
bazole than for mononitration of nor-harmane.
lated by AM1. As can be seen in Table 1, β-carbolines (1,
2, 3, 4, and 5) and cationic β-carbolines (β-carboline + H ;
1H, 2H, 3H, 4H and 5H) have higher charge density values
+
Formation of dimeric products from harmane and
harmine, which resemble the N,N'-bis-β-carboline sym-
metric dimers which we obtained by uv-irradiation of
these β-carbolines [12], can be explained taking into
account the formation of N-β-carbolinyl radicals which
then yield symmetric dimers. Formation of alkoxy radicals
by CAN induced oxidation of alcohols and benzylic alco-
hols has recently been demonstrated by epr [59] suggest-
ing that CAN works as one electron oxidation reagent. A
similar CAN induced radical reaction would account for
the dimerization mentioned above and also for the oxida-
tion of the methyl group at C-1 of harmane (2) and
harmine (3) affording 4d.
localized at 6-C and 8-C, thus predicting that the incoming
NO2 would preferentially attack these positions. In agree-
+
ment with this fact 6-nitro- and 8-nitro-β-carbolines were
obtained from 1, 2, 3, 5, 1H, 2H and 3H. Net atomic charge
values calculated by AM1 also predicted that the mononi-
+
trated-β-carbolines might react with NO2 to yield the 6,8-
dinitro derivative as we observed for harmine (Scheme 1,
compound 3c).
As can be seen in Table 1, the values obtained for β-car-
bolines 1, 2, 3, 4 and 5, calculated by the PM3 method,
predicted higher charge density values at 6-C and 8-C and
+
for cationic β-carbolines (β-carboline + H ; 1H, 2H, 3H,
4H and 5H) at 1-C and 3-C. The absence of the predicted
1
-nitro- and 3-nitro-β-carbolines in the reaction mixture
Theoretical Calculations.
when nitration was performed in acidic conditions
(HNO /H ) from 1H, 2H, 3H and 4H suggests that PM3
+
The substitution patterns for the nitration of β-carbolines
3
1
-5 were studied using the net atomic charge values calcu-
does not explain the substitution pattern experimentally
obtained.
lated by semiempirical methods (AM1, PM3 and
ZINDO/S) after geometry optimization with the HF level
of theory and 6-31G basis set.
Finally, as can be seen in Table 1, ZINDO/S calculations
for all the β-carbolines studied predicted similar charge
values for 4-C, 6-C and 8-C and do not explain the experi-
mental results obtained. 4-Nitro-β-carbolines were not
detected in any of the reactions studied.
The nitration patterns observed by using acidic nitration
+
reagents (HNO /H ) and neutral nitration reagents
3
(Cu(NO ) ; CAN) were predicted using the values calcu-
3
2
Table 2
Uv Absorption Sspectra of β-Carbolines in Solution at 298 K [a]
λ max (nm) (log ε)
Compound
Solvent
MeCN
1
346 (3.61)
374 (3.65)
366 (3.68)
366 (3.67)
396 (3.68)
392 (3.67)
344 (3.690
366 (3.73)
395 (3.69)
385 (3.72)
394 (3.67)
384 (3.98)
332 (3.68)
360 (3.92)
360 (3.75)
356 (3.91)
372 (3.65)
362 (3.65)
432 (3.57)
476 (3.58)
334 (3.65)
326 (4.30)
332 (3.68)
358 (3.93)
358 (3.90)
356 (3.93)
371 (3.66)
360 (3.65)
332 (3.60)
302 (4.22)
346 (3.69)
336 (3.68)
302 (3.60)
296 (3.61)
330 (3.68)
300 (4.27)
355 (s) (3.68)
345 (s) (3.70)
348(s) (3.15)
298 (4.25)
324 (3.72)
324 (4.30)
332 (3.90)
314 (s) (3.90)
292 (s) (3.73)
301 (4.20)
380 (3.60)
288 (4.01)
322 (3.77)
286 (4.15)
+
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
3
3
4
4
5
5
5
5
5
5
H
a
aH
b
bH
MeCN/H [b]
MeCN
332 (3.96)
286 (4.38)
270 (4.37)
268 (4.35)
286 (4.17)
288 (4.35)
+
MeCN/H
MeCN
+
MeCN/H
MeCN
H
a
aH
b
bH
MeCN/H+
MeCN
310 (3.93)
297 (4.86)
286 (3.85)
268 (4.55)
298 (4.21)
276 (4.10)
+
MeCN/H
278 (s) (3.88)
MeCN
+
MeCN/H
MeCN
+
H
a
aH
b
bH
c
MeCN/H
300 (3.94)
298 (4.10)
280 (3.98)
278 (4.15)
308 (3.60)
MeCN
+
MeCN/H
MeCN
+
MeCN/H
324 (3.59)
245 (4.10)
300 (4.18)
MeCN
+
cH
MeCN/H
MeCN
+
H
MeCN/H
323 (3.71)
324 (4.31)
332 (3.89)
319 (3.20)
292 (s) (3.74)
300 (4.10)
298 (4.20)
MeCN
+
H
a
aH
b
bH
MeCN/H
300 (3.94)
298 (4.12)
280 (3.99)
279 (4.18)
MeCN
+
MeCN/H
MeCN
+
MeCN/H
a] Concentration: 5.0 x 10-5 M; λ in nm; ε in M-1 cm . [b] MeCN with 1% sulfuric acid 0.1 M.
-1
[

Sep-Oct 2001
Nitro-β-carbolines Obtained by Nitration of Commercial β-Carboline Alkaloids
1075
The theoretical results predicted by AM1 calculations and
the reaction patterns obtained experimentally agree with
previous results reported for quinoline [55]. The products
arising from nitration clearly showed that one ring reacts
faster than the other, and the authors stated that the pyridine
ring reactivity was lowered by the factor 2.319 for elec-
trophilic substitution as compared to the other aromatic ring.
routes involving expulsion of the NO and NO species as
the primary processes. The extent and ease of valence iso-
merization reflected by the relative abundances of [M-
2
+
+
NO] (quinonoid structure) and [M-NO2] peaks is
strongly influenced by the structure of aromatic system,
the character of the other substituents and the position of
the nitro group [61]. Hydrogen transfer and oxygen migra-
tion are the main rearrangements that complicate the spec-
tra of substituted nitroarenes [61].
Absorption Spectra.
The absorption spectra of β-carbolines 1, 2, 3, 4 and 5
and the nitro-β-carbolines 1a, 1b, 2a, 2b, 3a, 3b, 3c, 5a
and 5b in acetonitrile were recorded (Table 2). The absorp-
tion spectra of the cationic species were obtained by addi-
tion of the appropriate quantity of sulfuric acid.
In the spectra of 1a and 1b the molecular ions (M) are
quite important (Table 3, ei-ms, 84% and 30%).
