Antimalarial and antitubercular nostocarboline and eudistomin derivatives: Synthesis, in vitro and in vivo biological evaluation

Article abstract of DOI:10.1016/j.bmc.2010.01.013

The synthesis of nine nostocarboline derivatives with substitutions of the 2-methyl group by alkyl, aryl and functionalized residues, 10 symmetrical bis cationic dimers linking 6-Cl-norharmane through the 2-position and fifteen derivatives of the marine a

Full text of DOI:10.1016/j.bmc.2010.01.013

Bioorganic & Medicinal Chemistry 18 (2010) 1464–1476
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antimalarial and antitubercular nostocarboline and eudistomin
derivatives: Synthesis, in vitro and in vivo biological evaluation
Simone Bonazzi a, Damien Barbaras b, Luc Patiny c, Rosario Scopelliti c, Patricia Schneider d, Stewart T. Cole d,
Marcel Kaiser e, Reto Brun e, Karl Gademann a,
*
a Chemical Synthesis Laboratory (SB-ISIC-LSYNC), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland
b Laboratorium für Organische Chemie, Swiss Federal Institute of Technology (ETH), CH-8093 Zürich, Switzerland
c Institute of Chemistry and Chemical Engineering (ISIC), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland
d Global Health Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland
e Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute (STPH), Socinstrasse 57, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of nine nostocarboline derivatives with substitutions of the 2-methyl group by alkyl, aryl
and functionalized residues, 10 symmetrical bis cationic dimers linking 6-Cl-norharmane through the 2-
position and fifteen derivatives of the marine alkaloids eudistomin N and O is reported. These compounds
were evaluated in vitro against four parasites (Trypanosoma brucei rhodesiense STIB 900, Trypanosoma cru-
zi Tulahuen C2C4, Leishmania donovani MHOM-ET-67/L82 axenic amastigotes, and Plasmodium falciparum
K1 strain), against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155 and Corynebac-
terium glutamicum ATCC13032, and cytotoxicity was determined against L6 rat myoblast cells. Nostocarb-
oline and derivatives displayed potent and selective in vitro inhibition of P. falciparum with weak
cytotoxicity. The dimers displayed submicromolar inhibition of L. donovani and T. brucei, and nanomolar
activity against P. falciparum, albeit with pronounced cytotoxicity. One dimer showed a MIC99 value
Received 30 November 2009
Revised 5 January 2010
Accepted 6 January 2010
Available online 11 January 2010
Keywords:
Natural products
Malaria
Antiplasmodial agents
against M. tuberculosis of 2.5 lg/ml. The alkylated eudistomin N and O derivatives displayed activities
down to 18 nM against P. falciparum for N-Me Eudistomin N. Four dimers, nostocarboline and three eudo-
stomin derivatives were evaluated in an in vivo Plasmodium berghei mouse model. No significant activity
was observed for the dimers, but a 50% reduction in parasitaemia was observed at 4 ꢀ 50 mg/kg ip for
nostocarboline.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
roughly 50% of all anti-infective drugs are natural products or
derivatives thereof.5 Against malaria, quinine and its derivatives
Infectious diseases such as malaria, tuberculosis, leishmaniasis
or sleeping sickness are considered major public health challenges
in developing countries.1 For malaria alone, over 1 million deaths
are estimated per year, and the number of clinical cases is thought
to be 100 times higher.2 Parasite chemotherapy remains the ther-
apeutic option of choice, given the absence of a vaccine and the dif-
ficulties associated with systematic vector control.3 However, the
emergence of malaria cases in non-endemic areas combined with
the rise of multidrug-resistant parasites calls for the development
of novel drugs against this disease.3 A similar situation is observed
related to tuberculosis, where a diminished efficacy of currently
used drugs combined with the emergence of resistant strains also
emphasizes the need to discover novel leads.4
are still used today, and combination therapies relying on artemis-
inin are currently the treatment of choice.3,5 The search for novel
lead structures from natural sources such as plants, bacteria or
marine organisms is thus warranted.6
Cyanobacteria are an interesting source of novel metabolites7
and a large diversity of compounds such as peptides, peptolides,
polyketides, terpenes and alkaloids is documented in the litera-
ture.7,8 These prokaryotic phototrophs face ecological pressure
from both grazers and competing organisms, which they address
by producing bioactive secondary metabolites. Several compounds
display allelochemical properties in inhibiting the growth of com-
peting phototrophs.9 Such phytotoxic or algicidal compounds have
been shown to display activity against Plasmodium,10 as this para-
site contains an organelle of algal (cyanobacterial) origin, the api-
coplast.11 Based on this experimental evidence, we have been
employing a chemical ecology strategy for the search of novel anti-
plasmodial compounds based on their allelochemical properties in
cyanobacteria. We have characterized the heterocyclic macrocyclic
Natural products have been successfully employed for decades
in the search for novel biologically active compounds, and to date,
* Corresponding author. Tel.: +41 21 693 93 15; fax: +41 21 693 97 00.
E-mail address: karl.gademann@epfl.ch (K. Gademann).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.013

S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
1465
peptide aerucyclamide B,8b which displays submicromolar activity
CH3
N
N
Cl
Br
against Plasmodium falciparum K1 and only weak cytotoxicity.8c
These results are in line with other studies of similar peptides such
as venturamides A and B.12 Interestingly, this class of peptides has
been shown earlier to possess allelochemical properties towards
other phototrophic organisms,13 thus validating our chemical ecol-
ogy approach. Another literature example is the antimalarial agent
calothrixin A,14a which also displays allelochemical properties.14b
Several other antiplasmodial natural products have been described
from cyanobacteria that might also involve allelochemical proper-
ties.14c–e
N
H
N
H
I
Nostocarboline
Eudistomin N (2)
Hydroiodide (1)
CH3
CH3
N
N
Cl
Cl
Another compound that underlines the value of this strategy is
nostocarboline (1) or N-methyl-6-chloro-carbolinium, which was
isolated from the cyanobacterium Nostoc 78-12A.15 Nostocarboline
was shown to inhibit hydrolytic enzymes such as acetyl and butyryl
cholinesterase, and trypsin,15,16 while only displaying very weak
toxicity against the sensitive freshwater crustacean Thamnocephalus
platyurus.16 We have shown earlier that nostocarboline (1) displays
allelochemicalpropertiesininhibitingthegrowthofbotheukaryotic
and prokaryotic photosynthetic organisms, while being inactive
N
N
Nostocarboline
3b
Anhydronium Base (3a)
(MIC >93 l
M) against non-photosynthetic pathogens.17a This
inhibition of the photosystem was confirmed by chlorophyll-a
fluorescence imaging, which demonstrated that inhibition of photo-
synthesis preceded cell death.17b Taking advantage of this selective
action against phototrophs,17 we tested nostocarboline and ten
dimeric derivatives in vitro against a series of protozoan parasites
and found selective and potent in vitro activity.18 In this article, we
report in full detail the synthesis and the biological in vitro evalua-
tion of additional nostocarboline derivatives, demonstrate in vivo
activity of nostocarboline (1) in the Plasmodium berghei mouse
model, evaluate mechanistic details and report additional activity
of the nostocarboline dimers against Mycobacterium tuberculosis. In
addition, we report on the synthesis of N-alkylated derivatives of
eudistomin N19 (2) and eudistomin O19 (34) and also their biological
in vitro and in vivo evaluation.
Figure 1. Formulae of nostocarboline hydroiodide (1), eudistomin N (2), resonance
structures 3a and 3b of nostocarboline anhydronium base, and X-ray crystal
structure of 3.
12. The dimers 13–22 are obtained by treating 6-Cl-norharmane
with a series of different symmetrical dihalogenated linkers. We
were able to employ alkyl, aryl, alkenyl and alkynyl linkers of vary-
ing length and the corresponding products were obtained in good
to excellent yields.
The preparation of the N-alkylated eudistomin derivatives fol-
lowed a similar short and straightforward synthesis. Norharmane
was brominated using NBS to afford eudistomin N (6-bromo-
9H-beta-carboline, 2)19 and a minor fraction of eudistomin O
(8-bromo-9H-beta-carboline, 34).19 Alkylation of both Br-carbo-
lines 2 and 34 using a series of electrophiles directly afforded
the N-alkylated derivatives 23–33 and 35–39 as crystalline com-
pounds in yields between 23% and quantitative (Scheme 1).
The in vitro biological evaluation of nostocarboline (1), its
derivatives 5–12 and dimers 13–22 was carried out against four
parasites, Trypanosoma brucei rhodesiense STIB 900, Trypanosoma
cruzi Tulahuen C2C4, Leishmania donovani MHOM-ET-67/L82 axe-
nic amastigotes, and P. falciparum K1 strain. The corresponding
IC50 values are given in Table 1. In addition, cytotoxicity was mea-
sured against L6 rat myoblasts and the IC50 value as well as the
selectivity index (SI = IC50(L6)/IC50(P.f.)) are reported in Table 1.
In general, nostocarboline (1) and the derivatives 5–12 are weakly
active against L. donovani, T. brucei and T. cruzi, with the most ac-
tive being the 3-phenylpropyl substituted compound 12. This com-
pound displayed single digit micromolar activity against T. brucei,
and was found to be roughly 3 times less active against the other
parasites. In contrast, nostocarboline (1) and the derivatives 5–12
are clearly more active against P. falciparum, where submicromolar
activities were determined for compound 1, 5 and 6. The most ac-
tive compound in this series was the parent nostocarboline (1),
which showed a pronounced activity of 194 nM. Cytotoxicity of
2. Results and discussion
Nostocarboline, which was originally prepared as the hydroio-
dide salt 1,15 constitutes an anhydronium base, which can be rep-
resented by two different resonance structures 3a and 3b. We have
prepared nostocarboline anhydronium base by treating nostocarb-
oline (1) with aqueous NaOH, and a change in color and fluores-
cence became visible: whereas nostocarboline (1) is yellow-
brown and emits blue fluorescence, the corresponding base 3a is
yellow and emits a strong yellow fluorescence. We were able to ob-
tain crystals of the anhydronium base that were subjected to X-ray
crystallographic analysis (Fig. 1). As the anhydronium base is rela-
tively weak (pKa around 10), it is reasonable to assume that at
physiological pH a minor fraction of 1 is present in the deproto-
nated state 3, which could be beneficial in crossing cellular mem-
branes. Moreover, nostocarboline (1) features a Cl group—unusual
for a freshwater metabolite, which certainly adds lipophilicity to
the compound. We think that these chemical features of the anhy-
dronium base in combination with the Cl substituent might be cru-
cial for antimalarial activity.
The synthesis of the new nostocarboline derivatives follows a
short and straightforward route, and relies on inexpensive starting
materials such as tryptophan that can be converted to norharman
in two steps (Scheme 1).15 We think that these parameters are an
important requirement for successful drugs against tropical infec-
tious diseases. The synthesis of analogs of nostocarboline starts
from 6-Cl-norharmane (4), which itself is accessible via chlorina-
tion of norharmane.15 Alkylation of carboline 4 with a series of
electrophiles directly afforded the nostocarboline derivatives 5–
nostocarboline was low, as an IC50 value of 120.9 lM was deter-
mined, resulting in a SI of 622. In order to study the influence of
structure on activity, we calculated a set of molecular descriptors,
of which total surface area and clog P are reported in Table 1. The
relationship of antiplasmodial activity compared to the total

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S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
R
R
R
N
N
N
Cl
Cl
Br
N
Br
RX
RX
CH3CN
CH3CN
N
H
N
H
N
H
N
H
X
X
2
23-33
6-chloro-norharmane (4)
5-12
N
N
XLinkerX
iPrOH
RX
H
N
N
H
CH3CN
N
X
H
X
Br
Br
34
35-39
linker
N
X
N
Cl
Cl
N
H
13-22
Scheme 1. Preparation of nostocarboline derivatives 5–12, dimers 13–22 and eudistomin derivatives 23–33 and 35–39.
surface area for nostocarboline (1) and derivatives 5–12 reveals an
interesting trend (Fig. 2). Clearly, increasing the total surface area
by replacing the CH3 substituent in nostocarboline (1) with larger
groups results in a loss of antiplasmodial activity while increasing
the cytotoxicity of the compounds. This trend is reflected in a
selectivity drop, where a change in the SI from 622 to 9 was ob-
served by replacing CH3 by a Bn group.
The dimeric series 13–22 was generally found to be more active
than the nostocarboline derivatives, displaying single digit
lM IC50
values against T. cruzi, and submicromolar values against L. dono-

