Cytotoxic hybrids between the aromatic alkaloids bauerine C and rutaecarpine

Article abstract of DOI:10.1515/znb-2007-1013

Two hybrids between the alkaloids bauerine C and rutaecarpine were prepared. Screening for cytotoxic activity revealed that introduction of two chlorine substituents to the quinazolinocarboline core of rutaecarpine strongly enhances cytotoxic activity, wh

Full text of DOI:10.1515/znb-2007-1013

Cytotoxic Hybrids Between the Aromatic Alkaloids Bauerine C and
Rutaecarpine
Kilian Huber and Franz Bracher
Department Pharmazie – Zentrum fu¨r Pharmaforschung, Ludwig-Maximilians-Universita¨t,
Butenandtstr. 5 – 13, 81377 Mu¨nchen, Germany
Reprint requests to Prof. Dr. Franz Bracher. Fax: +49-89-2180 77802.
E-mail: Franz.Bracher@cup.uni-muenchen.de
Z. Naturforsch. 2007, 62b, 1313 – 1316; received May 21, 2007
Two hybrids between the alkaloids bauerine C and rutaecarpine were prepared. Screening for cyto-
toxic activity revealed that introduction of two chlorine substituents to the quinazolinocarboline core
of rutaecarpine strongly enhances cytotoxic activity, whereas methylation at the indole nitrogen is
detrimental to activity.
Key words: Cytotoxic Activity, Hybrids, Bauerine C, Rutaecarpine, Alkaloids
Introduction
cytotoxic alkaloids bauerine C (2) and rutaecarpine (3)
as potential new anticancer agents. These hybrids can
be seen as 11,12-dichloro-rutaecarpine (7; in case the
indole nitrogen is not methylated) and as N-methyl-
11,12-dichloro-rutaecarpine(8). We were primarily in-
terested in exploring the benefit of the chloro sub-
stituents and the N-methyl group for the cytotoxic ac-
tivity of the hybrids.
A number of monohalogenated analogues of rutae-
carpine (3) have already been described as cyclooxy-
genase inhibitors by Lee et al. [8]. 10,11-Methylenedi-
oxyrutaecarpine and 11-methoxyrutaecarpine showed
cytotoxic activities superior to the native alkaloid 3 [9],
and 10-bromorutaecarpine showed a slight increase
(factor 2 – 3) in cytotoxic activity compared to alka-
loid 3, whereas the 10-methoxy analogue (identical to
the alkaloid hortiacine from Hortia arborea, Rutaceae
[10]) was found to be inactive [11]. Baruah et al. [12]
reported on the cytotoxic activity of 10-chloro deriva-
tives of rutaecarpine (3) and the related alkaloid evo-
diamine, but the results are not clear due to erroneous
molecular formulas in the publication.
Polycyclic aromatic alkaloids represent a common
source of lead structures for the development of new
anticancer drugs, mainly based on their ability to in-
teract with DNA by intercalation and/or inhibition of
topoisomerases. One of the most recent successful ex-
amples is topotecan, a topoisomerase I inhibitor de-
rived from the plant alkaloid camptothecin (1) [1].
In the course of our recent investigations in order
to develop new anticancer drugs, we worked out the
first total synthesis of the 1-oxo-β-carboline alkaloid
bauerine C (2) [2]. This alkaloid has been isolated
from the blue-green alga Dichotrix baueriana, and
showed very promising cytotoxic activitiy in prelimi-
nary screenings [3]. Typical structural elements of this
alkaloid are a 1,2-dichlorobenzene ring, an untypical
N-methylindole moiety, and a pyridone ring. The cor-
responding 1-oxo-β-carboline lacking the two chloro
substituents [4] does not show significant cytotoxicity.
Another relevant example for cytotoxic polycyclic
alkaloids is the quinazolinocarboline alkaloid rutae-
carpine (3), the major alkaloid from Evodia rutaecarpa
(Rutaceae) (Fig. 1) [5]. This alkaloid has been shown
to exhibit moderate cytotoxic activitiy against several
tumor cell lines, and its ability to inhibit topoisomerase
II has been reported recently [6].