Comparison of the relative intensities of M peaks with that
+
+
+
of [M-NO] and [M-NO ] (Table 3, [M-NO] : m/z 183,
2
+
8
0% and 7%; [M-NO ] : m/z 167, 85% and 23%) provides
2
As we previously described, in non-polar as well as in
polar protic and polar aprotic organic solvents β-carbolines
an explanation of the greater stability of the 6-quinonoid
structure obtained from 1a. This clear difference between
1
, 2, 3 and 4 exist in the neutral form in the S state, thus, the
+
0
the relative intensity of [M-NO] species is also observed
bands of the neutral species are observed and there is enough
evidence that the low energy band is due to a π→π* transi-
tion with typical λmax at 346, 344, 332 and 334 nm, respec-
tively (Table 2) [24-27,60]. These λmax values show a dra-
matic and clear bathochromic shift (red shift) when the spec-
tra are recorded in acidic medium (Table 2, λmax values for
in the spectra of the pairs 2a/2b and 3a/3b although the
+
abundances of the [M-NO] fragments are lower (Table 3,
+
[M-NO] : m/z 197, 2a, 10%; 2b, 5%; m/z 227, 3a, 9%, 3b,
3%). For each pair the main fragmentation route involving
the NO substituent is the expulsion of this group (Table 3,
2
+
[M-NO ] : m/z 181, 2a and 2b). For compounds 3a and 3b
2
1H, 2H, 3H and 4H) owing to the yield of the protonated
the susceptibility of the nitro-nitrite rearrangement is
caused by the ortho effect of the methoxy group at 7-C.
From the methoxy-group, the hydrogen transfer to the nitro
molecule. In the cationic species the stronger interaction of
the pyridine n electron pair involved in the proton bridge
with the aromatic ring π system (mesomeric effect; extension
of conjugation) would account for the λmax shifts observed.
For the 7-acetyl-harmol 5 the same spectroscopical behavior
was observed (Table 2, see λmax for 5 and 5H).
group followed by secondary decomposition ([M-HNO ]),
2
produces more abundant peaks in both spectra than the iso-
+
merization to yield [M-NO] and the simple expulsion of
+
NO to yield [M-NO ] (Table 3, [M-HNO ]: m/z 210, 3a,
2
2
2
As shown in Table 2, the absorption data of the nitro-β-
carboline derivatives in both neutral and protonated forms
+
6
3%; 3b, 93%; [M-NO] , m/z 227, see above).
Compound 5 (M , m/z 240), the 7-acetyl derivative of
+.
(1a and 1aH, 1b and 1bH, 2a and 2aH, 2b and 2bH, 3a and
harmol, mainly loses CH CO (ketene) (Table 3, m/z 198,
2
3aH, 3b and 3bH, 3c and 3cH, 5a and 5aH, 5b and 5bH) are
100%). The nitro derivatives of compound 5, 5a and 5b,
quite similar. Protonation of nitro-β-carbolines does not lead
to an important bathochromic shift of λmax. On the contrary,
in all cases minor to middle clear hypsochromic shifts (blue
shifts) were observed (2 to 10 nm). The protonation effect
observed on the lowest energy band of the absorption spectra
of nitro-β-carbolines suggests that these transitions are in
nature different from those of β-carbolines (compounds 1, 2,
containing the NO substituent next to the 7-acetyloxy
2
+
group do not show significant [M-HNO ] peaks (m/z
2
238). By comparison of the spectra of 3a, 3b, 5a and 5b in
connection with HNO expulsion it seems that hydrogen
2
transfer is a specific rearrangement only possible if the
intramolecular hydrogen bridge involves the formation of
stable 5 to 7 membered rings as an intermediate [61]. From
the molecular ions (Table 3, m/z 285, 5a, 4%; 5b, 6%) the
main fragmentation path is the subsequent expulsion of
3, 4 and 5) and that some of them clearly are n→π* (higher
blue shift). However, it is to be noted that in some cases the
protonation effect was low or there was no effect at all (Table
CH CO (Table 3, m/z 243, 5a, 100%; 5b, 37%) and NO
2
2
2
, compounds 1a, 1b and 3a) and that extinction coefficient
(Table 3, m/z 197, 5a, 56%; 5b, 39%). The differences in
4
values (ε) are always around 10 . Probably the close-lying
n→π* transitions having much lower ε values are likely hid-
den by the more intense π→π* ones, and their positions
could not be clearly detected even by the protonation effect.
the spectra of 5a and 5b are determined by the subsequent
expulsion of NO (Table 3, m/z 213, 5a, 10%; 5b, < 1%)
and CO (Table 3, m/z 185, 5a, 3%; 5b, < 1%) from the
fragment of m/z 243, which is favored when the stable
6-quinonid structure can be formed (compound 5a).
Mass Spectrometry.
+
.
The intensities of [M-O] ions are low or just do not
appear in the spectra of the nitro-β-carbolines studied.
After the expulsion of the nitro subtituent the main frag-
mentation path of 1a, 1b, 2a, 2b, 3a, 3b, 5a and 5b are
Electron Impact Mass Spectrometry (ei-ms).
As it is known, the electron impact induced fragmenta-
tion of nitroarene radical-cations proceeds by two basic

1076
M. A. Ponce and R. Erra-Balsells
Vol. 38
Table 3
Ld-tof-ms and Ei-ms Signals of β-Carbolines, Nitro-β-carbolines, Carbazole and Nitrocarbazoles [a]
ld-tof-ms
MW Meth. [a] Positive ion mode
ei-ms
Positive ion mode
Comp
Negative ion mode
1
168
213
A
B
A
334 (5), 169 (100)
335 (36), 169 (100), 132 (19)
214 (20), 198 (10), 184 (18),
225 (5), 167 (100), 112 (90)
225 (10), 167 (100), 112 (90)
496 (4), 422 (6), 212 (100), 196 (18),
182 (14), 166 (28), 112 (6)
+·
1a
213 (M , 84), 183 (80), 167 (85),
155 (44), 140 (100), 129 (8), 113 (82),
9 (11), 83 (16), 69 (35), 46 (36)
168 (100)
9
B
A
425 (8), 214 (22), 198 (10),
84 (18), 168 (100)
496 (14), 422 (10), 212 (100), 196 (20),
182 (10), 166 (20), 112 (16)
1
+·
1b
213
492 (5), 214 (100), 198 (18), 184 212 (100), 196 (25), 112(8)
8), 168 (70), 129 (30)
