Unexpected formation of pyridodiindole derivatives with cytotoxic activity via double nenitzescu reaction

Article abstract of DOI:10.1002/jhet.5570450546

(Chemical Equation Presented) The title compounds 10 and 11 were prepared by a one-step procedure from 1,4-benzoquinone (4) and pyridine-2,4,6-triamine (5) via an extension of the Nenitzescu reaction.

Full text of DOI:10.1002/jhet.5570450546

Sep-Oct 2008
1517
Unexpected Formation of Pyridodiindole Derivatives
with Cytotoxic Activity via Double Nenitzescu Reaction
Christine Willemanna, Reiner Waibela, Renate Grünertb,
Patrick Bednarskib, Reinhard Troschütza*
aDepartment Chemie und Pharmazie der Universität Erlangen-Nürnberg,
Lehrstuhl für Pharmazeutische Chemie,
Schuhstr. 19, D-91052 Erlangen, Germany
bInstitut für Pharmazie der Ernst-Moritz-Arndt-Universität,
Friedrich-Ludwig-Jahn-Str. 17, D-17487 Greifswald, Germany
Received February 27, 2008
HO
OH
HO
HO
NH2
N
NH2
N
NH2
N
1,4-benzoquinone,
ethanol, r.t.
OH
+
+
N
H2N
NH2
NH2
N
N
N
N
NH2
H
H
H
H
The title compounds 10 and 11 were prepared by a one-step procedure from 1,4-benzoquinone (4) and
pyridine-2,4,6-triamine (5) via an extension of the Nenitzescu reaction
J. Heterocyclic Chem., 45, 1517 (2008).
INTRODUCTION
RESULTS AND DISCUSSION
Dotzauer et al. synthesized 2,4-diamino-9H-pyri-
mido[4,5-b]indol-6-ole (1) and N-substituted derivatives
as a prototype of a series of ellipticine analogues by an
extension of the Nenitzescu reaction using 1,4-benzo-
quinone (4) and pyrimidine-2,4,6-triamines. These indole
derivatives showed interesting antitumor activity [1-3].
Related to these compounds, we planned pyridine-2,4,6-
triamine (5) to react as a new type of enamine component
with 1,4-benzoquinone (4) to achieve desired 6-hydroxy-
carbolines 2 and 3 (Scheme 1).
The preparation of pyridine-2,4,6-triamine (5) has been
reported several times. Meyer et al. started their multi step
synthesis using 2,6-dichloropyridin-4-amine [4] whereas
in a Schering patent a triple Chichibabin reaction using
sodium amide and high temperature is described [5,6].
Another exhausting multi step strategy is reported by
Coburn et al. [7]. For quick access to pyridine-2,4,6-
triamine (5), we planned to hydrolyze the nitrile group of
2,4,6-triamino-nicotinonitrile (6) to gain triamine 5 after
decarboxylation of the corresponding nicotinate. Nitrile 6
can be prepared via Pinner reaction of malononitrile and
following ammonolysis (Scheme 2) [8,9].
Scheme 1
Scheme 2
H2N
HO
NH2
NH2
N
KOH/H2O
reflux, 5h
CN
NH2
N
H
H2N
N
NH2
H2N
N
NH2
2
6
5
H2N
H2N
HO
HO
N
Alkaline hydrolysis of nitrile 6 and adjacent extraction
with diethyl ether led to pure pyridine-2,4,6-triamine (5)
in 32 % yield. Prior adjustment of the pH to 8-9 with
diluted sulfuric acid followed by evaporation of the
aqueous phase and extraction of the solid sample by cool
ethanol increased the yield to average 70 % even though
this time including small amounts of inorganic impurities.
For our Nenitzescu reaction [10-14], pyridine-2,4,6-
triamine (5) was dissolved in ethanol and 2 eq. of 1,4-
benzoquinone (4) added carefully [15]. The solution
turned to dark brown and after only a few minutes at least
three products could be detected by tlc beside quinone 4
and hydroquinone. A first mass spectrometrical analysis
NH2
NH2
N
N
N
N
H
H
1
3
NH2
O
+
O
H2N
N
NH2
4
5
In this paper we are going to present the unexpected
synthesis of pyridodiindole derivatives and their cytotoxic
activity.

1518
C. Willemann, R. Waibel, R. Grünert, P. Bednarski, R. Troschütz
Vol 45
Scheme 3
NH2
H2N
N
NH2
5
1,4-benzoquinone (4)
ethanol, r.t.