S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
1467
Table 1
Antiparasitic in vitro activities of nostocarboline 1, the derivatives 5–12, the dimers 13–22 and the eudistomin derivatives 23–33 and 35–39
Compound
Residue R
L.d.a
T.b.b
T.c.c
P.f.d
Cytotoxicitye
SIf
Surfaceg
clog Ph
1
5
6
7
8
–CH3
–C2H5
Allyl
–nBu
–(CH2)3COOCH3
Benzyl
p-Fluoro benzyl
p-Nitro benzyl
3-Phenyl propyl
2-Z-Butene-1,4-diyl
2-Butyn-1,4-diyl
Xylo-1,4-diyl
Xylo-1,3-diyl
4,40-Bis(methanyl)biphenyl
Bis(ethanyl)ether
Hexan-1,6-diyl
Octan-1,8-diyl
Decan-1,10-diyl
Dodecan-1,12-diyl
–CH3
–C2H5
Allyl
–nBu
–(CH2)3COOCH3
Benzyl
p-Fluoro benzyl
m-Fluoro benzyl
p-Nitro benzyl
3-Phenyl propyl
CH2naphthyl
–C2H5
34.3
251.0
196.1
112.1
116.6
112.6
110.5
145.6
18.6
9.6
34.7
19.9
0.2
68.4
8.6
70.5
36.8
33.5
11.6
105.6
6.2
18.1
21.3
6.2
1.1
6.4
1.0
1.2
2.5
1.2
1.2
0.9
1.1
1.2
47.2
26.4
17.4
11.7
111.0
5.2
4.7
5.6
10.8
4.5
4.1
25.0
26.2
7.0
3.8
4.4
>87.1
100.2
57.8
103.8
114.6
24.6
87.7
32.8
20.9
>56.6
10.6
>51.7
5.9
>45.7
51.1
36.2
31.4
36.6
10.0
131.9
90.5
86.6
35.3
64.6
16.3
13.3
25.9
14.1
12.6
7.5
0.194
0.452
0.831
1.616
3.143
2.997
4.672
2.209
1.608
0.113
0.738
0.223
0.121
0.056
0.018
0.020
0.018
0.014
0.023
0.018
0.032
0.492
1.151
2.330
2.368
1.761
2.153
1.330
0.459
0.481
6.624
6.738
2.583
3.279
0.502
120.9
113.0
126.5
74.0
207.1
27.0
111.3
42.2
71.3
61.1
28.5
17.4
3.8
23.0
47.9
36.2
7.5
8.2
4.3
86.1
78.8
130.9
68.7
162.3
61.6
22.6
41.5
18.1
27.5
5.1
153.4
112.2
28.7
17.5
3.7
622
250
152
46
66
9
24
19
44
540
39
204.97
222.48
235.23
254.52
300.72
276.2
285.7
307.03
310.29
412.14
409.43
455.58
456.21
527.38
430.05
449.74
478.10
510.41
542.17
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2.83
3.16
3.33
4.09
3.65
4.38
4.44
4.25
4.90
5.51
5.42
6.77
6.77
8.45
4.88
6.80
7.73
8.66
9.59
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
35
36
37
38
39
78
32
408
2625
1810
423
575
186
4783
2443
266
60
70
26
13
19
14
60
11
23
6.6
2.3
0.9
0.6
>230
>223
20.1
35.1
>203
21.2
16.4
11.0
38.2
20.4
17.7
>223
123.1
66.5
53.4
46.4
169.6
153.6
42.9
28.7
7.9
Allyl
Benzyl
p-Fluoro benzyl
CH2naphthyl
17
11
5
7
All results are reported as IC50 values in lM.
a
Leishmania donovani MHOM-ET-67/L82.
Trypanosoma brucei rhodesiense STIB 900.
Trypanosoma cruzi Tulahuen C2C4.
Plasmodium falciparum K1.
b
c
d
e
f
Rat myoblast L6 cells.
The selectivity index is calculated by IC50((L6)/IC50(P.f.)).
g
h
The surface in Å2 was calculated using moloc (http://www.moloc.ch).
cLog P was calculated using Osiris Property Explorer developed by Thomas Sander from Actelion (Basel). n.d. = not determined.
more restricted linkers such as 17. However, this trend again ap-
pears correlated to cytotoxicity, where also an increase can be
found. A drastic impact of structure on activity was found for xylyl
derivatives 15 and 16: While the para substituted 15 displays weak
activity (IC50 = 20 lM), a value of 0.2 lM was found for the corre-
sponding meta substituted derivative 16, giving rise to a two orders
of magnitude increase in activity upon change of aromatic substi-
tution from para to meta. These data suggest that the relative ori-
entation of the carbolinium structures in the dimer 16 directly
affects the inhibition of L. donovani. Consistent low micromolar
values were found against T. brucei and there appears to be little
influence of linker properties on bioactivity. When compared to
these values, activity against T. cruzi was decreased 10 to 50-fold,
and generally values almost equal or higher to those observed
against L6 rat myoblasts were measured, indicating little or no
selectivity.
The nostocarboline dimers 13–22 showed very potent activity
against P. falciparum, and values as low as 14 nM were determined
(for compound 21). Again, dimers employing a long and flexible
linker such as 17–22 display better activity, as IC50 values below
100 nM were determined. These derivatives, however, also display
increased cytotoxicity down to low micromolar values. From the
point of selectivity, shorter but flexible linkers are preferred, with
18 and 19 displaying selectivities for P. falciparum over L6 myo-
Figure 2. Antiplasmodial activity (d) and cytotoxicity (ꢀ) of nostocarboline (1) and
derivatives 5–12 reported as IC50 values plotted against the total surface area.
Increasing the surface results in decreased activity and decreased selectivity of the
compounds.
vani and T. brucei (Table 1). Against L. donovani, longer and flexible
linkers such as in 18–22 gave better IC50 values, reaching 0.6 lM
for the C12 derivative 22. This represents an increase in activity
of 2 orders of magnitude when compared to derivatives involving

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S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
blasts of >2600 and >1800-fold, respectively. Compound 18 proved
to be optimal in this series, while displaying very potent activity
(18 nM against P. falciparum) and a selectivity of >2600 against
L6 rat myoblasts. These in vitro data of nostocarboline dimers
13–22 complement earlier studies on bis cationic dimers, a class
of compounds known for antiprotozoal activity.26
The eudistomin derivatives 23–33 and 35–39 were evaluated
in vitro against the same four parasites: L. donovani, T. brucei rho-
desiense, T. cruzi and P. falciparum. These results and also the cyto-
toxicity against rat myoblast L6 cells and the selectivity index (SI)
are reported in Table 1. From the 6-bromo derivatives 23–33, the
2-naphthyl substituted compound 33 displayed the strongest
activity against L. donovani, T. brucei and T. cruzi with activities of
CH3
CH3
N
N
Cl
Br
N
H
N
H
Nostocarboline (1)
N-Methyl Eudistomin N (23)
0.018 µM (P. falciparum K1)
SI > 4783
0.194 µM (P. falciparum K1)
SI > 622
CH3
N
N
N
H
N
H
O
17.7 lM, 4.1 lM and 7.5 lM, respectively. Similar results were ob-
tained for the 8-bromo derivatives 35–39, with the 2-naphthyl
substituted compound 39 displaying the strongest activities of
46.4 lM, 4.4 lM and 7.9 lM against the same three parasites. In
Normelinonine F
Fascaplysin
13.6 µM (P. f. K1) ref. 20a 0.184 µM (P. falciparum K1)
0.34 µM (P. f. K1) ref. 20b
SI > 853 ref. 20b
SI = 50
contrast, stronger activities against P. falciparum were observed
with IC50 values reaching 18 nM and 32 nM for the 6-bromo deriv-
atives 23 (R = Me) and 24 (R = Et), respectively. In addition, these
H3C
N
CH3
N
two compounds exhibit
a low cytotoxicity of 86.1 lM and
78.8 M, resulting in a very high selectivity index of 4783 and
l
N
H
N
H
2443, respectively. Again, as discussed for the Cl-series above, for
both 6-bromo derivatives 23–33 and 8-bromo derivatives 35–39,
it was evident that increasing the size of the residue on the pyri-
dine nitrogen caused a loss of activity and an increase in cytotox-
icity. This trend directly influenced also the SI value, as for
example it drastically passed from 4783 for the methyl derivative
23 to 11 for the 2-naphthyl derivative 33. It is interesting also to
compare the compounds 24 and 35; both compounds had the ethyl
group as residue, but different positioning of the Br group on the
carboline. Compound 24 with the bromine at C(6) displayed an
IC50 values of 32 nM, while compound 35 with the bromine at
Cryptolepine
0.12 µM (P. falciparum K1)
SI = 9
Isoneocryptolepine
0.23 µM (P. falciparum K1)
SI = 18
Figure 3. Anhydronium bases such as normelinonine F,20 fascaplysin,21 cryptole-
pine20,22 and isoneocryptolepine23 display antiplasmodial activity and pronounced
cytotoxicity when compared to nostocarboline (1).
ization results in increased activity, although with increased
cytotoxicity.25
C(8) displayed an IC50 values of 6.6 lM, resulting in a loss of activ-
Nostocarboline (1), its ethyl derivative 5, four dimers 13, 17–19
and the eudistomin derivatives 23–25 that showed potent and
selective in vitro activity were selected for in vivo evaluation in a
P. berghei mouse model (Table 2). Compounds were administered
intraperitoneally as DMSO/water formulations on four consecutive
days (4, 24, 48 and 72 h post infection). Parasitaemia was deter-
mined on day 4 post infection (24 h after last treatment) by FACS
analysis. Activity was calculated as the difference between the
mean per cent parasitaemia for the control (n = 5 mice) and treated
groups expressed as a per cent relative to the control group. As can
be seen in Table 2, the dimers 13 and 17–19 and the eudistomin
derivatives 23–25 were found to be inactive in vivo. Interestingly,
nostocarboline (1) displayed roughly a 50% reduction in parasita-
emia when compared to the control group at a dose of 50 mg/kg,
and the mean survival time increased from 6.3 to 7.3 d. No toxicity
ity of more than 200-fold. This difference in activity disappeared of
the 6-bromo and 8-bromo derivatives with the same residue, but
of larger size. In general high cytotoxicity was again observed for
large substituents, and in particular for phenyl or naphthyl groups,
that is, compound 33 (IC50 = 5.1 lM) or 39 (IC50 = 3.7 lM). This is
in line with the Cl-series discussed above and can be explained
by similar factors.
The observed values of these simple quaternary carbolinium
alkaloids compare well with literature precedents (Fig. 3). For
example, normelinonine F (or deschloronostocarboline) was found
to display an IC50 value of 13.6
lM and no cytotoxicity was repor-
ted.20a A more recent study measured an IC50 value of 0.45
lM
against the K1 strain.20b Interestingly, addition of a Cl substituent
(to give 1) at the 6-position led to an increase of activity (Table 1
and Fig. 2). Tetra- and pentacyclic anhydronium bases such as for
example fascaplysin,21 cryptolepine20,22 or isoneocryptolepine23
display similar inhibition of the chloroquine resistant K1 strain of
P. falciparum, yet with increased cytotoxicity. Literature reports
attribute these cytotoxicity issues to DNA intercalation properties,
and cryptolepine was found to be a potent DNA intercalator, topo-
isomerase II inhibitor and cytotoxic agent.22b In addition, isoneo-
cryptolepine was found active in a DNA-methyl green assay
based on improved DNA intercalation.23 These results thus sub-
stantiate the hypothesis that larger lipophilic cations such as cry-
ptolepine or the aryl nostocarboline derivatives 9–12 or aryl
eudistomin N derivatives 29–33 display unselective cytotoxicity
which might be due to unfavorable interaction with DNA. Along
Table 2
Biological in vivo evaluation of nostocarboline (1), ethyl derivative 5, dimers 13, 17–
19, and eudistomin derivatives 23–25 in the P. berghei mouse model
Compound
Dose (mg/kg)
Route
% Activitya
Survival (d)
Nostocarboline (1)
5
13
17
18
19
23
24
4 ꢀ 50
4 ꢀ 50
4 ꢀ 50
4 ꢀ 50
4 ꢀ 50
4 ꢀ 50
4 ꢀ 50
4 ꢀ 50
4 ꢀ 50
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
49.6
15.36
11.53
0
2.6
0
7.3
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
6.3
0
13.25
3.57
25
Control
these lines, so-called
p-delocalized lipophilic cations (DLC) have
been shown to be powerful cytotoxic agents and antimalarial
agents.24 However, the observation that increased surface area re-
sults in increased cytotoxicity is also supported by these studies.24
This is also corroborated by reports on manzamine, where quatern-
The compounds were formulated in DMSO/water.
a
Activity means reduction of parasitaemia versus the untreated control group
n.d. = not determined due to inactivity, mice euthanized on day 4 (for 1, 13–19) or
on day 6 (for 5, 23–25) after parasitaemia determination.