Results
Chemistry
Several related strategies for the construction of
the pentacyclic quinazolinocarboline skeleton of rutae-
carpine (3) have been published over the years [5b].
Most of these approaches start from anthranilic acid
[12], anthranilic esters [13– 15], or isatoic acid, de-
Following our concept of combining typical struc-
tural elements of known antimicrobial and cytotoxic
compounds [7] in order to find new bioactive com-
pounds we envisaged to prepare hybrids between the
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1314
K. Huber – F. Bracher · Hybrids Between the Alkaloids Bauerine C and Rutaecarpine
Fig. 1. Polycyclic aromatic alka-
loids: Camptothecin (1), bauerine
C (2), rutaecarpine (3).
Scheme 1. Synthesis of the hy-
brids 7 and 8.
rived from anthranilate [12, 16, 17] as building blocks 24 h. Since large amounts of 6 were needed for our
for the quinazoline partial structure. The indole part is present investigations, we re-examined this cyclization
most commonly introduced using tryptamine [12, 15 – and found that the reaction can be dramatically accel-
17] or by Fischer indolization [8]. Moreover, 1-oxo- erated by microwaves [18]. Irradiation for 15 min gave
1,2,3,4-tetrahydro-β-carbolines can be applied as ver- the desired product in 40% yield when formic acid
satile tricyclic building blocks [12 – 14].
was used as the acidic solvent. Reaction in polyphos-
phoric acid trimethylsilyl ester [19] yielded 41 %, and
finally, reaction in polyphosphoric acid 47 % of 5. N-
Methylation at the indole nitrogen atom to give 6 was
performed as described previously [2]. Both 5 and 6
were converted to the quinazolinocarbolines 7 and 8
using Lee’s protocol [14]. Thus the lactams were con-
We decided to use the last mentioned strategy, since
the dichloro-oxocarbolines 5 and 6 were already in our
hands as intermediates of our total synthesis of bauer-
ine C (2) [2] (Scheme 1). Compound 5 was conve-
niently prepared in a Japp-Klingemann reaction by cy-
clization of arylhydrazone 4 in formic acid at 80 ◦C for
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K. Huber – F. Bracher · Hybrids Between the Alkaloids Bauerine C and Rutaecarpine
1315
Table 1. Cytotoxic ac-
tivities of the com-
pounds on HL-60 cells
(MTT assay).
Compound IC50 [µM] IC70 [µM]
were acquired on a Hewlett Packard 5989 A mass spectrome-
ter, electronic ionization (EI) 70 eV, chemical ionization (CI)
with CH4 (300 eV), (Agilent Technologies, Palo Alto, CA,
USA). IR spectra were measured on a Jasco FT/IR-410 spec-
trometer (Jasco Inc., Easton, MD, USA). Elemental analysis
was performed on a CHN-Rapid instrument (Heraeus Hold-
ing GmbH, Hanau, Germany). All microwave experiments
were performed using a CEM Discover apparatus (CEM Cor-
poration, Matthews, NC, USA).
2
3
5
6
7
8
11.9
5.4
5.9
31.0
0.15
0.67
27.7
12.6
13.7
72.1
0.36
1.58
verted to their hydrochloride salts with HCl gas in
chloroform, and then to the corresponding iminochlo-
rides with POCl3. After removal of excess POCl3 the
crude products were reacted with methyl anthranilate
to give 7 and 8 in 80 and 76 % yield, respectively. Ru-
taecarpine (3) was prepared for comparison in the same
manner starting from easily available 1-oxo-1,2,3,4-
tetrahydro-β-carboline [4].
MTT assay: This assay was performed on HL-60 cells as
described in ref. [20]. The experiments were carried out in
triplicate with each compound.