213 (M , 30), 183 (7), 167 (23),
155 (5), 140 (23), 129 (6), 113 (11),
(
97 (14), 83 (18), 69 (32), 46 (100)
B
492 (15), 214 (100), 198 (28),
84 (9), 168 (75), 129 (23)
223 (7), 212 (100), 196 (28), 112(10)
1
2
2
182
228
A
B
A
362 (4), 183 (100)
363 (14), 183 (100)
362 (5), 229 (10), 199 (8) 183
181 (100), 112 (35)
246 (21), 224 (35), 181 (88), 112 (100)
227 (100), 211 (24), 197 (20), 181 (18),
112 (78), 96 (64)
+·
a
227 (M , 100), 210 (2), 197 (10), 181 (63),
169 (21), 154 (22), 140 (8), 127 (34), 113
(100)
(
(
15), 97 (17), 84 (10), 78 (19), 73
11), 69(4), 63 (20), 57 (7), 45 (10 )
B
A
362 (20), 229 (8), 199 (8) 183
100)
444 (10), 408 (13), 229 (100),
13 (20)
324 (10), 248 (6), 227 (78), 211 (20), 197
(12), 181 (10), 134 (48), 113 (100), 96 (34)
453 (4), 405 (3), 227 (100), 211 (18),
112 (8)
(
+·
2b
228
227 (M , 80), 210 (2), 197 (5), 181
(41), 169 (5), 154 (11), 140 (3), 127
2
(12), 113 (6), 99 (4), 88 (3), 77 (9),
71 (16), 69 (100), 57 (33), 44 (58)
B
444 (15), 408 (10), 229 (100),
13 (20)
453 (10), 405 (8), 227 (100), 211 (10),
112 (16)
2
3
3
212
257
A
B
A
423 (3), 213 (100)
423 (6), 213 (100)
258 (100), 242 (20), 228 (60),
211 (100), 112 (35)
246 (20), 224 (80), 211 (75), 112 (100)
256 (100), 240 (14), 226 (16)
+
·
a
b
c
257 (M , 100), 227 (9), 212 (32), 210
(63), 199 (16), 197 (18), 181 (79), 169
(
(
6
212 (76), 182 (6)
42), 154 (28), 140 (23), 127 (36), 113
21), 99 (13), 88 (14), 77 (25), 71 (35),
9 (22), 57 (42), 45 (54 )
B
A
513 (3), 258 (100), 242 (15),
28 (56), 212 (68), 182 (4)
258 (100), 242 (70), 228 (12),
12 (3)
511 (20), 497 (8), 483 (12), 256 (100),
240 (24), 226 (10)
256 (100), 24 (24), 226 (24), 150 (14)
2
+·
3
3
257
302
257 (M , 63), 227 (3), 212 (12), 210
(93), 199 (11), 197 (11), 181 (93), 169
2
(
(
6
37), 154 (55), 140 (41), 127 (56), 113
40), 99 (30), 88 (37), 77 (48), 71 (43),
9 (30), 63 (100), 57 (17), 45 (75 )
B
A
512 (5), 280 (12), 271 (34), 258 300 (15), 271 (10), 256 (100), 240
100), 242 (64), 228 (14), 212 (6) (24), 226 (24), 150 (14)
346 (2), 324 (1), 303 (100), 287 301 (44), 285 (100), 271 (56), 255 (42),
(
+·
302 (M , 4), 285 (1), 270 (1), 257 (2),
255 (3), 243 (2), 225 (1), 213 (2), 147
(5), 136 (4), 121 (4), 97 (11), 83 (23), 77
(42), 273 (20), 257 (58), 227
197 (30), 179 (32)
28), 211 (10), 197 (16), 181 (3)
(
(
4), 71 (10), 69 (50), 63 (1), 59 (3), 58
2), 57 (13), 56 (56), 46 (30), 45 (100)
B
346 (14), 324 (10), 303 (100),
601 (8), 301 (100), 285 (100), 271 (10),
2
87 (22), 273 (12), 257 (38), 227 255 (10)
18), 211 (8), 197 (6), 181 (8)
212 (10), 199 (100), 39 (12), 23 393 (20), 233 (80), 197 (100), 113
(
4
5
198
240
A
B
A
(
26)
212 (5), 199 (100), 39 (10), 23
26)
479 (10), 241 (100), 212 (18),
98 (28), 39 (44)
(48), 97 (49), 62 (20), 34 (28)
393 (8), 233 (12) , 197 (24), 113 (100),
97 (7), 69 (58), 34 (26)
(
275 (100), 239 (72), 197 (12), 132 (70),
113 (28), 62 (58), 35 (36)
240 (M+· , 17), 198 (100), 182 (2), 170 (13),
154 (2),140 (4), 128 (2), 115 (6), 99 (3), 97
1
(3), 85 (3), 75 (5), 63 (5), 43 (46)
B
479 (10),241 (100), 212 (18),
98 (28), 39 (8)
275 (100), 239 (52), 197 (6), 132 (17),
62 (2)
1

Sep-Oct 2001
Nitro-β-carbolines Obtained by Nitration of Commercial β-Carboline Alkaloids
1077
Table 3 (continued)
ld-tof-ms
MW Meth. [a] Positive ion mode
ei-ms
Positive ion mode
Comp
Negative ion mode
+
·
5a
285
A
286 (100), 270 (14), 256 (28),
41 (26), 213 (6)
284 (100), 268 (14), 254 (22), 238 (28), 285 (M , 4), 243 (100), 225 (5), 213 (10),
223 (22)
2
197 (56), 185 (3), 182 (9), 169 (21), 167
24), 155 (7), 140 (18), 128 (8), 114 (13),
01 (9), 99 (9), 88 (8), 77 (9), 71 (18), 69
12), 57 (22), 43 (68 )
(
1
(
B
A
286 (100), 270 (14), 256 (28),
41 (26), 213 (6)
284 (100), 268 (14), 254 (22), 238 (28),
223 (22)
2
+·
5b
285
285 (M , 6), 243 (37), 225 (5), 197 (39),
1
1
84 (3), 182 (1), 169 (4), 167 (11), 155 (2),
40 (6), 128 (3), 114 (5), 101 (3), 99 (2), 88
(3), 77 (3), 71 (4), 69 (5), 57 (7), 43 (100)
B
6
6
167
212
A [b]
A [b]
334 (18), 167 (100)
213 (100), 197 (32), 183 (30),
166 (100), 79 (18), 63 (28)
211 (100), 195 (28), 181 (4)
+·
a
213 (1.7), 212 (M , 5), 181 (4), 166 (4), 164
(5), 154 (3), 140 (4), 139 (4), 128 (5), 113
167 (16)
(
3), 98 (15), 82 (22), 69 (31), 63 (19), 45 (100)
+.
6
b
212
257
A
213 (34), 197 (86), 183 (100),
67 (10)
211 (100), 195 (18), 181 (22)
213 (15), 212 (M , 100), 182 (26), 166 (63),
164 (14), 154 (13), 140 (18), 139 (53),
1
1
6
28 (4), 113 (6), 98 (2), 82 (7), 69 (24),
3 (11), 45 (14)
+.
6c
A [b]
Clusters (551, 494, 466, 422, 279, Clusters (510, 300), 256 (100)
50), 258 (14), 242 (14), 212
68), 196 (6), 182 (100), 166 (10)
258 (9), 257 (M , 57), 241 (4), 236 (3), 227
(17), 211 (21), 194 (3), 181 (12), 165 (20),
164 (27), 153 (9), 138 (12), 137 (10), 129 (3),
2
(
111 (12), 97 (22), 83 (28), 69 (45), 58 (61),
5
6 (60), 47 (54), 46 (87), 45 (100)
+·
6d
257
A [b]
Clusters (494, 302, 280, 250),
Clusters (574, 510, 300, 270) 256
(100), 240 (14), 226 (26)
258 (8), 257 (M , 56), 241 (1), 227 (15), 211
(15),197 (2), 181 (17), 165 (13), 164 (19),
152 (5), 138 (9), 137 (7), 129 (3), 111 (5), 97
258 (20), 242 (76), 228 (100),
212 (4), 196 (6), 182 (6), 166 (6)
(9), 83 (11), 71 (12), 69 (17), 58 (28), 56 (26),
47 (51), 46 (100), 45 (62)
[a] Spectra were obtained by loading two drop of the β-carboline solution (Method A: 1 mg per ml of DMSO; Method B: 0.5 mg per ml of acetonitrile –
water trifluoroacetic acid 0.1 % (3:1, v/v)) on the stainless steel probe. λexc = 337 nm; [b] Similar spectra were obtained by using Method B.
similar to those described for the corresponding β-carbo-
line [12,62].