OH
OH
OH
OH
OH
O
O
O
O
C
NH2
NH2
H2N
further 4
oxidation by 4
OH
H2N
NH2
H2N
NH2
B
N
N
H2N
NH2
N
A
9
7
8
1. oxidation by 4 ,
2. - H2O
1. - H2O
2. reduction by
A B
,
A C
,
hydroquinone
3. reduction by hydroquinone
HO
OH
HO
HO
HN
NH2
NH2
OH
1,4-benzoquinone (4 ),
ethanol, r.t.
+
N
H
N
N
H
N
N
NH2
N
N
NH2
H
H
11
10
3
of the raw sample indicated the new masses m/z 214 and
304.
were performed using same molar ratios of quinone 4 and
pyridine-2,4,6-triamine (5). The structure as an ꢀ-carbo-
line 3 is proved by Heteronuclear Multiple Bond Cor-
relation (HMBC). In this sample, pyridodiindoles 10 and
11 have only been isolated in very little amounts whereas
pyridine-2,4,6-triamine 5 was indicated in considerable
amounts by tlc but could not be quantified due to its
instability. ꢁ-Carboline 2 could not be detected in any
case.
To evaluate the in vitro cytotoxic activity of our indole
derivatives 3, 10 and 11, an established microtiter assay,
based on cell staining with crystal violet, was used to
determine the inhibition of cell growth caused by the test
compounds. In these studies, different human cancer cell
lines from solid tumors were used. Surprisingly,
ꢀ-carboline derivative 3 did not show any activity while
IC50 values from 3.1 to 10.5 μM were found for
pyridodiindoles 10 and 11 on cell lines 5637, RT-4 and
A-427 [16].
After isolation of the reaction products by column
chromatography, further spectral analysis confirmed our
guess that a 1:1 and 2:1 Nenitzescu reaction had occurred.
The following reaction sequence seems to be plausible.
After a first Michael type addition, resulting adduct 7 runs
through the typical Nenitzescu reaction steps: oxidation
by 1,4-benzoquinone (4) leading to a quinone inter-
mediate and intramolecular quinonimine formation
followed by a final reduction by hydroquinone resulting
in desired ꢀ-carbolinediamine 3. But compound 7, while
owning a rather nucleophilic position 5, might react with
another equivalent of 1,4-benzoquinone (4) to form
intermediate 8. Thereafter, oxidation by excess of
quinone 4 takes place leading to not isolated intermediate
9. Then, ring closure can proceed in two different ways:
Performing reaction pathway A and B, linear 12-amino-
5,7-dihydropyrido[2,3-b:6,5-b']diindole-2,10-diole (10),
and via pathway
A and C, 7-amino-5,12-dihydro-
pyrido[2,3-b:4,5- b']diindole-2,9-diole (11) is formed. The
diindoles 10 and 11 could not be prepared from
carbolinediamine 3 and 1,4-benzoquinone (4) (Scheme 3).
To accomplish better yields of ꢀ-carbolinediamine 3,
we carried out the same reaction with modified amounts
of 1,4-benzoquinone (4). The best yields of compound 3
EXPERIMENTAL
Starting materials were obtained from commercial sources
and were used without further purification. Reaction progress
was observed by thin-layer chromatography (tlc) making use of
commercial silica gel plates (Merck, silica gel F254 on

Sep-Oct 2008
Unexpected Formation of Pyridodiindole Derivatives with Cytotoxic Activity
1519
aluminum sheets). Column chromatography was done on silica
gel 60 and LiChroprep RP-18 (Merck). Melting points were
determined in open capillary tubes on a Buechi 510 melting
point apparatus and are uncorrected. Elemental analyses were
performed by the Institut für Organische Chemie (Universität
Erlangen-Nürnberg) using Carlo Erba Elemental Analyzer 1108.
1H nuclear magnetic resonance (1H nmr) spectra were
determined with a Bruker AM 360 (360 MHz) spectrometer in
appropriate deuterated solvents and are expressed in parts per
million (ꢀ, ppm) downfield from tetramethylsilane (internal
standard). In nmr data, all NH, OH and NH2 signals were
exchangeable with D2O. Mass spectra (ms, hrms) were taken
with a Finnigan MAT TSQ 700 mass spectrometer in the
electron impact mode (70 eV). Significant infrared (ir) spectra
were obtained on a Jasco FT/IR 410 spectrometer. HRMS on an
Jeol GC-Mate II.