S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
1469
Table 3
fied, spectroscopically pure compounds. Melting points (mp) were
determined on a Büchi 545 apparatus in open glass capillaries and
are uncorrected. 1H NMR spectra were recorded on Bruker DPX
400 MHz spectrometer or on Bruker Avance 500 MHz spectrometer
at 298 K in the indicated deuterated solvent. Data are reported as
follows: chemical shift (d, ppm), multiplicity (s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet or not resolved signal; br, broad
signal), coupling constants (J, Hz), integration. All signals were ref-
erenced to the internal solvent signal as standard (CD3OD, d 3.34).
IR spectra were recorded on a Varian 800 FT-IR ATR spectrometer
Antitubercular evaluation of nostocarboline (1) and dimers 13 and 15–22. MIC99
values are given in
lg/ml and in lM in brackets
Compound
MIC99 M.
tuberculosis
H37Rv
MIC99 M.
smegmatis
mc2155
MIC99 C. glutamicum
ATCC13032
13
15
16
17
18
19
20
21
22
5 [9]
>100 [>188]
>100 [>170]
25 [50]
100 [152]
25 [46]
12.5 [20]
100 [147]
>100 [>142]
12.5 [17]
100 [290]
2.5 [5]
>10 [>17]
10 [20]
>10 [>15]
5 [9]
5 [8]
5 [7]
10 [14]
10 [14]
>10 [>29]
>10 [>17]
10 [20]
>10 [>15]
10 [18]
5 [8]
5 [7]
2.5 [4]
5 [7]
and data are reported in terms of frequency of absorption (m,
cmꢁ1) with corresponding characteristic intensity (w, weak; m,
medium; s, strong; br, broad signal). Mass spectra were obtained
from the Mass Spectroscopy Service from the EPFL. High-resolution
measurements were recorded on an IonSpec 4.7 FT-ICR (ESI) using
the DHB (2,5-Dihydroxy-benzoic acid) matrix or on a MICROMASS
(ESI) Q-TOF Ultima API (Waters Corporation, Milford, MA, USA).
Data are reported as mass-to-charge ratio (m/z). Analytical
RP-HPLC was performed on a Dionex HPLC System (Interface ASI-
100 Chromeleon, UV detector PDA-100, Pump P-680, degaser).
The flow rate was 1 mL/min and the detector was fixed at the
indicated wavelength. Column: Phenomenex GeminiÒ RP-18
Nostocarboline (1)
>10 [>29]
was observed for all compounds at a dose of 50 mg/kg. In earlier
experiments, isoneocryptolepine was found to suppress parasita-
emia by 38.9% (50 mg/kg s.c.)23 and cryptolepine was found to be
toxic at a dose of 20 mg/kg ip,22c although the route of administra-
tion was found to be relevant,22a as earlier studies demonstrated
some efficacy in po administration at similar doses.20
The biological profile of nostocarboline (1) and the dimers
13–22 was further complemented by their biological in vitro eval-
uation against M. tuberculosis H37Rv, Mycobacterium smegmatis
mc2155 and Corynebacterium glutamicum ATCC13032 (Table 3).
Nostocarboline (1) was not found active against these three strains,
which is in line with an earlier study demonstrating no activity
against several strains of Staphylococcus, Enterococcus, Pseudomo-
nas, Streptococcus, Haemophilus, Moraxella and Escherichia.17a In
contrast, several dimers were active against M. tuberculosis, and a
(5 lm, 150 mm–4.6 mm), solvent A: CH3CN, solvent B: 0.1% TFA
in H2O. Conditions and retention times (tR) are indicated. All sepa-
rations were performed at ambient temperature.
4.1. X-ray crystal structure determination
The Data collections were performed at low temperature using
Mo Ka radiation on an Oxford Diffraction/Varian Sapphire/KM4
CCD, (3a, 23) and on a Bruker/Nonius APEX II CCD (25), both having
a kappa geometry goniometer. Data were reduced by means of Cry-
salis PRO28 (3a, 23) and EVALCCD29 and then corrected for absorp-
tion.30 The solutions and refinements were performed by SHELX.31
The crystal structures were refined using full-matrix least-squares
on F2 with all non hydrogen atoms anisotropically defined. Hydro-
gen atoms were placed in calculated positions by means of the ‘rid-
MIC99 value of 2.5
lg/ml was found for dimer 21 featuring a C10
linker. Dimers with different alkyl linkers were found active too,
although with a slightly reduced activity of 5 lg/ml.
3. Conclusion
In conclusion, we have reported the synthesis and biological
evaluation of nostocarboline (1), its derivatives 5–12, dimers
13–22 and eudistomin derivatives 23–33 and 35–39 against four
parasites and three bacteria relevant to tuberculosis research.
While some nostocarboline dimers displayed potent antiplasmo-
dial activity in vitro, very weak or no activity was observed in
the P. berghei mouse model. The best compound was the parent
natural product, nostocarboline (1). Benefits of this natural product
include (1) simple, inexpensive and straightforward preparation
(2) potent and selective in vitro activity against P. falciparum K1
and (3) moderate in vivo activity in the P. berghei mouse model.
Based on the structure/activity relationships reported in this study,
as well as literature precedents, we think that smaller carboline
anhydronium bases 3 or their salts 1 display favorable properties
and reduced cytotoxicity (due to decreased DNA intercalation).
These conclusions should help in the design of more efficient
carbolinium anhydronium bases as antiplasmodial agents.
ing’ model.
A twinning problem was discovered during the
refinement of 23 and treated by means of the TWINROTMAT rou-
tine found in PLATON.32 A new dataset was then created and used
in the final stages of refinement with cards HKLF 5, MERG 0 and
one refined BASF parameter [0.171(4)].
4.1.1. 6-Chloro-2-methyl-2H-beta-carboline (3a, nostocarboline
anhydronium base)
To a mixture of nostocarboline (1) (33.0 mg, 0.096 mmol,
1.00 equiv) in EtOAc (15.0 mL), a solution of NaOH (1 M, 7.5 mL)
was added dropwise. The starting material immediately dissolved
and generated a strongly yellow colored mixture that was stirred
at rt for 10 min. The mixture was extracted with EtOAc (3ꢀ) care-
fully recovering only the organic phases. The combined organic
phases could not be dried using standard salts (NaSO4 or MgSO4)
without reprotonation of the generated base and they were di-
rectly concentrated and dried under high vacuum to afford the
anhydronium base 3a (18.1 mg, 0.084 mmol, 88%) as a yellow so-
lid. An analytical sample was recrystallized (MeOH/Et2O/hexane)
for X-ray analysis. 1H NMR (400 MHz, CD3OD) d 8.79 (s, 1H), 8.25
(d, J = 6.2 Hz, 1H), 8.17 (d, J = 2.1 Hz, 1H), 7.92 (dd, J1 = 6.5 Hz,
J2 = 1.2 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.49 (dd, J1 = 8.8 Hz,
J2 = 2.1 Hz, 1H), 4.38 (s, 3H); 13C NMR (100 MHz, CD3OD) d 179.0,
153.6, 144.4, 131.6, 129.2, 126.3, 123.3, 121.4, 121.1, 118.2,
115.9, 46.2; HR-MS (ESI) calcd for C12H10ClN2: [M+H]+ 217.0533;
4. Experimental section
Solvents and reagents were purchased reagent-grade and used
without further purification, unless noted otherwise. 6-Chloro-nor-
harmane was prepared via tetrahydrocarboline27a in a modifica-
tion of the procedure reported by Nakano.27b Solvents for
extractions and recrystallizations were of analytical grade and
used as received. Deuterated solvents were obtained from Armar
Chemicals, Switzerland. All reactions were carried out under an
atmosphere of Ar or N2 and magnetically stirred unless otherwise
stated. Yields refer to recrystallized or chromatographically puri-
found 217.0525; FTIR (ATR)
1408s, 1335m, 1285m, 1246m, 1157m, 1092w, 1053m, 1015m,
922m, 872w, 806m, 783m, 752m, 702m cmꢁ1
m 3005w, 2932w, 2855w, 1570s,
.