Finally, although very clear ei-ms spectra have been
reported for dinitroaromatic compounds, e.g., dinitrocar-
bazoles [61], (see in Table 3 ei-ms data for 1,6-dinitro and
on the sample preparation method [32]. Spectra obtained
from both solutions are quite similar. As expected, those
obtained from DMSO showed a variable amount of clusters
with m/z two or three times the molecular mass whose inten-
sity depended simultaneously on the laser power and on the
effectiveness of the solvent evaporation process. In positive
3,6-dinitrocarbazole, compounds 6c and 6d, respectivley),
+
peaks of very low intensity were obtained from the dinitro
derivatives of harmine (Table 3, compound 3c) by ei-ms
even at very soft experimental conditions (low temperature
at the injection chamber; low voltage for the ionizing elec-
tron source).
and negative ion modes the peaks corresponding to [M+H]
-
and [M-H] were detected in general as one of the most abun-
dant signal (Table 3, ld-tof-ms data; see for each compound
+
-
values for [M+H] and [M-H] in the corresponding mode).
In both modes, the molecular ion generated suffers the expul-
+
sion of O and NO yielding abundant [M+H-O] and [M+H-
NO] signals in positive ion mode, and [M-H-O] and [M-H-
NO] signals in negative ion mode, respectively. These are the
Laser Desorption/Ionization Time-of-flight Mass
Spectrometry (ld-tof-ms).
+
-
-
The laser desorption/ionization (λexc = 337 nm) time-of-
flight mass spectra (ld-tof-ms) of nitro-β-carbolines have
been recorded in positive and negative ion modes. Samples
were prepared in two different solvents: dimethylsulfoxide
main fragmentation paths of 6-nitro and 8-nitro-β-carbolines
1a, 1b, 2a, 2b, 3a, 3b, 5a and 5b (see as example in Table 3,
ld-tof-ms; positive ion mode, Method A, [M+H-O] : 1a, 198
+
(10%), 1b, 198 (18%); 2b, 213 (20%); 3b, 242 (70%); 5a,
+
(DMSO) (Method A) and acetonitrile – water trifluoroacetic
270 (14%); [M+H-NO] : 1a, 184 (18%), 1b, 184 (8%); 2a,
acid 0.1% (3:1, v/v) (MeCN:H O TFA 0.1%) (Method B)
because as we have reported the quality of the ld-ms depends
199 (8%); 3a, 228 (60%); 3b, 228 (12%); 5a, 256 (28%)).
2
+
Besides [M+H-NO ] signals in positive ion mode and
2

1078
M. A. Ponce and R. Erra-Balsells
Vol. 38
-
[
M-H-NO ] signals in the negative ion mode together with
Studies in connection with the potential use of this fam-
ily of nitro-β-carbolines as matrices in the matrix-assisted
ultraviolet-laser desorption/ionization time-of-flight mass
spectrometry (uv-maldi-tof-ms) analysis of macromole-
cules such as synthetic polymers, biopolymers and pro-
teins, are in progress in our laboratory and the preliminary
results are quite good.
2
the above mentioned signals were observed.
The fragmentation processes of the dinitro derivative of
harmine (6,8-dinitro-harmine; 3c) is not very complicated
although two nitro groups are present. In this case,
although the signal corresponding to the molecular ion is
quite important (Table 3, ld-tof-ms; 3c, positve ion mode,
m/z 303 (100%); negative ion mode, m/z 301 (44 and
1
00%), the other most abundant ion in negative mode is
EXPERIMENTAL
-
the [M-H-O] (Table 3, ld-tof-ms; 3c, m/z 285, 100%)
being the peaks corresponding to [M-H-NO] (m/z 271
-
Dichloromethane, chloroform, acetic acid, ethanol, hexane,
ethyl acetate and other reagents used were of analytical grade.
Solvents were freshly distilled and dried before using. β-
Carbolines (9H-pyrido[3,4-b]indoles), i.e., nor-harmane (9H-
pyrido[3,4-b]indole, 1), harmane (1-methyl-9H-pyrido[3,4-
b]indole, 2), harmine (7-methoxy-1-methyl-9H-pyrido[3,4-b]-
indole, 3), harmol (1-methyl-9H-pyrido[3,4-b]indol-7-ol, 4), har-
maline (7-methoxy-1-methyl-3,4-dihydro-9H-pyrido[3,4-b]-
indole), carbazole (6), 2,5-dihydroxybenzoic acid (gentisic acid,
DHB) and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic
acid, SA) were purchased from Aldrich, Sigma and Wako Pure
Chemical Ind. Japan. Ceric ammonium nitrate (Ce(NH ) (NO ) ;
-
(
56%)) and [M-H-NO ] (m/z 255 (50%) also important.
2
The presence of the second NO group in the molecule,
placed at the same benzenic ring, would be the cause of
the higher stability shown by the [M-H-O] in the negative
ion mode. Comparison of the spectra obtained from the
2
-
6
,8-dinitro-harmine 3c by ei-ms and by ld-tof-ms tech-
niques shows that the latter is a very useful technique
because of its soft desorption/ionization conditions com-
pound 3c can be effectively ionized to intact monocharged
molecules in gas phase. Besides, the soft character of the
latter technique together with the different stability of ions
and radical-ions in the gas state would explain the notice-
4 2
3 6
CAN), ammonium ferric (II) sulfate hexahydrate
(NH ) Fe(SO ) .6H O, Mohr salt), EDTA, copper (II) nitrate tri-
(
4
2
4 2
2
hydrate (Cu(NO ) .3H O), sodium nitrite, acetic anhydride, nitric
3
2
2
+
-
able high abundance of the [M+H-O] and [M-H-O]
acid, glacial acetic acid were purchased from Aldrich.
species obtained in the respective ion mode, by ld-tof-ms
analysis.
Similar results were obtained when we compared the ld-
tof-ms and the ei-ms obtained from carbazole (6), 1-nitro-
carbazole (6a), 3-nitrocarbazole (6b), 1,6-dinitrocarbazole
Thin layer chromatography (tlc) analysis was performed with
aluminium silica gel sheets (0.2 layer thickness, silica-gel 60
F254). Products were isolated by preparative thick layer chro-
matography and column chromatography which was carried out
using silica gel 200-400 mesh 60Å and ethyl acetate and ethyl
1
acetate - ethanol as eluent. Melting points are uncorrected. H-
(
6c) and 3,6-dinitrocarbazole (6d). As is shown in Table 3,
13
and C-nmr spectra were run in deuterochloroform, deuterochlo-
+
-
[M+H-O] and [M-H-O] species are formed efficiently by
roform/methanol-d4 and dimethylsulfoxide-d6 at 200 and 500
MHz. Chemical shifts are reported in ppm values, using tetram-
ethylsilane as internal standard. The uv spectra were run in ace-
tonitrile and acetonitrile – sulfuric acid solutions of the β-carbo-
lines (10- M). Electron impact mass spectra (ei-ms) and high-res-
olution mass spectra (hrms) were obtained under electron impact
the former technique (Table 3, ld-tof-ms; positive ion
mode: 6a, m/z 197 (32%), 6b, m/z 197 (86%), 6c, m/z 242
(
(
14%), 6d, m/z 242 (76%); negative ion mode: 6a, m/z 195
28%), 6b, m/z 195 (18%), 6d, m/z 240 (14%)), while they
4
are not detected by using the latter (Table 3, ei-ms data for
(70 eV). The ratios m/z and the relative intensities are reported.