7,8-Dichloro-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
one (5)
0.50 g (1.84 mmol) 3-(2-(2,3-dichlorophenyl)hydrazono)-
piperidin-2-one (4) [2] and 1.0 g polyphosphoric acid were
stirred at 80 ◦C for 15 min using microwave irradiation
(20 W). After cooling the mixture was poured into ice wa-
ter (50 mL) and stirred at r. t. for 30 min. The suspension
was filtered and the residue recrystallized from EtOH to give
220 mg of 5 (47 %). The spectroscopic data were in close
Cytotoxic activity
Quinazolinocarbolines 7 and 8, alkaloids rutae-
carpine (3) and bauerine C (2), and both building
blocks 5 and 6, related to the alkaloid bauerine C, were
tested for cytotoxic activity in a standard MTT assay
on HL-60 cells [20]. The results are presented in Ta- agreement with literature values [2].
ble 1.
11,12-Dichloro-8,13-dihydro-7H-indolo[2ꢀ ,3ꢀ : 3,4]-
pyrido[2,1-b]quinazolin-5-one (7)
Discussion
Dry HCl gas was passed into a solution of 820 mg
(3.21 mmol) 7,8-dichloro-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indol-1-one (5) in 100 mL of CHCl3 until no further pre-
cipitation was observed. The lactam hydrochloride was col-
lected◦by filtration, suspended in 30 mL of POCl3 and stirred
at 50 C for 2 h. Excess POCl3 was removed in vacuo and
the residue dissolved in 100 mL of anhydrous THF. 900 mg
(5.95 mmol) methyl anthranilate was added and the mix-
ture was stirred at r. t. for 12 h. Then it was poured into
80 mL of water, 100 mL of aqueous ammonia (30 %) were
added, followed by extraction with toluene/ethyl acetate
(1 : 1; 3 × 100 mL). The combined organic layers were dried
over MgSO4 and the solvent was evaporated in vacuo. Re-
crystallization from toluene/ethanol afforded 916 mg (80 %)
of 7 as pale orange needles. M. p. 267 ◦C. – IR (KBr):
ν = 3377, 2929, 1655 (CO–NR2), 1599, 1560, 1471, 1306,
766 cm−1. – 1H NMR (400 MHz, [D6]DMSO): δ = 3.18 (t,
J = 6.8 Hz, 2 H, 8-H), 4.46 (t, J = 6.8 Hz, 2 H, 7-H), 7.30
(d, 3J = 8.5 Hz, 1 H, 10-H), 7.50 (ddd, 3J = 8.0 Hz, 8.0 Hz,
4J = 1.2 Hz, 1 H, 3-H), 7.67 (d, 3J = 8.5 Hz, 1 H, 9-H), 7.76
(ddd, 3J = 8.0 Hz, 4J = 1.2 Hz, 5J = 0.5 Hz, 1 H, 1-H), 7.83
(ddd, 3J = 8.0 Hz, 8.0 Hz, 4J = 1.5 Hz, 1 H, 2-H), 8.17 (ddd,
A convenient approach to rutaecarpine-bauerine C
hybrids 7 and 8 has been worked out. The cytotoxic ac-
tivities of these compounds were determined and com-
pared with those of the native alkaloids.
Alkaloids rutaecarpine (3) and bauerine C (2), as
well as the chlorinated oxo-β-carbolines 5 and 6
showed only poor cytotoxic activities (IC50 values be-
tween 5 and 31 µM) against HL-60 cells. In contrast,
the two hybrids 11,12-dichloro-rutaecarpine (7; IC50
=
0.15 µM) and N-methyl-11,12-dichloro-rutaecarpine
(8; IC50 = 0.67 µM) were found to exhibit high cyto-
toxic activities. Introduction of the chloro substituents
at 11- and 12-position obviously leads to a signifi-
cant increase in cytotoxic activity, whereas additional
methylation at the indole nitrogen atoms turns out to
be detrimental.
Work is in progress to screen the active hybrids 7
and 8 on a broad panel of tumor cell lines.
Experimental Section
Melting points were determined on a Bu¨chi Melting Point 3J = 8.0 Hz, 4J = 1.5 Hz, 5J = 0.5 Hz, 1 H, 4-H), 12.28 (br.