1470
S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
4.1.2. Crystallographic details for compound 3a
any precipitate removed by filtration. The filtrate was concentrated
and dried under high vacuum affording compound 7 (21.2 mg,
0.055 mmol, 37%) as a crystalline solid. Mp = 213.0–214.0 °C; 1H
NMR (500 MHz, CD3OD) d 9.37 (s, 1H), 8.73 (d, J = 6.8 Hz, 1H),
8.64 (d, J = 6.4 Hz, 1H), 8.51 (s, 1H), 7.81–7.80 (m, 2H), 4.80 (t,
J = 7.5 Hz, 2H), 2.12 (quint., J = 7.5 Hz, 2H), 1.49 (sext., J = 7.5 Hz,
2H), 1.06 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) d 143.0,
136.2, 132.3, 132.3, 132.2, 129.5, 127.3, 122.4, 120.6, 118.1, 114.1,
61.3, 33.6, 19.2, 12.5; HR-MS (ESI) calcd for C15H16ClN2: [M]+
A total of 8596 reflections (ꢁ7 6 h 6 7, ꢁ28 6 k 6 27, ꢁ8 6 l 6 9)
were collected at T = 140(2) K in the range of 3.03–26.36° of which
1978 were unique (Rint = 0.0795); Mo Ka radiation (k = 0.71073 Å).
The residual peak and hole electron densities were 0.400 and
ꢁ0.411 eÅꢁ3
,
respectively. The absorption coefficient was
0.354 mmꢁ1. The least squares refinement converged normally with
residuals of R(F) = 0.0644 (observed data), wR(F2) = 0.1456 (all data)
and a GoF = 1.000. C12H9ClN2, Mw = 216.66, space group P21/c, mono-
clinic, a = 6.0420(8), b = 22.907(3), c = 7.2662(11) Å, b = 104.655(14)°,
259.1002; found 259.0999; FTIR (ATR)
m 3445w, 3032s, 2997s,
V = 973.0(2) Å3, Z = 4,
q
calcd = 1.479 Mg/m3. CCDC-728844 contains
2959s, 2858m, 1651m, 1570w, 1516m, 1489s, 1450s, 1323m,
the supplementary crystallographic data for this paper. These data
can be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
1281s, 1165m, 1142s, 1065s, 903w, 872m, 806s, 756m, 725s cmꢁ1
.
4.1.6. 6-Chloro-2-(4-methoxycarbonyl-butyl)-9H-beta-
carbolin-2-ium bromide (8)
4.1.3. 6-Chloro-2-ethyl-9H-beta-carbolin-2-ium iodide (5)
To a mixture of 6-chloro-norharmane (4) (500 mg, 2.47 mmol,
To a solution of 6-chloro-norharmane (4) (30.0 mg, 0.15 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added methyl bromovalerate
1.00 equiv) in CH3CN (15 mL) was added ethyl iodide (490
lL,
(53 lL, 0.37 mmol, 2.50 equiv). The flask was sealed and heated
6.18 mmol, 2.50 equiv). The flask was sealed and heated at 85 °C
for 18 h. The reaction was concentrated; then the residue was dis-
solved in a minimum amount of CH3CN, the product precipitated
by addition of Et2O, collected by filtration and washed with a mix-
ture of CH3CN/Et2O. The product was dissolved in MeOH and any
precipitate was removed by filtration. The filtrate was concen-
trated and dried under high vacuum affording compound 5
at 85 °C overnight. The reaction was concentrated; then the residue
was triturated in a mixture of Et2O/CH3CN and the precipitate was
collected by filtration. The product was dissolved in MeOH and any
precipitate removed by filtration. The filtrated was concentrated
and dried under high vacuum affording compound 8 (14.5 mg,
0.036 mmol, 24%) as a crystalline solid. Mp = 159.0–160.0 °C; 1H
NMR (500 MHz, CD3OD) d 9.36 (s, 1H), 8.74 (d, J = 6.4 Hz, 1H),
8.64 (d, J = 6.0 Hz, 1H), 8.53 (s, 1H), 7.83 (s, 2H), 4.81–4.78
(m, 2H), 3.68 (s, 3H), 2.48 (t, J = 7.2 Hz, 2H), 2.19–2.13 (m, 2H),
1.76–1.72 (m, 2H); 13C NMR (125 MHz, CD3OD) d 173.8, 143.1,
136.2, 132.4, 132.3, 129.6, 127.4, 122.4, 120.6, 118.1, 114.1, 61.0,
50.7, 32.4, 30.8, 21.1; HR-MS (ESI) calcd for C17H18ClN2O2: [M]+
(384 mg, 1.07 mmol, 43%) as
a
crystalline brown solid.
Mp = 215.0–216.0 °C; 1H NMR (500 MHz, CD3OD) d 9.38 (s, 1H),
8.74 (d, J = 6.8 Hz, 1H), 8.66 (dd, J1 = 6.8 Hz, J2 = 1.2 Hz, 1H), 8.51–
8.50 (m, 1H), 7.81–7.80 (m, 2H), 4.85 (q, J = 7.2 Hz, 2H), 1.77 (t,
J = 7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) d 142.9, 136.1, 132.3,
132.3, 132.1, 129.3, 127.3, 122.4, 120.6, 118.2, 113.8, 56.9, 16.0;
HR-MS (ESI) calcd. for C13H12ClN2: [M]+ 231.0689; found
317.1057; found 317.1062; FTIR (ATR)
m 3426w, 3036w, 2994w,
2951m, 2905w, 1728s, 1647m, 1570w, 1520m, 1493m, 1439m,
231.0692; FTIR (ATR)
1570w, 1489s, 1447s, 1319m, 1281s, 1250m, 1169m, 1142s,
1065s, 937w, 876m, 806s, 725m, 706m cmꢁ1
m 3418w, 3098m, 3017m, 2288w, 1647m,
1350m, 1281s, 1227m, 1157s, 1126s, 1069s, 984s, 891m, 810s,
752s cmꢁ1
.
.
4.1.7. 2-Benzyl-6-chloro-9H-beta-carbolin-2-ium bromide (9)17a
To a solution of 6-chloro-norharmane (4) (50.0 mg, 0.25 mmol,
1.00 equiv) in iPrOH (8.0 mL) was added benzyl bromide (60 lL,
4.1.4. 2-Allyl-6-chloro-9H-beta-carbolin-2-ium bromide (6)17a
To a solution of 6-chloro-norharmane (4) (50.0 mg, 0.25 mmol,
1.00 equiv) in iPrOH (4.0 mL) was added allyl bromide (43
l
L,
0.50 mmol, 2.00 equiv). The flask was sealed and heated at 85 °C
for 15 h. The reaction was concentrated; then the residue was dis-
solved in a minimum amount of CH3CN, the product precipitated
by addition of Et2O, collected by filtration and washed with a mix-
ture CH3CN/Et2O. The product was dissolved in MeOH and any pre-
cipitate removed by filtration. The filtrated was concentrated and
dried under high vacuum affording (9) (54.0 mg, 0.14 mmol, 58%)
as a crystalline solid. Mp = 121–125 °C; 1H NMR (600 MHz, CD3OD)
d 9.41 (s, 1H), 8.71–8.67 (m, 2H), 8.45 (s, 1H), 7.89 (s, 1H), 7.76 (d,
J = 1.25, 2H), 7.55–7.44 (m, 5H), 5.96 (s, 2H); 13CNMR (150 MHz,
CD3OD) d 143.2, 136.4, 135.2, 133.3, 132.9, 130.3, 130.2, 130.2,
129.7, 127.8, 123.0, 120.8, 119.1, 114.7, 79.2, 65.1; LR-MS 295.08
(37), 293.08 (100), 259.12 (53); HR-MS (MALDI) Anal. Calcd for
C18H14ClN2: [M]+ 293.0846; found 293.0840; FTIR (ATR) 3399w,
3057m, 2970m, 1643m 1491s, 1284s, 730s; RP-HPLC:
tR = 19.35 min (C18, 5–95% A in 30 min; 95% A for 3 min, 95–5% A
in 1 min).
0.50 mmol, 2.00 equiv). The flask was sealed and heated at 85 °C
for 21 h. The reaction was concentrated; then the residue was dis-
solved in a minimum amount of CH3CN, the product precipitated
by addition on Et2O, collected by filtration and washed with a mix-
ture of CH3CN/Et2O. The product was dissolved in MeOH and any
precipitate was removed by filtration. The filtrate was concen-
trated and dried under high vacuum affording compound 6
(36.0 mg, 0.11 mmol, 45%) as
a crystalline solid. Mp = 174–
177 °C; 1H NMR (600 MHz, CD3OD) d 9.31 (s, 1H), 8.71 (d,
J = 6.6 Hz, 1H), 8.58 (dd, J1 = 6.6 Hz, J2 = 1.1, 1H), 8.47 (d,
J = 1.1 Hz, 1H), 7.78 (s, 2H), 6.33–6.20 (m, 1H), 5.55 (d, J = 1.4 Hz,
1H), 5.51 (d, J = 5.1 Hz, 1H), 5.39 (d, J = 6.3 Hz, 2H); 13C NMR
(150 MHz, CD3OD) d 143.5, 136.7, 133.3, 133.2, 133.1, 132.3,
130.4, 128.0, 123.2, 122.4, 121.2, 119.0, 114.9, 64.1; MS 245.06
(16), 243.06 (100), 209.10 (6); HR-MS (MALDI) Anal. Calcd for
C14H12ClN2: [M]+ 243.0689; found 243.0680; FTIR (ATR)
m
3007m, 2949m, 2855m, 1905w, 1813w, 1745s, 1651s, 1489s,
1285s, 951s, 806s. RP-HPLC: tR = 16.79 min (C18, 5–95% A in
30 min; 95% A for 3 min, 95–5% A in 1 min).
4.1.8. 6-Chloro-2-(4-fluoro-benzyl)-9H-beta-carbolin-2-ium
bromide (10)
To a solution of 6-chloro-norharmane (4) (30.0 mg, 0.15 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added 4-fluorobenzyl bromide
(69 lL, 0.37 mmol, 2.50 equiv). The flask was sealed and heated
4.1.5. 2-Butyl-6-chloro-9H-beta-carbolin-2-ium iodide (7)
To a solution of 6-chloro-norharmane (4) (30.0 mg, 0.15 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added iodobutane (42
l
L,
at 85 °C overnight. The reaction was concentrated; then the residue
triturated using a mixture of Et2O/CH3CN and the precipitate col-
lected by filtration. The product was dissolved in MeOH and any
precipitate removed by filtration. The filtrate was concentrated
and dried under high vacuum affording compound 10 (56.3 mg,
0.37 mmol, 2.50 equiv). The flask was sealed and heated at 85 °C
overnight. The reaction was concentrated and then the residue
was triturated using a mixture of Et2O/CH3CN and the precipitate
collected by filtration. The product was dissolved in MeOH and