+
compounds 6a, 6b, 6c and 6d). The species [M+H-NO] ,
Laser desorption/ionization time-of-flight mass spectrometry
+
-
-
[
M+H-NO ] , [M-H-NO] and [M-H-NO ] were also
2 2
(ld-tof-ms): measurements were performed using: i) Shimadzu
Kratos, Kompact MALDI III UV-laser desorption time-of-flight
mass spectrometer and ii) Shimadzu Kratos, Kompact MALDI 4
observed, especially in the negative ion mode. It is note-
worthy to emphasize that from the dinitrocarbazoles 6c and
(
Pulsed Extraction). Both spectrometers are equipped with a
6
d both NO substituents are expelled efficiently in the
2
pulsed nitrogen laser (λ = 337 nm; pulse width = 3 ns), the latter
only with tuneable time delay capability. The analyzer was used at
accelerating voltage of 20kV. The laser can be fired at any spot or
fired continuously along a selected length of the sample holder.
The sample was irradiated just above the threshold for obtaining
ions. Thus, the irradiance used for producing a mass spectrum
was analyte dependent. Usually 50 spectra were accumulated.
All mass spectra were taken (a) in the positive ion mode and in the
negative ion mode and (b) in the linear and in the reflectron mode.
positive ion mode, while in negative ion mode the species
are more stable keeping both (Table 3, ld-tof-ms, negative
ion mode: compound 6c) or at least one NO group (Table
2
3, ld-tof-ms, negative ion mode: compound 6d).
Finally, it is noteworthy to point the fact that after the
expulsion of the nitro substituent the β-carboline like
structure remaining does not suffer the typical cleavage
that β-carbolines show by ei-ms technique (expulsion of
-2
Stock solution of β-carbolines (10 M) were prepared by dis-
HCN and C H fragments inter alia). As expected they
2
2
-5
solving 10 moles of the compound in 1 ml of the selected sol-
showed at that m/z region, m/z ≤ β-carboline molecular
mass, signals quite similar to those observed in the ld-tof-
ms of the corresponding β-carboline (see in Table 3, ld-tof-
ms for 1, 2, 3, 4 and 5).
vent (Method A: dimethylsulfoxide (DMSO); Method B: ace-
tonitrile - water-trifluoroacetic acid 0.1% (3:1) (v:v)). The solu-
tions were kept in the dark at low temperature to prevent photo-
chemical decomposition.

Sep-Oct 2001
Nitro-β-carbolines Obtained by Nitration of Commercial β-Carboline Alkaloids
1079
Sample preparation method: ld-tof-ms experiments using
β-carbolines as analytes, a sample deposit, typically 0.5 µl of the
solution, was placed on the sample probe tip (2 mm diameter;
stainless steel). Thus the solvent was removed by room tempera-
ture evaporation by forced air (film). Then, additional 0.5 µl of
the same solution was placed on the same probe tip covering the
first deposited film, and again the solvent removed by forced air.
Spectra were calibrated by use of (a) internal calibrant reagent;
e.g.: gramicidin S, Cs, Na and (b) standard chemicals as external
calibrant reagent (gentisic acid and sinapinic acid) using Kratos
Kompact calibration program.
The aqueous solution was washed with dichloromethane (3 x 15
ml). The combined organic extracts were washed with water (100
ml), and the organic layer was dried over sodium sulfate, filtered
and evaporated in vacuo to give an almost colorless residue. The
residue was treated by flash column chromatography following
the procedure described.
Olah's Method.
A mixture of copper (II) nitrate trihydrate (200 mg, 0.83
mmole) and acetic anhydride (1.5 ml) was stirred at room tem-
perature for 1.5 hours. After that the β-carboline compound (0.72
mmole) was added to the mixture, and stirring was continued for
7-Acetyloxy-1-methyl-9H-pyrido[3,4-b]indole (5).
24 hours. Additional copper (II) nitrate trihydrate (100 mg, 0.83
A mixture of harmol (204.7 mg, 1.03 mmoles) (commercial
-methyl-9H-pyrido[3,4-b]indol-7-ol (harmol, 4)) and acetic
mmole) was added, and stirring was continued for another 24
hours. Then methanol (10 ml) was added. After 30 minutes of
stirring volatiles were evaporated in vacuo, 20 ml of water were
added to the remaining solution, and the mixture was stirred for
1
anhydride (1.0 ml) in pyridine (0.5 ml) was stirred at room tem-
perature during 20 hours. In order to prevent the hydrolysis of the
acetyloxy group, addition of methanol and/or sodium bicarbonate
aqueous solution was avoided. Instead of these chemicals,
toluene was added in order to yield the pyridine – toluene
azeotrope and then the system was evaporated in vacuo. The
solid residue obtained was purified by silica-gel column chro-
matography eluted with ethyl acetate and ethyl acetate - ethanol
mixtures. Compound 4 (205 mg, 83% yield) was obtained as
white needles (ethanol), mp 155°; 1H-nmr (deuteriochloro-
30 minutes the water solution was adjusted to pH 14 by potas-
sium carbonate. The aqueous solution was washed with
dichloromethane (3 x 15 ml). The combined extracts were
washed with water (100 ml), and the organic layer was dried over
sodium sulfate, filtered and evaporated in vacuo to give a solid
residue. The residue was separated by flash column chromatogra-
phy as described above.
Nitric Acid - Acetic Acid Method.
form/methanol-d ): δ 8.46 (broad singlet (bs), 1H, NH), 8.32 (d,
4
A slurry of the β-carboline (0.5 mmole) and 0.5 mmole of
sodium nitrite in glacial acetic acid (6 ml) was stirred at 30-40°
for 1.5 hours. Then, 1.4 ml of the mixture concentrated nitric acid
– glacial acetic acid (1:1, v:v) was slowly added. The resultant
mixture was stirred at 40-45° for additional 3.5 hours and left
overnight at room temperature. Water was added (100 ml), the
solution was neutralized with potassium carbonate and the
workup was conducted as described above.
1
4
6
H, 3-H, J = 5.4 Hz), 7.91 (d, 1H, 5-H, J = 8.7 Hz), 7.63 (d, 1 H,
-H, J = 5.4 Hz), 7.20 (d, 1 H, 8-H, J = 2.0 Hz), 6.96 (dd, 1 H,
-H, J = 8.7, 2.0 Hz), 2.77 (s, 3 H, CH ), 2.39 ppm (s, 3 H,
3
1
3
CH CO); C-nmr (deuteriochloroform/methanol-d ): δ 170.3
3
4
(
(
CO), 150.8 (7-C), 141.4 (1-C), 140.2 (9a-C), 136.6 (3-C), 135.2
8a-C), 127.9 (4b-C), 122.1 (5-C), 119.1 (4a-C), 113.8 (6-C),
112.6 (4-C), 104.7 (8-C), 20.9 (CH ), 18.9 ppm (CH CO); hrms:
3 3
Calcd. for C14H12 N O 240.089878, found: 240.089947.
2
2
Anal. Calcd. for C14H12N O : C, 69.98; H, 5.03; N, 11.66.
Found: C, 69.96; H, 5.04; N, 11.65.
6-Nitro-9H-pyrido[3,4-b]indole (6-Nitro-nor-harmane) (1a).