B-540 apparatus (Bu¨chi, Flawil, Switzerland) and are uncor- s, 1 H, N–H). – 13C NMR (100 MHz, [D6]DMSO): δ = 18.7
rected. NMR spectra were recorded on a Jeol JNMR-GX400 (C-8), 40.4 (C-7), 115.1 (C-12), 119.3 (C-8a), 119.7 (C-9),
instrument (Jeol, Peabody, MA, USA). Mass spectra data 120.8 (C-4a), 121.6 (C-10), 125.3 (C-8b), 126.3 (C-3), 126.4
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K. Huber – F. Bracher · Hybrids Between the Alkaloids Bauerine C and Rutaecarpine
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(C-4), 126.6 (C-1), 127.1 (C-11), 129.4 (C-13a), 134.4 (C-2), (t, J = 6.3 Hz, 2 H, 7-H), 7.35 (d, J = 8.6 Hz, 1 H, 10-H),
136.3 (C-12a), 144.4 (C-13b), 147.0 (C-14a), 160.4 (C=O). – 7.51 (d, 3J = 8.6 Hz, 1 H, 9-H), 7.70 (t, J = 8.0 Hz, 1 H,
MS (CI): m/z (%) = 360 (10) [M+5]+, 358 (57) [M+3]+, 3-H), 7.80 (d, 3J = 8.0 Hz, 1 H, 1-H), 7.96 (t, J = 8.0 Hz,
356 (100) [M+1]+. – MS (EI, 70 eV): m/z (%) = 359 (10) 1 H, 2-H), 8.35 (d, 3J = 8.0 Hz, 1 H, 4-H). – 13C NMR
[M+4]+, 357 (62) [M+2]+, 355 (100) M+. – C18H11Cl2N3O (100 MHz, CF3COOD): δ = 21.5 (C-8), 38.1 (N–CH3),
(356.2): calcd. C 60.69, H 3.11, N 11.80; found C 60.48, 43.7 (C-7), 119.5 (C-4a), 120.6 (C-12), 121.0 (C-1), 122.6
H 3.14, N 11.64.
(C-9), 126.9 (C-13a), 127.7 (C-8b), 128.3 (C-10), 130.7
(C-4), 132.2 (C-3), 136.9 (C-8a), 138.4 (C-14a), 139.5
(C-11), 140.2 (C-2), 146.1 (C-12a), 148.2 (C-13b), 162.5
(C=O). – MS (CI): m/z (%) = 374 (11) [M+5]+, 372 (61)
[M+3]+, 370 (100) [M+1]+. – MS (EI, 70 eV): m/z (%) =
372 (17) [M+3]+, 370 (74) [M+1]+, 368 (100) [M–1]+. –
C19H13Cl2N3O (370.2): calcd. C 61.64, H 3.54, N 11.35;
found C 61.52, H 3.34, N 11.30.
11,12-Dichloro-8,13-dihydro-13-methyl-7H-indolo-
[2ꢀ,3ꢀ : 3,4]pyrido[2,1-b]quinazolin-5-one (8)
This compound was prepared in the same manner
as described for 7 starting from 670 mg (2.51 mmol)
7,8-dichloro-9-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indol-1-one (6) and 605 mg (4.00 mmol) methyl anthrani-
late. Yield: 714 mg (77 %), as pale yellow needles after
recrystallization from toluene. M. p. 261 ◦C. – IR (KBr): ν =
3055, 2989, 2952, 2898, 2850, 1674 (CO–NR2), 1585, 1468,
1155, 766 cm−1. – 1H NMR (400 MHz, CF3COOD): δ =
3.27 (t, J = 6.3 Hz, 2 H, 8-H), 4.22 (s, 3 H, N–CH3), 4.65
Acknowledgements
We thank Ms. M. Stadler for performing the MTT assays,
and Ms. N. Iordanidis and Ms. C. Gra¨tsch for technical
assistance.
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