S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
1471
0.14 mmol, 96%) as a crystalline solid. 1H NMR (500 MHz, CD3OD) d
4.2.1. 2-(Z)-Butene-1,4-diyl homo-dimer (13)
(Z)-1,4-Dichlorobut-2-ene (14 L, 0.125 mmol, 1.00 equiv) was
9.50 (s, 1H), 8.75 (d, J = 6.8 Hz, 1H), 8.71 (dd, J1 = 6.8 Hz, J2 = 1.6 Hz,
1H), 8.51 (t, J = 1.2 Hz, 1H), 7.82 (d, J = 1.2 Hz, 2H), 7.67 (d,
J = 5.2 Hz, 1H), 7.65 (d, J = 5.2 Hz, 1H), 7.23 (t, J = 8.7 Hz, 2H), 6.00
(s, 2H); 13C NMR (125 MHz, CD3OD) d 163.4 (d, J = 248.4 Hz),
143.1, 136.1, 132.6, 132.5, 132.4, 130.9 (d, J = 9.7 Hz), 130.4 (d,
J = 3.7 Hz), 129.6, 127.4, 122.5, 120.5, 118.3, 116.0 (d, J = 22.0 Hz),
114.2, 63.2; HR-MS (ESI) calcd for C18H13ClFN2: [M]+ 311.0751;
l
transformed according to GP 1. Recrystallization from iPrOH/MeOH
gave 13 (48 mg, 0.090 mmol, 72%). Yellow powder. Mp >200 °C; 1H
NMR (400 MHz, CD3OD) d 9.50 (br s, 2H), 8.78 (d, J = 6.5 Hz, 2H),
8.73 (d, J = 6.5 Hz, 2H), 8.51 (br s, 2H), 7.81 (m, 4H), 6.30 (br s,
2H), 5.81 (br s, 4H); HR-MS (ESI) Anal. calcd for C26H20Cl2N4:
[M]++ 230.0605; found: 230.0603; FTIR (ATR)
m 3056w, 3011w,
found 311.0738; FTIR (ATR)
m
3460w, 3414w, 3044m, 2982m,
2964w, 2909w, 2865w, 2782w, 1649s, 1573m, 1520s, 1492s,
1453s, 1376w, 1320m, 1280s, 1253m, 1219w, 1170w, 1158w,
1133s, 1071s, 1023w, 963m, 919w, 882m, 828s, 806s, 782s, 704s,
652w, 620s; RP-HPLC: tR = 13.27 min (C18, 5–50% A in 17 min;
50% A for 3 min, 50–5% A in 1 min).
2893w, 1647m, 1605m, 1570w, 1512m, 1489m, 1454m, 1350w,
1281m, 1223m, 1161s, 1119s, 1069s, 883w, 826s, 779m,
760m cmꢁ1
.
4.1.9. 6-Chloro-2-(4-nitro-benzyl)-9H-beta-carbolin-2-ium
bromide (11)
4.2.2. 2-Butyn-1,4-diyl homo-dimer (14)
To a solution of 6-chloro-norharmane (4) (30.0 mg, 0.15 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added 4-nitrobenzyl bromide
(80.0 mg, 0.37 mmol, 2.50 equiv). The flask was sealed and heated
at 85 °C overnight. The reaction was concentrated; then the residue
triturated using a mixture of Et2O/CH3CN and the precipitate was
collected by filtration. The product was dissolved in MeOH and
any precipitate removed by filtration. The filtrate was concentrated
and dried under high vacuum affording (11) (34.6 mg, 0.083 mmol,
1,4-Dichlorobut-2-yne (13 lL, 0.125 mmol, 1.00 equiv) was
transformed according to GP 1. Recrystallization from iPrOH/MeOH
gave 14 (43 mg, 0.081 mmol, 65%). Dark brown powder. Mp
>200 °C; 1H NMR (400 MHz, CD3OD) d 9.15 (br s, 2H), 8.64 (br s,
2H), 8.38 (br s, 2H), 8.22 (br s, 2H), 7.71 (br s, 4H), 3.35 (br s,
4H); HR-MS (ESI) Anal. calcd for C26H18Cl2N4: [M]++ 229.0527;
found: 229.0525; FTIR (ATR)
m 3032w, 2928w, 2817w, 2712w,
1642s, 1570w, 1487s, 1452s, 1362w, 1321m, 1280s, 1247w,
1151m, 1069s, 1024w, 886m, 810s, 732m, 607w; RP-HPLC:
tR = 13.19 min (C18, 5–50% A in 17 min; 50% A for 3 min, 50–5% A
in 1 min).
55%) as
a
crystalline solid. Mp = 210.0–211.0 °C; 1H NMR
(500 MHz, CD3OD) d 9.55 (s, 1H), 8.80 (d, J = 6.8 Hz, 1H), 8.74 (d,
J = 6.8 Hz, 1H), 8.54 (s, 1H), 8.34 (d, J = 8.7 Hz, 2H), 7.84 (s, 2H),
7.76 (d, J = 8.7 Hz, 2H), 6.17 (s, 2H); 13C NMR (125 MHz, CD3OD)
d 144.6, 143.2, 141.2, 136.2, 132.8, 132.7, 132.7, 130.1, 129.3,
127.6, 124.0, 122.6, 120.5, 118.5, 114.2, 62.8; HR-MS (ESI) calcd
4.2.3. Xylo-1,4-diyl homo-dimer (15)
1,4-Bis-chloro-methylbenzene (22 mg, 0.125 mmol, 1.00 equiv)
was transformed according to GP 1. Recrystallization from iPrOH/
MeOH gave 15 (69 mg, 0.119 mmol, 95%). Highly hygroscopic yel-
low powder. Mp >200 °C; 1H NMR (400 MHz, CD3OD) d 9.43 (br s,
2H), 8.68 (d, J = 6.5 Hz, 2H), 8.66 (d, J = 6.5 Hz, 2H), 8.45 (br s, 2H),
7.76 (br s, 4H), 7.63 (br s, 4H), 5.99 (br s, 4H); HR-MS (ESI) Anal.
calcd for C30H22Cl2N4: [M]++ 255.0684; found: 255.0684; FTIR
for C18H13ClN3O2: [M]+ 338.0696; found 338.0686; FTIR (ATR)
m
3387w, 3059m, 3009m, 1647m, 1609m, 1574w, 1520s, 1493s,
1454m, 1342s, 1285s, 1161m, 1130m, 1069m, 1018w, 856m,
806s, 733s, 710m cmꢁ1
.
4.1.10. 6-Chloro-2-(3-phenyl-propyl)-9H-beta-carbolin-2-ium
bromide (12)
(ATR)
m 3352br, 3053w, 3002w, 2956w, 2900w, 2848w, 2757w,
To a solution of 6-chloro-norharmane (4) (30.0 mg, 0.15 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added 1-bromo-3-phenylpro-
pane (56 lL, 0.37 mmol, 2.50 equiv). The flask was sealed and
2651w, 1643s, 1571m, 1516m, 1490s, 1453s, 1403w, 1318s,
1282s, 1253s, 1213m, 1162s, 1124s, 1069s, 1024w, 945w, 875m,
813s, 747s, 705w, 628m; RP-HPLC: tR = 16.56 min (C18, 5–50% A
in 17 min; 50% A for 3 min, 50–5% A in 1 min).
heated at 85 °C overnight. The reaction was concentrated; then
the residue triturated using a mixture of Et2O/CH3CN and the pre-
cipitate collected by filtration. The product was dissolved in MeOH
and any precipitate removed by filtration. The filtrate was concen-
trated and dried under high vacuum affording compound 12
4.2.4. Xylo-1,3-diyl homo-dimer (16)
1,3-Bis-chloro-methylbenzene (22 mg, 0.125 mmol, 1.00 equiv)
was transformed according to GP 1. Recrystallization from iPrOH/
MeOH gave 16 (65 mg, 0.111 mmol, 89%). Highly hygroscopic yel-
low powder. Mp >200 °C; 1H NMR (400 MHz, CD3OD) d 9.44 (br s,
1H), 9.40 (br s, 1H), 8.72 (d, J = 6.5 Hz, 1H), 8.67 (m, 2H), 8.63 (d,
J = 6.5 Hz, 1H), 8.49 (br s, 1H), 8.44 (br s, 1H), 7.76 (br s, 4H),
7.63 (br s, 4H), 5.99 (br s, 4H); HR-MS (ESI) Anal. Calcd for
(18.7 mg, 0.047 mmol, 31%) as
a
crystalline solid. 1H NMR
(500 MHz, CD3OD) d 9.31 (s, 1H), 8.68 (d, J = 6.4 Hz, 1H), 8.60 (d,
J = 6.4 Hz, 1H), 8.49 (t, J = 1.2 Hz, 1H), 7.81 (d, J = 1.2 Hz, 2 H),
7.25 (s, 2H), 7.24 (s, 2H), 7.13–7.09 (m, 1H), 4.83 (t, J = 7.2 Hz,
2H), 2.83 (t, J = 7.2 Hz, 2H), 2.51–2.45 (m, 2H); 13C NMR
(125 MHz, CD3OD) d 143.2, 140.1, 136.2, 132.3, 132.2, 129.7,
128.2, 128.0, 127.2, 125.9, 122.3, 120.6, 118.0, 114.2, 61.2, 32.6,
32.1, 22.7; HR-MS (ESI) calcd. for C20H18ClN2: [M]+ 321.1158;
C30H22Cl2N4: [M]++ 255.0684; found: 255.0682; FTIR (ATR)
m
3141w, 3037w, 3004w, 2956w, 2906w, 2852w, 2761w, 2708w,
2657w, 1644s, 1572m, 1517w, 1491s, 1453s, 1321s, 1282s,
1253m, 1222w, 1162m, 1126m, 1070m, 1026m, 874m, 812s,
found 321.1146; FTIR (ATR)
m 3418w, 3024m, 2990m, 2943m,
2905m, 2843w, 1643m, 1574s, 1520m, 1493s, 1450s, 1412s,
749s, 708s, 636s; RP-HPLC: tR = 16.44 min (C18, 5–50% A in
1319m, 1285s, 1157s, 1123s, 1069s, 922m, 876m, 826s,
17 min; 50% A for 3 min, 50–5% A in 1 min).
741m cmꢁ1
.
4.2.5. 4,40-Bis(methanyl)biphenyl homo-dimer (17)
4,40-Bis(chloromethyl)-1,10-biphenyl
(31 mg,
4.2. General procedure 1 (GP 1) for dimerization
0.125 mmol,
1.00 equiv) was transformed according to GP 1. Recrystallization
from iPrOH/MeOH gave 17 (71 mg, 0.107 mmol, 86%). Highly
hygroscopic yellow powder. Mp >200 °C; 1H NMR (400 MHz,
CD3OD) d 9.43 (br s, 2H), 8.72 (d, J = 6.5 Hz, 2H), 8.69 (d,
J = 6.5 Hz, 2H), 8.49 (br s, 2H), 7.79 (m, 4H), 4.78 (br s, 4H), 2.14
(br s, 4H), 1.55 (br s, 4H); HR-MS (ESI) Anal. calcd for C36H26Cl2N4:
To a solution of 6-chloro-norharmane (4) (50.0 mg, 0.25 mmol,
2.00 equiv) in iPrOH (4 mL, 62.5 mM) was added the bis-halogeno
linker (0.125 mmol, 1.00 equiv) at 50 °C and the resulting sus-
pension was heated to reflux for 12–48 h. The resulting yellow
suspension was cooled to rt and filtered. The filtrate was washed
with ice-cooled iPrOH and dried under high vacuum. Purification
by recrystallization gave the desired bis-b-carbolinium compound.
[M]++ 293.0840; found: 293.0846; FTIR (ATR)
3004w, 2955w, 2902w, 2849w, 2758w, 2704w, 2647w, 1646s,
m 3339w, 3049w,