Compound 1a was obtained as yellow needles (ethanol), mp
2
2
1
Nitration of β-Carbolines.
255° [lit 325-326° [49]; 340-342° [52]); H-nmr (deuteriochloro-
form/methanol-d ): δ 10.17 (bs, 1H, NH), 9.14 (d, 1 H, 5-H, J =
4
-
Ce(NH ) (NO ) (H 0) (CAN) Method 1.
4 2 3 6 2 6
2
.2 Hz), 8.91 (s, 1H, 1-H), 8.47 (dd, 1 H, 7-H, J = 2.2, 9.1 Hz),
The β-carboline (0.62 mmole) and CAN (1.1 mmoles) were
8.44 (d, 1 H, 3-H, J = 5.2 Hz), 8.15 (d, 1 H, 4-H, J = 5.2 Hz), 7.65
1
3
separately dissolved in acetonitrile (100 ml) and then mixed
together. The resulting mixture was stirred in the dark for 6 hours
at 90º until tlc indicated that the reaction was complete. All these
reactions were carried out under normal (air) atmosphere.
Acetonitrile was evaporated in vacuo to give a light brown-
greenish oily residue. The residue was separated by column flash
chromatography (silica gel-ethyl acetate-ethanol mixtures) to
give 1a, 1b, 1c and polynitrated-β-carboline dimeric compounds
and cerium-polynitrated-β-carboline complexes.
ppm (d, 1 H, 8-H, J = 9.1 Hz); C-nmr (deuteriochloroform): δ
143.7 (6-C), 140.2 (9a-C), 139.6 (3-C), 138.0 (8a-C), 134.9
(1-C), 127.9 (4b-C), 123.2 (5-C), 121.6 (4a-C), 119.2 (7-C),
115.4 (4-C), 112.2 (8-C) ppm; hrms: Calcd. for C11H N O
7
3 2
213.053827, found 213.053932.
Anal. Calcd. for C11H N O : C, 63.97; H, 3.31; N, 17.71.
7
3 2
Found: C, 63.99; H, 3.30; N, 17.70.
8
-Nitro-9H-pyrido[3,4-b]indole (8-Nitro-nor-harmane) (1b).
Compound 1b was obtained as yellow needles (ethanol), mp
Ce(NH ) (NO ) (H 0) (CAN) Method 2.
4
2
3 6
2
6
1
2
15°; H-nmr (deuteriochloroform): δ 12.45 (bs, 1H, NH), 9.11
In order to remove more efficiently the remaining cerium to
obtain compounds of higher purity we proceeded as follows: 30
ml of water solution containing Mohr salt (1.1 mmoles,
(s, 1 H, 1-H), 8.78 (d, 1H, 3-H, J = 5.3 Hz), 8.49 (d, 2 H, 5-H and
7-H, J = 7.4), 8.26 (d, 1 H, 4-H, J = 5.3), 7.47 ppm (dd, 1 H, 6-H,
1
3
J = 7.4 Hz); C-nmr (dimethylsulfoxide-d ): δ 142.8 (9a-C),
6
(
NH ) Fe(SO ) •6H O)) was added to the solid residue obtained
139.7 (3-C), 133.4 (1-C), 132.5 (8a-C), 131.4 (8-C), 129.9 (5-C),
127.9 (4b-C), 124.3 (7-C), 123.6 (4a-C), 119.1 (6-C), 112.3 (4-C)
4
2
4 2
2
after acetonitrile evaporation. The mixture was stirred at room
temperature for one hour. Then ethylene diamine tetraacetic acid
EDTA (2.5 mmol) was added to complex both Fe and Ce
ppm; hrms: Calcd. for C11H7 N O 213.053827, found:
3
2
3
+
3+
.
213.054005.
Stirring was continued for another hour. After that, potassium
carbonate was added until the solution became neutral (pH = 7).
Anal. Calcd. for C11H N O : C, 63.97; H, 3.31; N, 17.71.
Found: C, 63.96; H, 3.32; N, 17.72.
7
3 2

1080
M. A. Ponce and R. Erra-Balsells
Vol. 38
6
-Nitro-1-methyl-9H-pyrido[3,4-b]indole (6-Nitroharmane) (2a).
Compound 2a was obtained as yellow needles (ethanol), mp
134.6 (3-C), 133.9 (8a-C), 128.3 (6-C), 127.9 (4b-C), 124.2 (5-
C), 119.4 (4a-C and 8-C), 112.8 (4-C), 65.1 (CH O), 29.5 ppm
3
(
CH ); hrms: Calcd. for C13H10N O 302.065120, found:
1
3
4
5
225° (d) [lit 300-301° [44,46]; 296-298° [48]); H-nmr (deuteri-
3
02.065368.
ochloroform): δ 10.17 (bs, 1H, NH), 9.08 (d, 1 H, 5-H, J = 1.8
Hz), 8.50 (d, 1H, 3-H, J = 5.5 Hz), 8.39 (d, 1 H, 4-H, J = 5.5 Hz),
Anal. Calcd. for C13H10N O : C, 51.66; H, 3.33; N, 18.54.
4
5
Found: C, 51.68; H, 3.32; N, 18.55.
7
2
.99 (dd, 1 H, 7-H, J = 1.8, 5.1 Hz), 7.91 (d, 1 H, 8-H, J = 5.1 Hz),
.87 ppm (s, 3 H, CH3); C-nmr (dimethylsulfoxide-d ): δ 143.1
1
3
6
6-Nitro-7-acetyloxy-1-methyl-9H-pyrido[3,4-b]indole (6-nitro-
(
1
1-C), 140.9 (6-C), 140.2 (9a-C), 138.9 (3-C), 135.6 (8a-C),
29.7 (5-C), 127.5 (4b-C), 122.9 (7-C), 120.7 (4a-C), 113.3 (8-
C), 112.4 (4-C), 20.2 ppm (CH ); hrms: Cal. for C H N O
12 9 3 2
7
-acetoxy-harmol) (5a).
Yellow needles (ethanol), mp 250° (d); H-nmr (deuteriochlo-
roform/methanol-d ): δ 14.4 (bs, 1H, NH), 9.25 (s, 1 H, 5-H),
1
3
4
2
27.069477, found: 227.069000.
Anal. Calcd. for C12H N O : C, 63.43; H, 3.99; N, 18.50.
8
.35 (d, 1H, 3-H, J = 5.5 Hz), 8.16 (d, 1 H, 4-H, J = 5.5 Hz), 7.51
9
3 2
(
s, 1 H, 8-H), 2.76 (s, 3 H, CH ), 2.33 ppm (s, 3 H, CH CO;
3
3
Found: C, 63.41; H, 3.98; N, 18.52.
1
3
C-nmr (dimethylsulfoxide-d ): δ 168.9 (C=O), 143.5 (7-C),
6
8-Nitro-1-methyl-9H-pyrido[3,4-b]indole (8-Nitroharmane) (2b).
143.0 (1-C), 142.8 (9a-C), 139.2 (3-C), 135.8 (8a-C), 134.4
6-C), 127.1 (4b-C), 121.1 (5-C), 118.4 (4a-C), 113.2 (4-C),
(
Compound 2b was obtained as yellow needles (ethanol), mp
1
107.1 (8-C), 20.6 (CH3), 20.3 ppm (CH3CO); hrms: Calcd. for
C14H N O 285.074956, found: 285.075046.