Pyridine-2,4,6-triamine (5). 2,4,6-Triaminonicotinonitrile
(6) (3.60 g, 24.1 mmol) was heated to reflux for 5 h in a KOH
solution (26.0 g KOH and 13.0 g H2O). After cooling to r.t.
pH 8-9 was adjusted with diluted H2SO4.
a) The aqueous solution was extracted five times with diethyl
ether, the organic phase dried over Na2SO4 and evaporated to
dryness. 0.98 g (32.8 %) of a beige powder was obtained, mp
185 °C. (Lit. 185 °C. [4]); ir: NH 3380, NH 3324, C=C 1604
cm-1; 1H nmr (DMSO-d6): ꢀ 5.07 (s, 2H, NH2), 5.01 (s, 2H, 3-H,
5-H), 4.84 (s, 4H, NH2), 13C nmr (DMSO-d6): ꢀ 159.0 (2-C,
6-C), 157.1 (4-C), 83.1 (3-C, 5-C); ms: m/z 124 (M+); hrms
calcd. for C5H8N4: 124.0749. Found: 124.0750.
benzoquinone (4) (605 mg, 5.60 mmol) for 2 h at r.t. Purification
was performed using two subsequent column chromatography
runs (first by MPLC on LiChroprep RP-18 with a methanol/H2O
gradient, followed by flash chromatography on silica gel with a
chloroform/methanol gradient). 19 mg (2.2 %) colourless solid
was obtained, mp 270 °C. (dec.); ir: OH 3561, NH 3392, NH
3245, C=C 1698 cm-1; 1H nmr (DMSO-d6): ꢀ 10.86 (s, 2H, NH),
8.71 (s, 2H, OH), 7.62 (d, 2H, J = 1.9 Hz, 1-H, 11-H), 7.13 (d,
2H, J = 8.3 Hz, 4-H, 8-H), 6.74 (dd, 2H, J1 = 1.9 Hz, J2 = 8.3 Hz,
3-H, 9-H), 6.29 (s, 2H, NH2), 13C nmr (DMSO-d6): ꢀ 153.5
(5a-C, 6a-C), 150.4 (2-C, 10-C), 145.4 (12-C), 130.4 (4a-C,
7a-C), 122.6 (11a-C, 12b-C), 110.6 (3-C, 9-C), 109.7 (4-C, 8-C),
106.2 (1-C, 11-C), 95.3 (11b-C, 12a-C); ms: m/z 304 (M+).
Anal. Calcd. for C17H12N4O2 (304.31): C, 67.10; H, 3.97; N,
18.41. Found: C, 67.50; H, 3.69; N, 18.22.
7-Amino-5,12-dihydropyrido[2,3-b:4,5-b']diindole-
2,9-diole (11). Preparation following the same method as
indicated for compound 10; 73 mg (8.6 %) of a grey powder, mp
1
270 °C. (dec.); ir: OH 3625, NH 3376, C=C 1614 cm-1, H nmr
(DMSO-d6): ꢀ 11.51 (s, 1H, 12-NH), 10.85 (s, 1H, 5-NH), 8.83
(s, 1H, 2-OH), 8.69 (s, 1H, 9-OH), 7.67 (d, 1H, J = 1.9 Hz, 1-H),
7.50 (d, 1H, J = 1.9 Hz, 8-H), 7.33 (d, 1H, J = 8.7 Hz, 4-H), 7.15
(d, 1H, J = 8.7 Hz, 11-H), 6.80 (dd, 1H, J1 = 1.9 Hz, J2 = 8.7 Hz,
3-H), 6.73 (dd, 1H, J1 = 1.9 Hz, J2 = 8.7 Hz, 10-H), 6.07 (s, 2H,
NH2), 13C nmr (DMSO-d6): ꢀ 153.5 (5a-C), 151.4 (2-C), 151.3
(9-C), 150.7 (12a-C), 140.9 (7-C), 132.4 (11a-C), 129.8 (4a-C),
123.6 (7b-C), 121.9 (12c-C), 111.1 (3-C), 110.6 (4-C, 10-C),
110.3 (11-C), 105.7 (8-C), 105.3 (1-C), 97.8 (7a-C), 91.8
(12b-C); ms: m/z 304 (M+). Anal. Calcd. for C17H12N4O2
(304.31): C, 67.10; H, 3.97; N, 18.41. Found: C, 66.86; H, 3.65;
N, 18.03.
b) The aqueous solution was evaporated to dryness. The
residue was pulverized and than washed five times with cold
ethanol. After filtration the alcoholic solution was evaporated to
dryness. Resulting colourless solid gave the same spectro-
scopical analysis as mentioned above but contained a small
amount of inorganic impurities. 2.10 g (70.2 %) was obtained.