1472
S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
1614w, 1571m, 1519m, 1492s, 1454s, 1404m, 1318s, 1284s,
1253m, 1211w, 1164m, 1122m, 1068s, 1006s, 946w, 874w, 813s,
756s, 706m, 645s; RP-HPLC: tR = 18.23 min (C18, 5–50% A in
17 min; 50% A for 3 min, 50–5% A in 1 min).
>200 °C; 1H NMR (400 MHz, CD3OD) d 9.31 (br s, 2H), 8.71 (d,
J = 6.5 Hz, 2H), 8.60 (d, J = 6.5 Hz, 2H), 8.50 (br s, 2H), 7.79 (m,
4H), 4.74 (t, J = 7.5 Hz, 4H), 2.08 (m, 4H), 1.41 (br s, 4H), 1.34 (br
s, 12H); HR-MS (ESI) Anal. calcd for C34H38Cl2N4: [M]++
287.1310; found: 287.1312; FTIR (ATR)
m 3398br, 3053w, 3017w,
2923s, 2852s, 2764w, 2699w, 2651w, 1649s, 1573m, 1519m,
1491s, 1455s, 1350w, 1322m, 1280s, 1252m, 1221w, 1171w,
1153m, 1130w, 1069s, 1022w, 873s, 816s, 762w, 727m, 692w,
630s; RP-HPLC: tR = 20.05 min (C18, 5–50% A in 17 min; 50% A for
3 min, 50–5% A in 11 min).
4.2.6. Bis(ethanyl)ether homo-dimer (18)
Bis(2-chloroethyl)ether (16 lL, 0.125 mmol, 1.00 equiv) was
transformed according to GP 1. Recrystallization from iPrOH/MeOH
gave 18 (41 mg, 0.075 mmol, 60%). Yellow powder. Mp >200 °C; 1H
NMR (400 MHz, CD3OD) d 9.30 (br s, 2H), 8.70 (d, J = 6.3 Hz, 2H),
8.62 (d, J = 6.3 Hz, 2H), 8.49 (br s, 2H), 7.78 (m, 4H), 4.95 (m, 4H),
4.06 (m, 4H); HR-MS (ESI) Anal. calcd for C26H22Cl2N4O: [M]++
4.2.11. 6-Bromo-2-methyl-9H-beta-carbolin-2-ium iodide (23)
To a solution of 6-bromo-norharmane (2) (500 mg, 2.47 mmol,
239.0658; found: 239.0651; FTIR (ATR)
m 3063m, 2925m, 2855m,
2763w, 1732w, 1647s, 1564s, 1518m, 1488s, 1454s, 1377w,
1326m, 1280s, 1245s, 1165w, 1117s, 1069s, 1037w, 838w, 872m,
816s, 735s, 705m, 663w; RP-HPLC: tR = 15.97 min (C18, 5–50% A
in 17 min; 50% A for 3 min, 50–5% A in 1 min).
1.00 equiv) in CH3CN (15 mL) was added methyl iodide (380 lL,
6.18 mmol, 2.50 equiv). The flask was sealed and heated at 85 °C
for 18 h. The reaction was cooled with an ice-bath, the precipitate
filtered, washed with CH3CN and Et2O. The product was dissolved
in MeOH and any precipitate removed by filtration. The filtrated
was concentrated and dried under high vacuum affording 23
(685 mg, 1.76 mmol, 71%) as a yellow solid. An analytical sample
was recrystallized (MeOH) for X-ray analysis. Mp = 292.0–
293.0 °C; 1H NMR (500 MHz, CD3OD) d 9.28 (s, 1H), 8.71 (d,
J = 6.4 Hz, 1H), 8.67 (d, J = 2.0 Hz, 1H), 8.57 (d, J = 6.4 Hz, 1H),
7.94 (dd, J1 = 8.7 Hz, J2 = 1.6 Hz, 1H), 7.75 (d, J = 9.1 Hz, 1H), 4.58
(s, 3H); 13C NMR (125 MHz, CD3OD) d 143.1, 135.8, 134.8, 133.3,
131.9, 130.4, 125.6, 121.1, 117.9, 114.4, 114.4, 48.5; HRMS-ESI
4.2.7. Hexan-1,6-diyl homo-dimer (19)
1,6-Dibromo-hexane (19 lL, 0.125 mmol, 1.00 equiv) was
transformed according to GP 1. Recrystallization from iPrOH/MeOH
gave 19 (75 mg, 0.116 mmol, 93%). Yellow powder. Mp >200 °C; 1H
NMR (400 MHz, CD3OD) d 9.39 (br s, 2H), 8.68 (d, J = 6.5 Hz, 2H),
8.63 (d, J = 6.5 Hz, 2H), 8.47 (br s, 2H), 7.78 (br s, 4H), 4.78 (br s,
4H), 2.14 (br s, 4H), 1.55 (br s, 4H); HR-MS (ESI) Anal. calcd for
C28H26Cl2N4: [M]++ 245.0840; found: 245.0845; FTIR (ATR)
m
calcd for C12H10N2Br: [M]+ 261.0027; found 261.0029; FTIR
m
3016w, 2987w, 2936w, 2884w, 2834w, 2764w, 2697w, 2647w,
1646s, 1571m, 1519m, 1488s, 1454s, 1320s, 1282s, 1247m,
1155s, 1067s, 1019w, 953w, 866m, 826s, 761w, 736s, 678w,
633s, 609w; RP-HPLC: tR = 16.47 min (C18, 5–50% A in 17 min;
50% A for 3 min, 50–5% A in 1 min).
3040m, 1643m, 1566w, 1520w, 1485s, 1447s, 1323m, 1277s,
1254s, 1146m, 1123m, 1053m, 872m, 810s, 729m, 694m cmꢁ1
.
4.2.12. Crystallographic details for compound 23
A
total of 2517 reflections (ꢁ13 6 h 6 13, ꢁ19 6 k 6 19,
ꢁ6 6 l 6 9) were collected at T = 140(2) K in the range of 3.03–
26.37° of which 2517 were unique; Mo radiation
4.2.8. Octan-1,8-diyl homo-dimer (20)
Ka
1,8-Dibromo-octane (23 lL, 0.125 mmol, 1.00 equiv) was trans-
(k = 0.71073 Å). The residual peak and hole electron densities were
4.346 and ꢁ1.602 eÅꢁ3, respectively. The absorption coefficient
was 5.772 mmꢁ1. The least squares refinement converged nor-
mally with residuals of R(F) = 0.0561 (observed data),
formed according to GP 1. Recrystallization from iPrOH/MeOH gave
20 (74 mg, 0.108 mmol, 87%). Yellow powder. Mp >200 °C; 1H NMR
(400 MHz, CD3OD) d 9.34 (br s, 2H), 8.69 (d, J = 6.5 Hz, 2H), 8.61 (d,
J = 6.5 Hz, 2H), 8.49 (br s, 2H), 7.79 (br s, 4H), 4.75 (br s, 4H), 2.10
(br s, 4H), 1.45 (br s, 8 H); HR-MS (ESI) Anal. calcd for C30H30Cl2N4:
wR(F2) = 0.1530 (all data) and
a GoF = 1.129. C12H10BrIN2,
[M]++ 259.0997; found: 259.0996; FTIR (ATR)
m 3009w, 2921m,
Mw = 389.03, space group P21/c, monoclinic, a = 10.7180(9), b =
15.5515(16), c = 7.4595(6) Å, b = 93.188(8)°, V = 1241.42(19) Å3,
2851m, 2764w, 2700w, 2652w, 1646s, 1572m, 1518m, 1491s,
1453s, 1401w, 1352w, 1318s, 1281s, 1253w, 1169w, 1155w,
1129m, 1072s, 1024w, 952w, 856m, 815s, 734m, 631s; RP-HPLC:
tR = 16.95 min (C18, 5–50% A in 17 min; 50% A for 3 min, 50–5% A
in 1 min).
Z = 4, q
calcd = 2.081 Mg/m3. CCDC-755505 contains the supplemen-
tary crystallographic data for this paper. These data can be ob-
tained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
4.2.13. 6-Bromo-2-ethyl-9H-beta-carbolin-2-ium iodide (24)
To a solution of 6-bromo-norharmane (2) (15.0 mg, 0.06 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added ethyl iodide (12 lL,
4.2.9. Decan-1,10-diyl homo-dimer (21)
1,10-Dibromo-decane (28 lL, 0.125 mmol, 1.00 equiv) was
transformed according to GP 1. Recrystallization from iPrOH/MeOH
gave 21 (69 mg, 0.097 mmol, 78%). Light yellow powder. Mp
>200 °C; 1H NMR (400 MHz, CD3OD) d 9.33 (br s, 2H), 8.69 (d,
J = 6.5 Hz, 2H), 8.61 (d, J = 6.5 Hz, 2H), 8.49 (br s, 2H), 7.79 (br s,
4H), 4.74 (br s, 4H), 2.08 (br s, 4H), 1.41 (br s, 8 H), 1.34 (br s, 4H);
HR-MS (ESI) Anal. Calcd for C32H34Cl2N4: [M]++ 273.1153; found:
0.15 mmol, 2.50 equiv). The flask was sealed and heated at 85 °C
for 15 h. The reaction was cooled to rt, the precipitate filtered,
washed with CH3CN and pentane. The product was dissolved in
MeOH and any precipitate removed by filtration. The filtrated
was concentrated and dried under high vacuum affording 24
(20.0 mg, 0.05 mmol, 83%) as a crystalline solid. Mp = 226.5–
227.5 °C; 1H NMR (500 MHz, CD3OD) d 9.37 (s, 1H), 8.74 (d,
J = 6.4 Hz, 1H), 8.68 (d, J = 1.6 Hz, 1H), 8.65 (dd, J1 = 6.4 Hz,
J2 = 1.2 Hz, 1H), 7.94 (dd, J1 = 8.7 Hz, J2 = 2.0 Hz, 1H), 7.76 (d,
J = 8.7 Hz, 1H), 4.84 (q, J = 7.2 Hz, 2H), 1.76 (t, J = 7.2 Hz, 3H); 13C
NMR (125 MHz, CD3OD) d 143.3, 136.0, 134.8, 132.2, 132.1,
129.3, 125.6, 121.2, 118.2, 114.4, 114.4, 56.9, 16.0; HRMS-ESI calcd
273.1150; FTIR (ATR)
m 2987w, 2921m, 2852m, 2759w, 2698w,
2652w, 1650s, 1570m, 1519m, 1490s, 1453s, 1401w, 1349w,
1326m, 1278s, 1249m, 1171w, 1155w, 1130m, 1068s, 873s, 826w,
812s, 765m, 730s, 685m, 630s; RP-HPLC: tR = 18.77 min (C18
5–50% A in 17 min; 50% A for 3 min, 50–5% A in 1 min).
,
for C13H12N2Br: [M]+ 275.0184; found 275.0192; FTIR
m 3514w,
4.2.10. Dodecan-1,12-diyl homo-dimer (22)
1,12-Dibromo-dodecane (41 mg, 0.125 mmol, 1.00 equiv) was
transformed according to GP 1. Recrystallization from iPrOH/MeOH
gave 22 (65 mg, 0.089 mmol, 71%). Light yellow powder. Mp
3055s, 2955m, 1647s, 1612w, 1516m, 1493s, 1450s, 1319m,
1281s, 1250s, 1165m, 1142s, 1053s, 937m, 868s, 814s, 802s,
725s cmꢁ1
.