205-207° [lit 210° [44]); H-nmr (dimethylsulfoxide-d ): δ 11.72
6
(bs, 1H, NH), 8.76 (d, 1 H, 7-H, J = 7.3 Hz), 8.49 (d, 1H, 5-H, J =
11 3 4
Anal. Calcd. for C14H N O : C, 58.94; H, 3.89; N, 14.73.
8.4 Hz), 8.37 (d, 1 H, 3-H, J = 5.1), 8.09 (d, 1 H, 4-H, J = 5.1), 7.48
11 3 4
13
Found: C, 58.92; H, 3.88; N, 14.74.
(dd, 1 H, 6-H, J = 8.4, 7.3 Hz), 2.92 ppm (s, 3 H, CH3); C-nmr
(
1
1
dimethylsulfoxide-d ): δ 143.8 (1-C), 140.4 (9a-C), 139.5 (3-C),
6
8-Nitro-7-acetyloxy-1-methyl-9H-pyrido[3,4-b]indole (8-Nitro-
35.0 (8a-C), 129.7 (8-C), 126.8 (5-C), 125.8 (4b-C), 124.2 (7-C),
21.6 (4a-C), 119.2 (6-C), 112.7 (4-C), 20.9 ppm (CH ); hrms:
7-acetoxyharmol) (5b).
3
Compound 5b was obtained as yellow needles (ethanol), mp
Calcd. for C H N O 227.069477, found: 227.069535.
12
9
3 2
1
1
90°; H-nmr (dimethylsulfoxide-d ): δ 11.76 (bs, 1H, NH), 8.67
Anal. Calcd. for C12H N O : C, 63.43; H, 3.99; N, 18.50.
6
9
3 2
(d, 1 H, 5-H, J = 8.4), 8.36 (d, 1H, 3-H, J = 5.5 Hz), 8.06 (d, 1 H,
Found: C, 63.41; H, 3.99; N, 18.52.
-Nitro-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (6-Nitro-
harmine) (3a).
4
-H, J = 5.5 Hz), 7.27 (s, 1 H, 6-H, J = 8.4 Hz), 2.87 (s, 3 H,
6
13
CH ), 2.40 ppm (s, 3 H, CH CO); C-nmr (deuteriochloro-
3
3
form/methanol-d ): δ 168.7 (C=O), 144.1 (7-C and 9a-C), 143.6
4
o
1
(1-C), 139.6 (3-C), 135.4 (8a-C), 133.7 (4b-C), 128.5 (5-C),
Yellow needles (ethanol), mp 265 (d) ; H-nmr (deuteriochlo-
1
26.5 (8-C), 123.0 (4a-C), 115.6 (6-C), 112.7 (4-C), 20.8 (CH3),
roform): δ 11.98 (bs, 1H, NH), 8.93 (s, 1 H, 5-H), 8.26 (d, 1H,
-H, J = 5.4 Hz), 7.99 (d, 1 H, 4-H, J = 5.4 Hz), 7.20 (s, 1 H, 8-H),
.03 (s, 3 H, CH O), 2.74 ppm (s, 3 H, CH ); C-nmr (dimethyl-
sulfoxide-d ): δ 155.1 (7-C), 146.1 (1-C), 140.2 (9a-C), 135.8 (8a-
2
0.6 ppm (CH CO); hrms: Calcd. for C H N O 285.074956,
3
4
3
14 11 3 4
13
found: 285.075075.
Anal. Calcd. for C14H N O : C, 58.94; H, 3.89; N, 14.73.
3
3
11 3 4
6
Found: C, 58.96; H, 3.88; N, 14.74.
C), 133.2 (3-C), 129.8 (6-C), 126.5 (4b-C), 122.3 (5-C), 115.1
(
4-C), 113.0 (4a-C), 96.1 (8-C), 57.5 (CH O), 17.5 ppm (CH );
3 3
1-Nitrocarbazole (6a).
hrms: Calcd. For C H N O 257.080041, found: 257.080451.
13
11
3 3
Compound 6a was obtained as yellow needles (ethanol), mp
Anal. Calcd. for C13H N O : C, 60.64; H, 4.31; N, 16.39.
Found: C, 60.62; H, 4.30; N, 16.41.
11
3 3
1
1
1
8
2
95° (lit 197-198° [61,62]); H-nmr (dimethylsulfoxide-d ): δ
6
2.15 (bs, 1H, NH), 8.62 (d, 1 H, 4-H, J = 7.3 Hz), 8.41 (d, 1 H,
-H, J = 8.4 Hz), 8.31 (dd, 1H, 5-H, J = 1.1, 7.8 Hz), 8.24 (d, 1 H,
-H, J = 8.0 Hz), 7.51 (dd, 1 H, 7-H, J = 7.3, 8.4 Hz), 7.35 (dd, 1
8
-Nitro-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (8-Nitro-
harmine) (3b).
Compound 3b was obtained as yellow needles (ethanol), mp
12-213° [lit 204-205° [43]); H-nmr (deuteriochloroform): δ
H, 3-H, J = 7.3, 8.0 Hz), 7.29 ppm (dd, 1 H, 6-H, J = 1.1, 7.3, 7.8
1
Hz); 13C-nmr (dimethylsulfoxide-d ): δ 140.6 (1-C), 132.8 (4-C),
2
9
6
.90 (bs, 1H, NH), 8.42 (d, 1 H, 3-H, J = 5.4 Hz), 8.3 (d, 1H, 5-H,
131.6 (9a-C), 130.9 (4a-C), 127.9 (3-C), 127.1 (7-C, 8a-C), 121.6
(2-C), 121.4 (4b-C), 120.5 (5-C), 118.2 (6-C), 112.6 ppm (8-C).
J = 8.7 Hz), 7.73 (d, 1 H, 4-H, J = 5.4), 7.00 (d, 1 H, 6-H, J = 8.7),
4
sulfoxide-d ): δ 153.7 (7-C), 142.9 (1-C, 9a-C), 139.4 (3-C),
1
3
.13 (s, 3 H, CH O), 2.86 ppm (s, 3 H, CH ); C-nmr (dimethyl-
3 3
3-Nitrocarbazole (6b).
6
134.3 (8a-C), 127.8 (5-C), 127.2 (4b-C), 117.6 (4a-C, 8-C), 112.3
Compound 6b was obtained as yellow needles (ethanol), mp
1
(4-C), 105.8 (6-C), 57.5 (CH O), 20.7 ppm (CH ); hrms: Calcd.
215° (lit 215-216° [61,62]); H-nmr (dimethylsulfoxide-d6): δ
12.02 (bs, 1H, NH), 9.15 (d, 1 H, 4-H, J = 1.8 Hz), 8.36 (d, 1H,
5-H, J = 8.0 Hz), 8.29 (dd, 1 H, 2-H, J = 1.8, 8.8 Hz), 7.65 – 7.58
(m, 2 H, 1-H and 8-H), 8.51 (dd, 1 H, 6-H, J = 6.9, 8.0 Hz), 7.29
3
3
for C13H11 N O 257.080041, found: 257.080146.
3
3
Anal. Calcd. for C13H N O : C, 60.64; H, 4.31; N, 16.39.
11
3 3
Found: C, 60.65; H, 4.30; N, 16.40.