2,4-Diamino-9H-pyrido[2,3-b]indol-6-ole (3). A solution of
pyridine-2,4,6-triamine (5) (310 mg, 2.50 mmol) in 10 mL
ethanol was reacted with 1,4-benzoquinone (4) (323 mg, 3.00
mmol) for 2 h at r.t. Purification was performed using two
subsequent column chromatography runs (first by MPLC on
LiChroprep RP-18 with a methanol/H2O gradient, followed by
flash chromatography on silica gel with a cyclohexane/ethyl
acetate/methanol gradient). 90 mg (17.0 %) of a grey powder
was obtained, mp 247-248 °C; ir: OH 3491, NH 3442, NH 3359,
REFERENCES AND NOTES
[1] Dotzauer, B.; Grünert, R.; Bednarski, P.J.; Lanig, H.;
Landwehr, J.; Troschütz, R. Bioorg. Med. Chem. 2006, 14, 7282.
[2] Dotzauer, B.; Troschütz, R. Synlett 2004, 1039.
[3] Harding, M.H.; Grummitt, A.R. Mini Reviews in Medicinal
Chemistry 2003, 3, 67.
[4] Meyer, H.; Ritter v. Beck, E. Monatsh. Chem. 1915, 36, 731.
[5] Schering AG, DE663891 (1983).
[6] Bergstrom, F.W.; Ferrnelius, W.C. Chem. Rev. 1933, 12, 43.
[7] Coburn, M.D.; Singleton, J.L. J. Heterocycl. Chem. 1972, 9,
1039.
1
C=C 1604 cm-1; H nmr (DMSO-d6): ꢀ 10.45 (s, 1H, NH), 8.54
[8] Pinner, A. Ber. Dtsch. Chem. Ges. 1895, 28, 473.
[9] Troschütz, R. Arch. Pharm. (Weinheim), 1989, 322, 285.
[10] Nenitzescu, C. Chem. Zentralbl. II, 1929, 2331.
[11] Allen, G.R.J. Organic Reactions (J. Wiley and Sons) New
York, 1973, Vol 20, pp 337-454.
(s, 1H, OH), 7.29 (d, 1H, J = 2.2 Hz, 5-H), 7.01 (d, 1H, J = 8.4
Hz, 8-H), 6.70 (dd, 1H, J1 = 2.2 Hz, J2 = 8.4 Hz, 7-H), 5.83 (s,
2H, 4-NH2); 5.48 (s, 1H, 3-H), 5.42 (s, 2H, 2-NH2); 13C nmr
(DMSO-d6): ꢀ 158.6 (2-C), 153.4 (9a-C), 151.1 (6-C), 150.3
(4-C), 129.7 (8a-C), 122.8 (4b-C), 109.8 (7-C), 109.6 (8-C),
105.4 (5-C), 94.1 (4a-C), 84.0 (3-C); ms: m/z 214 (M+); hrms
calcd. for C11H10N4O: 214.0855. Found: 214.0855.
12-Amino-5,7-dihydropyrido[2,3-b:6,5-b']diindole-
2,10-diole (10). A solution of pyridine-2,4,6-triamine (5) (347
mg, 2.80 mmol) in 10 mL ethanol was reacted with 1,4-
[12] Granik, V.G.; Lyubchanskaya, V.M.; Muhkanova, T.T.
Khim. Farm. Zh. 1993, 27, 37.
[13] Kuckländer, U. Tetrahedron 1973, 29, 921.
[14] Bernier, J.L.; Hénichart, J.P. J. Org. Chem. 1981, 46, 4197.
[15] Kinugawa,M.; Arai, H.; Nishikawa, H.; Sakaguchi, H.;
Ogasa, T.; Tomioka,S.; Kasai, M. J. Chem. Soc. Perk. Trans. 1, 1995,
21, 2677.