S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
1473
4.2.14. 2-Allyl-6-bromo-9H-beta-carbolin-2-ium bromide (25)
To a solution of 6-bromo-norharmane (2) (15.0 mg, 0.06 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added allyl bromide (13 lL,
in MeOH and any precipitate removed by filtration. The filtrated
was concentrated and dried under high vacuum affording 27
(20.9 mg, 0.047 mmol, 79%) as a crystalline solid. Mp = 203.5–
204.5 °C; 1H NMR (500 MHz, CD3OD) d 9.39 (s, 1H), 8.66–8.65
(m, 2H), 8.66 (dd, J1 = 6.8 Hz, J2 = 1.2 Hz, 1H), 7.94 (dd, J1 = 8.7 Hz,
J2 = 1.6 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 4.82 (t, J = 7.5 Hz, 2H),
3.68 (s, 3H), 2.48 (t, J = 7.2 Hz, 2H), 2.17 (quint, J = 7.5 Hz, 2H),
1.74 (quint, J = 7.2 Hz, 2H); 13C NMR (125 MHz, CD3OD) d 173.8,
143.2, 135.9, 134.9, 132.5, 132.2, 129.6, 125.7, 121.1, 118.1,
114.4, 114.4, 61.0, 50.8, 32.4, 30.8, 21.1; HRMS-ESI calcd for
0.15 mmol, 2.50 equiv). The flask was sealed and heated at 85 °C
for 15 h. The reaction was cooled to rt, the precipitate filtered,
washed with CH3CN and pentane. The product was dissolved in
MeOH and any precipitate removed by filtration. The filtrated
was concentrated and dried under high vacuum affording 25
(14.6 mg, 0.04 mmol, 66%) as a crystalline solid. An analytical sam-
ple was recrystallized (Et2O/hexane) for X-ray analysis.
Mp = 195.0–196.0 °C; 1H NMR (500 MHz, CD3OD) d 9.35 (s, 1H),
8.75 (d, J = 6.4 Hz, 1H), 8.68 (d, J = 1.6 Hz, 1H), 8.62 (dd,
J1 = 6.4 Hz, J2 = 1.2 Hz, 1H), 7.95 (dd, J1 = 8.7 Hz, J2 = 2.0 Hz, 1H),
7.76 (d, J = 8.7 Hz, 1H), 6.30 (m, 1H), 5.57 (dd, J1 = 9.9, J2 = 1.2 Hz),
5.56 (dd, J1 = 15.9 Hz, J2 = 1.2 Hz), 5.43 (d, J = 6.0 Hz, 3H); 13C
NMR (125 MHz, CD3OD) d 143.3, 135.9, 135.0, 132.5, 132.4,
131.4, 129.6, 125.7, 121.3, 121.1, 118.2, 114.5, 114.4, 63.0;
HRMS-ESI calcd for C14H12N2Br: [M]+ 287.0184; found 287.0179;
C17H18N2O2Br: [M]+ 361.0552; found 361.0555; FTIR
m 3024s,
2986s, 2947s, 2913s, 2843m, 1736s, 1643s, 1612w, 1574w,
1520m, 1489s, 1447s, 1366m, 1285s, 1242s, 1200s, 1153s, 1126s,
1092m, 972m, 922m, 872s, 826s, 741s cmꢁ1
.
4.2.18. 2-Benzyl-6-bromo-9H-beta-carbolin-2-ium bromide
(28)
To a solution of 6-bromo-norharmane (2) (15.0 mg, 0.06 mmol,
FTIR
1358w, 1315m, 1281s, 1254s, 1123s, 1053s, 991m, 937s, 833s,
814s, 768s, 725m cmꢁ1
m
3024m, 2997m, 1643s, 1570w, 1512m, 1489s, 1450s,
1.00 equiv) in CH3CN (0.5 mL) was added benzyl bromide (18 lL,
0.15 mmol, 2.50 equiv). The flask was sealed and heated at 85 °C
for 15 h. The reaction was cooled to rt, the precipitate filtered,
washed with CH3CN and pentane. The product was dissolved in
MeOH and any precipitate removed by filtration. The filtrated
was concentrated and dried under high vacuum affording 28
(25.0 mg, 0.06 mmol, quant.) as a crystalline solid. Mp = 235.5–
236.5 °C; 1H NMR (500 MHz, CD3OD) d 9.45 (s, 1H), 8.73 (d,
J = 6.0 Hz, 1H), 8.71 (dd, J1 = 6.4 Hz, J2 = 1.2 Hz, 1H), 8.66 (dd,
J1 = 2.0 Hz, J2 = 0.8 Hz, 1H), 7.93 (dd, J1 = 8.7 Hz, J2 = 2.0 Hz, 1H),
7.74 (d, J = 8.3 Hz, 1H), 7.57 (dd, J1 = 7.9 Hz, J2 = 2.0 Hz, 2H), 7.51–
7.47 (m, 3H), 5.99 (s, 2H); 13C NMR (125 MHz, CD3OD) d 143.3,
135.9, 135.0, 134.3, 132.5, 132.4, 129.5, 129.4, 129.2, 128.4,
125.7, 121.1, 118.3, 114.5, 114.4, 64.1; HRMS-ESI calcd for
.
4.2.15. Crystallographic details for compound 25
total of 51084 reflections (ꢁ10 6 h 6 10, ꢁ29 6 k 6 29,
ꢁ9 6 l 6 9) were collected at T = 100(2) K in the range of 3.37–
27.50° of which 3031 were unique (Rint = 0.0461); Mo K radiation
A
a
(k = 0.71073 Å). The residual peak and hole electron densities were
0.604 and ꢁ0.660 eÅꢁ3, respectively. The absorption coefficient
was 6.123 mmꢁ1. The least squares refinement converged nor-
mally with residuals of R(F) = 0.0260 (observed data), wR(F2) =
0.0625 (all data) and a GoF = 1.153. C14H12Br2N2, Mw = 368.08,
space group P21/c, monoclinic, a = 8.1536(7), b = 22.969(2),
c = 7.4050(6) Å, b = 107.832(8)°, V = 1320.2(2) Å3, Z = 4, qcalcd
=
C18H14N2Br: [M]+ 337.0340; found 337.0336; FTIR
m 3021m,
2986m, 2943m, 2889m, 2843m, 1647m, 1566w, 1520w, 1489s,
1.852 Mg/m3. CCDC-755506 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
1454s, 1319m, 1281s, 1254m, 1200w, 1161m, 1126m, 1053m,
1026w, 868m, 814s, 733s, 706s cmꢁ1
.
4.2.19. 6-Bromo-2-(4-fluoro-benzyl)-9H-beta-carbolin-2-ium
bromide (29)
To a solution of 6-bromo-norharmane (2) (25.0 mg, 0.10 mmol,
1.00 equiv) in CH3CN (1.5 mL) was added 4-fluorobenzylbromide
4.2.16. 2-Butyl-6-bromo-9H-beta-carbolin-2-ium iodide (26)
To a solution of 6-bromo-norharmane (2) (15.0 mg, 0.06 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added iodobutane (17 lL,
0.15 mmol, 2.50 equiv). The flask was sealed and heated at 85 °C
for 15 h. The reaction was cooled to rt, the precipitate filtered,
washed with CH3CN and pentane. The product was dissolved in
MeOH and any precipitate removed by filtration. The filtrated
was concentrated and dried under high vacuum affording 26
(11.0 mg, 0.026 mmol, 43%) as a crystalline solid. Mp = 231.5–
232.5 °C; 1H NMR (500 MHz, CD3OD) d 9.38 (s, 1H), 8.73 (d,
J = 6.4 Hz, 1H), 8.66 (d, J = 1.2 Hz, 1H), 8.65 (dd, J1 = 6.4 Hz,
J2 = 1.2 Hz, 1H), 7.93 (dd, J1 = 9.1 Hz, J2 = 2.0 Hz, 1H), 7.75 (d,
J = 8.3 Hz, 1H), 4.80 (t, J = 7.5 Hz, 2H), 2.12 (quint, J = 7.5 Hz, 2H),
1.50 (sext, J = 7.5 Hz, 2H), 1.06 (t, J = 7.5 Hz, 3H); 13C NMR
(125 MHz, CD3OD) d 143.2, 135.9, 134.9, 132.5, 132.2, 129.5,
125.6, 121.1, 118.1, 114.4, 114.4, 61.3, 33.6, 19.2, 12.5; HRMS-ESI
(31 lL, 0.25 mmol, 2.50 equiv). The flask was sealed and heated
at 85 °C for 1 h. The reaction was cooled to rt, the precipitate fil-
tered, washed with CH3CN and pentane. The product was dissolved
in MeOH and any precipitate removed by filtration. The filtrated
was concentrated and dried under high vacuum affording 29
(42.2 mg, 0.096 mmol, 96%) as a crystalline solid. Mp = 279.5–
280.0 °C; 1H NMR (500 MHz, CD3OD) d 9.43 (s, 1H), 8.73–8.66
(m, 3H), 7.94 (dd, J1 = 8.8 Hz, J2 = 1.7 Hz, 1H), 7.75 (d, J = 8.8 Hz,
1H), 7.62 (dd, J1 = 8.5 Hz, J2 = 5.4 Hz, 2H), 7.23 (t, J = 8.5 Hz, 2H),
5.97 (s, 2H); 13C NMR (125 MHz, CD3OD) d 163.8 (d, J = 248.0 Hz),
143.7, 136.3, 135.5, 132.9, 132.8, 131.2 (d, J = 8.8 Hz), 130.8 (d,
J = 3.2 Hz), 129.9, 126.1, 121.5, 118.7, 116.4 (d, J = 22.1 Hz), 114.9,
114.8, 63.6; HRMS-ESI calcd for C18H13FN2Br: [M]+ 355.0246;
calcd for C15H16N2Br: [M]+ 303.0497; found 303.0508; FTIR
m
found 355.0232; FTIR
1605w, 1508s, 1489s, 1454s, 1350m, 1277s, 1250m, 1223s,
1165s, 1119s, 1053m, 826s, 779s, 698s cmꢁ1
m 3040m, 2982m, 2943m, 2886m, 1647m,
3028s, 2994s, 2955s, 2855m, 1647m, 1570w, 1516m, 1489s,
1447s, 1319m, 1281s, 1254m, 1165m, 1138s, 1049s, 1022w,
.
903w, 864s, 802s, 725s cmꢁ1
.
4.2.20. 6-Bromo-2-(3-fluoro-benzyl)-9H-beta-carbolin-2-ium
bromide (30)
To a solution of 6-bromo-norharmane (2) (25.0 mg, 0.10 mmol,
1.00 equiv) in CH3CN (1.5 mL) was added 3-fluorobenzylbromide
4.2.17. 6-Bromo-2-(4-methoxycarbonyl-butyl)-9H-beta-
carbolin-2-ium bromide (27)
To a solution of 6-bromo-norharmane (2) (15.0 mg, 0.06 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added methyl bromovalerate
(31 lL, 0.25 mmol, 2.50 equiv). The flask was sealed and heated at
(22
l
L, 0.15 mmol, 2.50 equiv). The flask was sealed and heated
85 °C for 22 h. The reaction was cooled to rt, the precipitate filtered,
washed with CH3CN and pentane. The product was dissolved in
MeOH and any precipitate removed by filtration. The filtrated was
at 85 °C for 15 h. The reaction was cooled to rt, the precipitate fil-
tered, washed with CH3CN and pentane. The product was dissolved