1
3
ppm (dd, 1 H, 7-H, J = 6.9, 7.7 Hz); C-nmr (dimethylsulfoxide-
6
,8-Dinitro-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole (6,8-
d6): δ 143.1 (3-C), 140.8 (9a-C), 139.6 (8a-C), 127.3 (7-C), 122.2
Dinitroharmine) (3c).
(
4a-C), 121.9 (4b-C), 121.1 (2-C), 120.9 (5-C), 120.0 (6-C),
Compound 3c was obtained as yellow needles (ethanol), mp
116.9 (4-C), 111.7 (1-C), 110.9 ppm (8-C).
2
8
38° (d); 1H-nmr (deuteriochloroform): δ 9.94 (bs, 1H, NH),
1,6-Dinitrocarbazole (6c).
.93 (s, 1 H, 5-H), 8.56 (d, 1H, 3-H, J = 5.3), 7.84 (d, 1 H, 4-H,
1
3
J = 5.3), 4.21 (s, 3 H, CH O), 2.91 ppm (s, 3 H, CH ); C-nmr
Compound 6c was obtained as yellow needles (ethanol), mp
3
3
1
(
dimethylsulfoxide-d ): δ 158.6 (7-C), 145.7 (1-C), 140.8 (9a-C),
343° [lit 344-346° [61,62]); H-nmr (dimethylsulfoxide-d ): δ
6
6

Sep-Oct 2001
Nitro-β-carbolines Obtained by Nitration of Commercial β-Carboline Alkaloids
1081
1
4
8
2.71 (bs, 1H, NH), 9.30 (d, 1 H, 5-H, J = 2.1 Hz), 8.90 (d, 1H,
-H, J = 7.7 Hz), 8.58 – 8.39 (m, 2 H, 2-H and 7-H), 7.88 (d, 1 H,
-H, J = 9 Hz), 7.49 ppm (dd, 1 H, 3-H, J = 7.7, 7.9 Hz); C-nmr
UBA, Argentina) and Prof. Hiroshi Nonami (College of
Agriculture-Ehime University, Japan) with the facilities of the
High Resolution Liquid Chromatography-integrated Mass
Spectrometer System of the United Graduated School of
Agricultural Sciences (Ehime University, Japan).
1
3
(dimethylsulfoxide-d ): δ 144.0 (6-C), 132.4 (1-C), 132.1 (9a-C),
6
1
29.2 (4-C), 129.1 (8a-C), 124.6 (4a-C), 123.2 (3-C), 122.6
(7-C), 121.4 (4b-C), 119.9 (2-C), 116.5 (5-C), 112.9 ppm (8-C).
3,6-Dinitrocarbazole (6d).
REFERENCES AND NOTES
Compound 6b was obtained as yellow needles (ethanol), mp
[
1] L. Marion, The Alkaloids, eds. R. H. Manske and H. L.
Holmes, Academic Press, New York, 1952, Vol II.
2] J. R. F. Allen and B. R. Holmstedt, Phytochemistry, 19, 1573
1980).
1
3
1
4
8
85° [lit 386-387° [61,62]); H-nmr (dimethylsulfoxide-d ): δ
6
2.60 (bs, 1H, NH), 9.45 (d, 1 H, 5-H, J = 2.1 Hz), 8.90 (d, 1H,
-H, J = 7.7 Hz), 8.58 – 8.39 (m, 2 H, 2-H and 7-H), 7.88 (d, 1 H,
[
(
1
3
-H, J = 9 Hz), 7.49 ppm (dd, 1 H, 3-H, J = 7.7, 7.9 Hz); C-nmr
[3] G. H. N. Towers, Can. J. Bot., 62, 2900 (1984).
(
dimethylsulfoxide-d ): δ 149.8 (3-C and 6-C), 141.0 (8a- and
[4] A. C. Sen, N. Ueno and B. Chakrabarti, Photochem.
6
9
a-C), 122.7 (2-C, 4a-C, 4b-C and 7-C), 118.8 (4-C and 5-C),
Photobiol., 55, 753 (1992).
112.4 ppm (1-C and 8-C).
[5] E. H. Poindexter and R. D. Carpenter, Phytochemistry, 1, 215
(1962).
Nitration of 7-Hydroxy-1-methyl-9H-pyrido[3,4-b]indole (4,
Harmol).
[
[
6] R. E. Schultes, Science, 163, 245 (1969).
7] B. T. Ho, W. M. McIsaac, K. E. Walker and V. Estevez, J.
Pharm. Sci., 57, 269 (1968).
8] W. E. Müller, K. J. Fehske, H. O. Borbe, U. Wollert, C. Nanz
Nitric Acid - Acetic Acid – THF at 0° Method.
[
Harmol (4, 80 mg) was dissolved in a mixture of glacial acetic
acid (6 ml) and tetrahydrofuran (2 ml). To this solution, stirred
and kept at 0°, a mixture of nitric acid (1 ml) and glacial acetic
acid (1 ml) was added dropwise during 3 hours. After that the
system was stirred at room temperature overnight. Water was
added (100 ml), and the aqueous solution was neutralized with
potassium carbonate, then saturated with sodium bicarbonate and
finally washed with dichloromethane (3 x 15 ml). The combined
extracts were washed with water (100 ml), and the organic layer
was dried over sodium sulfate, filtered and evaporated in vacuo
to give a solid residue. The residue was separated by flash col-
umn chromatography as described above.
and H. Rommelspacher, Pharm. Biochem. Behav., 14, 693 (1981).
[9] R. A. Larson, K. A. Marley, R. W. Tuveson and M. R.
Berenbaum, Photochem. Photobiol., 48, 665 (1988).
[
10] J. B. Hudson, E. A. Graham and G. H. N. Towers, Photochem.
Photobiol., 43, 21 (1986).
11] K. H. Chae and H. S. Ham, Bull. Korean Chem. Soc., 7, 478
1986).
[
(
[
[
12] R. Erra-Balsells and A. R. Frasca, Tetrahedron, 39, 33 (1983).
13] M. A. Muñoz, C. Carmona, J. Hidalgo, P. Guardo and M.
Balon, Bioorg. Med. Chem., 3, 41 (1995).
[14] G. Duportail, Int. J. Macromol., 3, 188 (1981).
[15] K. P. Ghiggino, P. F. Skilton and P. J. Thistlethwaite, J.
Pohtochem., 31, 113 (1985).
[
16] A. Pardo, D. Reyman, J. M. L. Poyato and F. Medina, J.
Lumin., 42, 113 (1982).
17] O. S. Wolfbeis, E. Fuerlinger and R. Winstersteiger, Monatsh.
Chem., 113, 509 (1982).
18] O. S. Wolfbeis and E. Fuerlinger, Z. Phys. Chem. N.F., 129,
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[
3
85°; H-nmr (dimethylsulfoxide-d ): δ 10.63 (s, 1H, HCO), 8.52
6
[
(
8
s, 1 H, NH), 8.02 (d, 1H, 3-H, J = 6.6 Hz), 7.67 (d, 1 H, 5-H, J =
.7 Hz ), 7.50 (d, 1 H, 4-H, J = 6.6 Hz), 6.20 (d, 1 H, 6-H, J = 8.7
Hz ), 5.75 ppm (s, 1 H, 8-H); ms (ei): m/z 422 (2), 380 (4), 305
1
[
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[
(
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(
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