1474
S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
concentrated and dried under high vacuum affording 30 (19.3 mg,
0.044 mmol, 44%) as a crystalline solid. Mp = 237.0–238.0 °C; 1H
NMR (500 MHz, CD3OD) d 9.48 (s, 1H), 8.74 (d, J = 6.8 Hz, 1H),
8.72 (dd, J1 = 6.4 Hz, J2 = 1.2 Hz, 1H), 8.64 (d, J = 2.0 Hz, 1H), 7.91
(dd, J1 = 9.1 Hz, J2 = 2.0 Hz, 1H), 7.74 (d, J = 9.1 Hz, 1H), 7.54–7.50
(m, 1H), 7.40–7.36 (m, 2H), 7.22 (ddd, J1 = 9.1 Hz, J2 = 8.3 Hz,
J3 = 2.4 Hz, 1H), 6.02 (s, 2H); 13C NMR (125 MHz, CD3OD) d 165.4
(d, J = 247.4 Hz), 145.6, 138.9 (d, J = 8.2 Hz), 138.1, 137.3, 134.8,
134.7, 133.4 (d, J = 8.2 Hz), 131.9, 128.0, 126.5 (d, J = 2.7 Hz),
123.3, 120.6, 118.4 (d, J = 21.1 Hz), 117.6 (d, J = 23.9 Hz), 116.8,
116.7, 65.5; HRMS-ESI calcd for C18H13FN2Br: [M]+ 355.0246; found
33 (22.9 mg, 0.049 mmol, 82%) as a crystalline solid. Mp = 223.5–
224.0 °C; 1H NMR (500 MHz, CD3OD) d 9.49 (s, 1H), 8.78–8.73
(m, 2H), 8.67 (d, J = 1.6 Hz, 1H), 8.11 (s, 1H), 7.99–7.91 (m, 4H),
7.75 (d, J = 9.1 Hz, 1H), 7.60–7.58 (m, 3H), 6.15 (s, 2H); 13C NMR
(125 MHz, CD3OD) d 143.3, 136.0, 135.1, 133.6, 133.4, 132.6,
132.5, 131.5, 129.6, 129.3, 128.3, 127.9, 127.5, 127.0, 126.7,
125.7, 124.9, 121.1, 118.3, 114.5, 114.4, 64.3; HRMS-ESI calcd for
C22H16N2Br: [M]+ 387.0497; found 387.0499; FTIR
m 3615w,
3537w, 3368w, 3040m, 2986m, 2936m, 2882m, 2839m, 2797m,
2646w, 1643m, 1609w, 1520m, 1489s, 1450m, 1319m, 1281s,
1157m, 1126s, 1053m, 968w, 872m, 818s, 775s, 733s, 706m cmꢁ1
.
355.0232; FTIR
1643m, 1593m, 1516m, 1485s, 1450s, 1319m, 1281s, 1254s,
1150m, 1123s, 1053m, 876m, 806s, 752s cmꢁ1
m 3453w, 3040m, 2947m, 2893m, 2839m, 2696w,
4.2.24. 8-Bromo-2-ethyl-9H-beta-carbolin-2-ium iodide (35)
.
To
0.05 mmol, 1.00 equiv) in CH3CN (0.5 mL) was added ethyl iodide
(10 L, 0.13 mmol, 2.50 equiv). The flask was sealed and heated
a
solution of 8-bromo-norharmane (34) (10.0 mg,
4.2.21. 6-Bromo-2-(4-nitro-benzyl)-9H-beta-carbolin-2-ium
bromide (31)
l
at 85 °C overnight. The reaction was cooled to rt, the precipitate fil-
tered, washed with CH3CN and pentane. The product was dissolved
in MeOH and any precipitate removed by filtration. The filtrated
was concentrated and dried under high vacuum affording 35
(4.60 mg, 0.011 mmol, 23%) as a crystalline solid. Mp = 292.0–
293.0 °C; 1H NMR (500 MHz, CD3OD) d 9.28 (s, 1H), 8.77 (d,
J = 6.4 Hz, 1H), 8.70 (d, J = 6.4 Hz, 1H), 8.48 (d, J = 7.9 Hz, 1H),
8.06 (d, J = 7.5 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 4.86 (q, J = 7.9 Hz,
2H), 1.77 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) d 143.1,
135.9, 134.4, 133.7, 132.6, 129.3, 123.0, 122.4, 121.1, 118.5,
105.1, 57.0, 16.0; HRMS-ESI calcd for C13H12N2Br: [M]+ 275.0184;
To a solution of 6-bromo-norharmane (2) (15.0 mg, 0.06 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added 4-nitrobenzyl bromide
(32.4 mg, 0.15 mmol, 2.50 equiv). The flask was sealed and heated
at 85 °C for 5 h. The reaction was cooled to rt, the precipitate filtered,
washed with CH3CN and pentane. The product was dissolved in
MeOH and any precipitate removed by filtration. The filtrated was
concentrated and dried under high vacuum affording 31 (27.6 mg,
0.060 mmol, 99%) as a crystalline solid. Mp = 261.0–262.0 °C; 1H
NMR (500 MHz, CD3OD) d 9.52 (s, 1H), 8.79 (d, J = 6.4 Hz, 1H), 8.74
(d, J = 6.0 Hz, 1H), 8.70 (s, 1H), 8.34 (d, J = 8.3 Hz, 2H), 7.96 (d,
J = 8.3 Hz, 1H), 7.78–7.74 (m, 3 H), 6.16 (s, 2H); 13C NMR (125 MHz,
CD3OD) d 148.5, 143.5, 141.2, 136.0, 135.3, 132.8, 132.7, 130.1,
129.3, 125.8, 124.0, 121.1, 118.5, 114.7, 114.5, 62.8; HRMS-ESI calcd
found 275.0182; FTIR
1555m, 1497m, 1470s, 1327s, 1246m, 1215m, 1130s, 1034m,
837s, 791s, 748s cmꢁ1
m 3356w, 3048m, 3009m, 2326w, 1643m,
.
for C18H13N3O2Br: [M]+ 382.0191; found 382.0183; FTIR
m 3140w,
3048m, 3009m, 2955w, 2855w, 1643m, 1605w, 1516s, 1489s,
4.2.25. 2-Allyl-8-bromo-9H-beta-carbolin-2-ium bromide (36)
To a solution of 8-bromo-norharmane (34) (10.0 mg, 0.05 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added allyl bromide (11 lL,
1450m, 1339s, 1285s, 1258m, 1223m, 1161m, 1057w, 945m,
856m, 818s, 729s cmꢁ1
.
0.13 mmol, 2.50 equiv). The flask was sealed and heated at 85 °C
overnight. The reaction was cooled to rt, the precipitate filtered,
washed with CH3CN and pentane. The product was dissolved in
MeOH and any precipitate removed by filtration. The filtrated was
concentrated and dried under high vacuum affording 36 (4.50 mg,
0.012 mmol, 24%) as a crystalline solid. Mp = 220.0–221.0 °C; 1H
NMR (500 MHz, CD3OD) d 9.25 (s, 1H), 8.79 (d, J = 6.4 Hz, 1H), 8.66
(dd, J1 = 6.4 Hz, J2 = 1.2 Hz, 1H), 8.49 (dd, J1 = 7.9 Hz, J2 = 0.8 Hz,
1H), 8.07 (dd, J1 = 7.5 Hz, J2 = 0.8 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H),
6.34–6.26 (m, 1H), 5.58 (dd, J1 = 10.3 Hz, J2 = 1.2 Hz, 1H), 5.57 (dd,
J1 = 16.7 Hz, J2 = 1.2 Hz, 1H), 5.45 (d, J = 6.4 Hz, 2H); 13C NMR
(125 MHz, CD3OD) d 143.2, 135.8, 134.5, 134.0, 133.0, 131.4, 129.5,
123.1, 122.4, 121.4, 121.0, 118.5, 105.1, 63.1; HRMS-ESI calcd for
4.2.22. 6-Bromo-2-(3-phenyl-propyl)-9H-beta-carbolin-2-ium
bromide (32)
To a solution of 6-bromo-norharmane (2) (15.0 mg, 0.06 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added 1-bromo-3-phenylpro-
pane(23 lL, 0.15 mmol, 2.50 equiv). Theflaskwas sealedand heated
at 85 °C for 15 h. The reaction was cooled to rt, the precipitate fil-
tered, washed with CH3CN and pentane. The product was dissolved
in MeOH and any precipitate removed by filtration. The filtrated was
concentrated and dried under high vacuum affording 32 (25.2 mg,
0.056 mmol, 94%) as a crystalline solid. Mp = 257.5–258.0 °C; 1H
NMR (500 MHz, CD3OD) d 9.30 (s, 1H), 8.68 (d, J = 6.8 Hz, 1H), 8.65
(d, J = 1.6 Hz, 1H), 8.62 (dd, J1 = 6.4 Hz, J2 = 0.8 Hz, 1H), 7.93 (dd,
J1 = 9.1 Hz, J2 = 2.0 Hz, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.25 (s, 2H), 7.24
(s, 2H), 7.12 (m, 1H), 4.83 (t, J = 7.5 Hz, 2H), 2.83 (t, J = 7.2 Hz, 2H),
2.49 (quint, J = 7.5 Hz, 2H); 13C NMR (125 MHz, CD3OD) d 143.1,
140.0, 135.8, 134.9, 132.4, 132.2, 129.6, 128.2, 128.0, 125.9, 125.6,
121.1, 118.1, 114.4, 114.3, 61.2, 32.6, 32.1; HRMS-ESI calcd for
C14H12N2Br: [M]+ 287.0184; found 287.0178; FTIR
m 3352w,
3051m, 3017m, 2974m, 2905m, 2858m, 1647m, 1616w, 1558m,
1501m, 1470s, 1327s, 1300m, 1219m, 1138m, 1115m, 1034m,
1011m, 953m, 810m, 783s, 745s, 683m cmꢁ1
.
C20H18N2Br: [M]+ 365.0653; found 365.0653; FTIR
m
3410w, 3024s,
4.2.26. 2-Benzyl-8-bromo-9H-beta-carbolin-2-ium bromide
2986s, 2943s, 2839m, 1639s, 1609m, 1570w, 1516w, 1489s,
1450s, 1315m, 1281s, 1254s, 1157s, 1126s, 1049m, 972w, 907w,
(37)
To
0.05 mmol, 1.00 equiv) in CH3CN (0.5 mL) was added benzyl bro-
mide (15 L, 0.13 mmol, 2.50 equiv). The flask was sealed and
a
solution of 8-bromo-norharmane (34) (10.0 mg,
876s, 826s, 822s, 733s, 694s cmꢁ1
.
l
4.2.23. 6-Bromo-2-naphthalen-2-ylmethyl-9H-beta-carbolin-2-
ium bromide (33)
heated at 85 °C overnight. The reaction was cooled to rt, the precip-
itate filtered, washed with CH3CN and pentane. The product was
dissolved in MeOH and any precipitate removed by filtration. The
filtrated was concentrated and dried under high vacuum affording
37 (9.10 mg, 0.022 mmol, 44%) as a crystalline solid. Mp = 235.0–
236.0 °C; 1H NMR (500 MHz, CD3OD) d 9.34 (s, 1H), 8.77 (d,
J = 6.4 Hz, 1H), 8.75 (d, J = 6.4 Hz, 1H), 8.46 (d, J = 7.9 Hz, 1H),
8.04 (d, J = 7.5 Hz, 1H), 7.58–7.56 (m, 2H), 7.53–7.48 (m, 3H),
7.43 (t, J = 7.5 Hz, 1H), 6.02 (s, 2H); 13C NMR (125 MHz, CD3OD) d
To a solution of 6-bromo-norharmane (2) (15.0 mg, 0.06 mmol,
1.00 equiv) in CH3CN (0.5 mL) was added 2-bromomethyl naph-
thalene (33.2 mg, 0.15 mmol, 2.50 equiv). The flask was sealed
and heated at 85 °C for 5 h. The reaction was cooled to rt, the pre-
cipitate filtered, washed with CH3CN and pentane. The product was
dissolved in MeOH and any precipitate removed by filtration. The
filtrated was concentrated and dried under high vacuum affording

S. Bonazzi et al. / Bioorg. Med. Chem. 18 (2010) 1464–1476
1475
143.2, 135.8, 134.5, 134.2, 133.9, 133.1, 129.5, 129.4, 129.3, 128.5,
123.1, 122.4, 121.0, 118.6, 105.1, 64.1; HRMS-ESI calcd for
MHOM-ET/67/L82 axenic amastigotes as well as for cytotoxicity
using L6 cells were carried out as previously reported.34
C18H14N2Br: [M]+ 337.0340; found 337.0350; FTIR
m
3399w,
3055m, 3017m, 1643m, 1562w, 1520m, 1497m, 1470s, 1454m,
1327s, 1254m, 1119m, 1034w, 818m, 787m, 748s, 706s cmꢁ1
Acknowledgments
.
K.G. is a European Young Investigator (EURYI) and thanks the
Swiss National Science Foundation (SNF) for support (PE002-
117136/1). Project support by the ETH (TH Gesuch 13/04-3) and
the SNF (project no. 200021-115918/1) is gratefully acknowledged.
This investigation received financial support from the UNICEF/
UNDP/World Bank/WHO Special Program for Research and Train-
ing in Tropical Diseases (TDR).
4.2.27. 8-Bromo-2-(4-fluoro-benzyl)-9H-beta-carbolin-2-ium
bromide (38)
To
0.05 mmol, 1.00 equiv) in CH3CN (0.5 mL) was added 4-fluoroben-
zyl bromide (16 L, 0.13 mmol, 2.50 equiv). The flask was sealed
a
solution of 8-bromo-norharmane (34) (10.0 mg,
l
and heated at 85 °C overnight. The reaction was cooled to rt, the
precipitate filtered, washed with CH3CN and pentane. The product
was dissolved in MeOH and any precipitate removed by filtration.
The filtrated was concentrated and dried under high vacuum
affording 38 (5.50 mg, 0.013 mmol, 25%) as a crystalline solid.
Mp = 259.5–260.5 °C; 1H NMR (500 MHz, CD3OD) d 9.34 (s, 1H),
8.78 (d, J = 6.8 Hz, 1H), 8.74 (dd, J1 = 6.4 Hz, J2 = 0.8 Hz, 1H), 8.47
(d, J = 8.3 Hz, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.64 (dd, J1 = 8.7 Hz,
J2 = 5.2 Hz, 2H), 7.44 (t, J = 7.9 Hz, 1H), 7.25 (t, J = 8.7 Hz, 2H),
6.01 (s, 2H); 13C NMR (125 MHz, CD3OD) d 163.5 (d, J = 248.4 Hz),
143.2, 135.8, 134.6, 134.0, 133.0, 130.9 (d, J = 8.2 Hz), 130.23 (d,
J = 2.7 Hz), 129.4, 123.1, 122.5, 121.0, 118.7, 116.1 (d, J = 22.9 Hz),
105.1, 63.2; HRMS-ESI calcd for C18H13N2BrF: [M]+ 355.0246;
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.01.013.
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1562m, 1512m, 1497m, 1470s, 1327s, 1250m, 1138m, 1115m,
860m, 810m, 783s, 748s cmꢁ1
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39 (10.0 mg, 0.021 mmol, 43%) as a crystalline solid. Mp = 244.5–
245.0 °C; 1H NMR (500 MHz, CD3OD) d 9.40 (s, 1H), 8.81 (dd,
J1 = 6.4 Hz, J2 = 1.2 Hz, 1H), 8.77 (d, J = 6.4 Hz, 1H), 8.46 (d,
J = 7.9 Hz, 1H), 8.12 (s, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.99 (d,
J = 8.7 Hz, 1H), 7.97 (dd, J1 = 6.0 Hz, J2 = 3.6 Hz, 1H), 7.93 (dd,
J1 = 6.0 Hz, J2 = 3.6 Hz, 1H), 7.61–7.58 (m, 3H), 7.43 (t, J = 7.9 Hz,
1H), 6.19 (s, 2H); 13C NMR (125 MHz, CD3OD) d 143.2, 135.8,
134.5, 134.0, 133.7, 133.4, 133.2, 131.4, 129.5, 129.3, 128.4, 127.9,
127.5, 127.0, 126.8, 125.0, 123.1, 122.4, 121.0, 118.6, 105.1, 64.3;
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FTIR
m 3372m, 3051m, 3013m, 2928m, 1643m, 1562w, 1516m,
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.
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